Abstract: We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 μm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 μm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 μm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 μmGd3+, as well as 30–100 μm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 μm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 μm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+ entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid. We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 μm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 μm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 μm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 μmGd3+, as well as 30–100 μm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 μm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 μm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+ entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid. inositol 1,4,5-trisphosphate endoplasmic reticulum phospholipase A2 HEPES-buffered physiological saline solution 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 2-aminoethyoxydiphenyl borane sarcoplasmic-endoplasmic reticulum calcium ATPase An increase in the level of intracellular free Ca2+concentration ([Ca2+]i) plays a central role in signal transduction for a variety of cellular functions, including cellular secretion, muscle contraction, cell growth and differentiation, and apoptosis. Changes in [Ca2+]i in mammalian cells are mediated by mobilization of Ca2+ from internal Ca2+ stores and/or by entry of Ca2+ from the extracellular space. In many nonexcitable cells Ca2+ signaling by neurotransmitters or hormones is initiated through cell membrane receptors coupled to phospholipase C and the production of inositol 1,4,5-trisphosphate (IP3)1 (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar). IP3 as a second messenger produces a biphasic Ca2+ signal, comprised of an initial Ca2+release from endoplasmic reticulum (ER), followed by a sustained Ca2+ plateau due to Ca2+ entry across the plasma membrane. This Ca2+ entry usually results from the depletion of intracellular Ca2+ stores and in such instances is termed "capacitative Ca2+ entry" (2Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2109) Google Scholar, 3Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). This mode of entry presumably involves store-operated Ca2+channels in the plasma membrane. Although capacitative calcium entry has been documented in many different cell types, the signal by which store emptying activates store-operated Ca2+ channels remains uncertain (4Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1049) Google Scholar, 5Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In addition to the sustained elevation of [Ca2+]iseen with high agonist concentrations, a more complex and subtle repetitive cycling of [Ca2+]i, known as [Ca2+]i spiking or [Ca2+]ioscillations, often results from lower concentrations of agonists in some cell types (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 6Prince W.T. Berridge M.J. J. Exp. Biol. 1973; 58: 367-384Google Scholar, 7Thomas A.P. St J. Bird G. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB. J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar). The characteristics of [Ca2+]i oscillations vary widely among different cell types, and a single mechanism may be insufficient to account for the variety of observed responses (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 7Thomas A.P. St J. Bird G. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB. J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar, 8Tsunoda Y. Stuenkel E.L. Williams J.A. Am. J. Physiol. 1990; 258: C147-C155Crossref PubMed Google Scholar, 9Fewtrell C. Ann. Rev. Physiol. 1993; 55: 427-454Crossref PubMed Scopus (202) Google Scholar). Formation of IP3 and cyclical release of Ca2+ from IP3-sensitive stores may underlie the generation of oscillations induced by agonists (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 10De Young G.W. Keizer J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9895-9899Crossref PubMed Scopus (579) Google Scholar). However, a Ca2+-induced Ca2+ release pathway has been suggested in initiating oscillations by caffeine or other agents unrelated to IP3 generation (9Fewtrell C. Ann. Rev. Physiol. 