Pharmacological treatment of oro-facial pain - health technology assessment including a systematic review with network meta-analysis.

This health technology assessment evaluated the efficacy of pharmacological treatment in patients with oro-facial pain. Randomised controlled trials were included if they reported pharmacological treatment in patients ≥18 years with chronic (≥3 months) oro-facial pain. Patients were divided into subgroups: TMD-muscle [temporomandibular disorders (TMD) mainly associated with myalgia]; TMD-joint (TMD mainly associated with temporomandibular joint pain); and burning mouth syndrome (BMS). The primary outcome was pain intensity reduction after pharmacological treatment. The scientific quality of the evidence was rated according to GRADE. An electronic search in PubMed, Cochrane Library, and EMBASE from database inception to 1 March 2017 combined with a handsearch identified 1552 articles. After screening of abstracts, 178 articles were reviewed in full text and 57 studies met the inclusion criteria. After risk of bias assessment, 41 articles remained: 15 studies on 790 patients classified as TMD-joint, nine on 375 patients classified as TMD-muscle and 17 on 868 patients with BMS. Of these, eight studies on TMD-muscle, and five on BMS were included in separate network meta-analysis. The narrative synthesis suggests that NSAIDs as well as corticosteroid and hyaluronate injections are effective treatments for TMD-joint pain. The network meta-analysis showed that clonazepam and capsaicin reduced pain intensity in BMS, and the muscle relaxant cyclobenzaprine, for the TMD-muscle group. In conclusion, based on a limited number of studies, evidence provided with network meta-analysis showed that clonazepam and capsaicin are effective in treatment of BMS and that the muscle relaxant cyclobenzaprine has a positive treatment effect for TMD-muscle pain.

Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain.

Transient receptor potential vanilloid 1 (TRPV1), vanilloid 2 (TRPV2) and melastatin 8 (TRPM8) are thermosensitive cation channels expressed on primary sensory neurons. In contrast to TRPV1, which is present on nociceptive primary afferents and keratinocytes in human skin, less is known about the distribution of TRPV2 and TRPM8 in this tissue. Immunohistochemistry of human forearm skin identified TRPV2 and TRPM8 immunoreactive nerve fibers in epidermis-papillary dermis and around blood vessels and hair follicles in dermis, although these nerve fibers were less abundant than TRPV1 immunoreactive nerve fibers throughout the skin. The TRPV2 and TRPM8 immunoreactive nerve fibers also showed immunoreactivity for calcitonin gene-related peptide (CGRP) and to a lesser extent substance P (SP). Neither of the TRP ion channels co-localized with neurofilament 200 kDa (NF200), vasoactive intestinal peptide (VIP) or tyrosine hydroxylase (TH). Nerve fibers immunoreactive for TRPV1, TRPV2, TRPM8, CGRP and SP were absent or substantially reduced in number in individuals with Norrbottnian congenital insensitivity to pain, an autosomal disease selectively affecting the development of C-fiber and Adelta-fiber primary afferents. Quantitative real time PCR detected mRNA transcripts encoding TRPV1 and TRPV2, but not TRPM8, in skin from healthy volunteers, suggesting that these ion channels are also expressed extraneuronally. In conclusion, nerve fibers in human skin express TRPV1, TRPV2 and TRPM8 that co-localize with the sensory neuropeptides CGRP and SP, but not with NF200, VIP or TH. A dramatic loss of such nerve fibers was seen in skin from individuals with Norrbottnian congenital insensitivity to pain, further suggesting that these ion channels are expressed primarily on nociceptive primary sensory neurons in human skin.

TRPV1-mediated itch in seasonal allergic rhinitis.

