H2O2 activates redox- and 4-aminopyridine-sensitive Kv channels in coronary vascular smooth muscle

Previously, we demonstrated that coronary vasodilation in response to hydrogen peroxide (H(2)O(2)) is attenuated by 4-aminopyridine (4-AP), an inhibitor of voltage-gated K(+) (K(V)) channels. Using whole cell patch-clamp techniques, we tested the hypothesis that H(2)O(2) increases K(+) current in coronary artery smooth muscle cells. H(2)O(2) increased K(+) current in a concentration-dependent manner (increases of 14 +/- 3 and 43 +/- 4% at 0 mV with 1 and 10 mM H(2)O(2), respectively). H(2)O(2) increased a conductance that was half-activated at -18 +/- 1 mV and half-inactivated at -36 +/- 2 mV. H(2)O(2) increased current amplitude; however, the voltages of half activation and inactivation were not altered. Dithiothreitol, a thiol reductant, reversed the effect of H(2)O(2) on K(+) current and significantly shifted the voltage of half-activation to -10 +/- 1 mV. N-ethylmaleimide, a thiol-alkylating agent, blocked the effect of H(2)O(2) to increase K(+) current. Neither tetraethylammonium (1 mM) nor iberiotoxin (100 nM), antagonists of Ca(2+)-activated K(+) channels, blocked the effect of H(2)O(2) to increase K(+) current. In contrast, 3 mM 4-AP completely blocked the effect of H(2)O(2) to increase K(+) current. These findings lead us to conclude that H(2)O(2) increases the activity of 4-AP-sensitive K(V) channels. Furthermore, our data support the idea that 4-AP-sensitive K(V) channels are redox sensitive and contribute to H(2)O(2)-induced coronary vasodilation.     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways.

We hypothesized that hydrogen peroxide (H2O2) has a role in the local regulation of skeletal muscle blood flow, thus significantly affecting the myogenic tone of arterioles. In our study, we investigated the effects of exogenous H2O2 on the diameter of isolated, pressurized (at 80 mmHg) rat gracilis skeletal muscle arterioles (diameter of approximately 150 microm). Lower concentrations of H2O2 (10(-6)-3 x 10(-5) M) elicited constrictions, whereas higher concentrations of H2O2 (6 x 10(-5)-3 x 10(-4) M), after initial constrictions, caused dilations of arterioles (at 10(-4) M H2O2, -19 +/- 1% constriction and 66 +/- 4% dilation). Endothelium removal reduced both constrictions (to -10 +/- 1%) and dilations (to 33 +/- 3%) due to H2O2. Constrictions due to H2O2 were completely abolished by indomethacin and the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ-29548. Dilations due to H2O2 were significantly reduced by inhibition of nitric oxide synthase (to 38 +/- 7%) but were unaffected by clotrimazole or sulfaphenazole (inhibitors of cytochrome P-450 enzymes), indomethacin, or SQ-29548. In endothelium-denuded arterioles, clotrimazole had no effect, whereas H2O2-induced dilations were significantly reduced by charybdotoxin plus apamin, inhibitors of Ca(2+)-activated K+ channels (to 24 +/- 3%), the selective blocker of ATP-sensitive K+ channels glybenclamide (to 14 +/- 2%), and the nonselective K(+)-channel inhibitor tetrabutylammonium (to -1 +/- 1%). Thus exogenous administration of H2O2 elicits 1) release of PGH2/TxA2 from both endothelium and smooth muscle, 2) release of nitric oxide from the endothelium, and 3) activation of K+ channels, such as Ca(2+)-activated and ATP-sensitive K+ channels in the smooth muscle resulting in biphasic changes of arteriolar diameter. Because H2O2 at low micromolar concentrations activates several intrinsic mechanisms, we suggest that H2O2 contributes to the local regulation of skeletal muscle blood flow in various physiological and pathophysiological conditions.     

