Combination Of Charybdotoxin Research Articles

TRPM8 channel activation triggers relaxation of pudendal artery with increased sensitivity in the hypertensive rats

IntroductionErectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recent functional correlation between the expression of thermoreceptor channels TRPM8 (melastatin 8) and alterations in blood pressure in hypertension. The aim of this study was to investigate the function of cold-sensing TRPM8 channel in internal pudendal artery (IPA) in both normotensive and hypertensive rats. MethodsWe performed experiments integrating physiological, pharmacological, biochemical and cellular techniques. ResultsTRPM8 channels are expressed in the IPA and in vascular smooth muscle cells from IPA. In addition, TRPM8 activation, by both a cooling compound icilin (82.1 ± 3.0%, n = 6) and cold temperature [thermal stimulus, basal tone (25 °C, 41.2 ± 3.4%, n = 5) or pre-contracted tone induced by phenylephrine (25 °C, 87.0 ± 3.6%, n = 7)], induced relaxation in IPA. Furthermore, the results showed that the concentration-response curve to icilin was significantly shifted to the right in different conditions, such as: the absence of the vascular endothelium, in the presence of L-NAME (10−4 M), or indomethacin (10−5 M) or by a combination of charybdotoxin (10-7 M) and apamin (5 × 10-6 M), and Y27632 (10-6 M). Interestingly, icilin-induced vasodilation was significantly higher in IPA from spontaneously hypertensive (SHR, E10−4M = 75.3 ± 1.7%) compared to wistar rats (E10−4M = 56.4 ± 2.6%), despite no changes in the TRPM8 expression in IPA between the strains, suggesting that the sensitivity of TRPM8 channels is higher in SHR. ConclusionsThese data demonstrate for the first time, the expression and function of TRPM8 channels in the IPA involving, at least in part, endothelium-derived relaxing factors and ROCK inhibition. Overall, this channel could potentially be a new target for the treatment of hypertension associated-ED.

TRPM8 channel activation triggers relaxation of pudendal artery and corpus cavernosum with increased vascular sensitivity in spontaneously hypertensive rats: Is it a new target for erectile dysfunction?

Background and PurposeErectile dysfunction (ED) is frequently encountered in patients with arterial hypertension and there is a recently uncovered functional correlation between the expression of thermoreceptor channels TRPM8 (melastatin 8) and alterations in blood pressure. The aim of this study was to investigate the function of cold‐sensing TRPM8 channel in internal pudendal artery (IPA) in normotensive and hypertensive rats.Experimental ApproachWe performed experiments integrating physiological, pharmacological, biochemical, and cellular techniques.Key ResultsTRPM8 channels are expressed in the IPA and in vascular smooth muscle cells from IPA and corpus cavernosum (CC). In addition, TRPM8 activation, by both a cooling compound (icilin) and cold (thermal stimulus), induced relaxation in IPA and CC. In IPA, the concentration‐response curve to icilin was desensitized in different conditions, such as: the absence of vascular endothelium, the presence of L‐NAME (10−4 M) or indomethacin (10−5 M), a combination of charybdotoxin (10−7 M) and apamin (5×10−6 M), and Y27632 (10−6 M). Interestingly, icilin‐induced vasodilation was significantly higher in IPA from spontaneously hypertensive (SHR) compared to Wistar rats, despite no differences in the TRPM8 expression in IPA between the strains, suggesting that the sensitivity of TRPM8 channels is higher in SHR. In CC, the concentration‐response curve to icilin was shifted to the right in the presence of ROCK inhibitor Y27632, suggesting that ROCK, at least in part, is important to relaxation induced by TRPM8 activation. Supporting these functional data, we observed that the expression of RhoA and ROCK was not changed in CC treated with either vehicle, Phe, or Phe+icilin. However, MYPT1 phosphorylation was increased by Phe compared to control, and Phe+icilin reverted this effect, suggesting that TRPM8 activation inhibits RhoA/ROCK pathway in CC.ConclusionsThese data demonstrate, for the first time, the expression and function of TRPM8 channels in the IPA and CC involving, at least in part, endothelium‐derived relaxing factors and ROCK inhibition. Overall, this channel could potentially be a new target for the treatment of hypertension associated‐ED.Support or Funding InformationNational Council for Scientific and Technological Development (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES); NIDDK Diacomp Pilot & Feasibly Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Positive Feedback Regulation of Agonist-Stimulated Endothelial Ca 2+ Dynamics by K Ca 3.1 Channels in Mouse Mesenteric Arteries

Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca(2+) signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca(2+) transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1(-/-)) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca(2+) dynamics. Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca(2+) dynamics was not different between IK1(-/-) and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca(2+) dynamics was greatly decreased in IK1(-/-) endothelium (515±153 versus 1860±319 events; P<0.01). In IK1(-/-)/SK3(T/T) mice, ancillary suppression (+Dox) or overexpression (-Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca(2+) reduced acetylcholine-induced Ca(2+) dynamics to the same level in wild-type and IK1(-/-) arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 μmol/L) and apamin (0.5 μmol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 μmol/L) reduced acetylcholine Ca(2+) dynamics in wild-type arteries to the level of IK1(-/-)/SK3(T/T)+Dox arteries. These drug effects were not additive. IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca(2+) dynamics.

Endothelium-dependent mechanisms of the vasodilatory effect of the endocannabinoid, anandamide, in the rat pulmonary artery

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (KCa) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (NG-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CBx), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB1, CB2 and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CBx receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CBx receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve KCa or the NO pathway.

Inhibition of potassium (KATP) channels reduces the short-circuit current response of rat colonic mucosa to acetylcholine.

Intestinal secretion depends upon electrogenic chloride transport into the gut lumen, which requires maintenance of an electrically negative cell-membrane voltage. We have investigated whether secretory responses of rat colonic mucosa to acetylcholine were sensitive to inhibition of potassium channels and whether selective inhibition could indicate the nature of the channel involved. Rat colonic mucosa was set up in Ussing chambers, short-circuit current responses obtained to acetylcholine, and the sensitivity of such responses to inhibition of potassium channels was investigated. Non-selective potassium-channel blockade by barium induced concentration-dependent inhibition of responses to acetylcholine. Similar inhibitory effects were obtained using 4-aminopyridine and glibenclamide. 5-Hydroxydecanoate and phentolamine also inhibited the increase in short-circuit current. However, a combination of charybdotoxin plus apamin was without effect. We conclude that a basolateral outward movement of potassium ions is required for the secretory action of acetylcholine on rat colonic mucosa. The potassium channel involved seems to be ATP-dependent and calcium-insensitive.

Involvement of inwardly rectifying K+ channels in secretory responses of human ileal mucosa.

In acute secretory diarrhoea the primary event driving fluid secretion is a transcellular, electrogenic, serosal to mucosal transport of chloride ions. Such transport requires the maintenance of an electrically negative cell membrane voltage, which is achieved through a basolateral outward leakage of potassium ions. The aim of this study was to investigate the nature of K(+) channel involvement in facilitating secretory processes in the human ileum. Muscle-stripped mucosal preparations of human ileal mucosa were set up in Ussing chambers for recording short-circuit current and transmucosal conductance. Escherichia coli heat-stable toxin and vasoactive intestinal peptide (VIP) produced concentration-dependent increases in short-circuit current. Responses to the heat-stable toxin were unaffected by basolateral application of 4-aminopyridine (5 mM), glibenclamide (10 microM) or a combination of charybdotoxin (0.3 microM) plus apamin (0.3 microM). However, basolateral barium (0.2-5 mM) caused a concentration-dependent inhibition. Responses to VIP were similarly affected by barium (0.05-1 mM). These results suggested that electrogenic chloride transport by human ileal mucosa required the presence of basolateral K(+) channels. The use of selective K(+)-channel inhibitors and low concentrations of barium suggested that the channels involved might be of the inwardly rectifying type.

