Estradiol enhances T-type calcium channel activation in human myometrium telocytes

In the present study, we identified telocytes in sample sections in situ and primary cultures of the human myometrium. Fig. 1(A) Fig. 1. Identification of human myometrium telocytes and T-type calcium channel expression. (A) Immunohistochemistry for c-kit showed that telocytes positive for c-kit were shown in red. Nuclei were stained with DAPI. (B) In vitro primary culture of telocyte from human myometrium. The blackboard arrow points to the central oval-shaped cellular body containing the nucleus, and the thin black arrow points to several surrounding prolongations. Red arrow indicating the specific dilations in the process. (C) Double immunofluorescent staining for c-kit, CD34, and α subunits of the T-type calcium channel in telocytes cultured from human myometrium. Nuclei were stained with DAPI. provides direct visual evidence of the presence of telocytes in the human myometrium in situ. Telocytes are immunologically positive for c-kit and are shown in red. Under phase-contrast microscopy, telocytes showed the typical characteristics of several distinct prolongations emerging from the cell body, and bead-like specific dilations were observed ( Fig. 1(B) ). Identification of human myometrium telocytes and T-type calcium channel expression. (A) Immunohistochemistry for c-kit showed that telocytes positive for c-kit were shown in red. Nuclei were stained with DAPI. (B) In vitro primary culture of telocyte from human myometrium. The blackboard arrow points to the central oval-shaped cellular body containing the nucleus, and the thin black arrow points to several surrounding prolongations. Red arrow indicating the specific dilations in the process. (C) Double immunofluorescent staining for c-kit, CD34, and α subunits of the T-type calcium channel in telocytes cultured from human myometrium. Nuclei were stained with DAPI. Immunostaining of human myometrial cells in primary culture showed that c-kit and CD34 were detected in telocytes. Immunofluorescence double staining of telocytes confirmed that c-kit and CD34 were positive in cells with characteristic morphology. Double immunofluorescence staining also revealed co-expression of T-type calcium channel α subunits Ca v 3.1(α 1G )/Ca v 3.2(α 1H ) on c-kit/CD34 positive cells ( Fig. 1(C) ). To verify the existence of T-type calcium channels in telocytes in the human myometrium and their role in regulating intracellular calcium ions, intracellular calcium levels were measured using a Fluo-4 fluorescence indicator before and after NNC 55-0396 was dripped in. Estradiol was also used to examine its effect on T-type calcium channels and intracellular calcium levels. The effects of different concentrations of NNC 55-0396 and estradiol on intracellular calcium levels are shown in Fig. 2 Fig. 2. Intracellular calcium imaging shows real-time calcium changes in telocytes after adding NNC 55-0396 or estradiol. The intracellular calcium signals were recorded every 0.31 sec. We selected nine images from each group to represent the dynamic changes in intracellular calcium signals. The time lapse between images was 1.86 s except in picture (E), which showed rapid changes, and the time-lapse was 0.93 sec. (A) The intracellular calcium fluorescence intensity in the control group showed no significant change after adding DMEM. (B–E) As the concentration of NNC 55-0396 increased, the intracellular calcium fluorescence intensity decreased more rapidly, thoroughly, and lasted. (F–H) After the addition of estradiol, intracellular calcium fluorescence intensity was enhanced. When the estradiol concentration reached 40 nM, the green fluorescence flared and soon died. (I–J) Upon sequential adding NNC 55-0396 and estradiol, the inhibitory effect of NNC 55-0396 on intracellular calcium fluorescence intensity could be partially recovered by estradiol. (K) Relative calcium fluorescence intensity change curves for human uterine telocytes. Relative calcium fluorescence intensity was calculated by dividing each picture’s real-time calcium fluorescence intensity by the initial calcium fluorescence intensity of the first picture. . The results demonstrated that green fluorescence intensity was strongly attenuated by NNC 55-0396, and the inhibitory effect was concentration-dependent. NNC 55-0396 at 5 μM caused macroscopic changes in fluorescence intensity, starting from the prolongation and spreading to the cell body ( Video 1 in the supplementary data ). When increasing concentrations of NNC 55-0396, the inhibitory effect manifested more rapidly, thoroughly, and lasted ( Video 2 in the supplementary data ). For