1993; 55: 427-454Crossref PubMed Scopus (202) Google Scholar, 11Friel D.D. Tsien R.W. Neuron. 1992; 8: 1109-1125Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Ca2+influx from the external milieu is currently thought to be activated in such situations and appears to be needed to sustain [Ca2+]i oscillations (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 7Thomas A.P. St J. Bird G. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB. J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar). However, the mechanism whereby Ca2+ entry is triggered during [Ca2+]i oscillations is not altogether clear. Some models suggest that capacitative calcium entry provides Ca2+ entry during oscillations (7Thomas A.P. St J. Bird G. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB. J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar, 12Berridge M.J. Cell Calcium. 1991; 12: 63-72Crossref PubMed Scopus (142) Google Scholar). More recently a novel, noncapacitative mechanism has been proposed that involves agonist-activated generation of arachidonic acid and arachidonic acid-induced Ca2+ entry (13Shuttleworth T.J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar, 14Akagi K. Nagao T. Urushidani T. Jpn. J. Pharmacol. 1997; 75: 33-42Crossref PubMed Scopus (21) Google Scholar, 15Lankisch T.O. Nozu F. Owyang C. Tsunoda Y. Eur. J. Cell Biol. 1999; 78: 632-641Crossref PubMed Scopus (21) Google Scholar). Arachidonic acid is present in cell membranes esterified in phospholipids and can be released by phospholipase A2(PLA2) in response to various extracellular stimuli (16Dennis E.A. Rhee S.G. Billah M.M. Hannun Y.A. FASEB. J. 1991; 5: 2068-2077Crossref PubMed Scopus (475) Google Scholar,17Graber R. Sumida C. Nurez E.A. J. Lipid Mediat. Cell Signal. 1994; 9: 91-116PubMed Google Scholar). Arachidonic acid can also be generated from diacylglycerol, a product of phospholipase C or phospholipase D activation, by action of diglyceride lipase (16Dennis E.A. Rhee S.G. Billah M.M. Hannun Y.A. FASEB. J. 1991; 5: 2068-2077Crossref PubMed Scopus (475) Google Scholar). In recent years, an increasing number of reports have suggested that arachidonic acid directly modulates cellular responses, including Ca2+ signal transduction. As for IP3, Ca2+ release from ER and Ca2+ influx from the extracellular space induced by arachidonic acid have been demonstrated in a number of cell types (18Chow S.C. Jondal M. J. Biol. Chem. 1990; 265: 902-907Abstract Full Text PDF PubMed Google Scholar, 19van der Zee L. Nelemans A. Den Hertog A. Biochem. J. 1995; 305: 859-864Crossref PubMed Scopus (48) Google Scholar, 20Munaron L. Antoniotti S. Distasi C. Lovisolo D. Cell Calcium. 1997; 22: 179-188Crossref PubMed Scopus (62) Google Scholar, 21Shuttleworth T.J. J. Biol. Chem. 1997; 271: 21720-21725Abstract Full Text Full Text PDF Scopus (144) Google Scholar, 22Broad L.M. Cannon T.R. Taylor C.W. J. Physiol.(Lond.). 1999; 517: 121-134Crossref Scopus (195) Google Scholar, 23Mignen O. Shuttleworth T.J. J. Biol. Chem. 2000; 275: 9114-9119Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, the mechanisms underlying [Ca2+]i changes in response to arachidonic acid are not clear. In this study, we have used relatively specific pharmacological probes to analyze and compare capacitative, noncapacitative, and arachidonic acid-induced Ca2+ entry in HEK293 cells. We confirm earlier reports of a noncapacitative mechanism associated with [Ca2+]i oscillations in these cells. However, our findings indicate a possible role for the IP3 receptor in this signaling pathway and call into question the role of arachidonic acid, at least as a direct mediator of Ca2+ entry in this cell type. Human embryonic kidney 293 (HEK293) cells obtained from the ATCC were grown at 37 °C in Dulbecco's Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mm glutamine in a humidified 95% air, 5% CO2incubator. For Ca2+ measurements, cells were cultured to about 70% confluence, passaged onto glass coverslips, and used 24–48 h after plating. Fluorescence measurements were made with Fura2-loaded single or groups of HEK293 cells as described previously (24Bird G. St J. Putney Jr., J.W. J. Biol. Chem. 1996; 271: 6766-6770Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In brief, coverslips with attached cells were mounted in a Teflon chamber and incubated in Dulbecco's Eagle's medium with 1 μm acetoxymethyl ester of Fura2 (Fura2/AM, Molecular Probes) at 37 °C in the