Michael D. Randall

The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication

1 We have shown that the endocannabinoid anandamide and its stable analogue methanandamide relax rings of rabbit superior mesenteric artery through endothelium‐dependent and ‐independent mechanisms that are unaffected by blockade of NO synthase and cyclooxygenase. 2 The endothelium‐dependent component of the responses was attenuated by the gap junction inhibitor 18α‐glycyrrhetinic acid (18α‐GA; 50 μm), and a synthetic connexin‐mimetic peptide homologous to the extracellular Gap 27 sequence of connexin 43 (43Gap 27, SRPTEKTIFII; 300 μm). By contrast, the corresponding connexin 40 peptide (40Gap 27, SRPTEKNVFIV) was inactive. 3 The cannabinoid CB1 receptor antagonist SR141716A (10 μm) also attenuated endothelium‐dependent relaxations but this inhibition was not observed with the CB1 receptor antagonist LY320135 (10 μm). Furthermore, SR141716A mimicked the effects of 43Gap 27 peptide in blocking Lucifer Yellow dye transfer between coupled COS‐7 cells (a monkey fibroblast cell line), whereas LY320135 was without effect, thus suggesting that the action of SR141716A was directly attributable to effects on gap junctions. 4 The endothelium‐dependent component of cannabinoid‐induced relaxation was also attenuated by AM404 (10 μm), an inhibitor of the high‐affinity anandamide transporter, which was without effect on dye transfer. 5 Taken together, the findings suggest that cannabinoids derived from arachidonic acid gain access to the endothelial cytosol via a transporter mechanism and subsequently stimulate relaxation by promoting diffusion of an to adjacent smooth muscle cells via gap junctions. 6 Relaxations of endothelium‐denuded preparations to anandamide and methanandamide were unaffected by 43Gap 27 peptide, 18α‐GA, SR141716A, AM404 and indomethacin and their genesis remains to be established.

The complexities of the cardiovascular actions of cannabinoids

The cardiovascular actions of cannbinoids are complex. In general they cause vasorelaxation in isolated blood vessels, while in anaesthetised animals they cause multiphasic responses which involve an early bradycardia and long‐lasting hypotension. However, in conscious animals, the picture is one of bradycardia followed by pressor responses. Clearly, the responses to cannabinoids are dependent on the experimental conditions and synthetic cannabinoids and endocannabinoids exhibit different pharmacologies. In terms of mechanisms involved in the vascular responses to cannabinoids, the following have been implicated: the involvement of ‘classical’ cannabinoid receptors, the involvement of a novel endothelial cannabinoid receptor, the release of nitric oxide, the release of endothelium‐derived hyperpolarising factor (EDHF), the activation of vanilloid receptors, metabolism of endocannabinoids to vasoactive molecules, and both peripheral inhibition and central excitation of the sympathetic nervous system.

‘Entourage’ effects of N‐palmitoylethanolamide and N‐oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors

The endocannabinoid N‐arachidonoylethanolamide (anandamide) is co‐synthesized with other N‐acylethanolamides, namely N‐palmitoylethanolamide (PEA) and N‐oleoylethanolamide (OEA), which have been shown to potentiate anandamide responses (so‐called ‘entourage effects’) in non‐vascular tissues. It remains unclear whether such interactions occur in the circulation.

Characterization and modulation of EDHF-mediated relaxations in the rat isolated superior mesenteric arterial bed

