Pinocytosis in 2,5-di-tert-butylhydroquinone-stimulated hepatocytes and evaluation of its role in Ca²⁺ inflow

Abstract

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°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°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 Ca ²⁺ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°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 Mn²⁺-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.