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Calcium Release-activated Calcium Current (I CRAC) Is a Direct Target for Sphingosine

1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês

10.1074/jbc.273.39.25020

ISSN

1083-351X

Autores

Chris Mathes, Andrea Fleig, Reinhold Penner,

Tópico(s)

Calcium signaling and nucleotide metabolism

Resumo

Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current(I CRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits I CRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, includingN,N-dimethylsphingosine,dl-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also blockI CRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block I CRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit I CRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits I CRAC, and this inhibition can be antagonized by reducing sphingosine production withl-cycloserine, suggesting thatI CRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocksI CRAC, suggesting that the sphingomyelin pathway is involved in I CRAC regulation. Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current(I CRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits I CRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, includingN,N-dimethylsphingosine,dl-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also blockI CRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block I CRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit I CRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits I CRAC, and this inhibition can be antagonized by reducing sphingosine production withl-cycloserine, suggesting thatI CRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocksI CRAC, suggesting that the sphingomyelin pathway is involved in I CRAC regulation. phospholipase C calcium release-activated calcium current store-operated calcium protein kinase C sphingomyelin sphingosine 1-phosphate intracellular calcium concentration nerve growth factor carbamylcholine l-cycloserine bovine serum albumin inositol 3-phosphate sphingomyelinase N, N-dimethylsphingosine adenosine 5′-O-(thiotriphosphate) picofarads arachidonic acid phosphatidic acid ceramide 1-phosphate tetrodotoxin. Agonists that stimulate phospholipase C (PLC)1 and elevate InsP3 levels activate Ca2+ entry that is important for refilling depleted stores (for review see Refs. 1Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar, 2Penner R. Fasolato C. Hoth M. Curr. Opin. Neurobiol. 1993; 3: 368-374Crossref PubMed Scopus (126) Google Scholar, 3Putney Jr., J.W. Bird G.S. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (484) Google Scholar). In mast cells and T-lymphocytes, this store-operated calcium (SOC) current is highly selective for Ca2+ ions and has been termed calcium release-activated-calcium current (I CRAC) (4Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1486) Google Scholar, 5Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (692) Google Scholar). I CRAC has also been measured in the mast cell line RBL-2H3, in hepatocytes, in thyrocytes, 3T3 fibroblasts, and HL-60 cells (6Hoth M. Fasolato C. Penner R. Ann. N. Y. Acad. Sci. 1993; 70: 198-209Crossref Scopus (45) Google Scholar). Both protein kinase C (PKC) and a small guanosine triphosphate-binding protein regulateI CRAC (7Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 8Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (154) Google Scholar), although the exact mechanism for activation of I CRAC remains unknown. One of the difficulties in pin-pointing a definite mechanism for regulation of I CRAC may relate to the fact that PLC activation functions in parallel with other intracellular signaling processes. The sphingomyelin (SM) pathway, for example, "cross-talks" with the PLC pathway (9Spiegel S. Milstien S. Chem. Phys. Lipids. 