Hydrogen peroxide induces endothelium-dependent and -independent coronary arteriolar dilation: role of cyclooxygenase and potassium channels

Hydrogen peroxide, a relatively stable reactive oxygen species, is known to elicit vasodilation, but its underlying mechanism remains elusive. Here, we examined the role of endothelial nitric oxide (NO), prostaglandin, cytochrome P-450-derived metabolites, and smooth muscle potassium channels in coronary arteriolar dilation to abluminal H2O2. Pig subepicardial coronary arterioles (50-100 microm) were isolated and pressurized without flow for in vitro study. Arterioles developed basal tone and dilated dose dependently to H2O2 (1-100 microM). Disruption of th endothelium and inhibition of cyclooxygenase (COX) by indomethacin produced identical attenuation of vasodilation to H2O2. Conversely, the vasodilation to H2O2 was not affected by either the NO synthase inhibitor NG-nitro-l-arginine methyl ester or the cytochrome P-450 enzyme blocker miconazole. Inhibition of the COX-1, but not the COX-2 pathway, attenuated H2O2-induced dilation similarly to indomethacin. The production of prostaglandin E2 (PGE2), but not prostaglandin I2, from coronary arterioles was significantly increased by H2O2. Furthermore, inhibition of PGE2 receptors with AH-6809 attenuated vasodilation to H2O2 similar to that produced by indomethacin. In the absence of a functional endothelium, H2O2-induced dilation was attenuated, in an identical manner, by a depolarizing agent KCl and a calcium-activated potassium (KCa) channel inhibitor iberiotoxin. However, PGE2-induced dilation was not affected by iberiotoxin. The endothelium-independent dilation to H2O2 was also insensitive to the inhibition of guanylyl cyclase, lipoxygenase, ATP-sensitive potassium channels, and inward rectifier potassium channels. These results suggest that H2O2 induces endothelium-dependent vasodilation through COX-1-mediated release of PGE2 and also directly relaxes smooth muscle by hyperpolarization through KCa channel activation.     

Transmural difference in coronary arteriolar dilation to adenosine: effect of luminal pressure and K(ATP) channels

Coronary blood flow in the subendocardium is preferentially increased by adenosine but is redistributed to the subepicardium during ischemia in association with coronary pressure reduction. The mechanism for this flow redistribution remains unclear. Since adenosine is released during ischemia, it is possible that the coronary microcirculation exhibits a transmural difference in vasomotor responsiveness to adenosine at various intraluminal pressures. Although the ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in coronary arteriolar dilation to adenosine, its role in the transmural adenosine response remains elusive. To address these issues, pig subepicardial and subendocardial arterioles (60-120 micrometer) were isolated, cannulated, and pressurized to 20, 40, 60, or 80 cmH(2)O without flow for in vitro study. At each of these pressures, vessels developed basal tone and dilated concentration dependently to adenosine and the K(ATP) channel opener pinacidil. Subepicardial and subendocardial arterioles dilated equally to adenosine and pinacidil at 60 and 80 cmH(2)O luminal pressure. At lower luminal pressures (i.e., 20 and 40 cmH(2)O), vasodilation in both vessel types was enhanced. Enhanced vasodilatory responses were not affected by removal of endothelium but were abolished by the K(ATP) channel inhibitor glibenclamide. In a manner similar to reducing pressure, a subthreshold dose of pinacidil potentiated vasodilation to adenosine. In contrast to adenosine, dilation of coronary arterioles to sodium nitroprusside was independent of pressure changes. These results indicate that coronary microvascular dilation to adenosine is enhanced at lower intraluminal pressures by selective activation of smooth muscle K(ATP) channels. Since microvascular pressure has been shown to be consistently lower in the subendocardium than in the subepicardium, it is likely that the inherent pressure gradient in the coronary microcirculation across the ventricular wall may be an important determinant of transmural flow in vivo during resting conditions or under metabolic stress with adenosine release.     

Sodium azide dilates coronary arterioles via activation of inward rectifier K+ channels and Na+-K+-ATPase

