IMPAIRED FUNCTION OF INHIBITORY G-PROTEINS DURING ACUTE MYOCARDIAL-ISCHEMIA OF CANINE HEARTS AND ITS REVERSAL DURING REPERFUSION AND A 2ND PERIOD OF ISCHEMIA - POSSIBLE IMPLICATIONS FOR THE PROTECTIVE MECHANISM OF ISCHEMIC PRECONDITIONING

A brief antecedent period of myocardial ischemia and reperfusion can delay cellular injury during a subsequent ischemic condition. Recent observations suggest that this protective mechanism depends on the continued activation of adenosine A(1) receptors and G(i) proteins. During acute myocardial ischemia, sufficient amounts of adenosine for maximal activation of adenosine A(1) receptors are released, independent of a preconditioning ischemia. Hence, the protective mechanism of ischemic preconditioning may not exclusively be explained by activation of adenosine A(1) receptors. As a working hypothesis, an increased responsiveness of G(i) proteins toward receptor-mediated activation, leading to an increased response of G(i)-regulated effecters, was tested in this study. In 47 anesthetized dogs, ischemia was induced by proximal ligation of the left anterior descending coronary artery. Animals underwent either a single period of 5 minutes of ischemia (n=9), a single period of 15 minutes of ischemia (n=10), 5 minutes of ischemia followed by 15 minutes of reperfusion (n=8), 15 minutes of ischemia followed by 60 minutes of reperfusion (n=5), or 5 minutes of ischemia followed by 15 minutes of reperfusion and a second period of 5 minutes of ischemia (n=15). Sarcolemmal membranes were prepared from the central ischemic area and from the posterior left ventricular wall, which served as the control. During ischemia, carbachol-stimulated GTPase decreased by 38% (control, 33.5+/-17.7; ischemia, 24.2+/-15 pmol . min(-1) mg protein(-1); n=9; P<.001). The decrease in carbachol-stimulated GTPase activity was associated with a 45% decrease in carbachol-mediated inhibition of adenylyl cyclase (control, 28.9+/-2.4% maximal inhibition; ischemia, 15.1+/-2.6% maximal inhibition; n=5; P<.001). Prolongation of the ischemic period to 15 minutes did not lead to a further reduction of the G(i)-mediated signal transduction. The binding properties of muscarinic receptors were not affected by ischemia. Furthermore, as demonstrated by carbachol-stimulated binding of [gamma-S-35]GTP to sarcolemmal membranes, high- and low-affinity binding sites for the muscarinic antagonist carbachol, the EC(50) for carbachol-stimulated GTPase activity and the substrate dependency of the high-affinity GTPase, the interaction between muscarinic receptors and inhibitory G proteins, and GTP binding to G proteins were not altered (n=14). Immunoblotting with alpha(1)- and alpha(12)-specific antibodies did not indicate a loss of G(i) proteins during ischemia that could explain the reduced GTPase activity. During 15 minutes of reperfusion, carbachol-stimulated GTPase activity increased to 147% of the control value (control, 33.7+/-20.6; reperfusion 49.1+/-22.5 pmol . min(-1). mg protein(-1); n=7; P=.012). Maximal inhibition of adenylyl cyclase by carbachol increased similarly (control, 21+/-6.8% maximal inhibition; reperfusion, 26.4+/-7.6% maximal inhibition; n=8; P=.016). After 15 minutes of ischemia and 60 minutes of reperfusion, carbachol-stimulated GTPase activity remained increased. When the 5-minute ischemia and 15-minute reperfusion periods were followed by a second period of 5-minute ischemia, carbachol-stimulated GTPase activity and inhibition of adenylyl cyclase remained elevated (GTPase: control, 38.4+/-16.7; second ischemia, 49.2+/-20.1 pmol . min(-1). mg protein(-1); n=13; P=.009; adenylyl cyclase: control, 24.2+/-6.8% maximal inhibition; second ischemia, 28.6+/-8% maximal inhibition; n=15; P=.003). In conclusion, the responsiveness of G(i) proteins toward receptor activation decreased rapidly during the first 5 minutes of ischemia. During a following 15-minute period of reperfusion, this decreased responsiveness was reversed completely, exceeding control activities. The increased responsiveness of this signaling pathway was maintained during a subsequent second ischemic period. This suggests that the underlying mechanism of ischemic preconditioning is the increased responsiveness of G(i) proteins after a brief period of ischemia and reperfusion.