AJP - Gastrointestinal and Liver Physiology, Vol 272, Issue 3 638-G645, Copyright © 1997 by American Physiological Society

ARTICLES

Different effect of cold storage and rewarming on three pH regulating transporters in isolated rat hepatocytes

D. A. Forestal, J. Haimovici and P. Haddad
Departement de Pharmacologie, Universite de Montreal, Quebec, Canada.

Disturbances in hepatic pH homeostasis are thought to participate in the functional damage to liver grafts caused by the cold preservation and warm reperfusion necessitated by transplantation surgery. We have used an in vitro model of isolated rat hepatocytes suspended in cold University of Wisconsin (UW) solution and subsequently cultured at 37 degrees C to evaluate liver cell pH regulatory mechanisms after cold preservation and rewarming. Cells were kept for up to 72 h in cold UW solution, and at 24-h intervals intracellular pH (pHi) was measured after 60-90 min of warm culture by cytofluorometry using the fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. When challenged with an alkaline load by isohydric HCO(3)(-)-CO(2) steps, hepatocytes exhibited similar maximal pH(i) values and recovered at the same rate, irrespective of cold storage time, indicating that Cl-/HCO(3)- exchange activity is quite resistant to hypothermic storage and subsequent rewarming. In parallel studies, cells were subjected to an acid load by the NH4Cl pulse technique in bicarbonate buffer containing 50 microM ethylisopropylamiloride to block Na+/H+ exchange. Despite similar nadir pH(i) (lowest pH(i) values due to acid load), the subsequent pH(i) recovery rate that reflects Na+-(HCO(3)-)n cotransport activity was increased significantly after hypothermic preservation. Hepatocytes were also perfused with a bicarbonate-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, and Na+/H+ exchange activity was evaluated using the same acid load protocol. Although cells always exhibited similar steady-state initial pH(i) and nadir, the rate of pH(i) recovery decreased significantly as a function of cold storage time in UW solution. Finally, intracellular buffering capacity was calculated from the sudden pH(i) changes induced by HCO(3)(-)-CO(2) steps or NH4Cl pulses and was found to remain stable throughout the 72 h of cold preservation. Therefore, the results strongly suggest that cold preservation and rewarming disturb hepatocellular pH regulatory mechanisms by attenuating Na+/H+ exchange and increasing Na+-(HCO(3)-)n cotransport, whereas Cl-/HCO(3)- exchange is not affected.

This article has been cited by other articles:

				A. Elimadi and P. S. Haddad Cold preservation-warm reoxygenation increases hepatocyte steady-state Ca2+ and response to Ca2+-mobilizing agonist Am J Physiol Gastrointest Liver Physiol, September 1, 2001; 281(3): G809 - G815. [Abstract] [Full Text] [PDF]

				A. W. Jehle, H. Hilfiker, M. F. Pfister, J. Biber, E. Lederer, R. Krapf, and H. Murer Type II Na-Pi cotransport is regulated transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells Am J Physiol Renal Physiol, January 1, 1999; 276(1): F46 - F53. [Abstract] [Full Text] [PDF]