Role of the Phospholipase C-Inositol 1,4,5-Trisphosphate Pathway in Calcium Release-activated Calcium Current and Capacitative Calcium Entry
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m011571200
ISSN1083-351X
AutoresLisa M. Broad, Franz-Josef Braun, Jean-Philippe Lièvremont, Gary S. Bird, Tomohiro Kurosaki, James W. Putney,
Tópico(s)Venomous Animal Envenomation and Studies
ResumoWe investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP3) in capacitative calcium entry and calcium release-activated calcium current (Icrac) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry andIcrac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and Icrac. The membrane-permeant IP3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and Icrac. However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP3 receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP3 receptors had been disrupted. Intracellular application of another potent IP3 receptor antagonist, heparin, failed to inhibit activation of Icrac. Finally, the inhibition of Icrac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP3. These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP3 and IP3receptor in this mechanism, at least in these particular cell types. We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP3) in capacitative calcium entry and calcium release-activated calcium current (Icrac) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry andIcrac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and Icrac. The membrane-permeant IP3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and Icrac. However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP3 receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP3 receptors had been disrupted. Intracellular application of another potent IP3 receptor antagonist, heparin, failed to inhibit activation of Icrac. Finally, the inhibition of Icrac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP3. These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP3 and IP3receptor in this mechanism, at least in these particular cell types. phospholipase C calcium release-activated calcium current inositol 1,4,5-trisphosphate 2-aminoethoxydiphenyl borane HEPES-buffered physiological saline solution phosphatidylinositol 4-phosphate phosphatidylinositol 4,5-bisphosphate high pressure liquid chromatography 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate Activation of cell surface receptors coupled to phospholipase C (PLC)1 leads to generation of the second messenger inositol 1,4,5-trisphosphate (IP3). IP3 is known to bind to and activate receptors present on intracellular calcium stores, the endoplasmic reticulum, allowing calcium to be released into the cytosol (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). In most cells, the emptying of these calcium stores subsequently activates calcium influx across the plasma membrane through the "capacitative calcium entry" pathway (2Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2115) Google Scholar, 3Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). It is unclear how empty intracellular stores signal activation of plasma membrane capacitative calcium entry. However, a rise in cytosolic calcium is not required, nor is the activation of plasma membrane receptors, because agents such as the calcium-ATPase inhibitor thapsigargin and the calcium ionophore ionomycin, which deplete calcium stores independently of receptor-coupled events, can fully activate capacitative calcium entry (3Putney Jr., J.W. Capacitative Calcium Entry. Landes Biomedical Publishing, Austin, TX1997Crossref Google Scholar). Two general models are proposed as underlying mechanisms for capacitative calcium entry activation. One is based on the requirement for a diffusible messenger generated upon store depletion (4Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (789) Google Scholar, 5Trepakova E.S. Csutora P. Hunton D.L. Marchase R.B. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2000; 275: 26158-63261Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The other hypothesizes a conformational coupling between proteins on the intracellular stores (e.g. the IP3 receptor) and capacitative calcium entry channels or associated proteins in the plasma membrane (6Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar,7Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar). The diffusible messenger hypothesis proposes that a decrease in the concentration of stored calcium leads to the release of a factor that diffuses to the plasma membrane and activates capacitative calcium entry channels. Evidence in support of this model comes from reports of an unidentified calcium influx factor (4Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (789) Google Scholar, 5Trepakova E.S. Csutora P. Hunton D.L. Marchase R.B. Cohen R.A. Bolotina V.M. J. Biol. Chem. 2000; 275: 26158-63261Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 8Thomas D. Hanley M.R. J. Biol. Chem. 1995; 270: 6429-6432Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Csutora P. Su Z. Kim H.Y. Bugrim A. Cunningham K.W. Nuccitelli R. Keizer J.E. Hanley M.R. Blalock J.E. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 121-126Crossref PubMed Scopus (103) Google Scholar) and patch clamping experiments in Xenopus oocytes (10Parekh A.B. Terlau H. Stühmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (320) Google Scholar). Evidence from Xenopus oocytes indicates that if the signal is diffusible then its diffusion is somewhat limited, because the signal remains confined to the area of calcium release (11Petersen C.C.H. Berridge M.J. Pflüg. Arch. 1996; 432: 286-292Crossref PubMed Scopus (59) Google Scholar, 12Bird G. St J. Takahashi M. Tanzawa K. Putney Jr., J.W. J. Biol. Chem. 1999; 274: 20643-20649Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 13Jaconi M. Pyle J. Bortolon R. Ou J. Clapham D. Curr. Biol. 1997; 7: 599-602Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The conformational coupling model proposes the direct relay of a signal through protein-protein interactions. In the model's simplest form, IP3 receptors in the endoplasmic reticulum interact with capacitative calcium entry channels in the plasma membrane (6Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 7Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar). A change in the conformation of the IP3 receptor, which occurs after a drop in endoplasmic reticulum luminal calcium (14Oldershaw K.A. Taylor C.W. Biochem. J. 1993; 292: 631-633Crossref PubMed Scopus (68) Google Scholar), may then be transmitted directly to the capacitative calcium entry channels causing them to open (6Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar, 7Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar). This theory was originally proposed by analogy with ryanodine receptors on the sarcoplasmic reticulum stores, which bind directly to dihydropyridine calcium channels in the plasma membrane of skeletal muscle (15Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar). The theory has subsequently gained some experimental support. A number of studies suggest that the regulation of capacitative calcium entry is dependent upon an intimate interaction between the endoplasmic reticulum and plasma membrane. Pharmacological or physical dislocation of the plasma membrane away from the endoplasmic reticulum prevents activation of capacitative calcium entry by store depletion (16Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 17Rosado J.A. Sage S.O. J. Physiol. ( Lond. ). 2000; 526: 221-229Crossref PubMed Scopus (136) Google Scholar, 18Yao Y. Ferrer-Montiel A.V. Montal M. Tsien R.Y. Cell. 1999; 98: 475-485Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 19Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, there is more direct evidence that an IP3receptor-capacitative calcium entry channel complex bridges the gap between endoplasmic reticulum and plasma membrane. Calcium flux through endogenous capacitative calcium entry channels (20Zubov A.I. Kaznacheeva E.V. Alexeeno V.A. Kiselyov K. Muallem S. Mozhayeva G. J. Biol. Chem. 1999; 274: 25983-25985Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) or overexpressed TRP3 channels (a candidate capacitative calcium entry channel (21Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (563) Google Scholar)) can be recorded in the cell-attached configuration but ceases when the patch is excised. The addition of IP3 and the IP3 receptor to the patch (but not the IP3receptor alone) reconstitutes capacitative calcium entry activity. A similar requirement for the IP3 receptor is revealed by the use of the IP3 receptor inhibitors 2-aminoethoxydiphenyl borane (2-APB) and xestospongin C, which uncouple store depletion from the activation of capacitative calcium entry (22Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar). Thus, there is substantial evidence that IP3 receptors are required for activation of capacitative calcium entry channels and also evidence that these IP3 receptors need to be liganded with IP3. The requirement for IP3 for