CYTOSOLIC CALMODULIN IS INCREASED IN SK-N-SH HUMAN NEUROBLASTOMA-CELLS DUE TO RELEASE OF CALCIUM FROM INTRACELLULAR STORES

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastom a cell line SK-N-SH, Increasing the intracellular Ca2+ concentration w ith ionomycin also elevates cytosolic CaM, The aim of this study was t o investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N -SH cells, Stimulus-mediated changes in intracellular Ca2+ were monito red in fura-2-loaded cells, and CaM was measured by radioimmunoassay i n the 100,000-g cytosol and membrane fractions, The influx of extracel lular Ca2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carba chol-mediated increases in cytosolic CaM (168 +/- 18% of control Value s for carbachol treatment alone vs, 163 +/- 28% for Ni2+ and carbachol ) or decreases in membrane CaM, Similarly, removal of extracellular Ca 2+ from the medium did not affect carbachol-mediated increases in cyto solic CaM (168 +/- 26% of control), On the other hand, prevention of t he carbachol-mediated increase of intracellular free Ca2+ by pretreat ment with the cell-permeant Ca2+ chelator BAPTA/AM did attenuate the c arbachol-mediated increase in cytosolic CaM (221 +/- 37% of control wi thout BAPTA/AM vs. 136 +/- 13% with BAPTA/AM), The effect of direct en try of extracellular Ca2+ into the cell by K+ depolarization was asses sed, Incubation of SK-N-SH cells with 60 mM K+ elicited an immediate a nd persistent increase in intracellular free Ca2+ concentration, but t here was no corresponding alteration in CaM localization, On the contr ary, in cells where intracellular Ca2+ was directly elevated by thapsi gargin 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 Ca2+, which releases Ca2+ from intrace llular stores, induced an increase in cytosolic CaM (203 +/- 30% of co ntrol). The mechanism for the CaM release may involve activation of th e alpha isozyme of protein kinase C, which was translocated from cytos ol to membranes much more profoundly by thapsigargin than by K+ depola rization, These data demonstrate that release of Ca2+ from the intrace llular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.