A2B adenosine receptor-triggered intracellular calcium mobilization: Cell type-dependent involvement of Gi, Gq, Gs proteins and protein kinase C

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Gao, Clayton K. Meyer, Kenneth A. Jacobson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5442142/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2025 Read the published version in Purinergic Signalling → Version 1 posted 11 You are reading this latest preprint version Abstract Activation of PLCβ enzymes by G iβγ and G αq/11 proteins is a common mechanism to trigger cytosolic Ca 2+ increase. We and others reported that G αq/11 inhibitor FR900358 (FR) can inhibit both and G αq - and, surprisingly, G iβγ -mediated intracellular Ca 2+ mobilization. Thus, the G αi -G βγ -PLCβ-Ca 2+ signaling axis depends entirely on the presence of active G αq , which reasonably explained FR-inhibited G iβγ -induced Ca 2+ release. However, the conclusion that G iβγ signaling is controlled by G αq derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G αq/11 siRNA and G αq/11 inhibitors diminished Ca 2+ increase triggered by native G q -coupled P2Y 1 receptors, or by transfected G i -coupled A 1 - or G s -coupled A 2B adenosine receptors (ARs). However, in T24 bladder cancer cells, G i inhibitor PTX, but not G αq/11 inhibitors, FR, YM254890 (YM) or G q/11 siRNA, inhibited Ca 2+ increase triggered by native A 2B AR activation. Simultaneous inactivation of G i and G s further suppressed A 2B AR-triggered Ca 2+ increase in T24 cells. The G αq/11 inhibitor YM fully and partially inhibited endogenous P2Y 1 - and β 2 -adrenergic receptor-induced Ca 2+ increase in T24 cells, respectively. PKC activator PMA partially diminished A 2B AR-triggered but completely diminished β 2 -adrenergic receptor-triggered Ca 2+ increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected A 2B AR-mediated Ca 2+ increase. Unlike in T24 cells, YM inhibited native A 2B AR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G αq/11 is vital for Ca 2+ increase in some cell types, but G iβγ -mediated Ca 2+ signaling can be Gα q/11 -dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca 2+ increase depending on cell type and receptor. A2B adenosine receptor GPCR G protein calcium Gq Gi Gs. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Intracellular calcium mobilization triggered by G protein-coupled receptors (GPCRs), which ~ 34% of prescription drugs target [1], affects all aspects of cell functions including neurotransmitter release, insulin secretion, T cell activation, muscle contraction, immunity and cell survival and proliferation [2]. Activation of PLCβ enzymes by a GPCR is considered as a common mechanism to trigger Ca 2+ increase in cells. Both G iβγ and G αq , activated by G i -coupled and G q -coupled receptors, respectively, can trigger PLC activation [3] or intracellular calcium mobilization [4–7]. We previously compared the G protein-inhibitory effects of FR900358 (FR, UBO, UBO-QIC [8]) and pertussis toxin (PTX) on various GPCR signaling pathways [9]. For example, FR (UBO, at 300 nM), had no effect on A 2B adenosine receptor (AR)-G αs -mediated cAMP accumulation or A 1 AR- G αi -mediated inhibition of forskolin-stimulated cAMP accumulation in CHO cells [9]. Nevertheless, FR (100 nM) produced a nearly complete inhibition of G iβγ -mediated calcium mobilization, or IP1 accumulation, upon the activation of the G i -coupled A 1 AR, the M 2 muscarinic acetylcholine receptor, or the P2Y 12 receptor (P2Y 12 R, responds to ADP) overexpressed in either CHO or 1321N1 astrocytoma cells. However, 100 nM FR had no effect on A 1 AR-mediated Akt1/2/3 phosphorylation, which was completely inhibited by PTX. FR (100 nM) produced a small (< 30%) but significant inhibition of A 1 AR-mediated ERK1/2 activity [9]. It is also of note that FR, at both 100 and 300 nM, only partially inhibited ERK1/2 activity triggered by the G q/11 -coupled M 3 acetylcholine or P2Y 1 (responds to ADP) receptor, although the calcium response triggered by these two receptors was completely inhibited. In a follow-up study, Pfeil et al. [10] further explored the potential mechanism related to FR-inhibited G iβγ -triggered calcium signaling in one signaling pathway mentioned above, i.e. calcium signaling, but not Akt or ERK1/2 signaling. They confirmed our finding [9] and suggested that “the G αi -G βγ -PLCβ-Ca 2+ signaling axis is entirely dependent on the presence of active G αq . If G αq is pharmacologically inhibited or genetically ablated, G αq can bind to PLCβ but does not elicit Ca 2+ signals.” The finding related to G αq/11 control of G iβγ -mediated Ca 2+ signaling is an important discovery, since the mechanisms related to Ca 2+ increase in cells is vital to the discovery of drugs targeting different receptor classes. However, the conclusion that G αq controls G iβγ -mediated Ca 2+ increases via removal of PLC auto-inhibition was drawn mostly from the study of native and G protein-knockout HEK293 cells [10]. We have previously shown the critical role of G αq/11 in A 2B AR-triggered calcium mobilization in HEK293 cells overexpressing the recombinant human (h) A 2B AR [12,13]. In A 2B AR-overexpressing HEK293 cells, both CRISPR-Cas9-based G q/11 knockout and the G q inhibitor FR completely eliminated the A 2B AR-mediated Ca 2+ increase [12,13]. It is of note that the native A 2B AR in HEK293 cells is capable of inducing cAMP accumulation via G s and ERK1/2 activity increase via G i , but it is unable to trigger a robust Ca 2+ signal [12,13]. However, when hA 2B AR is overexpressed in HEK293 cells, a robust intracellular Ca 2+ mobilization signal mediated via G αq/11 is induced [12–14], which suggests that A 2B AR overexpression could enable the coupling to G αq/11 . The A 2B AR has emerged as an important target for various conditions, including cancer, cardiac ischemia and sarcopenia [15–20]. Calcium signaling is a major signaling pathway involved in these critical conditions. Thus, it is important to better understand the A 2B AR downstream signaling, including Ca 2+ mobilization. The cytosolic Ca 2+ increase triggered by the nonselective AR agonist NECA has been explored using T24 bladder cancer cells expressing the native hA 2B AR [12,21]. NECA-induced Ca 2+ increase in T24 cells can be completely blocked by a selective A 2B AR antagonist PSB603 and diminished by A 2B AR siRNA [12]. In addition, Panjehpour et al. [22] reported that a NECA-triggered Ca 2+ signal in MDA-MB-231 breast cancer cells also occurs via the A 2B AR. In the present study we further examined the mechanisms of A 2B AR-triggered Ca 2+ release, especially whether G iβγ -triggered Ca 2+ release can be blocked by G αq/11 siRNA or pharmacological inhibitors, in HEK293 cells overexpressing the recombinant A 2B AR, and in T24 bladder cancer cells expressing a native A 2B AR. Thus, we examined the effects of two G αq/11 inhibitors: FR and YM-254890 (YM [23]), as well as G αq/11 siRNA, the G i inactivator PTX, and the G s inactivator CTX. We found that, unlike in HEK293 cells, G iβγ , but not G q/11 , plays a critical role in A 2B AR-triggered Ca 2+ increase in T24 bladder cancer cells. We also analyzed the role of G s (relative to G i ) in A 2B AR-mediated Ca 2+ release, the synergistic effects between G i and G s , and cell type- and receptor-dependent modulation by protein kinase C (PKC). In some cases, we compared the A 2B AR-mediated effects with those of other purinergic receptors or biogenic amine receptor. Materials and Methods Materials BAY60-6583 (LUF6210) was provided by Prof. Ad IJzerman at Leiden-Amsterdam Center for Drug Research (Leiden, The Netherlands). Nonselective AR agonist adenosine-5′- N -ethyluronamide (NECA), β 2 adrenergic receptor agonists formoterol and isoproterenol, lysophosphatidic acid (LPA) receptor agonist LPA, YM254890, PSB603, ESI-09, H89, PKC activator phorbol 12-myristate 13-acetate (PMA), and PKC inhibitor Go6983 were from Tocris (Minneapolis, MN, USA). FR900359 (UBO-QIC) was purchased from University of Bonn (Germany). HEK293 cells, T24 bladder cancer cells, and MDA-MB-231 cells were from ATCC (Manassas, VA, USA). HEK293-A2B cells were made at the Laboratory of Bioorganic Chemistry, NIDDK, NIH (Bethesda, MD, USA). An AlphaScreen cAMP kit was purchased from PerkinElmer (Waltham, MA). Calcium assay kits were from Molecular Devices (Sunnyvale, CA, USA). All other reagents were from standard commercial sources and of analytical grade. RNA extraction and quantitative real-time PCR detection of gene expression level Total RNA was extracted from 10 7 cells using a RNeasy kit (Qiagen, Redwood City, CA, USA) and was reversed-transcribed using the SuperScript™ III First-Strand Synthesis SuperMix kit (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocol and as described [24]. Briefly, the thermocycles were as follows: 25 ○ C for 10 min; 50 ○ C for 30 min; 85 ○ C for 5 min and then chilled on ice; 1 µl (2 U) of E Coli RNase H was added and incubation continued for 20 min before storage at -20 ○ C until use. The cDNA was then amplified with TaqMan gene expression assays (ThermoFisher, Waltham, MA USA) for four AR subtypes and GAPDH on a CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) according to the manufacturer’s protocol. The temperature cycles were: 50 ○ C for 2 min; 95 ○ C for 10 min; 95 ○ C 15s and 60 ○ C for 30s for 40 cycles. The ΔΔCt method was used to conduct quantitative analysis of data. Values were normalized to GAPDH and then expressed as relative expression levels. Cell culture and measurement of cyclic AMP levels HEK293, T24 bladder cancer and MDA-MB-231 breast cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 µmol/ml glutamine. For the assay of 3′,5′-cyclic adenosine monophosphate (cAMP), cells were plated in 96-well (4x10 4 cells/well) clear plates in 100 µl of medium overnight. Cell culture medium was then replaced with 80 µl HBSS buffer containing 20 mM HEPES, phosphodiesterase inhibitor rolipram (10 µM), and 3 units/ml adenosine deaminase (Worthington Biochemical, Lakewood, NJ, USA) for 30 min followed by agonist addition to the mixture, which was then incubated for 20 min. The treatment of YM or FR was 20 min before agonist addition. The treatment with CTX (500 ng/ml or PTX (200 ng/ml) was for overnight before agonist addition. The reaction was terminated by aspirating the reaction mixture and the addition of 100 µl cold 0.3% Tween-20 to each well. Cells were then shaken at room temperature for 10 min. For the determination of cAMP production, an AlphaScreen cAMP kit was used according to the manufacturer’s instructions (Revvity/PerkinElmer, Waltham, MA, USA). Measurement of intracellular calcium increase The measurement of calcium mobilization was essentially as described previously [19,24]. We utilized a calcium assay kit as directed without washing cells and with probenecid added to the loading dye at a final concentration of 2.5 mM to increase dye retention. Briefly, cells were grown at 37°C/5% CO 2 in a set of 96-well black-wall, clear-bottom plates overnight, or until they reach