Disruption of inositol phosphate accumulation in cerebellar granule cells by polychlorinated biphenyls: A consequence of altered Ca2+ homeostasis

The present study examined the activation of protein kinase C (PKC) and disruption of Ca2+ homeostasis as potential mechanisms underlying effects of the polychlorinated biphenyl (PCB) congener 2,2'-dichlorobiphenyl (DCB) on inositol phosphate (IP) signaling in cerebellar granule cells. DCB (100 mu M) increased basal IP accumulation in cerebellar granule cells when the extracellular free Ca2+ concentration ([Ca2+](e)) was 0.75 mM but not when [Ca2+](e) was 1 mu M. Ionomycin (0.1 to 30 mu M), a Ca2+ ionophore, also increased basal IP accumulation and [Ca2+](i) in a concentration-dependent manner in cerebellar granule cells in the absence of DCB; increases in basal IP accumulation induced by 100 mu M DCB were not additive with ionomycin. Ionomycin also disrupted carbachol (CARB, 1 mM)-stimulated IP accumulation, A 30-min preincubation with 0.3 or 1.0 mu M ionomycin decreased CARB-stimuIated IP accumulation, whereas simultaneous addition of 1.0 and 10 mu M ionomycin with CARB increased and decreased, respectively, IP accumulation. DCB caused concentration-dependent increases in intracellular free Ca2+ concentration ([Ca2+](i)) in cerebellar granule cells under experimental conditions identical to those used to measure IP accumulation. Following a one-half hour exposure to DMSO, 50 or 100 mu M DCB, the [Ca2+](i) was 36, 103, and 453 nM, respectively. We examined whether direct or indirect activation of PKC underlies DCB-induced inhibition of agonist-stimulated IP accumulation. DCB (100 mu M) did not alter PKC activity in cytosolic or membrane fractions of granule cell homogenates. In intact cells, 50 nM phorbol 12-myristate, 13-acetate (PMA) inhibited CARB-stimulated IP accumulation by 80%, an effect which was blocked completely by the PKC inhibitor bisindolylmaleimide (2 mu M; BIM). However, inhibition of CARB-stimulated IP accumulation (90%) induced by 100 mu M DCB was not relieved by BIM. These results suggest that (1) perturbations of Ca2+ homeostasis may underlie DCB effects on IP accumulation, (2) at a time which corresponds to addition of agonists in IP accumulation assays, [Ca2+](i) is elevated in cerebellar granule cells exposed to DCB, and (3) activation of PKC is not a mechanism by which DCB inhibits agonist-stimulated IP accumulation.