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• W2145735359 abstract "Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca2+ ([Ca2+]i) to stimulate cholecystokinin release from enteroendocrine cells. Using the murine enteroendocrine cell line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca2+ directly from the intracellular store. STC-1 cells loaded with fura-2 were briefly (3 min) exposed to saturated FA above and below the threshold length (C8, C10, and C12). C12, but not C8 or C10, induced a dose-dependent increase in [Ca2+]i, in the presence or absence of extracellular Ca2+. Various signaling inhibitors, including d-myo-inositol 1,4,5-triphosphate receptor antagonists, all failed to block FA-induced Ca2+ responses. To identify direct effects of cytosolic FA on the intracellular Ca2+ store, [Ca2+]i was measured in STC-1 cells loaded with the lower affinity Ca2+ dye magfura-2, permeabilized by streptolysin O. In permeabilized cells, again C12 but not C8 or C10, induced release of stored Ca2+. Although C12 released Ca2+ in other permeabilized cell lines, only intact STC-1 cells responded to C12 in the presence of extracellular Ca2+. In addition, 30 min exposure to C12 induced a sustained elevation of [Ca2+]i in the presence of extracellular Ca2+, but only a transient response in the absence of extracellular Ca2+. These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (≥C12) transiently induce Ca2+ release from intracellular Ca2+ stores. However, they also induce sustained Ca2+ entry from the extracellular medium to maintain an elevated [Ca2+]i. Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca2+ ([Ca2+]i) to stimulate cholecystokinin release from enteroendocrine cells. Using the murine enteroendocrine cell line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca2+ directly from the intracellular store. STC-1 cells loaded with fura-2 were briefly (3 min) exposed to saturated FA above and below the threshold length (C8, C10, and C12). C12, but not C8 or C10, induced a dose-dependent increase in [Ca2+]i, in the presence or absence of extracellular Ca2+. Various signaling inhibitors, including d-myo-inositol 1,4,5-triphosphate receptor antagonists, all failed to block FA-induced Ca2+ responses. To identify direct effects of cytosolic FA on the intracellular Ca2+ store, [Ca2+]i was measured in STC-1 cells loaded with the lower affinity Ca2+ dye magfura-2, permeabilized by streptolysin O. In permeabilized cells, again C12 but not C8 or C10, induced release of stored Ca2+. Although C12 released Ca2+ in other permeabilized cell lines, only intact STC-1 cells responded to C12 in the presence of extracellular Ca2+. In addition, 30 min exposure to C12 induced a sustained elevation of [Ca2+]i in the presence of extracellular Ca2+, but only a transient response in the absence of extracellular Ca2+. These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (≥C12) transiently induce Ca2+ release from intracellular Ca2+ stores. However, they also induce sustained Ca2+ entry from the extracellular medium to maintain an elevated [Ca2+]i. The ability to sense luminal nutrients after a meal is of fundamental importance in the gut epithelium. This serves to orchestrate digestion and so optimize nutrient assimilation. In addition, epithelial nutrient sensing is central to the short term control of food intake via gut to brain signaling pathways. After a meal, several gastrointestinal peptides are secreted by epithelial enteroendocrine cells (EEC). 1The abbreviations used are: EEC, enteroendocrine cells; CCK, cholecystokinin; 2-APB, 2-aminoethyldiphenyl borate; SLO, streptolysin-O; ER, endoplasmic reticulum; TG, thapsigargin. The pattern of secretion from EEC in vivo is complex, being encoded both chemically and anatomically, responding to the presence of specific macronutrient molecules in each luminal region (1Buchan A. Am. J. Physiol. 1999; 277: G1103-G1107PubMed Google Scholar, 2Furness J.B. Kunze W. Clerc N. Am. J. Physiol. 1999; 277: G922-G928PubMed Google Scholar, 3Raybould H.E. Am. J. Physiol. 