BACKGROUND: Patients with allergic rhinitis may be abnormally sensitive to stimulation of the ion channel transient receptor potential vanilloid-1 (TRPV1). AIM OF THE STUDY: To examine effects of various TRP ion channel activators on sensory symptoms in allergic rhinitis prior to and during seasonal allergen exposure. METHODS: Nasal challenges were carried out with the TRPV1-activators capsaicin, anandamide and olvanil. Moreover, challenges were performed with mustard oil (allylisothiocyanate) and cinnamaldehyde as well as menthol, activators of TRPA1 and TRPM8, respectively. Nasal symptoms were monitored after each challenge and compared with symptoms reported following corresponding sham challenges. Symptoms recorded after challenge prior to pollen season were also compared with challenge-induced symptoms during pollen season. RESULTS: The TRPV1, TRPA1 and TRPM8-activators produced sensory symptoms dominated by pain and smart. During seasonal allergen exposure, but not prior to season, TRPV1-activators also induced itch. Furthermore, the seasonal challenge to the TRPV1-activator olvanil was associated with rhinorrhoea. CONCLUSION: Patients with allergic rhinitis feature an increased itch response to TRPV1 stimulation at seasonal allergen exposure. We suggest that this reflects part of the hyperresponsiveness that characterizes on-going allergic rhinitis. Intervention with the TRPV1-signalling pathway may offer potential treatments of this condition.

Vasodilator effects of KRN2391, levcromakalim and 3-morpholino-sydnonimin in human pial and omental arteries.

The vasodilator action of KRN2391 (10 nM-10 microM), a combined ATP-sensitive potassium channel (KATP) opener and organic nitrate, was investigated in human pial and omental arteries. Previous animal studies have suggested that opening of KATP and activation of guanylate cyclase may contribute to varying extents to the vasodilator action of KRN2391, depending on the origin and size of the vascular preparation. Vasodilator responses were studied in isolated vascular segments (diameter 0.4-0.8 mm) pre-contracted with endothelin-1 in the presence or absence of glibenclamide (inhibitor of KATP), LY83583 (inhibitor of guanylate cyclase), zaprinast (inhibitor of cyclic GMP phosphodiesterase V) and NG-nitro-L-arginine (inhibitor of nitric oxide synthase). KRN2391 induced concentration-dependent vasodilator responses of similar potency in arteries from the two vascular regions. While glibenclamide (1 microM) had no effect in omental arteries, this compound produced a tenfold rightwards shift of the concentration-response curve for KRN2391 in pial arteries without affecting the maximal response (Emax). LY83583 (10 microM), zaprinast (10 microM) and NG-nitro-L-arginine (0.1 mM) all failed to affect the vasodilator responses to KRN2391 significantly in either artery. However, in ring segments of rat aorta LY83583 displaced the concentration-response curve for the nitric oxide donor 3-morpholino-sydnonimin (10 nM-0.1 mM) to the right, while zaprinast produced a leftwards shift. The prototype KATP opener levcromakalim (0.01-10 microM) elicited a larger relaxation in pial (Emax 80+/-6%) than in omental (Emax 47+/-13%) arteries, whereas 3-morpholino-sydnonimin produced a smaller relaxation in pial (Emax 50+/-18%) than in omental (Emax 90+/-4%) arteries. These results suggest that the vasodilator response to KRN2391 is mediated by KATP in human cerebral arteries, but dependent on neither KATP nor guanylate cyclase in human omental arteries. The results with levcromakalim and 3-morpholino-sydnonimin indicate that opening of KATP may be a more effective mechanism of vasodilatation in pial than in omental arteries from man, whereas the reverse appears to be true for guanylate cyclase activation.

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 (pEC(50)=6.0+/-0.1, E(max)=87+/-3%) and arterial segments (pEC(50)=6.4+/-0.1, E(max)=93+/-3%). In arterial beds, in vitro capsaicin pre-treatment blocked vasorelaxation to 1 and 3 microM methanandamide, and reduced to 12+/-7% vasorelaxation to 10 microM 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 microM did not evoke vasorelaxation, and relaxation at 10 microM methanandamide was reduced to 26+/-4%. Ruthenium red (0.1 microM), an inhibitor of vanilloid responses, attenuated vasorelaxation to methanandamide in arterial beds (pEC(50)=5.6+/-0.1, E(max)=89+/-1%). Ruthenium red at 1 microM abolished the response to 1 microM methanandamide, and greatly attenuated relaxation at 3 and 10 microM methanandamide in arterial beds. In arterial segments, ruthenium red (0.15 microM) blocked vasorelaxation to methanandamide, but not to CGRP. In arterial segments, the vanilloid receptor antagonist capsazepine (1 microM) inhibited, and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8 - 37) (3 microM) abolished, methanandamide-induced relaxations. CGRP(8 - 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, CGRP(8 - 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.

