Pinocytosis in 2,5-di-tert-butylhydroquinone-stimulated hepatocytes and evaluation of its role in Ca2+ inflow.
In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca2+ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20 degrees C and used to predict the amount of extracellular Ca2+ taken up by pinocytosis. This was compared with the measured rate of Ca2+ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca2+ + Mg2+)ATP-ase. Fluid uptake by pinocytosis was measured using [14C]sucrose. In hepatocytes incubated at 20 degrees C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca2+ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20 degrees C by measuring the uptake of 45Ca2+. The degree of stimulation of Ca2+ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn(2+)-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd3+. It was calculated that the process of pinocytosis accounts for at least 15% of Ca2+ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca2+ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca2+ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca2+ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca2+ inflow.
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
K C
KC
Fernando
G J
GJ
Barritt
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008674:Metals, Rare Earth / Q000494:pharmacology; D010873:Pinocytosis / Q000187:drug effects; D051381:Rats; D013395:Sucrose / Q000378:metabolism
D004791:Enzyme Inhibitors; D006873:Hydroquinones; D008674:Metals, Rare Earth; C019359:2,5-di-tert-butylhydroquinone; D008345:Manganese; D013395:Sucrose; D000252:Calcium-Transporting ATPases; D002118:Calcium