Beta-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells.
We have investigated the intracellular signaling mechanisms underlying the release of nitric oxide (NO) evoked by beta-adrenoceptor (AR) agonists in urinary bladder strips and cultured bladder urothelial cells from adult rats. Reverse transcription-PCR revealed that inducible NO synthase and endothelial NOS but not neuronal NOS genes were expressed in urothelial cells. NO release from both urothelial cells and bladder strips was decreased (37-42%) in the absence of extracellular Ca2+ (100 microm EGTA) and was ablated after incubation with BAPTA-AM (5 microm) or caffeine (10 mm), indicating that the NO production is mediated in part by intracellular calcium stores. NO release was reduced (18-24%) by nifedipine (10 microm) and potentiated (29-32%) by incubation with the Ca2+ channel opener BAYK8644 (1-10 microm). In addition, beta-AR-evoked NO release (isoproterenol; dobutamine; terbutaline; 10(-9) to 10(-5) m) was blocked by the NOS inhibitors N(G)-nitro-L-arginine methyl ester (30 microm) or N(G)-monomethyl-L-arginine (50 microm), by beta-adrenoceptor antagonists (propranol, beta1/beta2; atenolol, beta1; ICI 118551; beta2; 100 microm), or by the calmodulin antagonist trifluoperazine (50 microm). Incubating cells with the nonhydrolyzable GTP analog GTPgammaS (1 microm) or the membrane-permeant cAMP analog dibutyryl-cAMP (10-100 microm) directly evoked NO release. Forskolin (10 microm) or the phosphodiesterase IBMX (50 microm) enhanced (39-42%) agonist-evoked NO release. These results indicate that beta-adrenoceptor stimulation activates the adenylate cyclase pathway in bladder epithelial cells and initiates an increase in intracellular Ca2+ that triggers NO production and release. These findings are considered in light of recent reports that urothelial cells may exhibit a number of "neuron-like" properties, including the expression of receptors/ion channels similar to those found in sensory neurons.
Birder, LA; Nealen, ML; Kiss, S; de Groat, WC; Caterina, MJ; Wang, E; Apodaca, G; Kanai, AJ
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