Differential actions of anandamide, potassium ions and endothelium-derived hyperpolarizing factor in guinea-pig basilar artery.

Vasodilator responses to anandamide (arachidonylethanolamide) and potassium ions were compared with those mediated by endothelium-derived hyperpolarizing factor (EDHF) in guinea-pig isolated basilar artery contracted with prostaglandin F2alpha. In this artery, EDHF-mediated responses can be evoked by acetylcholine in the presence of both indomethacin (10 microM) and NG-nitro-L-arginine (0.3 mM). In endothelium-denuded arterial segments, which failed to respond to acetylcholine, anandamide was still able to evoke a complete relaxation. Anandamide (10 microM) did not affect the resting membrane potential, whereas acetylcholine (10 microM) hyperpolarized the smooth muscle cells by 23 mV in the presence of indomethacin and NG-nitro-L-arginine. Pre-treatment with capsaicin (10 microM) or resiniferatoxin (0.1 microM) abolished the anandamide-induced relaxation, but had no effect on the EDHF-mediated relaxation induced by acetylcholine. Treatment with a mixture of the calcium-sensitive potassium channel inhibitors, apamin and charybdotoxin, which abolishes EDHF-mediated relaxation in this artery, did not affect the relaxation evoked by anandamide. The additional presence of glibenclamide or ciclazindol, inhibitors of ATP-sensitive and voltage-dependent potassium channels, also had no effect on the anandamide-induced relaxation. Increasing the potassium ion concentration by 2-10 mM induced inconsistent vasodilator responses. However, re-admission of potassium ions to preparations incubated in potassium-free solution elicited almost complete and sustained relaxations. A short incubation period with ouabain (10 microM for 10 min) or cooling (18-22 degrees C) abolished these responses, whereas the acetylcholine-induced relaxation in the presence of indomethacin and NG-nitro-L-arginine was unaffected (ouabain) or partially reduced (cooling). The anandamide-induced relaxation was also abolished by ouabain and cooling. Furthermore, ouabain inhibited the vasodilator response to capsaicin, but not that to calcitonin gene-related peptide (CGRP), and per se evoked a release of CGRP from the artery. The gap junction uncoupler, 18alpha-glycyrrhetinic acid (100 microM), affected neither the EDHF-mediated relaxation induced by acetylcholine nor the vasodilator responses to anandamide and potassium ions. Thus, EDHF-mediated vasorelaxation in the guinea-pig basilar artery does not seem to involve Na+/K+-ATPase, sensory nerves or gap junctions. These results indicate that EDHF is neither anandamide nor potassium ions in this artery.

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.

Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery.

1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl-bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel.

Involvement of sensory nerves in vasodilator responses to acetylcholine and potassium ions in rat hepatic artery.

In the presence of ouabain (1 mM), acetylcholine and KCl (5 mM) evoked endothelium-independent relaxations in rat hepatic arteries. Treatment with capsaicin (10 microM), scopolamine (1 microM) or CGRP(8 - 37) (3 microM) prevented these relaxations. Acetylcholine-induced relaxations in intact arterial segments in the presence of indomethacin (10 microM) and N(G)-nitro-L-arginine (0.3 mM) were only partially inhibited by ouabain plus BaCl(2) (30 microM). However, ouabain plus BaCl(2) almost abolished such relaxations in capsaicin-pre-treated preparations. In arteries without endothelium, the neurosecretagogue alpha-latrotoxin (1 nM) induced complete relaxations, which were abolished by CGRP(8 - 37) or pre-treatment with capsaicin. alpha-Latrotoxin also induced a smooth muscle hyperpolarization (12+/-2 mV), which was abolished by CGRP(8 - 37). The ability of ouabain to disclose a CGRP-mediated neurogenic relaxation must be considered when this agent is used as a pharmacological tool. The results further suggest that CGRP is a nerve-derived hyperpolarizing factor in the rat hepatic artery.

Vasodilator action in the nitroxylated cyanoamidine derivative, KRN2391, in rabbit basilar artery.