Mechanism Underlying Endothelium-Dependent Relaxation by 2-Methylthio-ADP in Monkey Cerebral Artery

We recently reported endothelium-dependent relaxation caused by nucleotides in the non-human primate cerebral artery. Here, we investigated the endothelium-dependent, nitric oxide- and prostanoid-independent relaxation induced by 2-methylthio-ADP (2MeSADP) in monkey cerebral artery. Mechanical responses of isolated monkey cerebral arteries to the agents were isometrically recorded. In endothelium-intact arterial strips treated with indomethacin plus NG-nitro-l-arginine and partially contracted with prostaglandin F2α, 2MeSADP (1 nM – 10 μM) induced concentration-dependent relaxation that was abolished by removal of endothelium but was not influenced by either carboxy PTIO or 18α-glycyrrhetinic acid. The 2MeSADP-induced relaxation was inhibited by MRS2179 and U73122. The relaxation was markedly suppressed by exposure of the strips to high K+ media, but was not affected by glibenclamide. Combination of charybdotoxin plus apamin markedly suppressed the relaxation, whereas iberiotoxin partially attenuated it. Relaxation induced by 2MeSADP was inhibited by arachidonyl trifluoromethyl ketone, ketoconazole, and 14,15-epoxyeicosa-5(Z)-enoic acid. The inhibitors that affected the 2MeSADP-induced relaxation did not influence relaxation caused by sodium nitroprusside or forskolin. These findings indicate that 2MeSADP elicits ‘endothelium-derived hyperpolarizing factor (EDHF)-type’ relaxation via stimulation of endothelial P2Y1 receptors in monkey cerebral artery. Furthermore, phospholipase A2, cytochrome P450–derived epoxyeicosatrienoic acids and Ca2+-activated K+ channels appear to be involved in the relaxation.

Epoxyeicosatrienoic Acids Regulate Trp Channel–Dependent Ca 2+ Signaling and Hyperpolarization in Endothelial Cells

An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells. In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca(2+) influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition. Similar effects were observed in cultured endothelial cells overexpressing CYP2C9, but not in CYP2C9-deficient cells, and were prevented by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid as well as by the cAMP antagonist, Rp-cAMPS. The effects on Ca(2+) were mirrored by prolongation of the bradykinin-induced hyperpolarization. Ruthenium red and the combination of charybdotoxin and apamin prevented the latter effect, suggesting that Trp channel activation increases Ca(2+) influx and prolongs the activation of Ca(2+)-dependent K(+) (K(Ca)) channels. Indeed, overexpression of CYP2C9 enhanced the agonist-induced translocation of a TrpC6-V5 fusion protein to caveolin-1-rich areas of the endothelial cell membrane, which was prevented by Rp-cAMPS and mimicked by 11,12-EET. Elevated EET levels regulate Ca(2+) influx into endothelial cells and the subsequent activation of K(Ca) channels, via a cAMP/PKA-dependent mechanism that involves the intracellular translocation of Trp channels.

Effects of Increased Intracellular Cl− Concentration on Membrane Responses to Acetylcholine in the Isolated Endothelium of Guinea Pig Mesenteric Arteries

ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than -10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K(+) and Cl(-) currents simultaneously. The K(+) current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK(Ca) and SK(Ca) channels. The Cl(-) current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca(2+)-activated Cl(-) channels. When the pipettes contained 20 mM Cl(-), the ACh-induced K(+) conductance started decreasing during a 1-min application of ACh while the Cl(-) conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl(-), both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl(-)](i) is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl(-)](i) may be one of the reasons for the variations in the ACh-induced membrane response.

Role of endothelial large conductance Ca 2+ ‐activated potassium channels in blunted myogenic responsiveness of small skeletal muscle arteries after 48‐hour hypoxic exposure

Chronic hypoxia (CH) results in an endothelium-dependent blunting of myogenic responsiveness in isolated rat skeletal muscle arteries. We hypothesized that this attenuated myogenic responsiveness is due to an alteration of large conductance Ca2+-activated K+ (BKCa) channel activity in the endothelial cell of CH arteries. Third order branches of the caudal femoral artery (150–200 μm) from normoxic control rats and rats exposed to hypoxia (PB = 380 mmHg for 48 hours), were isolated, cannulated and pressurized. Vessel inner diameter was recorded at intraluminal pressures of 20–200 mmHg under normoxic conditions. Blunted myogenic responsiveness of CH arteries persisted in the presence of N-Nitro-L-arginine (L-NNA) (100 μM) and indomethacin (10 μM) and after further treatment with the luminally administered small and intermediate conductance potassium channel blockers, apamin (100 nM) and TRAM 34 (1 μM), respectively. However, myogenic responsiveness of CH arteries was restored to control levels by luminal administration of either a combination of charybdotoxin (100 nM) and apamin (100 nM) (n=5, both groups) or iberiotoxin (100 nM) alone (n=5, both groups), in the continued presence of L-NNA and indomethacin. These latter treatments did not affect myogenic reactivity in arteries from control rats. We conclude that endothelial cell BKCa channel activity is increased in CH arteries resulting in hyperpolarization that is conducted to the underlying vascular smooth muscle thereby attenuating myogenic responsiveness.

