Calcium compartmentation and exchange rates in primary hepatocyte culture.
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 45Ca2+ 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 scintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium (45Ca2+), to apparent asymptote, the washout of 45Ca2+ 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 (Ca2+) was determined by its displacement with 1 mM La3+ after asymptote had been reached during 45Ca2+ labeling (1.59 mmol Ca2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (greater than 3.4 min-1 with a t1/2 less than 12 s). The rate constants for the fast and slow exchangeable compartments were 0.11 min-1 (t1/2 = 6.5 min) and 0.013 min-1 (t1/2 = 56 min), respectively. The fast compartment contained 0.40 mmol Ca2+/kg dry wt and the slow compartment contained 0.27 mmol Ca2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca2+ in response to 100 microM phenylephrine, 10 nM angiotensin II, and 100-microM 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca2+/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 45Ca2+ 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.
Gish
R G
RG
Liver Research Laboratory, Wadsworth V.A. Hospital, Los Angeles, California.
Garcia
C
C
Reedy
T
T
Kaplowitz
N
N
Langer
G A
GA
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D006873:Hydroquinones / Q000494:pharmacology; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D006873:Hydroquinones; D000804:Angiotensin II; D010656:Phenylephrine; D007811:Lanthanum; C018855:2-tert-butylhydroquinone; D002118:Calcium