Studies on the effects of anandamide in rat hepatic artery

Abstract

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.

Characterisation of the vasorelaxant properties of the novel endocannabinoid N‐arachidonoyl‐dopamine (NADA)

“…Since the endothelium-derived relaxing factor stimulated by NADA has been shown in the present study to be sensitive to a combination of drugs that are believed to inhibit EDHF activity (Randall & Kendall, 1998;Zygmunt et al, 1997), stimulation of the novel endothelial receptor may be coupled Effects of the CB 1 receptor antagonists SR141716A at 100 nM (n ¼ 5) and 1 mM (n ¼ 6, a) and AM251 at 100 nM (n ¼ 6) and at 1 mM (n ¼ 6, b) on vasorelaxation to NADA in small-resistance vessels. Data are given as means, with error bars representing s.e.m.…”

“…In these experiments, L-NAME (300 mM) and indomethacin (10 mM) were present in the KREBS solution throughout the experiments to inhibit nitric oxide and prostanoid synthesis, respectively. Preparations were then incubated with charybdotoxin (ChTX) to block large calcium-activated K þ channels and voltagesensitive K þ channels (ChTX, 100 nM) and apamin to block small calcium-activated K þ channels (500 nM), as this combination inhibits EDHF responses (Randall & Kendall, 1998;Zygmunt et al, 1997). Concentration-response curves were then constructed to NADA.…”

Prejunctional and peripheral effects of the cannabinoid CB1 receptor inverse agonist rimonabant (SR 141716)

“…Moreover, rimonabant inhibits cannabinoid-induced activation of extracellular signalregulated kinase (Su and Vo 2007) and nitrous oxide (NO) formation in blood vessels (Poblete et al 2005), and NO synthase inhibitors can attenuate cannabinoid-induced vasodilatation (Ho and Hiley 2003). However, rimonabant-sensitive cannabinoid-induced vasodilatation has also been observed in the presence of NO synthase inhibition (Zygmunt et al 1997). Taken together, these findings demonstrate that cannabinoids and rimonabant can affect vascular function not only by prejunctional inhibition of neurotransmitter release but also via direct effects on the endothelium.…”

Endothelium‐independent relaxation to cannabinoids in rat‐isolated mesenteric artery and role of Ca2+ influx

“…However, R-(+)-WIN 55,212-2 was a much less effective vasorelaxant than expected from its affinities towards both CB 1 and CB 2 receptors and its relaxation was not affected by the CB 1 receptor-selective antagonist SR 141716A(N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) (White & Hiley, 1998a;Wagner et al, 1999). Interestingly, SR 141716A has been shown to attenuate anandamide relaxations, but this required higher concentrations of the antagonist than expected from its affinity for the classical CB 1 receptor (White & Hiley, 1997;Zygmunt et al, 1997). Thus, although CB 1 receptor mRNA and CB 1 -like immunoreactivity have been detected in rat mesenteric arteries (Darker et al, 1998), the involvement of cannabinoid receptors in relaxation to R-(+)-WIN 55,212-2 and anandamide remains unresolved.…”

“…In addition, the relaxation of KCl-precontracted vessels and the inhibition of CaCl 2 contractions induced by anandamide (unlike that induced by JWH 015) were not affected by SR 141716A (3 mM). Thus, the SR 141716A-sensitive component of anandamide relaxation might involve actions on any contractile mechanism that is bypassed by direct membrane depolarisation, such as Ca 2+ release from intracellular stores and opening of Ca 2+ -activated Cl À channels (Zygmunt et al, 1997;White & Hiley, 1998a), or activation of K + channels, although earlier reports indicated that K + channels were unlikely to be involved (White & Hiley, 1997;Zygmunt et al, 1997). These observations suggest that there are both SR 141716A-sensitive and -insensitive vasorelaxation mechanisms which mediate the relaxation to anandamide in capsaicin-pretreated, small mesenteric artery of the rat.…”