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Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery.In addition to nitric oxide (NO) and prostacyclin (PGI2), the vascular endothelium can influence local vascular tone by a mechanism involving the hyperpolarization of vascular smooth muscle cells. This response is attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF). The present study was performed to determine the characteristics of the EDHF that mediates the NO/PGI2-independent hyperpolarization and relaxation of human renal interlobar arteries.Acetylcholine-induced, EDHF-mediated hyperpolarization and relaxation were assessed using sharp microelectrodes impaled into interlobar smooth muscle cells and in organ chamber experiments, respectively. All experiments were performed in the combined presence of NO synthase (NOS) and cyclooxygenase inhibitors and the thromboxane analog U46619.Interlobar arteries demonstrated pronounced NO/PGI2-independent relaxations and hyperpolarizations that were sensitive to the blockade of calcium-activated K+-channels (KCa+ channels) by the combination of charybdotoxin and apamin and to the inhibition of the Na-K-ATPase by ouabain. Exogenously applied KCl also exhibited relaxations and hyperpolarizations that were sensitive to ouabain but insensitive to the combined inclusion of charybdotoxin and apamin. Relaxations induced by KCl were also observed in endothelium-denuded interlobar arteries.These results indicate that in the human renal interlobar artery, EDHF-mediated responses display the pharmacologic characteristics of K+ ions released through endothelial KCa+ channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of ouabain-sensitive subunits of the Na-K-ATPase.

Evidence that inward rectifier K+ channels mediate relaxation by the PGI2 receptor agonist cicaprost via a cyclic AMP-independent mechanism

We investigated the role of the inward rectifier potassium (KIR) channel and the cyclic AMP-dependent pathway in mediating vasorelaxation induced by the prostacyclin analogue cicaprost. Small vessel myography was used to assess responses to cicaprost in segments of rat tail artery contracted with phenylephrine. Microelectrode recordings were made from helical strips to assess effects on membrane potential. Cicaprost caused relaxation and hyperpolarisation that were significantly inhibited by Ba2+ (30-100 microM), a known blocker of KIR channels. Raising extracellular K+ from 5 to 15 mM elicited membrane hyperpolarisation and an endothelium-independent relaxation that was blocked by Ba2+ (30-100 microM), suggesting the existence of functional KIR channels on the smooth muscle. In contrast, neither glibenclamide (10 microM), a blocker of ATP-sensitive K+ channels, nor fluoxetine hydrochloride (100 microM), a blocker of G-protein-gated inward rectifier K+ channels, nor pertussis toxin (PTX; 1 microg/ml), which irreversibly inhibits Gi/Go, reduced relaxation to cicaprost. Indeed, PTX significantly potentiated responses. Relaxation to cicaprost was not mediated by NO but was partially endothelium-dependent, consistent with a similar inhibition by a combination of charybdotoxin (0.1 microM) and apamin (0.5 microM), blockers of endothelium-derived hyperpolarising factor (EDHF). However, relaxation was unaffected by adenylyl cyclase (SQ22536, dideoxyadenosine) or protein kinase A (Rp-2-O-monobutyryl-cAMP) inhibitors, consistent also with Ba2+ only weakly inhibiting relaxation to the adenylyl cyclase activator forskolin. We conclude that cicaprost relaxes rat tail artery by activating KIR channels with some involvement from EDHF. The mechanism appears to be largely independent of cyclic AMP and Gi/Go, although the latter appears to counteract relaxation through an unknown pathway and/or receptor.