confluence. Then, media was aspirated, and 100 µl of dye (Calcium 6) was added to each well. Afterward, the plates were subsequently maintained at room temperature in the dark for 60 min. Finally, 50 µl of compound or a control agonist was added into respective assay plate wells during the determination of intracellular Ca 2+ using a FLIPR (Molecular Devices, San Jose, CA, USA). The treatment of YM and FR was 20 min before agonist addition. The treatment with CTX (500 ng/ml) or PTX (200 ng/ml) was for overnight before agonist addition. The compound plate was prepared using dilutions of various compounds in HBSS buffer (pH 7.4) without added calcium. Samples were run in duplicate or triplicate at room temperature. Cell fluorescence (excitation = 485 nm; emission = 525 nm) was monitored following compound exposure. Increases in intracellular Ca 2+ are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure. Statistical and data analyses Functional parameters were calculated using Prism 10.2.3 software (GraphPad, San Diego, CA, USA). Data was expressed as mean ± standard error. A Student’s t-test (between two conditions) or a One-Way Analysis of Variance (ANOVA) followed by Tukey’s or Bonferroni’s multiple comparison tests (between multiple conditions) was used to compare statistically significant differences. Differences yielding P values < 0.05 are considered statistically significant. Results HEK293 cells: G αq/11 protein contribution to A 2B AR-mediated intracellular calcium mobilization Here, we further explore the G αi -G βγ -PLCβ-Ca 2+ signaling axis, previously demonstrated to be inhibited by G αq/11 inhibitor FR and Gq/11 knockout [10,13]. In addition to FR, G q/11 siRNA and another G q/11 inhibitor, YM, inhibited calcium mobilization in HEK293 cells, endogenously expressing the G q -coupled P2Y 1 R, or overexpressing the recombinant human G i -coupled A 1 or G i - and G s -coupled A 2B ARs (Fig. 1 ). These findings are consistent with previous pharmacological results [9] and align with the proposed mechanism that G iβγ -mediated calcium signaling is under G q/11 control in HEK293 cells [10]. Thus, the results from the present study using FR, YM, G q/11 siRNA knockdown and prior CRISPR-Cas9-based G q/11 knockout research [10] are all consistent. Unlike the G q inhibitors FR and YM, treatment with the G i inhibitor PTX had no effect on G q/11 -coupled P2Y 1 R agonist MRS2365-induced Ca 2+ release in HEK293 cells (Fig. 2 D), but completely inhibited the A 1 AR agonist CCPA-induced Ca 2+ signaling in HEK293-A1 cells (Fig. 2 C). The A 2B AR-mediated increase of ERK1/2 activity in HEK293-A2B cells has been reported to be G i -dependent by Yang et al. [25] based on PTX sensitivity, which was later confirmed with both PTX and CRISPR-Cas9-based Gq/11 knockout [13]. However, A 2B AR-mediated Ca 2+ mobilization in HEK293-A2B cells is G i -independent, as incubation with 200 ng/ml PTX for 18 hours did not affect the NECA-induced Ca 2+ increase [14]. It is of note that the native A 1 AR expression level in HEK293 cells is extremely low, and A 1 AR agonist CCPA does not trigger Ca 2+ signaling [12,13]. The native A 2B AR in HEK293 cells can induce robust cAMP accumulation and ERK1/2 activity but not sufficient to trigger a robust calcium signaling [12,13,19]. In HEK293 cells, there is a possibility that the overexpression of G i -coupled A 1 or G s -coupled A 2B ARs enable their stronger coupling to G αq/11 , and thus G αq/11 can subsequently control the A 1 AR-mediated G iβγ -Ca 2+ signaling. FR inhibition of native G q/11 -coupled P2Y 1 R-induced calcium release is expected. In addition, LPA (lysophosphatidic acid)-induced intracellular calcium mobilization (EC 50 = 4.59 µM) was also completely inhibited by 300 nM YM (Fig. 2 E). HEK293 cells: Similar potencies of G αq/11 inhibitors on G iβγ - versus G αq -mediated stimulation of intracellular calcium mobilization The A 1 AR and P2Y 1 R are G i -coupled and G q -coupled receptors, respectively. We compared the potencies of FR and YM in inhibiting A 1 AR (overexpressed)-triggered calcium mobilization by A 1 AR agonist CCPA (1 µM) and native P2Y 1 R-mediated Ca 2+ increase by P2Y 1 R agonist MRS2365 (1 µM) in HEK293 cells (Fig. 2 A,B). The IC 50 values FR and YM to inhibit P2Y 1 R-mediated calcium mobilization were 13.2 ± 3.2 and 12.8 ± 4.1 nM, respectively. The IC 50 values for the inhibition of the A 1 AR-mediated effect were 8.66 ± 3.23 and 12.9 ± 2.52 nM, respectively. Additionally, the IC 50 values of YM in inhibiting the effects of 10 µM carbachol and 1 µM CCPA in CHO cells overexpressing the recombinant G i -coupled M 2 muscarinic or A 1 receptor, respectively, were determined to be 9.53 and 11.6 nM, respectively (Fig. 2 C,D). Thus, the potencies of FR and YM for inhibition of G q - and G iβγ -mediated calcium signaling are similar, and the use of either inhibitor at 100–300 nM should completely inhibit both G iβγ - and G αq -triggered Ca 2+ signaling. HEK293 cells: G inhibitors lack effects on G-mediated inhibition or G-mediated stimulation of cAMP accumulation As previously demonstrated in other cell types [9], FR and/or YM did not affect A 1 AR-mediated inhibition or A 2A AR- and A 2B AR-mediated stimulation of cAMP accumulation in HEK293 cells (Fig. 3 ), although they inhibited Ca 2+ signaling induced by those receptors. T24 bladder cancer cells: G q/11 proteins do not play a major role in native A 2B AR-mediated intracellular calcium mobilization As described above, the G q/11 pharmacological inhibitors and siRNA diminished the A 2B AR-mediated calcium signaling in HEK293 cells overexpressing the recombinant hA 2B AR, which is consistent with results from CRISPR-Cas9-based G q/11 knockout HEK293 cells [13]. We next carefully examined the intracellular calcium mobilization triggered by the native A 2B AR in T24 bladder cancer cells [12,21]. Figure 4 A shows that, unlike in HEK293 cells, G q/11 siRNA suppressed P2Y 1 R agonist MRS2365-induced calcium mobilization but not A 2B AR agonist NECA-triggered calcium mobilization (Fig. 4 B). The EC 50 s of NECA in the absence and presence of G q/11 siRNA were 234 ± 61 and 247 ± 46 nM, respectively (n = 3), which are not significantly different ( P > 0.05, Student’s t-test ). Similar to the effect of G q/11 siRNA, Fig. 4 C shows that the G q/11 inhibitor YM (300 nM) also completely inhibited the effect of P2Y 1 R agonist MRS2365 but had little if any effect on A 2B AR-induced calcium mobilization in T24 cells. The EC 50 s of NECA in the absence and presence of YM were 427 ± 76 and 524 ± 88 nM, respectively (n = 3), which are not significantly different ( P > 0.05, Student’s t-test ). Figure 4 D shows that, similarly to YM in T24 cells, another G q/11 inhibitor FR (300 nM) also completely inhibited calcium mobilization triggered by the native P2Y 1 R but not by the native A 2B AR. T24 bladder cancer cells: Contribution of both G i and G s proteins to intracellular calcium mobilization triggered by native A 2B ARs Figure 5 A shows that, unlike YM and FR, PTX diminished A 2B AR agonist NECA-mediated calcium mobilization in T24 cells but had no effect on the P2Y 1 R agonist MRS2365. Thus, it is suggested that G i plays a prominent role in A 2B AR-mediated intracellular calcium mobilization in T24 bladder cancer cells. Considering that G iα isoforms do not play a significant role in activation PLCβ [26], it is assumed that G βγ is responsible for this effect (Gao and Jacobson, 2016). Interestingly, simultaneous inactivation of G i and G s by PTX and CTX almost completely eliminated A 2B AR-triggered Ca 2+ release (Fig. 5 A), suggesting both G iβγ and G s are responsible for A 2B AR-mediated Ca 2+ mobilization in T24 cells. The EC 50 s of MRS2365 in the absence and presence of PTX were 1.38 ± 0.26 and 1.55 ± 0.47 nM, respectively, which are not significantly different ( P > 0.05, Student’s t-test ). The EC 50 s of NECA for Control group, PTX group and PTX + CTX group were 538 ± 112, 3550 ± 890 and 12,300 ± 4600 nM, respectively, which are significantly different ( P < 0.05, One-Way Analysis of Variance followed by Bonferroni’s multiple comparison tests). The maximal effect of NECA (in terms of relative fluorescence units) in Control group, PTX group and PTX + CTX group were 505 ± 78, 173 ± 39 and 88 ± 15, respectively, which are significantly different ( P < 0.05, One-Way Analysis of Variance followed by Bonferroni’s multiple comparison tests). To examine whether the pathway downstream of G s , PKA or EPAC, contributes to A 2B -Gs-mediated calcium mobilization in T24 cells, PKA inhibitor H89 and EPAC inhibitor ESI09 were used. Figure 5 B shows that both EPAC and PKA are involved in A 2B AR-G s -mediated calcium mobilization. However, the combination of ESI09 and H89 did not produce an effect larger that ESI09 alone (P > 0.05, Student’s t-test ). The inactivation of G s by CTX produced a similar effect to that of ESI09. In addition to the A 2B AR, T24 cells also endogenously express the β 2 -adrenergic receptor. Figure 6 shows that the β 2 adrenergic agonist formoterol and isoproterenol induced a robust calcium response albeit a little less efficacious compared to the A 2B AR agonist NECA. However, unlike the effects of FR on the A 2B AR agonist NECA-triggered Ca 2+ release, FR (300 nM) diminished Ca 2+ increase triggered by both β 2 -adrenergic receptor agonists formoterol and isoproterenol, suggesting a role of G q/11 (Fig. 6 A,B). In a separate set of experiments, YM (1 µM) was shown to inhibit 78% of the maximum effect (E max ) of formoterol (Fig. 6 C). Unlike its effect on the A 2B AR, PTX treatment did not produce significant attenuation of the E max in β 2 agonist formoterol-induced calcium mobilization (Fig. 6 c). However, CTX treatment produced an effect larger than it was on the NECA-induced Ca 2+ response (Fig. 6 C; Fig. 5 B) in the same cell type, i.e. T24 bladder cancer cells. Thus, even in the same cell type, the involvement of G proteins in intracellular calcium mobilization is dependent on receptor type, although both the A 2B AR and the β 2 -adrenergic receptor have been reported to couple to both G s and G i [13,27]. We were not able to observe formoterol-triggered Ca 2+ increase in HEK293 cells. In addition to T24 cell, the A 2B AR has been reported to trigger intracellular calcium mobilization in a human breast cancer cell line, MDA-MB-231 [22], although the G proteins involved have not been examined. In the present study, we compared the effect of CTX, PTX and YM. Figure 7 shows that, unlike in T24 bladder cancer cells, YM, CTX, and PTX all inhibited NECA-induced Ca 2+ increase although to a different extent. Thus, G i , G s and G q are all involved in the A 2B AR-mediated Ca 2+ increase in MDA-MB-231 cells. Furthermore, CTX and PTX together produced an effect larger either alone (Fig. 7 B). The selective A 2B AR antagonist PSB603 (1 µM) completely blocked the effect of NECA (Fig. 7 B). Thus, we demonstrated that intracellular calcium mobilization in two cell types, i.e. T24 bladder cancer and MDA-MB-231 breast cancer, both expressing the native A 2B ARs, is independent