1999; 277: G751-G755PubMed Google Scholar). This precision implies that a highly specific, nutrient-sensing apparatus must exist at a cellular and molecular level to produce appropriate EEC responses. However, the molecular bases for nutrient sensing by individual EEC are largely uncharacterized. In the proximal small intestinal epithelium, cholecystokinin (CCK) is a major EEC product and is secreted in response to free fatty acid. Also in this gut region, glucose evokes glucagon-like peptide 1 and 5-hydroxytryptamine secretion, amino acids induce gastrin release, and luminal acid causes secretin release. This categorization is a little oversimplified; for instance, dietary proteins can also stimulate CCK release (4Hira T. Hara H. Aoyama Y. Biosci. Biotechnol. Biochem. 1999; 63: 1192-1196Crossref PubMed Scopus (9) Google Scholar, 5Nishi T. Hara H. Hira T. Tomita F. Exp. Biol. Med. 2001; 226: 1031-1036Crossref PubMed Scopus (45) Google Scholar). Nonetheless, specific information about the nutrient environment in the lumen is transduced across the epithelium by EEC to activate local and distant reflexes. Of the various EEC cellular mechanisms, those involved in glucose-induced glucagon-like peptide 1 secretion by L-cells (6Reimann F. Gribble F.M. Diabetes. 2002; 51: 2757-2763Crossref PubMed Scopus (239) Google Scholar, 7Gribble F.M. Williams L. Simpson A.K. Reimann F. Diabetes. 2003; 52: 1147-1154Crossref PubMed Scopus (324) Google Scholar) are best understood. Lipid sensing is less well explained. Our earlier studies demonstrate that 12 or more carbon atoms (C12) are required in the acyl chain for saturated fatty acids to stimulate CCK release in humans (8McLaughlin J.T. Grazia Luca M. Jones M.N. D'Amato M. Dockray G.J. Thompson D.G. Gastroenterology. 1999; 116: 46-53Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and in the murine enteroendocrine cell line STC-1, where fatty acid exposure causes a reversible increase in intracellular Ca2+ concentrations ([Ca2+]i) (9McLaughlin J.T. Lomax R.B. Hall L. Dockray G.J. Thompson D.G. Warhurst G. J. Physiol. 1998; 513: 11-18Crossref PubMed Scopus (118) Google Scholar). Fatty acids are the most difficult nutrients to study, because they display complex physicochemical behavior in an aqueous environment. On account of their hydrophobic nature, once they exceed their limit of solubility they preferentially form insoluble aggregates unless a detergent such as bile is present. Bile salt secretion and, hence, this dissolution step, only occurs as a secondary event in response to the detection of fatty acids. The delivery of bile to the duodenum by gall bladder contraction is mediated by CCK. Therefore the system must initially be able to identify these unsolubilized fatty acids to secrete CCK in the first place. Our recent data have demonstrated that STC-1 cells are indeed able to respond to such unsolubilized particulate material, either lipid or nonlipid in origin, and this may underpin a component of the fatty acid response (10Benson R.S. Sidhu S. Jones M.N. Case R.M. Thompson D.G. J. Physiol. 2002; 538: 121-131Crossref PubMed Scopus (10) Google Scholar, 11Kazmi S. Sidhu S.S. Donohoe T.J. Wickham M. Jones M.N. Thompson D.G. Case R.M. Benson R.S. J. Physiol. 2003; 553: 759-773Crossref PubMed Scopus (6) Google Scholar). However, this cannot be the sole mechanism because, under different physicochemical conditions (e.g. a Ca2+-free milieu), the same saturated fatty acids are far more soluble and effectively nonparticulate, yet still evoke a response from STC-1 cells. Hydrophobic lipids will rapidly leave aqueous solution to enter the lipid plasma membrane and, as our previous data show, are rapidly accumulated in the cytoplasm of STC-1 cells (12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar). This is germane to the critical but unresolved issue as to whether fatty acids act on EEC at an extracellular or intracellular site. The relevant but uncharacterized signal transduction pathways activated by fatty acids clearly require identification. Intracellular fatty acid effects are the focus of the current study. We have analyzed in STC-1 cells the role of extracellular Ca2+ and intracellular Ca2+ pools in the C12-induced increase in [Ca2+]i, and the possible involvement of candidate signal transduction pathways. In light of the results from the