Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump

Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca2+ signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1: the assay of the in vitro activity of the plasma membrane Ca2+ pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 muM did not change the basal level of intracellular calcium ([Ca2+](i)), whereas it affected the [Ca2+](i) response to thapsigargin (TG), a powerful inhibitor of microsomal Ca2+ pump. In control studies, 5 muM TG produced a biphasic response: [Ca2+](i) peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca2+](i), which remains above basal. However, it became evident that CsA strengthened the Ca2+ response to TG because the addition of 5 muM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca2+](i) = 156 +/- 4.84 versus 130 +/- 3.28 nmol, mean +/- SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 muM calmodulin (CaM), a significant decrease of plasma membrane Ca2+ pump (PMCA) activity (46.96 +/- 0.26 versus 53.48 +/- 1.96 nmol . mg of protein(-1). min(-1), n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immumosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca2+ export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca2+](i) response after exposure to agents that trigger intracellular Ca2+ release. The increased cell sensitivity during Ca2+ signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA.