Discriminating between Capacitative and Arachidonate-activated Ca2+ Entry Pathways in HEK293 Cells

Trevor J Shuttleworth,Jill L Thompson

doi:10.1074/jbc.274.44.31174

Trevor J Shuttleworth, Jill L Thompson

Open Access

https://doi.org/10.1074/jbc.274.44.31174

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Abstract

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca(2+) entry can account for the influx of Ca(2+) seen at low agonist concentrations, such a those typically producing [Ca(2+)](i) oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca(2+) entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca(2+) under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca(2+)-stimulated type VIII adenylyl cyclase for Ca(2+) entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca(2+)](i) or to the release of Ca(2+) from internal stores but was specifically dependent on the induced capacitative entry of Ca(2+). In marked contrast, the arachidonate-induced entry of Ca(2+) completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca(2+) occurs via an entirely distinct influx pathway.

Highlights

The agonist-stimulated entry of extracellular Ca2ϩ plays a critical role in the generation and maintenance of intracellular Ca2ϩ ([Ca2ϩ]i)1 signals resulting from activation of receptors coupled to the phospholipase C/inositol trisphosphate signaling pathway
Our studies have shown that 1) arachidonic acid is generated at the relevant agonist concentrations that are known to produce [Ca2ϩ]i oscillations in the same cells; 2) the addition of low concentrations of exogenous arachidonic acid induces an entry of Ca2ϩ that is entirely independent of store depletion; 3) the inhibition of the agonist-induced generation of arachidonic acid and rapidly blocks the Ca2ϩ entry associated with [Ca2ϩ]i oscillations, yet it is without effect on capacitative Ca2ϩ entry; 4) inhibition of the metabolism of arachidonic acid converts agonist-induced oscillatory [Ca2ϩ]i signals into sustained “plateau” signals, as might be expected if Ca2ϩ entry was further increased by the accumulating arachidonic acid [10, 11]
We have previously shown that the [Ca2ϩ]i signal produced by capacitative Ca2ϩ entry and that produced by the addition of exogenous arachidonic acid demonstrate differences in their sensitivity to a reduction in extracellular pH, suggesting that they may represent distinct entry pathways [11]

Summary

Introduction

The agonist-stimulated entry of extracellular Ca2ϩ plays a critical role in the generation and maintenance of intracellular Ca2ϩ ([Ca2ϩ]i) signals resulting from activation of receptors coupled to the phospholipase C/inositol trisphosphate signaling pathway. Our studies have shown that 1) arachidonic acid is generated at the relevant agonist concentrations that are known to produce [Ca2ϩ]i oscillations in the same cells; 2) the addition of low concentrations of exogenous arachidonic acid induces an entry of Ca2ϩ that is entirely independent of store depletion; 3) the inhibition of the agonist-induced generation of arachidonic acid and rapidly blocks the Ca2ϩ entry associated with [Ca2ϩ]i oscillations, yet it is without effect on capacitative Ca2ϩ entry; 4) inhibition of the metabolism of arachidonic acid converts agonist-induced oscillatory [Ca2ϩ]i signals into sustained “plateau” signals, as might be expected if Ca2ϩ entry was further increased by the accumulating arachidonic acid [10, 11] Based on these findings, it is our contention that arachidonic acid fulfills all the generally accepted criteria for being the second messenger responsible for the regulation of the agonist-activated entry of Ca2ϩ during [Ca2ϩ]i oscillations. This is the same adenylyl cyclase and cell line used by Fagan et al [13] who showed that its activity was markedly and increased by Ca2ϩ entering via the capacitative or store-operated pathway

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