and {Department of Biomedical Sciences, University of She eld, Western Bank, She eld S10 2TN1 The e ects of anandamide on K + currents and membrane potential have been examined in freshlyisolated smooth muscle cells from rat hepatic artery and the results compared with the e ects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery. 2 In the presence of 0.3 mM L-NOARG and 10 mM indomethacin, anandamide (0.1 ± 100 mM) and endothelium-derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01 ± 10 mM) each relaxed endothelium-intact segments of hepatic artery precontracted with phenylephrine. These e ects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 mM).3 The relaxant e ects of anandamide were una ected by a toxin combination (apamin plus charybdotoxin, each 0.3 mM) which abolishes EDHF relaxations and were essentially unchanged in endothelium-denuded arteries. The relaxant e ects of anandamide in endothelium-intact arteries were signi®cantly reduced in a physiological salt solution containing 30 mM KCl and abolished when the K + concentration was raised to 60 mM. 4 Anandamide (10 mM), acetylcholine (1 mM, via release of EDHF) and levcromakalim (10 mM) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium-intact arteries. However, when the endothelium was removed, the hyperpolarizing e ects of both anandamide (10 mM) and acetylcholine were essentially abolished whereas those of levcromakalim (10 mM) were una ected. 5 Under voltage-clamp conditions, anandamide (10 mM) abolished spontaneous transient outward currents (STOCs) in freshly-isolated single hepatic artery cells held at 0 mV but had no e ect on the holding current at this potential. In current-clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no signi®cant change in basal membrane potential. 6 Anandamide (10 mM) abolished the iberiotoxin-sensitive K + current (I BK(Ca) ) produced by ca eine and the corresponding hyperpolarizations generated by this xanthine derivative in current-clamp mode. In contrast, anandamide had no e ect on I BK(Ca) generated on exposure to NS1619 (30 mM). 7 It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide-induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrinecontracted vessels.

No abstract

The rat hepatic artery responds to acetylcholine (ACh) with an endothelium-dependent relaxation, which is unaffected by nitric oxide (NO) synthase and cyclooxygenase inhibition. The purpose of this study was to investigate whether the NO-independent relaxation is caused by hyperpolarization of the smooth muscle cells. In vessels with endothelium ACh induced a hyperpolarization in the presence of 0.3 mM N omega-nitro-L-arginine (L-NOARG) and 10 microM indomethacin. The hyperpolarization, which slowly decayed after an initial maximum, generally lasted for at least 20 min. ACh in contrast to levcromakalim failed to hyperpolarize the smooth muscle cells in endothelium-denuded vessels. In vessels contracted by phenylephrine (PhE) ACh caused a concentration-dependent hyperpolarization and relaxation, and both events occurred over the same concentration interval. Curve fitting using the Hill equation showed a close correlation between the hyperpolarization and the relaxation. Exposure to a 30 mM K+ solution abolished the hyperpolarization and suppressed the relaxation induced by ACh. Nimodipine did not affect the ACh-induced hyperpolarization, whereas the relaxation induced by ACh and levcromakalim, but not that evoked by the NO donor 3-morpholino-sydnonimin, were attenuated. Glibenclamide had no effect on the ACh-induced hyperpolarization and relaxation, but abolished the corresponding responses to levcromakalim. The results demonstrate a NO-independent hyperpolarization and relaxation in the rat hepatic artery. The hyperpolarization and relaxation were endothelium-dependent, and apparently causally related to each other, since interference with the hyperpolarization or the subsequent effector pathway inhibited the relaxation.

We conclude that oxybutynin and N-desethyl-oxybutynin have a similar antimuscarinic effect in the human detrusor, and the same binding characteristics in detrusor and parotid gland, respectively.

1 Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle. 2 A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO 2 Me) and a recently developed human chymase substrate Pro 11 -,D-Ala 12 )-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used. 3 Angiotensin I (AI) (0.3 mM) induced a contractile response amounting to 58+5% (n=12) of the initial K + (124 mM)-induced contractions. This response was reduced to 36+3% (n=8) by the ACEinhibitor enalaprilat (10 mM), while pretreatment with soybean trypsin inhibitor (STI 200 mg ml 71 ) or CH 5450 (10 mM) had no eect. However, the combination of enalaprilat and STI reduced the AIinduced contractions to 19+5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1+1% (n=6). )-AI-induced contractions to 34+5% (n=6) and 24+4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition. 5 Experiments with detrusor membrane fractions incubated with AI (50 mM) were performed. In the presence of enalaprilat (100 mM), carboxypeptidase inhibitor CPI (10 mg ml 71 ) and aprotinin (15 mM), CH 5450 (10 nM ± 1 mM) caused a concentration-dependent inhibition of AII formation. 6 The results con®rm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.