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 d elay cellular injury during a subsequent ischemic condition. Recent ob servations suggest that this protective mechanism depends on the conti nued 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 ade nosine A(1) receptors. As a working hypothesis, an increased responsiv eness 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 liga tion of the left anterior descending coronary artery. Animals underwen t either a single period of 5 minutes of ischemia (n=9), a single peri od 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 6 0 minutes of reperfusion (n=5), or 5 minutes of ischemia followed by 1 5 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 activi ty was associated with a 45% decrease in carbachol-mediated inhibition of adenylyl cyclase (control, 28.9+/-2.4% maximal inhibition; ischemi a, 15.1+/-2.6% maximal inhibition; n=5; P<.001). Prolongation of the i schemic period to 15 minutes did not lead to a further reduction of th e G(i)-mediated signal transduction. The binding properties of muscari nic receptors were not affected by ischemia. Furthermore, as demonstra ted by carbachol-stimulated binding of [gamma-S-35]GTP to sarcolemmal membranes, high- and low-affinity binding sites for the muscarinic ant agonist carbachol, the EC(50) for carbachol-stimulated GTPase activity and the substrate dependency of the high-affinity GTPase, the interac tion between muscarinic receptors and inhibitory G proteins, and GTP b inding to G proteins were not altered (n=14). Immunoblotting with alph a(1)- and alpha(12)-specific antibodies did not indicate a loss of G(i ) proteins during ischemia that could explain the reduced GTPase activ ity. During 15 minutes of reperfusion, carbachol-stimulated GTPase act ivity increased to 147% of the control value (control, 33.7+/-20.6; re perfusion 49.1+/-22.5 pmol . min(-1). mg protein(-1); n=7; P=.012). Ma ximal inhibition of adenylyl cyclase by carbachol increased similarly (control, 21+/-6.8% maximal inhibition; reperfusion, 26.4+/-7.6% maxim al inhibition; n=8; P=.016). After 15 minutes of ischemia and 60 minut es of reperfusion, carbachol-stimulated GTPase activity remained incre ased. When the 5-minute ischemia and 15-minute reperfusion periods wer e followed by a second period of 5-minute ischemia, carbachol-stimulat ed GTPase activity and inhibition of adenylyl cyclase remained elevate d (GTPase: control, 38.4+/-16.7; second ischemia, 49.2+/-20.1 pmol . m in(-1). mg protein(-1); n=13; P=.009; adenylyl cyclase: control, 24.2/-6.8% maximal inhibition; second ischemia, 28.6+/-8% maximal inhibiti on; n=15; P=.003). In conclusion, the responsiveness of G(i) proteins toward receptor activation decreased rapidly during the first 5 minute s of ischemia. During a following 15-minute period of reperfusion, thi s 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 incre ased responsiveness of G(i) proteins after a brief period of ischemia and reperfusion.