Bradykinin Mediation of Ca2+-Activated K+ Channels Regulates Coronary Blood Flow in Ischemic Myocardium
Background Endothelium-dependent hyperpolarizing factor relaxes vascular smooth muscles by opening the Ca2+-activated K+ (KCa) channels. The role of the opening of KCa channels in coronary vasodilation during myocardial ischemia was investigated.
Methods and Results The left anterior descending coronary arteries of open-chest dogs were perfused with blood through an extracorporeal bypass tube from the carotid artery. Intracoronary administration of bradykinin increased coronary blood flow (CBF) in dogs treated with NG-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase; this effect was completely inhibited by the KCa channel blocker iberiotoxin. In dogs treated with L-NAME, the bypass tube was occluded to reduce CBF to one third of the baseline value, after which coronary perfusion pressure was maintained constant. Intracoronary administration of iberiotoxin for 20 minutes further decreased CBF (from 33±2 to 19±2 mL·100 g−1·min−1, P<.01), fractional shortening, and lactate extraction ratio during coronary hypoperfusion. Bradykinin was released, and the bradykinin receptor antagonist HOE-140 blocked the effects of iberiotoxin on coronary hemodynamic and metabolic parameters during myocardial ischemia. Although the combination of L-NAME and the adenosine receptor antagonist 8-sulfophenyltheophylline reduced reactive hyperemic flow after 20 seconds of coronary occlusion, the additional presence of iberiotoxin resulted in a further decrease in this parameter.
Conclusions The opening of KCa channels in response to endogenous bradykinin contributed to coronary vasodilation and reduced contractile and metabolic dysfunction during myocardial ischemia in open-chest dogs.
Vascular endothelial cells not only produce vasoconstrictive substances such as endothelin but also mediate the endothelium-dependent relaxation of smooth muscle.1 Endothelium-mediated vasorelaxation is partially attributable to PGI2 and to NO,1 which increases the concentration of cGMP in smooth muscle cells by stimulating soluble guanylate cyclase.1 However, other agents may contribute to the endothelium-dependent relaxation of coronary arteries.2 3 One candidate is EDHF, which induces membrane hyperpolarization and subsequent relaxation of vascular smooth muscle.4 5 Bradykinin has been shown to be released in the ischemic heart in vivo6 and to induce coronary vasodilation mediated by EDHF,7 NO, and PGI2. The EDHF-induced hyperpolarization of vascular smooth muscle cells is mediated by activation of K+ channels and the consequent increase in the K+ conductance of the cell membrane.4 5 Endothelium-dependent hyperpolarization and subsequent vascular relaxation are inhibited by blockers of KCa channels in coronary arteries,5 suggesting that the activation of KCa channels plays an important role in coronary vasodilation in the ischemic myocardium.
We have investigated whether the opening of KCa channels (1) reduces coronary vascular resistance and improves myocardial contractile and metabolic function during coronary hypoperfusion and (2) contributes to the extent of reactive hyperemia after a brief period of myocardial ischemia in open-chest dogs in the canine heart. We also investigated the role of bradykinin in the opening of KCa channels in the ischemic canine heart.
Mongrel dogs (body mass, 16 to 24 kg) were anesthetized with sodium pentobarbital (30 mg/kg body mass IV), intubated, and ventilated with room air mixed with oxygen (100% O2 at a flow rate of 1.0 to 1.5 L/min). The chest was opened through the left fifth intercostal space, and the heart was suspended in a pericardial cradle. After administration of heparin (500 U/kg IV), we cannulated the LAD and perfused it with blood from the left carotid artery through an extracorporeal bypass tube. CBF in the perfused area was measured with an electromagnetic flow probe attached to the bypass tube, and CPP was monitored at the tip of the coronary artery cannula. A small-caliber (1-mm), short (70-mm) collecting tube was introduced into a small coronary vein near the center of the perfused area to sample coronary venous blood. Drained venous blood was collected in a reservoir placed at the level of the left atrium and was returned to the jugular vein. The left atrium was catheterized for microsphere injection. Hydration was maintained by slow infusion of normal saline. LVP was measured with a high-fidelity micromanometer (model p-5, Konigsberg) placed in the left ventricular cavity through the apex. A pair of ultrasonic crystals was placed in the inner one third of the myocardium ≈10 mm apart to measure myocardial segment length with an ultrasonic dimension gauge (5 MHz, 2-mm diameter; Schuessler). Hemodynamic parameters were recorded on a multichannel recorder (Rm-6000, Nihon Kohden). EDL was determined at the R wave of the ECG, and ESL was determined at the minimal dP/dt. We calculated FS from the equation FS=[(EDL−ESL)/EDL]×100%.
