Impaired Function of Inhibitory G Proteins During Acute Myocardial Ischemia of Canine Hearts and Its Reversal During Reperfusion and a Second Period of Ischemia
Authors: Feraydoon Niroomand, Christof Weinbrenner, Angelika Weis, Matthias Bangert, Carsten Schwencke, Rainer Marquetant, Thorsten Beyer, Ruth H. Strasser, Wolfgang Kübler, Bernhard Rauch
Journal: Circulation Research (1995)

Abstract 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 effectors, 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 &lt;.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 &lt;.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 [γ- 35 S]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 α i - and α i2 -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.