We have used the isolated, buffer‐perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mm K+) to characterize nitric oxide (NO)‐independent vasorelaxation which is thought to be mediated by the endothelium‐derived hyperpolarizing factor (EDHF). The muscarinic agonists carbachol, acetylcholine (ACh) and methacholine caused dose‐related relaxations in preconstricted preparations with ED50 values of 0.18±0.04 nmol (n=8), 0.05±0.02 nmol (n=6) and 0.26±0.16 nmol (n=5), respectively. In the same preparations NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) significantly (P<0.05) decreased the potency of all the agents (ED50 values in the presence of l‐NAME: carbachol, 0.66±0.11 nmol; ACh, 0.28±0.10 nmol; methacholine, 1.97±1.01 nmol). The maximal relaxation to ACh was also significantly (P<0.05) reduced (from 85.3±0.9 to 73.2±3.7%) in the presence of l‐NAME. The vasorelaxant effects of carbachol were not significantly altered by the cyclo‐oxygenase inhibitor indomethacin (10 μm; n=4). The K+ channel blocker, tetraethylammonium (TEA, 10 mm) also significantly (P<0.001) reduced both the potency of carbachol (ED50=1.97±0.14 nmol in presence of TEA) and the maximum relaxation (Rmax=74.6±3.2% in presence of TEA, P<0.05, n=3). When TEA was added in the presence of l‐NAME (n=4), there was a further significant (P<0.001) decrease in the potency of carbachol (ED50=22.4±13.5 nmol) relative to that in the presence of l‐NAME alone, and Rmax was also significantly (P<0.05) reduced (74.6±4.2%). The ATP‐sensitive K+ channel inhibitor, glibenclamide (10 μm), had no effect on carbachol‐induced relaxation (n=9). High extracellular K+ (60 mm) significantly (P<0.01) reduced the potency of carbachol (n=5) by 5 fold (ED50: control, 0.16±0.04 nmol; high K+, 0.88±0.25 nmol) and the Rmax was also significantly (P<0.01) reduced (control, 83.4±2.7%; high K+, 40.3±9.2%). The residual vasorelaxation to carbachol in the presence of high K+ was abolished by l‐NAME (100 μm; n=5). In preparations preconstricted with high K+, the potency of sodium nitroprusside was not significantly different from that in preparations precontracted with methoxamine, though the maximal response was reduced (62.4±3.4% high K+, n=7; 83.1±3.1% control, n=7). In the presence of the cytochrome P450 inhibitor, clotrimazole (1 μm, n=5 and 10 μm, n=4), the dose‐response curve to carbachol was significantly shifted to the right 2 fold (P<0.05) and 4 fold (P<0.001) respectively, an effect which was further enhanced in the presence of l‐NAME. Rmax was significantly (P<0.01) reduced by the presence of 10 μm clotrimazole alone, being 86.9±2.5% in its absence and 61.8±7.8% in its presence (n=6). In the presence of the cell permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (6 μm), the inhibitory effects of l‐NAME on carbachol‐induced relaxation were substantially enhanced (ED50: l‐NAME alone, 0.52±0.11 nmol, n=5; l‐NAME+8‐bromo cyclic GMP, 1.42±0.28 nmol, n=7. Rmax: l‐NAME alone, 82.2±2.4%; l‐NAME+8‐bromo cyclic GMP, 59.1±1.8%. P<0.001). These results suggest that the magnitude of the NO‐independent component of vasorelaxation is reduced when functional cyclic GMP levels are maintained, suggesting that basal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium‐dependent relaxations becomes functionally greater. The present investigation demonstrates that muscaranic receptor‐induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO‐dependent and independent mechanisms. The l‐NAME‐insensitive mechanism, most probably occurs via activation of a K+ conductance and shows the characteristics of EDHF‐mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and this may compensate for the loss of NO.

Cardiovascular effects of cannabinoids

The prototypic endocannabinoid, anandamide, and synthetic analogues have been shown to elicit pressor and depressor effects, bradycardia, vasorelaxation, and inhibition of neurotransmission in the central and peripheral nervous systems. Cannabinoid-mediated inhibition of neurotransmission is mediated by inhibition of voltage-gated Ca(2+) channels and adenylyl cyclase and activation of inwardly rectifying K(+) channels. The precise mechanisms underlying the vasorelaxant actions of cannabinoids are currently unclear, but might involve both receptor-dependent and -independent and endothelium-dependent and -independent pathways. Mechanisms proposed have included the release of endothelial autacoids, activation of myoendothelial gap junctions, activation of the Na(+) pump, activation of K(+) channels, inhibition of Ca(2+) channels, and activation of vanilloid receptors, leading to the release of sensory neurotransmitters. Pathophysiologically, the vasodilator actions of endocannabinoids have been implicated in the hypotension associated with both septic and haemorrhagic shock, but their physiological significance remains to be determined.

Endocannabinoids: a new class of vasoactive substances

Endogenous cannabinoids (endocannabinoids) have recently been identified in the CNS and attention has now turned to their cardiovascular actions. The prototypic endocannabinoid, anandamide, derived from arachidonic acid, has been shown to be a vasorelaxant, particularly in the resistance vasculature. This vasorelaxation has been shown to be both endothelium-independent and -dependent, depending on the vascular bed. It has been proposed that an endocannabinoid may mediate the nitric oxide- and prostanoid-independent component of endothelium-dependent relaxations, as these responses are sensitive to a cannabinoid receptor antagonist and show similarities to anandamide-induced relaxations. This hypothesis has generated much controversy and the emerging conflicts in the literature are discussed in this article by Michael Randall and David Kendall. Despite this controversy, it has recently been shown that anandamide is produced by endothelial cells. Clearly, much work is required to adequately define the physiological significance of endocannabinoids in the cardiovascular system.