and dependent on G q/11 , respectively. A cAMP accumulation assay confirmed the A 2B AR rather than A 2A AR is the dominant AR subtype (Fig. 7 C). The gene expression levels of four AR subtypes in MDA-MB-231 cells are shown in Fig. 7 D with A 2B AR being the highest expressed. T24 bladder cancer cells: Involvement of PKC in A 2B AR-mediated intracellular calcium mobilization We have demonstrated previously in HEK293-A2B cells that the activation of PKC by PMA completely diminished the Ca 2+ mobilization induced by the A 2B AR agonist NECA [19]. Figure 8 shows that in addition to G s , G iβγ , and G αq/11 , PKC also plays a role in receptor-triggered cytosolic calcium increase. The PKC activator PMA completely eliminated P2Y 1 R-mediated calcium mobilization, but only partially diminished A 2B AR-mediated calcium mobilization in T24 cells (Fig. 8 A). Unlike the effect of PKC on the A 2B AR, but similar to its effect on the P2Y 1 R, cell treatment with PKC activator PMA (1 µM) completely diminished the intracellular calcium mobilization induced by β 2 agonists formoterol (Fig. 8 B) and isoproterenol (Fig. 8 C). Thus, the effect of PKC on calcium intracellular mobilization in T24 cells is receptor type-dependent. Pretreatment of cells with the PKC inhibitor GO for 20 min eliminated the effect of PMA at both A 2B AR and P2Y 1 R (Fig. 8 D). Interestingly, the PKC inhibitor GO alone significantly enhanced the E max of calcium mobilization induced by P2Y 1 R agonist MRS2365 ( P < 0.05, One-Way ANOVA) but not by the A 2B AR agonist NECA, which suggests that PKC could act as an endogenous suppressor of P2Y 1 R-calcium signaling in T24 bladder cancer cells. T24 bladder cancer cells: β-Arrestins 1 and 2 are not involved in A 2B AR-mediated intracellular calcium mobilization We have shown previously that β-arrestin2 siRNA diminished P2Y 1 R agonist MRS2365-induced and β-arrestin2-mediated ERK1/2 activity [28]. Here it is shown that neither β-arrestin1 nor β-arrestin2 siRNA affected A 2B AR agonist NECA-induced Ca 2+ increase (Fig. 9 ). T24 bladder cancer cells: L-type calcium channels are not involved in A 2B AR-mediated intracellular calcium mobilization Figure 10 . Effects of the L-type calcium channel blocker nifedipine, A 2B antagonist PSB603 and the PLC inhibitor U73122 on A 2B agonist NECA-triggered Ca 2+ increase in T24 cells. Results are from three experiments. Inhibitors were incubated with cells for 20 min in 96-well black plates before addition of NECA. Discussion We have probed the mechanisms of GPCR-triggered calcium fluctuations in specific cell types, in which a GPCR is either endogenous or overexpressed, and may be coupled to G i -, G s - or G q -protein. In the present study, we expanded the previous work and further explored A 2B AR-mediated calcium transients, showing that G i , G s , G q and PKC play different roles in different cell types. We also compare results with the A 2B AR, either overexpressed or endogenously expressed in HEK293 cells, with several other GPCRs (Table 1 ). Table 1 Purinergic (i.e. A 2B , A 1 , and P2Y 1 ) and biogenic amine receptor signaling leading to calcium mobilization, showing results of treatment with pharmacological inhibitors or activators. The related figure is shown in italic parentheses. Modulation of G protein, arrestin or PKC - and effect Cell type and receptor G αq/11 G iβγ G s β-arrestin1/2 PKC A 2B AR HEK293 (A 2B overexpressing) FR and YM and Gα q/11 siRNA sensitive (1) PTX insensitive a ND ND PMA completely sensitive (Gao et al., 2023) T24 (native A 2B ) FR and YM insensitive (4) ; Gα q/11 siRNA insensitive (4) PTX sensitive (5) CTX sensitive (5) , Variable, depending on cell type b, c β-arrestin1 or β-arrestin2 siRNA insensitive (9) PMA partially sensitive (8) MDA-MB-231 (native A 2B ) YM sensitive (7) PTX partly sensitive (7) CTX partly sensitive (7) ND ND A 1 AR HEK293 (A 1 overexpressing) YM and FR – fully sensitive (2) PTX sensitive (1) ND ND ND P2Y 1 R HEK293 (native P2Y 1 ) Gα q/11 siRNA partially sensitive (1) ; YM and FR – fully sensitive (2) PTX insensitive (1) ND ND ND 1321N1 astro-cytoma (P2Y 1 -overexpressing) d YM sensitive PTX insensitive ND ND ND T24 (native P2Y 1 ) YM – fully sensitive (4) PTX insensitive (5) ND ND ND β 2 R e T24 (native b 2 ) YM – partially sensitive (6) PTX not sensitive (6) CTX sensitive (6) ND PMA completely sensitive (8) a Linden et al., 1999 [14]. b NECA-induced Ca 2+ increase in HMC-1 cells was insensitive to CTX and PTX, Feoktistov et al., 1995 [29]. c NECA-induced Ca 2+ increase in human erythroleukemia cells was sensitive to CTX, Feoktistov et al., 1994 [30]. d Gao and Jacobson, 2017 [28]. e G q/11 inhibitors blocked calcium mobilization by activation of the G i -coupled M 2 muscarinic receptor overexpressed in CHO cells. ND, not determined. Abbreviations CCPA, 2-chloro- N 6 -cyclopentyladenosine ; CTX, cholera toxin; EPAC, exchange protein activated by cAMP; FLIPR, Fluorometric Imaging Plate Reader; FR, FR900358; GPCR, G protein-coupled receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLP, glucagon-like peptide; GRK, G protein-coupled receptor kinases; HBSS, Hanks balanced salt solution; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid; LPA, lysophosphatidic acid; NECA, adenosine-5′- N -ethyluronamide; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; YM, YM254890. Declarations Ethical Approval Not applicable, does not involve any human or animal studies. Competing Interest Zhan-Guo Gao declares that he/she has no conflict of interest. Ray Gao declares that he/she has no conflict of interest. Clayton Meyer declares that he/she has no conflict of interest. Kenneth A. Jacobson declares that he/she has no conflict of interest. Inclusion and Diversity We support inclusive, diverse and equitable conduct of research. Funding This work was supported by the Intramural Research Program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (ZIADK031117). Author Contribution ZGG and KAJ conceptualized and wrote the text. ZGG wrote the first draft. ZGG, RRG, and CKM performed the pharmacological experiments. Acknowledgements: We thank the NIDDK Intramural Research for support (ZIADK031117). Data Availability Primary data for this study is available from the authors upon reasonable request. References Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017;16(12):829–842. doi: 10.1038/nrd.2017.178. Clapham DE. Calcium signaling. Cell. 2007;131(6):1047-58. doi: 10.1016/j.cell.2007.11.028. 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Cite Share Download PDF Status: Published Journal Publication published 11 Feb, 2025 Read the published version in Purinergic Signalling → Version 1 posted Editorial decision: Revision requested 15 Jan, 2025 Reviews received at journal 15 Jan, 2025 Reviewers agreed at journal 08 Jan, 2025 Reviews received at journal 05 Jan, 2025 Reviewers agreed at journal 04 Jan, 2025 Reviewers agreed at journal 23 Dec, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers invited by journal 18 Nov, 2024 Editor assigned by journal 14 Nov, 2024 Submission checks completed at journal 14 Nov, 2024 First submitted to journal 12 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5442142","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381902176,"identity":"3abc78d8-9ef8-4050-8fdd-e1d21ffa46ff","order_by":0,"name":"Zhan-Guo Gao","email":"","orcid":"","institution":"NIDDK, National Institutes of Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhan-Guo","middleName":"","lastName":"Gao","suffix":""},{"id":381902177,"identity":"9403ef0c-96bb-4a38-86e6-5ab86016d0ec","order_by":1,"name":"Ray R. 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Jacobson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACCQh1gJmfgbFBgucAAwObBFFaEg4wSzaQqoXBAKgarIWBkBbJ9t5jDz7+uMNufLu58cabMwx5fNINzC8+tuHWIs1zLt1wRsIzZrM7B5st59xgKGaTOcBmOROPFjmJHDNpnoTDzGY3EtukeT4wJLZJJLAZ85zBo0X+jZn0H6AW4xnEapGW4DGTZgBqMZAAabkB1sL8mKcCj/d7cswke9IOM0vcSAT65QxQIxAxzsCjReL4GTOJHzaHk/lnpD+88eaYTeL8GcmHP3wwwK0FBpJhRgAxYxuhqAEDO2QO8wditIyCUTAKRsGIAQDa8FGnJS4hfwAAAABJRU5ErkJggg==","orcid":"","institution":"NIDDK, National Institutes of Health","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"A.","lastName":"Jacobson","suffix":""}],"badges":[],"createdAt":"2024-11-12 20:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5442142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5442142/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11302-025-10070-1","type":"published","date":"2025-02-11T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71341059,"identity":"d787b673-235c-406f-98ab-3e1ca40efd0c","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116234,"visible":true,"origin":"","legend":"\u003cp\u003eG\u003csub\u003eq/11\u003c/sub\u003e siRNA knockdown and effects of G\u003csub\u003eq/11\u003c/sub\u003e inhibitors in HEK293 cells. A. Effects of G\u003csub\u003eq/11\u003c/sub\u003e siRNA (250 nM) on A\u003csub\u003e2B\u003c/sub\u003e-, A\u003csub\u003e1\u003c/sub\u003e-, and P2Y\u003csub\u003e1\u003c/sub\u003e receptor-mediated intracellular calcium mobilization in HEK293 cells. Data are mean ± SEM from three independent experiments. B,C,D,E. The recombinant A\u003csub\u003e1\u003c/sub\u003e and A\u003csub\u003e2B\u003c/sub\u003eARs are stably expressed in HEK293 cells. The P2Y\u003csub\u003e1\u003c/sub\u003e and LPA receptors are endogenously expressed in HEK293 cells. CCPA, A\u003csub\u003e1\u003c/sub\u003e agonist; NECA, A\u003csub\u003e2B\u003c/sub\u003eAR agonist; MRS2365, P2Y\u003csub\u003e1\u003c/sub\u003eR agonist. Data expressed as mean ± SEM from 2–4 independent experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/72cbb7b5923db3217e24df93.png"},{"id":71341962,"identity":"df8bc16a-fd0c-4b4c-9447-79bc0ef342cc","added_by":"auto","created_at":"2024-12-13 13:24:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57071,"visible":true,"origin":"","legend":"\u003cp\u003eActions of FR and YM at G\u003csub\u003eq\u003c/sub\u003e-coupled P2Y\u003csub\u003e1\u003c/sub\u003e and G\u003csub\u003ei \u003c/sub\u003ecoupled A\u003csub\u003e1\u003c/sub\u003e and M\u003csub\u003e2\u003c/sub\u003e receptors. Potencies of FR and YM in inhibition of G\u003csub\u003eαq\u003c/sub\u003e- (HEK293 a native P2Y\u003csub\u003e1\u003c/sub\u003eR) or G\u003csub\u003eiβγ\u003c/sub\u003e-mediated calcium increase. A. MRS2365 (1 µM)-induced Ca\u003csup\u003e2+\u003c/sup\u003e in HEK293 cells expressing a native P2Y\u003csub\u003e1\u003c/sub\u003eR (G\u003csub\u003eq\u003c/sub\u003e coupled). B. CCPA (1 µM)-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in HEK293 cells expressing the recombinant A\u003csub\u003e1\u003c/sub\u003eAR (G\u003csub\u003ei\u003c/sub\u003e-coupled). C. CCPA (1 µM)-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in CHO cells expressing the recombinant A\u003csub\u003e1\u003c/sub\u003eAR. D. Carbachol (10 µM)-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in CHO cells expressing the recombinant M\u003csub\u003e2\u003c/sub\u003e muscarinic receptors. Data are from at three independent experiments. The IC\u003csub\u003e50\u003c/sub\u003e values (nM) are listed in the text.