GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.FAPESPCNP

The soluble guanylyl cyclase activator BAY 60-2770 ameliorates overactive bladder in obese mice

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOActivators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. Materials and Methods: C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. Results: Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase alpha 1 and beta 1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. Conclusions: Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase alpha 1 and beta 1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction1912539547FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770

CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOObesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b] pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice(P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one] (10 mM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactiveoxygen species (ROS) production was enhanced, but protein expression of beta 1 second guanylate cyclase subunit was reduced in USM from obesemice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese miceAmerican Society for Pharmacology and Experimental Therapeutics349129CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãoBethesda, M

Menthol Inhibits Detrusor Contractility Independently Of Trpm8 Activation

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.911BBSRC; 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. 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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. 1276-128