Testosterone-induced vasorelaxation in the rat mesenteric arterial bed is mediated predominantly via potassium channels

We have investigated the involvement of nitric oxide and K+ channels in the vasorelaxant responses to physiologically‐relevant concentrations of testosterone in the rat isolated mesenteric arterial bed. Testosterone (100 pM – 10 μM) elicited concentration‐dependent relaxations in the isolated mesenteric arterial bed (pEC50=9.47 (9.22 – 9.73, 95% CI), maximal relaxation, Rmax=62.8±2.0%, n=6). A nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μM) or removal of the endothelium significantly inhibited maximal relaxations to testosterone (L‐NAME: Rmax=51.4±1.1%, P<0.01, n=6; endothelium‐denuded: Rmax=46.9±2.8%, P<0.001, n=5). Raising the extracellular K+ concentration to 30 and 60 mM, or pre‐treatment with 300 μM tetrabutylammonium chloride (TBA), a calcium‐activated K+ channel inhibitor, abolished vasorelaxations induced by testosterone. A selective inhibitor of ATP‐sensitive K+ (KATP) channels, glibenclamide (10 μM) and an inhibitor of voltage‐sensitive K+ (KV) channels, 4‐aminopyridine (4‐AP, 1 mM) did not affect testosterone‐induced responses. Vasorelaxation to 1 μM testosterone was significantly (P<0.05) inhibited by 100 nM charybdotoxin (ChTx), an inhibitor of large conductance calcium‐activated K+ (BKCa) channels (control: 63.3±9.9%, n=6; ChTx: 11.9±12.7%, n=3). Neither the testosterone receptor antagonist, flutamide (10 μM) nor an aromatase inhibitor, aminoglutethimide (10 μM) inhibited testosterone‐induced responses. In conclusion, the present findings demonstrate, in the rat isolated mesenteric arterial bed, that testosterone causes acute vasorelaxations at physiologically relevant concentrations which are, in part, mediated via NO‐ and endothelium‐dependent pathways. However, the activation of BKCa channels plays a substantial role in testosterone‐induced vasorelaxation.

Sex differences in the relative contributions of nitric oxide and EDHF to agonist‐stimulated endothelium‐dependent relaxations in the rat isolated mesenteric arterial bed

We have used the isolated, buffer‐perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium‐derived hyperpolarizing factor (EDHF) to endothelium‐dependent relaxations to carbachol. Carbachol caused dose‐related relaxations of methoxamine‐induced tone in mesenteric vascular beds from male rats described by an ED50(M) of 0.43±0.15 nmol and a maximum relaxation (Rmax(M) of 89.6±1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED50(F)=0.72±0.19 nmol and Rmax(F)=90.7±0.9%; n=22). In the males, the addition of 100 μM NG‐nitro‐L‐arginine methyl ester (L‐NAME) caused the dose‐response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED50(M)=6.45±3.53 nmol) and significantly (P<0.01) reduced Rmax(M) (79.7±2.8%, n=13). By contrast, L‐NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED50(F)=0.89±0.19 nmol, Rmax(F)=86.9±2.3%, n=9). Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (Rmax(M)=40.3±9.2%, n=4; P<0.001) and female rats by 1.5 fold (Rmax(F)=55.3±3.3%, n=6; P<0.001), compared with methoxamine‐induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED50(M)=0.87±0.26 nmol; P<0.01) but not the females (ED50(F)=4.04±1.46 nmol). In the presence of both 60 mM KCl and L‐NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups. The cannabinoid receptor antagonist SR141716A (1 μM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED50(M)=1.41±0.74 nmol, Rmax(M)=89.4±2.5%, n=7; ED50(F)=2.17±0.95 nmol, Rmax(F)=89.9±1.8%, n=9). In mesenteries from male rats, in the presence of 100 μM L‐NAME, SR141716A significantly (P<0.05) shifted the dose‐response curve to carbachol 8 fold further to the right than that seen in the presence of L‐NAME alone (ED50(M)=53.8±36.8 nmol) without affecting Rmax(M) (72.4±4.8%, n=10). In mesenteries from female rats, the combined presence of L‐NAME and SR141716A, significantly (P<0.01) shifted the dose‐response curve to carbachol 7.5 fold, (ED50(F)=6.66±2.46 nmol), as compared to L‐NAME alone and significantly (P<0.001) decreased Rmax(F) (70.1±5.5%, n=8). Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP‐sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds. The continuous presence of sodium nitroprusside (SNP; 20–60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L‐NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED50(M)=40.9±19.6 nmol, Rmax(M)=79.4±2.5%, n=11). Similarly in mesenteries from female rats, the ED50(F) was also significantly (P<0.01) increased 7 fold (6.24±2.02 nmol), while the Rmax(F) was unaffected (81.9±11.0%; n=4). The results of the present investigation demonstrate that the relative contributions of agonist‐stimulated NO and EDHF to endothelium‐dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium‐dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males.