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/c7a5b67870f156e99ea02eb5.png"},{"id":71341058,"identity":"8cddfc89-e3f8-49b1-9570-6f215623d120","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92467,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of FR and/or YM on G\u003csub\u003ei\u003c/sub\u003e-mediated inhibition or G\u003csub\u003es\u003c/sub\u003e-mediated stimulation of cAMP accumulation in HEK293 cells. Data are from three separate experiments. A. Inhibition by CCPA of forskolin-stimulated cAMP accumulation. B. NECA-induced cAMP accumulation. C. CGS21680-induced cAMP accumulation.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/003d839c2d76da555d4cac50.png"},{"id":71341061,"identity":"2824b302-733e-4609-9971-e379b0b0ca73","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93957,"visible":true,"origin":"","legend":"\u003cp\u003eRoles of G\u003csub\u003eq/11\u003c/sub\u003e proteins in intracellular calcium mobilization in endogenously expressed GPCRs in T24 cells. A,B. G\u003csub\u003eq/11\u003c/sub\u003e siRNA on P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365- and A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-induced a Ca\u003csup\u003e2+\u003c/sup\u003e increase mediated by native P2Y\u003csub\u003e1\u003c/sub\u003e and A\u003csub\u003e2B\u003c/sub\u003e receptors, respectively. C,D. The effect of G\u003csub\u003eq/11\u003c/sub\u003e chemical inhibitor YM (C) FR (D). Results are from 2–4 independent experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/9867bf07b6456e447e9b3332.png"},{"id":71341963,"identity":"ae10461f-89f0-4c0c-9f93-c5d29c40c8bf","added_by":"auto","created_at":"2024-12-13 13:24:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51428,"visible":true,"origin":"","legend":"\u003cp\u003eRoles of G\u003csub\u003ei\u003c/sub\u003e and G\u003csub\u003es\u003c/sub\u003e proteins in intracellular calcium mobilization mediated by endogenous GPCRs in T24 cells. Data are from three independent experiments. A. Effects of G\u003csub\u003ei\u003c/sub\u003e inactivator PTX (200 ng/ml) and the combination of PTX and Gs inactivator CTX (500 ng/ml). B. effects of G\u003csub\u003es\u003c/sub\u003e inactivator CTX (500 ng/ml), EPAC inhibitor ESI09 (10 µM), or PKA inhibitor H89 (10 µM).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/d2d09fbf59558dcfa6f03768.png"},{"id":71341062,"identity":"7d56f6d8-7acc-491a-8282-de20d5a204ab","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82113,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of G\u003csub\u003eαq\u003c/sub\u003e inhibitors FR and YM, G\u003csub\u003ei\u003c/sub\u003e inactivator PTX and G\u003csub\u003es\u003c/sub\u003e inactivator CTX on endogenous GPCR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. A,B. FR (300 nM) on triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase triggered by β-adrenergic receptor agonists isoproterenol- and formoterol in T24 cells. C. Effects of YM (1 µM), CTX (500 ng/ml) and PTX (200 ng/ml). Data are from three independent experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/48a194b6d82d9ffbdd19e32c.png"},{"id":71341063,"identity":"06937f55-6b83-4a5a-9721-8cd91a52545d","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81663,"visible":true,"origin":"","legend":"\u003cp\u003eEffects on agonist-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in MDA-MB-231 breast cells expressing endogenous adenosine receptors. A. Effects of YM (300 nM), CTX (500 ng/ml) and PTX (200 ng/ml) on the response to NECA. B. Effects of A\u003csub\u003e2B\u003c/sub\u003e antagonist PSB603 (1 µM) and the combination of CTX and PTX on NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase. C. Comparison of cAMP accumulation induced by A\u003csub\u003e2A\u003c/sub\u003e agonist CGS21680 and A\u003csub\u003e2B\u003c/sub\u003e agonist NECA. D. Gene expression level of four ARs on MDA-MB-231 cells. Results are from three experiments performed in duplicate.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/7b69b29994191772f97b1e45.png"},{"id":71341067,"identity":"733847e8-15a2-479f-ac0e-a8ddf7b4c13e","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":140428,"visible":true,"origin":"","legend":"\u003cp\u003eRole of protein kinase C in A\u003csub\u003e2B\u003c/sub\u003eAR-, P2Y\u003csub\u003e1\u003c/sub\u003e- and β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. A. Effects of PKC activator PMA on MRS2365- or NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase. B. Effects of PMA or PKC blocker GO (Go6983). C. Effects of PMA. D. Effects of preincubation of GO for 20 min followed by addition of PMA for 20 min before addition of agonists. E,F. Effects of PKC inhibitor on P2Y\u003csub\u003e1\u003c/sub\u003e- or A\u003csub\u003e2B\u003c/sub\u003e-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase. Results are from three experiments. #Significantly different from control (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/bd1b99ad43aca39649c3d1a6.png"},{"id":71341065,"identity":"dfb38e76-94da-4158-a28a-5ea9354eed66","added_by":"auto","created_at":"2024-12-13 13:16:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":79289,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of β-arrestin siRNA on A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. Data are expressed as mean ± SEM from three experiments. Results are from three experiments. The transfection of siRNA was performed using Lipofectamine 2000.\u003c/p\u003e","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/4bd0b26e6116079b04d57f1b.png"},{"id":71341964,"identity":"dd8b79a4-d1ea-430d-9071-5a11b972dc09","added_by":"auto","created_at":"2024-12-13 13:24:05","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":101310,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the L-type calcium channel blocker nifedipine, A\u003csub\u003e2B\u003c/sub\u003e antagonist PSB603 and the PLC inhibitor U73122 on A\u003csub\u003e2B\u003c/sub\u003e agonist NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. Results are from three experiments. Inhibitors were incubated with cells for 20 min in 96-well black plates before addition of NECA.\u003c/p\u003e","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/05e1547906c6875dca58029e.png"},{"id":76487696,"identity":"71e81f1c-3e48-4242-b580-7484fa87b998","added_by":"auto","created_at":"2025-02-17 16:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3059002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5442142/v1/d93f4a94-5ed9-4b17-9d68-b630e82a2e40.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A2B adenosine receptor-triggered intracellular calcium mobilization: Cell type-dependent involvement of Gi, Gq, Gs proteins and protein kinase C","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntracellular calcium mobilization triggered by G protein-coupled receptors (GPCRs), which\u0026thinsp;~\u0026thinsp;34% of prescription drugs target [1], affects all aspects of cell functions including neurotransmitter release, insulin secretion, T cell activation, muscle contraction, immunity and cell survival and proliferation [2]. Activation of PLCβ enzymes by a GPCR is considered as a common mechanism to trigger Ca\u003csup\u003e2+\u003c/sup\u003e increase in cells. Both G\u003csub\u003eiβγ\u003c/sub\u003e and G\u003csub\u003eαq\u003c/sub\u003e, activated by G\u003csub\u003ei\u003c/sub\u003e-coupled and G\u003csub\u003eq\u003c/sub\u003e-coupled receptors, respectively, can trigger PLC activation [3] or intracellular calcium mobilization [4\u0026ndash;7].\u003c/p\u003e \u003cp\u003eWe previously compared the G protein-inhibitory effects of FR900358 (FR, UBO, UBO-QIC [8]) and pertussis toxin (PTX) on various GPCR signaling pathways [9]. For example, FR (UBO, at 300 nM), had no effect on A\u003csub\u003e2B\u003c/sub\u003e adenosine receptor (AR)-G\u003csub\u003eαs\u003c/sub\u003e-mediated cAMP accumulation or A\u003csub\u003e1\u003c/sub\u003eAR- G\u003csub\u003eαi\u003c/sub\u003e-mediated inhibition of forskolin-stimulated cAMP accumulation in CHO cells [9]. Nevertheless, FR (100 nM) produced a nearly complete inhibition of G\u003csub\u003eiβγ\u003c/sub\u003e-mediated calcium mobilization, or IP1 accumulation, upon the activation of the G\u003csub\u003ei\u003c/sub\u003e-coupled A\u003csub\u003e1\u003c/sub\u003eAR, the M\u003csub\u003e2\u003c/sub\u003e muscarinic acetylcholine receptor, or the P2Y\u003csub\u003e12\u003c/sub\u003e receptor (P2Y\u003csub\u003e12\u003c/sub\u003eR, responds to ADP) overexpressed in either CHO or 1321N1 astrocytoma cells. However, 100 nM FR had no effect on A\u003csub\u003e1\u003c/sub\u003eAR-mediated Akt1/2/3 phosphorylation, which was completely inhibited by PTX. FR (100 nM) produced a small (\u0026lt;\u0026thinsp;30%) but significant inhibition of A\u003csub\u003e1\u003c/sub\u003eAR-mediated ERK1/2 activity [9]. It is also of note that FR, at both 100 and 300 nM, only partially inhibited ERK1/2 activity triggered by the G\u003csub\u003eq/11\u003c/sub\u003e-coupled M\u003csub\u003e3\u003c/sub\u003e acetylcholine or P2Y\u003csub\u003e1\u003c/sub\u003e (responds to ADP) receptor, although the calcium response triggered by these two receptors was completely inhibited.\u003c/p\u003e \u003cp\u003eIn a follow-up study, Pfeil et al. [10] further explored the potential mechanism related to FR-inhibited G\u003csub\u003eiβγ\u003c/sub\u003e-triggered calcium signaling in one signaling pathway mentioned above, i.e. calcium signaling, but not Akt or ERK1/2 signaling. They confirmed our finding [9] and suggested that \u0026ldquo;the G\u003csub\u003eαi\u003c/sub\u003e-G\u003csub\u003eβγ\u003c/sub\u003e-PLCβ-Ca\u003csup\u003e2+\u003c/sup\u003e signaling axis is entirely dependent on the presence of active G\u003csub\u003eαq\u003c/sub\u003e. If G\u003csub\u003eαq\u003c/sub\u003e is pharmacologically inhibited or genetically ablated, G\u003csub\u003eαq\u003c/sub\u003e can bind to PLCβ but does not elicit Ca\u003csup\u003e2+\u003c/sup\u003e signals.\u0026rdquo; The finding related to G\u003csub\u003eαq/11\u003c/sub\u003e control of G\u003csub\u003eiβγ\u003c/sub\u003e-mediated Ca\u003csup\u003e2+\u003c/sup\u003e signaling is an important discovery, since the mechanisms related to Ca\u003csup\u003e2+\u003c/sup\u003e increase in cells is vital to the discovery of drugs targeting different receptor classes. However, the conclusion that G\u003csub\u003eαq\u003c/sub\u003e controls G\u003csub\u003eiβγ\u003c/sub\u003e-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increases via removal of PLC auto-inhibition was drawn mostly from the study of native and G protein-knockout HEK293 cells [10].