H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation.

RATIONALE: Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. OBJECTIVE: Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization. METHODS AND RESULTS: Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches. CONCLUSIONS: H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK(Ca) channels.

Epicardial perivascular adipose-derived leptin exacerbates coronary endothelial dysfunction in metabolic syndrome via a protein kinase C-beta pathway.

OBJECTIVE: Factors released by perivascular adipose tissue (PVAT) disrupt coronary endothelial function via phosphorylation of endothelial NO synthase by protein kinase C (PKC)-beta. However, our understanding of how PVAT potentially contributes to coronary disease as a complication of obesity/metabolic syndrome (MetS) remains limited. The current study investigated whether PVAT-derived leptin impairs coronary vascular function via PKC-beta in MetS. METHODS AND RESULTS: Coronary arteries with and without PVAT were collected from lean or MetS Ossabaw miniature swine for isometric tension studies. Endothelial-dependent vasodilation to bradykinin was significantly reduced in MetS. PVAT did not affect bradykinin-mediated dilation in arteries from lean swine but significantly exacerbated endothelial dysfunction in arteries from MetS swine. PVAT-induced impairment was reversed by inhibition of either PKC-beta with ruboxistaurin (Eli Lilly and Company, Indianapolis, Ind) or leptin receptor signaling with a recombinant, pegylated leptin antagonist. Western blot and immunohistochemical analyses demonstrated increased PVAT-derived leptin and coronary leptin receptor density with MetS. Coronary PKC-beta activity was increased in both MetS arteries exposed to PVAT and lean arteries exposed to leptin. Finally, leptin-induced endothelial dysfunction was reversed by ruboxistaurin. CONCLUSIONS: Increases in epicardial PVAT leptin exacerbate coronary endothelial dysfunction in MetS via a PKC-beta-dependent pathway. These findings implicate PVAT-derived leptin as a potential contributor to coronary atherogenesis in MetS.

Metabolic syndrome reduces the contribution of K+ channels to ischemic coronary vasodilation.

This investigation tested the hypothesis that metabolic syndrome decreases the relative contribution of specific K(+) channels to coronary reactive hyperemia. Ca(2+)-activated (BK(Ca)), voltage-activated (K(V)), and ATP-dependent (K(ATP)) K(+) channels were investigated. Studies were conducted in anesthetized miniature Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) for 20 wk. The latter diet induces metabolic syndrome, increasing body weight, fasting glucose, total cholesterol, and triglyceride levels. Ischemic vasodilation was determined by the coronary flow response to a 15-s occlusion before and after cumulative administration of antagonists for BK(Ca) (penitrem A; 10 microg/kg iv), K(V) (4-aminopyridine; 0.3 mg/kg iv) and K(ATP) (glibenclamide; 1 mg/kg iv) channels. Coronary reactive hyperemia was diminished by metabolic syndrome as the repayment of flow debt was reduced approximately 30% compared with lean swine. Inhibition of BK(Ca) channels had no effect on reactive hyperemia in either lean or metabolic syndrome swine. Subsequent inhibition of K(V) channels significantly reduced the repayment of flow debt ( approximately 25%) in both lean and metabolic syndrome swine. Additional blockade of K(ATP) channels further diminished ( approximately 45%) the repayment of flow debt in lean but not metabolic syndrome swine. These data indicate that the metabolic syndrome impairs coronary vasodilation in response to cardiac ischemia via reductions in the contribution of K(+) channels to reactive hyperemia.

Contribution of BK(Ca) channels to local metabolic coronary vasodilation: Effects of metabolic syndrome.

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MVo(2)). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BK(Ca) channels with penitrem A (10 microg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po(2) and MVo(2) (P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MVo(2) was increased with exercise (P < 0.005). Inhibition of BK(Ca) channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po(2) and MVo(2) in lean or MetS swine. These data indicate that BK(Ca) channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BK(Ca) channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.

Impaired function of coronary BK(Ca) channels in metabolic syndrome.

The role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3-6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK(Ca) opener NS-1619 in vivo (30-100 microg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1-100 microM). MetS reduced whole cell penitrem A (1 microM)-sensitive K(+) current and NS-1619-activated (10 microM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca(2+) and augmented coronary vasoconstriction to the L-type Ca(2+) channel agonist BAY K 8644 (10 pM-10 nM). BK(Ca) channel alpha and beta(1) protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK(Ca) channel function and is accompanied by significant increases in L-type Ca(2+) channel-mediated coronary vasoconstriction.

Periadventitial adipose tissue impairs coronary endothelial function via PKC-beta-dependent phosphorylation of nitric oxide synthase.

