Norepinephrine, vasopressin, glucagon, and A23187 induce efflux of calcium from an exchangeable pool in isolated rat hepatocytes

Abstract

Isolated rat hepatocytes do not actively accumulate Ca2+ during prolonged incubation in vitro; however, these cells do exhibit a limited exchange of intracellular with extracellular Ca2+. The exchangeable pool represents about 2 nmol of CaS+ per mg of protein. In medium containing either a low (20 MM) or high (1 mM) concentration of Ca2+, the divalent cation ionophore, A23187 (at concentrations of 0.03-0.1 nmol/ mg of protein), causes release of 42Cad+ from this exchangeable pool but does not allow net influx of extracellular CaZ+ detectable by the use of a Ca2+-sensitive electrode. Like A23187, the hormones norepinephrine, vasopressin, and glucagon (at concentrations that stimulate gluconeogenesis) each induces a similar net efflux of Ca2+. Treatment with one hormone decreases the subsequent reponse to the others, whereas treatment with A23187 abolishes the hormonal effects upon both Ca2. release and gluconeogenesis. The action of norepinephrine, but not of glucagon, upon CaO+ efflux is prevented by the a-adrenergic antagonist, phenoxybenzamine. The action of norepinephrine is not prevented by the ,B-adrenergic antagonist, propranolol Together these results indicate that the release of Ca2+ from a common pool of exchangeable Ca2+ is important to the action of a variety of hormones on hepatocytes. This Ca2+ pool in the isolated hepatocyte is characterized as being similar in size and having exchange kinetics that are com arable to those reported for the major intracellular pool of Ca + in the intact liver. The possibility that this pool is intramitochondrial calcium is discussed.The effects of glucagon upon gluconeogenesis in intact, perfused liver are mediated through alterations in cyclic nucleotide levels (1,2 Zahlten and Stratman (11). Routinely, cells were suspended at 6.5 X 106 cells per ml in Krebs-Henseleit bicarbonate medium containing 1.5% bovine serum albumin, 10 mM Na lactate, and 1 mM Na pyruvate without added calcium (the free calcium in the cell suspension was estimated at 15-30 gM). Cell suspensions (1.0 or 2.5 ml) were incubated in plastic scintillation vials at 370 in a Dubnoff shaker with agitation (110 cycle/min) under 95% 02/5% CO2.45Ca2+ Exchange Studies. After 40 min of equilibration, 10 gCi of 45Ca2+ was added to each 1.0 ml of cell suspension. Subsequently 20-,ul aliquots were removed for determination of isotopic Ca uptake. Each aliquot was layered on the top of 0.35 ml of 7% bovine serum albumin in 5 mM CaCl2/150 mM NaCl all contained in a 0.45-ml polyethylene centrifuge tube (no. 47/7, Sarstedt Co., Princeton, NJ). The tubes were then centrifuged for 15 sec at 2000 X g. The tips containing the cell pellet were cut off and their contents were flushed into scintillation vials by injecting 0.5 ml of 1% sodium dodecyl sulfate solution. The cells were solubilized with 5 ml of Aquasol and the associated radioactivity was determined.Ca2+ Electrode Studies. Cell suspensions of 2.5 ml were prepared as described above with each vial containing a Ca2+-sensitive and a reference electrod...

Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability

“…The present paper describes the effects of the same agents on the efflux ofenergetically accumulated Ca2+ from rat heart mitochondria. Another thiolspecific reagent, namely N-ethylmaleimide, has also been reported as inducing a loss of Ca2+ from energized mitochondria (Lofrumento & Zanotti, 1978;Chen et al, 1978). These effects of mercurials and N-ethylmaleimide suggest that free thiol groups are required to maintain the membrane in a nonleaky state, and it is this that we have investigated.…”

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

“…These results led to the suggestion that an increased inflow of Ca to the cell is responsible for the increased concentration of cytoplasmic Ca (Assimacopoulos-Jeannet et al 1977;Keppens et al 1977). However, the results of further experiments showed that a-agonists can stimulate glycogen phosphorylase in the absence of extracellular Ca (Blackmore, Brumley, Marks & Exton, 1978), and induce a loss of Ca from liver cells Blackmore, Dehaye, Strickland & Exton, 1979a;Blackmore, Dehaye & Exton, 1979b;Chen, Babcock& Lardy, 1978) and, in particular, from the mitochondria (Blackmore et al 1979a b; Babcock, Chen, Yip & Lardy, 1979). Consequently, Blackmore et al (1979b) have proposed that x-agonists increase the concentration of cytoplasmic Ca principally by stimulating the release of Ca from mitochondria.…”

“…An increase in the cytoplasmic Ca concentration is consistent with the proposal that the activation of glycogen phosphorylase kinase by x-agonists is mediated by an increase in the concentration of Ca in the cytoplasm which surrounds this enzyme . a-Agonists have been shown to cause a net loss of Ca from liver cells Chen et al 1978;Banks, Brown, Burgess, Burnstock, Claret, Cocks & Jenkinson, 1979), and evidence that mitochondrial Ca contributes to the bulk of this loss has been presented (Blackmore et al 1979a, b;Babcock et al 1979). The present results, in which a completely different technique has been used to show that adrenaline causes a loss of Ca from a kinetically defined intracellular compartment of exchangeable Ca, which includes mitochondrial Ca, are consistent with the observations of Blackmore et al (1979b) and Babcock et al (1979).…”

“…When studying the effect of a-agonists on 45Ca exchange with isolated hepatocytes, other authors have generally not commenced sampling until 1 min after addition of 45Ca (Assimacopoulos-Jeannet et al 1977;Foden & Randle, 1978;Chen et al 1978). Sampling at intervals shorter than 1 min after addition of tracer was necessary to define adequately the second exponential term in eqn.…”

Exogenous phospholipase enzymes mimic effects of phenylephrine on Ca2+ transport in hepatocytes

“…A large body of evidence indicates that this is due to the release of Ca2+ from the mitochondria and endoplasmic reticulum (Chen et al, 1978;Babcock et al, 1979;Blackmore et al, 1979;Murphy et al, 1980;Barritt et al, 1981b;Berthon et al, 1981) and enhancement of Ca2+ inflow across the plasma membrane (Keppens et al, 1977;Assimacopoulos-Jeannet et al, 1977;Foden & Randle. 1978;Barritt et al, 1981b).…”