Gen. Pharmac. Vol. 23, No. 5, pp. 921-923, 1992 Printed in Great Britain.All fights reserved
0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
INFLUENCE OF ATP-SENSITIVE POTASSIUM CHANNEL BLOCKER ON HYPOXIA-INDUCED DAMAGE OF ISOLATED GUINEA PIG HEART T. A. MCKEAN and A. J. BRANZ Department of BiologicalSciencesand WAMI Medical Program, Universityof Idaho, Moscow, ID 83843, U.S.A. [Tel. (208) 885-6280; Fax (208) 885-7905] (Received 25 November 1991)
Abstract--l. Isolated guinea pig hearts were perfused under constant flow conditions with Krebs--Henseleitbuffer. Hearts were subjected to 15 rain of hypoxia followed by reoxygenation in the presence and in the absence of I00/~m glyburide, an ATP-sensitivepotassium channel blocker. Heart rate, left ventricular pressure and lactate dehydrogenase (LDH) release were measured at 5 rain intervals. 2. Small decreases in heart rate and left ventrieular pressure were observed during glyburide infusion with hypoxia, however LDH release, which was used as an index of cellular damage, was dramatically elevated. 3. Neither glyburide, nor the vehiclein which it was dissolved,appeared to produce myocardial damage under normoxic conditions. 4. It is concluded that ATP-sensitive potassium channels are important in the protection of the myocardium during hypoxia in isolated guinea pig hearts perfused under constant flow conditions.
INTRODUCTION ATP-sensitive potassium channels were discovered in cardiac muscle (Noma, 1983) but are also found in pancreatic r-cells, skeletal muscle, smooth muscle and brain (Ashcroft, 1988). These channels are closed in mM concentrations of ATP and open when ATP levels fall. The role of ATP-sensitive potassium channels is well established in the control of insulin secretion by the pancreas (Petersen, 1990); however, its importance in cardiac tissue is less well established. The duration of the cardiac action potential is decreased during metabolic stress and this may be mediated by opening of the ATP-sensitive potassium channel (Elliot et al., 1989). An abbreviated action potential duration might limit sarcolemmal calcium influx and reduce the metabolic requirements of the cell during metabolic stress. The open ATP-sensitive potassium channels are also thought to mediate the extracellular potassium accumulation which occurs during the first few minutes of ischemia in perfused rabbit, rat, and guinea pig hearts (Wilde et al., 1990). Increases in extracellular potassium have both a negative inotropic and chronotropic effect on the heart (McKean, 1990) and might serve to further limit the metabolic demand on the heart during an episode of metabolic stress. Recent studies indicate that ATP-sensitive channels agonists protect the heart from ischemic damage when the agonists are administered prior to the onset of global ischemia. The protective effect is blocked by > 1/tM glyburide, an ATP-sensitive potassium channel blocker (Grover et al., 1990). When glyburide (10/~M) is given alone, the severity of the ischemic damage is not affected (Grover et aL, 1989). Activation of ATP-sensitive potassium channels produces large increases (up to 50%) in coronary flow. Some portion of the
preservation of reperfusion function might be due to changes in coronary perfusion since ATP-sensitive potassium channels found in the smooth muscle of the coronary vascular bed would also be activated. Cole et al. (1991), however, showed that prior treatment with ATP-sensitive potassium channel openers (pinacidil) have a protective effect and channel closers (glyburide) have a deleterious effect on the myocardium in a guinea pig right ventricular preparation in which flow was under experimental control. Various periods of global ischemia were followed by reperfusion in which the reperfusion flows were not allowed to increase. The purpose of this study was to test the hypothesis that the ATP-sensitive potassium channels have a protective effect on the myocardium during hypoxia. These experiments were performed under conditions of constant coronary perfusion. This constraint was utilized