__________________________________________________________ VANILLOID (VR1) RECEPTORS ARE EXPRESSED BY PARIETAL CELLS IN THE RAT STOMACH FUNDUS AND ANTRUM __________________________________________________________

__________________________________________________________ VANILLOID (VR1) RECEPTORS ARE EXPRESSED BY PARIETAL CELLS IN THE RAT STOMACH FUNDUS AND ANTRUM __________________________________________________________ | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 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Schubert Authors Info & Affiliations https://doi.org/10.22541/au.175491821.10576299/v1 247 views 66 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract We describe a simple technique using in-house-designed forward and reverse primers to detect the receptors for vanilloid (VR-1) in the fundus and antrum of the rat stomach. Using RT-PCR followed by agarose gel electrophoresis, DNA band purification, sequencing followed by GeneBlast confirmed the presence of these receptors. VANILLOID (VR1) RECEPTORS ARE EXPRESSED BY PARIETAL CELLS IN THE RAT STOMACH FUNDUS AND ANTRUM __________________________________________________________ 1, 2, 3 Shyamal Premaratne, MD, PhD, 4 Hansa Doppalapudi, BA, 1 Morgan B. Edwards III, BS, 1 Vjaughn Ingraham, MS, and 2, 3 Mitchell L. Schubert, MD, FACP 1 Virgina Union University, Richmond, Virginia; 2 Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia; 3 Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, Virginia; 4 George Washington University School of Medicine and Health Sciences, Washington, District of Columbia Running Title: Detecting VR-1 Receptors Key Words: Vanilloid, Primers, Gene Sequencing Address for Correspondence and requests for reprints: Shyamal Premaratne, MD, PhD Mail Stop 151 Hunter Holmes McGuire Veterans Administration Medical Center 1201 Broad Rock Boulevard Richmond, Virginia 23249-0001 (804) 675 5000 (Voice); (804) 937 5673 (Mobile / Text) ABSTRACT We describe a simple technique using in-house-designed forward and reverse primers to detect the receptors for vanilloid (VR-1) in the fundus and antrum of the rat stomach. Using RT-PCR followed by agarose gel electrophoresis, DNA band purification, sequencing followed by GeneBlast confirmed the presence of these receptors. INTRODUCTION The endocannabinoid system (ECS) is a complex signaling network that is integral to regulating gastrointestinal homeostasis, influencing inflammation, digestive motility, and secretory functions throughout the gastrointestinal tract (1, 2). Among the receptors influenced by endocannabinoids is the transient receptor potential vanilloid type 1 (TRPV1 or VR1), a non-selective cation channel activated by heat, capsaicin, and certain lipid mediators (3). VR1 is expressed in various components of the gastrointestinal tract, particularly in gastric tissues, where it plays a critical role in sensory signaling, mucosal protection, and inflammatory response modulation (4, 5). Although VR1 has been detected in various gastric cell types, its specific localization within parietal cells remains unclear (6). This study aims to detect VR1 receptor expression in rat stomach tissues, specifically the fundus and antrum, utilizing a molecular approach involving RT-PCR with custom-designed primers. Confirmation through sequencing supports the molecular identity of VR1 transcripts, contributing to our understanding of cannabinoid effects on gastric function. Determining whether VR1 receptors are expressed in parietal cells could provide insight into novel mechanisms of acid regulation and neuroendocrine signaling in the stomach. Furthermore, understanding this localization may help enhance VR1’s potential as a therapeutic target in gastrointestinal disorders (7). MATERIALS AND METHODS Tissue Collection Gastric tissues, including the fundus and antrum, were harvested from adult male rats under appropriate ethical guidelines. Tissues were immediately frozen in liquid nitrogen and stored at -80°C until further processing. RNA Extraction and cDNA Synthesis Total RNA was extracted from the gastric fundic and antral tissues using a standard TRIzol-based method. RNA concentration and purity were assessed using spectrophotometry. Reverse transcription was performed using 1 µg of total RNA and a cDNA synthesis kit, yielding complementary DNA (cDNA) for subsequent amplification (8). Primer Design and PCR Amplification PCR amplification targeted the VR1 receptor gene using uniquely designed primers: Upstream Primer: 5’-GACATGCCACCCAGCAGG-3’ Downstream Primer: 5’-TCAATTCCCACACACCTCCC-3’ PCR was carried out in a 25 µL reaction volume using standard cycling parameters optimized for the VR1 