Human Urinary Bladder Strip Relaxation by the β-Adrenoceptor Agonist Isoprenaline: Methodological Considerations and Effects of Gender and Age

The present study was primarily designed to explore various methodological aspects related to organ bath experiments evaluating human detrusor relaxation by the β-adrenoceptor agonist isoprenaline. Data are based upon a series of 30 consecutive patients, and this cohort was also used to explore possible effects of gender and age. KCl-induced contraction was related to strip length but not weight or cross-sectional area, indicating that the former is most suitable for data normalization. Storage of detrusor strips in cold buffer for up to 2 days did not affect contractile responses to KCl or efficacy of isoprenaline to cause relaxation but significantly affected the isoprenaline potency. No such alterations were observed with up to 1 day of cold storage. The type (KCl vs. passive tension) or strength of contractile stimulus had only minor effects on isoprenaline responses although these differences reached statistical significance in some cases. Similarly, gender and age had only minor if any effects on KCl-induced contraction or isoprenaline-induced relaxation, but the current data are too limited for robust conclusions. In summary we have evaluated experimental conditions for the testing of human detrusor strip contraction and relaxation which should be useful for future larger studies

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. II. Protein kinases

We have investigated the role of several protein kinases in carbachol-stimulated, M-3 muscarinic receptor-mediated contraction of rat urinary bladder. Concentration-response curves for the muscarinic receptor agonist carbachol were generated in the presence of multiple concentrations of inhibitors of various protein kinases, their inactive controls, or their vehicles. Bladder contraction was not significantly inhibited by three protein kinase C inhibitors (chelerythrine, 1-10 muM; calphostin C, 0.1-1 muM; and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]3-(1H-indol-3-yl)-maleimide (Go 6850), 1- 10 muM), by the tyrosine kinase inhibitor genistein or its inactive control daidzein (3-30 muM each), or by two inhibitors of activation of mitogen-activated protein kinase [10-100 muM 2'-amino-3'-methoxyflavone (PD 98,059) and 3-30 muM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 124)] or their negative control 1,4-diamino-2,3- dicyano-1,4-bis(methylthio) butadiene (U 126) (3-30 muM). Although high concentrations of wortmannin (3- 30 muM) inhibited bladder contraction, this was not mimicked by another inhibitor of phosphatidylinositol-3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294,002) (3-30 muM) and, hence, was more likely due to direct inhibition of myosin light chain kinase by wortmannin than to an involvement of phosphatidylinositol-3-kinase. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 muM), an inhibitor of rho-associated kinases, concentration-dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of rat urinary bladder does not involve protein kinase C, phosphatidylinositol-3-kinase, tyrosine kinases, or extracellular signal-regulated kinases; in contrast, rho-associated kinases appear to play an important role in the regulation of bladder contractio

Transient relaxation of rat mesenteric microvessels by ceramides

We have investigated the vasodilating effects of D-erythro-C2-ceramide (C2-ceramide) in methoxamine-contracted rat mesenteric microvessels. C2-ceramide (10 - 100 microM) caused a concentration-dependent, slowly developing relaxation which reached maximum values after approximately 10 min and partially abated thereafter. Endothelium removal or inhibitors of guanylyl cyclase (3 microM ODQ), protein kinase A (10 microM H7, 1 microM H89) and various types of K(+) channels (10 microM BaCl(2), 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 microM glibenclamide) had only small if any inhibitory effects against C2-ceramide-induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2-ceramide-induced vasodilation. A second administration of C2-ceramide caused a detectable but weaker relaxation. L-threo-C2-ceramide (100 microM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (100 microM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2-ceramide. The late-developing reversal of C2-ceramide-induced vasodilation was absent when alpha-adrenergic tone was removed by addition of 10 microM phentolamine. We conclude that C2-ceramide relaxes rat resistance vessels in an endothelium-independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxin-sensitive K(+) channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2-ceramide to an inactive metabolit

M(3) muscarinic receptors mediate contraction of human urinary bladder

Since muscarinic receptors appear to be the physiologically most important control system for urinary bladder contraction, we have characterized the receptor subtype mediating contraction in response to the muscarinic agonist carbachol in the human bladder. Experiments were based on four antagonists, the non-selective atropine, the M(1)-selective pirenzepine, the M(2)-selective methoctramine and the M(3)-selective darifenacin. All antagonists yielded Schild-plots with a slope close to unity. The order of potency (atropine⩾darifenacin>pirenzepine>methoctramine) as well as the estimated antagonist affinities suggested that contraction of the human bladder occurs predominantly if not exclusively via the M(3) receptor