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.