Heterogeneity in the mechanisms of vasorelaxation to anandamide in resistance and conduit rat mesenteric arteries

1 In order to address mechanistic differences between arterial vessel types, we have compared the vasorelaxant actions of anandamide in resistance (G3) and conduit (G0) mesenteric arteries. 2 Anandamide produced concentration-dependent relaxations of pre-constricted G3 arteries with a maximal response that was significantly greater than seen in G0. 3 The CB1 receptor selective antagonists SR141716A (100 nm) and AM251 (100 nm) caused reductions in the vasorelaxant responses to anandamide in both arteries. Maximal vasorelaxant responses to anandamide were reduced in both arteries after treatment with capsaicin to deplete sensory neurotransmitters (10 microm for 1 h). 4 Vasorelaxation to anandamide was not affected by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 300 microm) in either artery. Only responses in G3 arteries were sensitive to removal of the endothelium. In G3 vessels only, vasorelaxation to anandamide was reduced by inhibition of EDHF activity with a combination of charybdotoxin (100 nm) and apamin (500 nm) in the presence of L-NAME (300 microm) and indomethacin (10 microm). 5 Antagonism of the novel endothelial cannabinoid receptor (O-1918, 1 microm) caused a reduction in the sensitivity to anandamide in G3 but not G0. 6 G3, but not G0, vessels showed a small reduction in vasorelaxant responses to anandamide after inhibition of gap junctional communication with 18alpha-GA (100 microm). 7 These results demonstrate that there are differences in the mechanisms of vasorelaxation to anandamide between conduit and resistance mesenteric arteries. In small resistance vessels, vasorelaxation occurs through stimulation of vanilloid receptors, CB1 receptors, and an endothelial receptor coupled to EDHF release. By contrast, in the larger mesenteric artery, vasorelaxation is almost entirely due to stimulation of vanilloid receptors and CB1 receptors, and is endothelium-independent.

Chronic treatment with progesterone but not medroxyprogesterone acetate restores the endothelial control of vascular tone in the mesenteric artery of ovariectomized rats.

To examine whether chronic administration of the natural hormone progesterone or a synthetic progestogen, medroxyprogesterone acetate, to ovariectomized rats affects the endothelial control of arterial tone in the isolated mesenteric artery. Sham-operated rats received a daily subcutaneous injection of solvent (sesame oil), whereas ovariectomized rats received either sesame oil, progesterone (22 mg kg/day), or medroxyprogesterone acetate (22 mg kg/day) for 4 weeks, according to their respective group. Phenylephrine-induced contractions were significantly increased (about 200% at 10 microM) by N-nitro-L-arginine, a nitric oxide synthase inhibitor, in intact mesenteric arterial rings from the sham-operated but not from the ovariectomized group. The progesterone but not the medroxyprogesterone treatment restored the potentiating effect of N-nitro-L-arginine on phenylephrine-induced contraction (about 180% at 10 microM). Contractions to phenylephrine were not affected by the combination of charybdotoxin plus apamin, two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses, in all groups. Acetylcholine induced endothelium-dependent relaxations, which were partially inhibited by N-nitro-L-arginine and abolished by the combination of N-nitro-L-arginine plus charybdotoxin and apamin, in all groups. Acetylcholine induced similar charybdotoxin and apamin-sensitive hyperpolarizations in intact mesenteric artery segments from all groups. Chronic administration of progesterone, but not medroxyprogesterone, to ovarictomized rats restores the endothelium-dependent attenuation of contractile responses to phenylephrine in mesenteric arterial rings through the endothelial formation of nitric oxide. Thus, an enhancement of the protective effect of endothelial cells on the arterial wall might contribute to the beneficial effect of certain progestogen-containing preparations during hormonal treatment.

Developmental changes in myoendothelial gap junction mediated vasodilator activity in the rat saphenous artery.

A role for myoendothelial gap junctions (MEGJs) has been proposed in the action of the vasodilator endothelium-derived hyperpolarizing factor (EDHF). EDHF activity varies in disease and during ageing, but little is known of the role of EDHF during development when, in many organ systems, gap junctions are up-regulated. The aims of the present study were therefore to determine whether an up-regulation of heterocellular gap junctional coupling occurs during arterial development and whether this change is reflected functionally through an increased action of EDHF. Results demonstrated that in the saphenous artery of juvenile WKY rats, MEGJs were abundant and application of acetylcholine (ACh) evoked EDHF-mediated hyperpolarization and relaxation in the presence of N(omega)-nitro-l-arginine methyl ester (L-NAME) and indomethacin to inhibit nitric oxide and prostaglandins, respectively. Responses were blocked by a combination of charybdotoxin plus apamin, or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) plus apamin, or by blockade of gap junctions with the connexin (Cx)-mimetic peptides, (43)Gap26, (40)Gap27 and (37,43)Gap27. On the other hand, we found no evidence for the involvement of the putative chemical mediators of EDHF, eicosanoids, L-NAME-insensitive nitric oxide, hydrogen peroxide or potassium ions, since 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), hydroxocobalamin, catalase or barium and ouabain were without effect. In contrast, in the adult saphenous artery, MEGJs were rare, EDHF-mediated relaxation was absent and hyperpolarizations were small and unstable. The present study demonstrates that MEGJs and EDHF are up-regulated during arterial development. Furthermore, the data show for the first time that this developmentally regulated EDHF is dependent on direct electrotonic coupling via MEGJs.

