Inhibitor Of Large-conductance Ca2 Research Articles

Role of Epoxyeicosatrienoic Acids in Acetylcholine-Induced Dilation of Rat Retinal Arterioles in Vivo.

CYP epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent hyperpolarization (EDH)-related dilation in multiple vascular beds. The present study aimed to determine the role of EETs in the acetylcholine (ACh)-induced dilation of retinal arterioles in rats in vivo. The vasodilator responses were assessed by determining the change in diameter of the retinal arterioles on images of the ocular fundus. The intravitreal injection of 17-octadecynoic acid (1.4 nmol/eye), an inhibitor of CYP epoxygenase, and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E; 2 nmol/eye), an antagonist of EETs, reduced the ACh (0.3-10 µg/kg/min)-induced dilation of the retinal arterioles. The EET antagonist attenuated the vasodilator response to ACh under blockade of nitric oxide (NO) synthases and cyclooxygenases with NG-nitro-L-arginine methyl ester (30 mg/kg) plus indomethacin (5 mg/kg). Intravitreal injection of 14,15-EET (0.5 nmol/eye) dilated retinal arterioles and the response was prevented by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BKCa channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.

Activation of transient receptor potential vanilloid 4 channels dilates rat retinal arterioles through nitric oxide- and BKCa channel-dependent mechanisms in vivo.

Transient receptor potential vanilloid 4 (TRPV4) channel, a cation channel expressed in nearly all cell types, plays an important role in the regulation of vascular tone. In the present study, we examined the effect of GSK1016790A, an activator of TRPV4 channels, on the diameter of retinal blood vessels in rats and the underlying mechanisms. Ocular fundus images were captured with an original high-resolution digital fundus camera in vivo and diameters of retinal blood vessels were measured. Intravenous infusion of GSK1016790A (0.2-2μgkg-1min-1) increased retinal arteriolar diameter in a dose-dependent manner. The higher dose of GSK1016790A (2μgkg-1min-1) slightly decreased blood pressure. These responses to GSK1016790A were significantly attenuated by intravenous injection of GSK2193874 (0.3mg/kg), an antagonist of TRPV4 channels. Intravitreal injection of Nω-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase or iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channel, significantly attenuated the GSK1016790A-induced increases in retinal arteriolar diameter. These results suggest that activation of TRPV4 channels dilates rat retinal arterioles through NO- and BKCa channel-dependent mechanisms in vivo.

Involvement of Ca2+ Signaling in the Synergistic Effects between Muscarinic Receptor Antagonists and β₂-Adrenoceptor Agonists in Airway Smooth Muscle.

Long-acting muscarinic antagonists (LAMAs) and short-acting β2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 μM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors.

Sufentanil attenuates impairment of the endothelium-dependent vasodilation induced by hypoxia-reoxygenation in the rat coronary artery.

Sufentanil has been used broadly in cardiac surgery, but the mechanisms by which it modulates coronary vascular tone after ischemia-reperfusion injury are largely unknown. Effects of sufentanil on coronary tone and on the relaxation of rat coronary arteries (CAs) in response to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) relaxing agents in the presence of hypoxia-reoxygenation (H/R) was studied in an in vitro organ chamber setup. Sufentanil (10-7-10-4 mol/L) relaxed rat CA rings in endothelium-dependent and endothelium-independent manners. In endothelium-intact rings, preincubation of H/R-treated CAs with sufentanil (10-5 mol/L) significantly increased the acetylcholine response, but did not augment sodium nitroprusside-induced relaxation. Sufentanil-mediated potentiation of acetylcholine-induced relaxation was not affected by a nitric oxide synthase inhibitor or by intermediate- or small-conductance Ca2+-activated K+ channel blockers. However, potentiation was abolished by iberiotoxin (100 nmol/L), a selective inhibitor of large-conductance Ca2+-activated K+ channels, as well as Rp-cAMPS (30 μmol/L), a cyclic AMP-dependent protein kinase (PKA) inhibitor. Sufentanil induced endothelium-dependent and endothelium-independent relaxation and attenuated H/R-induced impairment of endothelium-dependent vasodilation in the rat CAs. The potentiating effect of sufentanil may involve activation of large-conductance Ca2+-activated K+ channels via cAMP-dependent mechanisms.