Effects of KCa Channel Blockers on Myocardial Hemodynamic and Metabolic Parameters in the Nonischemic Myocardium
To examine whether the opening of KCa channels regulates coronary vascular tone in the nonischemic heart, we administered IBTX (1 μg·kg−1·min−1; n=7; Research Biochemicals), a selective inhibitor of high-conductance KCa channels; CTX (1 μg·kg−1·min−1; n=8; Research Biochemicals), also an inhibitor of high-conductance KCa channels; TEA (10 μg·kg−1·min−1, n=8; Research Biochemicals), an inhibitor of intermediate-type KCa channels; or saline (the control group; n=8) into the LAD for 20 minutes at an infusion rate of 0.0167 mL·kg−1·min−1. After hemodynamic stabilization, coronary arterial and venous blood was sampled for blood gas analysis and the determination of plasma lactate and norepinephrine concentrations. Hemodynamic parameters (LVP, dP/dt, and FS) were also measured. Samples and measurements were taken before, 20 minutes after the onset of, and 20 minutes after the discontinuation of infusion of IBTX, CTX, TEA, or saline.
Effects of KCa Channel Blockers on Myocardial Hemodynamic and Metabolic Parameters in the Ischemic Myocardium
After hemodynamic stabilization, CPP was reduced until the CBF decreased to one third of the control value with the use of an occluder attached to the extracorporeal bypass tube. To exclude the coronary vasodilatory effect of NO, we administered L-NAME (10 μg·kg−1·min−1, at an infusion rate of 0.0167 mL·kg−1·min−1), an inhibitor of NO synthase, into the LAD 10 minutes before reduction of CBF. After the low CPP was set, the occluder was adjusted precisely to maintain a constant CPP. Hemodynamic parameters were measured 5 and 10 minutes after the onset of hypoperfusion. The serum concentrations of norepinephrine and lactate were determined, and blood gas analysis was performed with coronary arterial and venous blood sampled at 10 minutes. After these measurements were obtained, IBTX (n=7), CTX (n=7), TEA (n=7), or saline (n=8; the control group) was infused into the LAD, and the hemodynamic and metabolic parameters were again measured. Infusion of each inhibitor was discontinued after 20 minutes, and the hemodynamic and metabolic parameters were measured after stabilization of the hemodynamic parameters. In other dogs, microspheres were injected into the left atrium before and during coronary hypoperfusion and infusion of KCa channel blockers or saline. Endocardial and epicardial tissue was also sampled in these dogs to assess the endocardial to epicardial flow ratio. We measured the concentration of end products of NO metabolism (nitrate and nitrite) in coronary arterial and venous blood before and during the reduction of CPP and with and without L-NAME and KCa channel blockers. To evaluate the dose dependency of the effects of KCa channel blockers on CBF, we administered IBTX and CTX at doses of 0.2, 0.4, 1, and 5 μg·kg−1·min−1 each and TEA at doses of 2, 4, 10, and 50 μg·kg−1·min−1 into the LAD during coronary hypoperfusion.
