Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca 2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca 2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca 2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000- g cytosol and membrane fractions. The influx of extracellular Ca 2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca 2+ channel blocker Ni 2+ . Blocking the influx of extracellular Ca 2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 18% of control values for carbachol treatment alone vs. 163 28% for Ni 2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca 2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca 2+ by pretreatment with the cell-permeant Ca 2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 37% of control without BAPTA/AM vs. 136 13% with BAPTA/AM). The effect of direct entry of extracellular Ca 2+ into the cell by K + depolarization was assessed. Incubation of SK-N-SH cells with 60 m M K + elicited an immediate and persistent increase in intracellular free Ca 2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca 2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca 2+ , which releases Ca 2+ from intracellular stores, induced an increase in cytosolic CaM (203 30% of control). The mechanism for the CaM release may involve activation of the isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K + depolarization. These data demonstrate that release of Ca 2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.