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(Circulation Research. 1995;76:861-870.)
© 1995 American Heart Association, Inc.

Articles

Impaired Function of Inhibitory G Proteins During Acute Myocardial Ischemia of Canine Hearts and Its Reversal During Reperfusion and a Second Period of Ischemia

Possible Implications for the Protective Mechanism of Ischemic Preconditioning

Presented in part at the 63rd to 66th Scientific Sessions of the American Heart Association (1990-1993).

Feraydoon Niroomand, Christof Weinbrenner, Angelika Weis, Matthias Bangert, Carsten Schwencke, Rainer Marquetant, Thorsten Beyer, Ruth H. Strasser, Wolfgang Kübler, Bernhard Rauch

From the Department of Cardiology, University of Heidelberg (Germany).

Correspondence to Dr Feraydoon Niroomand, Universität Heidelberg, Innere Medizin III, Bergheimerstr 58, 69115 Heidelberg, Germany.

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 A₁ receptors and G_i proteins. During acute myocardial ischemia, sufficient amounts of adenosine for maximal activation of adenosine A₁ receptors are released, independent of a preconditioning ischemia. Hence, the protective mechanism of ischemic preconditioning may not exclusively be explained by activation of adenosine A₁ 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<.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 [-³⁵S]GTP to sarcolemmal membranes, high- and low-affinity binding sites for the muscarinic antagonist carbachol, the EC₅₀ 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 _i- and _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.

Key Words: myocardial ischemia • reperfusion • ischemic preconditioning • G proteins • muscarinic receptors • adenylyl cyclase

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