Roles of Endogenous Bradykinin, Adenosine, PGI2, and the Opening of KATP Channels in the Effects of KCa Channel Blockers on CBF During Coronary Hypoperfusion
To investigate the roles of bradykinin, adenosine, PGI2, and the opening of KATP channels in the effects of KCa channel blockers on CBF during coronary hypoperfusion, we infused IBTX, together with the selective B2 bradykinin receptor antagonist HOE-140 (0.5 ng·kg−1·min−1; n=6; Sigma Chemical Co), the adenosine receptor antagonist 8-SPT (25 μg·kg−1·min−1; n=6; Research Biochemicals), the cyclooxygenase inhibitor indomethacin (10 μg·kg−1·min−1; n=6; Sigma), or the KATP channel blocker glibenclamide (5 μg·kg−1·min−1; n=6; Sigma) into the LAD at a rate of 0.0617 mL·kg−1·min−1 during coronary hypoperfusion. Glibenclamide was dissolved in 1.5 mL dimethyl sulfoxide, to which 30 mEq of NaHCO3 was added after dilution to 1 L with normal saline. Glibenclamide (0.30 mg/mL) was infused into the perfusion line at a rate of 0.0167 mL/min. At the indicated dose, glibenclamide abolished the coronary vasodilatory effect of the KATP channel opener cromakalim (CBF during intracoronary infusion of cromakalim [0.2 μg·kg−1·min−1] with and without glibenclamide: 117±3 and 271±3 mL·100 g−1·min−1, respectively, from a baseline of 93±3 mL·100 g−1·min−1; n=5; P<.01). Administration of HOE-140, 8-SPT, indomethacin, or glibenclamide was initiated 10 minutes before coronary hypoperfusion. Inhibition of cyclooxygenase was demonstrated by prevention of the coronary dilatory effect of arachidonic acid (600 mg IC).
Effects of KCa Channel Blockers on Bradykinin-Induced Coronary Vasodilation in the Nonischemic Heart
Because bradykinin induces hyperpolarization as a result of the opening of KCa channels, we investigated whether bradykinin increases CBF through the activation of KCa channels in nonischemic hearts. After hemodynamic stabilization, coronary arterial and venous blood was sampled for blood gas analysis and determination of the concentrations of lactate, end products of NO metabolism, and norepinephrine. Hemodynamic parameters (LVP, dP/dt, and FS) were also measured. Bradykinin (20 ng·kg−1·min−1 at an infusion rate of 0.0167 mL· kg−1·min−1; Sigma) was then infused into the LAD under conditions of intracoronary infusion of L-NAME (10 μg·kg−1·min−1) in the absence (n=9) or presence of IBTX (1 μg·kg−1·min−1; n=8), CTX (1 μg·kg−1·min−1; n=7), or TEA (10 μg·kg−1·min−1; n=7). In the control group (n=9), bradykinin was administered into the LAD in the absence of other treatment. In a preliminary study, we confirmed that these doses of IBTX, CTX, and TEA are the minimal effective doses for maximal inhibition of bradykinin-induced coronary vasodilation and that L-NAME 10 μg·kg−1·min−1 abolishes the increase in the concentration of end products of NO induced by infusion of bradykinin. Administration of these inhibitors was initiated 10 minutes before infusion of bradykinin and continued throughout the experimental protocol. All hemodynamic and metabolic parameters were measured 10 minutes after the onset of bradykinin infusion. Bradykinin infusion was then discontinued, and hemodynamic and metabolic parameters were measured after stabilization of the former.
Effects of KCa Channel Blockers on Bradykinin-Induced Coronary Vasodilation in the Ischemic Heart
To examine the role of the opening of KCa channels in bradykinin-induced coronary vasodilation during coronary hypoperfusion, we reduced CPP so that CBF decreased to one third of the control value. Hemodynamic parameters were measured 5, 10, and 20 minutes after the onset of hypoperfusion, and both coronary arterial and venous blood was sampled at 10 minutes. Bradykinin (20 ng·kg−1·min−1) was infused into the LAD during coronary hypoperfusion in the presence of L-NAME alone or L-NAME plus a KCa channel blocker. Bradykinin infusion was discontinued after 10 minutes, and hemodynamic and metabolic parameters were measured. We also measured the concentration of end products of NO metabolism in coronary arterial and venous blood before, during, and after infusion of bradykinin. The difference in nitrate plus nitrite concentration between coronary venous and arterial blood [ΔVa(NO)] reflects the amount of NO released from the myocardium.
In other dogs, we measured cGMP in epicardial coronary arteries in the ischemic heart. After the low CPP was maintained for 10 minutes and bradykinin infusion was maintained for 10 minutes in the presence of L-NAME alone or L-NAME plus a KCa channel blocker, the LAD (ischemic region) and left circumflex coronary artery (nonischemic control region) were removed rapidly with the use of precooled stainless steel scissors and tongs and were stored in liquid nitrogen. In five other dogs, CPP was reduced for 20 minutes, and the epicardial LAD (ischemic region) and left circumflex coronary artery were rapidly removed and stored in liquid nitrogen.
To examine the roles of adenosine, PGI2, and KATP channels in bradykinin-induced coronary vasodilation in the nonischemic and ischemic myocardium, we infused bradykinin (20 ng·kg−1·min−1) into the LAD under condition of intracoronary administration of 8-SPT (n=6), indomethacin (n=6), or glibenclamide (n=6) initiated 10 minutes before infusion of bradykinin.
Effects of KCa Channel Blockers on Reactive Hyperemia After a Brief Period of Ischemia
We investigated the effects of IBTX or TEA on the extent of reactive hyperemic flow after 20 seconds of myocardial ischemia. Saline (n=8), IBTX (n=7), or TEA (n=7) was administered 10 minutes before coronary occlusion. Coronary hemodynamic parameters were measured before and during reactive hyperemia. The duration of reactive hyperemia was defined as the time between the release of coronary occlusion and the return of CBF to the preocclusion value. Flow debt was defined as preocclusion baseline flow rate multiplied by the duration of occlusion, and flow repayment was defined as the area under the curve of flow versus time during reactive hyperemia minus the product of preocclusion baseline flow and the duration of reactive hyperemia.
To examine the role of KCa channels in the reactive hyperemia that appears to be partially attributable to adenosine, NO,8 9 KATP channels, and PGI2, we compared the effects of 8-SPT+L-NAME (n=7), L-NAME+8-SPT+IBTX (n=8), L-NAME+8-SPT+glibenclamide+indomethacin (n=5), and L-NAME+8-SPT+glibenclamide+indomethacin+IBTX (n=5) on the reactive hyperemia.
Regional myocardial blood flow was determined by the microsphere technique as previously described.10
Myocardial oxygen consumption (milliliters per 100 g per minute) was calculated as the product of CBF (milliliters per 100 g per minute) and coronary arteriovenous blood oxygen difference (milliliters per deciliters). Lactate was measured by enzymatic assay, and the LER was calculated as the coronary AV difference in lactate concentration multiplied by 100 and divided by the arterial lactate concentration. NO,11 bradykinin, cGMP, and norepinephrine12 were measured by use of the methods as described previously.
Data are presented as mean±SEM and were analyzed by ANOVA. Statistical significance of the differences was assessed by Bonferroni’s multiple comparison test. A value of P<.05 was considered statistically significant.
Effects of KCa Channel Blockers on Coronary Hemodynamic and Metabolic Function in the Nonischemic Myocardium
Systemic hemodynamic parameters were not affected by intracoronary infusion of the KCa channel blockers IBTX, CTX, or TEA under nonischemic conditions. Intracoronary infusion of IBTX, CTX, or TEA also had no effects on CPP or CBF (Fig 1A⇓) in the nonischemic myocardium. Furthermore, LER, the pH of coronary venous blood, and FS were not affected by IBTX, CTX, and TEA, respectively.
Effects of KCa Channel Blockers on Coronary Hemodynamic and Metabolic Function in the Ischemic Myocardium
Heart rate (142±5 bpm) and systolic (140±2 mm Hg) and diastolic (82±4 mm Hg) blood pressures were not affected by coronary hypoperfusion before or during the infusion of KCa channel blockers. Coronary hemodynamic and metabolic parameters did not differ significantly among the groups before the onset of coronary hypoperfusion. Ten minutes after reduction of CPP (102±2 to 42±2 mm Hg), CBF (91±2 to 31±1 mL·100 g−1·min−1), FS (23.5±1.7% to 4.7±1.3%), LER (24.3±2.3% to −40.6±2.1%), and the pH of the coronary venous blood (7.41±0.01 to 7.22±0.01) were all decreased. In dogs not pretreated with L-NAME, ΔVa(NO) increased from 3.6±0.4 to 14.7±1.7 μm (P<.01) 10 minutes after reduction of CPP; this increase was prevented by L-NAME treatment [ΔVa(NO), 2.4±0.7 μmol/L; P<.001 versus the untreated group]. After 20 minutes of infusion with IBTX, CTX, or TEA, CBF (Fig 1B⇑), FS (Fig 2A⇓), LER (Fig 2B⇓), and the pH of coronary venous blood (Fig 2C⇓) were all decreased, despite a constant CPP; the pH of coronary arterial blood remained unchanged throughout the experimental protocol (7.41±0.01). Thus, intracoronary infusion of KCa channel blockers reduced CBF and worsened metabolic and contractile function of the ischemic myocardium. These parameters returned to their baseline values after discontinuation of drug administration. The effects of the KCa channel blockers on CBF during coronary hypoperfusion were dose dependent; the effects of IBTX and CTX were maximal at 1 μg·kg−1·min−1 and those of TEA at 10 μg·kg−1·min−1 (Fig 3⇓). Although CPP was maintained constant, intracoronary infusion of KCa channel blockers significantly reduced the endocardial/epicardial flow ratio (untreated, 0.78±0.03, n=6; IBTX, 0.69±0.04, n=5; CTX, 0.71±0.03, n=5; TEA, 0.70±0.02, n=5; P<.05 versus the untreated group) in the ischemic myocardium, indicating that the opening of KCa channels preferentially increases endocardial flow. Norepinephrine release (coronary AV difference in norepinephrine multiplied by CBF) was not affected by L-NAME or by infusion of IBTX, CTX, or TEA during coronary hypoperfusion (con- trol, 3.02±1.23 ng·100 g−1·min−1; L-NAME, 2.94±1.01 ng·100 g−1·min−1; L-NAME+IBTX, 2.98±1.01 ng·100 g−1·min−1; L-NAME+CTX, 2.96±0.08 ng·100 g−1·min−1; and L-NAME+TEA, 2.92±0.06 ng·100 g−1·min−1). The difference in bradykinin concentration between coronary venous and arterial blood was 2.1±0.5 and 34±3.5 pg/mL under the nonischemic and ischemic conditions, respectively (P<.001).
The difference in bradykinin concentration between coronary venous and arterial blood was 2.1±0.5 and 34±3.5 pg/mL under the nonischemic and ischemic conditions, respectively (P<.001). Administration of HOE-140 prevented the decreases in CBF (28.6±1.4 versus 30.2±2.4 mL·100 g−1·min−1), LER (−49.3±3.1% versus −46.5±3.9%), FS, and the pH of coronary venous blood (data not shown) normally induced by IBTX during coronary hypoperfusion. In contrast, 8-SPT, indomethacin, and glibenclamide did not affect the IBTX-induced changes in hemodynamic and metabolic parameters during coronary hypoperfusion (data not shown).
Effects of KCa Channel Blockers on Bradykinin-Induced Coronary Vasodilation in the Nonischemic Myocardium
Neither systolic and diastolic blood pressures or heart rate differed significantly among the various experimental groups before, during, or after infusion of bradykinin. Similarly, neither L-NAME nor KCa channel blockers affected coronary hemodynamic and metabolic parameters before infusion of bradykinin. Both ΔVa(NO) (from 3.1±1.1 to 13.4±1.8 μmol/L, P<.001) and CBF (Fig 4A⇓) were increased significantly 10 minutes after the onset of bradykinin infusion. These parameters [ΔVa(NO), 3.9±0.8 μmol/L] returned to baseline values 10 minutes after discontinuation of bradykinin infusion. L-NAME prevented the bradykinin-induced increase in ΔVa(NO) [ΔVa(NO) 10 minutes after onset of bradykinin infusion, 2.2±0.4 μmol/L], and it reduced the bradykinin-induced increase in CBF by 55% (Fig 4A⇓). The combination of L-NAME and each KCa channel blocker completely prevented bradykinin-induced coronary vasodilation (Fig 4A⇓).
Effects of KCa Channel Blockers on Bradykinin-Induced Coronary Vasodilation in the Ischemic Myocardium
Baseline coronary hemodynamic and metabolic parameters did not differ significantly among the various experimental groups before the onset of coronary hypoperfusion. FS decreased (from 24.8±1.8% to 4.7±0.9%) within 1 minute of the reduction in CPP associated with coronary hypoperfusion and then remained constant for 10 minutes. CPP was consistently maintained at the value of its initial reduction. Both CBF (Fig 4B⇑) and FS (Fig 4C⇑) increased during the 10-minute infusion of bradykinin, effects that were inhibited partially by L-NAME alone and completely by the combination of L-NAME and a KCa channel blocker. The LER and pH of coronary venous blood decreased from 24.7±1.2% and 7.41±0.01 under the baseline condition to −58.6±3.3% and 7.22±0.01 during coronary hypoperfusion, respectively. Bradykinin increased both LER (−27.2±2.3%, P<.01 versus before infusion of bradykinin) and the pH of coronary venous blood (7.31±0.01, P<.01 versus before infusion of bradykinin), indicating reduced myocardial anaerobic metabolism in response to bradykinin infusion. The effects of bradykinin were partially inhibited by L-NAME (LER, −41.4±2.3%; pH of coronary venous blood, 7.26±0.01) and abolished by L-NAME combined with a KCa channel blocker (LER and pH of coronary venous blood: IBTX, −61.4±3.7% and 7.22±0.01; CTX, −56.7±3.1% and 7.23±0.01; and TEA, −57.3±2.1% and 7.21±0.01, respectively). Norepinephrine release in the baseline condition did not differ significantly from that before, during, or after administration of bradykinin (2.51±0.43, 1.32±0.25, 1.02±0.85, and 1.11±0.73 ng·100 g−1·min−1, respectively). Similarly, norepinephrine release in the ischemic myocardium during administration of bradykinin was not significantly affected by L-NAME in the absence or presence of KCa channel blockers (control, 2 .83±0.15 ng·100 g−1·min−1; L-NAME, 2.91±0.43 ng·100 g−1·min−1; L-NAME+IBTX, 2.88±0.62 ng·100 g−1·min−1; L-NAME+CTX, 2.72±0.32 ng·100 g−1·min−1; and L-NAME+TEA, 2.91±0.53 ng·100 g−1·min−1).
Myocardial ischemia (CPP, 101±3.2 to 45.1± 2.1 mm Hg; CBF, 89±2 to 29±1 mL 100·g−1·min−1) increased ΔVa(NO) and the cGMP content of the epicardial LAD; bradykinin further increased parameters in an L-NAME–sensitive manner (Table 1⇓). These results indicate that the beneficial effects of bradykinin in the ischemic myocardium are mostly attributable to augmentation of NO release and the opening of KCa channels.
Indomethacin, 8-SPT, and glibenclamide had no effect on the bradykinin-induced coronary vasodilation in the nonischemic myocardium (CBF, 132±2.8, 131±3.4, and 134±4.1 mL·100 g−1·min−1, respectively; P<.01 versus baseline) or ischemic (CBF, 46.2±1.8, 44.2±1.4, and 45.8±1.4 mL·100 g−1·min−1; LER, −29.5±2.2, −29.2±2.3, and −31.2±3.3%, respectively; P<.05 versus baseline).
Effects of KCa Channel Blockers on Reactive Hyperemia
Reactive hyperemic flow after 20 seconds of coronary occlusion was reduced by IBTX or TEA (Table 2⇓). Although the combination of L-NAME and 8-SPT also reduced reactive hyperemic flow, the additional presence of IBTX resulted in a further decrease in this parameter. Moreover, IBTX further decreased reactive hyperemia in the presence of L-NAME, 8-SPT, indomethacin, and glibenclamide, indicating that this portion of reactive hyperemia is attributable to the opening of KCa channels, possibly induced by EDHF.
Endothelium-dependent relaxation in coronary arteries is reported to be attributable to at least three different mechanisms: NO, PGI2, and EDHF.4 Bradykinin is thought to stimulate the release of EDHF and NO in various endothelium-containing tissues,3 7 13 and EDHF relaxes smooth muscles by opening KCa channels.14 However, the possible role of the opening of KCa channels in coronary vasodilation in the ischemic heart has not been previously determined. Here, we have provided in vivo evidence that KCa channels are an important component in the regulation of CBF in the ischemic myocardium and that endogenous bradykinin may activate KCa channels to induce coronary vasodilation in open-chest dogs. We have shown that IBTX, CTX, or TEA reduces CBF in the ischemic myocardium. There are several possible explanations for our observations. First, TEA may have vasoconstrictive effects attributable to inhibition of muscarinic acetylcholine receptors.15 TEA preferentially blocks KCa channels at low concentrations; at higher concentrations, however, it blocks other types of K+ channels such as inward rectifier K+ channels and KATP channels16 in smooth muscle. In the present study, glibenclamide had no effect on the bradykinin-induced coronary vasodilation. Because the effects of IBTX and CTX, both of which are highly specific blockers of large-conductance KCa channels, on CBF during coronary hypoperfusion resembled that of TEA, the effects of all three agents are likely attributable to inhibition of KCa channels.
Second, TEA may reduce the production of NO.17 In the present study, however, TEA did not reduce NO production during an infusion of bradykinin under conditions of coronary hypoperfusion, and TEA reduced CBF in the presence of L-NAME, which abolished NO production in response to ischemia.
Third, IBTX, CTX, and TEA may reduce myocardial contractility in the ischemic region, resulting in a decrease in CBF, given that both CTX and IBTX are potential cardiodepressant factors. However, such a mechanism is unlikely because these inhibitors did not reduce FS, myocardial oxygen consumption, or LER in the nonischemic condition.
Fourth, intracoronary infusion of IBTX, CTX, or TEA may close functional collateral vessels to the ischemic areas, thereby increasing the severity of ischemia. We cannot exclude this possibility.
Fifth, the opening of KCa channels may reduce sympathetic neural activity and thereby induce coronary vasodilation; inhibition of KCa channels in adrenal chromaffin cells increases catecholamine secretion.18 However, the administration of inhibitors of KCa channels during coronary hypoperfusion did not increase the concentration of norepinephrine in the coronary venous blood, suggesting that a decrease in norepinephrine release in response to the opening of KCa channels does not contribute to the observed coronary vasodilation.
Our results suggest that the opening of KCa channels is important in the preservation of CBF and myocardial contractile and metabolic function in the ischemic myocardium.
Mechanism of KCa Channel Opening During Ischemia
KCa channels contribute to the decrease in coronary vascular resistance in the ischemic heart, but they do not play a major role in regulating CBF in the nonischemic myocardium. Although our data suggest that KCa channels are activated and contribute to the regulation of coronary vascular tone during coronary hypoperfusion, the mechanism of KCa channel opening during ischemia remains unknown. One possibility is that KCa channel opening results from an increase in the intracellular Ca2+ concentration induced by ischemia per se.19 A second possibility is that cellular acidosis20 or a decrease in the intracellular ATP concentration21 may open KCa channels. Patch-clamp recordings have shown that cAMP and cAMP-dependent protein kinase activate KCa channels in cultured smooth muscle cells from porcine coronary artery and that ATP inhibits the opening of KCa channels, resulting in membrane depolarization and vascular contraction.21 A third possibility is that an endothelium-derived vasoactive factor such as bradykinin, NO, prostaglandin, adenosine, or EDHF may open KCa channels. PGI2 induces relaxation of isolated guinea pig coronary arteries by increasing cAMP accumulation in smooth muscle cells and hyperpolarizing the cell membrane,22 suggesting that hyperpolarization of smooth muscle cells may occur through cyclic nucleotide-dependent protein kinase–mediated modulation of K+ channels.23 Adenosine also hyperpolarizes smooth muscle cells in the canine saphenous vein denuded of endothelium,24 and other activators of adenylate cyclase induce hyperpolarization by opening KATP channels.25 In the present study, pretreatment with indomethacin, glibenclamide, or 8-SPT did not inhibit bradykinin-induced coronary vasodilation in nonischemic or ischemic hearts, nor did it affect the changes in CBF and LER induced by IBTX in the ischemic heart. Therefore, PGI2, KATP channels, and adenosine appear not to be responsible for the coronary vasodilation induced by bradykinin and the opening of KCa channels during coronary hypoperfusion in the canine heart. NO in solution or nitrovasodilators can hyperpolarize smooth muscle of the aorta or coronary arteries of the guinea pig.26 Because NO production is increased during ischemia,27 it is possible that NO released tonically from the endothelium can regulate membrane potential by a KCa channel–dependent mechanism. In the present study, however, KCa channel blockers reduced both CBF during coronary hypoperfusion and bradykinin-induced coronary vasodilation in the presence of L-NAME. Thus, NO does not contribute to the opening of KCa channels induced by bradykinin or myocardial ischemia. In contrast, bradykinin was released during coronary hypoperfusion, and HOE-140 prevented the effects of IBTX on coronary hemodynamic and metabolic parameters during myocardial ischemia, indicating that the beneficial effects of KCa channel opening are caused mainly by the accumulation of bradykinin in the ischemic myocardium. However, it remains unclear whether endogenous bradykinin directly opens KCa channels during ischemia or whether it triggers the release of EDHF. Therefore, the present study did not reveal the site of the action of bradykinin, ie, coronary smooth muscle cells or endothelial cells.
In the present study, we could not clarify the mechanisms by which the cardiac bradykinin levels are increased in the ischemic heart. Because cardiac bradykinin production is increased during anaphylaxis,27 cardiac anaphylaxis is associated with marked ischemia, and ischemia increases bradykinin outflow from the heart, the release of bradykinin may be attributable to cardiac inflammatory responses during ischemia in open-chest dogs that underwent acute surgery. Thus, important factors during cardiac inflammatory responses in the ischemic heart, eg, bradykinin, may control the tones of coronary vessels. In addition, we need to be careful in extending the present experimental results to the clinical setting because these results were obtained in open-chest dogs that received acute surgery. Therefore, further investigation is necessary to apply this hypothesis between bradykinin and KCa channels to the coronary circulation in patients with ischemic heart disease.
Selected Abbreviations and Acronyms
|CBF||=||coronary blood flow|
|CPP||=||coronary perfusion pressure|
|EDHF||=||endothelium-dependent hyperpolarizing factor|
|L-NAME||=||NG-nitro-L-arginine methyl ester|
|LAD||=||left anterior descending coronary artery|
|LER||=||lactate extraction ratio|
|LVP||=||left ventricular pressure|
We thank Kayoko Yoshida, Yukiyo Nomura, and Makoto Hasegawa for their technical assistance.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996.
- Received August 26, 1996.
- Revision received October 23, 1996.
- Accepted November 4, 1996.
- Copyright © 1997 by American Heart Association
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