KRN2391 is a cyanoamidine derivative with a pyridine ring and a nitroxyl group. This gives the molecule a dual pharmacological action as both an ATP-sensitive K channel (K(ATP)) opener and an organic nitrate. In cerebrovascular disease with endothelial dysfunction, such a compound could be advantageous to prevent the negative consequences of a reduced synthesis of endogenous nitric oxide and endothelium-derived hyperpolarizing factor. The objective of this study was to characterise the vasodilator action of KRN2391 in a cerebral artery. As shown in the rabbit basilar artery contracted by endothelin-1 KRN2391 elicited a concentration-dependent relaxation. KRN2391 was unable to relax arteries contracted by a 60 mM K solution. The KRN2391-induced relaxation of endothelin-1-contracted arteries was unaffected by N(G)-nitro-L-arginine (0.1 mM), indomethacin (10 microM) or removal of the endothelium. The guanylate cyclase inhibitors ODQ (10 microM) and LY53583 (10 microM), and the cGMP phosphodiesterase inhibitor zaprinast (10 microM) each had no effect on the KRN2391-induced relaxation. Glibenclamide (1 microM), a blocker of K(ATP), caused a rightward shift of the concentration-response curve for KRN2391. The relaxation induced by the prototype K(ATP) opener levcromakalim was inhibited to a similar extent by glibenclamide. Addition of ODQ or LY53583, or the calcium-sensitive K channel blockers apamin (0.1 microM) and charybdotoxin (0.1 microM) in the presence of glibenclamide did not produce a significant further inhibition of the KRN-induced relaxation. KRN2391 (10 microM) did not influence the content of cGMP in the basilar artery, whereas the nitric oxide donor 3-morpholino-sydnonimine (0.1 mM) increased the cGMP level three-fold. Thus, KRN2391 is an effective vasodilator of the rabbit basilar artery, acting mainly through opening of KATP . The nitro-moiety of the molecule does not seem to contribute to the relaxant effect in this artery.

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, but not the cannabinoid CB1 receptor blocker SR141716A, 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.

Endothelial P2Y receptors induce hyperpolarisation of vascular smooth muscle by release of endothelium-derived hyperpolarising factor.

The effects of P2Y receptor agonists on smooth muscle membrane potential in isolated ring segments of rat mesenteric artery were examined by intracellular microelectrodes. In the presence of inhibitors of nitric oxide-synthase and cyclo-oxygenase, the selective P2Y1 receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS) induced endothelium-dependent membrane hyperpolarisations, which were abolished by a combination of the K+ channel inhibitors charybdotoxin and apamin, providing direct evidence that ADPbetaS releases endothelium-derived hyperpolarising factor (EDHF). 2-MethylthioATP and ATP, each which stimulates both endothelial P2Y receptors and P2X receptors on the smooth muscle cells, also elicited hyperpolarisation, but only after desensitisation of P2X receptors with alphabeta-methylATP indicating that simultaneous activation of P2X receptors may counteract the action of EDHF. In conclusion, activation of endothelial P2Y receptors induce release of EDHF.

A non-nitrergic smooth muscle relaxant factor released from rat urinary bladder by muscarinic receptor stimulation.

PURPOSE: To study the possible release of a relaxant factor from isolated rat bladder tissue. MATERIALS AND METHODS: Thoracic aortae and urinary bladders were obtained from 55 female Sprague-Dawley rats. The bladder body was used in its original tubular shape as the donor tissue in a co-axial bioassay system, and the aorta served as acceptor tissue. RESULTS: In a co-axial bioassay system with endothelium-free, norepinephrine-contracted, rat aortic preparations mounted within urothelium-intact urinary bladder, carbachol caused a concentration-dependent relaxation, amounting to 64+/-7% (n = 10) of the induced contraction, suggesting release of a relaxing factor. The relaxant effect of carbachol was lost if the urinary bladder segment was removed. However, the relaxation was affected neither by removal of the urothelium, nor by bladder segment inversion. It was resistant to inhibition of the L-arginine/nitric oxide and cyclo-oxygenase pathways, and unaffected by beta-adrenoceptor blockade and K+ channel inhibition. The relaxation was not associated with any significant changes in the intracellular levels of cGMP or cAMP. CONCLUSION: A previously unrecognized non-adrenergic, non-nitrergic, non-prostanoid inhibitory mediator is released from the rat urinary bladder by muscarinic receptor stimulation. The physiological importance of such a factor remains to be established.

