The activation of Calcium-Sensing Receptors (CaSRs) reduces detrusor activity in bladder tissues. Also, hydrogen sulfide (H2S) produces in bladder tissue and regulates the bladder smooth muscles tone. However, there is no evidence of the interaction between CaSRs and H2S in bladder tissue. The aim of this study is to investigate the possible contribution of L-cysteine/H2S pathway in CaSRs-mediated relaxation responses in isolated mouse bladder tissue. CaCl2 (1, 2, 3, 5, 10 mM) was applied to isolated mouse bladder tissues pre-contracted with carbachol (1 microM). CaCl2-induced relaxations were performed in the presence of PAG (10 mM), AOAA (1 mM), and Calhex-231 (5 microM), cystathionine-gamma-lyase (CSE), cystathionine-beta-synthase (CBS) and CaSR inhibitor, respectively. L-cysteine (1 microM-10 mM), an H2S substrate, was used to induced a concentration-dependent relaxant response in isolated bladder tissues pre-contracted with carbachol. L-cysteine induced relaxations were performed in the presence of PAG (CSE inhibitor, 10 mM), AOAA (CBS inhibitor, 1 mM) and Calhex-231 (CaSR inhibitor, 5 microM). CaCl2-induced relaxations were decreased by PAG and AOAA. Also, Calhex-231 decreased the CaCl2-induced relaxant responses. L-cysteine-induced relaxant responses were reduced in the presence of PAG (10 mM) and AOAA (1 mM). Calhex-231 (5 microM) caused a significant decrease in L-cysteine-induced relaxations. Also, Calhex-231 reduced the increase in H2S production in the presence of L-cysteine. In addition, CaCl2 increased basal H2S generation, and PAG (10 mM), AOAA (1 mM) and Calhex-231 (5 microM) reduced the increase in H2S production stimulated with CaCl2. In conclusion, CSE and CBS-derived endogenous H2S formation may, at least in part, contribute to CaSR-mediated relaxation responses, and CaSRs involve in endogenous H2S relaxation responses in isolated mouse bladder tissue. Key words Bladder o CaSRs " Calhex-231 " Hydrogen sulfide " L-cysteine " Mouse.

Department of Pharmacology Pharmacy Faculty Cukurova University Adana Republic of Türkiye

See more in PubMed

Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev. 2001;81:239–297. doi: 10.1152/physrev.2001.81.1.239. PubMed DOI

Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, et al. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature. 1993;366:575–580. doi: 10.1038/366575a0. PubMed DOI

Ward DT. Calcium receptor-mediated intracellular signalling. Cell Calcium. 2004;35:217–228. doi: 10.1016/j.ceca.2003.10.017. PubMed DOI

Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: a review. Crit Rev Clin Lab Sci. 2005;2:35–70. doi: 10.1080/10408360590886606. PubMed DOI

Hannan FM, Kallay E, Chang W, Brandi ML, Thakker RV. The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol. 2018;15:33–51. doi: 10.1038/s41574-018-0115-0. PubMed DOI PMC

Schepelmann M, Yarova PL, Lopez-Fernandez I, Davies TS, Brennan SC, Edwards PJ, Aggarwal A, et al. The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure. Am J Physiol Cell Physiol. 2016;310:C193–C204. doi: 10.1152/ajpcell.00248.2015. PubMed DOI PMC

Weston AH, Absi M, Ward DT, Ohanian J, Dodd RH, Dauban P, Petrel C, et al. Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231. Circ Res. 2005;97:391–398. doi: 10.1161/01.RES.0000178787.59594.a0. PubMed DOI

Greenberg HZE, Jahan KS, Shi J, Vanessa Ho WS, Albert AP. The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels. Eur J Pharmacol. 2016;791:659–668. doi: 10.1016/j.ejphar.2016.10.008. PubMed DOI PMC

Berra Romani R, Raqeeb A, Laforenza U, Scaffino MF, Moccia F, Avelino-Cruz JE, Oldani A, et al. Cardiac microvascular endothelial cells express a functional Ca+-sensing receptor. J Vasc Res. 2009;46:73–82. doi: 10.1159/000140677. PubMed DOI

Wonneberger K, Scofield MA, Wangemann P. Evidence for a calcium-sensing receptor in the vascular smooth muscle cells of the spiral modiolar artery. J Membr Biol. 2000;175:203–212. doi: 10.1007/s002320001068. PubMed DOI

Molostvov G, Fletcher S, Bland R, Zehnder D. Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis. Cell Physiol Biochem. 2008;22:413–422. doi: 10.1159/000185484. PubMed DOI

Alam MU, Kirton JP, Wilkinson FL, Towers E, Sinha S, Rouhi M, Vizard TN, et al. Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells. Cardiovasc Res. 2009;81:260–268. doi: 10.1093/cvr/cvn279. PubMed DOI

Bukoski RD, Bian K, Wang Y, Mupanomunda M. Perivascular sensory nerve Ca2+ receptor and Ca2+-induced relaxation of isolated arteries. Hypertension. 1997;30:1431–1439. doi: 10.1161/01.HYP.30.6.1431. PubMed DOI

Thakore P, Ho WS. Vascular actions of calcimimetics: role of Ca2(+) -sensing receptors versus Ca2(+) influx through L-type Ca2(+) channels. Br J Pharmacol. 2011;162:749–762. doi: 10.1111/j.1476-5381.2010.01079.x. PubMed DOI PMC

Petrel C, Kessler A, Maslah F, Dauban P, Dodd RH, Rognan D, Ruat M. Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-sensing receptor. J Biol Chem. 2003;278:49487–49494. doi: 10.1074/jbc.M308010200. PubMed DOI

Awumey EM, Bridges LE, Williams CL, Diz DI. Nitric-oxide synthase knockout modulates Ca2+-sensing receptor expression and signaling in mouse mesenteric arteries. J Pharmacol Exp Ther. 2013;346:38–47. doi: 10.1124/jpet.113.205534. PubMed DOI PMC

Greenberg HZ, Shi J, Jahan KS, Martinucci MC, Gilbert SJ, Vanessa Ho WS, Albert AP. Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels. Vascul Pharmacol. 2016;80:75–84. doi: 10.1016/j.vph.2016.01.001. PubMed DOI PMC

Loot AE, Pierson I, Syzonenko T, Elgheznawy A, Randriamboavonjy V, Zivković A, Stark H, Fleming I. Ca2+-sensing receptor cleavage by calpain partially accounts for altered vascular reactivity in mice fed a high-fat diet. J Cardiovasc Pharmacol. 2013;61:528–535. doi: 10.1097/FJC.0b013e31828d0fa3. PubMed DOI

Smajilovic S, Sheykhzade M, Holmegard HN, Haunso S, Tfelt-Hansen J. Calcimimetic, AMG 073, induces relaxation on isolated rat aorta. Vascul Pharmacol. 2007;47:222–228. doi: 10.1016/j.vph.2007.06.010. PubMed DOI

Ziegelstein RC, Xiong Y, He C, Hu Q. Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells. Biochem Biophys Res Commun. 2006;342:153–163. doi: 10.1016/j.bbrc.2006.01.135. PubMed DOI

Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci. 1996;16:1066–1071. doi: 10.1523/JNEUROSCI.16-03-01066.1996. PubMed DOI PMC

Shibuya N, Tanaka M, Yoshida M, Ogasawara Y, Togawa T, Ishii K, Kimura H. 3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane sulfur in the brain. Antioxid Redox Signal. 2009;11:703–714. doi: 10.1089/ars.2008.2253. PubMed DOI

Tang C, Li X, Du J. Hydrogen sulfide as a new endogenous gaseous transmitter in the cardiovascular system. Curr Vasc Pharmacol. 2006;4:17–22. doi: 10.2174/157016106775203144. PubMed DOI

Matsunami M, Miki T, Nishiura K, Hayashi Y, Okawa Y, Nishikawa H, Sekiguchi F, et al. Involvement of the endogenous hydrogen sulfide/Ca(v) 3.2 T-type Ca2+ channel pathway in cystitis-related bladder pain in mice. Br J Pharmacol. 2012;167:917–928. doi: 10.1111/j.1476-5381.2012.02060.x. PubMed DOI PMC

Gai JW, Wahafu W, Guo H, Liu M, Wang XC, Xiao YX, Zhang L, et al. Further evidence of endogenous hydrogen sulphide as a mediator of relaxation in human and rat bladder. Asian J Androl. 2013;15:692–696. doi: 10.1038/aja.2013.32. PubMed DOI PMC

Fernandes VS, Ribeiro AS, Barahona MV, Orensanz LM, Martínez-Sáenz A, Recio P, Martínez AC, et al. Hydrogen sulfide mediated inhibitory neurotransmission to the pig bladder neck: role of KATP channels, sensory nerves and calcium signaling. J Urol. 2013;190:746–756. doi: 10.1016/j.juro.2013.02.103. PubMed DOI

