A kinetic analysis of the effects of adrenaline on calcium distribution in isolated rat liver parenchymal cells

Gregory J. Barritt, Janice C. Parker, John C. Wadsworth

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    Abstract

    1. The effects of adrenaline on Ca distribution in isolated rat liver parenchymal cells were studied using a 45Ca exchange technique under steady‐state conditions with respect to the net movement of Ca. 45Ca was initially introduced into the extracellular medium. The amount of cellular 45Ca was determined after separation of the cells from the medium by centrifugation through a solution which contained LaCl3 (to displace 45Ca bound to sites on the outside of the cell membrane) and silicon oil. At 1·3 and 2·4 m m‐extracellular Ca, a stimulation of the initial rate of 45Ca exchange was observed in the presence of 10−7 m‐adrenaline (or 10−6 m‐phenylephrine) with a 7% decrease, and no change, respectively, in the plateau of the exchange curve. The same degree of stimulation was observed when 45Ca was added at 1, 15, 30 or 45 min after the adrenaline. 2. No stimulation of the initial rate of exchange was observed at 0·1 m m‐extracellular Ca, or at 2·4 m m‐extracellular Ca in the presence of antimycin A and oligomycin. At 0·1 m m‐Ca, a 60% decrease in the plateau of the exchange curve was observed in the presence of adrenaline. The concentration of adrenaline (10−7 m) which caused half‐maximal stimulation of the initial rate of 45Ca exchange at 1·3 m m‐Ca was similar to that (2 × 10−7 m) which caused half‐maximal decrease in the plateau at 0·1 m m‐Ca. 3. The addition of adrenaline to cells equilibrated with 45Ca at either 2·4 or 1·3 m m‐Ca caused a transient loss of 45Ca followed by a return to a new steady state after 1 or 10 min, respectively. A loss of 45Ca was also observed at 0·1 m m‐Ca, but the 45Ca content of the cells remained maximally depressed for at least 30 min. 4. A non‐linear least‐squares iterative curve‐fitting technique was used to demonstrate that (a) an equation which includes two exponential terms and (b) a parallel or series arrangement of three compartments of exchangeable Ca (the medium and two compartments associated with the cell) are consistent with each set of data obtained at 1·3 or 2·4 m m‐Ca in the presence or absence of adrenaline (or phenylephrine). At 1·3 m m‐Ca, the quantities of exchangeable Ca in the two kinetically defined cellular compartments were 0·04‐0·07 and 0·34‐0·37 nmol per mg wet weight with rate constants for Ca outflow of 1·2‐1·5 and 0·06‐0·08 min−1, respectively. 5. Analysis of the changes induced by adrenaline or phenylephrine showed that at 1·3 and 2·4 m m‐extracellular Ca these agents caused a 75‐150% increase in the quantity of exchangeable Ca in the small kinetically defined compartment and a 20% decrease in the quantity of exchangeable Ca in the large kinetically defined compartment. These changes were mediated by an 80‐160% increase in the rate constant for the inflow of Ca from the medium to the small kinetically defined compartment, and either a 20‐60% decrease in the rate constant for inflow to, or a 20% increase in the rate constant for outflow from, the large compartment. 6. Replacement of the LaCl3 in the solution used to separate the cells from the incubation medium with either 5 m m‐EGTA or 5 m m‐CaCl2 did not alter the kinetics of 45Ca exchange or the stimulation by adrenaline. This, together with the observation that at 1·3 m m‐extracellular Ca, adrenaline increases the initial rate of exchange in the absence, but not in the presence, of antimycin A plus oligomycin, indicates that both cellular compartments of exchangeable Ca are intracellular. 7. The addition of antimycin A plus oligomycin to cells equilibrated with 45Ca at 2·4 m m‐extracellular Ca in the presence or absence of adrenaline displaced 0·09 and 0·14 nmol 45Ca. mg−1, respectively. 8. Subcellular fractionation of cells equilibrated with 45Ca at 0·1 m m‐extracellular Ca revealed that the mitochondria and microsomes contained significant amounts of 45Ca. The amounts of 45Ca in these fractions decreased by 50 and 40%, respectively, in the presence of adrenaline. 9. In 45Ca exchange experiments conducted with isolated mitochondria at 37 °C at 1·5 × 10−7 m and 0·9 × 10−7 m free Ca in the presence of 2 m m‐Mg2+, one kinetically defined compartment of exchangeable mitochondrial Ca was detected. The rate constants for Ca outflow were found to be 0·15±0·03 and 0·12±0·04 min−1, respectively, in reasonable agreement with the value obtained for the rate constant for the outflow of Ca from the large kinetically defined compartment of exchangeable Ca observed in cells. 10. It is concluded that adrenaline has two effects on Ca movement in the liver cell. These are to cause a loss of Ca from an intracellular compartment, which includes the mitochondria and microsomes, and to increase the transport of Ca from the extracellular medium to an intracellular site. This results in an increase in the amount of Ca in a small intracellular compartment which may represent cytoplasmic Ca, or Ca bound to sites on the inside of the plasma membrane.

    Original languageEnglish
    Pages (from-to)29-55
    Number of pages27
    JournalThe Journal of Physiology
    Volume312
    Issue number1
    DOIs
    Publication statusPublished - 1 Mar 1981

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