Cold preservation and rewarming differentially affects na+/h+, na+(
hco3-)n and cl-/hco3- transport activity in isolated rat
hepatocytes.
Forestal, Doris A., Judith Haimovici, and Pierre Haddad.
Groupe de Recherche en Transport Membranaire and D[acute]epartement
de pharmacologie, Universit[acute]e de Montr[acute]eal,
Montr[acute]eal, Qu[acute]ebec
APStracts 3:0219G, 1996.
Disturbances in hepatic pH homeostasis are thought to participate in
the functional damages caused to liver grafts 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 hrs in cold UW solution and, at 24 hr
intervals, intracellular pH (pHi) was measured after 60-90 min of
warm culture by cytofluorometry using the fluorochrome BCECF. When
challenged with an alkaline load by isohydric HCO3-/CO2 steps,
hepatocytes exhibited similar maximal pHi values and recovered at the
same rate irrespective of cold storage time indicating that Cl-/HCO3-
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 [mu]M EIPA to block Na+/H+ exchange. Despite similar
nadir pHi (lowest pHi values due to acid load), the subsequent pHi
recovery rate which reflects Na+(HCO3-)n co-transport activity was
increased significantly after hypothermic preservation. Hepatocytes
were also perfused with a bicarbonate-free HEPES buffer and Na+/H+
exchange activity evaluated using the same acid load protocol.
Although cells always exhibited similar steady-state initial pHi and
nadir, the rate of pHi recovery decreased significantly as a function
of cold storage time in UW solution. Finally, intracellular buffering
capacity was calculated from the sudden pHi changes induced by HCO3
-/CO2 steps or NH4Cl pulses and was found to remain stable throughout
the 72 hrs of cold preservation. Therefore, the results strongly
suggest that cold preservation and rewarming disturbs hepatocellular
pH regulatory mechanisms by attenuating Na+/H+ exchange and
increasing Na+(HCO3-)n co-transport while Cl-/HCO3- exchange is not
affected.
Received 14 February 1996; accepted in final form 3 October 1996.
APS Manuscript Number G60-6.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996