Comparative pharmacology of endothelium-derived hyperpolarizing factor and anandamide in rat isolated mesentery

Abstract

We have recently proposed that anandamide, or a related cannabinoid, is the endothelium-derived hyperpolarizing factor (EDHF) and have now compared EDHF-mediated responses (induced by carbachol in the presence of both nitric oxide and prostanoid synthesis inhibitors) with those induced by anandamide in the rat isolated superior mesenteric arterial bed. Both EDHF-mediated and anandamide-induced relaxations were inhibited in the presence of high K⁺ (60 mM) and opposed by blockade of K⁺ channels with 10 mM tetraethylammonium. The cytochrome P450 inhibitors, and putative EDHF inhibitors, clotrimazole (10 μM) and proadifen (SKF 525A) (10 μM), opposed both anandamide-induced and EDHF-mediated relaxations and also relaxant responses to the K⁺ channel activator levcromakalim. Therefore, EDHF-mediated and anandamide-induced vasorelaxations show very similar pharmacological characteristics, with both responses being mediated via K⁺ channel activation. Further, the actions of EDHF and anandamide are both sensitive to proadifen and clotrimazole, EDHF antagonists which appear to act through K⁺ channel inhibition. Accordingly, these results support our proposal that an endocannabinoid is an EDHF.

Introduction

In 1980 Furchgott and Zawadzki demonstrated that the endothelium, in response to agonist stimulation, released the endothelium-derived relaxant factor (EDRF) (Furchgott and Zawadzki, 1980). Nitric oxide (NO) was identified as an important mediator of endothelium-dependent relaxations by Palmer et al. (1987). However, it has become clear that NO does not account for all EDRF activity, and that there is an additional factor, the endothelium-derived hyperpolarizing factor (EDHF), which contributes to these responses by hyperpolarizing the vascular smooth muscle through K⁺ channel activation (Taylor and Weston, 1988; Garland et al., 1995). Indeed, EDHF is now thought to assume greatest importance in resistance beds and may be upregulated in response to impairment of NO activity (Kilpatrick and Cocks, 1994; Kemp et al., 1995; McCulloch et al., 1997).

The identity of EDHF has so far remained elusive, but it is thought to be a non-prostanoid arachidonic acid metabolite (Cohen and Vanhoutte, 1995). It has been suggested that EDHF may be a cytochrome P450-derived arachidonate metabolite, as some inhibitors of this enzyme system oppose endothelium-dependent relaxations (Singer et al., 1984; Pinto et al., 1987) and in particular those mediated by EDHF (Bauersachs et al., 1994; Hecker et al., 1994; Fulton et al., 1995; Campbell et al., 1996). However, more recent evidence casts severe doubt on this contention, since not all cytochrome P450 inhibitors inhibit EDHF-mediated responses (Corriu et al., 1996; Zygmunt et al., 1996). In addition, the selectivity of these agents must be questioned because some cytochrome P450 inhibitors, which inhibit EDHF responses, are also K⁺ channel blockers and have been shown to inhibit K⁺ channel activation (Zygmunt et al., 1996) and may therefore inhibit EDHF at its site of action rather than its synthesis.

More recently we have proposed that arachidonoylethanolamide (anandamide), an endogenous cannabinoid derived from arachidonic acid (Di Marzo et al., 1994), may represent EDHF (Randall et al., 1996). This contention is based on the finding that in the rat isolated mesentery and the conscious rat, the highly selective cannabinoid receptor antagonist SR 141716A inhibits NO-independent endothelium-dependent relaxations mediated by EDHF. Furthermore, under conditions which evoke EDHF release, we have detected an arachidonic acid metabolite which is similar to or identical with anandamide. We have also reported that exogenous anandamide causes endothelium-independent relaxations which are blocked by raising extracellular K⁺, consistent with these responses being mediated via K⁺ channel activation (Randall et al., 1996).

In view of our proposal that anandamide, or a related substance, is EDHF we have now carried out a comparison of the vasorelaxant responses to carbachol, which releases both NO and EDHF, with those to anandamide. In order to define EDHF-mediated responses we have used carbachol to evoke endothelium-dependent relaxations in the presence of both nitric oxide synthase and cyclo-oxygenase inhibitors. In the present investigation we have compared the sensitivity of EDHF-mediated and anandamide-induced responses to K⁺ channel blockade and elevation of extracellular K⁺ (Adeagbo and Triggle, 1993). Furthermore, we have examined the effects of the cytochrome P450 inhibitors proadifen (SKF 525A) and clotrimazole on EDHF-mediated and anandamide responses as there is now evidence that these agents are EDHF antagonists, acting through K⁺ channel blockade (Zygmunt et al., 1996).