Characterization of endothelium- dependent relaxation in guinea pig basilar artery - effect of hypoxia and role of cytochrome P450 mono-oxygenase.

In the guinea pig basilar artery, acetylcholine and the calcium ionophore A23187 induced endothelium-dependent relaxations, which were not significantly affected by the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine (L-NOARG; 0.3 mM) or the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; 1-10 microM), or by these inhibitors combined. However, acetylcholine (10 microM) and A23187 (3 microM) each significantly increased the tissue level of cGMP in the absence but not in the presence of L-NOARG, suggesting that NO is released from the vascular endothelium in this blood vessel. Treatment with the potassium (K) channel inhibitors charybdotoxin (0.1 microM) plus apamin (0.1 microM), a toxin mixture previously shown to inhibit relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF) in this artery, had no effect on the A23187-induced relaxation but slightly inhibited the response to acetylcholine (Emax was reduced by 24%). When the action of EDHF was prevented by these K channel inhibitors, the remaining relaxation was abolished by either ODQ (1 microM) or L-NOARG (0.3 mM), indicating that NO, apart from EDHF, contributes to the endothelium-dependent relaxations. Furthermore, ODQ (10 microM) abolished the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. Thus, activation of soluble guanylate cyclase seems to be the only mechanism through which NO causes relaxation in this artery. When vessels were exposed to grave hypoxia (pO2 = 6 mm Hg), the NO-mediated relaxation (induced by acetylcholine in the presence of charybdotoxin plus apamin) disappeared. In contrast, EDHF-mediated responses (elicited by acetylcholine in the presence of L-NOARG) were only marginally affected by hypoxia (Emax was reduced by 16%). 17-Octadecynoic acid (50 microM) and 5,8,11,14-eicosatetraynoic acid (10 microM), inhibitors of cytochrome P450-dependent oxidation of arachidonic acid, failed to inhibit the acetylcholine-induced relaxation in the presence of L-NOARG. The cytochrome P450-dependent arachidonic acid metabolite 11,12-epoxyecosatrienoic acid (0.3-3.0 microM) had no relaxant effect per se. In conclusion, EDHF and NO are both mediators of endothelium-dependent relaxations in the guinea pig basilar artery. However, during grave hypoxia, EDHF alone mediates acetylcholine-induced relaxation. The results further suggest that EDHF is not a metabolite of arachidonic acid formed by cytochrome P450 mono-oxygenase or generated by another oxygen-dependent enzyme in this artery.

Interactions between endothelium-derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF.

1. In rat isolated hepatic arteries contracted with phenylephrine, acetylcholine and the calcium ionophore A23187 each elicit endothelium-dependent relaxations, which involve both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). However, the contribution of prostanoids to these responses, and the potential interaction between EDHF and other endothelium-derived relaxing factors have not been examined. 2. In the presence of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG, 0.3 mM) and a mixture of charybdotoxin (0.3 microM) and apamin (0.3 microM), inhibitors of the target potassium (K) channel(s) for EDHF, acetylcholine and A23187 each induced a concentration-dependent and almost complete relaxation, which was abolished in the additional presence of indomethacin (10 microM). Thus, in addition to EDHF and NO, a relaxing factor(s) generated by cyclo-oxygenase (COX) contributes to endothelium-dependent relaxation in the rat hepatic artery. 3. The resting membrane potentials of endothelium-intact and endothelium-denuded vascular segments were -57 mV and -52 mV, respectively (P>0.05). In intact arteries, the resting membrane potential was not affected by L-NOARG plus indomethacin, but reduced to -47 mV in the presence of charybdotoxin plus apamin. Acetylcholine and A23187 (10 microM each) elicited a hyperpolarization of 13 mV and 15 mV, respectively. The hyperpolarization induced by these agents was not affected by L-NOARG plus indomethacin (12 mV and 14 mV, respectively), but reduced in the presence of charybdotoxin plus apamin (7 mV and 10 mV, respectively), and abolished in the combined presence of charybdotoxin, apamin and indomethacin. 4. The NO donor 3-morpholino-sydnonimine (SIN-1) induced a concentration-dependent relaxation, which was unaffected by charybdotoxin plus apamin, but abolished by the selective soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM). SIN-1 (10 microM) did not alter the resting membrane potential in endothelium-denuded vascular segments. 5. The COX-dependent relaxation induced by acetylcholine was abolished following exposure to 30 mM KCl, but unaffected by glibenclamide (10 microM). The prostacyclin analogue iloprost induced a concentration-dependent relaxation, which was also abolished in 30 mM KCl and unaffected by the combined treatment with glibenclamide, charybdotoxin and apamin. Iloprost (10 microM) induced a glibenclamide-resistant hyperpolarization (8 mV with and 9 mV without glibenclamide) in endothelium-denuded vascular segments. 6. Exposure to SIN-1 or iloprost did not affect the EDHF-mediated relaxation induced by acetylcholine (i.e. in the presence of L-NOARG and indomethacin). Replacement of L-NOARG with the NO scavenger oxyhaemoglobin (10 microM) or the soluble guanylate cyclase inhibitor ODQ (10 microM) or methylene blue (10 microM), which all significantly inhibited responses to endothelium-derived NO, did not affect the acetylcholine-induced relaxation in the presence of indomethacin, indicating that endogenous NO also does not suppress EDHF-mediated responses. 7. These results show that, in addition to EDHF and NO, an endothelium-derived hyperpolarizing factor(s) generated by COX contributes significantly to endothelium-dependent relaxation in the rat heptic artery. Neither this factor nor NO seems to regulate EDHF-mediated responses. Thus, EDHF does not serve simply as a 'back-up' system for NO and prostacyclin in this artery. However, whether EDHF modulates the NO and COX pathways remains to be determined.

