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

Receptor-enhanced entry of Ca2+in non-excitable cells is generally ascribed to a capacitative mechanism in which the activation of the entry pathway is specifically dependent on the emptying of agonist-sensitive intracellular Ca2+ stores. Although such entry can be clearly demonstrated under conditions of maximal or near-maximal stimulation, it is uncertain whether such a mechanism can operate during the oscillatory [Ca2+]i signals that are frequently seen following stimulation with low concentrations of agonists. In this study, we report that the stimulation of human m3 muscarinic receptors stably transfected into HEK293 cells results in the appearance of a novel arachidonate-mediated Ca2+ entry pathway. We show that the generation of arachidonic acid and the activation of this pathway are specifically associated with stimulation at the low agonist concentrations that typically give rise to oscillatory [Ca2+]i signals. At such agonist concentrations, however, the generation of arachidonic acid is independent of the simultaneous activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway. We further show that the arachidonate-mediated Ca2+ entry demonstrates characteristics that distinguish it from the corresponding capacitative pathway in the same cells and therefore is likely to represent an entirely distinct pathway that is specifically responsible for the receptor-enhanced entry of Ca2+ during [Ca2+]i oscillations. Receptor-enhanced entry of Ca2+in non-excitable cells is generally ascribed to a capacitative mechanism in which the activation of the entry pathway is specifically dependent on the emptying of agonist-sensitive intracellular Ca2+ stores. Although such entry can be clearly demonstrated under conditions of maximal or near-maximal stimulation, it is uncertain whether such a mechanism can operate during the oscillatory [Ca2+]i signals that are frequently seen following stimulation with low concentrations of agonists. In this study, we report that the stimulation of human m3 muscarinic receptors stably transfected into HEK293 cells results in the appearance of a novel arachidonate-mediated Ca2+ entry pathway. We show that the generation of arachidonic acid and the activation of this pathway are specifically associated with stimulation at the low agonist concentrations that typically give rise to oscillatory [Ca2+]i signals. At such agonist concentrations, however, the generation of arachidonic acid is independent of the simultaneous activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway. We further show that the arachidonate-mediated Ca2+ entry demonstrates characteristics that distinguish it from the corresponding capacitative pathway in the same cells and therefore is likely to represent an entirely distinct pathway that is specifically responsible for the receptor-enhanced entry of Ca2+ during [Ca2+]i oscillations. inositol 1,4,5-trisphosphate intracellular free calcium ion concentration muscarinic receptor phospholipase C phospholipase D phospholipase A2 protein kinase C 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 5,8,11,14-eicosatetraynoic acid phorbol 12-myristate 13-acetate. 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 (InsP3),1generated 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 (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2107) Google Scholar, 2Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1261) Google Scholar). 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 (3Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1048) Google Scholar). 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 (4Penner R. Fasolato C. Hoth M. Curr. Opin. Neurobiol. 1993; 3: 368-374Crossref PubMed Scopus (126) Google Scholar, 5Fasolato C. Innocenti B. Pozzan T. Trends Pharmacol. Sci. 1994; 15: 77-83Abstract Full Text PDF PubMed Scopus (438) Google Scholar). For example, many cells show an oscillatory [Ca2+]i signal when stimulated at low agonist concentrations (6Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6173) Google Scholar, 7Berridge M.J. Biochem. J. 1994; 302: 545-550Crossref PubMed Scopus (32) Google Scholar), and such signals are associated with an enhanced entry of Ca2+. However, evidence indicates that the activation of capacitative Ca2+ entry generally requires significantly higher levels of agonist-generated InsP3 than does the release of Ca2+ from the bulk of the agonist-sensitive internal stores (i.e. at low concentrations of InsP3, substantial release of Ca2+ from agonist-sensitive stores can occur without any activation of capacitative entry) (8Parekh A. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9Hartzell C. J. Gen. Physiol. 1996; 108: 157-175Crossref PubMed Scopus (87) Google Scholar, 10Liu K.