1996; 80: 27-36Crossref PubMed Scopus (24) Google Scholar). Activation of the SM pathway produces important second messengers such as sphingosine, sphingosine 1-phosphate (S1P), and ceramide (10Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1491) Google Scholar). Generally, sphingosine and S1P mediate cell growth and proliferation, whereas ceramide elevations cause programmed cell death (apoptosis) or cell cycle arrest (9Spiegel S. Milstien S. Chem. Phys. Lipids. 1996; 80: 27-36Crossref PubMed Scopus (24) Google Scholar). Although several direct targets have been characterized for ceramide (10Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1491) Google Scholar), the picture is less clear for sphingosine. PKC has been shown to be a target for sphingosine (11Hannun Y.A. Loomis C.R. Merrill Jr., A.H. Bell R.M. J. Biol. Chem. 1986; 261: 12604-12609Abstract Full Text PDF PubMed Google Scholar), but the action of sphingosine on other proteins and intracellular processes may be mediated by conversion to S1P and/or ceramide (10Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1491) Google Scholar, 12Spiegel S. Olivera A. Carlson R.O. Adv. Lipid Res. 1993; 25: 105-129PubMed Google Scholar,13Merrill Jr., A.H. Hannun Y.A. Bell R.M. Adv. Lipid Res. 1993; 25: 1-24PubMed Google Scholar). Intracellular Ca2+ elevations may be an important connection for cross-talk between the PLC and SM pathways. Indeed, it has been demonstrated that elevations in [Ca2+]ican modulate cell growth (14Conklin B.R. Brann M.R. Buckley N.J. Ma A.L. Bonner T.I. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8698-8702Crossref PubMed Scopus (129) Google Scholar, 15Felder C.C. MacArthur L. Ma A.L. Gusovsky F. Kohn E.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1706-1710Crossref PubMed Scopus (45) Google Scholar, 16Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (248) Google Scholar), and sphingosine has been shown to alter Ca2+ homeostasis in various preparations (17Fatatis A. Miller R.J. J. Biol. Chem. 1996; 271: 295-301Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Pandol S.J. Schoeffield-Payne M.S. Gukovskaya A.S. Rutherford R.E. Biochim. Biophys. Acta. 1994; 1195: 45-50Crossref PubMed Scopus (16) Google Scholar, 19Sakano S. Takemura H. Yamada K. Imoto K. Kaneko M. Ohshika H. J. Biol. Chem. 1996; 271: 11148-11155Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Sugiya H. Furuyama S. FEBS Lett. 1991; 286: 113-116Crossref PubMed Scopus (28) Google Scholar, 21Breittmayer J.P. Bernard A. Aussel C. J. Biol. Chem. 1994; 269: 5054-5058Abstract Full Text PDF PubMed Google Scholar). We have therefore investigated the role of the SM pathway in regulating intracellular calcium concentration, focusing on the major Ca2+ influx pathway provided by the SOC currentI CRAC in the mast cell line RBL-2H3. We surveyed the sphingomyelin pathway for possible regulators of I CRAC and found that sphingosine and structurally related compounds (but not S1P nor ceramides) are inhibitors. None of the agents tested activateI CRAC. A requirement for inhibition of ICRAC is incorporation of the lipid molecule into the plasma membrane. Sphingolipid incorporation was measured as an elevation of the whole cell capacitance, which represents a new approach for monitoring lipid or possibly drug accumulation in the plasma membrane. To provide a link between cellular sphingosine and inhibition of I CRAC, we tested several growth factors, which are known to activate sphingomyelinase and/or to elevate sphingosine levels (9Spiegel S. Milstien S. Chem. Phys. Lipids. 1996; 80: 27-36Crossref PubMed Scopus (24) Google Scholar, 22Blàchl A. Sirrenberg C. J. Biol. Chem. 1996; 271: 21100-21107Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 23Dobrowsky R.T. Jenkins G.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 22135-22142Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 24Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 25Grabbe J. Welker P. Dippel E. Czarnetzki B.M. Arch. Dermatol. Res. 1994; 287: 78-84Crossref PubMed Scopus (116) Google Scholar, 26Jacobs L.S. Kester M. Am. J. Physiol. 