Mechanisms of endothelium-dependent relaxation evoked by anandamide in isolated human pulmonary arteries

Endocannabinoids contract, relax or do not affect vessels with different calibre and tone in the pulmonary circulation in four species. The aim of the present study was to determine the mechanisms involved in the anandamide-induced relaxation of human pulmonary arteries (hPAs). Studies were performed in the isolated hPAs pre-constricted with the prostanoid TP receptor agonist, U-46619. To detect fatty acid amide hydrolase (FAAH) expression, Western blots were used. Anandamide concentration dependently relaxed the endothelium-intact hPAs pre-constricted with U-46619. The anandamide-induced relaxation was virtually abolished by removal of the endothelium and strongly attenuated by inhibitors of cyclooxygenases (indomethacin, COX-1/COX-2, and nimesulide, COX-2), nitric oxide synthase (N G-nitro-l-arginine methyl ester) given separately or in combination, FAAH (URB597), and the prostanoid IP receptor antagonist, RO1138452. The anandamide-evoked relaxation in the endothelium-intact vessels was attenuated in KCl pre-constricted preparations or by the inhibitor of large-conductance Ca2+-activated K+ channels, iberiotoxin. In experiments performed in the presence of URB597 to exclude effects of anandamide metabolites, the antagonist of the endothelial cannabinoid receptor, O-1918, diminished the anandamide-evoked relaxation whereas the antagonists of cannabinoid CB1, CB2 and vanilloid TRPV1 receptors, AM251, SR144528 and capsazepine, respectively, had no effect. Western blot studies revealed the occurrence of FAAH protein in the hPAs. The present study shows that anandamide breakdown products, cyclooxygenase pathways, nitric oxide, potassium channels and the O-1918-sensitive cannabinoid receptor play a role in the anandamide-induced relaxation of the hPAs with intact endothelium.

Mechanisms involved in the vasodilator effect induced by diosgenin in rat superior mesenteric artery

The aim of this study was to investigate the vasorelaxant effect induced by diosgenin in superior mesenteric rings. In rings pre-contracted with phenylephrine (10 microM), diosgenin caused concentration-dependent relaxations [EC(50) = (3.3 +/- 1.2) x 10(- 4)M, E(max) = 94.2 +/- 2.6 %]. Vascular relaxation induced by diosgenin was significantly inhibited after removal of the endothelium (E(max) = 46 +/- 8.8%, p < 0.001) or after pre-treatment of the rings with N-nitro-L-arginine methyl esther (l-NAME) 100 or 300 microM (E(max) = 35.3 +/- 4%; 28.1 +/- 3.3%, respectively, p < 0.001), atropine 1 microM (E(max) = 24.6 +/- 3.4%, p < 0.001), hydroxocobalamin 30 microM (E(max) = 54.0 +/- 9.6%, p < 0.001), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) 10 microM (E(max) = 46.0 +/- 8.0%, p < 0.001) or indomethacin 1 microM (E(max) = 22.6 +/- 11.8%, p < 0.001). Vasorelaxation evoked by diosgenin was significantly inhibited after pre-treatment of preparations with both selective and non-selective inhibitors of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, iberiotoxin 100 nM or tetraethylammonium (TEA) 1mM, respectively (E(max) = 62.5 +/- 9.1%; 65.7 +/- 1.1%, p < 0.001). Conversely, in endothelium-denuded vessels, none of BK(Ca) channel blockers modified the relaxant effect induced by diosgenin. In mesenteric endothelial cells loaded with FURA-2 diosgenin was able to increase intracellular calcium concentrations, which were significantly decreased by atropine 1 microM. In addition, in isolated mesenteric rings, diosgenin induced marked increase in nitric oxide (NO) levels, which was completely abolished after functional endothelium removal. The results obtained here demonstrated that diosgenin-induced relaxation appears to involve endothelial muscarinic receptor activation with increase in intracellular calcium concentrations and consequent release of endothelium-derived relaxing factors (EDRFs), mainly NO and cyclooxygenase derivatives, which activate BK(Ca) channels. Nevertheless, further studies are necessary to clearly elucidate residual endothelium-independent relaxation induced by diosgenin.

