Cyclosporine A Amplifies Ca2+ Signaling Pathway in LLC-PK1 Cells through the Inhibition of Plasma Membrane Ca2+ Pump

Abstract

ABSTRACT. 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 μM 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 μM 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 μM 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 μM 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-immunosuppressive 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. E-mail: vincenzo.calderaro@unina2.it

SERCA2b Activity Is Regulated by Cyclophilins in Human Platelets

“…This finding is different from previous results, but these discrepancies might be attributed to a different effect of CsA on distinct PMCA isoforms (PMCA1 and PMCA4), with PMCA4 being the isoform predominantly expressed in platelets, more likely insensitive to CsA. 17,33,34 To identify the SERCA isoform regulated by CsA, we stimulated platelets with Thr in the presence of specific inhibitors of the different SERCA isoforms in these cells (SERCA2b and SERCA3). 18 -21 When SERCA3 was inhibited by TBHQ, CsA was still able to alter Thr-induced Ca 2ϩ mobilization, including the ability of platelets to accumulate Ca 2ϩ into intracellular stores; however, when SERCA2b was inhibited by TG, CsA was unable to modify the Thr-evoked response, thus indicating that SERCA2b activity was inhibited in the presence of CsA.…”

Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein?Related Activities

“…In microsomal membrane vesicles, the ATPase activity was reduced to about 12% by orthovanadate (37). Cyclosporin A is frequently used as P-gp inhibitor but inhibits also other ATPases such as Na + / K + -ATPases (43) and Ca 2+ -pumps (44,45). One hundred M cyclosporin A reduced the basal ATPase activity of P388/ ADR and P388 rafts to about 60% and 70%, respectively.…”

Comparative Study on the Effects of Cyclosporin A in Renal Cells in Culture

“…Due to the contrast between the efficacy of CSA as an immunosuppressant and its high nephrotoxicity, many studies have been conducted to understand the mechanisms of CSA-induced nephrotoxicity. It has been shown that renal arteriolar constriction [4], direct fibrogenic effect on human tubulointerstitial cells [5], amplification of Ca 2+ signaling pathway [36], inhibition of COX-2 expression in response to low salt intake [37], inhibition of the adaptive responses to hypertonicity occurring during the urinary concentrating mechanism [38]and apoptosis involving mitochondrial injury [39]may contribute to CSA-induced nephrotoxicity.…”