Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 μM), CaCl2 (1 μM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 mM) or nifedipine (1 mM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 μM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 μM apamin, 10 mM glibenclamide and 1 μM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.911 BBSRC; Higher Education Funding Council for England; HEFCE; Higher Education Funding Council for England; MRC; Higher Education Funding Council for EnglandChapple, C., Khullar, V., Gabriel, Z., Dooley, J.A., The effects of antimuscarinic treatments in overactive bladder: A systematic review and meta-analysis (2005) Eur Urol, 48, pp. 5-26Jiang, C.H., Mazieres, L., Lindstrom, S., Cold- and menthol-sensitive C afferents of cat urinary bladder (2002) J Physiol, 543, pp. 211-220Nomoto, Y., Yoshida, A., Ikeda, S., Kamikawa, Y., Harada, K., Effect of menthol on detrusor smooth-muscle contraction and the micturition reflex in rats (2008) Urology, 72, pp. 701-705Lei, Z., Ishizuka, O., Imamura, T., Noguchi, W., Yamagishi, T., Functional roles of transient receptor potential melastatin 8 (TRPM8) channels in the cold stress-induced detrusor overactivity pathways in conscious rats (2013) Neurourol Urodyn, 504, pp. 500-504McKemy, D.D., Neuhausser, W.M., Julius, D., Identification of a cold receptor reveals a general role for TRP channels in thermosensation (2002) Nature, 416, pp. 52-58Calixto, J.B., Kassuya, C.A., André, E., Ferreira, J., Contribution of natural products to the discovery of the transient receptor potential (TRP) channels family and their functions (2005) Pharmacol Ther, 106 (2), pp. 179-208Peier, A.M., Moqrich, A., Hergarden, A.C., Reeve, A.J., Andersson, D., A TRP channel that senses cold stimuli and menthol (2002) Cell, 108, pp. 705-715Patel, T., Ishiuji, Y., Yosipovitch, G., Menthol: A refreshing look at this ancient compound (2007) J Am Acad Dermatol, 57, pp. 873-878Baylie, R.L., Cheng, H., Langton, P.D., James, A.F., Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol (2010) J Physiol Pharmacol, 61, pp. 543-550Hans, M., Wilhelm, M., Swandulla, D., Menthol suppresses nicotinic acetylcholine receptor functioning in sensory neurons via allosteric modulation (2012) Chem Senses, 37 (5), pp. 463-469Gaudioso, C., Hao, J., Martin-Eauclaire, M.F., Gabriac, M., Delmas, P., Menthol pain relief through cumulative inactivation of voltage-gated sodium channels (2012) Pain, 153, pp. 473-484Stein, R.J., Santos, S., Nagatomi, J., Hayashi, Y., Minnery, B.S., Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract (2004) J Urol, 172, pp. 1175-1178Mukerji, G., Yiangou, Y., Corcoran, S.L., Selmer, I.S., Smith, G.D., Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations (2006) BMC Urol, 6, p. 6Kullmann, F.A., Shah, M.A., Birder, L.A., De Groat, W.C., Functional TRP and ASIC-like channels in cultured urothelial cells from the rat (2009) Am J Physiol Renal Physiol, 296, pp. F892-F901Everaerts, W., Vriens, J., Owsianik, G., Appendino, G., Voets, T., Functional characterization of transient receptor potential channels in mouse urothelial cells (2010) Am J Physiol Renal Physiol, 298, pp. F692-701Tsukimi, Y., Mizuyachi, K., Yamasaki, T., Niki, T., Hayashi, F., Cold response of the bladder in Guinea pig: Involvement of transient receptor potential channel, TRPM8 (2005) Urology, 65, pp. 406-410Vahabi, B., Parsons, B.A., Doran, O., Rhodes, A., Dean, S., TRPM8 agonists modulate contraction of the pig urinary bladder (2013) Can J Physiol Pharmacol, 91, pp. 503-509Lashinger, E.S.R., Steiginga, M.S., Hieble, J.P., Leon, L.A., Gardner, S.D., AMTB, a TRPM8 channel blocker: Evidence in rats for activity in overactive bladder and painful bladder syndrome (2008) Am J Physiol Renal Physiol, 939, pp. 803-810Ramos-Filho, A.C., Mónica, F.Z., Franco-Penteado, C.F., Rojas-Moscoso, J.A., Báu, F.R., Characterization of the urinary bladder dysfunction in renovascular hypertensive rats (2011) Neurourol Urodyn, 30 (7), pp. 1392-1402Mendes-Silverio, C.B., Leiria, L.O., Morganti, R.P., Anhê, G.F., Marcondes, S., Activation of haem-oxidized soluble guanylyl cyclase with BAY 60-2770 in human platelets lead to overstimulation of the cyclic GMP signaling pathway (2012) PLoS One, 7 (11)Zimmermann, H., ATP and acetylcholine, equal brethren (2008) Neurochem Int., 52 (4-5), pp. 634-648Tong, Y.C., Hung, Y.C., Lin, S.N., Cheng, J.T., Pharmacological characterization of the muscarinic receptor subtypes responsible for the contractile response in the rat urinary bladder (1997) J Auton Pharmacol, 17 (1), pp. 21-25Birder, L., Andersson, K.E., Urothelial signaling (2013) Physiol Rev, 93, pp. 653-680Petkov, G.V., Role of potassium ion channels in detrusor smooth muscle function and dysfunction (2011) Nat Rev Urol, 9, pp. 30-40Gopalakrishnan, M., Buckner, S.A., Whiteaker, K.L., Shieh, C.C., Molinari, E.J., (-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637): A novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions. I. In vitro characterization (2002) J Pharmacol Exp Ther, 303 (1), pp. 379-386Soder, R.P., Parajuli, S.P., Hristov, K.L., Rovner, E.S., Petkov, G.V., SK channel-selective opening by SKA-31 induces hyperpolarization and decreases contractility in human urinary bladder smooth muscle (2013) Am J Physiol Regul Integr Comp Physiol, 304 (2), pp. R155-63Wegener, J.W., Schulla, V., Lee, T.S., Koller, A., Feil, S., An essential role of cav1.2 L-type calcium channel for urinary bladder function (2004) FASEB J, 18 (10), pp. 1159-1161Fry, C.H., Meng, E., Young, J.S., The physiological function of lower urinary tract smooth muscle (2010) Auton Neurosci 19, 154 (1-2), pp. 3-13Rivera, L., Brading, A.F., The role of ca2+ influx and intracellular ca2+ release in the muscarinic-mediated contraction of Mammalian urinary bladder smooth muscle (2006) BJU Int, 98, pp. 868-875Leiria, L.O., Mónica, F.Z., Carvalho, F.D., Claudino, M.A., Franco-Penteado, C.F., Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: Evidence of a role for L-type voltage-operated ca2+ channels (2011) Br J Pharmacol, 163 (6), pp. 1276-1288