telocytes supplemented with estradiol, the fluorescence intensity increased rapidly and in a concentration-dependent manner. When the estradiol concentration reached 40 nM, green fluorescence flared and soon died ( Video 3 in the supplementary data ). Interestingly, when the fluorescence intensity was inhibited by NNC 55-0396, it was partially recovered by adding estradiol ( Video 4 in the supplementary data ). Intracellular calcium imaging shows real-time calcium changes in telocytes after adding NNC 55-0396 or estradiol. The intracellular calcium signals were recorded every 0.31 sec. We selected nine images from each group to represent the dynamic changes in intracellular calcium signals. The time lapse between images was 1.86 s except in picture (E), which showed rapid changes, and the time-lapse was 0.93 sec. (A) The intracellular calcium fluorescence intensity in the control group showed no significant change after adding DMEM. (B–E) As the concentration of NNC 55-0396 increased, the intracellular calcium fluorescence intensity decreased more rapidly, thoroughly, and lasted. (F–H) After the addition of estradiol, intracellular calcium fluorescence intensity was enhanced. When the estradiol concentration reached 40 nM, the green fluorescence flared and soon died. (I–J) Upon sequential adding NNC 55-0396 and estradiol, the inhibitory effect of NNC 55-0396 on intracellular calcium fluorescence intensity could be partially recovered by estradiol. (K) Relative calcium fluorescence intensity change curves for human uterine telocytes. Relative calcium fluorescence intensity was calculated by dividing each picture’s real-time calcium fluorescence intensity by the initial calcium fluorescence intensity of the first picture. The T-type calcium current in the telocytes was successfully evoked and recorded by the patch-clamp technique using a step-depolarizing pulse protocol. The pulses ranged from −90 mV to +60 mV for 100 msec duration and 10 mV increments from a holding potential of −70 mV. Depolarizing pulses above −60 mV elicited an apparent inward current, indicating the presence of a low voltage-activated calcium current (T-type calcium current) in myometrium telocytes. T-type calcium channels in telocytes were highly sensitive to NNC 55-0396 and were fully blocked at a concentration of 10 μM ( Fig. 3(A) Fig. 3. T-type calcium current changes with estradiol in Telocytes. (A) T-type calcium current amplitude of telocytes in different estradiol groups after applying NNC 55-0396 (10 μM). An asterisk indicates the difference between the two groups was significant (P < 0.01). (B) T-type calcium currents in telocytes were evoked by depolarizing voltage steps from −90 mV to +60 mV with a holding potential of −70 mV in the absence and presence of different estradiol concentrations. (a) T-type calcium current amplitude gradually enhanced due to increasing estradiol concentration in the culture medium. (b) T-type calcium current in telocytes of different groups evoked at 30 mV was composed in the same picture. (c) T-type calcium current amplitude curve of telocytes cultured in different concentrations of estradiol. (d) The T-type calcium current of a telocyte was recorded in whole-cell configuration under the voltage-clamp mode during patch-clamp recording. (C) T-type and L-type calcium currents were recorded in different telocytes cultured with estradiol (40 nM) in human myometrium. (a) A telocyte displayed a typical T-type calcium current at the beginning after step-depolarizing pulse stimulation. After 5 stimulations, T-type calcium current gradually converted to L-type calcium current, with slow and long-lasting activation. (b) High-voltage activated calcium currents were also detected in another telocyte with long-lasting activation (L-type calcium current). ). The T-type calcium current amplitude in different estradiol groups were both significantly attenuated after adding NNC 55-3096 (N = 4 for each group: control: −269.9 ± 22.1 pA vs . −111.7 ± 49.1 pA, P = 0.001, E 2 10 nM: −607.8 ± 57.5 pA vs . −40.7 ± 23.2 pA, P < 0.001, E 2 20 nM: −685.4 ± 71.7 pA vs . −123.7 ± 26.3 pA, P < 0.001, E 2 40 nM: −1520.3 ± 96.7 pA vs . −72.6 ± 104.6 pA, P < 0.001, respectively). However, by incubating with estradiol for 24 h, the amplitude of T-type calcium current was significantly increased (control: N = 16, −358.3 ± 59.9 pA, E 2 10 nM: N = 30, −623.5 ± 104.0 pA, E 2 20 nM: N = 16, −750.6 ± 214.5 pA, and E 2 40 nM: N = 21, −1125.3 ± 373.4 pA, P < 0.001) ( Fig. 3(B) ). In addition to T-type currents, high-voltage activated calcium currents were detected in a few telocytes with long-lasting activation (L-type calcium current) ( Fig. 