Sodium azide (NaN(3)), a potent vasodilator, causes severe hypotension on accidental exposure. Although NaN(3) has been shown to increase coronary blood flow, the direct effect of NaN(3) on coronary resistance vessels and the mechanism of the NaN(3)-induced response remain to be established. To address these issues without confounding influences from systemic parameters, subepicardial coronary arterioles were isolated from porcine hearts for in vitro study. Arterioles developed basal tone at 60 cmH(2)O intraluminal pressure and dilated acutely, in a concentration-dependent manner, to NaN(3) (0.1 microM to 50 microM). The NaN(3) response was not altered by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester or endothelial removal. Neither inhibition of phosphoinositol 3-kinase and tyrosine kinases nor blockade of ATP-sensitive, Ca(2+)-activated, and voltage-dependent K(+) channels affected NaN(3)-induced dilation. However, the vasomotor action of NaN(3) was significantly attenuated in a similar manner by the inward rectifier K(+) (K(IR)) channel inhibitor Ba(2+), the Na(+)-K(+) ATPase inhibitor ouabain, or the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Ba(2+), in combination with either ouabain or ODQ, nearly abolished the vasodilatory response. However, there was no additive inhibition by combining ouabain and ODQ. The NaN(3)-mediated vasodilation was also attenuated by morin, an inhibitor of phosphatidylinositolphosphate (PIP) kinase, which can regulate K(IR) channel activity. With the use of whole cell patch-clamp methods, NaN(3) acutely enhanced Ba(2+)-sensitive K(IR) current in isolated coronary arteriolar smooth muscle cells. Collectively, this study demonstrates that NaN(3), at clinically toxic concentrations, dilates coronary resistance vessels via activation of both K(IR) channels and guanylyl cyclase/Na(+)-K(+)-ATPase in the vascular smooth muscle. The K(IR) channels appear to be modulated by PIP kinase.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gender-specific K(+)-channel contribution to adenosine-induced relaxation in coronary arterioles.

We examined the contribution of K(+)-channel activity on basal tone and adenosine-mediated relaxation of coronary arterioles isolated from sexually mature male and female miniature swine. Arterioles (approximately 100-200 microm ID) isolated from the apical region of the heart were cannulated and studied using videodimensional analysis under constant intraluminal pressure. Coronary arterioles from male and female pigs demonstrated similar levels of basal tone and reductions in basal diameter in response to the K(+)-channel blockers 4-aminopyridine (4-AP; 1 mM), tetraethylammonium (1 mM), and glibenclamide (Glib; 10 microM), with 4-AP producing significantly greater constriction than tetraethylammonium or Glib. After endothelin-induced preconstriction, relaxation responses to adenosine were not significantly different between coronary arterioles of male and female pigs. Inhibition of 4-AP-sensitive channels significantly impaired adenosine-mediated relaxation in arterioles from male but not female pigs. However, inhibition of K(+) channels with iberiotoxin (100 nM) or Glib had no effect on adenosine-induced relaxation in either sex. Results obtained in the presence of nitric oxide synthase inhibition suggest a potential interaction of 4-AP-sensitive channels and nitric oxide at low adenosine concentrations. In conclusion, our data indicate that 4-AP-sensitive channels 1) contribute significantly to basal tone in coronary arterioles of both male and female pigs, 2) contribute to adenosine-mediated relaxation in male but not female pigs, and 3) can contribute to adenosine-induced relaxation independent of nitric oxide production in male pigs. These data are consistent with a significant role for voltage-dependent K(+) channels in adenosine-mediated relaxation of coronary arterioles from males.     

Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane KATP channels

Hydrogen sulfide (H(2)S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H(2)S exist. Here, we tested the hypothesis that piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K(+) (K(ATP)) currents and that H(2)S induces vasodilation by activating K(ATP) currents. Gas chromatography/mass spectrometry data demonstrated that after placing Na(2)S, an H(2)S donor, in solution, it rapidly (1 min) converts to H(2)S. Patch-clamp electrophysiology indicated that pinacidil (a K(ATP) channel activator), Na(2)S, and NaHS (another H(2)S donor) activated K(+) currents at physiological steady-state voltage (-50 mV) in isolated cerebral arteriole smooth muscle cells. Glibenclamide, a selective K(ATP) channel inhibitor, fully reversed pinacidil-induced K(+) currents and partially reversed (～58%) H(2)S-induced K(+) currents. Western blot analysis indicated that piglet arterioles expressed inwardly rectifying K(+) 6.1 (K(ir)6.1) channel and sulfonylurea receptor 2B (SUR2B) K(ATP) channel subunits. Pinacidil dilated pressurized (40 mmHg) piglet arterioles, and glibenclamide fully reversed this effect. Na(2)S also induced reversible and repeatable vasodilation with an EC(50) of ～30 μM, and this effect was partially reversed (～55%) by glibenclamide. Vasoregulation by H(2)S was also studied in pressurized resistance-size cerebral arteries of mice with a genetic deletion in the gene encoding SUR2 (SUR2 null). Pinacidil- and H(2)S-induced vasodilations were smaller in arterioles of SUR2 null mice than in wild-type controls. These data indicate that smooth muscle cell K(ATP) currents control newborn cerebral arteriole contractility and that H(2)S dilates cerebral arterioles by activating smooth muscle cell K(ATP) channels containing SUR2 subunits.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Time course of flow-induced vasodilation in skeletal muscle: contributions of dilator and constrictor mechanisms