Role of Ca 2+-Activated K + Channels on Adrenergic Responses of Human Saphenous Vein

We studied the participation of K(+) channels on the adrenergic responses in human saphenous veins as well as the intervention of dihydropyridine-sensitive Ca(2+) channels on modulation of adrenergic responses by K(+) channels blockade. Saphenous vein rings were obtained from 40 patients undergoing coronary artery bypass surgery. The vein rings were suspended in organ bath chambers for isometric recording of tension. Iberiotoxin (10(-7) mol/L), an inhibitor of large conductance Ca(2+)-activated K(+) channels, and charybdotoxin (10(-7) mol/L), an inhibitor of both large and intermediate conductance Ca(2+)-activated K(+) channels, enhanced the contractions elicited by electrical field stimulation and produced a leftward shift of the concentration-response curve to norepinephrine. In contrast, the inhibitor of small conductance Ca(2+)-activated K(+) channels apamin (10(-6) mol/L) did not modify the contractile response to electrical field stimulation or norepinephrine. In the presence of the dihydropyridine Ca(2+)-channel blocker nifedipine (10(-6) mol/L), iberiotoxin and charybdotoxin failed to enhance the contractile responses to electrical field stimulation and norepinephrine. The results suggest that large conductance Ca(2+)-activated K(+) channels are activated by stimulation with norepinephrine to counteract the adrenergic-induced contractions of human saphenous vein. Thus, inhibition of these channels increases significantly the contraction, an effect that appears to be mediated by an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels.

Mechanisms of Paracrine Regulation by Fetal Membranes of Human Uterine Quiescence

To test the hypothesis that fetal membranes (chorion or amnion) release one or more factors responsible for myometrial quiescence. Myometrial samples were excised from women at elective term cesarean delivery prior to the onset of labor. Fetal membranes were obtained after cesarean delivery either before or during labor, and either term (greater than 37 weeks) or preterm (less than or equal to 36 weeks). Myometrial strips were placed in organ baths and contractions stimulated by oxytocin (10(-8) M). Contractility was measured under isometric conditions before and after exposure to fetal membranes or conditioned medium. The impact of either membrane or conditioned media on contractility was determined before and after myometrial K+ channel blockade. Both chorion and amnion and their respective conditioned mediums decrease oxytocin-stimulated myometrial contraction. The inhibitory effect was greatest with membranes from preterm pregnancies (mean gestation 32 weeks, P <.05). The inhibitory effect was detectable in the presence of term labor, but was absent when the fetal membranes were obtained after preterm labor. Iberiotoxin, an inhibitor of large conductance Ca2+-activated K+ channels (BK(Ca)) reduced the effect of fetal membranes by 50% (P <.05). We conclude that human fetal membranes release one or more factors that inhibit oxytocin-induced myometrial contractility. We suggest this factor (or factors) acts mainly by opening myometrial BK(Ca). The findings further support our hypothesis that the fetal membranes release a factor (or factors) that is central to myometrial quiescence and its premature loss leads to preterm delivery.

Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo

We have recently demonstrated that endogenous H2O2 plays an important role in coronary autoregulation in vivo. However, the role of H2O2 during coronary ischemia-reperfusion (I/R) injury remains to be examined. In this study, we examined whether endogenous H2O2 also plays a protective role in coronary I/R injury in dogs in vivo. Canine subepicardial small coronary arteries (>or=100 microm) and arterioles (<100 microm) were continuously observed by an intravital microscope during coronary I/R (90/60 min) under cyclooxygenase blockade (n=50). Coronary vascular responses to endothelium-dependent vasodilators (ACh) were examined before and after I/R under the following seven conditions: control, nitric oxide (NO) synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA), catalase (a decomposer of H2O2), 8-sulfophenyltheophylline (8-SPT, an adenosine receptor blocker), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA, an inhibitor of large-conductance Ca2+-sensitive potassium channels), and L-NMMA+catalase+8-SPT. Coronary I/R significantly impaired the coronary vasodilatation to ACh in both sized arteries (both P<0.01); L-NMMA reduced the small arterial vasodilatation (both P<0.01), whereas it increased (P<0.05) the ACh-induced coronary arteriolar vasodilatation associated with fluorescent H2O2 production after I/R. Catalase increased the small arterial vasodilatation (P<0.01) associated with fluorescent NO production and increased endothelial NOS expression, whereas it decreased the arteriolar response after I/R (P<0.01). L-NMMA+catalase, L-NMMA+TEA, or L-NMMA+catalase+8-SPT further decreased the coronary vasodilatation in both sized arteries (both, P<0.01). L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT significantly increased myocardial infarct area compared with the other four groups (control, L-NMMA, catalase, and 8-SPT; all, P<0.01). These results indicate that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo.

Involvement of guanylyl cyclase, protein kinase A and Na +K + ATPase in relaxations of bovine isolated bronchioles induced by GEA 3175, an NO donor

The present study was designed to investigate the role of the sodium potassium adenosine triphosphatase (the Na +K + ATPase) in relaxation of bovine isolated bronchioles by a new NO donor, GEA 3175 (3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulphonyl]amino]-)hydroxide)). Bronchioles were mounted in a wire myograph for isometric tension recordings and contracted with 5-hydroxytryptamine (5-HT) or a K + rich solution. Concentration-dependent relaxations evoked by GEA 3175 were inhibited by ouabain or K + free solution. The guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 μM) and ouabain (10 nM) reduced GEA 3175-evoked relaxations to the same extent without any additive effect. Iberiotoxin (10 nM), an inhibitor of large conductance Ca 2+-activated K + channels inhibited GEA 3175-evoked relaxations to the same extent as ouabain. Combining ouabain and iberiotoxin completely abolished GEA 3175 relaxation. An inhibitor of protein kinase G (PKG), Rp-β-phenyl-1,N 2-etheno-8-bromo-guanosine-3′-5′-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPs), slightly reduced GEA 3175-induced relaxations. An inhibitor of cyclic AMP-dependent kinase (PKA), Rp-adenosine-3′-5′-cyclic phosphorothioate (Rp-cAMPs), inhibited the GEA 3175-induced relaxations to the same extent as ouabain. Inhibition of both PKG and PKA abolished GEA 3175 relaxation. The study provides evidence that the NO donor GEA 3175 causes guanylyl cyclase-dependent relaxations, taking place through cyclic GMP and cyclic AMP-dependent protein kinases followed by opening of large conductance Ca 2+-activated K + channels and activation of smooth muscle Na +K + ATPase.

Reduced molecular expression of K+channel proteins in vascular smooth muscle from rats made hypertensive withNω-nitro-l-arginine

Smooth muscle membrane potential (E(m)) depends on K(+) channels, and arteries from rats made hypertensive with N(omega)-nitro-l-arginine (LHR) are depolarized compared with control. We hypothesized that decreased K(+) channel function, due to decreased K(+) channel protein expression, underlies E(m) depolarization. Furthermore, K(+) channel blockers should move control E(m) (-46 +/- 1 mV) toward that in LHR (-37 +/- 2 mV) and normalize contraction. The E(m) vs. K(+) relationship was less steep in LHR (23 +/- 2 vs. 28 +/- 1 mV/log K(+) concentration), and contractile sensitivity to K(+) was increased (EC(50) = 37 +/- 1 vs. 23 +/- 1 mM). Iberiotoxin (10 nM), an inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, depolarized control and LHR E(m) to -35 +/- 1 and -30 +/- 2 mV, respectively; however, effects on K(+) sensitivity were more profound in LHR (EC(50) = 25 +/- 2 vs. 15 +/- 3 mM). The voltage-dependent K(+) (K(V)) channel blocker 4-aminopyridine (3 mM) depolarized control E(m) to the level of LHR (-28 +/- 1 vs. -28 +/- 1 mV); however, effects on K(+) sensitivity were greater in LHR (EC(50) = 17 +/- 4 vs. 4 +/- 4 mM). Western blots revealed reduced BK(Ca) and K(V)1.5 channel expression in LHR arteries. The findings suggest that diminished expression of K(+) channels contributes to depolarization and enhanced contractile sensitivity. These conclusions are supported by direct electrophysiological assessment of BK(Ca) and K(V) channel function in control and LHR smooth muscle cells.

