Modulation of liver cell membrane NHE-1, Na+-K+ATPase, and GLUT-2 protein content after cold preservation and rewarming

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Abstract
Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+-H+exchanger isoform NHE-1 and the Na+-K+ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37°C did not further reduce this parameter. On the other hand, expression of Na+-K+ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins.Key words: pH regulation, volume regulation, sodium-hydrogen antiport, cryopreservation, isolated rat hepatocytes.