[Calcium antagonists. Great tissue selectivity is the basis of their therapeutic use]. / Kalciumantagonister. Stor vävnadsselektivitet grunden för den terapeutiska användningen.

Calcium antagonists are established drugs for the treatment of a number of cardiovascular disorders. A specific subtype of voltage-sensitive calcium channel (the L-channel) is the target of all these drugs. The main subgroups of calcium antagonists (phenylalkylamines, benzothiazepines and dihydropyridines) differ in their binding sites on the L-channel, and are characterised by differences in their selectivity of action vis-a-vis vascular smooth muscle and the cardiac conduction system. At a molecular level, each subgroup of calcium antagonists is unique in its mechanism of action, though individual dihydropyridines also differ from each other in this respect. The structure, function and distribution of L-channels, as well as the molecular basis of the unique tissue selectivity of calcium antagonists are described in the article, and the question of serious side-effects is also discussed.

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

1. 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. 2. The KV inhibitors, agitoxin-2 (0.3 microM), kaliotoxin (0.3 microM), beta-dendrotoxin (0.3 microM), dofetilide (1 microM) and terikalant (10 microM), each in combination with apamin (0.3 microM) had no effect on the EDHF-mediated relaxation induced by acetylcholine in the presence of N omega-nitro-L-arginine (0.3 mM) and indomethacin (10 microM), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n = 2-3). Although the KV inhibitor margatoxin (0.3 microM) 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). 3. Ciclazindol (10 microM) 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 microM; n = 6). This combination did not inhibit acetylcholine-induced relaxations mediated by endothelium-derived NO (n = 5). 4. 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 microM; n = 3) nor ciclazindol (10 microM; n = 5), alone or in combination with apamin (0.3 microM; 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 microM; n = 2). 5. 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 microM, 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 microM; n = 6). 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.

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

1. In the presence of NG-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 microM), 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. 2. Inhibitors of adenosine 5'-triphosphate (ATP)-sensitive potassium (K)-channels (KATP; glibenclamide, 10 microM), voltage-sensitive K-channels (Kv; dendrotoxin-1, 0.1 microM or 4-aminopyridine, 1 mM), small (SKCa; apamin, 0.1 microM) and large (BKCa; iberiotoxin, 0.1 microM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine. 3. Synthetic charybdotoxin (0.1 microM), 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. 4. A combination of charybdotoxin (0.1 microM) and apamin (0.1 microM) 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 microM) and apamin (0.1 microM). 5. Ciclazindol (10 microM), 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 microM) was combined with apamin (0.1 microM). 6. 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 microM) and apamin (0.1 microM), whereas each toxin had little effect alone. 7. 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.