Multiple effects of 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxy-phenethyl]-1H-imidazole hydrochloride [SKF 96365) on Ca2+ signaling in MDCK cells:depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[beta-[3-(4-methoxyphenyl)propoxyl-4-methoxyphenethyl]-1H-i midazole hydrochloride (SKF 96365) on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25-100 mu M evoked a robust [Ca2+](i) transient in a dose-dependent manner, measured by fura-2 fluorime try. A concentration of 10 mu M SKF 96365 did not have an effect. The trans ient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca2+ medium. Removal of extracellular Ca2+ reduced the Ca2+ s ignals evoked by 50-100 mu M SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca2+ release and al so extracellular Ca2+ influx. A concentration of 100 mu M SKF 96365 caused significant Mn2+ quench of fura-2 fluorescence, which was partly inhibited by La3+ (1 mM) or Gd3+ (0.1 mM), indicating that the SKF 96365-induced Ca2 influx had two components: one is sensitive to La3+ (1 mM) or Gd3+ (0.1 mM ), the other is not. The internal Ca2+ source for the SKF 96365-induced [Ca 2+](i) transient was the endoplasmic reticulum Ca2+ store because, pretreat ment with thapsigargin and cyclopiazonic acid, two inhibitors of the endopl asmic reticulum Ca2+ pump nearly abolished the SKF 96365-induced [Ca2+](i) increase in Ca2+- free medium. In contrast, pretreatment with 100 CIM SKF 9 6365 only partly depleted the thapsigargin-sensitive Ca2+ store. Addition o f 10 mM Ca2+ induced a significant [Ca2+](i) increase after prior incubatio n with 100 mu M SKF 96365 in Ca2+-free medium, demonstrating that SKF 96365 induced capacitative Ca2+ entry. This capacitative Ca2+ entry was about 40 % of that induced by 1 mu M thapsigargin. Additional to inducing its own ca pacitative Ca2+ entry, 100 mu M SKF 96365 partly inhibited thapsigargin- or uridine trisphosphate (UTP)-induced capacitative Ca2+ entry. We also inves tigated the mechanisms underlying the decay of the SKF 96365-induced [Ca2+] (i) transient. Inhibition of the plasma membrane Ca2+ pump with La3+ or Gd3 +, or lowering extracellular Na+ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca2+](i) transient. In contrast, the mitochondrial u ncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca2-free medium, the thapsigargin-induced [Ca2+](i) increase was greatly reduc ed by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca2+ entry in many cell t ypes, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca2signaling, it induced a considerable increase in [Ca2+](i) by releasing Ca2 + from the endoplasmic reticulum store followed by capacitative Ca2+ entry. It also caused a direct Ca2+ entry. The decay of the SKF 96365 response wa s significantly governed by efflux via the plasma membrane Ca2+ pump or Na/Ca2+ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacit ative Ca2+ entry.