The purpose of this study was to determine the time course of flow-induced vasodilation in soleus and gastrocnemius muscle arterioles and the mechanisms that underlie vasodilatory responses to an increase in intraluminal flow. Vasodilation was assessed during 20 min of continuous exposure to intraluminal flow. Both soleus and gastrocnemius muscle arterioles dilated in response to flow, although the magnitude of vasodilation was greater in arterioles from the gastrocnemius muscle. Neither blockade of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester (L-NAME) nor blockade of cyclooxygenase with indomethacin inhibited the initial vasodilation (0-2 min) in arterioles from either muscle. In contrast, vasodilation to sustained exposure to flow (2-20 min) was eliminated by treatment with L-NAME in arterioles from both muscles. Both depolarization with 40 mM KCl and blockade of Ca(2+)-activated K(+) channels inhibited the initial flow-induced dilation, and the inhibition was greater in gastrocnemius muscle arterioles than soleus muscle arterioles. In the presence of L-NAME, prolonged exposure to flow resulted in constriction in soleus and gastrocnemius muscle arterioles. This constriction was abolished by endothelin receptor blockade. These results indicate that the time course and magnitude of flow-induced vasodilation differs between arterioles from soleus and gastrocnemius muscles. The immediate response to increased flow is greater in gastrocnemius muscle arterioles and involves activation of K(+) channels. In arterioles from both soleus and gastrocnemius muscles, vasodilation to sustained flow exposure occurs primarily through production of nitric oxide. In the absence of nitric oxide, sustained exposure to flow results in pronounced constriction that is mediated by endothelin.     

Hypercholesterolemia abolishes voltage-dependent K+ channel contribution to adenosine-mediated relaxation in porcine coronary arterioles

Hypercholesterolemic patients display reduced coronary flow reserve in response to adenosine infusion. We previously reported that voltage-dependent K+ (Kv) channels contribute to adenosine-mediated relaxation of coronary arterioles isolated from male miniature swine. For this study, we hypothesized that hypercholesterolemia attenuates Kv channel contribution to adenosine-induced vasodilatation. Pigs were randomly assigned to a control or high fat/high cholesterol diet for 20-24 wk, and then killed. After completion of the experimental treatment, arterioles (approximately 150 microm luminal diameter) were isolated from the left-ventricular free wall near the apical region of the heart, cannulated, and pressurized at 40 mmHg. Adenosine-mediated relaxation was significantly attenuated in both endothelium-intact and -denuded arterioles from hypercholesterolemic compared with control animals. The classic Kv channel blocker, 4-aminopyridine (1 mM), significantly attenuated adenosine-mediated relaxation in arterioles isolated from control but not hypercholesterolemic animals. Furthermore, the nonselective K+ channel blocker, tetraethylammonium (TEA; 1 mM) significantly attenuated adenosine-mediated relaxation in arterioles from control but not hypercholesterolemic animals. In additional experiments, coronary arteriolar smooth muscle cells were isolated, and whole cell Kv currents were measured. Kv currents were significantly reduced (approximately 15%) in smooth muscle cells from hypercholesterolemic compared with control animals. Furthermore, Kv current sensitive to low concentrations of TEA was reduced (approximately 45%) in smooth muscle cells from hypercholesterolemic compared with control animals. Our data indicate that hypercholesterolemia abolishes Kv channel contribution to adenosine-mediated relaxation in coronary arterioles, which may be attributable to a reduced contribution of TEA-sensitive Kv channels in smooth muscle of hypercholesterolemic animals.     