3(C) ). It is worth noting that telocytes cultured with estradiol (40 nM) displayed typical T-type calcium current at the beginning after step-depolarizing pulse stimulation. As stimuli increased, the T-type calcium current gradually converted to an L-type calcium current, with slow and long-lasting activation ( Fig. 3(C) ). T-type calcium current changes with estradiol in Telocytes. (A) T-type calcium current amplitude of telocytes in different estradiol groups after applying NNC 55-0396 (10 μM). An asterisk indicates the difference between the two groups was significant (P < 0.01). (B) T-type calcium currents in telocytes were evoked by depolarizing voltage steps from −90 mV to +60 mV with a holding potential of −70 mV in the absence and presence of different estradiol concentrations. (a) T-type calcium current amplitude gradually enhanced due to increasing estradiol concentration in the culture medium. (b) T-type calcium current in telocytes of different groups evoked at 30 mV was composed in the same picture. (c) T-type calcium current amplitude curve of telocytes cultured in different concentrations of estradiol. (d) The T-type calcium current of a telocyte was recorded in whole-cell configuration under the voltage-clamp mode during patch-clamp recording. (C) T-type and L-type calcium currents were recorded in different telocytes cultured with estradiol (40 nM) in human myometrium. (a) A telocyte displayed a typical T-type calcium current at the beginning after step-depolarizing pulse stimulation. After 5 stimulations, T-type calcium current gradually converted to L-type calcium current, with slow and long-lasting activation. (b) High-voltage activated calcium currents were also detected in another telocyte with long-lasting activation (L-type calcium current).

In the present study, we demonstrated that telocytes in the human uterus function as estradiol sensors and transform hormone messages into electrobiological signals through a T-type calcium channel. Our study found that immunologically positive telocytes were present in the human myometrium. In vitro , the primary culture of telocytes presented a unique Cajal-like morphology of a consensus, such as a small oval-shaped cellular body containing a nucleus surrounded by several distinct prolongations with a small amount of cytoplasm [ 9 , 13 ]. The shape of telocytes depends on the number of cellular extensions: piriform for one prolongation, spindle for two telopodes, triangular for three, stellate, etc. [ 24 ]. These specific structural properties of telocytes enable them to form a network that contacts smooth muscle cells, nerve cells, and capillaries. In long prolongations, bead-like dilated portions containing mitochondria and the endoplasmic reticulum have been reported [ 13 ]. Evidence points towards the role of these long cellular extensions in coordinating the surrounding cells by exosome/ectosome release [ 11 , 25 ]. Telocyte-dominated cell communication in the uterus might play a key role in managing uterine peristalsis and orchestrating embryo implantation. The ICC in the digestive tube is responsible for gastrointestinal peristalsis and plays an essential role in pacemakers and neurotransmission. Similarly, telocytes in the myometrium may be the counterpart of ICC and participate in the initiation and propagation of uterine peristalsis. Research on uterine contraction has found that electrical activity is controlled by changes in the membrane potential of smooth muscle cells [ 15 ], and fluctuations in the membrane potential and the subsequent action potential are responsible for the consequent smooth muscle contractions. In a previous study, telocytes in the human myometrium exhibited spontaneous electrical activity, which might be a possible role of the pacemaker [ 9 ]. The next step of our study was to verify that the cells possessed the immunohistochemical features of telocytes. They were positive for c-kit and CD34, which are widely accepted and reliable markers of telocytes [ 9 , 13 , 24 , 25 ]. Double immunofluorescence also proved that the α-subunit of T-type calcium channels was expressed in telocytes in the human myometrium. This provides evidence of the presence of T-type calcium channels in telocytes and is the basis of electrophysiological activity. T-type channels controlling Ca 2+ influx in excitable cells during small depolarizations around resting potential have been demonstrated to be closely related to low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, and cell growth and differentiation [ 26 ]. Further confirmation of T-type calcium channel functions in modulating intracellular calcium levels was performed by