\u003c/p\u003e \u003cp\u003eWe have previously shown the critical role of G\u003csub\u003eαq/11\u003c/sub\u003e in A\u003csub\u003e2B\u003c/sub\u003eAR-triggered calcium mobilization in HEK293 cells overexpressing the recombinant human (h) A\u003csub\u003e2B\u003c/sub\u003eAR [12,13]. In A\u003csub\u003e2B\u003c/sub\u003eAR-overexpressing HEK293 cells, both CRISPR-Cas9-based G\u003csub\u003eq/11\u003c/sub\u003e knockout and the G\u003csub\u003eq\u003c/sub\u003e inhibitor FR completely eliminated the A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increase [12,13]. It is of note that the native A\u003csub\u003e2B\u003c/sub\u003eAR in HEK293 cells is capable of inducing cAMP accumulation via G\u003csub\u003es\u003c/sub\u003e and ERK1/2 activity increase via G\u003csub\u003ei\u003c/sub\u003e, but it is unable to trigger a robust Ca\u003csup\u003e2+\u003c/sup\u003e signal [12,13]. However, when hA\u003csub\u003e2B\u003c/sub\u003eAR is overexpressed in HEK293 cells, a robust intracellular Ca\u003csup\u003e2+\u003c/sup\u003e mobilization signal mediated via G\u003csub\u003eαq/11\u003c/sub\u003e is induced [12\u0026ndash;14], which suggests that A\u003csub\u003e2B\u003c/sub\u003eAR overexpression could enable the coupling to G\u003csub\u003eαq/11\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe A\u003csub\u003e2B\u003c/sub\u003eAR has emerged as an important target for various conditions, including cancer, cardiac ischemia and sarcopenia [15\u0026ndash;20]. Calcium signaling is a major signaling pathway involved in these critical conditions. Thus, it is important to better understand the A\u003csub\u003e2B\u003c/sub\u003eAR downstream signaling, including Ca\u003csup\u003e2+\u003c/sup\u003e mobilization. The cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e increase triggered by the nonselective AR agonist NECA has been explored using T24 bladder cancer cells expressing the native hA\u003csub\u003e2B\u003c/sub\u003eAR [12,21]. NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells can be completely blocked by a selective A\u003csub\u003e2B\u003c/sub\u003eAR antagonist PSB603 and diminished by A\u003csub\u003e2B\u003c/sub\u003eAR siRNA [12]. In addition, Panjehpour et al. [22] reported that a NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e signal in MDA-MB-231 breast cancer cells also occurs via the A\u003csub\u003e2B\u003c/sub\u003eAR. In the present study we further examined the mechanisms of A\u003csub\u003e2B\u003c/sub\u003eAR-triggered Ca\u003csup\u003e2+\u003c/sup\u003e release, especially whether G\u003csub\u003eiβγ\u003c/sub\u003e-triggered Ca\u003csup\u003e2+\u003c/sup\u003e release can be blocked by G\u003csub\u003eαq/11\u003c/sub\u003e siRNA or pharmacological inhibitors, in HEK293 cells overexpressing the recombinant A\u003csub\u003e2B\u003c/sub\u003eAR, and in T24 bladder cancer cells expressing a native A\u003csub\u003e2B\u003c/sub\u003eAR. Thus, we examined the effects of two G\u003csub\u003eαq/11\u003c/sub\u003e inhibitors: FR and YM-254890 (YM [23]), as well as G\u003csub\u003eαq/11\u003c/sub\u003e siRNA, the G\u003csub\u003ei\u003c/sub\u003e inactivator PTX, and the G\u003csub\u003es\u003c/sub\u003e inactivator CTX. We found that, unlike in HEK293 cells, G\u003csub\u003eiβγ\u003c/sub\u003e, but not G\u003csub\u003eq/11\u003c/sub\u003e, plays a critical role in A\u003csub\u003e2B\u003c/sub\u003eAR-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 bladder cancer cells. We also analyzed the role of G\u003csub\u003es\u003c/sub\u003e (relative to G\u003csub\u003ei\u003c/sub\u003e) in A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e release, the synergistic effects between G\u003csub\u003ei\u003c/sub\u003e and G\u003csub\u003es\u003c/sub\u003e, and cell type- and receptor-dependent modulation by protein kinase C (PKC). In some cases, we compared the A\u003csub\u003e2B\u003c/sub\u003eAR-mediated effects with those of other purinergic receptors or biogenic amine receptor.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eBAY60-6583 (LUF6210) was provided by Prof. Ad IJzerman at Leiden-Amsterdam Center for Drug Research (Leiden, The Netherlands). Nonselective AR agonist adenosine-5\u0026prime;-\u003cem\u003eN\u003c/em\u003e-ethyluronamide (NECA), β\u003csub\u003e2\u003c/sub\u003e adrenergic receptor agonists formoterol and isoproterenol, lysophosphatidic acid (LPA) receptor agonist LPA, YM254890, PSB603, ESI-09, H89, PKC activator phorbol 12-myristate 13-acetate (PMA), and PKC inhibitor Go6983 were from Tocris (Minneapolis, MN, USA). FR900359 (UBO-QIC) was purchased from University of Bonn (Germany). HEK293 cells, T24 bladder cancer cells, and MDA-MB-231 cells were from ATCC (Manassas, VA, USA). HEK293-A2B cells were made at the Laboratory of Bioorganic Chemistry, NIDDK, NIH (Bethesda, MD, USA). An AlphaScreen cAMP kit was purchased from PerkinElmer (Waltham, MA). Calcium assay kits were from Molecular Devices (Sunnyvale, CA, USA). All other reagents were from standard commercial sources and of analytical grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction and quantitative real-time PCR detection of gene expression level\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from 10\u003csup\u003e7\u003c/sup\u003e cells using a RNeasy kit (Qiagen, Redwood City, CA, USA) and was reversed-transcribed using the SuperScript\u0026trade; III First-Strand Synthesis SuperMix kit (ThermoFisher, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s protocol and as described [24]. Briefly, the thermocycles were as follows: 25\u003csup\u003e○\u003c/sup\u003eC for 10 min; 50\u003csup\u003e○\u003c/sup\u003eC for 30 min; 85\u003csup\u003e○\u003c/sup\u003eC for 5 min and then chilled on ice; 1 \u0026micro;l (2 U) of E Coli RNase H was added and incubation continued for 20 min before storage at -20\u003csup\u003e○\u003c/sup\u003eC until use. The cDNA was then amplified with TaqMan gene expression assays (ThermoFisher, Waltham, MA USA) for four AR subtypes and GAPDH on a CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) according to the manufacturer\u0026rsquo;s protocol. The temperature cycles were: 50\u003csup\u003e○\u003c/sup\u003eC for 2 min; 95\u003csup\u003e○\u003c/sup\u003eC for 10 min; 95\u003csup\u003e○\u003c/sup\u003eC 15s and 60\u003csup\u003e○\u003c/sup\u003eC for 30s for 40 cycles. The ΔΔCt method was used to conduct quantitative analysis of data. Values were normalized to GAPDH and then expressed as relative expression levels.\u003c/p\u003e\n\u003ch3\u003eCell culture and measurement of cyclic AMP levels\u003c/h3\u003e\n\u003cp\u003eHEK293, T24 bladder cancer and MDA-MB-231 breast cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 \u0026micro;g/ml streptomycin, and 2 \u0026micro;mol/ml glutamine. For the assay of 3\u0026prime;,5\u0026prime;-cyclic adenosine monophosphate (cAMP), cells were plated in 96-well (4x10\u003csup\u003e4\u003c/sup\u003e cells/well) clear plates in 100 \u0026micro;l of medium overnight. Cell culture medium was then replaced with 80 \u0026micro;l HBSS buffer containing 20 mM HEPES, phosphodiesterase inhibitor rolipram (10 \u0026micro;M), and 3 units/ml adenosine deaminase (Worthington Biochemical, Lakewood, NJ, USA) for 30 min followed by agonist addition to the mixture, which was then incubated for 20 min. The treatment of YM or FR was 20 min before agonist addition. The treatment with CTX (500 ng/ml or PTX (200 ng/ml) was for overnight before agonist addition. The reaction was terminated by aspirating the reaction mixture and the addition of 100 \u0026micro;l cold 0.3% Tween-20 to each well. Cells were then shaken at room temperature for 10 min. For the determination of cAMP production, an AlphaScreen cAMP kit was used according to the manufacturer\u0026rsquo;s instructions (Revvity/PerkinElmer, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eMeasurement of intracellular calcium increase\u003c/h3\u003e\n\u003cp\u003eThe measurement of calcium mobilization was essentially as described previously [19,24]. We utilized a calcium assay kit as directed without washing cells and with probenecid added to the loading dye at a final concentration of 2.5 mM to increase dye retention. Briefly, cells were grown at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e in a set of 96-well black-wall, clear-bottom plates overnight, or until they reach confluence. Then, media was aspirated, and 100 \u0026micro;l of dye (Calcium 6) was added to each well. Afterward, the plates were subsequently maintained at room temperature in the dark for 60 min. Finally, 50 \u0026micro;l of compound or a control agonist was added into respective assay plate wells during the determination of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e using a FLIPR (Molecular Devices, San Jose, CA, USA). The treatment of YM and FR was 20 min before agonist addition. The treatment with CTX (500 ng/ml) or PTX (200 ng/ml) was for overnight before agonist addition. The compound plate was prepared using dilutions of various compounds in HBSS buffer (pH 7.4) without added calcium. Samples were run in duplicate or triplicate at room temperature. Cell fluorescence (excitation\u0026thinsp;=\u0026thinsp;485 nm; emission\u0026thinsp;=\u0026thinsp;525 nm) was monitored following compound exposure. Increases in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure.\u003c/p\u003e\n\u003ch3\u003eStatistical and data analyses\u003c/h3\u003e\n\u003cp\u003eFunctional parameters were calculated using Prism 10.2.3 software (GraphPad, San Diego, CA, USA). Data was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. A Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e (between two conditions) or a One-Way Analysis of Variance (ANOVA) followed by Tukey\u0026rsquo;s or Bonferroni\u0026rsquo;s multiple comparison tests (between multiple conditions) was used to compare statistically significant differences. Differences yielding \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 are considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHEK293 cells: G\u003csub\u003eαq/11\u003c/sub\u003e protein contribution to A\u003csub\u003e2B\u003c/sub\u003eAR-mediated intracellular calcium mobilization\u003c/h2\u003e \u003cp\u003eHere, we further explore the G\u003csub\u003eαi\u003c/sub\u003e-G\u003csub\u003eβγ\u003c/sub\u003e-PLCβ-Ca\u003csup\u003e2+\u003c/sup\u003e signaling axis, previously demonstrated to be inhibited by G\u003csub\u003eαq/11\u003c/sub\u003e inhibitor FR and Gq/11 knockout [10,13]. In addition to FR, G\u003csub\u003eq/11\u003c/sub\u003e siRNA and another G\u003csub\u003eq/11\u003c/sub\u003e inhibitor, YM, inhibited calcium mobilization in HEK293 cells, endogenously expressing the G\u003csub\u003eq\u003c/sub\u003e-coupled P2Y\u003csub\u003e1\u003c/sub\u003eR, or overexpressing the recombinant human G\u003csub\u003ei\u003c/sub\u003e-coupled A\u003csub\u003e1\u003c/sub\u003e or G\u003csub\u003ei\u003c/sub\u003e- and G\u003csub\u003es\u003c/sub\u003e-coupled A\u003csub\u003e2B\u003c/sub\u003e ARs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings are consistent with previous pharmacological results [9] and align with the proposed mechanism that G\u003csub\u003eiβγ\u003c/sub\u003e-mediated calcium signaling is under G\u003csub\u003eq/11\u003c/sub\u003e control in HEK293 cells [10]. Thus, the results from the present study using FR, YM, G\u003csub\u003eq/11\u003c/sub\u003e siRNA knockdown and prior CRISPR-Cas9-based G\u003csub\u003eq/11\u003c/sub\u003e knockout research [10] are all consistent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnlike the G\u003csub\u003eq\u003c/sub\u003e inhibitors FR and YM, treatment with the G\u003csub\u003ei\u003c/sub\u003e inhibitor PTX had no effect on G\u003csub\u003eq/11\u003c/sub\u003e-coupled P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365-induced Ca\u003csup\u003e2+\u003c/sup\u003e release in HEK293 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), but completely inhibited the A\u003csub\u003e1\u003c/sub\u003eAR agonist CCPA-induced Ca\u003csup\u003e2+\u003c/sup\u003e signaling in HEK293-A1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The A\u003csub\u003e2B\u003c/sub\u003eAR-mediated increase of ERK1/2 activity in HEK293-A2B cells has been reported to be G\u003csub\u003ei\u003c/sub\u003e-dependent by Yang et al. [25] based on PTX sensitivity, which was later confirmed with both PTX and CRISPR-Cas9-based Gq/11 knockout [13]. However, A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in HEK293-A2B cells is G\u003csub\u003ei\u003c/sub\u003e-independent, as incubation with 200 ng/ml PTX for 18 hours did not affect the NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase [14].