-Q. Bunnell S.C. Gurniak C.B. Berg L.J. J. Exp. Med. 1998; 187: 1721-1727Crossref PubMed Scopus (273) Google Scholar). Consequently, it is far from clear that the transitory (and/or incomplete) nature of calcium store depletion during [Ca2+]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 Ca2+ entry during [Ca2+]ioscillations in cells from the exocrine avian nasal gland. In these studies, we showed that such Ca2+ entry was non-capacitative in nature (11Shuttleworth T.J. Thompson J.L. Biochem J. 1996; 316: 819-824Crossref PubMed Scopus (50) Google Scholar) and appeared to involve a novel arachidonate-activated pathway (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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 show that the receptor-mediated generation of arachidonic acid is independent of the simultaneous activation of the PLC-InsP3 pathway, indicating that the m3-mAChR is capable of coupling both to the activation of PLC and the generation of arachidonic acid in a separate but parallel manner. Furthermore, we show that the arachidonate-mediated calcium entry pathway demonstrates characteristics that distinguish it from the more well known capacitative or store-operated entry of calcium. Cultures of the human embryonic kidney cell line HEK293 that were stably transfected with the human m3 muscarinic receptor (m3-HEK cells) were obtained from Dr. Craig Logsdon (University of Michigan, Ann Arbor, MI) (see Ref. 13Yang J. Williams J.A. Yule D.I. Logsdon C.D. Mol. Pharmacol. 1995; 48: 477-485PubMed Google Scholar for details). These cells were cultured under standard conditions in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and antibiotics. Changes in [Ca2+]i in single individual cells were determined following loading with the fluorescent probe indo-1. Loading was achieved in for in were for a further at to for of the [Ca2+]i was as the of the as at and at as previously S.C. T.J. 1994; PubMed Scopus Google T.J. Cell Calcium. 1994; 15: PubMed Scopus Google Scholar). The for the simultaneous of in [Ca2+]i and was as previously T.J. Cell Calcium. 1994; 15: PubMed Scopus Google Scholar). In the experiments to the of increases in cells were with the Ca2+ in for experiments showed that this was to any [Ca2+]i signals in cells stimulated with concentrations of to of arachidonic acid release were as previously (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). cells were cultured in acid was to and was The cells were with serum to of agonist and/or as acid during a was determined of the and to the in the cells, determined following with inositol were determined in a manner. cultured in were in the presence of in To of inositol generation and experiments were in the presence of following a in the same concentration of At the of the the was and with of for on the was with and with and the was to The were with the inositol with in of the inositol were inositol generation is as in the determined from of the following with 5,8,11,14-eicosatetraynoic acid and were from and the phorbol phorbol 12-myristate was from acid and were from We determined whether activation of the transfected m3 muscarinic receptor in cells was to the generation of arachidonic as previously for the muscarinic receptor in avian nasal cells (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). that of the muscarinic receptor agonist to cells with acid in a increase in arachidonic acid increase was dependent on agonist concentration and was clearly at agonist concentrations to the for [Ca2+]i signals in these cells of were on our earlier (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), we that the receptor activation of arachidonic acid generation and the associated increase in calcium entry were specifically associated with stimulation at low agonist concentrations, oscillatory [Ca2+]i signals are To the of arachidonic acid on [Ca2+]i signaling in cells, low concentrations of arachidonic acid were to cells. of as as acid a of of in a increase in [Ca2+]i Such an increase in [Ca2+]i result from an increase in calcium entry from the extracellular medium or from release of calcium from intracellular stores. To distinguish between these two was used as a of calcium entry In the presence of of arachidonic acid to increase and only when the was was an increase in [Ca2+]i The to any increase in [Ca2+]i in the presence of that such an increase an increase in These the that arachidonic acid was an increase in [Ca2+]i of the plasma membrane calcium At concentrations or is known to the plasma membrane but such an would be to further increase to such an To that arachidonic acid was calcium simultaneous of in [Ca2+]i and were In extracellular is used as a for Ca2+ at low concentrations, it many of Ca2+ the the to and the of such as and the of can be used as an of the of at to in C, the increase in [Ca2+]i was associated with a simultaneous increase in the of indicating that Ca2+ entry is To the of the generation of arachidonic acid and the increase in Ca2+ entry on [Ca2+]i signals in cells, we the of the as an specific and of arachidonic acid generation T. T. Biochem. Pharmacol. PubMed Scopus Google Scholar, T. T. T. Biochem. Pharmacol. PubMed Scopus Google Scholar), as demonstrated in our earlier on the avian nasal (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). with studies, had on inositol generation in cells, but the simultaneous generation of arachidonic acid of to cells an oscillatory [Ca2+]i signal in to low concentrations of in an of the which with and on of the is to the seen in these cells Ca2+ entry was during an oscillatory [Ca2+]i For example, of to a cell during an oscillatory [Ca2+]i to in the of the which on of the To that to during oscillations was Ca2+ we used the previously reported us (11Shuttleworth T.J. Thompson J.L. Biochem J. 1996; 316: 819-824Crossref PubMed Scopus (50) Google Scholar, S.C. T.J. 1994; PubMed Scopus Google Scholar) and R. J. Physiol. 1990; Scopus Google Scholar, J. Physiol. 