fluorescence calcium imaging in living cells and was effectively inhibited by NNC 55-0396. The inhibitory effect of NNC 55-0396 gradually spread from the prolongations to the cell body, indicating that the prolongations were more susceptible to T-type calcium channel regulation and may enable signals to be effectively conducted downstream. When increasing concentrations of NNC 55-0396, green fluorescence was more effectively attenuated rapidly, thoroughly, and more prolonged in telocytes. This was in line with previous studies that identified an inhibitory effect of Mibefradil on T-type calcium channels [ 11 , 25 , 27 ]. In contrast, estradiol displayed an enhanced effect on intracellular calcium concentrations in telocytes. Low estradiol concentrations (10–20 nM) caused imperceptible changes in the fluorescence intensity to the naked eye. Quantitative analysis of the relative fluorescence intensity showed that the curve rose after adding estradiol. Estradiol at 40 nM induced a remarkable flash of calcium fluorescence, which quickly darkened ( Video 3 in the supplementary data ). We speculated that this might be attributed to the instant intracellular calcium increase resulting in calcium overload, which finally causes cell death. Cells inhibited by NNC 55-0396 (10 μM) could be partially recovered by adding estradiol (20 nM) but did not return to the previous state ( Video 4 in the supplementary data ). D. Cretoiu revealed that estrogen (ER) and progesterone (PR) receptors are located in the human myometrium, indicating the possible role of hormonal sensors in the regulation of human myometrial contractions [ 21 ]. Our findings corroborate these results by showing the influence of estradiol on telocytes. These findings provide a reasonable explanation for the clinical phenomenon that uterine peristalsis peaks during ovulation and is positively correlated with estradiol [ 5 ]. Our exploration of the electrophysiological characteristics of telocytes in the human myometrium further verified our conjecture. First, the recording of T-type calcium currents indicated that T-type calcium channels exist in telocytes and function as regulators of intracellular calcium ions. In addition, T-type calcium currents were abolished by the highly selective T-type calcium channel blocker NNC 55-0396. These results indicate that T-type calcium channels are involved in the electrical excitability of telocytes in the human myometrium. T-type calcium channels can participate in the generation of electrophysiological signals responsible for myometrial contraction. Estradiol displayed an enhanced effect on T-type calcium channels in a dose-dependent manner by increasing the current amplification, allowing more calcium ions to flow inward. L-type calcium currents were also recorded in telocytes with a relatively high current amplitude. Interestingly, we noticed that under high concentrations of E 2 , some T-type calcium currents tended to transform into L-type calcium currents when more stimulation was applied. We speculate that as the cytosolic free calcium increases, L-type calcium current might be activated and act as a messenger downstream for myometrium contractions. One limitation of our study was that all samples were collected from pregnant women during cesarean sections and lacked data from the non-pregnant uterus. Previous studies [ 9 , 20 , 21 ] have reached a consensus on the existence of telocytes and the expression of T-type calcium channels in both pregnant and non-pregnant myometrium. Considering fertility preservation for women of reproductive age, surgical indications for hysterectomy in the childbearing period were mostly under severe pathological conditions such as malignant tumors, severe adenomyosis, et al . Therefore, we chose the normal pregnant myometrium to avoid malignant cell interference. In addition, the myometrium at cesarean section was from the lower uterine segment, which was the counterpart of the uterine isthmus in the non-pregnant uterus, and the latter was also the origin of cervicofundal uterine peristalsis, the dominant type of uterine peristalsis during the entire menstrual cycle [ 5 ]. Further studies focusing on the non-pregnant myometrium could use multi-point sample collection of the uteri isthmus, body, and fundus to investigate their role in regulating uterine peristalsis. In conclusion, telocytes in the human myometrium express T-type calcium, functioning as a regulator of intracellular calcium ions. Estradiol could enhance T-type calcium channel activation in human myometrium telocytes, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

The authors declared no competing interests.