capacitative calcium entry presents something of a paradox, because, as discussed above, many laboratories have confirmed that store depletion alone, in the absence of phospholipase C activation, is capable of full activation of capacitative calcium entry. Thus, it has been suggested that the requirement for IP3 must normally be fulfilled by its basal production, presumably by a phospholipase C located in close proximity to the channel-IP3 receptor complex (23Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The experiments in the present study were therefore designed to address the question of whether basal PLC activity and basal levels of IP3 in resting cells are required and are sufficient to support a role for the IP3-liganded IP3 receptor in the context of the conformational coupling capacitative calcium entry model. To this end, we monitored capacitative calcium entry channel activity directly by measuring the calcium release-activated calcium current (Icrac) in RBL cells and indirectly by measuring the cytosolic calcium concentration in RBL, mouse lacrimal, and DT40 cells. In the last case, we used both wild-type DT40 cells and cells whose IP3 receptor genes were disrupted by targeted homologous recombination (24Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). IP3 receptor function was inhibited pharmacologically with the membrane-permeant IP3receptor inhibitor 2-APB and the membrane-impermeant inhibitor, low molecular weight heparin. Basal IP3 formation was prevented with a phospholipase C inhibitor (U73122), and the levels of the precursor polyphosphoinositides were decreased by use of a phosphatidylinositol 4-kinase inhibitor (wortmannin). Intracellular calcium stores were subsequently depleted, independent of PLC and the IP3 receptor, using thapsigargin and ionomycin, to test whether capacitative calcium entry could still be activated by store depletion. Our results show that maneuvers that are expected to disrupt basal PLC activity prevent activation of capacitative calcium entry upon store depletion. However, the specific function of PLC is uncertain, since we are unable to demonstrate a role for IP3 or IP3 receptors in this pathway. Rat basophilic leukemia cells stably expressing the muscarinic m1 receptor (RBL-2H3 m1) were a gift from Dr. M. Beaven (National Institutes of Health, Bethesda, MD) (25Choi 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). Cells were cultured in Earle's minimal essential medium with Earle's salts, 10% fetal bovine serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin, (37 °C, 5% CO2). For experiments, cells were passaged onto glass coverslips (number 1 ½) and used 12–36 h after plating. Mouse lacrimal cells were isolated as described previously (26Parod R.J. Leslie B.A. Putney Jr., J.W. Am. J. Physiol. 1980; 239: G99-G105PubMed Google Scholar). Briefly, the excised glands from three mice (male CD-1; 30–40 g) were finely minced and treated for 1 min with 0.2 mg/10 ml trypsin (Sigma). The cells were then removed from the trypsin by centrifugation, followed by a 5-min incubation with 2.5 mg/10 ml soybean trypsin inhibitor (Sigma) in the presence of 2.5 mm EGTA. Finally, the acinar cells were isolated after treating the tissue with 5 mg/10 ml collagenase (Roche Molecular Biochemicals) for 10 min. Throughout, all enzyme solutions were prepared in Dulbecco's modified Eagle's medium. Following isolation, the cells were washed and suspended in sterile Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm glutamine, 50 units/ml penicillin, and 50 units/ml streptomycin. The cells were allowed to attach to glass coverslips (number 1) coated with Matrigel. Lacrimal cells were incubated on glass coverslips at least 3 h before use. Lacrimal cells attached to glass coverslips were mounted in a Teflon chamber and incubated with 0.5 μm Fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at room temperature. The cells were then washed and bathed in a HEPES-buffered physiological saline solution (HBSS; 120 mm NaCl, 5.3 mm KCl, 0.8 mm MgSO4, 1.8 mm CaCl2, 11.1 mm glucose, 20 mm HEPES, pH 7.4) at room temperature at least 20 min before Ca2+ measurements were made. In some experiments, a nominally Ca2+-free medium was used, which was identical in composition except for the omission of added CaCl2. The fluorescence of Fura-2-loaded lacrimal cells was monitored with a photomultiplier-based system, mounted on a Nikon Diaphot 300 inverted microscope equipped with a Nikon × 40 (1.3 NA) Neofluor objective. The fluorescence light source was a Deltascan D101 (Photon