Edward D. Högestätt

Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide

The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP (ref. 5), but not the cannabinoid CB1 receptor blocker SR141716A (ref. 7), inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective ‘vanilloid receptor’ antagonist capsazepine, inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems.

Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery

1 In the presence of indomethacin (IM, 10μm) and Nω‐nitro‐L‐arginine (L‐NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium‐dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4‐aminopyridine (4‐AP, 1 mM) inhibited the L‐NOARG/IM‐resistant relaxation induced by ACh, whereas apamin (0.1‐0.3 μm), charybdotoxin (0.1‐0.3 μm), iberiotoxin (0.1 μm) and dendrotoxin (0.1 μm) each had no effect. TBA also inhibited the relaxation induced by the receptor‐independent endothelial cell activator, A23187. 2 When combined, apamin (0.1 μm) + charybdotoxin (0.1 μm), but not apamin (0.1 μm) + iberiotoxin (0.1 μm) or a triple combination of 4‐AP (1 mM) + apamin (0.1 μm) + iberiotoxin (0.1 μm), inhibited the L‐NOARG/IM‐resistant relaxation induced by ACh. At a concentration of 0.3 μm, apamin+charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L‐NOARG/IM‐resistant relaxation induced by A23187. 3 In the absence of L‐NOARG, TBA (1 mM) inhibited the ACh‐induced relaxation, whereas charybdotoxin (0.3 μm) + apamin (0.3 μm) had no effect, indicating that the toxin combination did not interfere with the L‐arginine/NO pathway. 4 The gap junction inhibitors halothane (2 mM) and 1‐heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L‐NOARG/IM‐resistant relaxation induced by ACh. 5 Inhibition of Na+/K+‐ATPase by ouabain (1 mM) had no effect on the L‐NOARG/IM‐resistant relaxation induced by ACh. Exposure to a K+‐free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6 The results suggest that the L‐NOARG/IM‐resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K‐channels sensitive to TBA and a combination of apamin+charybdotoxin. Chloride channels, Na+/K+‐ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium‐derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo‐oxygenase products, which activates more than one type of K‐channel on the smooth muscle cells. Alternatively, a single type of K‐channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.

The anandamide transport inhibitor AM404 activates vanilloid receptors

The possibility that the anandamide transport inhibitor N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404), structurally similar to the vanilloid receptor agonists anandamide and capsaicin, may also activate vanilloid receptors and cause vasodilation was examined. AM404 evoked concentration-dependent relaxations in segments of rat isolated hepatic artery contracted with phenylephrine. Relaxations were abolished in preparations pre-treated with capsaicin. The calcitonin-gene related peptide (CGRP) receptor antagonist CGRP-(8-37) also abolished relaxations. The vanilloid receptor antagonist capsazepine inhibited vasodilation by AM404 and blocked AM404-induced currents in patch-clamp experiments on Xenopus oocytes expressing the vanilloid subtype 1 receptor (VR1). In conclusion, AM404 activates native and cloned vanilloid receptors.

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Delta(9)-tetrahydrocannabiorcol

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1(-/-) mice. The electrophilic metabolites N-acetyl-p-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-p-benzoquinoneimine metabolite L-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1(-/-) mice. Intrathecal injection of a non-electrophilic cannabinoid, Δ(9)-tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. Our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists.

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB1 receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV1) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV1 on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV1 gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV1. Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB1 receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV1 and the cannabinoid CB1 receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV1 and thus may play a role as endogenous TRPV1 modulators.

Characterization of the potassium channels involved in EDHF‐mediated relaxation in cerebral arteries

In the presence of NG‐nitro‐l‐arginine (l‐NOARG, 0.3 mm) and indomethacin (10 μm), the relaxations induced by acetylcholine and the calcium (Ca) ionophore A23187 are considered to be mediated by endothelium‐derived hyperpolarizing factor (EDHF) in the guinea‐pig basilar artery. Inhibitors of adenosine 5′‐triphosphate (ATP)‐sensitive potassium (K)‐channels (KATP; glibenclamide, 10 μm), voltage‐sensitive K‐channels (KV; dendrotoxin‐I, 0.1 μm or 4‐aminopyridine, 1 mm), small (SKCa; apamin, 0.1 μm) and large (BKCa; iberiotoxin, 0.1 μm) conductance Ca‐sensitive K‐channels did not affect the l‐NOARG/indomethacin‐resistant relaxation induced by acetylcholine. Synthetic charybdotoxin (0.1 μm), an inhibitor of BKCa and KV, caused a rightward shift of the concentration‐response curve for acetylcholine and reduced the maximal relaxation in the presence of l‐NOARG and indomethacin, whereas the relaxation induced by A23187 was not significantly inhibited. A combination of charybdotoxin (0.1 μm) and apamin (0.1 μm) abolished the l‐NOARG/indomethacin‐resistant relaxations induced by acetylcholine and A23187. However, the acetylcholine‐induced relaxation was not affected by a combination of iberiotoxin (0.1 μm) and apamin (0.1 μm). Ciclazindol (10 μm), an inhibitor of KV in rat portal vein smooth muscle, inhibited the l‐NOARG/indomethacin‐resistant relaxations induced by acetylcholine and A23187, and the relaxations were abolished when ciclazindol (10 μm) was combined with apamin (0.1 μm). Human pial arteries from two out of four patients displayed an l‐NOARG/indomethacin‐resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 μm) and apamin (0.1 μm), whereas each toxin had little effect alone. The results suggest that KV, but not KATP and BKCa, is involved in the EDHF‐mediated relaxation in the guinea‐pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both KV and SKCa are activated by EDHF. However, a single type of K‐channel, which may be structurally related to KV and allosterically regulated by apamin, could also be the target for EDHF.

