Attenuation of calmodulin regulation evokes Ca²⁺ oscillations: evidence for the involvement of intracellular arachidonate-activated channels and connexons

Abstract

Intracellular Са²⁺ controls its own level by regulation of Ca²⁺ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca²⁺ concentration in different cells. We have found persistent Са²⁺ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1 μM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5 μM CMZ resulted from the release of stored Ca²⁺ and were supported by Са²⁺ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A₂ inhibited by AACOCF₃ was responsible for initial Ca²⁺ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA₂ by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са²⁺ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca²⁺ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са²⁺ release. Incubation with La³⁺ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca²⁺ rise. These results suggest that Ca²⁺-CaM regulation suppresses La³⁺-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca²⁺ oscillations, and connexons are intimately implicated in their generation mechanism.