initial studies, we then formulated and tested a novel hypothesis, that intracellular fatty acids can act directly and independently to induce Ca2+ release from intracellular Ca2+ stores. Materials—Cell culture consumables (Dulbecco's modified Eagle's medium, horse serum, fetal bovine serum, penicillin/streptomycin, trypsin, and EDTA solution) were purchased from Invitrogen. Saturated fatty acids (C8, C10, and C12), bombesin, poly-l-lysine solution (0.1% solution), U-73122, 2-aminoethyldiphenyl borate (2-APB), ryanodine, dantrolene, ruthenium red, thapsigargin, d-myo-inositol 1,4,5-triphosphate sodium salt (IP3), antimycin, and oligomycin were purchased from Sigma. Fura-2-AM, magfura-2-AM, and pluronic F-127 were obtained from Molecular Probes (Leiden, Netherlands). Genistein, adenosine 3′,5′-cyclic monophosphorothioate, 8-bromo-, Rp-isomer ((Rp)-8-Br-cAMPs), and xestospongin C were from Calbiochem (San Diego, CA). ONO-RS-082 was obtained from Biomol (Plymouth Meeting, PA). Streptolysin O was provided from Murex Diagnostics, NorCross, GA. Cell Culture—STC-1 cells (a gift from D. Hanahan, University of California, San Francisco, CA) were grown in Dulbecco's modified Eagle's medium (Invitrogen, number 41965-039) supplemented with 15% horse serum, 2.5% fetal bovine serum, 50 IU/ml penicillin, and 500 μg/ml streptomycin in a humidified 5% CO2 atmosphere at 37 °C. Cells were routinely subcultured by trypsinization upon reaching 80–90% confluency. For fluorescence imaging of intracellular Ca2+, cells were grown on 0.025% poly-l-lysine-coated coverslips (1.3 cm2) at a density of 1–2 × 105 cells/cm2 in 24-well plates and used 24–48 h after seeding. Cells between passages 50 and 60 were used. Other cell lines, PC12 (rat pheochromocytoma, ATCC), BON (human enterochromaffin, Dr. C. M. Townsend Jr., University of Texas Medical Branch, Galveston, TX), Caco-2 (human colon epithelial, ATCC), and IIC9 (Chinese hamster embryo fibroblast, ATCC) cells, were handled similarly to STC-1 cells. Cell viability was estimated by trypan blue exclusion and was always greater than 95%. Preparation of Fatty Acids—Fatty acids were dissolved in 99.6% ethanol and then diluted into extracellular or intracellular buffer (compositions described below), so as to produce working solutions with fatty acid concentrations between 100 and 500 μm. Each fatty acid solution was sonicated prior to use (SONICATOR XL, Misonix Inc., Farmingdale, NY) at level 9 for 3 min. Measurement of Intracellular Ca2+Concentration in Intact STC-1 Cells—The cytoplasmic free Ca2+ concentration ([Ca2+]i) was determined using dual-excitation fluorescence microscopy with the calcium-sensitive ratiometric dye fura-2-AM, as previously described (10Benson R.S. Sidhu S. Jones M.N. Case R.M. Thompson D.G. J. Physiol. 2002; 538: 121-131Crossref PubMed Scopus (10) Google Scholar, 11Kazmi S. Sidhu S.S. Donohoe T.J. Wickham M. Jones M.N. Thompson D.G. Case R.M. Benson R.S. J. Physiol. 2003; 553: 759-773Crossref PubMed Scopus (6) Google Scholar, 12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar). Briefly, cells were loaded with 2 μm fura-2-AM dissolved in extracellular buffer, containing 0.015% Pluronic F-127 at 37 °C for 20 min. After loading, coverslips were mounted into a perfusion chamber and washed with extracellular buffer or Ca2+-free extracellular buffer. The chamber was placed on the stage of an inverted epifluorescence microscope (Nikon Diaphot, Tokyo, Japan). Fluorescence images were observed via a ×40 oil immersion lens, at an emission wavelength of 510 nm and were captured by a cooled slow scan CCD camera (Digital Pixel Ltd., Brighton, UK) at excitation wavelengths of 340 and 380 nm. Images were acquired every 15 or 20 s, and the 340/380 nm ratio images were constructed and analyzed using Lucida 3.5 software (Kinetic Imaging Ltd., Bromborough, Wirral, UK). Normal extracellular buffer (pH 7.4) had the following composition (in mm): 140 NaCl, 4.5 KCl, 10 Hepes, 10 Hepes salt, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose. In Ca2+-free extracellular buffer, CaCl2 was omitted and 0.2 mm EGTA was included. The pH of both buffers was adjusted to 7.4. Measurement of Ca2+Release from Intracellular