gene. Amplification products were resolved on a 1.5% agarose gel to confirm the expected fragment size. Cloning and Sequencing PCR products were subcloned into pGEM-T vectors and transformed into competent E. coli cells. Positive colonies were selected, and plasmids were extracted and sequenced to confirm the identity of the amplified VR1 receptor gene fragment. RESULTS AND DISCUSSION Figure 1: RT-PCR Analysis of VR1 Expression in Rat Gastric Fundus and Antrum RT-PCR using rat VR1-specific primers yielded a band at the expected size for rat VR1 (987-bp) in both antral and fundic mucosa. The PCR product was cloned, and sequence analysis revealed complete homology with the known VR1 sequence. The successful amplification and sequencing of VR1 receptor transcripts from both fundic and antral regions of the rat stomach confirm the presence of these receptors in these gastric tissues (Figure 1). The use of unique primers ensured specificity in detecting the target gene, minimizing off-target amplification. The subcloning and sequencing steps provided robust validation of the PCR results, affirming that the observed products corresponded to VR1 receptor cDNA. This detection has significant physiological implications. The localization of VR1 receptors in the gastric mucosa supports the hypothesis that cannabinoids may directly influence gastrointestinal function. One possible pathway is through the modulation of somatostatin, a key inhibitory hormone involved in the regulation of gastric secretions and motility. The molecular evidence provided here lays the groundwork for future functional studies investigating the specific roles of cannabinoid signaling in gastric hormone dynamics and mucosal health. BIBLIOGRAPHY 1. Cuddihey H, MacNaughton WK, Sharkey KA. Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis. Cell Mol Gastroenterol Hepatol 2022; 14(No.4): 947-63. doi: 10.1016/j.jcmgh.2022.05.015. 2. Lu HC, Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry 2016; 79(No.7): 516-25. doi: 10.1016/j.biopsych.2015.07.028. 3. Chen J, Sun W, Zhu Y, Zhao F, Deng S, Tian M, Wang Y, Gong Y. TRPV1: The Key Bridge in Neuroimmune Interactions. J Intensive Med 2024; 4(No.4): 442-52. doi: 10.1016/j.jointm.2024.01.008. PMID: 39310069; PMCID: PMC11411435. 4. McIntyre P, McLatchie LM, Chambers A, Phillips E, Clarke M, Savidge J, Toms C, Peacock M, Shah K, Winter J, Weerasakera N, Webb M, Rang HP, Bevan S, James IF. Pharmacological Differences Between the Human and Rat Vanilloid Receptor 1 (VR1). Br J Pharmacol 2001; 132(No.5): 1084-94. doi: 10.1038/sj.bjp.0703918. 5. Sheikh NK, Dua A. Cannabinoids. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2024 Jan. PMID: 32310522. 6. Mezey É, Tóth ZE, Cortright DN, Arzubi MK, Krause JE, Elde R, Guo A, Blumberg PM, Szallasi A. Distribution of mRNA for Vanilloid Receptor Subtype 1 (VR1), and VR1-Like Immunoreactivity, in the Central Nervous System of the Rat and Human. Proc Natl Acad Sci USA 2000; 97(No.7): 3655-60. doi: 10.1073/pnas.97.7.3655. PMID: 10725386 7. Gao N, Yang F, Chen S, Wan H, Zhao X, Dong H. The Role of TRPV1 Ion Channels in the Suppression of Gastric Cancer Development. J Exp Clin Cancer 2020; 39(No.1): 206. doi: 10.1186 /s13046-020-01707-7. 8. Premaratne S, Xue C, McCarty JM, Zaki M, McCuen RW, Johns RA, Schepp W, Neu B, Lippman R, Melone PD, Schubert ML. Neuronal Nitric Oxide Synthase: Expression in Rat Parietal Cells. Am J Physiol Gastrointest Liver Physiol 2001; 280(No.2): G308-13. doi: 10.1152/ajpgi.2001.280.2.G308.PMID: 11208555. Information & Authors Information Version history V1 Version 1 11 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Shyamal Premaratne [email protected] Virginia Union University View all articles by this author Hansa Doppalapudi The George Washington University Department of Biochemistry and Molecular Medicine View all articles by this author Morgan B. Edwards Virginia Union University View all articles by this author Vjaughn Ingraham Virginia Union University View all articles by this author Mitchell L. Schubert Virginia Commonwealth University School of Medicine View all articles by this author Metrics & Citations Metrics Article Usage 247 views 66 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shyamal Premaratne, Hansa Doppalapudi, Morgan B. Edwards, et al. __________________________________________________________ VANILLOID (VR1) RECEPTORS ARE EXPRESSED BY PARIETAL CELLS IN THE RAT STOMACH FUNDUS AND ANTRUM __________________________________________________________. Authorea . 11 August 2025. DOI: https://doi.org/10.22541/au.175491821.10576299/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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