Chorion-induced myometrial relaxation is mediated by large-conductance Ca2+-activated K+ channel opening in the guinea pig

Objective: We previously demonstrated that chorion releases a factor that inhibits both spontaneous and oxytocin-stimulated myometrial contractility. Here, we investigate the mechanism of action of this unidentified substance. Study Design: Myometrial strips from pregnant guinea pigs were mounted in an organ bath and contractility stimulated with oxytocin. Results: Guinea pig chorion produced a time-dependent decrease in oxytocin-induced myometrial contractility. The ability of the chorion to reduce contractility was unaltered by inhibiting chorionic synthesis of either nitric oxide (N [ω]-nitro-L-arginine), carbon monoxide (tin-protoporphyrin), prostaglandins (indomethacin), or the myometrial cyclic guanosine monophosphate pathway (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalime-1-one and Rp-8Br-cGMP). In contrast, iberiotoxin, an inhibitor of large conductance Ca2+-activated K+ channels reduced the quiescent effect of chorion by 40%; in contrast, inhibition of adenosine triphosphate–sensitive (glibenclamide) and voltage-gated K+ channels (4amynopyridine) had no effect. Conclusion: Chorion-induced relaxation of oxytocin-stimulated myometrial contraction is, in great part, the product of a paracrine substance that opens myometrial large conductance Ca2+-activated K+ channels. (Am J Obstet Gynecol 2003;188:84-91.)

Sodium nitroprusside-induced rat fundus relaxation is ryanodine-sensitive and involves L-type Ca2+ channel and small conductance Ca(2+)-sensitive K+ channel components.

1 The aim of this study was to examine whether sodium nitroprusside (SNP)-induced relaxation of rat fundus longitudinal smooth muscle involves ryanodine-sensitive Ca2+ release. 2 SNP (300 nM-30 microM) elicited concentration-dependent relaxation of precontracted (1 microM carbachol) rat fundus, an effect almost abolished by the selective guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM). 3 SNP-mediated relaxations were almost abolished by 10 microM ryanodine. 4 SNP-mediated relaxations were also reduced by either 1 microM apamin (a selective small conductance Ca(2+)-sensitive K+ channel, SKCa, inhibitor) or the selective L-type Ca2+ channel inhibitor, nicardipine (3 microM). 5 SNP-induced relaxations were insensitive to 1 mM tetraethylammonium chloride (an inhibitor of large-conductance Ca(2+)-sensitive K+ channels) and 1 microM glibenclamide (an ATP-sensitive K+ channel inhibitor). 6 These data suggest that SNP-mediated fundus relaxation occurs via a cGMP-mediated and ryanodine-sensitive mechanism which requires, at least in part, SKCa and L-type Ca2+ channel activity.

Potentiated endothelium-derived hyperpolarizing factor-mediated dilations in cerebral arteries following mild head injury.

Evidence in the literature suggests that endothelium-derived hyperpolarizing factor (EDHF) may act in a compensatory manner such that during conditions of compromised nitric oxide (NO), EDHF serves as a back-up mechanism. Given that constitutive NO synthase is chronically downregulated after head trauma, we tested the hypothesis that EDHF is potentiated following injury. Male adult rats were subjected to either sham injury (n = 27) or mild controlled cortical impact (CCI) injury (n = 26). Branches of the middle cerebral artery (MCA) directly within the contusion site were harvested either 1 or 24 h later, pressurized to 60 mm Hg in a vessel chamber and allowed to develop spontaneous tone. Relaxation to luminal application of adenosine triphosphate (ATP) was similar in all groups. Relaxation to ATP in the presence of L-NAME (N(G)-nitro-L-arginine methyl ester) and indomethacin was similar in all groups except for vessels isolated at 24 h following mild CCI injury. In this case, L-NAME and indomethacin had no effect on the ATP-mediated dilation. The ATP-mediated dilation in L-NAME and indomethacin-treated MCA branches was inhibited by charybdotoxin, an inhibitor of large conductance Ca2+-sensitive K+ channels. These findings suggest that there is a significant potentiation of the EDHF-mediated dilation to ATP in cerebral arteries isolated at 24 h following mild CCI injury.

Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery.

We tested the hypothesis that endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant role in the female cerebrovasculature. The contribution of EDHF to the ATP-mediated dilation was determined in middle cerebral arteries (MCAs) isolated from male and female rats. Four groups of rats were tested: intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20) rats. Maximal dilation to ATP was similar in all groups. However, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-5) M) and indomethacin (10(-5) M), the maximal dilation to ATP was significantly reduced in intact female (24 +/- 9%) and estrogen-treated ovariectomized female (29 +/- 9%) rats compared with intact male (95 +/- 4%) and vehicle-treated ovariectomized female (96 +/- 2%) rats. The ATP-mediated dilation in L-NAME- and indomethacin-treated MCAs isolated from male and ovariectomized female rats was inhibited by charybdotoxin (10(-7) M), an inhibitor of large-conductance Ca2+-sensitive K+ channels. We have defined EDHF as the L-NAME- and indomethacin-insensitive component of the ATP-mediated dilation. Our findings indicate that EDHF-mediated dilations are negligible in the female rat MCA; these dilations can be significantly enhanced after ovariectomy, suggesting that this effect is mediated by estrogen.

Insulin-induced relaxation of rat mesenteric artery is mediated by Ca2+-activated K+ channels

We tested the hypothesis that relaxation of the rat mesenteric artery in response to insulin is mediated by K+ channels. Two concentrations of insulin (10 and 100 mU/ml) induced relaxation of the artery by 6±1%, 24±3% (mean±S.E.M.). Denudation of the endothelium or precontraction by KCl (30 mM), clotrimazole (10 μM), a cytochrome P450 inhibitor, charybdotoxin (30 nM) an inhibitor of large-conductance Ca2+-activated K+ channels, abolished the relaxation of the artery in response to insulin. However, Nω-nitro-l-arginine methyl ester (l-NAME; 100 μM), an inhibitor of nitric oxide synthase, apamin (1 μM), an inhibitor of small-conductance Ca2+-activated K+ channels, or glibenclamide (10 μM), an ATP-sensitive K+ channels blocker, did not attenuate the relaxation of the artery caused by insulin. These results suggest that the relaxation of rat mesenteric artery in response to insulin is mediated mostly by large-conductance Ca2+-activated K+ channels, perhaps an endothelium-derived hyperpolarizing factor (EDHF).

An indirect influence of phenylephrine on the release of endothelium-derived vasodilators in rat small mesenteric artery.

1. The possibility that stimulation of smooth muscle alpha(1)-adrenoceptors modulates contraction via the endothelium was examined in rat small mesenteric arteries. 2. N(omega)-nitro-L-arginine methyl ester, (L-NAME, 100 microM to inhibit NO synthase) increased contraction to single concentrations of phenylephrine (1 - 3 microM) by approximately 2 fold (from a control level of 14.2+/-3.0 to 34. 1+/-4.2% of the maximum contraction of the artery, n=20). The action of L-NAME was abolished by disrupting the endothelium. 3. The subsequent addition of apamin (to inhibit small conductance Ca(2+)-activated K(+) channels, 50 nM) further augmented phenylephrine contractions, in an endothelium-dependent manner, to more than 3 fold above control (50.4+/-5.3% of the maximum contraction, n=11). 4.Charybdotoxin (non-selective inhibitor of large conductance Ca(2+)-activated K(+) channels, BK(Ca), 50 nM) plus L-NAME augmented the level of phenylephrine contraction to 4 - 5-fold above control (64.1+/-3.1%, n=5), but this effect was independent of the endothelium. The potentiation of contraction by charybdotoxin could be mimicked with the selective BK(Ca) inhibitor, iberiotoxin,. 5. Apamin together with L-NAME and charybdotoxin further significantly increased the phenylephrine contraction by 5 - 6-fold, to 79.9+/-3.5% of the maximum contraction of the artery (n=13). 6. Phenylephrine failed directly to increase the intracellular Ca(2+) concentration in endothelial cells freshly isolated from the small mesenteric artery. 7. Stimulation of smooth muscle alpha(1)-adrenoceptors in the mesenteric artery induces contraction that is markedly suppressed by the endothelium. The attenuation of contraction appears to reflect both the release of NO from the endothelium and the efflux of K(+) from both endothelial and smooth muscle cells. This suggests that the release of NO and endothelium-derived hyperpolarizing factor can be evoked indirectly by agents which act only on the smooth muscle cells.