Functional contribution of P2Y1 receptors to the control of coronary blood flow

Activation of ADP-sensitive P2Y(1) receptors has been proposed as an integral step in the putative "nucleotide axis" regulating coronary blood flow. However, the specific mechanism(s) and overall contribution of P2Y(1) receptors to the control of coronary blood flow have not been clearly defined. Using vertically integrative studies in isolated coronary arterioles and open-chest anesthetized dogs, we examined the hypothesis that P2Y(1) receptors induce coronary vasodilation via an endothelium-dependent mechanism and contribute to coronary pressure-flow autoregulation and/or ischemic coronary vasodilation. Immunohistochemistry revealed P2Y(1) receptor expression in coronary arteriolar endothelial and vascular smooth muscle cells. The ADP analog 2-methylthio-ADP induced arteriolar dilation in vitro and in vivo that was abolished by the selective P2Y(1) antagonist MRS-2179 and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. MRS-2179 did not alter baseline coronary flow in vivo but significantly attenuated coronary vasodilation to ATP in vitro and in vivo and the nonhydrolyzable ATP analog ATPγS in vitro. Coronary blood flow responses to alterations in coronary perfusion pressure (40-100 mmHg) or to a brief 15-s coronary artery occlusion were unaffected by MRS-2179. Our data reveal that P2Y(1) receptors are functionally expressed in the coronary circulation and that activation produces coronary vasodilation via an endothelium/nitric oxide-dependent mechanism. Although these receptors represent a critical component of purinergic coronary vasodilation, our findings indicate that P2Y(1) receptor activation is not required for coronary pressure-flow autoregulation or reactive hyperemia.     

KCa+ channels contribute to exercise-induced coronary vasodilation in swine

Coronary blood flow is controlled via several vasoactive mediators that exert their effect on coronary resistance vessel tone through activation of K(+) channels in vascular smooth muscle. Because Ca(2+)-activated K(+) (K(Ca)(+)) channels are the predominant K(+) channels in the coronary vasculature, we hypothesized that K(Ca)(+) channel activation contributes to exercise-induced coronary vasodilation. In view of previous observations that ATP-sensitive K(+) (K(ATP)(+)) channels contribute, in particular, to resting coronary resistance vessel tone, we additionally investigated the integrated control of coronary tone by K(Ca)(+) and K(ATP)(+) channels. For this purpose, the effect of K(Ca)(+) blockade with tetraethylammonium (TEA, 20 mg/kg iv) on coronary vasomotor tone was assessed in the absence and presence of K(ATP)(+) channel blockade with glibenclamide (3 mg/kg iv) in chronically instrumented swine at rest and during treadmill exercise. During exercise, myocardial O(2) delivery increased commensurately with the increase in myocardial O(2) consumption, so that myocardial O(2) extraction and coronary venous Po(2) (Pcv(O(2))) were maintained constant. TEA (in a dose that had no effect on K(ATP)(+) channels) had a small effect on the myocardial O(2) balance at rest and blunted the exercise-induced increase in myocardial O(2) delivery, resulting in a progressive decrease of Pcv(O(2)) with increasing exercise intensity. Conversely, at rest glibenclamide caused a marked decrease in Pcv(O(2)) that waned at higher exercise levels. Combined K(Ca)(+) and K(ATP)(+) channel blockade resulted in coronary vasoconstriction at rest that was similar to that caused by glibenclamide alone and that was maintained during exercise, suggesting that K(Ca)(+) and K(ATP)(+) channels act in a linear additive fashion. In conclusion, K(Ca)(+) channel activation contributes to the metabolic coronary vasodilation that occurs during exercise. Furthermore, in swine K(Ca)(+) and K(ATP)(+) channels contribute to coronary resistance vessel control in a linear additive fashion.     

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.     

H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice

Our previous study showed that arteriolar tone is enhanced in Type 2 diabetes mellitus (T2-DM) due to an increased level of constrictor prostaglandins. We hypothesized that, in mice with T2-DM, hydrogen peroxide (H(2)O(2)) is involved in the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles ( approximately 100 microm in diameter) of mice with T2-DM (C57BL/KsJ-db(-)/db(-)) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg) than that of control vessels (at 80 mmHg, control: 25 +/- 5%; db/db: 34 +/- 4%, P < 0.05), which was reduced back to control level by catalase (db/db: 24 +/- 4%). Correspondingly, in carotid arteries of db/db mice, the level of dichlorofluorescein-detectable and catalase-sensitive H(2)O(2) was significantly greater. In control arterioles, exogenous H(2)O(2) (0.1-100 micromol/l) elicited dilations (maximum, 58 +/- 10%), whereas in arterioles of db/db mice H(2)O(2) caused constrictions (-28 +/- 8%), which were converted to dilations (maximum, 16 +/- 5%) by the thromboxane A(2)/prostaglandin H(2) (TP) receptor antagonist SQ-29548. In addition, arteriolar constrictions in response to the TP receptor agonist U-46619 were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H(2)O(2)-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus we propose that, in mice with T2-DM, arteriolar production of H(2)O(2) is enhanced, which leads to increased synthesis of the constrictor prostaglandins thromboxane A(2)/prostaglandin H(2) in the smooth muscle cells, which enhance basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in Type 2 diabetes mellitus.     