Biopsies of the human myometrium in the lower uterine segment were obtained from 18 healthy women of reproductive age (23–36 years old) between May 2019 and October 2020. Women with anatomical disorders of the female reproductive system, leiomyomas, and endometriosis were excluded. The approximate size of the myometrial biopsy specimen was 1 cm 3 . All procedures were conducted in accordance with the guidelines of the Declaration of Helsinki and with a protocol approved by the Institutional Ethics Committee Review Board of the First Affiliated Hospital of Army Medical University (Approval No.: KY201920 ). Informed written consent for sample collection was obtained before surgery. Myometrium samples were collected from the upper margin (in the midline) of the transverse incision of the lower uterine segment during cesarean sections. Four specimens were used for immunohistochemical staining with a c-kit to confirm the presence of telocytes in the human myometrium. Immunohistochemistry was performed on 3 μm-thick sections of formalin-fixed paraffin-embedded specimens. Briefly, the procedure comprised deparaffinization in xylene and alcohol series (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), rehydration, washing in phosphate-buffered saline (PBS), and microwave antigen retrieval for c-kit. After blocking with bovine serum albumin (BSA, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) for 30 min, the sample sections were incubated with primary antibody c-kit (mouse monoclonal, 1:800, #3308, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight, followed by incubation with a secondary antibody (Cy3 conjugated Goat Anti-mouse IgG, 1:400, GB21301, Wuhan Servicebio Technology Co., Ltd.) for 50 min according to the manufacturer’s instructions. Nuclei were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Boster Biological Technology Co., Ltd., Wuhan, China), and antifade solution (Boster Biological Technology Co., Ltd.) was added. Human myometrium samples (N = 14) were collected under sterile conditions during the surgery and immediately placed in sterile tubes containing ice-cold Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2% fetal bovine serum (FBS) (all from Corning, Manassas, VA, USA). The samples were transported to the cell culture laboratory within 30 min of isolation and primary culture. Briefly, after rinsing in sterile DMEM, tissue samples were minced into fragments of 0.5 mm pieces and digested in DMEM containing collagenase Ia (1 mg/ml, Sigma Chemical, St. Louis, MO, USA) and deoxyribonuclease I (2000 IU/ml, Sigma Chemical) for 30 min at 37ºC with agitation. The dispersed cells were separated using a 100 μm sterile cell strainer and collected by centrifugation at 250 × g for 10 min. The deposit was suspended in a culture medium of DMEM containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin and distributed on glass coverslips in 6-well plates in a humidified 5% CO 2 /95% air environment at 37ºC. The culture medium was changed every other day, and the cells were passaged using 0.25% trypsin and 0.02% EDTA. Experiments were performed in primary cultures between passages 1 and 2. Primary cultured cells grown on coverslips were used for immunofluorescence double staining for c-kit, CD34, and T-type calcium channel α subunits Ca v 3.1(α 1G ) and Ca v 3.2(α 1H ). After fixing in 2% paraformaldehyde for 10 min, the cells were washed in PBS and incubated in PBS containing 0.5% BSA and 0.075% saponin for 15 min (all reagents from Sigma Chemical). Incubation with primary antibodies was performed at room temperature for 1 h using anti-human antibodies: c-kit, mouse monoclonal (Ab 81), 1:800 (#3308, Cell Signaling Technology, Inc.), CD34, rabbit monoclonal (SI16-01), 1:50 (#MA5-32059, Invitrogen, Carlsbad, CA, USA), anti-CACNA1G (Cav3.1) antibody, rabbit polyclonal, 1:200 (#ACC-021, Alomone Labs, Jerusalem, Israel), and T-type Ca ++ CP α1H, mouse monoclonal (G-10), 1:50 (sc-377510, Santa Cruz Biotechnology, Inc., CA, USA). For double-staining experiments, after three serial rinses, the second reaction was detected using polyclonal FITC-labeled goat anti-mouse antibodies (working dilution 1:250, bs-0296G-FITC, Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) and Cy3 conjugated Goat Anti-Rabbit IgG (GB21301, Wuhan Servicebio Technology Co., Ltd.). Nuclei were counterstained with DAPI (Boster Biological Technology Co., Ltd.), and antifade solution (Boster Biological Technology Co., Ltd.) was added. The same protocol without primary antibodies was used for the negative controls. Samples were examined under a Zeiss LSM880 Airyscan microscope equipped with a Plan-Apochromat 40× and 60× objectives and the appropriate fluorescence filters. Intracellular calcium signals were measured using the fluorescent calcium indicator Fluo-4 AM (Beyotime, Shanghai, China) according to the manufacturer’s guidelines. Cells were seeded onto glass-bottom culture dishes and incubated