\u003c/p\u003e \u003cp\u003eIt is of note that the native A\u003csub\u003e1\u003c/sub\u003eAR expression level in HEK293 cells is extremely low, and A\u003csub\u003e1\u003c/sub\u003eAR agonist CCPA does not trigger Ca\u003csup\u003e2+\u003c/sup\u003e signaling [12,13]. The native A\u003csub\u003e2B\u003c/sub\u003eAR in HEK293 cells can induce robust cAMP accumulation and ERK1/2 activity but not sufficient to trigger a robust calcium signaling [12,13,19]. In HEK293 cells, there is a possibility that the overexpression of G\u003csub\u003ei\u003c/sub\u003e-coupled A\u003csub\u003e1\u003c/sub\u003e or G\u003csub\u003es\u003c/sub\u003e-coupled A\u003csub\u003e2B\u003c/sub\u003e ARs enable their stronger coupling to G\u003csub\u003eαq/11\u003c/sub\u003e, and thus G\u003csub\u003eαq/11\u003c/sub\u003e can subsequently control the A\u003csub\u003e1\u003c/sub\u003eAR-mediated G\u003csub\u003eiβγ\u003c/sub\u003e-Ca\u003csup\u003e2+\u003c/sup\u003e signaling. FR inhibition of native G\u003csub\u003eq/11\u003c/sub\u003e-coupled P2Y\u003csub\u003e1\u003c/sub\u003eR-induced calcium release is expected. In addition, LPA (lysophosphatidic acid)-induced intracellular calcium mobilization (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.59 \u0026micro;M) was also completely inhibited by 300 nM YM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHEK293 cells: Similar potencies of G\u003c/b\u003e \u003csub\u003e \u003cb\u003eαq/11\u003c/b\u003e \u003c/sub\u003e \u003cb\u003einhibitors on G\u003c/b\u003e\u003csub\u003e\u003cb\u003eiβγ\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e- versus G\u003c/b\u003e\u003csub\u003e\u003cb\u003eαq\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-mediated stimulation of intracellular calcium mobilization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe A\u003csub\u003e1\u003c/sub\u003eAR and P2Y\u003csub\u003e1\u003c/sub\u003eR are G\u003csub\u003ei\u003c/sub\u003e-coupled and G\u003csub\u003eq\u003c/sub\u003e-coupled receptors, respectively. We compared the potencies of FR and YM in inhibiting A\u003csub\u003e1\u003c/sub\u003eAR (overexpressed)-triggered calcium mobilization by A\u003csub\u003e1\u003c/sub\u003eAR agonist CCPA (1 \u0026micro;M) and native P2Y\u003csub\u003e1\u003c/sub\u003eR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increase by P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365 (1 \u0026micro;M) in HEK293 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). The IC\u003csub\u003e50\u003c/sub\u003e values FR and YM to inhibit P2Y\u003csub\u003e1\u003c/sub\u003eR-mediated calcium mobilization were 13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 and 12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 nM, respectively. The IC\u003csub\u003e50\u003c/sub\u003e values for the inhibition of the A\u003csub\u003e1\u003c/sub\u003eAR-mediated effect were 8.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23 and 12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52 nM, respectively. Additionally, the IC\u003csub\u003e50\u003c/sub\u003e values of YM in inhibiting the effects of 10 \u0026micro;M carbachol and 1 \u0026micro;M CCPA in CHO cells overexpressing the recombinant G\u003csub\u003ei\u003c/sub\u003e-coupled M\u003csub\u003e2\u003c/sub\u003e muscarinic or A\u003csub\u003e1\u003c/sub\u003e receptor, respectively, were determined to be 9.53 and 11.6 nM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D). Thus, the potencies of FR and YM for inhibition of G\u003csub\u003eq\u003c/sub\u003e- and G\u003csub\u003eiβγ\u003c/sub\u003e-mediated calcium signaling are similar, and the use of either inhibitor at 100\u0026ndash;300 nM should completely inhibit both G\u003csub\u003eiβγ\u003c/sub\u003e- and G\u003csub\u003eαq\u003c/sub\u003e-triggered Ca\u003csup\u003e2+\u003c/sup\u003e signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHEK293 cells: G inhibitors lack effects on G-mediated inhibition or G-mediated stimulation of cAMP accumulation\u003c/h3\u003e\n\u003cp\u003eAs previously demonstrated in other cell types [9], FR and/or YM did not affect A\u003csub\u003e1\u003c/sub\u003eAR-mediated inhibition or A\u003csub\u003e2A\u003c/sub\u003eAR- and A\u003csub\u003e2B\u003c/sub\u003eAR-mediated stimulation of cAMP accumulation in HEK293 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), although they inhibited Ca\u003csup\u003e2+\u003c/sup\u003e signaling induced by those receptors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eT24 bladder cancer cells: G\u003c/b\u003e \u003csub\u003e \u003cb\u003eq/11\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eproteins do not play a major role in native A\u003c/b\u003e\u003csub\u003e\u003cb\u003e2B\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eAR-mediated intracellular calcium mobilization\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs described above, the G\u003csub\u003eq/11\u003c/sub\u003e pharmacological inhibitors and siRNA diminished the A\u003csub\u003e2B\u003c/sub\u003eAR-mediated calcium signaling in HEK293 cells overexpressing the recombinant hA\u003csub\u003e2B\u003c/sub\u003eAR, which is consistent with results from CRISPR-Cas9-based G\u003csub\u003eq/11\u003c/sub\u003e knockout HEK293 cells [13]. We next carefully examined the intracellular calcium mobilization triggered by the native A\u003csub\u003e2B\u003c/sub\u003eAR in T24 bladder cancer cells [12,21]. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows that, unlike in HEK293 cells, G\u003csub\u003eq/11\u003c/sub\u003e siRNA suppressed P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365-induced calcium mobilization but not A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-triggered calcium mobilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The EC\u003csub\u003e50\u003c/sub\u003es of NECA in the absence and presence of G\u003csub\u003eq/11\u003c/sub\u003e siRNA were 234\u0026thinsp;\u0026plusmn;\u0026thinsp;61 and 247\u0026thinsp;\u0026plusmn;\u0026thinsp;46 nM, respectively (n\u0026thinsp;=\u0026thinsp;3), which are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar to the effect of G\u003csub\u003eq/11\u003c/sub\u003e siRNA, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows that the G\u003csub\u003eq/11\u003c/sub\u003e inhibitor YM (300 nM) also completely inhibited the effect of P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365 but had little if any effect on A\u003csub\u003e2B\u003c/sub\u003eAR-induced calcium mobilization in T24 cells. The EC\u003csub\u003e50\u003c/sub\u003es of NECA in the absence and presence of YM were 427\u0026thinsp;\u0026plusmn;\u0026thinsp;76 and 524\u0026thinsp;\u0026plusmn;\u0026thinsp;88 nM, respectively (n\u0026thinsp;=\u0026thinsp;3), which are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD shows that, similarly to YM in T24 cells, another G\u003csub\u003eq/11\u003c/sub\u003e inhibitor FR (300 nM) also completely inhibited calcium mobilization triggered by the native P2Y\u003csub\u003e1\u003c/sub\u003eR but not by the native A\u003csub\u003e2B\u003c/sub\u003eAR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eT24 bladder cancer cells: Contribution of both G\u003c/b\u003e \u003csub\u003e \u003cb\u003ei\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand G\u003c/b\u003e\u003csub\u003e\u003cb\u003es\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eproteins to intracellular calcium mobilization triggered by native A\u003c/b\u003e\u003csub\u003e\u003cb\u003e2B\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eARs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows that, unlike YM and FR, PTX diminished A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-mediated calcium mobilization in T24 cells but had no effect on the P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365. Thus, it is suggested that G\u003csub\u003ei\u003c/sub\u003e plays a prominent role in A\u003csub\u003e2B\u003c/sub\u003eAR-mediated intracellular calcium mobilization in T24 bladder cancer cells. Considering that G\u003csub\u003eiα\u003c/sub\u003e isoforms do not play a significant role in activation PLCβ [26], it is assumed that G\u003csub\u003eβγ\u003c/sub\u003e is responsible for this effect (Gao and Jacobson, 2016). Interestingly, simultaneous inactivation of G\u003csub\u003ei\u003c/sub\u003e and G\u003csub\u003es\u003c/sub\u003e by PTX and CTX almost completely eliminated A\u003csub\u003e2B\u003c/sub\u003eAR-triggered Ca\u003csup\u003e2+\u003c/sup\u003e release (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting both G\u003csub\u003eiβγ\u003c/sub\u003e and G\u003csub\u003es\u003c/sub\u003e are responsible for A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in T24 cells. The EC\u003csub\u003e50\u003c/sub\u003es of MRS2365 in the absence and presence of PTX were 1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 and 1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 nM, respectively, which are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e). The EC\u003csub\u003e50\u003c/sub\u003es of NECA for Control group, PTX group and PTX\u0026thinsp;+\u0026thinsp;CTX group were 538\u0026thinsp;\u0026plusmn;\u0026thinsp;112, 3550\u0026thinsp;\u0026plusmn;\u0026thinsp;890 and 12,300\u0026thinsp;\u0026plusmn;\u0026thinsp;4600 nM, respectively, which are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, One-Way Analysis of Variance followed by Bonferroni\u0026rsquo;s multiple comparison tests). The maximal effect of NECA (in terms of relative fluorescence units) in Control group, PTX group and PTX\u0026thinsp;+\u0026thinsp;CTX group were 505\u0026thinsp;\u0026plusmn;\u0026thinsp;78, 173\u0026thinsp;\u0026plusmn;\u0026thinsp;39 and 88\u0026thinsp;\u0026plusmn;\u0026thinsp;15, respectively, which are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, One-Way Analysis of Variance followed by Bonferroni\u0026rsquo;s multiple comparison tests).