1995; Scopus Google Scholar), oscillations in [Ca2+]i were associated with a enhanced of a Ca2+ entry. C that of an of this of which was on of the The on Ca2+ entry) were associated with the of the be that the to of Ca2+ entry during oscillatory [Ca2+]i signals seen is a in cell of which to for of extracellular Such likely in and in the of the Ca2+ on the plasma membrane and on the intracellular stores well as levels of However, such be as indicating that Ca2+ entry or role in oscillatory [Ca2+]i signals in in Ca2+ entry the of [Ca2+]i oscillations 1993; PubMed Scopus (144) Google of on [Ca2+]i oscillations. cell with was stimulated with to an oscillatory in as in the the was of Ca2+ entry of on C, of on the of in an for In cells, arachidonic acid frequently resulting in the of a variety of that known to signaling To whether the were to arachidonic acid or to one of the many of arachidonic acid we the of of the pathway, and the pathway, of of these to cells [Ca2+]i signals an increase in which into a of [Ca2+]i and the activation of Ca2+ entry was dependent on the of arachidonic acid these would be to result in the or of the [Ca2+]i as seen when arachidonic acid generation was the are clearly with the that of these for arachidonic acid are responsible for the increases in Ca2+ entry during oscillations. The seen however, with a increase in arachidonic acid levels in stimulated cells to of the responsible for its Such increases be to further increase Ca2+ resulting in a of as The direct of arachidonic acid was further in experiments that of the arachidonic acid to cells an increase in to that seen with arachidonic acid In these it was that the of were frequently more in than seen with arachidonic but higher concentrations Such are with the to arachidonic acid in the for arachidonic acid its the that it is arachidonic acid that is the responsible for the on Ca2+ entry. that both and are in HEK293 cells in to increases in levels of arachidonic The m3-mAChR is known to via a of the of to the activation of phospholipase C and the generation of and inositol The activates protein kinase C, the is to the of [Ca2+]i signals. To whether the increased generation of arachidonic acid was a of the simultaneous activation of this PLC pathway, were The possible of increases in arachidonic acid generation in cells with the calcium under and to stimulation with in the generation of arachidonic acid was in such cells, indicating that this was dependent on the receptor-activated [Ca2+]i signals. We the possible of the generation of and the activation of in the arachidonic acid stimulation of of the phorbol for in the presence of in a but of arachidonic acid release in cells in in with the arachidonic acid release seen following of These that the of activation to be to the stimulation of arachidonic acid release Although the precise for this is these clearly that the activation of does result in the stimulation of arachidonic acid on the from cells and cells, it that the increase in [Ca2+]i the activation of is involved in the activation of arachidonic acid we were that the experiments may entirely a of the PLC pathway. For example, it has been reported in cell that the stimulation of can a on receptor-activated PLC was therefore possible that the of to arachidonic acid release may the of PLC on cells that with for significantly the increase in inositol an with a of PLC Alternatively, it is possible that the activation of arachidonic acid generation requires an increase in both We therefore an of experiments the of the J. Pharmacol. Exp. 1990; Google Scholar) on activation of arachidonic acid Although entirely has been used as an of PLC and with we that in the presence of inositol generation was However, under arachidonic acid generation was These that the receptor-activated arachidonic acid generation is a of any PLC of on inositol generation and arachidonic acid release inositol generation and arachidonic acid release were as under and to stimulation with at the concentrations cells were to for are as the increase in inositol generation or arachidonic acid release the corresponding in the or presence of are the acid and inositol the activation of the capacitative mechanism of Ca2+ entry is dependent on the emptying of intracellular stores of calcium how this is However, under of receptor stimulation, the of the intracellular Ca2+ stores as a result of the generation of inositol which activates channels on the stores. The that activation of arachidonic acid generation is a of the simultaneous activation of PLC (see that the arachidonate-activated Ca2+ entry is to be of a capacitative mechanism. is further the that as is an of receptor-activated arachidonic acid has on the capacitative entry of Ca2+ were obtained in our earlier on avian nasal cells (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and been reported for the in cells 1993; PubMed Scopus Google Scholar). However, this does entirely the that arachidonic acid is involved in in the capacitative mechanism. For example, the depletion of the stores may result in such a stimulation of the capacitative mechanism that is able to it Alternatively, a single of Ca2+ entry may be capable of activated the capacitative its or arachidonic acid on the To these we the of extracellular on the Ca2+ entry activated and arachidonic in extracellular been to a on capacitative Ca2+ entry in a of different cell J. Physiol. PubMed Google Scholar, J. Physiol. 1995; Google Scholar, Biochem. 1996; PubMed Scopus Google Scholar, M.J. C. J. Physiol. 