with Fluo-4 AM (5 μM) for 30 min at 37ºC in the dark. The cells were then washed three times with PBS and incubated for another 20 min to ensure that Fluo-4 AM had completely transformed into Fluo-4. The fluorescence intensity and images were obtained by acquiring emission at 488 nm with a laser scanning confocal microscope (Zeiss LSM880 with Airyscan microscope). Different concentrations of NNC 55-0396 (MedChemExpress, Monmouth Junction, NJ, USA) (3 μM, 5 μM, 10 μM, and 20 μM, N = 6 for each group) [ 22 , 23 ], a mibefradil derivative and a highly selective T-type calcium channel blocker were added to test its inhibitory action on T-type calcium channels and intracellular calcium concentration. Estradiol (17-beta estradiol in DMSO, MedChemExpress) at 10 nM, 20 nM, and 40 nM (N = 6 for each group) was also used to investigate its effects on intracellular calcium signaling. Relative calcium fluorescence intensity was calculated by dividing each picture’s real-time calcium fluorescence intensity by the initial calcium fluorescence intensity of the first picture. Telocytes from the human myometrium in primary cultures were recorded in the whole-cell configuration under the voltage-clamp mode with an AxonPatch 200 B amplifier (Molecular Devices, San Jose, CA, USA). Telocytes grown on coverslips were placed in a cell chamber mounted on the stage of an inverted microscope (IX71; Olympus, Tokyo, Japan). MX7600 (Siskiyou Design Instruments, Grants Pass, OR, USA) micromanipulators with MC2000 controllers were used to position the microelectrodes. The closed-loop connection ensures a 0.2 μm resolution. On-site made glass microelectrodes were pulled from a borosilicate glass capillary with filament (O.D.:1.50 mm, I.D.:0.86 mm) (Sutter Instrument, Novato, CA, USA, catalog number: BF150-86-10) using a Horizontal Micropipette puller (programmable Flaming/Brown type micropipette puller, Sutter Instrument, model: P-97) with platinum filament and heat polished. When filled with the intracellular solution, the final resistance of the pipette was 6–8 MΩ. The liquid junction potential was set to zero before attaching the cell using the pipette offset button. Following the formation of a gigaseal, the membrane was ruptured by gentle suction to obtain the whole-cell configuration in voltage-clamp mode. The inward current was elicited by depolarizing voltage steps from −90 mV to +60 mV in 10 mV increments with a holding potential (HP) of −70 mV. Membrane currents were low-pass filtered at 5 kHz and sampled with an Axon Digidata 1440 data acquisition system (Molecular Devices) using pClamp 10.7 software in episodic stimulation. All electrophysiological experiments were performed at room temperature (22–24°C). The bath solution contained (mM) tetraethylammonium (TEA)-Cl 130, BaCl2 10, MgCl 2 1, HEPES 10, and glucose 10, adjusted to pH 7.4, at 25°C with TEA-OH. The pipette solution contained (mM) CsCl 137, MgCl 2 1, HEPES 10, 1,2-bis(2- aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) 10, and Mg-ATP 1, adjusted to pH 7.3 at 25°C with CsOH. Solutions for T-type calcium channel recordings were prepared as previously described [ 20 , 24 ]. To evaluate the effect of estradiol on T-type calcium of telocytes in the human myometrium, three groups of telocytes incubated with different concentrations of estradiol (17-beta estradiol in DMSO, MedChemExpress) at 10 nM, 20 nM, and 40 nM for 24 h as well as the control group, were used to investigate the differences in T-type calcium currents. A highly selective T-type calcium channel blocker NNC 55-0396 (10 μM), was applied during the step depolarization protocol to prove that T-type calcium currents were elicited on telocytes and could be blocked by NNC 55-0396. Statistical analysis was performed using SPSS (v19) and GraphPad Prism (v5.01). Continuous data were presented as mean and standard deviation. A paired samples t -test with two tails was used to determine whether the T-type current amplitude differed before and after adding NNC 55-0396. One-way analysis of variance (ANOVA) was used to analyze the effect of estradiol on the T-type calcium current amplitude in telocytes. P < 0.05 was considered statistically significant.

Video 1 shows that the green fluorescence intensity was slightly attenuated by NNC 55-0396 at 5 μM, starting from prolongation and spreading to the cell body. However, the cell body remained fluorescent during observation. Video 2 shows that after adding higher concentrations of NNC 55-0396 (10 μM), the inhibiting effect of calcium fluorescence intensity manifested more rapidly, thoroughly, and lasted the whole cell. Video 3 shows that 40 nM estradiol increased the calcium fluorescence intensity with a remarkable flash and soon darkened. Video 4 shows that intracellular calcium fluorescence intensity was first inhibited by NNC 55-0396 (10 μM) and then partially recovered by adding estradiol (20 nM) but no longer back to the previous state.