1993; 265: C740-C747Crossref PubMed Google Scholar, 27Oral H. Dorn G.W., II Mann D.L. J. Biol. Chem. 1997; 272: 4836-4842Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). We observed that nerve growth factor (NGF 2.5 S subunit; 1 μg/ml) inhibits ICRAC. NGF-induced inhibition of ICRAC was reduced by treatment withl-cycloserine (LCS; 2 mm), which lowers SM levels (26Jacobs L.S. Kester M. Am. J. Physiol. 1993; 265: C740-C747Crossref PubMed Google Scholar) and hinders sphingosine production by NGF. Implications for sphingosine-dependent block of I CRACare discussed. Rat basophilic leukemia cells (RBL-2H3) were plated on glass coverslips at low density and incubated at 37 °C with 10% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 45 mm sodium bicarbonate, 5 mm glucose, 0.12 mg/ml streptomycin, and 0.60 mg/ml penicillin. For the recordings, coverslips were transferred into the recording chamber containing the desired external solution. RBL-2H3 cells stably transfected with m1 muscarinic receptors (RBL-m1 (28Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Crossref PubMed Scopus (58) Google Scholar, 29Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595PubMed Google Scholar)) were a kind gift from Dr. O. H. Choi (National Institutes of Health, Bethesda). RBL-m1 cells were cultured identically to RBL-2H3 cells, except 400 μg/ml G418 was added to the RBL-m1 growth medium to select for transfected cells. The muscarinic receptor agonist carbamylcholine (CCh; 100 μm) activated calcium release (as measured by fura-2) or I CRAC (in patch-clamp experiments) in every RBL-m1 cell tested. The tight-seal whole cell configuration was used for patch-clamp. Experiments were conducted at room temperature (20–27 °C) in standard external saline solution containing the following (in mm): 140 NaCl, 2.8 KCl, 10 CaCl2, 2 MgCl2, 10 CsCl, 11 glucose, and 10 HEPES·NaOH (pH 7.2). Sylgard-coated, fire-polished patch pipettes had resistances between 1.5 and 3 megohms after filling with the standard internal solution which contained the following (in mm): 145 Cs·Glu, 8 NaCl, 1 MgCl2, 10 Cs·EGTA, 0.02 InsP3, and 10 mm HEPES·CsOH (pH 7.2). Whole cell break-in with this standard internal solution always led to activation of I CRAC. Whole cell break-in without activation of I CRACwas achieved by excluding InsP3, clamping intracellular Ca2+ to ∼80 nm with 2 to 1 ratio of Cs·EGTA/Ca·EGTA, and including nucleotides (4 mmMg·ATP and 300 μm Na2·GTP) to maintain intracellular integrity and to prevent depletion of internal Ca2+ stores. This internal solution was used when testing for activation of I CRAC by various sphingomyelin pathway intermediates. High resolution current recordings were acquired by a computer-based patch-clamp amplifier system (EPC-9). Currents due to cellular membrane capacitance were recorded and canceled before each voltage ramp using the automatic capacitance compensation of the EPC-9. In this manner, whole cell capacitance was measured throughout the experiment. Series resistance was between 2 and 10 megohms, and inhibition of I CRAC did not correlate with series resistance. Currents were filtered at 3.3 kHz and digitized at 100 μs. Currents elicited by voltage ramps in Fig. 1 were filtered at 1 kHz off-line. Ramps were given every 2 s (−100 to +100 mV in 50 ms) from a holding potential of 0 mV, and the Ca2+ current was analyzed at the ramp segment corresponding to −80 mV. This protocol minimized Ca2+ entry because of the delay between ramps and because of the relatively small driving force for Ca2+ at 0 mV. Minimizing Ca2+ entry is important to limit Ca2+-dependent inactivation of I CRAC by local accumulation of Ca2+(30Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (658) Google Scholar, 31Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). All currents were leak-subtracted by averaging the first two to six ramps after breaking in and then subtracting this from all subsequent traces. For display purposes, averaged time courses of I CRAC were graphed at slightly lower temporal resolution (i.e. 0.25–0.125 Hz). Extracellular solution changes were made by local pressure application from a wide-tipped micropipette placed within 20 μm of the cell. A liquid-junction potential of −10 mV was corrected. We have eliminated all experiments from our analysis in which nonspecific membrane breakdown by sphingosine occurred within the typical observation window of 5–10 min. During patch-clamp recordings, breakdown was characterized by sudden increases in linear ramp currents, and in fluorescence measurements, increases in [Ca2+] as well as dye leakage occurred. Such breakdown was seen in approximately 50% of the cells, both in patch-clamp and fluorescence experiments. Intracellular [Ca2+] was monitored with a photomultiplier-based system and calculated from the fluorescence ratio (360/390). For single cell Ca2+ measurements, coverslips were incubated in a Ringer's solution containing the following (in mm): 140 NaCl, 2.8 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, and 10 Na·HEPES (pH 7.2), to which 5 μm fura-2/acetoxymethyl ester had been added. Following incubation at 37°C for 15–20 min the coverslips were washed five times in the Ringer's solution. Experiments were done at room temperature. Inward rectifier potassium currents were measured in RBL-2H3 cells. The external saline solution contained the following (in mm): 140 NaCl, 20 KCl, 2 CaCl2, 2 MgCl2, 11 glucose, and 10 HEPES·NaOH (pH 7.2). The internal saline solution contained the following (in mm): 145 K·Glu, 8 NaCl, 1 MgCl2, 2 Mg·ATP, 0.3 Na2·GTP, 10 K·EGTA, and 10 HEPES·KOH (pH 7.2). Ramps were given every 2 s (−100 to +100 mV in 50 ms), and cells were held at 0 mV between ramps. Currents were not leak-subtracted. Under these conditions, the K+current (measured at −80 mV) did not run down, but in fact the amplitude steadily increased in control experiments over a period of approximately 200 s. Delayed rectifier potassium currents were measured in rat skeletal myoballs isolated from newborn rats (2–5 days old) as described (32Fleig A. Penner R. J. Physiol. (Lond.). 1995; 489: 41-53Crossref Scopus (21) Google Scholar). The external solution contained the following (in mm): 140 NaCl, 2.8 KCl, 1 CaCl2, 2 MgCl2, 0.5 CdCl2, 0.03 TTX, 11 glucose, and 10 HEPES·NaOH (pH 7.2). The internal solution contained the following (in mm): 140 K·Glu, 8 NaCl, 1 MgCl2, 2 Mg·ATP, 0.3 Na2·GTP, 20 K·EGTA, and 10 HEPES·KOH (pH 7.2). Voltage pulses to +60 mV were applied every 20 s from a holding potential of −70 mV. N-type Ca2+ channel currents were measured in bovine adrenal chromaffin cells isolated as described previously (33Fenwick E.M. Marty A. Neher E. J. Physiol. (Lond.). 1982; 331: 577-597Crossref Scopus (579) Google Scholar). The external solution contained the following (in mm): 135 NaCl, 2.8 KCl, 2 MgCl2, 10 CaCl2, 10 TEA·Cl, 0.03 TTX, and 10 HEPES·NaOH (pH 7.4). The internal solution contained (in mm): 140 Cs·Glu, 8 NaCl, 1 MgCl2, 10 Cs·EGTA, 4 Mg·ATP, 0.3 Na2·GTP, and 10 HEPES·CsOH (pH 7.4). Voltage pulses to +10 mV were applied every 2 s from a holding potential of −70 mV. L-type Ca2+ channel currents were measured in rat skeletal myoballs. The external solution contained the following (in mm): 140 NaCl, 2.8 KCl, 2 MgCl2, 10 CaCl2, 10 TEA·CL, 0.03 TTX, and 10 HEPES·NaOH (pH 7.2). The internal solution contained the following (in mm): 145N-methyl-d-glucamine, 8 NaCl, 1 MgCl2, 20 Cs·EGTA, 4 Mg·ATP, 0.3 Na2·GTP, and 10 HEPES·CsOH (pH 7.2). Voltage pulses to 0 mV were applied every 20 s from a holding potential of −70 mV. Na+ currents were measured in bovine adrenal chromaffin cells. The external solution contained the following (in mm): 135 NaCl, 2.8 KCl, 2 MgCl2, 2 CaCl2, 1 CdCl2, 10 TEA·Cl, and 10 HEPES·NaOH (pH 7.4). The internal solution contained the following (in mm): 140 Cs·Glu, 8 NaCl, 1 MgCl2, 10 Cs·EGTA, 4 Mg·ATP, 0.3 Na2·GTP, and 10 HEPES·CsOH (pH 7.4). Voltage pulses to +10 mV were applied every 2 s from a holding potential of −70 mV. Currents were filtered at 8 kHz and sampled at 25 μs. Current records obtained from all experiments in which voltage-activated currents were studied were leak-subtracted by a standard