Endogenous periadventitial adipose-derived factors have been shown to contribute to coronary vascular regulation by impairing endothelial function through a direct inhibition of endothelial nitric oxide synthase (eNOS). However, our understanding of the underlying mechanisms remains uncertain. Accordingly, this study was designed to test the hypothesis that periadventitial adipose tissue releases agents that attenuate coronary endothelial nitric oxide production via a protein kinase C (PKC)-beta-dependent mechanism. Isometric tension studies were conducted on isolated canine circumflex coronary arteries with and without natural amounts of periadventitial adipose tissue. Adipose tissue significantly diminished coronary endothelial-dependent vasodilation and nitric oxide production in response to bradykinin and acetylcholine. The selective inhibition of endothelial PKC-beta with ruboxistaurin (1 microM) abolished the adipose-induced impairment of bradykinin-mediated coronary vasodilation and the endothelial production of nitric oxide. Western blot analysis revealed a significant increase in eNOS phosphorylation at the inhibitory residue Thr(495) in arteries exposed to periadventitial adipose tissue. This site-specific phosphorylation of eNOS was prevented by the inhibition of PKC-beta. These data demonstrate that periadventitial adipose-derived factors impair coronary endothelial nitric oxide production via a PKC-beta-dependent, site-specific phosphorylation of eNOS at Thr(495).

Altered mechanism of adenosine-induced coronary arteriolar dilation in early-stage metabolic syndrome.

Onset of the combined metabolic syndrome (MetS) is a complex progressive process involving numerous cardiovascular risk factors. Although patients with established MetS exhibit reduced coronary flow reserve and individual components of the MetS reduce microvascular vasodilation, little is known concerning the impact of early-stage MetS on the mechanisms of coronary flow control. Therefore, we tested the hypothesis that coronary arteriolar dilation to adenosine is attenuated in early-stage MetS by reduced A2 receptor function and diminished K+ channel involvement. Pigs were fed control or high-fat/cholesterol diet for 9 weeks to induce early-stage MetS. Coronary atheroma was determined in vivo with intravascular ultrasound. In vivo coronary dilation was determined by intracoronary adenosine infusion. Further, apical coronary arterioles were isolated, cannulated and pressurized to 60 cmH2O for in vitro pharmacologic assessment of adenosine dilation. Coronary atheroma was not different between groups, indicating early-stage MetS. Coronary arteriolar dilation to adenosine (in vivo) and 2-chloroadenosine (2-CAD; in vitro) was similar between groups. In control arterioles, 2-CAD-mediated dilation was reduced only by selective A(2A) receptor inhibition, whereas only dual A(2A/2B) inhibition reduced this response in MetS arterioles. Arteriolar A(2B), but not A(2A), receptor protein expression was reduced by MetS. Blockade of voltage-dependent K+ (K(v)) channels reduced arteriolar sensitivity to 2-CAD in both groups, whereas ATP-sensitive K+ (K(ATP)) channel inhibition reduced sensitivity only in control arterioles. Our data indicate that the mechanisms mediating coronary arteriolar dilation to adenosine are altered in early-stage MetS prior to overt decrements in coronary vasodilator reserve.

Leptin and mechanisms of endothelial dysfunction and cardiovascular disease.

Leptin, a product of the obesity gene, is a molecule that has received much attention since its cloning in 1994. Initially, most work centered around the effects of leptin on satiety and energy balance. However, in recent years there has been an intense focus on leptin as it relates to the cardiovascular system. Plasma leptin concentration is markedly elevated in obesity and the metabolic syndrome, both of which are associated with increased incidence of cardiovascular pathologies. In many studies, hyperleptinemia has been linked to endothelial dysfunction (a known precursor to atherosclerotic cardiovascular disease) and activation of the sympathetic nervous system. Additionally, recent evidence suggests that leptin released from perivascular adipose tissue may also have deleterious effects on the underlying vasculature, including the coronary circulation. This report reviews pertinent literature on leptin-mediated endothelial dysfunction, leptin-mediated sympathetic activation, and leptin as a significant perivascular adipose-derived factor.

Endogenous adipose-derived factors diminish coronary endothelial function via inhibition of nitric oxide synthase.

Adipocytokines may be the molecular link between obesity and vascular disease. However, the effects of these factors on coronary vascular function have not been discerned. Accordingly, the goal of this investigation was to delineate the mechanisms by which endogenous adipose-derived factors affect coronary vascular endothelial function. Both isolated canine coronary arteries and coronary blood flow in anesthetized dogs were studied with and without exposure to adipose tissue. Infusion of adipose-conditioned buffer directly into the coronary circulation did not change baseline hemodynamics; however, endothelial-dependent vasodilation to bradykinin was impaired both in vitro and in vivo. Coronary vasodilation to sodium nitroprusside was unaltered by adipose tissue. Oxygen radical formation did not cause the impairment because quantified dihydroethidium staining was decreased by adipose tissue and neither a superoxide dismutase mimetic nor catalase improved endothelial function. Inhibition of nitric oxide (NO) synthase with L-NAME diminished bradykinin-mediated relaxations and eliminated the subsequent vascular effects of adipose tissue. In vitro measurement of NO demonstrated that adipose tissue exposure quickly lowered baseline NO and abolished bradykinin-induced NO production. The results indicate that adipose tissue releases factor(s) that selectively impair endothelial-dependent dilation via inhibition of NO synthase-mediated NO production.

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.