Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca²⁺ in insulin-secreting -cells

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP₂). Here, we have investigated the influence of Ca²⁺ on receptor-triggered PLC activity in individual insulin-secreting -cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP₂/IP₃-binding pleckstrin homology domain from PLC1, and the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 -cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca²⁺ or in the presence of the non-selective Ca²⁺ channel inhibitor La³⁺. The Ca²⁺-dependent sustained phase of PLC activity did not require voltage-gated Ca²⁺ influx, as hyperpolarization with diazoxide or direct Ca²⁺ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca²⁺ stores with the sarco(endo)plasmic reticulum Ca²⁺-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca²⁺ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca²⁺-deficient medium. Analogous suppressions were observed after loading cells with the Ca²⁺ chelator BAPTA. Stimulation of primary mouse pancreatic -cells with glucagon elicited pronounced [Ca²⁺]_i spikes, reflecting protein kinase A-mediated activation of Ca²⁺-induced Ca²⁺ release via IP₃ receptors. These [Ca²⁺]_i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca²⁺ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.