P/n procedure, where 4 leak pulses of −0.1× the actual test pulse amplitude were averaged, scaled, and subtracted from the current evoked by the test pulse. Palmitoyl-co-enzyme A, sphingomyelinase (Staphylococcus aureus), sphingosylphosphorylcholine (lysosphingomyelin), and Long R3 insulin growth factor were purchased from Sigma. N-Acetylsphingosine (C2-ceramide), N-octanoylsphingosine (C8-ceramide), N-octanoylsphingosine 1-phosphate (ceramide 1-phosphate), sphingosine 1-phosphate, phospholipase A2 (Trimeresurus flavoviridis), phospholipase D (Streptomyces chromofuscus), sphinganine (d-erythro-dihydrosphingosine), sphingomyelin, stem cell factor, platelet-derived growth factor, NGF 2.5S (murine), and tumor necrosis factor-α were from Calbiochem. Sphingosine (d-erythro-sphingosine),N, N-dimethylsphingosine,dl-threo-dihydrosphingosine,l-cycloserine, and fumonisin B1 were from Biomol. All other chemicals were from Sigma. Sphingosine and palmitoyl coenzyme A were dissolved in dimethyl sulfoxide (Me2SO) at 10 mm.dl-threo-Dihydrosphingosine, sphingosylphosphorylcholine, sphingosine 1-phosphate, and sphingomyelin were dissolved in methanol at 10 mm. C2-ceramide, C8-ceramide, and ceramide 1-phosphate were dissolved in either Me2SO or methanol at 10 mm. In most experiments, the vehicles Me2SO or methanol were diluted in external saline by a ratio of 1:1000 and neither vehicles significantly altered membrane currents when applied alone (up to 0.5%). Sphingomyelinase, LCS, and phospholipase A2 were dissolved directly in external saline. Phospholipase D was dissolved in special buffer containing 10 mm Tris·HCl (pH 8.0), 0.05% bovine serum albumin, and 0.1% Triton X-100 to a concentration of 500 units/ml (pH 8.0) and was further diluted in external solution by a factor of 1:1000. Growth factors, including NGF, were dissolved in external saline containing 0.1% essentially fatty acid-free BSA (Sigma). The external solution used for experiments with sphingomyelinase and palmitoyl coenzyme A contained 2 mm Ca2+ (instead of the usual 10 mm) to increase the solubility of these molecules. In RBL cells, InsP3 indirectly activatesI CRAC by depleting internal Ca2+stores. Fig. 1 A shows the time course of I CRAC following whole cell break-in with 20 μm InsP3 in the pipette solution (left panel). Voltage ramps (−100 to +100 mV in 50 ms;right panel) were applied every 2 s, and I CRAC was measured from the region on the ramp trace corresponding to −80 mV. Averaged values from 22 cells are shown (left panel). The Ca2+ current activates and peaks in approximately 50 s. Then I CRAC is sustained, decaying only 9.1 ± 2.5% (mean ± S.E.;n = 21) 300 s after break-in. The experiments were done without ATP in the pipette solution to minimize endogenous kinase-dependent inactivation of I CRAC (8Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (154) Google Scholar). Externally applied sphingosine (5 μm) blocks InsP3-evoked I CRAC (Fig. 1 B). Sphingosine was applied 50 s after whole cell break-in, and the onset of block follows a delay of 18 ± 3 s (n = 6). The delay probably relates to the time taken for sphingosine to accumulate in the plasma membrane (see "Elevation of Membrane Capacitance," below). Complete inhibition of the current occurs within 200 s after addition of sphingosine, and the effect is not reversible within measurement time (5–10 min). Sphingosine also inhibits activation of I CRAC when applied at least 1 min before break-in with InsP3 (n = 2, not shown). Furthermore, sphingosine was also able to suppressI CRAC when activated by thapsigargin, which represents an alternative pool-emptying protocol (Fig. 1 C). Sphingosine when co-applied with thapsigargin essentially abolished thapsigargin-induced I CRAC (n = 3), whereas control cells that were only stimulated with thapsigargin consistently activated regular-sized I CRAC after a delay of about 60 s (n = 3). In these experiments, cells were perfused with the standard internal solution (buffered to resting [Ca2+]i and without InsP3), and either