Fusco F, di Villa Bianca Rd, Mitidieri E, Cirino G, Sorrentino R, Mirone V. Sildenafil effect on the human bladder involves the L-cysteine/hydrogen sulfide pathway: a novel mechanism of action of phosphodiesterase type 5 inhibitors. Eur Urol. 2012;62:1174–1180. doi: 10.1016/j.eururo.2012.07.025. PubMed DOI

di Villa Bianca Rd, Cirino G, Sorrentino R. Hydrogen Sulfide and Urogenital Tract. Handb Exp Pharmacol. 2015;230:111–136. doi: 10.1007/978-3-319-18144-8_5. PubMed DOI

Dalkir FT, Aydinoglu F, Ogulener N. The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Naunyn Schmiedebergs Arch Pharmacol. 2023;396:2023–2038. doi: 10.1007/s00210-023-02440-6. PubMed DOI

Eto K, Kimura H. A novel enhancing mechanism for hydrogen sulfide-producing activity of cystathionine beta-synthase. J Biol Chem. 2002;277:42680–42685. doi: 10.1074/jbc.M205835200. PubMed DOI

Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science. 2008;322:587–590. doi: 10.1126/science.1162667. PubMed DOI PMC

Fu M, Zhang W, Wu L, Yang G, Li H, Wang R. Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production. Proc Natl Acad Sci U S A. 2012;109:2943–2948. doi: 10.1073/pnas.1115634109. PubMed DOI PMC

Zhong X, Wang Y, Wu J, Sun A, Yang F, Zheng D, Li T, et al. Calcium sensing receptor regulating smooth muscle cells proliferation through initiating cystathionine-gamma-lyase/hydrogen sulfide pathway in diabetic rat. Cell Physiol Biochem. 2015;35:1582–1598. doi: 10.1159/000373973. PubMed DOI

Wang Y, Wang X, Liang X, Wu J, Dong S, Li H, Jin M, et al. Inhibition of hydrogen sulfide on the proliferation of vascular smooth muscle cells involved in the modulation of calcium sensing receptor in high homocysteine. Exp Cell Res. 2016;347:184–191. doi: 10.1016/j.yexcr.2016.08.004. PubMed DOI

Wu WY, Lee SP, Chiang BJ, Lin WY, Chien CT. Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats. Pharmaceuticals (Basel) 2021;14:960. doi: 10.3390/ph14100960. PubMed DOI PMC

Aydinoglu F, Erdem EN, Toyran T, Ogulener N. Impairment of Endogenous H2S Pathway due to Aging and Endothelium Denudation in Mouse Isolated Thoracic Aorta. Physiol Res. 2025;74:59–68. doi: 10.33549/physiolres.935419. PubMed DOI PMC

Molostvov G, James S, Fletcher S, Bennett J, Lehnert H, Bland R, Zehnder D. Extracellular calcium-sensing receptor is functionally expressed in human artery. Am J Physiol Renal Physiol. 2007;293:F946–F955. doi: 10.1152/ajprenal.00474.2006. PubMed DOI

Smajilovic S, Hansen JL, Christoffersen TE, Lewin E, Sheikh SP, Terwilliger EF, Brown EM, et al. Extracellular calcium sensing in rat aortic vascular smooth muscle cells. Biochem Biophys Res Commun. 2006;348:1215–1223. doi: 10.1016/j.bbrc.2006.07.192. PubMed DOI

Zou S, Shimizu T, Shimizu S, Higashi Y, Nakamura K, Ono H, Aratake T, et al. Possible role of hydrogen sulfide as an endogenous relaxation factor in the rat bladder and prostate. Neurourol Urodyn. 2018;37:2519–2526. doi: 10.1002/nau.23788. PubMed DOI

Patacchini R, Santicioli P, Giuliani S, Maggi CA. Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle. Eur J Pharmacol. 2005;509:171–177. doi: 10.1016/j.ejphar.2005.01.005. PubMed DOI

Nemeth EF, Delmar EG, Heaton WL, Miller MA, Lambert LD, Conklin RL, Gowen M, et al. Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone. J Pharmacol Exp Ther. 2001;299:323–331. doi: 10.1016/S0022-3565(24)29333-2. PubMed DOI

Kessler A, Faure H, Roussanne MC, Ferry S, Ruat M, Dauban P, Dodd RH. N(1)-Arylsulfonyl-N(2)-(1-(1-naphthyl)ethyl)-1,2-diaminocyclohexanes: a new class of calcilytic agents acting at the calcium-sensing receptor. Chembiochem. 2004;5:1131–1136. doi: 10.1002/cbic.200400049. PubMed DOI

Wang W, Bo Q, Du J, Yu X, Zhu K, Cui J, Zhao H, et al. Endogenous H2S sensitizes PAR4-induced bladder pain. Am J Physiol Renal Physiol. 2018;14:F1077–F1086. doi: 10.1152/ajprenal.00526.2017. PubMed DOI