1996; PubMed Scopus Google Scholar), and in the of the store-operated this has been to a direct of extracellular on the A. J. Biol. 1995; PubMed Scopus Google Scholar). in the of the to a in the [Ca2+]i seen in cells, with an of capacitative entry. was on of extracellular in extracellular however, without on the in cells to arachidonic acid in the of extracellular indicates that the Ca2+ entry activated arachidonic acid is to be the same as that activated depletion of intracellular stores. In the reported here, we that activation of the m3-mAChR stably transfected in HEK293 cells results in the generation of arachidonic acid and that this specifically activates a Ca2+ entry pathway that is to the regulation of the oscillatory [Ca2+]i signals low concentrations of appropriate agonists. Although for the generation of arachidonic acid were it is clear that this a to muscarinic that is to that demonstrated the activation of PLC and is therefore with a role in the regulation or generation of [Ca2+]i signals. We that of the generation of arachidonic acid results in the of receptor-enhanced Ca2+ entry and the of the oscillatory [Ca2+]i The further that this is an of arachidonic acid and of a of the for arachidonic our on the activation of Ca2+ entry of arachidonic such has been reported to a variety of on [Ca2+]i signals in a many different cells, on Ca2+ release from intracellular stores Cell Calcium. 1995; PubMed Scopus Google Scholar) as well as on Ca2+ entry A. A. Biochem. J. 1995; PubMed Scopus Google Scholar). However, of these involved the of concentrations of the of a variety of In the on Ca2+ entry we are seen at low concentrations of arachidonic acid that extracellular via the medium is only a of the specific receptor-mediated intracellular generation of arachidonic this is likely to represent a of the of the Ca2+ entry pathway to this The we the receptor-activated generation of arachidonic with for its We that low concentrations of arachidonic acid a Ca2+ entry pathway and that of its generation a parallel of the receptor-activated entry of Ca2+ seen during of these are with the widely as a as a and us to that arachidonic acid is the intracellular signal responsible for the activation of Ca2+ entry during We previously reported a entry of Ca2+ during oscillatory [Ca2+]i to muscarinic receptor activation in the exocrine cells of the avian nasal (12Shuttleworth T.J. J. Biol. Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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Chem. 1996; 271: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), we showed that its activation was independent of any release of Ca2+ from intracellular stores. In the at the low agonist concentrations that typically give rise to oscillatory [Ca2+]i levels of InsP3 were adequate to any release of Ca2+ from the and such release was dependent on the of the arachidonate-mediated entry of Ca2+. However, the that the entry we in dependent on a capacitative mechanism that was activated the emptying of a specific of the agonist-sensitive store was in our evidence this is as from a variety of different cells that such a its would higher concentrations of InsP3 than to the bulk of the agonist-sensitive stores in the cell (8Parekh A. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9Hartzell C. J. Gen. Physiol. 1996; 108: 157-175Crossref PubMed Scopus (87) Google Scholar, 10Liu K.-Q. Bunnell S.C. 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We showed with from different cell such capacitative entry in HEK293 cells is to in extracellular In the Ca2+ entry activated arachidonic acid in the same cells is entirely to such in extracellular that the on the capacitative entry are reported to a direct on the store-operated Ca2+ in the of such a clear indicates that the arachidonate-activated pathway be distinct from that activated store is further our that the receptor-mediated increase in arachidonic acid generation we is independent of the simultaneous activation of the PLC-InsP3 pathway (see such a is with the that the muscarinic receptor stimulation of the arachidonate-mediated pathway is of any release of Ca2+. from a wide of different cell that are generally two of receptor-mediated increases in arachidonic acid The the of on in is from the of as a result of increases in J. Google Scholar, T. T. T. 1993; PubMed Scopus Google Scholar) or PLC J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. 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In the we clearly show that an increase in [Ca2+]i the activation of or was for the generation of arachidonic In this it is to that the of generally maximal concentrations of the Consequently, the the presence of these activation in HEK293 cells, it is clear that a a can any role in the activation of arachidonic acid generation under the specific conditions at low concentrations of muscarinic agonists. was further the obtained the which that the stimulation of arachidonic acid generation we is of PLC the HEK293 cell line only a single muscarinic receptor the show that this receptor is capable of coupling both to the activation of PLC and the generation of arachidonic acid in a separate but parallel manner. We therefore that the two signaling are activated the the role that such arachidonic acid generation in the activation of the Ca2+ entry to receptor-activated oscillatory [Ca2+]i signals in these and cells, the of the mechanism responsible for coupling of muscarinic receptors to arachidonic acid generation at these levels of stimulation is clearly of We Dr. Craig Logsdon for the HEK293 cells stably transfected with the human m3 muscarinic