dark for 25 min. Before [Ca2+]i measurements, cells were washed three times and incubated for 30 min at room temperature (25 °C) in HEPES-buffered physiological saline solution (HPSS: NaCl, 120 mm; KCl, 5.4 mm; Mg2SO4, 0.8 mm; HEPES, 20 mm; CaCl2, 1.8 mm; and glucose, 10 mm; with pH 7.4 adjusted by NaOH). Ca2+-free solutions contained no added CaCl2 in the HPSS. In preliminary experiments, we observed that [Ca2+]i oscillations were not reproducibly observed in cells loaded with 1 μm Fura2/AM, presumably due to excessive cytoplasmic Ca2+ buffering. Thus, for these experiments we used 100 nm Fura2/AM for loading and 1.5 mm extracellular CaCl2 as previously described (25Wayman G.A. Hinds T.R. Storm D.R. J. Biol. Chem. 1995; 270: 24108-24115Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Fluorescence was monitored by placing the Teflon chamber with Fura2-loaded cells onto the stage of a Nikon Diaphot microscope (40× Neofluor objective). The cells were excited alternatively by 340 and 380 nm wavelength light from a Deltascan D101 (Photon Technology International Ltd.,) light source equipped with a light path chopper and dual excitation monochromators. Emission fluorescence intensity at 510 nm was recorded by a photomultiplier tube (Omega Optical). All experiments were conducted at room temperature (25 °C) and carried out within 2 h of loading for each coverslip. Changes in [Ca2+]i are reported for one single cell in oscillation experiments or a group of cells (6Prince W.T. Berridge M.J. J. Exp. Biol. 1973; 58: 367-384Google Scholar, 7Thomas A.P. St J. Bird G. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB. J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar, 8Tsunoda Y. Stuenkel E.L. Williams J.A. Am. J. Physiol. 1990; 258: C147-C155Crossref PubMed Google Scholar, 9Fewtrell C. Ann. Rev. Physiol. 1993; 55: 427-454Crossref PubMed Scopus (202) Google Scholar, 10De Young G.W. Keizer J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9895-9899Crossref PubMed Scopus (579) Google Scholar) in other protocols. The data are expressed as the ratio of Fura2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm (F 340/F 380). Mn2+quench experiments were performed with a group of HEK293 cells in nominally Ca2+-free medium containing 0.1 or 2 mm MnCl2. F tot, which is independent of [Ca2+]i responses (26Shuttleworth T.J. Cell Calcium. 1994; 15: 457-466Crossref PubMed Scopus (29) Google Scholar), was obtained by a weighted summing of the fluorescence of 340 and 380 nm, and expressed as the percentage of the initial value in the absence of extracellular Mn2+. Arachidonic acid and 5,8,11,14-eicosatetraenoic acid were obtained from BioMol (Plymouth Meeting, PA). Carbachol, thapsigargin, and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were purchased from Calbiochem (La Jolla, CA). 2-Aminoethyoxydiphenyl borane (2-APB) was synthesized as previously described (27Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar). For some experiments, average peak responses (F 340/F 380) were calculated and expressed as mean ± S.E. for the indicated number (n) of experiments. Statistical significance was determined with the Student's t test (p < 0.05). In Ca2+-containing HPSS, 100 μm carbachol induced a large, somewhat transient increase in [Ca2+]i(F 340/F 380) followed by a slowly declining but generally sustained elevated level of [Ca2+]i (Fig.1 A). In nominally Ca2+-free medium, this same concentration of carbachol induced a transient increase in [Ca2+]i; following re-addition of Ca2+ to the medium, a second, sustained entry of Ca2+ was observed (Fig. 1 B), indicating release of Ca2+ from internal sites and Ca2+ entry. [Ca2+]i oscillations have been reported to be induced by 1 μm carbachol in HEK293 cells transfected with the M3 muscarinic receptors (28Shuttleworth T.J. Thompson J.L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, in the current study in which wild type HEK293 cells were used, we failed to consistently produce repetitive transient responses of [Ca2+]i with 1–3 μm carbachol. When the cells were stimulated with 5 μm carbachol, about 50% of the tested cells (101 of 205) showed oscillatory [Ca2+]i responses at a frequency of ∼0.5–1/min in the presence of 1.5 mm Ca2+ (Fig.1 C). These robust spikes could last up to 1 h, but the frequency progressively slowed with time. Because all cells did not oscillate and the frequency varied somewhat