β-Adrenoceptor Agonists Stimulate Endothelial Nitric Oxide Synthase in Rat Urinary Bladder Urothelial Cells

Abstract

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.

Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases

“…The cAMP-PKA pathway is coupled to ADM receptors in various types of cells (Kitamura et al, 1993;Shimekake et al, 1995), including neural cells (Xu and Krukoff, 2005). In addition, PKA mediates nNOS activation induced by diverse stimuli, such as electrical field stimulation (Ferrer et al, 2004) and application of endothelin (Jaureguiberry et al, 2004) or ␤-adrenoceptor agonists (Birder et al, 2002). We now show that ADM increases cAMP levels in hypothalamic neurons and that the cAMP-PKA pathway mediates the ADM-induced NO production, demonstrated by the blockade of NO responses in the presence of the PKA inhibitors H-89 and Rp-cAMP.…”

Mechanisms of Disease: involvement of the urothelium in bladder dysfunction

“…receptors and ion channels) that have been identified in the urothelium include receptors for bradykinin, neurotrophins (Trk-A and p75), purines (P2X and P2Y), norepinephrine (α and β), acetylcholine (nicotinic and muscarinic receptors), protease-activated receptors, amiloride-sensitive and mechanosensitive Na + channels, and a number of transient receptor potential (TRP) channels (TRPV1, TRPV2, TRPV4, TRPM8) ( Table 1). [28][29][30][31][32][33][34][35][36][37][38] …”

“…28,66 It is possible that neurally released norepinephrine might have an effect on the urothelium, and might influence bladder function, because of the close proximity of adrenergic nerves that innervate blood vessels in the suburothelial region. 22,23,67 Patients with a variety of lower urinary tract disorders including overactive bladder, benign prostatic hyperplasia and even interstitial cystitis-are often treated with selective α 1 -adrenergic antagonists.…”

“…It has been shown that stimulation of both α-adrenergic and β-adrenergic receptors on urothelial cells evokes the release of NO. 28,66 It has also been shown that cats diagnosed with feline interstitial cystitis exhibit an increase in norepinephrine content in the urinary bladder, compared with healthy cats. 72 In addition, urothelial cells from cats with feline interstitial cystitis exhibit an augmented release of mediators including ATP and NO, which is similar to that observed in human interstitial cystitis.…”

“…74 The localization of neuronal nitric oxide synthase (NOS) and/or NADPH diaphorase (a marker for neuronal NOS) in efferent and afferent nerve fibers and the expression of multiple NOS isoforms (neuronal, endothelial, and inducible) within the urothelium indicate that NO has a role in the micturition reflex pathway. 28,74 The release of NO from the urothelium or other cells is thought to either facilitate or inhibit the activity of bladder afferent nerves. For example, a reduction in NO levels adjacent to the urothelium (caused by intravesical administration of oxyhemoglobin) results in bladder hyperactivity, which suggests that there is an inhibitory role for NO in the control of bladder function.…”