Ca2+Stores in Streptolysin O-permeabilized STC-1 Cells—To measure the release of Ca2+ from intracellular Ca2+ stores, cells were loaded with the low affinity Ca2+ indicator magfura-2 and then permeabilized with the bacterial protein streptolysin-O (SLO) as previously described (13van de Put F.H. Elliott A.C. J. Biol. Chem. 1996; 271: 4999-5006Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). STC-1 cells on coverslips were exposed to extracellular buffer containing 2 μm magfura-2-AM and 0.015% Pluronic F-127 at 37 °C for 20 min. The coverslip was mounted into the perfusion chamber and washed with permeabilization buffer on the stage of the microscope as described above. Permeabilization buffer contained 135 mm KCl, 1.2 mm KH2PO4, 0.5 mm EGTA, and 20 mm Hepes/KOH (pH 7.1). The free Mg2+ concentration was calculated as 0.9 mm, being adjusted with MgCl2 according to a previously described method (14Schoenmakers T.J. Visser G.J. Flik G. Theuvenet A.P. BioTechniques. 1992; 12: 870, 876-874, 879Google Scholar). To observe the real time process of cytosolic dye leakage, cells loaded with magfura-2 were excited at 360 nm, the isosbestic wavelength for magfura-2, and the permeabilization procedure was performed on the microscope stage in permeabilization buffer containing 0.5 units/ml SLO. After exposure to SLO solution for 5–10 min, cytosolic magfura-2 was visibly lost and the fluorescence intensity at 360 nm significantly dropped. Permeabilized cells were then washed with Ca2+-free intracellular buffer containing: 1 mm ATP, 135 mm KCl, 1.2 mm KH2PO4, 0.5 mm EGTA, 20 mm Hepes/KOH (pH 7.1), and 0.9 mm free Mg2+, for 5 min to remove residual SLO. To load Ca2+ into the intracellular Ca2+ stores, permeabilized cells were exposed to intracellular buffer containing 0.2 μm free Ca2+, 0.9 mm free Mg2+, 1 mm ATP, 135 mm KCl, 1.2 mm KH2PO4, 0.5 mm EGTA, 20 mm Hepes/KOH (pH 7.1). For Ca2+ uptake experiments, cells were washed with the permeabilization buffer (free of ATP and Ca2+), then exposed to the buffer containing Ca2+ but devoid of ATP for 2 min. Uptake of Ca2+ was then initiated by exposure to the intracellular buffer containing ATP and Ca2+. After Ca2+ loading for 5 min, permeabilized cells were exposed to test agents to examine stimulatory effects on Ca2+ release from intracellular Ca2+ stores. Because magfura-2 has spectral properties similar to fura-2, images were acquired and analyzed as described above for fura-2. Data Analysis—Data were calculated by determining ratio values for each of the individual cells (10–40 cells) in a microscope field. All data are representative of at least three individual experiments. Significant differences were determined by Student's unpaired t test. Fatty Acids Elevate [Ca2+]i in the Absence of Extracellular Ca2+—To investigate the contribution of intracellular Ca2+ stores to fatty acid-induced [Ca2+]i responses, STC-1 cells were exposed to fatty acids in the presence or absence of extracellular Ca2+. In both cases, C12 at 500 μm induced a rise in [Ca2+]i. However, in the absence of extracellular Ca2+, the rise was more rapid (Fig. 1A). This response was dose dependent (Fig. 1B). STC-1 cells were also exposed to different chain length fatty acids. As shown in Fig. 1C, in the absence of extracellular Ca2+, C12 but not C8 or C10, induced an increase in [Ca2+]i. This chain length specificity corresponds to that reported by us both in humans (8McLaughlin J.T. Grazia Luca M. Jones M.N. D'Amato M. Dockray G.J. Thompson D.G. Gastroenterology. 1999; 116: 46-53Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and in STC-1 cells under Ca2+ containing conditions (9McLaughlin J.T. Lomax R.B. Hall L. Dockray G.J. Thompson D.G. Warhurst G. J. Physiol. 