The heart is uniquely responsible for providing its own blood supply through the coronary circulation. Regulation of coronary blood flow is quite complex and, after over 100 years of dedicated research, is understood to be dictated through multiple mechanisms that include extravascular compressive forces (tissue pressure), coronary perfusion pressure, myogenic, local metabolic, endothelial as well as neural and hormonal influences. While each of these determinants can have profound influence over myocardial perfusion, largely through effects on end-effector ion channels, these mechanisms collectively modulate coronary vascular resistance and act to ensure that the myocardial requirements for oxygen and substrates are adequately provided by the coronary circulation. The purpose of this series of Comprehensive Physiology is to highlight current knowledge regarding the physiologic regulation of coronary blood flow, with emphasis on functional anatomy and the interplay between the physical and biological determinants of myocardial oxygen delivery.

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.

Estrogens and xenoestrogens have non-genomic effects mediated by plasma membrane receptors unrelated to the nuclear estrogen receptor (1). One of these is to increase the NP o of BK 1 channels (2), Ca 2ϩ -sensitive members of the voltage-gated K ϩ channel superfamily with important functions in many cells (3). BK channels are composed of ␣ and ␤ subunits. The ␣ subunit forms the K ϩ -selective pore, while ␤ subunits influence the pharmacology, kinetics, and voltage/Ca 2ϩ -sensitivity of BK channels. The ␤1 subunit of smooth muscle BK channels is physiologically important because knockout mice lacking this subunit are hypertensive and demonstrate altered vascular reactivity (4, 5). Recent studies suggest that BK channels are potential targets for 17␤E and xenoestrogens. BK channel NP o is increased by 17␤E, an effect that requires the regulatory ␤1 subunit (2). 17␤E and xenoestrogens reduce coronary vascular tone by inhibiting L-type Ca 2ϩ channels and activating BK channels (6). The pharmacological nature of the putative 17␤E-binding site on the smooth muscle BK ␤1 subunit is unknown. The xenoestrogen Tx, a commonly used chemotherapeutic agent, is an antagonist of the nuclear estrogen receptor (7). It is not known, however, if this clinically important drug increases BK channel NP o . We investigated whether Tx increases BK channel NP o in smooth muscle cells and whether the ␤1 subunit is important for this effect. These findings give insight into BK channel structure and function, non-genomic regulation by xenoestrogens, and Tx-induced side effects. MATERIALS AND METHODSCell Isolation and Preparation-Smooth muscle cells were isolated by enzymatic dispersion described previously (8). Dogs were anesthetized with ketamine, and the colon was removed via a midline incision. Mice were anesthetized with chloroform and killed by cervical dislocation. Human tissue samples were obtained from consenting patients undergoing gastric bypass for the treatment of morbid obesity. Circular muscle of the canine colon and human jejunum was dissected free of mucosa, submucosa, and longitudinal muscle in Ca 2ϩ -free Hanks solution. Strips of muscle were treated with collagenase (345 units/ml; Worthington Biochemical Corp.; Freehold, NJ) in Ca 2ϩ -free Hanks at 37°C to produce suspensions of single cells by gentle stirring. Mouse colon (circular and longitudinal muscle layers) was dissected free of mucosa prior to enzymatic dispersion. Mouse aorta and canine mesenteric vein were enzymatically digested without further dissection.HEK293 cells (ATCC cell line number CRL-1573; Manassas, VA) were grown in glutamax-supplemented RPMI medium (Life Technologies, Inc., Manassas, VA) with 10% heat-inactivated horse serum (Summit Biotechnology; Fort Collins, CO) in a humidified atmosphere with 5% CO 2 at 37°C. cDNA encoding the ␣ and ␤1 subunits from canine colonic smooth was cloned into the pZEOSV mammalian expression vector (Invitrogen; Carlsbad, CA) as described previously (9). Cells were transiently transfected via electroporation with a tot...

