Calcium compartmentation and exchange rates in primary hepatocyte culture

Abstract

We utilized a technique, previously used to study myocardial cells ( G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239–H246), to study 45Ca 2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with seintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium ( 45Ca 2+), to apparent asymptote, the washout of 45Ca 2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca 2+) was determined by its displacement with 1 m m La 3+ after asymptote had been reached during 45Ca 2+ labeling (1.59 mmol Ca 2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (>3.4 min −1 with a t 1 2 <12 s ). The rate constants for the fast and slow exchangeable compartments were 0.11 min −1 ( t 1 2 = 6.5 min ) and 0.013 min −1 ( t 1 2 = 56 min ), respectively. The fast compartment contained 0.40 mmol Ca 2+/kg dry wt and the slow compartment contained 0.27 mmol Ca 2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca 2+ in response to 100 μ m phenylephrine, 10 n m angiotensin II, and 100-μ m 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca 2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca 2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca 2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast “microsomal” hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.