DIFFERENTIAL-EFFECTS OF ENDOPLASMIC-RETICULUM CA2-ATPASE INHIBITORS ON MADIN-DARBY CANINE KIDNEY-CELLS()

Endoplasmic reticulum (ER) Ca2+-ATPases regulate intracellular free ca lcium ([Ca2+](in)) in renal epithelial cells. Interestingly, treatment of certain cell types with ER Ca2+-ATPase inhibitors such as thapsiga rgin (TG), cyclopiazonic acid (CPA), and 2,5-di-tert-butyl-1,4-benzohy droquinone (BHQ) may result in distinct effects on [Ca2+](in) homeosta sis. In this study, we investigated whether these distinct effects are due to Ca2+ entry after Ca2+ depletion of the ER. Therefore, we coloa ded Madin-Darby canine kidney cells with SNARF-1 and Fura-2 (see text) to simultaneously measure intracellular pH (pH(in)) and [Ca2+](in), r espectively. Subsequently, Madin-Darby canine kidney cells were treate d both in the presence and absence of extracellular Ca2+([Ca2+](ex)) w ith ER-Ca2+-ATPase inhibitors (TG, CPA, and BHQ). In the presence of [ Ca2+](ex), both TG and BHQ caused a sustained increase in [Ca2+](in) a nd a decrease in pH(in). In contrast, CPA induced a transient increase in [Ca2+](in) in the presence of [Ca2+](ex). Acute [Ca2+](ex) removal resulted in a decrease in [Ca2+](in). In the absence of [Ca2+](ex), t he [Ca2+](in) increase induced by either TG, CPA, or BHQ was suppresse d. These data suggest that the TG-, BHQ-, and CPA-sensitive-Ca2+ pool is readily depleted by removal of[Ca2+](ex). Readdition of[Ca2+](ex) i ncreased [Ca2+](in) transiently in the absence of drugs. In the presen ce of CPA, the readdition of [Ca2+](ex) increased [Ca2+](in) also tran siently. In contrast, readdition of [Ca2+](ex) resulted in a sustained increase in [Ca2+](in) when either TG or BHQ was present. This latter effect was reversed by subsequent addition of CPA (in the presence of [Ca2+](ex)), or when CPA was added prior to the readdition of [Ca2+]( ex). These data suggest that CPA, unlike TG and BHQ, also decreases Ca 2+ entry across the plasma membrane. Altogether, our data indicate tha t ER Ca2+-ATPase inhibitors exhibit differential effects on [Ca2+](in) homeostasis in Madin-Darby canine kidney cells.