Red wine polyphenols cause endothelium-dependent EDHF-mediated relaxations in porcine coronary arteries via a redox-sensitive mechanism

Moderate consumption of wine is associated with cardiovascular protection most likely by increasing the endothelial formation of nitric oxide (NO). The present study investigated whether red wine polyphenolic compounds (RWPCs) increase the formation of endothelium-derived hyperpolarizing factor (EDHF) in arteries and, if so, to characterize the underlying mechanism. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension and membrane potential in the presence of indomethacin and N ω-nitro- l-arginine. RWPCs caused pronounced endothelium-dependent relaxations and hyperpolarizations, which were reduced by the combination of charybdotoxin plus apamin (two inhibitors of EDHF-mediated responses). Both responses to RWPCs were also reduced by antioxidants, membrane permeant analogues of superoxide dismutase, and diphenylene iodonium, an inhibitor of flavin-dependent enzymes. RWPCs induced the formation of superoxide in cultured endothelial cells. These findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries, which are critically dependent on a redox-sensitive mechanism involving a flavin-dependent enzyme.

Determinants of renal afferent arteriolar actions of bradykinin: evidence that multiple pathways mediate responses attributed to EDHF.

The determinants of bradykinin (BK)-induced afferent arteriolar vasodilation were investigated in the in vitro perfused hydronephrotic rat kidney. BK elicited a concentration-dependent vasodilation of afferent arterioles that had been preconstricted with ANG II (0.1 nmol/l), but this dilation was transient in character. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 micromol/l) and the cyclooxygenase inhibitor ibuprofen (10 micromol/l) did not prevent this dilation when tone was established by ANG II but fully blocked the response when tone was established by elevated extracellular KCl, which suggests roles for both NO and endothelium-derived hyperpolarizing factor (EDHF). We had previously shown that the EDHF-like response of the afferent arteriole evoked by ACh was fully abolished by a combination of charybdotoxin (ChTX;10 nmol/l) and apamin (AP; 1 micromol/l). However, in the current study, treatment with ChTX plus AP only reduced the EDHF-like component of the BK response from 98 +/- 5 to 53 +/- 6% dilation. Tetraethylammonium (TEA; 1 mmol/l), which had no effect on the EDHF-induced vasodilation associated with ACh, reduced the EDHF-like response to BK to 88 +/- 3% dilation. However, the combination of TEA plus ChTX plus AP abolished the response (0.3 +/- 1% dilation). Similarly, 17-octadecynoic acid (17-ODYA) did not prevent the dilation when it was administered alone (77 +/- 9% dilation) but fully abolished the EDHF-like response when added in combination with ChTX plus AP (-0.5 +/- 4% dilation). These findings suggest that BK acts via multiple EDHFs: one that is similar to that evoked by ACh in that it is blocked by ChTX plus AP, and a second that is blocked by either TEA or 17-ODYA. Our finding that a component of the BK response is sensitive to TEA and 17-ODYA is consistent with previous suggestions that the EDHF released by BK is an epoxyeicosatrienoic acid.