among the cells, which did oscillate, we adopted the protocol shown in Fig. 1 C. In this protocol, a cell was stimulated for about 20 min, the carbachol was removed by three changes of incubation medium, and then, after an additional period of about 25 min (data acquisition was halted during this period), the same cell was again stimulated with 5 μm carbachol for an additional 20 min, generally under an altered experimental condition. As shown in Fig. 1 C, in normal HPSS, the second stimulation always resulted in an oscillatory response that was similar to, although somewhat slower than the first. This protocol was utilized for the experiment illustrated in Fig.1 D. In this experiment, 5 min before, and during the second exposure to carbachol, the cell was bathed in a nominally Ca2+-free medium containing 200 μm BAPTA. With this protocol, carbachol induced one or two spikes, but sustained oscillations were not observed. These results confirm that, in wild type HEK293 cells, as shown previously for cells transfected with the M3 muscarinic receptor, [Ca2+]ioscillations produced by a low concentration of carbachol depend on extracellular Ca2+, presumably indicating a role for Ca2+ influx. Gd3+ is a potent inhibitor of agonist-activated calcium entry (29Fernando K.C. Barritt G.J. Biochim. Biophys. Acta. 1994; 1222: 383-389Crossref PubMed Scopus (33) Google Scholar) and has been shown to discriminate between capacitative and noncapacitative calcium entry (22Broad L.M. Cannon T.R. Taylor C.W. J. Physiol.(Lond.). 1999; 517: 121-134Crossref Scopus (195) Google Scholar). In experiments utilizing the same protocol as in Fig. 1 B, the effects of Gd3+ on Ca2+ entry due to the SERCA inhibitor, thapsigargin, were determined. Thapsigargin depletes Ca2+stores passively by virtue of its ability to inhibit the SERCA pumps on the endoplasmic reticulum, and the ensuing entry of Ca2+ is therefore assumed to be the very definition of capacitative calcium entry (3Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar, 30Takemura H. Hughes A.R. Thastrup O. Putney Jr., J.W. J. Biol. Chem. 1989; 264: 12266-12271Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.2 A, Gd3+ inhibited Ca2+ entry induced by the thapsigargin in a concentration-dependent manner, and with no significant effect on the Ca2+ release phase (Fig. 2 A and results not shown). Gd3+ had no significant effect on basal [Ca2+]i (data not shown). The sensitivity of thapsigargin-induced capacitative calcium entry to inhibition by Gd3+ is similar to that reported by Broad et al.(22Broad L.M. Cannon T.R. Taylor C.W. J. Physiol.(Lond.). 1999; 517: 121-134Crossref Scopus (195) Google Scholar). Similar experiments were carried out utilizing a maximal concentration of carbachol, and such an experiment is shown in Fig.2 B. Ca2+ entry due to maximal muscarinic receptor activation appeared similarly sensitive to inhibition by Gd3+, leading to the conclusion that the entry is largely or entirely capacitative. However, significantly different results were obtained when cells were stimulated to oscillate with the lower, 5 μmconcentration of carbachol. As Fig. 3 illustrates, concentrations of 1, 10, 30, 100, or 500 μm Gd3+ produced little or no effect on the [Ca2+]i oscillations; only at the highest concentrations tested, 100 and 500 μmGd3+, was there even partial suppression of the oscillatory frequency. The failure of even these very high concentrations of Gd3+ to block the oscillations was surprising. However, it is known that another lanthanide, La3+, can inhibit active membrane extrusion of Ca2+ at higher concentrations (31Van Breemen C. Farinas B. Gerba P. McNaughton E.D. Circ. Res. 1972; 30: 44-54Crossref PubMed Scopus (362) Google Scholar). We determined whether Gd3+ might have a similar action by examining the time course of the [Ca2+]i response to thapsigargin in the absence of extracellular Ca2+ and in the presence of varying concentrations of Gd3+. The decay of the [Ca2+]i response to thapsigargin under these conditions is due almost entirely to plasma membrane extrusion (32Kwan C.Y. Takemura H. Obie J.F. Thastrup O. Putney Jr., J.W. Am. J. Physiol. 1990; 258: C1006-C1015Crossref PubMed Google Scholar). As shown in Fig. 4, Gd3+ concentrations of 30 μm or greater caused an augmentation of the thapsigargin-induced [Ca2+]i signal and a slowing of its decay. Thus, at these higher concentrations, oscillations may continue due to "trapping" of intracellular Ca2+, despite an inhibition of Ca2+ entry. However, at 10 μmGd3+, there was no significant augmentation of the response, indicating that the Ca2+ entry channels supporting the oscillations are truly less sensitive to Gd3+ than are capacitative calcium entry channels.Figure 4Effects of Gd3+on Ca2+ extrusion in HEK293 cells. To assess inhibitory actions of Gd3+ on Ca2+ extrusion, cells were activated by 1 μmthapsigargin (TG) in the absence of external Ca2+ and in the presence of the indicated concentrations of Gd3+. Concentrations of Gd3+ of 30 μm or greater delayed the decay of the thapsigargin transient, indicating a degree of inhibition of plasma membrane Ca2+ extrusion.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Shuttleworth and his coworkers (13Shuttleworth T.J. Cell Calcium. 1999; 25: 237-246Crossref PubMed Scopus (94) Google Scholar, 21Shuttleworth T.J. J. Biol. Chem. 1997; 271: 21720-21725Abstract Full Text Full Text PDF Scopus (144) Google Scholar, 28Shuttleworth T.J. Thompson J.L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) have suggested that the noncapacitative calcium entry occurring in HEK293 cells and other cell types in response to low concentrations of muscarinic agonists is mediated by arachidonic acid, released from membrane lipids by phospholipase A2. Thus, we next examined the effects of Gd3+ on Ca2+ mobilization in HEK293 cells in response to arachidonic acid. In preliminary experiments, we found that between 30 and 300 μmarachidonic acid could reproducibly induce both Ca2+release and Ca2+ entry in a concentration-dependent manner. However, arachidonic acid at concentrations > 100 μm occasionally resulted in [Ca2+]i levels that saturated the indicator likely due to a nonselective increase in membrane permeability (33Meves H. Prog. Neurobiol. 1994; 43: 175-186Crossref PubMed Scopus (195) Google Scholar). Concentrations in the range of 5 to 10 μm did not induce increases in [Ca2+]i in all cells. As shown in Fig. 5 A, 30 μmarachidonic acid slowly increased the fluorescence ratio, and the response appeared to occur in two phases. In nominally Ca2+-free medium, arachidonic acid induced a transient [Ca2+]i rise followed by a sustained elevation of [Ca2+]i after restoration of Ca2+ to the medium (Fig. 5 B), indicating that both Ca2+release and Ca2+ entry are activated by arachidonic acid in HEK293 cells. To examine the possible involvement of metabolites of arachidonic acid in the [Ca2+]i responses, we employed 5,8,11,14-eicosatetraenoic acid, an inhibitor of cyclooxygenase, lipoxygenases, and cytochrome P450 arachidonic acid-metabolizing enzymes (34Collins D.R. Davies S.N. Eur. J. Pharmacol. 1998; 342: 213-216Crossref PubMed Scopus (4) Google Scholar). 20 μm5,8,11,14-eicosatetraenoic acid had no effect on either Ca2+ release or Ca2+ entry induced by 30 μm arachidonic acid, indicating that the [Ca2+]i changes induced by 30 μmarachidonic acid are unlikely to result from an arachidonic acid metabolite (data not shown). A similar conclusion was reached by Shuttleworth and Thompson based on a somewhat different strategy (28Shuttleworth T.J. Thompson J.L. J. Biol. Chem. 1998; 273: 32636-32643Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The pattern of [Ca2+]i signaling induced by arachidonic acid is reminiscent of that due to thapsigargin; a release of stored Ca2+ followed by activation of Ca2+entry across the plasma membrane. Thus, we next examined the effects of Gd3+ on arachidonic acid-induced signaling, because this lanthanide appears to have relatively selective effects on store-operated or capacitative calcium entry. At a concentration of 1 μm, which completely blocked Ca2+ entry due to carbachol and thapsigargin, Gd3+ had no significant effect on Ca2+ entry in response to 30 μmarachidonic acid (Fig. 5, B and C). At concentrations of 3 and 10 μm, Gd3+ inhibited Ca2+ influx induced by 30 μm arachidonic acid with complete blockade at 10 μm. Surprisingly, 10 μm Gd3+ also caused a complete abolishment of arachidonic acid-induced Ca2+ release (Fig. 5, Band C). After complete inhibition with 10 μmGd3+ of Ca2+ release due to 30 μmarachidonic acid in nominally Ca2+-free medium, a normal release of [Ca2+]i could be evoked on addition of 1 μm thapsigargin or 100 μm carbachol (not shown). These results indicate that arachidonic acid induces both Ca2+ release and