thapsigargin alone (1 μm) or in combination with sphingosine (5 μm) were applied externally. This block of I CRAC by sphingosine seems to be PKC-independent for the following reasons: 1) the PKC inhibitor staurosporine (5 μm; co-applied externally) has no effect on sphingosine block of I CRAC (n= 3, not shown), and 2) exogenous sphingosine inhibits PKC (11Hannun Y.A. Loomis C.R. Merrill Jr., A.H. Bell R.M. J. Biol. Chem. 1986; 261: 12604-12609Abstract Full Text PDF PubMed Google Scholar). Because PKC inhibitors sustain I CRAC (8Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (154) Google Scholar), an explanation dependent on PKC inhibition by sphingosine would be opposite to the blocking effect illustrated in Fig. 1 B. Another possible explanation for inhibition might be that sphingosine maximally activates I CRAC, in a manner similar to ionomycin (4Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1486) Google Scholar). Maximal activation could, in principle, cause intracellular Ca2+ accumulation during long applications and subsequent inhibition of I CRAC channels by calcium-dependent inactivation (30Hoth M. Penner R. J. Physiol. (Lond.). 1993; 465: 359-386Crossref Scopus (658) Google Scholar, 31Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). This possibility was ruled out by the following experiment: whole cell break-in without activation of I CRAC was achieved by excluding InsP3 from the pipette solution, including ATP, and buffering internal Ca2+ to ∼80 nm. With these conditions, applying sphingosine 50 s after break-in never activated I CRAC (n = 6, not shown), indicating that sphingosine block is not mediated by "over activation." It could still be possible, however, that sphingosine interacts with the [Ca2+]i inactivation site of CRAC channels thereby mimicking Ca2+-induced inactivation of I CRAC. Interestingly, sphingosine fails to inhibitI CRAC when applied from the inside (Fig. 1 D). Whole cell break-in was conducted with or without sphingosine in the pipette solution (omitting InsP3 and including 10 mm EGTA), and a short application of ionomycin (2 μm) was used to activate I CRAC. The time course of I CRAC in control cells (n = 3) versus the time course in cells dialyzed internally with 10 μm sphingosine (n = 4) is similar (Fig. 1 D). Block of I CRAC by external sphingosine is dose-dependent (Fig. 2). Concentrations between 10 and 30 μm block ∼100% of the Ca2+ current within 100 s. Lower concentrations inhibit more slowly. For example, 1 μm sphingosine blocks approximately 70% of I CRAC in 400 s. Average I CRAC time courses during application of 300 nm (n = 5), 1 μm(n = 5), and 10 μm (n = 4) sphingosine are plotted in Fig. 2 A. The averageI CRAC time course during treatment with 300 nm sphingosine is indistinguishable from the control (cf. Fig. 1 A). The large variability observed at 1 μm is due to variations of endogenous decay of I CRAC. Concentrations of sphingosine between 100 nm and 30 μm were tested, and a dose-response curve was constructed by measuring the percentage of inhibition at 50 s after application (Fig. 2 B). The corresponding apparent half-maximal inhibitory concentration (IC50) for sphingosine is at 6 μm. Because full block occurs at lower concentrations, but over a much longer time, we also constructed a dose-response curve using the time course of decay as the dependent variable (Fig. 2 C). To measure the blocking time, single exponentials were fit to theI CRAC time course during inhibition by sphingosine. The time constants of inhibition ranged from 24 ± 4 s with 30 μm sphingosine (n = 3) to 1,529 ± 400 s with 100 nm sphingosine (n = 4). The apparent IC50 using this method is at 1 μm. To determine whether sphingosine blocks I CRACactivated by agonist, we used muscarinic type 1 receptors stably transfected in RBL-2H3 cells (RBL-m1). Muscarinic receptor activation with carbamylcholine (CCh) elevates InsP3 levels which release Ca2+ from internal stores (Fig. 3 B (28Jones S.V. Choi O.H. Beaven M.A. FEBS Lett. 1991; 289: 47-50Crossref PubMed Scopus (58) Google Scholar, 29Choi O.H. Lee J.H. Kassessinoff T. Cunha-Melo J.R. Jones S.V. Beaven M.A. J. Immunol. 