Hydrogen peroxide (H(2)O(2)) is a proposed endothelium-derived hyperpolarizing factor and metabolic vasodilator of the coronary circulation, but its mechanisms of action on vascular smooth muscle remain unclear. Voltage-dependent K(+) (K(V)) channels sensitive to 4-aminopyridine (4-AP) contain redox-sensitive thiol groups and may mediate coronary vasodilation to H(2)O(2). This hypothesis was tested by studying the effect of H(2)O(2) on coronary blood flow, isometric tension of arteries, and arteriolar diameter in the presence of K(+) channel antagonists. Infusing H(2)O(2) into the left anterior descending artery of anesthetized dogs increased coronary blood flow in a dose-dependent manner. H(2)O(2) relaxed left circumflex rings contracted with 1 muM U46619, a thromboxane A(2) mimetic, and dilated coronary arterioles pressurized to 60 cmH(2)O. Denuding the endothelium of coronary arteries and arterioles did not affect the ability of H(2)O(2) to cause vasodilation, suggesting a direct smooth muscle mechanism. Arterial and arteriolar relaxation by H(2)O(2) was reversed by 1 mM dithiothreitol, a thiol reductant. H(2)O(2)-induced relaxation was abolished in rings contracted with 60 mM K(+) and by 10 mM tetraethylammonium, a nonselective inhibitor of K(+) channels, and 3 mM 4-AP. Dilation of arterioles by H(2)O(2) was antagonized by 0.3 mM 4-AP but not 100 nM iberiotoxin, an inhibitor of Ca(2+)-activated K(+) channels. H(2)O(2)-induced increases in coronary blood flow were abolished by 3 mM 4-AP. Our data indicate H(2)O(2) increases coronary blood flow by acting directly on vascular smooth muscle. Furthermore, we suggest 4-AP-sensitive K(+) channels, or regulating proteins, serve as redox-sensitive elements controlling coronary blood flow.

Objective-We tested the hypothesis that hydrogen peroxide (H 2 O 2 ), the dismutated product of superoxide (O 2 ⅐Ϫ ), couples myocardial oxygen consumption to coronary blood flow. Accordingly, we measured O 2 ⅐Ϫ and H 2 O 2 production by isolated cardiac myocytes, determined the role of mitochondrial electron transport in the production of these species, and determined the vasoactive properties of the produced H 2 O 2 . Methods and Results-The production of O 2⅐Ϫ is coupled to oxidative metabolism because inhibition of complex I (rotenone) or III (antimycin) enhanced the production of O 2 ⅐Ϫ during pacing by about 50% and 400%, respectively; whereas uncoupling oxidative phosphorylation by decreasing the protonmotive force with carbonylcyanide-ptrifluoromethoxyphenyl-hydrazone (FCCP) decreased pacing-induced O 2 ⅐Ϫ production. The inhibitor of cytosolic NAD(P)H oxidase assembly, apocynin, did not affect O 2 ⅐Ϫ production by pacing. Aliquots of buffer from paced myocytes produced vasodilation of isolated arterioles (peak response 67Ϯ8% percent of maximal dilation) that was significantly reduced by catalase (5Ϯ0.5%, PϽ0.05) or the antagonist of Kv channels, 4-aminopyridine (18Ϯ4%, PϽ0.05). In intact animals, tissue concentrations of H 2 O 2 are proportionate to myocardial oxygen consumption and directly correlated to coronary blood flow. Intracoronary infusion of catalase reduced tissue levels of H 2 O 2 by 30%, and reduced coronary flow by 26%. Intracoronary administration of 4-aminopyridine also shifted the relationship between myocardial oxygen consumption and coronary blood flow or coronary sinus pO 2 . Conclusions-Taken together, our results demonstrate that O 2⅐Ϫ is produced in proportion to cardiac metabolism, which leads to the production of the vasoactive reactive oxygen species, H 2 O 2 . Our results further suggest that the production of H 2 O 2 in proportion to metabolism couples coronary blood flow to myocardial oxygen consumption. Key Words: reactive oxygen species Ⅲ coronary circulation Ⅲ vasodilation Ⅲ microcirculation T he coupling of blood flow to metabolism is the most important vasomotor adjustment for the regulation of oxygen delivery to metabolically active organ systems. This matching, termed metabolic dilation, or metabolic or active hyperemia, is critical to ensure adequate oxygen delivery for aerobic metabolism and adequate organ function. 1 Although the factor or factors responsible for the coupling of flow to metabolism have been actively pursued for decades, no metabolite has been casually linked to the process of metabolic hyperemia or has withstood critical evaluation. 1-3 Most investigations have pursued the idea that the metabolic regulation of flow is a negative feedback pathway, in which an imbalance between oxygen supply (delivered via flow) and oxygen demands, ie, demands exceed supply, results in the production of a metabolic dilator. The adenosine hypothesis was such a scheme, in which oxygen demands, in excess of supply would increase the production of adenosine through hydro...

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.

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...