Ca2+ entry in HEK293 cells, and both of these responses are sensitive to inhibition by Gd3+; however, this pathway is at least 10-fold less sensitive to Gd3+ than capacitative calcium entry. Recent studies have indicated that capacitative calcium entry involves interactions between IP3 receptors and the plasma membrane (35Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (561) Google Scholar). One piece of evidence for this idea is the sensitivity of capacitative calcium entry to inhibition by 2-APB (36Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (533) Google Scholar), a membrane-permeant inhibitor of the IP3 receptor (27Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar). We next examined the actions of this reagent as a potential inhibitor of Ca2+entry responses to agonists, to thapsigargin, and to arachidonic acid. In unstimulated cells, and in the absence of extracellular Ca2+, 2-APB at 100 μm slightly augmented the baseline fluorescence ratio in about 80% of HEK293 cells tested (n = 48). The increment in the baseline was 8.6 ± 3.2% of that of Ca2+ release by 1 μmthapsigargin in nominally Ca2+-free medium (n = 9). A weak inhibitory effect on Ca2+-ATPase in the ER has been suggested to account for the rise of [Ca2+]i by high concentrations of 2-APB (27Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar). Utilizing a similar protocol as for the Gd3+ experiments, 2-APB produced a concentration-dependent inhibition of Ca2+ influx induced by 100 μm carbachol (Fig.6, top) or 1 μmthapsigargin (Fig. 6, bottom) when Ca2+ was restored to the bath. Like Gd3+, 2-APB altered the Ca2+ entry phase with almost the same potency among the two agonists, with 30 μm 2-APB producing essentially complete block of Ca2+ entry for both modes of activation. However, 30 μm 2-APB attenuated the Ca2+ release peak induced by 100 μm carbachol only weakly, and this inhibition was still incomplete with 100 μm 2-APB (Fig.6). With 100 μm 2-APB, an approximate 20% reduction of Ca2+ release due to 1 μm thapsigargin could also be seen (Fig. 6), which may be due to the inhibition of Ca2+-ATPase in the endoplasmic reticulum and a partial reduction of the size of the pool sensitive to thapsigargin. 2-APB at 100 μm, a concentration that caused complete inhibition of capacitative calcium entry, did not alter Ca2+ entry due to 30 μm arachidonic acid (Fig. 7). As for thapsigargin, 100 μm 2-APB caused a slight reduction of [Ca2+]i release in response to 30 μm arachidonic acid (Fig. 7 and results not shown). These data, including the data obtained with Gd3+, provide evidence that the mechanisms by which arachidonic acid activates Ca2+ release and Ca2+ influx are different from those of the store-depleting agents, thapsigargin and carbachol. As first suggested by Shuttleworth, capacitative calcium entry appears not to be involved in Ca2+ entry due to arachidonic acid in HEK293 cells (21Shuttleworth T.J. J. Biol. Chem. 1997; 271: 21720-21725Abstract Full Text Full Text PDF Scopus (144) Google Scholar). As shown in Fig. 8, 2-APB inhibited the repetitive transient [Ca2+]iresponses in a concentration-dependent manner and 100 μm 2-APB completely blocked the sustained oscillatory response of HEK293 cells to 5 μm carbachol. The inhibition by 2-APB of the [Ca2+]i response to 5 μm carbachol was unexpected, because arachidonic acid-induced Ca2+ signaling was unaffected by this drug. However, we considered the possibility that this concentration of carbachol might induce a small influx of Ca2+ that is only detectable when amplified through calcium-induced calcium release, and this might depend on functional IP3 receptors. Therefore, to assess more directly the actions of 2-APB on Ca2+ entry during [Ca2+]i oscillations, we utilized Mn2+ quench measurements. Mn2+ enters cells through divalent cation channels, but quenches Fura2 fluorescence at all wavelengths (37Merritt J.E. Jacob R. Hallam T.J. J. Biol. Chem. 1989; 264: 1522-1527Abstract Full Text PDF PubMed Google Scholar). Thus, the activity of Ca2+ influx channels is reported by the rate of Mn2+ quench of Fura2. In the presence of 0.1 mm Mn2+, in nominally Ca2+-free medium, a resting rate of Mn2+ quench was seen in unstimulated cells, and this was blocked when the cells were pretreated with 100 μm 2-APB (Fig.9 A). 5 μmcarbachol increased Mn2+ quench, and again the rate of quench in the presence of carbachol was completely blocke