Technology International Ltd.), equipped with a light path chopper and dual excitation monochromators. The light path chopper enabled rapid interchange between two excitation wavelengths (340 and 380 nm), and a photomultiplier tube monitored the emission fluorescence at 510 nm, selected by a barrier filter (Omega). All experiments were performed at room temperature. The data are expressed as a ratio of Fura-2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm (F340/F380). The immortalized chicken B-lymphocyte cell line, DT-40 (RIKEN Cell Bank number RCB1464), and a mutant version with genes for all three IP3 receptor types disrupted (RIKEN Cell Bank number RCB1467) were maintained in suspension with RPMI 1640 supplemented with 10% fetal bovine serum, 1% chicken serum, 4 mm glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 50 μg/ml 2-mercaptoethanol. The cells were maintained in culture at 40 °C in a humidified 95% air, 5% CO2 incubator, and at a cell density that ranged between 25 × 104 and 1 × 106 cells/ml. Both cell types were allowed to attach to glass coverslips (number 1) coated with Matrigel, and maintained in the RPMI 1640 medium described above. Both DT-40 cell types, attached to glass coverslips, were mounted in a Teflon chamber and incubated with either 1 μm Fura-2/AM (Molecular Probes) for 30 min at 40 °C or 1 μm fluo-4/AM (Molecular Probes) for 15 min at 40 °C. The cells were then washed and bathed in a modified HBSS (136.9 mm NaCl, 5.4 mm KCl, 0.81 mm MgSO4, 0.44 mmKH2PO4, 0.34 mmNa2HPO4, 0.34 mmNaHCO3, 1.8 mm CaCl2, 5.5 mm glucose, 10 mm HEPES, pH 7.4) at room temperature at least 20 min before Ca2+ measurements were made. In some experiments, a nominally Ca2+-free medium was used, which was identical in composition except for the omission of added CaCl2. In addition, calcium entry in DT-40 cells loaded with Fura-2 was determined in the presence of 0.5 mmCaCl2, whereas calcium entry with fluo-4 was determined in the presence of 0.25 mm CaCl2, in both instances to avoid saturation of the fluorescence signal due to the very large increases in [Ca2+]i in this cell type. The fluorescence intensities of Fura-2- and fluo-4-loaded DT-40 cells were monitored with a camera-based imaging system (Universal Imaging) mounted on a Zeiss Axiovert 35 inverted microscope equipped with a Zeiss × 40 (1.3 NA) fluor objective. Both fluorescence excitation and emission wavelengths were selected by filters (Chroma). For Fura-2 measurements, a Sutter Instruments filter changer enabled alternative excitation at 340 and 380 nm, and the emission fluorescence was monitored at 510 nm with a Paultek Imaging camera (model PC-20) equipped with a GenIISys intensifier (Dage-MTI, Inc.). The images of multiple cells collected at each excitation wavelength were subsequently processed using the MetaFluor software (Universal Imaging Corp., West Chester, PA) to provide ratio images. After background fluorescence correction, these images were further processed to convert the fluorescence ratios into [Ca2+] values (Kd(Fura-2) = 135 nm). Individual cells in the field of view were selected with suitable regions of interest, and their calcium changes with time were extracted. All experiments were performed at room temperature. Fluo-4 measurements were performed with the same imaging system, except single wavelength excitation was performed at 485 nm, and the emission fluorescence was monitored at 510 nm. Regions of interest were used to select Individual cells in the field of view so that the time course of changes in fluorescence intensity could be monitored and extracted. Patch clamp experiments were conducted in the standard whole cell recording configuration, using RBL-2H3 m1 cells (27Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflüg. Arch. 1981; 391: 85-100Crossref PubMed Scopus (15174) Google Scholar). Patch pipette (2–4 megaohms; Garner glass, type 7052) solutions contained 140 mm Cs-Asp, 2 mmMgCl2, 10 mm HEPES, 1 mm MgATP, and 10 mm1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-Cs4 (with free calcium set to 100 nm, calculated using MaxChelator software, version 6.60), pH 7.2. Bath solution (HBSS) was as described above, except CaCl2 was increased to 10 mm for calcium-HBSS, or omitted for nominally calcium-free HBSS (10 mm MgCl2 was included in nominally calcium-free HBSS). In all experiments, upon forming the whole cell configuration, the cell membrane potential was held at 0 or +20 mV. Once every 5 s, the membrane potential was stepped to −100 mV (for 20 ms to assessIcrac), and then a voltage ramp to +60 mV over a period of 160 ms was applied. Currents are normalized to cell capacitance. All voltages are corrected for a 10-mV liquid junction potential. Membrane currents were amplified with an Axopatch-1C amplifier (Axon Instruments, Burlingham, CA). Voltage clamp protocols were implemented, and data acquisition was performed with PCLAMP 6.1 software (Axon Instruments). Currents were filtered at 1 kHz and digitized at 200-μs intervals. RBL-2H3 cells were labeled with [3H]inositol and incubated in the presence or absence of methacholine, wortmannin, or both according to the protocol for examining effects on Icrac (see "Results"). 