1998; 513: 11-18Crossref PubMed Scopus (118) Google Scholar). In experiments measuring [Ca2+]i, the uncalibrated 340/380 nm ratio signal is generally presented as a surrogate for [Ca2+]i, because absolute estimates of [Ca2+]i are not routinely derived from ratio values. However, a two-point calibration of the fura-2 signal was carried out on a limited number of STC-1 cells as described elsewhere (12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar). Individual cells were initially treated with 1 μm thapsigargin and 1 μm ionomycin in Ca2+-free medium (containing 2 mm EGTA) to obtain fluorescence parameters for fura-2 under Ca2+-free conditions (Rmin). The cells were subsequently superfused with thapsigargin and ionomycin in Ca2+-supplemented medium to obtain fluorescence parameters for Ca2+-saturated fura-2 (Rmax). The 340/380 nm ratio signal was 0.99 ± 0.10 in resting cells (n = four experiments), corresponding to an estimated [Ca2+]i of 134 ± 22 nm (the Kd of fura-2 at 22 °C was taken as 135 nm (15Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar)). In Ca2+-containing conditions C12 (500 μm) typically raised the 340/380 fluorescence ratio by 0.25 ± 0.03, which corresponds to an estimated increase in [Ca2+]i of 68 ± 7 nm, which is around 200 nm, a value similar to those in our previous STC-1 studies (9McLaughlin J.T. Lomax R.B. Hall L. Dockray G.J. Thompson D.G. Warhurst G. J. Physiol. 1998; 513: 11-18Crossref PubMed Scopus (118) Google Scholar, 12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar) and in other endocrine cell types (16Menezes A. Zeman R. Sabban E. J. Neurochem. 1996; 67: 2316-2324Crossref PubMed Scopus (32) Google Scholar, 17Karlsson S. Sundler F. Ahren B. Biochem. Biophys. Res. Commun. 2001; 280: 610-614Crossref PubMed Scopus (5) Google Scholar, 18Hoenig M. Sharp G.W. Endocrinology. 1986; 119: 2502-2507Crossref PubMed Scopus (49) Google Scholar). In Ca2+-free conditions C12 (500 μm) typically raised the 340/380 fluorescence ratio by 0.35 ± 0.02, which corresponds to an estimated increase in [Ca2+]i of 83 ± 2 nm, which is around 220 nm. Finally, KCl (70 mmol) typically raised the 340/380 fluorescence ratio by 0.45 ± 0.05, which corresponds to an estimated increase in [Ca2+]i of 116 ± 11 nm, which is around 250 nm (Fig. 7A). C12Mobilizes Ca2+from Thapsigargin- and IP3-sensitive Intracellular Ca2+Stores—When intracellular Ca2+ stores were depleted by the Ca2+-ATPase inhibitor thapsigargin (TG) (Fig. 2A), C12 failed to induce [Ca2+]i responses in the absence of extracellular Ca2+. A similar result was obtained using the neuroendocrine peptide bombesin, which is known to activate the IP3 pathway in STC-1 cells (19Chang C.H. Chey W.Y. Erway B. Coy D.H. Chang T.M. Am. J. Physiol. 1998; 275: G192-G202PubMed Google Scholar). As expected, bombesin induced a rapid increase in [Ca2+]i, in the absence of extracellular Ca2+, indicating release of Ca2+ from intracellular Ca2+ stores. After Ca2+ release by bombesin, C12 failed to increase [Ca2+]i (Fig. 2B). These data suggest that C12 releases Ca2+ from the same intracellular store mobilized by IP3. Potential Pathways Involved in Fatty Acid-induced Ca2+Release from Intracellular Stores—To explore the signaling pathways involved in fatty acid-induced release of Ca2+ from intracellular stores, we examined the effect of pretreatment with several agents known to block intracellular signal transduction pathways linked to Ca2+ store mobilization. In the first instance, since C12 mobilizes Ca2+ from IP3-sensitive stores (Fig. 2A), pretreatment was undertaken with a PLC inhibitor, U73122 (10 μm, Fig. 3B), or an IP3 receptor antagonist, 2-APB (100 μm, Fig. 3C). However, both agents failed to block the C12-induced [Ca2+]i response although, as expected, both fully blocked the effects of 10 nm bombesin, a positive control of PLC/IP3-dependent Ca2+ release. Several other agents tested (data not shown), namely the IP3 antagonist xestospongin C, and a panel of ryanodine receptor antagonists (dantrolene, ruthenium red, and ryanodine used at >10 μm) also failed to block C12-induced [Ca2+]i responses, as did pertussis toxin, which inhibits Gi- and Go-coupled pathways, and genistein, which inhibits tyrosine kinase-linked receptor pathways. Although previous papers have demonstrated cAMP-dependent CCK release in STC-1 cells (20Chang C.H. Chey W.Y. Sun Q. Leiter A. Chang T.M. Biochim. Biophys. Acta. 1994; 1221: 339-347Crossref PubMed Scopus (56) Google Scholar, 21Prpic V. Basavappa S. Liddle R.A. Mangel A.W. Biochem. Biophys. Res. Commun. 