Muscarinic Receptor Activation of Arachidonate-mediated Ca2+ Entry in HEK293 Cells Is Independent of Phospholipase C

Abstract

Receptor] i oscillations.Calcium signaling in non-excitable cells is composed of two components: a release of calcium from intracellular stores and an increased entry of calcium from the extracellular medium. The role of inositol 1,4,5-trisphosphate (InsP 3 ), 1 generated as a result of the receptor activation of phospholipase C, in the release of calcium from specific intracellular stores is well established, but the nature of the calcium entry pathway and its regulation is far from clear. To date, discussion of such calcium entry has generally focused on the so-called "capacitative model," in which calcium entry is activated as a direct consequence of the emptying of the intracellular calcium stores and is independent of how this emptying is actually achieved (1, 2). The precise nature of the mechanism for the activation of capacitative entry is, as yet, unclear, but it may involve the release and/or generation of a diffusible signaling molecule within the cell that activates the plasma membrane channels ("store-operated channels") responsible for calcium entry. Alternatively, a more direct molecular coupling between the stores and the plasma membrane channels may occur (3). Although such capacitative entry can be clearly demonstrated in a wide variety of different cells, it is far from certain that such a mechanism is the only one involved in the increase in calcium entry in non-excitable cells following receptor activation (4, 5). For example, many cells show an oscillatory [Ca 2ϩ ] i signal when stimulated at low agonist concentrations (6, 7), and such signals are associated with an enhanced entry of Ca 2ϩ . However, evidence indicates that the activation of capacitative Ca 2ϩ entry generally requires significantly higher levels of agonist-generated InsP 3 than does the release of Ca 2ϩ from the bulk of the agonist-sensitive internal stores (i.e. at low concentrations of InsP 3 , substantial release of Ca 2ϩ from agonistsensitive stores can occur without any activation of capacitative entry) (8 -10). Consequently, it is far from clear that the transitory (and/or incomplete) nature of calcium store depletion during [Ca 2ϩ ] i oscillations would provide an adequate or appropriate signal for the activation of calcium entry via a capacitative mechanism. Such considerations led us previously to investigate the nature of receptor-activated increases in Ca 2ϩ entry during [Ca 2ϩ ] i oscillations in cells from the exocrine avian nasal gland. In these studies, we showed that such Ca 2ϩ entry was non-capacitative in nature (11) and appeared to involve a novel arachidonate-activated pathway (12).In the experiments reported here, we extend our earlier findings to another, more widely used and functionally less highly specialized cell type, namely HEK293 cells. The specific cell line chosen had been stably transfected with the human m3 muscarinic receptor (m3-mAChR), thereby avoiding possible complications resulting from the presence of multiple muscarinic receptor subtypes. Using this cell line, we were able to s...

Ca2+ Selectivity and Fatty Acid Specificity of the Noncapacitative, Arachidonate-regulated Ca2+ (ARC) Channels

“…Cell Distribution-As noted previously, arachidonic acid-dependent noncapacitative Ca 2ϩ entry has been described in a wide variety of different cells, including avian exocrine nasal gland cells (13), Balb-c 3T3 fibroblasts (14), HEK293 cells (15), and A7r5 smooth muscle cells (16). However, it is unclear whether ARC channels, as characterized and defined in our earlier studies on HEK293 cells (17,19), are specifically responsible for the observed Ca 2ϩ entry in all these cases.…”

“…Cell Culture-Cells from the human embryonic kidney cell line HEK293 stably transfected with the human m3 muscarinic receptor (m3-HEK cells; generous gift from Dr. Craig Logsdon, University of Michigan) were cultured in Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics in a 5% CO 2 incubator at 37°C as reported previously (15). Rat basophilic leukemia (RBL-1) cells, COS cells, and HeLa cells were all obtained from the American Type Culture Collection.…”

“…However, previous studies using cyclooxygenase and lipoxygenase inhibitors have indicated that the effects of exogenous arachidonic acid on the noncapacitative entry of Ca 2ϩ reflect the actions of the fatty acid itself and not one of its many bioactive metabolites (15). To confirm that this is true for the activation of the ARC channels themselves, the ability of ETYA (20:4, yne -5,8,11,14), to activate I ARC was examined.…”

“…In particular, a Ca 2ϩ entry pathway that is independent of store-depletion and is regulated by receptoractivated increases in arachidonic acid has been identified in a wide variety of different cell types (13)(14)(15)(16). The conductance underlying this novel noncapacitative Ca 2ϩ entry in HEK293 cells has been described recently (17) and defined as the arachidonate-regulated Ca 2ϩ (ARC) channel.…”

Arachidonic Acid-Induced Channel- and Carrier-Type Ion Transport across Planar Bilayer Lipid Membranes

“…14 AA-regulated Ca 2+ entry has also been reported for non-excitable cells at physiological levels of stimulation, although the mechanism for Ca 2+ transport is unclear. [15][16][17][18] It is now recognized that AA interacts with artificial BLMs and cell membranes to induce AA-facilitated ion transports. The approaches used by these studies detect ions which were transported into the inner aqueous phase of liposomes or cells and, therefore, the transmembrane movement of ions is measured indirectly.…”