3H-Labeled lipids were extracted, deacylated, and separated by HPLC as previously described (28Anderson K.E. Stephens L.R. Hawkins P.T. Milligan G. Signal Transduction: A Practical Approach. Oxford University Press, London1999: 283-300Google Scholar, 29Auger K.R. Serunian L.A. Cantley L.C. Irvine R.F. Methods in Inositide Research. Raven Press, New York1990: 159-166Google Scholar). The [3H]inositol-labeled polyphosphoinositide levels were determined by liquid scintillation spectroscopy of the HPLC fractions corresponding to the retention times of authentic PIP and PIP2 standards. IP3, caged IP3, ionomycin, U73122, and U73343 were from Calbiochem. Thapsigargin was from LC laboratories. Heparin and wortmannin were from Sigma. Cs4-1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and Fura-2 were from Molecular Probes. RBL-2H3 cells have a well defined calcium release-activated calcium current (Icrac), activated upon intracellular calcium store depletion (30Hoth M. Penner R. Nature. 1992; 355: 353-355Crossref PubMed Scopus (1495) Google Scholar). We used the whole cell patch clamp technique to measure Icrac in RBL-2H3 cells, stably transfected with the muscarinic m1 receptor (25Choi 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). Extracellular application of ionomycin (500 nm) led to rapid and full activation of Icrac (Fig.1 A). Subsequently, extracellular application of the IP3 receptor inhibitor 2-APB (100 μm) rapidly blockedIcrac. Cells incubated with 2-APB for 3 min before exposure to ionomycin failed to show any detectable activation of Icrac (Fig. 1 B). The inhibition was not readily reversible upon removal of 2-APB (Fig. 1,A and B). In contrast to the results with 2-APB, blockade of IP3receptor function with the competitive IP3 receptor antagonist heparin (10 mg/ml) failed to block ionomycin-activatedIcrac (Fig. 1 C). Heparin is cell-impermeant and was delivered to the cell interior by inclusion in the patch pipette. An effective concentration clearly entered the cell, because 200 s after forming the whole cell mode, flash photolysis of caged IP3 (30 μm) failed to release stored calcium or activate Icrac in cells exposed to heparin (Fig. 1 D, open circles). In the absence of heparin, Icrac was readily activated by UV flash photolysis of caged IP3 (Fig.1 D, closed circles). Heparin has previously been reported to have no effect on the rise in cytosolic calcium induced by thapsigargin-activated capacitative calcium entry in mouse lacrimal cells (31Bird G. St J. Rossier M.F. Hughes A.R. Shears S.B. Armstrong D.L. Putney Jr., J.W. Nature. 1991; 352: 162-165Crossref PubMed Scopus (128) Google Scholar). However, the IP3 receptor antagonist 2-APB (30 μm) does prevent capacitative calcium entry in response to store depletion with thapsigargin in mouse lacrimal cells (Fig. 1 E). Methacholine, a muscarinic receptor agonist, evokes a sustained rise in intracellular calcium in mouse lacrimal cells, when added at both a low (1 μm) and high (100 μm) concentration (Fig.2 A). The calcium signal is composed of the IP3-mediated release of stored calcium and plasma membrane capacitative calcium entry (32Kwan C.Y. Takemura H. Obie J.F. Thastrup O. Putney Jr., J.W. Am. J. Physiol. 1990; 258: C1006-C1015Crossref PubMed Google Scholar). We aimed to prevent this agonist response by blocking activation of PLC and IP3generation with a membrane-permeable PLC inhibitor, U73122 (33Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar). A 3–5-min pretreatment of cells with 10 μmU73122 has previously been documented to fully and irreversibly prevent PLC activation upon agonist stimulation (33Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar, 34Broad L.M. Cannon T.R. Taylor C.W. J. Physiol. ( Lond. ). 1999; 517: 121-134Crossref PubMed Scopus (196) Google Scholar). Likewise, a 5-min preincubation of mouse lacrimal cells with 10 μmU73122was sufficient to prevent the intracellular calcium response to low or high doses of methacholine, consistent with a blockade of agonist-activated PLC (Fig. 2 A). A 5-min pretreatment of cells with 1–5 μmU73122 resulted in variable degrees of inhibition of the calcium signal (data not shown). A 5-min preincubation of cells with 10 μmU73343, a less potent analogue of U73122, had no effect on the agonist-evoked responses (not shown). The addition of the Ca2+-ATPase inhibitor thapsigargin to lacrimal cells in the absence of extracellular calcium caused a transient increase in intracellular calcium, due to release of calcium from stores. Upon the addition of extracellular calcium, a second more sustained rise in intracellular calcium concentration occurs, due to calcium influx via capacitative calcium entry (Fig. 2 B). A 5-min pretreatment of cells with 10 μmU73122 had no effect on the ability of thapsigargin to release intracellular calcium stores but fully prevented the rise in intracellular calcium concentration upon the readdition of extracellular calcium (Fig.2 B). U73343 had no effect on thapsigargin-induced capacitative calcium entry (Fig. 2 B). Thus, inhibition of PLC activity with U73122 appears to result in a specific block of capacitative calcium entry after store depletion. To ensure the effects of U73122 were not indirect (for example, due to changes in cell membrane potential or stimulation of the calcium removal processes), we examined the effects of this reagent on Icrac in RBL-2H3 m1 cells, measured under voltage-clamped conditions. The extracellular addition of either thapsigargin (1 μm) or ionomycin (500 nm) was sufficient to deplete intracellular calcium stores and fully activateIcrac in control cells (Fig.3, black circles). A 5-min pretreatment of cells with U73122 (10 μm), prevented activation of Icrac upon store depletion with either thapsigargin (Fig. 3 A, open circles) or ionomycin (Fig. 3 B, open circles). A 5-min pretreatment of cells with 5 μmU73122 (a dose that failed to consistently block either agonist or thapsigargin-induced calcium signals in mouse lacrimal cells) failed to block activation ofIcrac in RBL-2H3 m1 cells, althoughIcrac was inhibited to 38 ± 4% (n = 9) of controls (see also Fig.4 B). A 5-min pretreatment of cells with the inactive analogue U73343 (10–15 μm) did not affect activation of Icrac upon store depletion with either thapsigargin or ionomycin (Fig. 3 B,gray circles).Figure 4The addition of exogenous IP3 fails to relieve the inhibition of Icracproduced by inhibition of PLC. A, RBL-2H3 m1 cells were pretreated for 5 min with either 10 μmU73122(n = 18, open symbols) or 10 μmU73343 (n = 5, gray symbols) or were untreated (n = 21,black symbols). After pretreatment, the whole cell configuration was established (time 0), and 50 or 500 μm (not shown) F-IP3 was delivered to the cell interior through the patch pipette. B, RBL-2H3 m1 cells were exposed to 5 μmU73122 for 5 min (black symbols, n = 10), after which time 500 nm ionomycin (IONO) was applied extracellularly (as indicated). After a further 200 s, 100 μmmethacholine (MeCh) was added. In control cells, not treated with U73122 (broken trace, no symbols), the addition of 100 μm methacholine 600 s after forming the whole cell mode rapidly activated Icrac. HBSS containing 10 mm calcium was replaced with nominally calcium-free HBSS at the times indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The addition of 10 μmU73122 to RBL-2H3 cells onceIcrac had been activated resulted in a much smaller inhibition of Icrac and never caused a complete block. Icrac in U73122-treated cells averaged 73 ± 12% of controls. This suggests thatIcrac is much less sensitive to inhibition of PLC once it is initiated (Fig. 3, C and D). In the context of the conformational coupling model, the apparent dependence of capacitative calcium entry on PLC might reflect a requirement for IP3 on the channel-associated IP3 receptor (6Irvine R.F. FEBS Lett. 1990; 263: 5-9Crossref PubMed Scopus (580) Google Scholar,7Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1050) Google Scholar, 23Putney Jr., J.W. Cell. 1999; 99: 5-8Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Thus, we next addressed whether the addition of exogenous IP3 to the patch pipette would overcome the block ofIcrac activation in cells treated with U73122. Intracellular delivery of F-IP3 (50 or 500 μm), a slowly metabolizable analogue of IP3, rapidly activated Icrac in control cells after forming the whole cell mode (Fig. 4 A). However, a 5-min pretreatm
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