1993; 151: 5586-5595PubMed Google Scholar)). Following store depletion, I CRAC activates and provides a pathway for Ca2+ entry. In whole cell recordings, CCh (100 μm) activated ICRAC in 9 of 9 cells tested (Fig. 3 A). Co-application of sphingosine (5 μm) and CCh decreased the amplitude of ICRACby greater than 50%, and the current decayed to base line with a time constant of 105 ± 53 s (n = 5). This rate of inhibition is similar to the rate measured for sphingosine-mediated block of InsP3-evoked I CRAC at the same concentration (76 ± 20 s; n = 6). Therefore, sphingosine can block I CRAC activated by a physiologically relevant stimulus. These results were confirmed by measuring intracellular calcium concentration ([Ca2+]i) changes in single RBL-m1 cells loaded with fura-2/acetoxymethyl ester. Fig. 3 B shows averaged muscarinic receptor-mediated Ca2+ signals in the presence of 10 mm extracellular Ca2+ (control), 0 Ca2+, or 10 mm Ca2+ + 5 μm sphingosine. In the control cells, CCh elevates Ca2+ levels to 1.3 ± 0.1 μm(n = 17) following a short delay. The [Ca2+]i remained elevated for longer than 100 s before declining slightly. I CRACcontributes to this sustained elevation of Ca2+. Following removal of CCh the [Ca2+]i returns toward base line (not shown). The Ca2+ dependence of the sustained phase is demonstrated by similar experiments in nominally Ca2+-free external saline where the [Ca2+]i returns quickly toward base-line values following the peak elevation (1.3 ± 0.1 μm;n = 5). When sphingosine was co-applied with CCh, the sustained phase was dramatically reduced (n = 5). Clearly, the time course of sphingosine inhibition is similar to the time course of I CRAC block shown in Fig. 3 A. Assessment of the mechanism by which sphingosine blocksI CRAC depends on whether this sphingolipid alters calcium homeostasis; however, the reported effects of sphingosine on intracellular calcium homeostasis are highly variable. In some preparations, sphingosine elevates intracellular InsP3 (34Chao C.P. Laulederkind S.J. Ballou L.R. J. Biol. Chem. 1994; 269: 5849-5856Abstract Full Text PDF PubMed Google Scholar, 35Sugiya H. Furuyama S. Cell Calcium. 1990; 11: 469-475Crossref PubMed Scopus (15) Google Scholar) and [Ca2+]i (17Fatatis A. Miller R.J. J. Biol. Chem. 1996; 271: 295-301Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Pandol S.J. Schoeffield-Payne M.S. Gukovskaya A.S. Rutherford R.E. Biochim. Biophys. Acta. 1994; 1195: 45-50Crossref PubMed Scopus (16) Google Scholar, 19Sakano S. Takemura H. Yamada K. Imoto K. Kaneko M. Ohshika H. J. Biol. Chem. 1996; 271: 11148-11155Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Sugiya H. Furuyama S. FEBS Lett. 1991; 286: 113-116Crossref PubMed Scopus (28) Google Scholar). In other cell types, exogenous sphingosine has no effect on resting Ca2+ levels (21Breittmayer J.P. Bernard A. Aussel C. J. Biol. Chem. 1994; 269: 5054-5058Abstract Full Text PDF PubMed Google Scholar, 36Hudson P.L. Pedersen W.A. Saltsman W.S. Liscovitch M. MacLaughlin D.T. Donahoe P.K. Blusztajn J.K. J. Biol. Chem. 1994; 269: 21885-21890Abstract Full Text PDF PubMed Google Scholar). We observed variability within the population of RBL cells tested. In 15 of 21 cells tested, sphingosine (5 μm) by itself elevated Ca2+ levels following a delay of 81 ± 11 s (data not shown). In nominally Ca2+-free external solution, sphingosine elevated Ca2+ levels in 8 of 16 cells after a delay of 49 ± 10 s. However, including 0.1% fatty acid-free bovine serum albumin (BSA) to the perfusion solution along with sphingosine prevented [Ca2+]i changes in Ca2+-containing (n = 7) and Ca2+-free (n = 8) solutions. BSA had no effect on sphingosine-mediated block of I CRAC(n = 2). When co-applied with sphingosine, BSA prevents cell permeabilization and subsequent dye leakage (37Pittet D. Krause K.H. Wollheim C.B. Bruzzone R. Lew D.P. J. Bio

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