Effects of paxilline on K + channels in rat mesenteric arterial cells

The effects of paxilline, a mycotoxin, on whole-cell outward currents from freshly isolated cells of the rat mesenteric artery were studied. Paxilline inhibited a component of the outward current that was also sensitive to iberiotoxin. Inhibition could be observed at a concentration of 10 nM and complete inhibition of the iberiotoxin-sensitive current was achieved at 300 nM. The inhibition could be described by a single site of interaction with a K i of 35.7 nM. Paxilline had no effect on the component of the current that was sensitive to 4-aminopyridine. It is concluded that paxilline is a potent inhibitor of large conductance Ca 2+-activated K + currents in vascular smooth muscle cells.

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels.

Hydrogen peroxide (H2O2) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H2O2 was markedly attenuated by 10 microM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100 nM charybdotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels. A combination of the ODQ and charybdotoxin abolished the H2O2-induced relaxation. Pretreatment with 25 microM of an Rp stereoisomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), 20 microM glibenclamide, or 1 mM 4-aminopyridine did not affect the vascular response to H2O2. The presence of catalase at 1000 U/ml significantly attenuated the H2O2-induced relaxation. Exposure of cultured smooth muscle cells to H2O2 activated KCa channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of KCa channels. Rp-cAMPS did not inhibit the H2O2-induced activation of KCa channels. The activation of KCa channels by H2O2 was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H2O2 activated KCa channels in a concentration-dependent manner. In inside-out patches, H2O2 significantly activated KCa channels through a process independent of cyclic guanosine 3',5'-monophosphate (cGMP). In conclusion, H2O2 elicits vascular relaxation due to activation of KCa channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.

Bradykinin, lemakalim and sodium nitroprusside relax the mouse trachea in vitro by different mechanisms

The role of K + channels in the relaxations induced by bradykinin, lemakalim, an activator of ATP-sensitive K + channels and sodium nitroprusside (SNP), a nitric oxide (NO) donor was examined in the isolated mouse trachea precontracted by methacholine (1 μM). 4-aminopyridine (4-AP, 0.1–2 mM), an inhibitor of 4-AP sensitive delayed rectifier channels, did not alter relaxations induced by bradykinin, lemakalim or SNP. Glibenclamide and glipizide (10–33 μM), inhibitors of ATP-sensitive K + channels, inhibited relaxation to lemakalim without affecting responses to bradykinin and SNP. Charybdotoxin (10–100 nM) and iberiotoxin (10–100 nM), inhibitors of large conductance Ca 2+-activated K + channels, failed to inhibit relaxation to bradykinin, lemakalim or SNP. Apamin (0.1–1 μM), an inhibitor of small conductance Ca 2+-activated K + channels, did not alter responses to bradykinin, lemakalim and SNP. The results implicate that the mechanism of relaxation induced by bradykinin and SNP is different from that of lemakalim. Relaxation of the isolated mouse trachea by lemakalim appears to be mediated by ATP-sensitive K + channels. Bradykinin and SNP induced relaxations are not mediated via Ca 2+-activated K + channels.