Differential Effects of Endoplasmic Reticulum Ca<sup>2+</sup>-ATPase Inhibitors on Madin-Darby Canine Kidney Cells

Abstract

Endoplasmic reticulum (ER) Ca2+-ATPases regulate intracellular free calcium ([Ca2+]in) in renal epithelial cells. Interestingly, treatment of certain cell types with ER Ca2+-ATPase inhibitors such as thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-tert-butyl-l,4-benzohydroquinone (BHQ) may result in distinct effects on [Ca2+]m homeostasis. In this study, we investigated whether these distinct effects are due to Ca2+ entry after Ca2+ depletion of the ER. Therefore, we coloaded Madin-Darby canine kidney cells with SNARF-1 and Fura-2 (see text) to simultaneously measure intracellular pH (pHin) and [Ca2+]in, respectively. Subsequently, Madin-Darby canine kidney cells were treated both in the presence and absence of extracellular Ca2+ ([Ca2+]ex) with 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 and a decrease in pHιn. In contrast, CPA induced a transient increase in [Ca2+]m in the presence of [Ca2+]ex. Acute [Ca2+]ex removal resulted in a decrease in [Ca2+]in. In the absence of [Ca2+]ex, the [Ca2+]in increase induced by either TG, CPA, or BHQ was suppressed. These data suggest that the TG-, BHQ-, and CPA-sensitive-Ca2+ pool is readily depleted by removal of [Ca2+]ex. Readdition of [Ca2+]ex increased [Ca2+]ιn transiently in the absence of drugs. In the presence of CPA, the readdition of [Ca2+]ex increased [Ca2+]in also transiently. 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 Ca2+ entry across the plasma membrane. Altogether, our data indicate that ER Ca2+-ATPase inhibitors exhibit differential effects on [Ca2+]in homeostasis in Madin-Darby canine kidney cells.