Agonist-specific participation of SOC and ARC channels and iPLA₂ in the regulation of Ca²⁺ entry during oscillatory responses in adipocytes

Abstract

Activation of Ca²⁺ entry upon cell stimulation by agonists can be accomplished by different mechanisms, including store-operated calcium entry (SOCE) and the action of phospholipase A₂ (PLA₂) products. In adipocytes there are two relatively independent pathways for Ca²⁺ mobilization from intracellular stores. In the present work we studied, whether these pathways are coupled with particular mechanisms of Ca²⁺ entry into the cell. It is shown that acetylcholine (ACh) induces oscillatory responses in cytosolic Ca²⁺ concentration independently of SOCE inhibition by YM-58483 or 2-APB. These oscillations were abolished by the addition of La³⁺, which inhibits both store-operated calcium (SOC) and ARC channels (regulated by arachidonic acid, AA). The responses to ACh were suppressed by AACOCF₃ inhibiting PLA₂ of type IV and VIA (iPLA₂). Oscillations evoked by fetal bovine serum (FBS) were distinguished by the baseline spiking and, in contrast, were terminated by YM-58483 and La³⁺ but were not dependent on AACOCF₃. The same cell could respond to ACh and FBS at their sequential addition in any order with the intermediate wash. Oscillatory responses of a similar (base or elevated line) form to phenylephrine decayed only gradually after the inhibition of phospholipase C or inositol 1,4,5-trisphosphate receptor, and were partially attenuated by the inhibitors YM-58483 and La³⁺ without appreciable influence of AACOCF₃. AA at concentrations 1–10 μM caused oscillations when added after spontaneous cessation of ACh-induced oscillations or itself, with a discernible effect produced at lower concentrations after ACh. Calmodulin inhibitor R24571 caused oscillations, which could be suppressed by YM-58483 or AACOCF₃ suggesting activation of SOCE and iPLA₂, respectively. Taken together, these results indicate that the mechanism of Ca²⁺ entry activation depends on the signaling pathway involved by an agonist. ACh does not employ SOCE but activates PLA₂ with probable participation of the form VIA, which entails the action of its product(s) on ARC channels and likely on lysophospholipid-activated channels. FBS acts through SOCE without participation of PLA₂. These two versions can coexist in the case of phenylephrine.