Characterization of the endothelium-derived hyperpolarizing factor (EDHF) response in the human interlobar artery

In addition to nitric oxide (NO) and prostacyclin (PGI2), the vascular endothelium can influence local vascular tone by a mechanism involving the hyperpolarization of vascular smooth muscle cells. This response is attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF). The present study was performed to determine the characteristics of the EDHF that mediates the NO/PGI2-independent hyperpolarization and relaxation of human renal interlobar arteries. Acetylcholine-induced, EDHF-mediated hyperpolarization and relaxation were assessed using sharp microelectrodes impaled into interlobar smooth muscle cells and in organ chamber experiments, respectively. All experiments were performed in the combined presence of NO synthase (NOS) and cyclooxygenase inhibitors and the thromboxane analog U46619. Interlobar arteries demonstrated pronounced NO/PGI2-independent relaxations and hyperpolarizations that were sensitive to the blockade of calcium-activated K+-channels (KCa+ channels) by the combination of charybdotoxin and apamin and to the inhibition of the Na-K-ATPase by ouabain. Exogenously applied KCl also exhibited relaxations and hyperpolarizations that were sensitive to ouabain but insensitive to the combined inclusion of charybdotoxin and apamin. Relaxations induced by KCl were also observed in endothelium-denuded interlobar arteries. These results indicate that in the human renal interlobar artery, EDHF-mediated responses display the pharmacologic characteristics of K+ ions released through endothelial KCa+ channels. Smooth muscle cell hyperpolarization and relaxation appear to be dependent on the activation of ouabain-sensitive subunits of the Na-K-ATPase.

Inhibition of EDHF by two new combinations of K+-channel inhibitors in rat isolated mesenteric arteries.

It is widely established that in rat mesenteric arteries, endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation evoked by acetylcholine is abolished by a combination of charybdotoxin plus apamin. 4-Aminopyridine, an inhibitor of voltage-gated (Kv) K(+)-channels, in combination with apamin had moderate effects on the EDHF-mediated relaxation. Maurotoxin (MTX), an inhibitor of Kv and intermediate-conductance Ca(2+)-activated K(+)-channels (IK), had no effect on EDHF-mediated relaxation. However, MTX in combination with apamin completely abolished EDHF-mediated relaxation and endothelial cell hyperpolarization. The selective IK inhibitor 2-(2-chlorophenyl)-2,2-diphenyl acetonitrile (TRAM-39) had no significant effect on EDHF-mediated relaxation. EDHF-mediated vasorelaxation and hyperpolarization was abolished by a combination of TRAM-39 and apamin. These data demonstrate two new combinations of K(+)-channel inhibitors for the investigation of EDHF. Furthermore, by using TRAM-39, a potent selective inhibitor of IK channels, we provide the first direct evidence that abolition of EDHF requires the simultaneous presence of intermediate- and small-conductance Ca(2+)-activated K(+)-channel inhibitors.

NO contributes to EDHF-like responses in rat small arteries: a role for NO stores.

Responses to EDHF are usually characterised in the presence of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors. The contribution of NO to endothelium-dependent relaxation in the presence of NOS inhibitors was assessed using NO scavengers with the objective of testing (i) whether any residual NO produces endothelium-dependent relaxation in a manner similar to EDHF and (ii) to identify the source of the residual NO. Small rat hepatic and mesenteric arteries were mounted in a tension myograph for either isometric or membrane potential measurements. Relaxation to ACh was unaffected by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM), and indomethacin (Indo, 5 microM) in the absence or presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), nitro-L-arginine (300 microM) or L-nitro-mono-methyl-arginine (L-NMMA, 300 microM). Addition of OxyHb (20 microM) or carboxy-PTIO (300 microM) produced a significant suppression of ACh-induced relaxations ( approximately 40%). In L-NAME+Indo treated arteries ACh-induced hyperpolarisation (delta16.3+/-2.1 mV, n=8) was significantly suppressed with the addition of OxyHb (Delta10.2+/-1.6 mV, n=12). ACh-induced relaxation, in the presence of L-NAME+Indo+OxyHb, was abolished by raised extracellular K(+), or the combination of charybdotoxin (CTX, 100 nM)+apamin (100 nM). In contrast whilst L-NAME+indo+barium+ouabain suppressed ACh-induced relaxation, the presence of OxyHb had no additional effect. Ultraviolet light induced a relaxation in arteries treated with L-NMMA+Indo (37.0+/-5.2%, n=9) which was sensitive to OxyHb (15.2+/-10.9%, n=4), and barium+ouabain (6.39+/-2.7%, n=4), but not CTX+apamin (37.8+/-2.4%, n=4). These findings suggest that NO contributes significantly to the "EDHF-like" response seen in rat small arteries and that the source of this NO may be preformed vascular stores.