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine whether the pathway downstream of G\u003csub\u003es\u003c/sub\u003e, PKA or EPAC, contributes to A\u003csub\u003e2B\u003c/sub\u003e-Gs-mediated calcium mobilization in T24 cells, PKA inhibitor H89 and EPAC inhibitor ESI09 were used. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows that both EPAC and PKA are involved in A\u003csub\u003e2B\u003c/sub\u003eAR-G\u003csub\u003es\u003c/sub\u003e-mediated calcium mobilization. However, the combination of ESI09 and H89 did not produce an effect larger that ESI09 alone (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Student\u0026rsquo;s \u003cem\u003et-test\u003c/em\u003e). The inactivation of G\u003csub\u003es\u003c/sub\u003e by CTX produced a similar effect to that of ESI09.\u003c/p\u003e \u003cp\u003eIn addition to the A\u003csub\u003e2B\u003c/sub\u003eAR, T24 cells also endogenously express the β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that the β\u003csub\u003e2\u003c/sub\u003e adrenergic agonist formoterol and isoproterenol induced a robust calcium response albeit a little less efficacious compared to the A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA. However, unlike the effects of FR on the A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e release, FR (300 nM) diminished Ca\u003csup\u003e2+\u003c/sup\u003e increase triggered by both β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor agonists formoterol and isoproterenol, suggesting a role of G\u003csub\u003eq/11\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a separate set of experiments, YM (1 \u0026micro;M) was shown to inhibit 78% of the maximum effect (E\u003csub\u003emax\u003c/sub\u003e) of formoterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Unlike its effect on the A\u003csub\u003e2B\u003c/sub\u003eAR, PTX treatment did not produce significant attenuation of the E\u003csub\u003emax\u003c/sub\u003e in β\u003csub\u003e2\u003c/sub\u003e agonist formoterol-induced calcium mobilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). However, CTX treatment produced an effect larger than it was on the NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) in the same cell type, i.e. T24 bladder cancer cells. Thus, even in the same cell type, the involvement of G proteins in intracellular calcium mobilization is dependent on receptor type, although both the A\u003csub\u003e2B\u003c/sub\u003eAR and the β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor have been reported to couple to both G\u003csub\u003es\u003c/sub\u003e and G\u003csub\u003ei\u003c/sub\u003e [13,27]. We were not able to observe formoterol-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in HEK293 cells.\u003c/p\u003e \u003cp\u003eIn addition to T24 cell, the A\u003csub\u003e2B\u003c/sub\u003eAR has been reported to trigger intracellular calcium mobilization in a human breast cancer cell line, MDA-MB-231 [22], although the G proteins involved have not been examined. In the present study, we compared the effect of CTX, PTX and YM. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows that, unlike in T24 bladder cancer cells, YM, CTX, and PTX all inhibited NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase although to a different extent. Thus, G\u003csub\u003ei\u003c/sub\u003e, G\u003csub\u003es\u003c/sub\u003e and G\u003csub\u003eq\u003c/sub\u003e are all involved in the A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increase in MDA-MB-231 cells. Furthermore, CTX and PTX together produced an effect larger either alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The selective A\u003csub\u003e2B\u003c/sub\u003eAR antagonist PSB603 (1 \u0026micro;M) completely blocked the effect of NECA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, we demonstrated that intracellular calcium mobilization in two cell types, i.e. T24 bladder cancer and MDA-MB-231 breast cancer, both expressing the native A\u003csub\u003e2B\u003c/sub\u003eARs, is independent and dependent on G\u003csub\u003eq/11\u003c/sub\u003e, respectively. A cAMP accumulation assay confirmed the A\u003csub\u003e2B\u003c/sub\u003eAR rather than A\u003csub\u003e2A\u003c/sub\u003eAR is the dominant AR subtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The gene expression levels of four AR subtypes in MDA-MB-231 cells are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD with A\u003csub\u003e2B\u003c/sub\u003eAR being the highest expressed.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eT24 bladder cancer cells: Involvement of PKC in A\u003csub\u003e2B\u003c/sub\u003eAR-mediated intracellular calcium mobilization\u003c/h2\u003e \u003cp\u003eWe have demonstrated previously in HEK293-A2B cells that the activation of PKC by PMA completely diminished the Ca\u003csup\u003e2+\u003c/sup\u003e mobilization induced by the A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA [19]. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows that in addition to G\u003csub\u003es\u003c/sub\u003e, G\u003csub\u003eiβγ\u003c/sub\u003e, and G\u003csub\u003eαq/11\u003c/sub\u003e, PKC also plays a role in receptor-triggered cytosolic calcium increase. The PKC activator PMA completely eliminated P2Y\u003csub\u003e1\u003c/sub\u003eR-mediated calcium mobilization, but only partially diminished A\u003csub\u003e2B\u003c/sub\u003eAR-mediated calcium mobilization in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Unlike the effect of PKC on the A\u003csub\u003e2B\u003c/sub\u003eAR, but similar to its effect on the P2Y\u003csub\u003e1\u003c/sub\u003eR, cell treatment with PKC activator PMA (1 \u0026micro;M) completely diminished the intracellular calcium mobilization induced by β\u003csub\u003e2\u003c/sub\u003e agonists formoterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and isoproterenol (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Thus, the effect of PKC on calcium intracellular mobilization in T24 cells is receptor type-dependent. Pretreatment of cells with the PKC inhibitor GO for 20 min eliminated the effect of PMA at both A\u003csub\u003e2B\u003c/sub\u003eAR and P2Y\u003csub\u003e1\u003c/sub\u003eR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Interestingly, the PKC inhibitor GO alone significantly enhanced the E\u003csub\u003emax\u003c/sub\u003e of calcium mobilization induced by P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, One-Way ANOVA) but not by the A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA, which suggests that PKC could act as an endogenous suppressor of P2Y\u003csub\u003e1\u003c/sub\u003eR-calcium signaling in T24 bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eT24 bladder cancer cells: β-Arrestins 1 and 2 are not involved in A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2B\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eAR-mediated intracellular calcium mobilization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe have shown previously that β-arrestin2 siRNA diminished P2Y\u003csub\u003e1\u003c/sub\u003eR agonist MRS2365-induced and β-arrestin2-mediated ERK1/2 activity [28]. Here it is shown that neither β-arrestin1 nor β-arrestin2 siRNA affected A\u003csub\u003e2B\u003c/sub\u003eAR agonist NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eT24 bladder cancer cells: L-type calcium channels are not involved in A\u003csub\u003e2B\u003c/sub\u003eAR-mediated intracellular calcium mobilization\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Effects of the L-type calcium channel blocker nifedipine, A\u003csub\u003e2B\u003c/sub\u003e antagonist PSB603 and the PLC inhibitor U73122 on A\u003csub\u003e2B\u003c/sub\u003e agonist NECA-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. Results are from three experiments. Inhibitors were incubated with cells for 20 min in 96-well black plates before addition of NECA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have probed the mechanisms of GPCR-triggered calcium fluctuations in specific cell types, in which a GPCR is either endogenous or overexpressed, and may be coupled to G\u003csub\u003ei\u003c/sub\u003e-, G\u003csub\u003es\u003c/sub\u003e- or G\u003csub\u003eq\u003c/sub\u003e-protein. In the present study, we expanded the previous work and further explored A\u003csub\u003e2B\u003c/sub\u003eAR-mediated calcium transients, showing that G\u003csub\u003ei\u003c/sub\u003e, G\u003csub\u003es\u003c/sub\u003e, G\u003csub\u003eq\u003c/sub\u003e and PKC play different roles in different cell types. We also compare results with the A\u003csub\u003e2B\u003c/sub\u003eAR, either overexpressed or endogenously expressed in HEK293 cells, with several other GPCRs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePurinergic (i.e. A\u003csub\u003e2B\u003c/sub\u003e, A\u003csub\u003e1\u003c/sub\u003e, and P2Y\u003csub\u003e1\u003c/sub\u003e) and biogenic amine receptor signaling leading to calcium mobilization, showing results of treatment with pharmacological inhibitors or activators. The related figure is shown in italic parentheses.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eModulation of G protein, arrestin or PKC - and effect\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell type and receptor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG\u003csub\u003eαq/11\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\u003csub\u003eiβγ\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003csub\u003es\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-arrestin1/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePKC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eA\u003csub\u003e2B\u003c/sub\u003eAR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHEK293 (A\u003csub\u003e2B\u003c/sub\u003e overexpressing)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFR and YM and Gα\u003csub\u003eq/11\u003c/sub\u003e siRNA sensitive \u003cb\u003e(1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX insensitive\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePMA completely sensitive (Gao et al., 2023)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT24 (native A\u003csub\u003e2B\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFR and YM insensitive \u003cb\u003e(4)\u003c/b\u003e;\u003c/p\u003e \u003cp\u003eGα\u003csub\u003eq/11\u003c/sub\u003e siRNA insensitive \u003cb\u003e(4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX sensitive \u003cb\u003e(5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTX sensitive \u003cb\u003e(5)\u003c/b\u003e,\u003c/p\u003e \u003cp\u003eVariable, depending on cell type \u003csup\u003eb, c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ-arrestin1 or β-arrestin2 siRNA insensitive \u003cb\u003e(9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePMA partially sensitive \u003cb\u003e(8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA-MB-231 (native A\u003csub\u003e2B\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYM sensitive \u003cb\u003e(7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX partly sensitive \u003cb\u003e(7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTX partly sensitive \u003cb\u003e(7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003eAR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHEK293 (A\u003csub\u003e1\u003c/sub\u003e overexpressing)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYM and FR \u0026ndash; fully sensitive \u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX sensitive \u003cb\u003e(1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eP2Y\u003csub\u003e1\u003c/sub\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHEK293 (native P2Y\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGα\u003csub\u003eq/11\u003c/sub\u003e siRNA partially sensitive \u003cb\u003e(1)\u003c/b\u003e; YM and FR \u0026ndash; fully sensitive \u003cb\u003e(2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX insensitive (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1321N1 astro-cytoma (P2Y\u003csub\u003e1\u003c/sub\u003e-overexpressing)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYM sensitive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX insensitive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT24 (native P2Y\u003csub\u003e1\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYM \u0026ndash; fully sensitive \u003cb\u003e(4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX insensitive \u003cb\u003e(5)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eβ\u003csub\u003e2\u003c/sub\u003eR\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT24 (native b\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYM \u0026ndash; partially sensitive \u003cb\u003e(6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePTX not sensitive \u003cb\u003e(6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTX sensitive \u003cb\u003e(6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePMA completely sensitive \u003cb\u003e(8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Linden et al., 1999 [14].\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003e NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in HMC-1 cells was insensitive to CTX and PTX, Feoktistov et al., 1995 [29].\u003c/p\u003e \u003cp\u003e \u003csup\u003ec\u003c/sup\u003e NECA-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in human erythroleukemia cells was sensitive to CTX, Feoktistov et al., 1994 [30].\u003c/p\u003e \u003cp\u003e \u003csup\u003ed\u003c/sup\u003e Gao and Jacobson, 2017 [28].\u003c/p\u003e \u003cp\u003e \u003csup\u003ee\u003c/sup\u003e G\u003csub\u003eq/11\u003c/sub\u003e inhibitors blocked calcium mobilization by activation of the G\u003csub\u003ei\u003c/sub\u003e-coupled M\u003csub\u003e2\u003c/sub\u003e muscarinic receptor overexpressed in CHO cells.\u003c/p\u003e \u003cp\u003eND, not determined.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCPA, 2-chloro-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e6\u003c/sup\u003e-cyclopentyladenosine ; CTX, cholera toxin; EPAC, exchange protein activated by cAMP; FLIPR, Fluorometric Imaging Plate Reader; FR, FR900358; GPCR, G protein-coupled receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLP, glucagon-like peptide; GRK, G protein-coupled receptor kinases; HBSS, Hanks balanced salt solution; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid; LPA, lysophosphatidic acid; NECA, adenosine-5\u0026prime;-\u003cem\u003eN\u003c/em\u003e-ethyluronamide; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; YM, YM254890.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable, does not involve any human or animal studies.\u003c/p\u003e\n\u003ch2\u003eCompeting Interest\u003c/h2\u003e\n\u003cp\u003eZhan-Guo Gao declares that he/she has no conflict of interest. Ray Gao declares that he/she has no conflict of interest. Clayton Meyer declares that he/she has no conflict of interest. Kenneth A. Jacobson declares that he/she has no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eInclusion and Diversity\u003c/h2\u003e\n\u003cp\u003eWe support inclusive, diverse and equitable conduct of research.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Intramural Research Program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (ZIADK031117).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eZGG and KAJ conceptualized and wrote the text. ZGG wrote the first draft. ZGG, RRG, and CKM performed the pharmacological experiments.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\n\u003cp\u003eWe thank the NIDDK Intramural Research for support (ZIADK031117).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003ePrimary data for this study is available from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHauser AS, Attwood MM, Rask-Andersen M, Schi\u0026ouml;th HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017;16(12):829\u0026ndash;842. doi: 10.1038/nrd.2017.178.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClapham DE. Calcium signaling. Cell. 2007;131(6):1047-58. doi: 10.1016/j.cell.2007.11.028.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeda N, I\u0026ntilde;iguez-Lluhi JA, Lee E, Smrcka AV, Robishaw JD, Gilman AG. 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J Biol Chem. 2001;276(35):32648\u0026ndash;32656. doi: 10.1074/jbc.M104143200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Pascali F, Inoue A, Benovic JL, Diverse pathways in GPCR-mediated activation of Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in HEK293 cells. J Biol Chem. 2024;300:107882, https://doi.org/10.1016/j.jbc.2024.107882.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"A2B adenosine receptor, GPCR, G protein, calcium, Gq, Gi, Gs.","lastPublishedDoi":"10.21203/rs.3.rs-5442142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5442142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivation of PLCβ enzymes by G\u003csub\u003eiβγ\u003c/sub\u003e and G\u003csub\u003eαq/11\u003c/sub\u003e proteins is a common mechanism to trigger cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e increase. We and others reported that G\u003csub\u003eαq/11\u003c/sub\u003e inhibitor FR900358 (FR) can inhibit both and G\u003csub\u003eαq\u003c/sub\u003e- and, surprisingly, G\u003csub\u003eiβγ\u003c/sub\u003e-mediated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e mobilization. Thus, the G\u003csub\u003eαi\u003c/sub\u003e-G\u003csub\u003eβγ\u003c/sub\u003e-PLCβ-Ca\u003csup\u003e2+\u003c/sup\u003e signaling axis depends entirely on the presence of active G\u003csub\u003eαq\u003c/sub\u003e, which reasonably explained FR-inhibited G\u003csub\u003eiβγ\u003c/sub\u003e-induced Ca\u003csup\u003e2+\u003c/sup\u003e release. However, the conclusion that G\u003csub\u003eiβγ\u003c/sub\u003e signaling is controlled by G\u003csub\u003eαq\u003c/sub\u003e derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G\u003csub\u003eαq/11\u003c/sub\u003e siRNA and G\u003csub\u003eαq/11\u003c/sub\u003e inhibitors diminished Ca\u003csup\u003e2+\u003c/sup\u003e increase triggered by native G\u003csub\u003eq\u003c/sub\u003e-coupled P2Y\u003csub\u003e1\u003c/sub\u003e receptors, or by transfected G\u003csub\u003ei\u003c/sub\u003e-coupled A\u003csub\u003e1\u003c/sub\u003e- or G\u003csub\u003es\u003c/sub\u003e-coupled A\u003csub\u003e2B\u003c/sub\u003e adenosine receptors (ARs). However, in T24 bladder cancer cells, G\u003csub\u003ei\u003c/sub\u003e inhibitor PTX, but not G\u003csub\u003eαq/11\u003c/sub\u003e inhibitors, FR, YM254890 (YM) or G\u003csub\u003eq/11\u003c/sub\u003e siRNA, inhibited Ca\u003csup\u003e2+\u003c/sup\u003e increase triggered by native A\u003csub\u003e2B\u003c/sub\u003eAR activation. Simultaneous inactivation of G\u003csub\u003ei\u003c/sub\u003e and G\u003csub\u003es\u003c/sub\u003e further suppressed A\u003csub\u003e2B\u003c/sub\u003eAR-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. The G\u003csub\u003eαq/11\u003c/sub\u003e inhibitor YM fully and partially inhibited endogenous P2Y\u003csub\u003e1\u003c/sub\u003e- and β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor-induced Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells, respectively. PKC activator PMA partially diminished A\u003csub\u003e2B\u003c/sub\u003eAR-triggered but completely diminished β\u003csub\u003e2\u003c/sub\u003e-adrenergic receptor-triggered Ca\u003csup\u003e2+\u003c/sup\u003e increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected A\u003csub\u003e2B\u003c/sub\u003eAR-mediated Ca\u003csup\u003e2+\u003c/sup\u003e increase. Unlike in T24 cells, YM inhibited native A\u003csub\u003e2B\u003c/sub\u003eAR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G\u003csub\u003eαq/11\u003c/sub\u003e is vital for Ca\u003csup\u003e2+\u003c/sup\u003e increase in some cell types, but G\u003csub\u003eiβγ\u003c/sub\u003e-mediated Ca\u003csup\u003e2+\u003c/sup\u003e signaling can be Gα\u003csub\u003eq/11\u003c/sub\u003e-dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e increase depending on cell type and receptor.\u003c/p\u003e","manuscriptTitle":"A2B adenosine receptor-triggered intracellular calcium mobilization: Cell type-dependent involvement of Gi, Gq, Gs proteins and protein kinase C","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-13 13:16:00","doi":"10.21203/rs.3.rs-5442142/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-15T17:46:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-15T16:50:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53137617278442315308905541159711701200","date":"2025-01-08T17:22:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-05T17:11:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175847862814980698283914384284702919970","date":"2025-01-04T16:36:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33777316183640164683758098204735584152","date":"2024-12-23T17:46:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262560965075506164213162761531973666912","date":"2024-11-20T17:10:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-18T14:47:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-14T09:05:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-14T09:01:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Purinergic Signalling","date":"2024-11-12T20:41:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"purinergic-signalling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pusi","sideBox":"Learn more about [Purinergic Signalling](http://link.springer.com/journal/11302)","snPcode":"11302","submissionUrl":"https://submission.nature.com/new-submission/11302/3","title":"Purinergic Signalling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ba298f3-fa81-4fb0-b8ea-063ea507fe6f","owner":[],"postedDate":"December 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-17T16:06:06+00:00","versionOfRecord":{"articleIdentity":"rs-5442142","link":"https://doi.org/10.1007/s11302-025-10070-1","journal":{"identity":"purinergic-signalling","isVorOnly":false,"title":"Purinergic Signalling"},"publishedOn":"2025-02-11 15:57:35","publishedOnDateReadable":"February 11th, 2025"},"versionCreatedAt":"2024-12-13 13:16:00","video":"","vorDoi":"10.1007/s11302-025-10070-1","vorDoiUrl":"https://doi.org/10.1007/s11302-025-10070-1","workflowStages":[]},"version":"v1","identity":"rs-5442142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5442142","identity":"rs-5442142","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}