Characterization of H+ efflux pathways in rat hepatocytes.
A pH-stat method was used to characterize H+ efflux pathways in hepatocytes in order to determine if Na+/H+ and Ca++/H+ exchange are involved in H+ efflux from hepatocytes under basal conditions and if cyclic AMP analogs affect Na+/H+ exchange. Total H+ efflux of freshly prepared hepatocytes ranged from 10 to 15 nmoles per min per mg protein. A part of total H+ efflux (35 to 50%) was dependent on extracellular Na+. This Na+-dependent H+ efflux was (i) inhibited by amiloride with a half-maximal effect at 0.3 mM, (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a Km of 15 +/- 3 mM Na+ and a Vmax of 9 +/- 0.07 nmoles per min per mg protein. Amiloride, ouabain and replacement of Na+ by choline also decreased intracellular pH determined from equilibrium distribution of dimethyloxazolidinedione. Li+ could partially substitute for Na+ in Na+-dependent H+ efflux and in maintaining intracellular pH. Efflux of CO2 and lactic acid from hepatocytes represented 80% of Na+-independent H+ efflux. Efflux of H+ in the presence and absence of Na+ was not significantly altered by extracellular Ca++ (less than 10 microM and 1.0 mM). Thus, Ca++/H+ exchange is unlikely to contribute significantly to total H+ efflux from hepatocytes. Cyclic AMP analogs, dibutyryl cyclic AMP and 8-bromo cyclic AMP, inhibited amiloride-sensitive Na+-dependent H+ efflux, and dibutyryl cyclic AMP decreased intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Anwer
M S
MS
Department of Veterinary Medicine and Pharmacology, Tufts University, School of Veterinary Medicine, North Grafton, Massachusetts 01536.
Nolan
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002245:Carbon Dioxide / Q000378:metabolism; D002794:Choline / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D008094:Lithium / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology; D013997:Time Factors
D007773:Lactates; D011506:Proteins; D002245:Carbon Dioxide; D015124:8-Bromo Cyclic Adenosine Monophosphate; D019344:Lactic Acid; D010042:Ouabain; D003994:Bucladesine; D000584:Amiloride; D006859:Hydrogen; D008094:Lithium; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D002794:Choline; D002118:Calcium