Characterization of calcium oscillations in normal and benzo[a]pyrene-treated clone 9 cells

Intracellular Ca²⁺ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[α]pyrene (BaP) alters Ca²⁺ signaling patterns in these cells. Clone 9 cells exhibit an initial Ca²⁺ spike, followed by Ca²⁺ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca²⁺ generated from the inositol trisphosphate (InsP₃)-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca²⁺ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca²⁺ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP₃)-sensitive stores and that extra-cellular Ca²⁺ contributes to refilling of this intracellular Ca²⁺ pool. The frequency of Ca²⁺ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca²⁺ levels were elevated and the frequency of Ca²⁺ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca²⁺ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K⁺ channels. This was confirmed by showing that inhibition of the K⁺ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca²⁺ oscillations, and potentially decrease the toxicity of BaP.