Activator of calcium influx proves a slippery customer.

Abstract

On stimulation almost all cells produce an elevation of cytosolic calcium that then acts on a broad range of targets. The sources of the calcium are twofold: intracellular stores usually identified with the endoplasmic reticulum (ER) and the extracellular fluid. We have a clear understanding of at least the basics of the mechanism and molecular basis of calcium release from the ER. Inositol trisphosphate (InsP3) liberated by hydrolysis of the membrane lipid phosphatidylinositol bisphosphate by phospholipase C (PLC) opens the InsP3 receptor (InsP3R), a calcium channel, allowing calcium to flood out. The vast majority of signal-associated calcium release from ER occurs through the InsP3R or the related ryanodine receptor. The mechanism of influx from the extracellular fluid is more variable between cells and is less clearly understood. Some systems are well defined - electrically excitable cells use voltage-operated calcium channels (VOCCs). A few use ionotropic receptor calcium channels (IRCCs); that is, one protein acts both as receptor and channel. Most cells use other mechanisms for receptor-stimulated calcium influx, and it is here that things get murky. In 1993 Putney & Bird pointed out that in many cells calcium influx was secondary to InsP3R activation and was a direct consequence of ER emptying. This process, known as capacitative calcium influx, is easy to study in isolation since directly emptying the stores by agents like thapsigargin will open the channels, which are therefore called store-operated calcium channels (SOCCs). It is now clear that at least some SOCCs are members of the trp family of ion channels opened by a direct physical interaction with InsP3Rs, the latter perhaps adopting an activating configuration when the ER calcium concentration falls (Kiselyov et al. 1998). What remains of agonist-induced calcium influx when the capacitative component is discounted is a ragbag of pathways that can be designated second messenger-operated calcium channels (SMOCCs). Every known second messenger has at some time been proposed to activate a SMOCC. However, only for cAMP and cGMP gated channels are the function and molecular biology well characterized. In this issue of The Journal of Physiology, Broad, Cannon & Taylor examine receptor-stimulated calcium influx in A7r5 smooth muscle cells. By clever use of strontium and gadolinium they are able to observe the operation of SOCCs and SMOCCs independently, and estimate that at physiological agonist concentrations influx through SMOCCs dominates over capacitative calcium influx. The second messenger they implicate is polyunsaturated fatty acid (PUFA). PUFAs, of which arachidonic acid dominates at the mammalian plasma membrane, are found at the two position on the glycerol backbone of phospholipids. Free PUFAs can be directly liberated by phospholipase A; however, Broad et al. implicate a different route in which diacylglycerol (DAG) generated by PLC is further hydrolysed by DAG lipase. PUFAs are the building blocks of eicosanoids. Indeed, eicosanoids open some SMOCCs (Peppelenbosch et al. 1992). However, Broad et al. are confident that their SMOCCs are activated directly by PUFAs because inhibitors of cyclooygenase and lipoxygenase augment rather than block agonist-induced influx. Similarly DAG itself, reported to open some trp family SMOCCs (Hofmann et al. 1999), cannot be the active agent because inhibiting DAG lipase blocks the influx. Broad et al. performed their experiments in the presence of nimodipine or verapamil to block VOCCs of the L type. Paradoxically, exactly that channel acts as a PUFA-activated SMOCC in nerve cells, triggering calcium influx when PLC is activated by the fibroblast growth factor receptor tyrosine kinase (RTK) (Archer et al. 1997). Our realization of the complex relationship between lipids and calcium signalling continues to advance. The result reported by Broad et al., together with similar results in Drosophila photoreceptors (Chyb et al. 1999) and the findings in nerve cells, point the finger squarely at PUFAs as an important part of this story. The pharmacological tools Broad et al. characterize will help all of us find our way through this oily maze.