Abstract
The immunosuppressive agent, cyclosporine, has been found to augment receptor-stimulated calcium fluxes in isolated hepatocytes. After treatment of Quin 2-loaded hepatocytes with cyclosporine, both the amplitude and duration of the vasopressin-induced rise in the cytosolic free Ca2+ are increased. These effects are dependent upon the concentration and time of exposure of the cells to cyclosporine. Cyclosporine increases both 45Ca2+ influx across the plasma membrane and the cellular calcium content. The total cellular magnesium, sodium, and potassium contents are not affected by cyclosporine. However, cyclosporine treatment, per se, has no apparent effect on the cytosolic free Ca2+ concentration as assayed by Quin 2 fluorescence. The increase in total cell calcium is associated with progressive increases in the calcium content of the endoplasmic reticular and mitochondrial calcium pools. The vasopressin-induced net efflux of Ca2+ from hepatocytes was 2-fold greater after treatment with 10 micrograms/ml cyclosporine for 10 min, but the lag time prior to the onset of Ca2+ efflux was not affected. These results are interpreted on the basis of cyclosporine having a primary effect on increasing the permeability of the plasma membrane to Ca2+, thereby leading to an increase of the calcium content of the hormone-sensitive intracellular calcium pool.
Highlights
From the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, The immunosuppressive agent, cyclosporine, has been found to augment receptor-stimulated calcium fluxes in isolated hepatocytes
The vasopressin-induced net efflux of Ca2’ from hepatocytes was 2-fold greater after treatment with 10 pg/ml cyclosporine for 10 min, but the lag time prior to the onset of Ca” efflux was not affected. These results are interpreted on the basis of cyclosporine having a primary effect on increasing the permeability of the plasma membrane to Ca2’, thereby leading to an increase of the calcium content of the hormone-sensitive intracellular calcium pool
Because of the high affinity of Quin 2 for Ca2+ hormone addition is independent of the hepatocyte calcium (Kd = 115 nM), the fluorescence response of this dye is content over the range from 0.5 to 2.5 nmol/mg cell, dry essentially saturated at free ca2+concentrations above 1p~ weight, of total releasable calcium (25)
Summary
Hepatocyte Preparation-Hepatocytes were prepared from fed, male Sprague-Dawley rats (250-350g) as previously described (23). Resuspended in a modified Hanks' medium containing 137 mM NaCl, 5.4 mM KCl, 0.44 mM KHzPO,, 4.2 mM NaHC03, 0.33 mM Na2HP04,1.3 mM CaC12, 1.0 mMMgC12, 10 mM glucose, and 20 mM Hepes (pH 7.4). Quin 2 Studies-Isolated hepatocytes were loaded with the fluorescent Ca2+indicator Quin 2 as previously described (6). Cells of cellular Ca2+distribution and hormone-sensitive Ca2+release in isolated hepatocytes has been presented in a previous publication from this laboratory (25). Materials-Arg-vasopressin, FCCP, A23187, and Arsenazo I11were purchased from Sigma. Cyclosporine (Sandoz, NJ) was kindly provided by Dr Richard Cheney, Department of Pathology and Laboratory Medicine, University of Pennsylvania. (10 mg, dry weight/ml) were suspended in modified Hanks' medium supplemented with 2% dialyzed bovine serum albumin and equili-
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