so that the channel antagonist would have continuous access to myocardium throughout the period of metabolic stress and recovery. Also changes in coronary perfusion as a result of ATP-sensitive potassium channel activity would be minimized. METHODS AND RESULTS Hearts were removed from ether anaesthetized guinea pigs and perfused with Krebs-Henseleit buffer equilibrated at 37°C. The buffer was bubbled with 95% 02 and 5% CO2 (normoxia) or 95% N2 and 5% CO2 (hypoxia). Left ventricular pressure was measured with a fluid-filledballoon connected to a Statham pressure transducer. Balloon volume was adjusted for an end-diastolic pressure of 5-10mmHg. Aortic perfusion pressure was adjusted to 50 mmHg by means of a roller pump and the heart perfused for a 30 rain equilibration period. At the end of this period flow was readjusted and then maintained at this same value throughout the remainder of the study. Figure 1 shows data
921
T. A. McKEAN and A.J. BRANZ
922
250
200
O •
- hypoxia - glyburide
RATE ~
I
the 800% increase observed with glyburide infusion in the presence of hypoxia (P = 0.026). A final control study was performed which was designed to determine if perfusion time alone was responsible for the increased LDH release. In this group hearts (n = 5) were subjected to 15 min hypoxia followed by 35min of reoxygenation. Heart rate, left ventricular pressure did not change during the reoxygenation while the LDH release decreased significantly (P = 0.017). DISCUSSION
0
I
0
I
I
5 i0 rain of hypoxia
I
15
///,~
RECOV
Fig. 1. Change in heart rate, left ventricular pressure and LDH release during hypoxia with and without glyburide infusion. Methods described in text. Units are heart rate in beats/min; left ventricular pressure in mmHg; and LDH release in milliunits/min/g of heart. Hypoxia began at time zero and was present for 15min. The recovery is a 10min reoxygenation period. Data are from the same 6 hearts. Data are presented as the mean ___SEM. from hearts which were perfused with the hypoxic buffer for 15min and then reperfused with oxygenated buffer for 10 rain. Samples were taken during the indicated time periods and are labelled "hypoxia". These same hearts were then continuously infused with glyburide dissolved in ethanol so the concentration of glyburide in the perfusate was about 100/zM depending on coronary flow. This concentration has been shown to have no effect on the cardiac action potential under normoxic conditions (Cole et al., 1991). The hearts were again perfused with the hypoxic buffer followed by reoxygenation. The data from these hearts are labelled "glyburide". The changes in heart rate and lactate dehydrogenase (LDH) release (by method of Wroblewski and LaDue, 1955) between hypoxia with and without glyburide are significant (P
0306-3623/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd
INFLUENCE OF ATP-SENSITIVE POTASSIUM CHANNEL BLOCKER ON HYPOXIA-INDUCED DAMAGE OF ISOLATED GUINEA PIG HEART T. A. MCKEAN and A. J. BRANZ Department of BiologicalSciencesand WAMI Medical Program, Universityof Idaho, Moscow, ID 83843, U.S.A. [Tel. (208) 885-6280; Fax (208) 885-7905] (Received 25 November 1991)
Abstract--l. Isolated guinea pig hearts were perfused under constant flow conditions with Krebs--Henseleitbuffer. Hearts were subjected to 15 rain of hypoxia followed by reoxygenation in the presence and in the absence of I00/~m glyburide, an ATP-sensitivepotassium channel blocker. Heart rate, left ventricular pressure and lactate dehydrogenase (LDH) release were measured at 5 rain intervals. 2. Small decreases in heart rate and left ventrieular pressure were observed during glyburide infusion with hypoxia, however LDH release, which was used as an index of cellular damage, was dramatically elevated. 3. Neither glyburide, nor the vehiclein which it was dissolved,appeared to produce myocardial damage under normoxic conditions. 4. It is concluded that ATP-sensitive potassium channels are important in the protection of the myocardium during hypoxia in isolated guinea pig hearts perfused under constant flow conditions.