Involvement of a cannabinoid in endothelium-derived hyperpolarizing factor-mediated coronary vasorelaxation

We have recently proposed that an endocannabinoid is the endothelium-derived hyperpolarizing factor (EDHF) and have now tested this hypothesis in the rat isolated perfused heart. In this preparation bradykinin gave rise to nitric oxide- and prostanoid-independent relaxations, assessed as reductions in coronary perfusion pressure (ED50 = 14.9 +/- 5.9 pmol; Rmax = 25.2 +/- 2.2%), which are thought to be mediated by EDHF. These relaxations were antagonised by both the highly selective cannabinoid antagonist, SR141716A (1 microM) (Rmax = 8.3 +/- 1.2%, P < 0.001) and by the calcium-dependent potassium channel blocker tetrabutylammonium (300 microM) (Rmax = 6.7 +/- 3.4%, P < 0.01) and were abolished by the EDHF inhibitor clotrimazole (3 microM). The endogenous cannabinoid, anandamide, similarly caused coronary vasorelaxation (Rmax = 32.3 +/- 2.3%), which was abolished by clotrimazole (3 microM) and antagonised by both 300 microM tetrabutylammonium (Rmax = 18.2 +/- 2.8%, P < 0.01) and 1 microM SR141716A (Rmax = 16.4 +/- 3.3%, P < 0.01). Accordingly, these results suggest that EDHF-mediated responses in the rat coronary vasculature are due to an endogenous cannabinoid and that anandamide causes vasorelaxation through potassium channel activation. These findings are, therefore, consistent with our recent proposal that EDHF is an endogenous cannabinoid.

Vanilloid receptors on capsaicin‐sensitive sensory nerves mediate relaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arteries

In the present study, the vasodilator actions of methanandamide and capsaicin in the rat isolated mesenteric arterial bed and small mesenteric arterial segments were investigated. Methanandamide elicited concentration‐dependent relaxations of preconstricted mesenteric arterial beds (pEC50=6.0±0.1, Emax=87±3%) and arterial segments (pEC50=6.4±0.1, Emax=93±3%). In arterial beds, in vitro capsaicin pre‐treatment blocked vasorelaxation to 1 and 3 μM methanandamide, and reduced to 12±7% vasorelaxation to 10 μM methanandamide. Methanandamide failed to relax arterial segments pre‐treated in vitro with capsaicin. In arterial beds from rats treated as neonates with capsaicin to cause destruction of primary afferent nerves, methanandamide at 1 and 3 μM did not evoke vasorelaxation, and relaxation at 10 μM methanandamide was reduced to 26±4%. Ruthenium red (0.1 μM), an inhibitor of vanilloid responses, attenuated vasorelaxation to methanandamide in arterial beds (pEC50=5.6±0.1, Emax=89±1%). Ruthenium red at 1 μM abolished the response to 1 μM methanandamide, and greatly attenuated relaxation at 3 and 10 μM methanandamide in arterial beds. In arterial segments, ruthenium red (0.15 μM) blocked vasorelaxation to methanandamide, but not to CGRP. In arterial segments, the vanilloid receptor antagonist capsazepine (1 μM) inhibited, and the calcitonin gene‐related peptide (CGRP) receptor antagonist CGRP8–37 (3 μM) abolished, methanandamide‐induced relaxations. CGRP8–37, but not capsazepine, attenuated significantly relaxation to exogenous CGRP. These data show that capsaicin and ruthenium red attenuate vasorelaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments. In addition, CGRP8–37 and capsazepine antagonize responses to methanandamide in mesenteric arterial segments. In conclusion, vanilloid receptors on capsaicin‐sensitive sensory nerves play an important role in the vasorelaxant action of methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments.