Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms

Pflugers Arch. 2000 Mar;439(5):627-33. doi: 10.1007/s004249900205.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Animals
  • Arginine Vasopressin / pharmacology
  • Biological Transport / drug effects
  • Biological Transport / physiology
  • Calcineurin Inhibitors
  • Calcium / pharmacokinetics*
  • Cyclosporine / toxicity*
  • Estrenes / pharmacology
  • Fluorescent Dyes
  • Fura-2
  • Immunosuppressive Agents / toxicity*
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Kidney Tubules, Proximal / cytology
  • LLC-PK1 Cells / drug effects
  • LLC-PK1 Cells / enzymology
  • Nicardipine / pharmacology
  • Phosphodiesterase Inhibitors / pharmacology
  • Pyrrolidinones / pharmacology
  • Renal Agents / pharmacology
  • Signal Transduction / drug effects
  • Swine
  • Tacrolimus / pharmacology
  • Type C Phospholipases / antagonists & inhibitors
  • Vasodilator Agents / pharmacology


  • Calcineurin Inhibitors
  • Estrenes
  • Fluorescent Dyes
  • Immunosuppressive Agents
  • Phosphodiesterase Inhibitors
  • Pyrrolidinones
  • Renal Agents
  • Vasodilator Agents
  • 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
  • Arginine Vasopressin
  • Cyclosporine
  • Inositol 1,4,5-Trisphosphate
  • Adenosine Triphosphate
  • Nicardipine
  • Type C Phospholipases
  • Calcium
  • Fura-2
  • Tacrolimus