Characterization of vascular neuropeptide Y receptors.

1 In the present study we compared neuropeptide Y (NPY) and NPY‐related analogues for their ability to activate or bind to vascular NPY receptors in four experimental set‐ups. Previous results have suggested the existence of different receptor subtypes, Y1 receptors requiring full‐length NPY (1–36) or [Pro34]‐NPY, and Y2 receptors recognizing also N‐terminally truncated forms of NPY but not [Pro34]‐NPY. 2 NPY 1–36 and [Pro34]‐NPY dose‐dependently increased arterial pressure in the anaesthetized rat with a similar magnitude and potency. NPY 2–36 was much less potent than NPY 1–36. NPY 4–36 and NPY 11–36 were inactive even at a dose as high as 10 nmol kg−1. 3 NPY 1–36, [Pro34]‐NPY, NPY 2–36 and NPY 5–36 concentration‐dependently increased the coronary resistance in the Langendorff preparation of the rat. NPY 1–36 and [Pro34]‐NPY were equipotent, while NPY 2–36 and NPY 5–36 were about 7 and 20 times less potent. At 0.3 μm, NPY 11–36, NPY 20–36 and NPY 22–36 induced a slight contraction while NPY 23–36 was inactive. 4 NPY 1–36, [Pro34]‐NPY, NPY 2–36, NPY 4–36, NPY 5–36 and NPY 11–36 evoked concentration‐dependent contractions in the isolated inferior caval vein of the rat and guinea‐pig. [Pro34]‐NPY was more potent than NPY 1–36. NPY 2–36 was equipotent with NPY 1–36, while NPY 4–36, NPY 5–36 and NPY 11–36 were approximately 30 times less potent. 5 [Pro34]‐NPY was equipotent with NPY 1–36 in displacing the 125I‐labelled gut hormone peptide ([125I]‐PYY) from rat aortic smooth muscle cells, while NPY 2–36 and shorter forms of NPY were much less potent or inactive. 6 In caval vein smooth muscle cells of the rat, the displacement pattern was more complex than in aortic smooth muscle cells, in that both [Pro34]‐NPY and NPY 13–36 effectively displaced the radioligand, albeit none of them completely. 7 In conclusion, the NPY‐evoked pressor response in the whole rat and coronary vessels seems to be mediated by vascular Y1 receptors and the binding characteristics of the NPY‐related peptides in the aortic smooth muscle cells correspond to a population of such receptors. In the caval vein, the profile of the bioactivity and the binding affinity of the NPY‐related peptides suggest a mixed population of Y1/Y2 receptors.

Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery

In the rat hepatic artery, the acetylcholine‐induced relaxation mediated by endothelium‐derived hyperpolarizing factor (EDHF) is abolished by a combination of apamin and charybdotoxin, inhibitors of small (SKCa) and large (BKCa) conductance calcium‐sensitive potassium (K)‐channels, respectively, but not by each toxin alone. The selective BKCa inhibitor iberiotoxin cannot replace charybdotoxin in this combination. Since delayed rectifier K‐channels (KV) represent another target for charybdotoxin, we explored the possible involvement of KV in EDHF‐mediated relaxation in this artery. The KV inhibitors, agitoxin‐2 (0.3 μM), kaliotoxin (0.3 μM), β‐dendrotoxin (0.3 μM), dofetilide (1 μM) and terikalant (10 μM), each in combination with apamin (0.3 μM) had no effect on the EDHF‐mediated relaxation induced by acetylcholine in the presence of Nω‐nitro‐L‐arginine (0.3 mM) and indomethacin (10 μM), inhibitors of nitric oxide (NO) synthase and cyclo‐oxygenase, respectively (n=2–3). Although the KV inhibitor margatoxin (0.3 μM) was also without effect (n=5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC50 and Emax values were 7.5±0.0 and 95±1% in the absence and 7.0±0.1 and 81±6% in the presence of margatoxin plus apamin, respectively; n=6; P<0.05). Ciclazindol (10 μM) partially inhibited the EDHF‐mediated relaxation by shifting the acetylcholine‐concentration‐response curve 12 fold to the right (n=6; P<0.05) and abolished the response when combined with apamin (0.3 μM; n=6). This combination did not inhibit acetylcholine‐induced relaxations mediated by endothelium‐derived NO (n=5). A 4‐aminopyridine‐sensitive delayed rectifier current (IK(V)) was identified in freshly‐isolated single smooth muscle cells from rat hepatic artery. None of the cells displayed a rapidly‐activating and ‐inactivating A‐type current. Neither charybdotoxin (0.3 μM; n=3) nor ciclazindol (10 μM; n=5), alone or in combination with apamin (0.3 μM; n=4–5), had an effect on IK(V). A tenfold higher concentration of ciclazindol (0.1 mM, n=4) markedly inhibited IK(V), but this effect was not increased in the additional presence of apamin (0.3 μM; n=2). By use of membranes prepared from rat brain cortex, [125I]‐charybdotoxin binding was consistent with an interaction at a single site with a KD of approximately 25 pM. [125I]‐charybdotoxin binding was unaffected by iberiotoxin (0.1 μM, n=6), but was increased by apamin in a concentration‐dependent manner (Emax 43±10%, P<0.05 and pEC50 7.1±0.2; n=7–8). Agitoxin‐2 (10 nM) displaced [125I]‐charybdotoxin binding by 91±3% (n=6) and prevented the effect of apamin (1 μM; n=6). It is concluded that the EDHF‐mediated relaxation in the rat hepatic artery is not mediated by the opening of either KV or BKCa. Instead, the target K‐channels for EDHF seem to be structurally related to both KV and BKCa. The possibility that a subtype of SKCa may be the target for EDHF is discussed.

Effects of cytochrome P450 inhibitors on EDHF‐mediated relaxation in the rat hepatic artery

1 The possibility that the endothelium‐derived hyperpolarising factor (EDHF) in the rat hepatic artery is a cytochrome P450 mono‐oxygenase metabolite of arachidonic acid was examined in the present study. In this preparation, acetylcholine elicits EDHF‐mediated relaxations in the presence of the nitric oxide (NO) synthase and cyclo‐oxygenase inhibitors Nω‐nitro‐L‐arginine (L‐NOARG) and indomethacin, respectively. 2 17‐Octadecynoic acid (17‐ODYA, 50 μm), a suicide‐substrate inhibitor of the cytochrome P450 mono‐oxygenases responsible for the production of 5,6‐, 8,9‐, 11,12‐ and 14,15‐epoxyeicosatrienoic acids (EETs), had no effect on acetylcholine‐induced relaxations in the presence of L‐NOARG (0.3 mM) plus indomethacin (10 μm). Furthermore, 5,6‐, 8,9‐, 11,12‐ and 14,15‐ EETs failed to relax arteries without endothelium in the presence of L‐NOARG plus indomethacin. 3 Proadifen and clotrimazole, which are inhibitors of several isoforms of cytochrome P450 mono‐oxygenases, inhibited acetylcholine‐induced relaxations in the presence of L‐NOARG plus indomethacin. The concentration of acetylcholine which caused half‐maximal relaxation was about 3 and 30 times higher in the presence than in the absence of clotrimazole (3 μm) and proadifen (10 μm), respectively. The maximal relaxation was reduced by proadifen but not by clotrimazole. Proadifen (10 μm) also inhibited acetylcholine‐induced hyperpolarization in the presence of L‐NOARG plus indomethacin. 4 In the presence of 30 mM K+ plus indomethacin (10 μm), acetylcholine induced an L‐NOARG‐sensitive relaxation mediated via release of NO. Under these conditions, proadifen (10 μm) shifted the acetylcholine concentration‐response curve 6 fold to the right without affecting the maximal relaxation. Clotrimazole (3 μm) was without effect on these responses. The relaxant actions of the NO donor, 3‐morpholino‐sydnonimine, were unaffected by proadifen (10 μm). 5 The relaxant effects of the opener of ATP‐sensitive potassium channels, levcromakalim, were abolished by proadifen (10 μm) and strongly attenuated by clotrimazole (3 μm). Proadifen (10 μm) also abolished the hyperpolarization induced by levcromakalim (1 μm). 6 The lack of effect of 17‐ODYA on relaxations mediated by EDHF, together with the failure of extracellularly‐applied EETs to produce relaxation, collectively suggest that EDHF is not an EET in the rat hepatic artery. It seems likely that inhibition of ion channels in the smooth muscle rather than reduced EDHF formation in the endothelium offers a better explanation for the actions of the cytochrome P450 inhibitors proadifen and clotrimazole.