INTRODUCTION ATP-sensitive potassium channels were discovered in cardiac muscle (Noma, 1983) but are also found in pancreatic r-cells, skeletal muscle, smooth muscle and brain (Ashcroft, 1988). These channels are closed in mM concentrations of ATP and open when ATP levels fall. The role of ATP-sensitive potassium channels is well established in the control of insulin secretion by the pancreas (Petersen, 1990); however, its importance in cardiac tissue is less well established. The duration of the cardiac action potential is decreased during metabolic stress and this may be mediated by opening of the ATP-sensitive potassium channel (Elliot et al., 1989). An abbreviated action potential duration might limit sarcolemmal calcium influx and reduce the metabolic requirements of the cell during metabolic stress. The open ATP-sensitive potassium channels are also thought to mediate the extracellular potassium accumulation which occurs during the first few minutes of ischemia in perfused rabbit, rat, and guinea pig hearts (Wilde et al., 1990). Increases in extracellular potassium have both a negative inotropic and chronotropic effect on the heart (McKean, 1990) and might serve to further limit the metabolic demand on the heart during an episode of metabolic stress. Recent studies indicate that ATP-sensitive channels agonists protect the heart from ischemic damage when the agonists are administered prior to the onset of global ischemia. The protective effect is blocked by > 1/tM glyburide, an ATP-sensitive potassium channel blocker (Grover et al., 1990). When glyburide (10/~M) is given alone, the severity of the ischemic damage is not affected (Grover et aL, 1989). Activation of ATP-sensitive potassium channels produces large increases (up to 50%) in coronary flow. Some portion of the
preservation of reperfusion function might be due to changes in coronary perfusion since ATP-sensitive potassium channels found in the smooth muscle of the coronary vascular bed would also be activated. Cole et al. (1991), however, showed that prior treatment with ATP-sensitive potassium channel openers (pinacidil) have a protective effect and channel closers (glyburide) have a deleterious effect on the myocardium in a guinea pig right ventricular preparation in which flow was under experimental control. Various periods of global ischemia were followed by reperfusion in which the reperfusion flows were not allowed to increase. The purpose of this study was to test the hypothesis that the ATP-sensitive potassium channels have a protective effect on the myocardium during hypoxia. These experiments were performed under conditions of constant coronary perfusion. This constraint was utilized so that the channel antagonist would have continuous access to myocardium throughout the period of metabolic stress and recovery. Also changes in coronary perfusion as a result of ATP-sensitive potassium channel activity would be minimized. METHODS AND RESULTS Hearts were removed from ether anaesthetized guinea pigs and perfused with Krebs-Henseleit buffer equilibrated at 37°C. The buffer was bubbled with 95% 02 and 5% CO2 (normoxia) or 95% N2 and 5% CO2 (hypoxia). Left ventricular pressure was measured with a fluid-filledballoon connected to a Statham pressure transducer. Balloon volume was adjusted for an end-diastolic pressure of 5-10mmHg. Aortic perfusion pressure was adjusted to 50 mmHg by means of a roller pump and the heart perfused for a 30 rain equilibration period. At the end of this period flow was readjusted and then maintained at this same value throughout the remainder of the study. Figure 1 shows data
921
T. A. McKEAN and A.J. BRANZ
922
250
200
O •
- hypoxia - glyburide
RATE ~
I
the 800% increase observed with glyburide infusion in the presence of hypoxia (P = 0.026). A final control study was performed which was designed to determine if perfusion time alone was responsible for the increased LDH release. In this group hearts (n = 5) were subjected to 15 min hypoxia followed by 35min of reoxygenation. Heart rate, left ventricular pressure did not change during the reoxygenation while the LDH release decreased significantly (P = 0.017). DISCUSSION
0
I
0
I
I
5 i0 rain of hypoxia
I
15
///,~
RECOV
Fig. 1. Change in heart rate, left ventricular pressure and LDH release during hypoxia with and without glyburide infusion. Methods described in text. Units are heart rate in beats/min; left ventricular pressure in mmHg; and LDH release in milliunits/min/g of heart. Hypoxia began at time zero and was present for 15min. The recovery is a 10min reoxygenation period. Data are from the same 6 hearts. Data are presented as the mean ___SEM. from hearts which were perfused with the hypoxic buffer for 15min and then reperfused with oxygenated buffer for 10 rain. Samples were taken during the indicated time periods and are labelled "hypoxia". These same hearts were then continuously infused with glyburide dissolved in ethanol so the concentration of glyburide in the perfusate was about 100/zM depending on coronary flow. This concentration has been shown to have no effect on the cardiac action potential under normoxic conditions (Cole et al., 1991). The hearts were again perfused with the hypoxic buffer followed by reoxygenation. The data from these hearts are labelled "glyburide". The changes in heart rate and lactate dehydrogenase (LDH) release (by method of Wroblewski and LaDue, 1955) between hypoxia with and without glyburide are significant (P