1994; 201: 1483-1489Crossref PubMed Scopus (15) Google Scholar), the cAMP antagonist (Rp)-8-Br-cAMPs also failed to block C12-induced [Ca2+]i responses. Finally, thromboxane A2, an arachidonic acid cascade product generated by phospholipase A2 has been reported to induce Ca2+ release from intracellular stores (22Hertelendy F. Molnar M. Jamaluddin M. Mol. Cell. Endocrinol. 1992; 83: 173-181Crossref PubMed Scopus (15) Google Scholar). Therefore the phospholipase A2 inhibitor ONO-RS-082 was tested, but it too failed to block the C12-evoked Ca2+ responses. This evolving mass of negative data raised the alternative hypothesis: that C12 itself, which gains rapid access to the intracellular compartment (12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar), was transducing its own signal. To assess this possibility, we developed a permeabilized STC-1 cell system in which the effect of C12 on the intracellular Ca2+ store can be assessed directly. Ca2+Stores Remain Functional in Permeabilized STC-1 Cells—The fluorescence image of magfura-2-loaded STC-1 cells was monitored while excited at 360 nm, the dye isosbestic wavelength, before and after treatment of cells with SLO. In intact cells, all cell compartments including the cytosol and nucleus were stained with magfura-2 after 20 min of loading. Three minutes after exposure to 0.5 units/ml SLO, fluorescence intensity began to decrease, and full permeabilization was achieved within 10 min. As a consequence of the loss of cytosolic magfura-2, together with the soluble cytosolic contents (including soluble signaling molecules), magfura-2 fluorescence became punctate, indicating that residual dye was compartmentalized into cell organelles including the endoplasmic reticulum (ER) Ca2+ stores. To demonstrate that stores retained physiological functions in permeabilized cells, Ca2+ uptake and Ca2+ release were evaluated under several conditions. The experimental protocol was adapted by initially exposing cells to Ca2+-free intracellular buffer containing 0.9 mm free Mg2+ and 1 mm ATP to rule out the possibility that the changes in the magfura-2 ratio were because of changes in intraorganelle [Mg2+], rather than [Ca2+]. Cells were then exposed to the same buffer, but containing in addition 0.2 μm free Ca2+, which resulted in an appropriate increase in the magfura-2 ratio as Ca2+ was sequestered into the organelles (data not shown). Because free Mg2+ concentration was maintained constant at 0.9 mm, any increase in magfura-2 ratio indicates an increase in [Ca2+] in cell organelles. The magfura-2 ratio was not changed by adding Ca2+ in the absence of ATP, but was increased in the presence of ATP, an effect that was appropriately prevented by 1 μm thapsigargin pretreatment (data not shown). These results confirmed that Ca2+ was sequestered into the organelles by a sarco/endoplasmic reticulum calcium ATPase-type Ca2+-ATPase. Exposing permeabilized cells to IP3 resulted in a rapid, dose-dependent release of stored Ca2+ (Fig. 4, A and D). By contrast, thapsigargin induced a slow and continuous decrease in the magfura-2 ratio, indicating the existence of a slow efflux of Ca2+ from intracellular stores that is normally masked by sarco/endoplasmic reticulum calcium ATPase-mediated Ca2+ re-uptake (Fig. 4B). When applied in combination with thapsigargin, IP3 induced a larger Ca2+ release than did IP3 or thapsigargin alone, almost completely depleting the stores (Fig. 4, C and D). These validation studies confirm that in permeabilized cells Ca2+ is still functionally sequestered into intracellular Ca2+ stores via Ca2+-ATPase, and that these stores are the source of the fluorescence we measured. Intracellular Ca2+ stores are not damaged by SLO permeabilization, as they are still appropriately responsive to physiological and pharmacological stimuli. C12 Induces Ca2+Release from Intracellular Ca2+Stores in Permeabilized STC-1 Cells—Having validated the model of permeabilized STC-1 cells, we next examined whether intracellular fatty acids can induce Ca2+ release. Permeabilized cells were exposed to C12 after loading Ca2+ into the stores. Exposure to C12 at ≥250 μm induced a rapid decrease in magfura-2 ratio (corresponding to release of 50% of loaded Ca2+), and the ratio dropped irreversibly to the basal value on exposure to 500 μm C12 (Fig. 5A). The magfura-2 ratio did not recover after removing C12. This might suggest that C12 has a nonspecific permeabilization effect on the ER membrane. To exclude this possibility, we isolated and analyzed the time course data for cells excited at 380 nm. As the denominator in the 340/380 ratio, this signal appropriately falls as calcium is loaded into the stores. Simple leakage of store contents would have caused this signal to fall upon C12 exposure because of dye loss. However, the 380 nm fluorescence rose upon C12 exposure, so that the overall 340/380 ratio fell. This is in keeping with a selective transmembrane flux of calcium (Fig. 5B). The effect of C12 on magfura-2 ratio was dose dependent (Fig. 5C). In contrast to the result in intact cells (Fig. 3C), exposure to 10 nm bombesin did not change the magfura-2 ratio in permeabilized cells (Fig. 5D) even though C12 induced a decrease in magfura-2 ratio in the same cell preparations. Mitochondrial Ca2+ uptake inhibitors (23van de Put F.H. Elliott A.C. J. Biol. Chem. 1997; 272: 27764-27770Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) antimycin (5 μm) and oligomycin (5 μm) did not impair the decrease in magfura-2 ratio induced by C12 (Fig. 5E). As in intact cells (Fig. 1B), C8 and C10 (500 μm) did not affect magfura-2 ratio, but subsequent C12 was still effective (Fig. 6, A and B). In summary, C12 evokes Ca2+ release from non-mitochondrial stores in permeabilized STC-1 cells.Fig. 6C8 and C10 do not induce Ca2+ release from intracellular stores in permeabilized STC-1 cells. Permeabilized cells were washed with the intracellular buffer containing 1 mm ATP and 0.9 mm free Mg2+ in the absence of Ca2+, then cells were loaded with 0.2 μm free Ca2+ for 5 min. Ca2+-loaded cells were sequentially exposed to: A, 500 μm C8 for 5 min and 500 μm C12 for 2 min; B, 500 μm C10 for 5 min and 500 μm C12 for 2 min. Values are mean fluorescence ratio ± S.E. of 10–30 cells obtained from a single experiment, and data are representative of at least three different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Extracellular Ca2+Changes the Response Pattern to C12 and Is Necessary for Continuous [Ca2+]i Elevation—The above data indicate a direct effect of C12 on intracellular calcium stores. However, previous data have also shown that responses to C12 in Ca2+-containing medium depend largely on Ca2+ entry (9McLaughlin J.T. Lomax R.B. Hall L. Dockray G.J. Thompson D.G. Warhurst G. J. Physiol. 1998; 513: 11-18Crossref PubMed Scopus (118) Google Scholar, 12Sidhu S.S. Thompson D.G. Warhurst G. Case R.M. Benson R.S. J. Physiol. 2000; 528: 165-176Crossref PubMed Scopus (76) Google Scholar), suggesting that fatty acids may influence intracellular Ca2+ homeostasis in more than one manner, probably depending on their mode of presentation. During short term exposure of calcium-free C12 to STC-1 cells, the [Ca2+]i response showed a very brisk rate of onset and decline. We went on to examine the Ca2+ dependence of C12 responses during longer exposures. Accordingly, STC-1 cells were exposed to 500 μm C12 for 30 min in the presence or absence of extracellular Ca2+. In the presence of extracellular Ca2+, the C12-induced elevation of [Ca2+]i was maintained throughout the exposure (Fig. 7A). This response was also reversible, [Ca2+]i rapidly returning to basal values when C12 was washed out. In addition, STC-1 cells tolerate prolonged C12 exposure, responding promptly to depolarization (70 mm KCl) even after 30 min exposure to C12. In contrast, in the absence of extracellular Ca2+, C12 induced only a transient Ca2+ spike, and the elevated [Ca2+]i returned to basal value within 10 min of starting continuous exposure to C12 (Fig. 7B)" @default.
• W2145735359 created "2016-06-24" @default.
• W2145735359 creator A5000406768 @default.
• W2145735359 creator A5021618915 @default.
• W2145735359 creator A5025591995 @default.
• W2145735359 creator A5035878357 @default.
• W2145735359 creator A5085869885 @default.
• W2145735359 date "2004-06-01" @default.
• W2145735359 modified "2023-10-15" @default.
• W2145735359 title "Multiple Fatty Acid Sensing Mechanisms Operate in Enteroendocrine Cells" @default.
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