Pulses of extracellular K⁺ produce two cytosolic Ca²⁺ transients that display different temperature dependence and carbonyl cyanide m-chlorophenyl sensitivity in SH-SY5Y cells

In SH-SY5Y cells we have shown that stimulation with high extracellular K⁺ ([K⁺]_e) evokes a transient increase in cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) (K⁺_on) that is triggered by the opening of voltage-dependent Ca²⁺ channels and followed by Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum (Xu, F., Zhang, J., Recio-Pinto, E. and Blanck, T.J., Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients, Anesthesiology, 92 (2000) 1746-56). The removal of high-[K⁺]_e results in a second transient increase in [Ca²⁺]_cyt (K⁺_off) that is independent of extracellular Ca²⁺ (Corrales, A., Montoya, G.J., Sutachan, J.J., Cornillez-Ty, G., Garavito-Aguilar, Z., Xu, F., Blanck, T.J. and Recio-Pinto, E., Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients, Brain Res., 1031 (2005) 174-184). In this study we further characterize the properties of K⁺_off. We found that K⁺_off was detectable at near physiological temperatures (34-36 °C) but, depending on the level of [K⁺]_e, it was undetectable or highly diminished at room temperature. In contrast, K⁺_on was increased by lowering the temperature. Extracellular Na⁺-replacement with K⁺ did not affect K⁺_off, indicating that K⁺_off was not generated by osmolarity changes. Replacement of extracellular Na⁺ with choline⁺ did not affect K⁺_off, indicating that K⁺_off did not result from activity changes of the plasma membrane Na⁺/Ca²⁺ exchanger. Caffeine decreased K⁺_on but not K⁺_off. CCCP (carbonyl cyanide m-chlorophenyl), a protonophore uncoupler that decreases mitochondrial Ca²⁺ uptake, decreased K⁺_on but not K⁺_off. CGP37157, an inhibitor of the mitochondria Na⁺/Ca²⁺ exchanger, decreased K⁺_off when added alone but not when added simultaneously with CCCP. Clonazepam had similar effects as CGP37157. These findings indicate that the generation of K⁺_off is strongly temperature-dependent and its pharmacology is distinct from the [Ca²⁺]_cyt changes observed previously at room temperature.