Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms.
Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50(: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10(-6) mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10(-6) mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52+/-16% (n=10) and 13+/-10% (n=13) of control respectively. Diltiazem and verapamil (10(-6) mol/l) were also effective, but flufenamate (10(-4) mol/l), Gd3+ (10(-5) mol/l) and La3+ (10(-5) mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate-(InsP3-) sensitive Ca2+ stores by extracellular ATP (10(4) mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP3 concentration. Furthermore, inhibition of phospholipase-Cbeta (PLCbeta) by U73122 (2x10(-5) mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP3-sensitive Ca2+ stores, the InsP3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCbeta activity and InsP3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.
Gordjani
N
N
Universitäts-Kinderklinik, Freiburg, Germany. gordjani@kkl200.ukl.uni-freiburg.de
Epting
T
T
Fischer-Riepe
P
P
Greger
R F
RF
Brandis
M
M
Leipziger
J
J
Nitschke
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000493:pharmacokinetics*; D016572:Cyclosporine / Q000633:toxicity*; D004963:Estrenes / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D007166:Immunosuppressive Agents / Q000633:toxicity*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology; D018374:LLC-PK1 Cells / Q000187:drug effects / Q000201:enzymology; D009529:Nicardipine / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012076:Renal Agents / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D016559:Tacrolimus / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014665:Vasodilator Agents / Q000494:pharmacology
D065095:Calcineurin Inhibitors; D004963:Estrenes; D005456:Fluorescent Dyes; D007166:Immunosuppressive Agents; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012076:Renal Agents; D014665:Vasodilator Agents; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D001127:Arginine Vasopressin; D016572:Cyclosporine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D009529:Nicardipine; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2; D016559:Tacrolimus