Objective Factors released by perivascular adipose tissue (PVAT) disrupt coronary endothelial function via phosphorylation of eNOS by PKC-β. 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-β 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-β with ruboxistaurin or leptin receptor signaling with a recombinant, pegylated leptin antagonist. Western and immunohistochemical analysis demonstrated increased PVAT-derived leptin and coronary leptin receptor (ObR) density with MetS. Coronary PKC-β 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-β-dependent pathway. These findings implicate PVAT-derived leptin as a potential contributor to coronary atherogenesis in MetS.

Rationale Hydrogen peroxide (H2O2) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H2O2 elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H2O2 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 H2O2 dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca2+-activated K+ (BKCa) channel and subsequent smooth muscle hyperpolarization. Methods and Results Flow and H2O2 induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BKCa channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H2O2-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H2O2 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 H2O2 into the bath solution significantly increased the probability of BKCa single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H2O2 exhibited an attenuated stimulatory effect on BKCa channel open probability in inside-out membrane patches. Conclusions H2O2 dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BKCa channels.

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.

ϩ (KV) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H2O2, as responses were attenuated by the KV channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that KV channels participate in coronary reactive hyperemia and examined the role of KV 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 Ϯ 5% and inhibited hyperemic volume 32 Ϯ 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 Ϯ 6% block at 9 g/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 M correolide, a selective KV1 antagonist (76 Ϯ 7% and 47 Ϯ 2% block, respectively, at 1 M). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 Ϯ 6% block at 10 g/min) and by correolide in vitro (29 Ϯ 4% block at 1 M). KV current in smooth muscle cells was inhibited by 4-AP (IC50 1.1 Ϯ 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for KV1 family members was detected in coronary arteries. Our data indicate that KV channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine-and NO-induced vasodilation. ischemic vasodilation; adenosine; 4-aminopyridine; delayed rectifier potassium channel; vascular smooth muscle IN THE CORONARY CIRCULATION, a brief period of ischemia is normally followed by a large and transient compensatory increase in blood flow. This phenomenon of reactive hyperemia, different from active (also known as functional or metabolic) hyperemia, is thought to represent a repayment of blood flow debt and is attributed to the accumulation of ischemic vasodilator metabolites. Evidence supports both adenosine and nitric oxide (NO) as mediators of reactive hyperemia (2, 4, 12, 52). Importantly, however, neither block of adenosine nor NO signaling can completely abolish reactive hyperemia (56). Thus the mechanisms of reactive hyperemia remain incompletely understood. Moreover, other mediators have been suggested, and it is likely that future studies will identify additional candidates. Rather than focus on putative metabolites underlying reactive hyperemia, we have turned our attention to possible end-effectors in vascular smooth muscle. K ϩ channels are likely targets of vasodilator metabolites, because K ϩ channels determine membrane potential and thus vascular tone (27,35). Previous studies have focused on Ca 2ϩ /voltage-sensitive (BK Ca ) and ATP-dependent (K ATP ) K ϩ channels. To date, only one study suggests a role for BK C...

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).