Abstract
A nondisruptive technique developed by Bellomo et al. (Bellomo, G., Jewell, S. A., Thor, H., and Orrenius, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6842-6846) has been used to examine the distribution of calcium ions between mitochondrial and extramitochondrial compartments in the perfused rat liver. The amount of calcium released by the uncoupler 2,4-dinitrophenol from the mitochondrial compartment was 19 +/- 2 nmol X g-1, wet weight, which is equivalent to a total calcium concentration of 3.5 X 10(-4) M in the mitochondria and is by several orders of magnitude smaller than the concentration thought to be present in these organelles. The amount of calcium released from the liver in the presence of the divalent cation ionophore A 23187 was 96 +/- 7 nmol X g-1, wet weight, which is of the same order of magnitude as the amount released by the calcium-dependent hormone vasopressin (97 +/- 11 nmol X g-1, wet weight). Experiments with different sequential combinations of hormone with uncoupler or ionophore reveal that in the perfused liver, in contrast to isolated hepatocytes or isolated mitochondria, the amount of calcium attributable to the mitochondria is too small to account for the calcium released during hormonal stimulation. Consequently extramitochondrial calcium stores are the main source of cellular calcium mobilized under this condition. In addition these findings imply that in the liver several mitochondrial enzymes, e.g. alpha-oxoglutarate dehydrogenase, can be effectively regulated by calcium and that the role of mitochondria in buffering the cytosolic free calcium in vivo has to be reconsidered.
Highlights
Jochen Kleinekeand Hans-Dieter Soling From the Departmentof Clinical Biochmistry, Divisionof Internal Medicine, Universityof Gottingen, 3400 Gottingen, Federal Republicof Germany
It is most likely that ionophore A 23187was 96f7 nmol X g-’, wet weight, under these conditions calcium is taken up mainly by mitowhich is of the same ordeorf magnitude as the amount chondria, fromwhere it can bereleased by uncoupling agents released by the calcium-dependent hormone vasopres- or ionophores [10,11,12, 20, 21]
In the experiments reported here the nondisruptive technique of compartmental analysis [24, 25] has been used for the first time in the intact liver to determine the amount of exchangeable calcium in mitochondria andto investigate whether, and to what extent, mitochondrial calcium stores are involved in calcium release following stimulation with calcium-dependent hormones
Summary
Show that the distribution of calcium between mitochondria and microsomes (as themainextramitochondrial A23187 sensitive ,calcium pool) depends on thecalcium “load” of the cellswhen tested overa range between 0.13 and 1.3 mM exchangeable calcium They concluded that underphysiological conditions, a t a calcium load above 1 nmol x mg”, dry weight (0.26 pmol X g-’, wet weight [17]), mitochondria are the predominant source of cellular calcium mobilized by calcium-dependenthormonessuchasphenylephrineor vasopressin. The onset of the A23187 infusion was immediatelyfollowed by a release of calcium whereas in the presence of DNP a lag phase of 1-2 min was observed Under both conditions,maximum rates of calcium release were approached after 6 min (Fig. 1).Thereafter, thecalcium extrusion from the liver declined to values corresponding to background (not shown) indicating that the calcium stores sensitive toward uncoupler or ionophore had been depleted. Only 18% of total releasable calcium is liberated by uncoupler indicating that in the perfused liver onlythis portion idserived fromthe mitochondria
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