Yonago Acta medica 1999;42:31–39
Activated Protein Kinase C Attenuates Ca2+ Overloading and Reoxygenation Hypercontracture in Isolated Rat Cardiomyocytes following Chemical Hypoxia
Shigeto Miyasaka, Takashi Ichiba, Hiroaki Kuroda and Shigetsugu Ohgi
Second Department of Surgery, Faculty of Medicine, Tottori University, Yonago 683-0826 Japan
The aims of this study were i) to test the effects of protein kinase C (PKC) activation on the intracellular Ca2+ concentration of rat cardiomyocytes during chemical hypoxia-reoxygenation, ii) to determine the contribution of the sarcoplasmic reticulum (SR) and the Na+-Ca2+ exchange on the regulation of intracellular Ca2+ following PKC activation and iii) to test the role of PKC-dependent intracellular pH changes in intracellular Ca2+ regulation. We used the isolated adult rat cardiomyocyte perfusion model. Cardiomyocytes were loaded with the Ca2+-fluorescent probe Fluo-3 and the pH-fluorescent probe SNARF1. Cells were subjected to 50 min of glucose-free and NaCN chemical hypoxia followed by 30 min of simulated reoxygenation. The activation of PKC significantly inhibited the hypoxia-induced increase of Fluo-3 fluorescent intensity (control; 692 ± 100% and activated PKC; 322 ± 43%: P < 0.05). This inhibitory effect was not affected by the inhibition of the SR Ca2+ uptake induced by thapsigargin, but was cancelled by the inhibition of the Na+-Ca2+ exchange with dichlorobenzamil (thapsigargin 337 ± 47%; dichlorobenzamil 609 ± 100%). PKC activation also attenuated the decrease in intracellular pH during chemical hypoxia, even in the presence of the Na+-H+ exchange inhibitor amiloride (control; 6.54 ± 0.02, PKC; 6.72 ± 0.03, PKC + amiloride; 6.73 ± 0.03). We concluded that the PKC attenuation of Ca2+ overloading to rat cardiomyocytes during chemical hypoxia-reoxygenation does not depend on the Ca2+ uptake by the SR, but does require a Na+-Ca2+ exchange. Since PKC attenuated the increasing intracellular H+ during chemical hypoxia, a low H+ concentration may be important for the maintenance of Ca2+ extrusion via the Na+-Ca2+ exchange.
Key words: Ca2+ overload; intracellular pH; isolated rat cardiomyocytes; Na+-Ca2+ exchanger; protein kinase C