Effects of exercise training and hypercholesterolemia on adenosine activation of voltage-dependent K+ channels in coronary arterioles

Coronary arterioles from hypercholesterolemic swine display attenuated adenosine-mediated vasodilatation that is attributable to the elimination of voltage-dependent K(+) (Kv) channel stimulation. For the present study, we tested the hypotheses that exercise training would correct impaired adenosine-induced dilatation in coronary arterioles from hypercholesterolemic pigs through restoration of adenosine activation of Kv channels and that vasodilatation to the receptor-independent adenylyl cyclase activator, forskolin, would also be attenuated in arterioles from hypercholesterolemic pigs. Pigs were randomly assigned to a control (NC) or high-fat, high-cholesterol (HC) diet for 20 wk. Four weeks after the diet was initiated, pigs from both groups were assigned to exercise training (Ex; 5 days/wk for 16 wk) or sedentary (Sed) protocols, resulting in four groups of pigs: NC-Sed, NC-Ex, HC-Sed, and HC-Ex. Arterioles ( approximately 150 mum) from both HC-Sed and HC-Ex pigs displayed impaired adenosine-mediated dilatation that was attributable to the elimination of 4-aminopyridine (4-AP; 1 mM)-sensitive Kv channel activation compared with NC counterparts. Arteriolar smooth muscle whole cell Kv currents were significantly reduced in HC-Sed compared with NC-Sed, although HC-Ex and NC-Ex did not differ. Forskolin-mediated dilatation was attenuated by 4-AP (1 mM) and in a concentration-dependent manner by tetraethylammonium (TEA; 0.1-1 mM) in NC-Sed but not HC-Sed. Further, TEA-sensitive Kv currents were diminished in cells of HC-Sed compared with NC-Sed pigs. Quantitative RT-PCR revealed similar expression levels of Kv3.1 and 3.3 in arterioles of NC-Sed and HC-Sed swine with undetectable expression of Kv1.1, 3.2, and 3.4. Taken together, these results suggest that hypercholesterolemia-mediated attenuation of adenosine-induced vasodilatation in coronary arterioles is not corrected by exercise training and is likely attributable to an impairment in the pathway coupling adenylyl cyclase with a highly TEA-sensitive Kv channel isoform(s).     

C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K(+) channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K(+) channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nonprotein contaminant. Sodium azide (NaN(3), 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN(3) elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K(+) channels. NaN(3)-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone. Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN(3) and NaN(3)-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN(3) (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone.     

Potassium channels modulate cerebral autoregulation during acute hypertension

We tested the hypothesis that constriction of cerebral arterioles during acute increases in blood pressure is attenuated by activation of potassium (K(+)) channels. We tested the effects of inhibitors of calcium-dependent K(+) channels [iberiotoxin (50 nM) and tetraethylammonium (TEA, 1 mM)] on changes in arteriolar diameter during acute hypertension. Diameter of cerebral arterioles (baseline diameter = 46 +/- 2 microm, mean +/- SE) was measured using a cranial window in anesthetized rats. Arterial pressure was increased from a control value of 96 +/- 1 mmHg to 130, 150, 170, and 200 mmHg by intravenous infusion of phenylephrine. Increases in arterial pressure from baseline to 130 and 150 mmHg decreased the diameter of cerebral arterioles by 5-10%. Greater increases in arterial pressure produced large increases in arteriolar diameter (i.e., "breakthrough of autoregulation"). Iberiotoxin or TEA inhibited increases in arteriolar diameter when arterial pressure was increased to 170 and 200 mmHg. The change in arteriolar diameter at 200 mmHg was 20 +/- 3% and -1 +/- 4% in the absence and presence of iberiotoxin, respectively. These findings suggest that calcium-dependent K(+) channels attenuate cerebral microvascular constriction during acute increases in arterial pressure, and that increases in arteriolar diameter at high levels of arterial pressure are not simply a passive phenomenon.