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Originally published In Press as doi:10.1074/jbc.M400098200 on April 5, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26082-26089, June 18, 2004
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Multiple Fatty Acid Sensing Mechanisms Operate in Enteroendocrine Cells

NOVEL EVIDENCE FOR DIRECT MOBILIZATION OF STORED CALCIUM BY CYTOSOLIC FATTY ACID*

Tohru Hira{ddagger}§, Austin C. Elliott{ddagger}, David G. Thompson¶, R. Maynard Case{ddagger}, and John T. McLaughlin¶||

From the {ddagger}School of Biological Sciences, G.38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom, the §Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, 060-8589, Japan, and Gastrointestinal Sciences, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom

Received for publication, January 6, 2004 , and in revised form, March 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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).1 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 (1–3). 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 (4, 5). 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 (6, 7) 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 (8) and in the murine enteroendocrine cell line STC-1, where fatty acid exposure causes a reversible increase in intracellular Ca2+ concentrations ([Ca2+]i) (9).

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 (10, 11). 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 (12). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 x 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 (10–12). 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 x40 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 (13). 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 (14). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (8) and in STC-1 cells under Ca2+ containing conditions (9).



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  		FIG. 1.
Fatty acid (C12) induces intracellular Ca2+ mobilization in the absence of extracellular Ca2+ in STC-1 cells. A, STC-1 cells were exposed to 500 µM C12 for 3 min (horizontal bars) in the presence of extracellular Ca2+ (1.2 mM), then cells were exposed to Ca2+-free extracellular buffer for 5 min followed by exposure to C12 solution for 3 min. B, cells were exposed to various concentrations (100–500 µM) of C12 for 2 min in the absence of extracellular Ca2+. C, STC-1 cells were sequentially exposed to 500 µM C8, C10, or C12 for 2 min (horizontal bars) in the absence of extracellular Ca2+. Values are mean fluorescence ratio ± S.E. of 10–30 cells obtained from a single experiment, and data are representative of at least three separate experiments.

 
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 (12). 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 (15)). 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 (9, 12) and in other endocrine cell types (16–18). 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).



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  		FIG. 7.
Changes in [Ca2+]i in response to C12 exposure for 30 min in the presence or absence of extracellular Ca2+ in STC-1 cells. STC-1 cells were exposed to 500 µM C12 for 30 min in the presence (A) or absence (B) of extracellular Ca2+. In the presence of extracellular Ca2+, cells were depolarized with 70 mM KCl after C12 exposure. Values are mean fluorescent ratio value ± S.E., and data are representative of three individual experiments.

 
C12 Mobilizes 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 (19). 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.



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  		FIG. 2.
C12 induces Ca2+ release from intracellular Ca2+ stores. In the absence of extracellular Ca2+, STC-1 cells were exposed to (A)10 µM TG for 3 min, or (B) 10 nM bombesin (BBS) for 5 min before exposure to 500 µM C12. Values are mean fluorescence ratio ± S.E. of 10–30 cells obtained from a single experiment, and the data are representative of at least four separate experiments.

 
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 [GenBank] (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.



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  		FIG. 3.
Phospholipase C-IP3 pathway is not involved in C12-induced [Ca2+]i responses. STC-1 cells were exposed to 500 µM C12 (open circles) or 10 nM bombesin (BBS; no symbols)) for 2 min in the presence of 0.1% (v/v) dimethyl sulfoxide (DMSO) (A), 10 µM U73122 [GenBank] (B), or 100 µM 2-APB (C). Cells were exposed to each drug for 3–10 min prior to exposure to C12 or bombesin in the presence of Me2SO, U73122 [GenBank] , or 2-APB. Compounds were dissolved in Me2SO to make a stock solution (10 mM for U73122 [GenBank] , 100 mM for 2-APB), respectively, and were diluted into extracellular buffer at 1:1000. Values are mean fluorescence ratio ± S.E. of 10–30 cells obtained from a single experiment, and data are representative of at least three separate experiments.

 
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 (20, 21), 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 (22). 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 (12), 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).



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  		FIG. 4.
IP3 and thapsigargin 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. A, Ca2+-loaded cells were sequentially exposed to 0.5, 1.0, and 5.0 µM IP3 for 3 min. B, Ca2+-loaded cells were exposed to 1.0 µM TG for 35 min. C, Ca2+-loaded cells were exposed to 1.0 µM TG for 5 min, and exposed to 5.0 µM IP3 in the presence of 1.0 µM TG. 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. D, mean percent value (%) of Ca2+ released by various concentrations of IP3 (in 3 min), by a combination of IP3 and TG (in 3 min), and by 1.0 µM TG alone (in 35 min). Values are mean ± S.E. obtained from different experiments (n = 6 for 1–5 µM IP3, n = 5 for 10 µM IP3, n = 9 for IP3 + TG, n = 5 for TG, respectively).

 
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 (23) 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.



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  		FIG. 5.
C12 induces 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. Changes in fluorescence ratio (A, 340:380) and fluorescence intensity excited by 380 nm (B) in Ca2+-loaded cells exposed to 100, 250, and 500 µM C12 for 3 min sequentially. C, mean percent value (%) of Ca2+ released from intracellular stores by exposure to various concentrations of C12 for 3 min. Values are mean ± S.E. obtained from different experiments (n = 5 for 100 µM, n = 4 for 250 µM, n = 7 for 500 µM C12, respectively). D, Ca2+-loaded cells were sequentially exposed to 10 nM bombesin (BBS) and 500 µM C12. E, permeabilized cells were washed and loaded with 0.2 µM free Ca2+ in the buffer containing mitochondrial Ca2+ uptake inhibitors (5 µM antimycin and 5 µM oligomycin). Ca2+-loaded cells were exposed to 500 µM C12 in the presence of mitochondria Ca2+ uptake inhibitors. Values in A, B, and D are mean fluorescence ratio or fluorescence intensity ± S.E. of 10–30 cells obtained from a single experiment, and data are representative of at least three different experiments. E presents mean data pooled from three experiments.

 



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  		FIG. 6.
C8 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.

 
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 (9, 12), 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).

Depletion of Intracellular Ca2+ Stores Does Not Prevent C12-induced Ca2+ Response in the Presence of Extracellular Ca2+—In calcium-free conditions, intracellular store depletion prevents a rise in [Ca2+]i in response to C12 (Fig. 2). However, we have previously suggested that external Ca2+ entry through a L-type Ca2+ channel is involved in fatty acid-induced responses (9, 12). The dual kinetics presented in Fig. 7 suggested that both mechanisms may in fact operate. To further investigate the involvement of extracellular Ca2+ in the fatty acid sensing mechanism, the intracellular Ca2+ store was depleted by thapsigargin (TG) as before (Fig. 2), but this time in the presence of extracellular Ca2+ and before fatty acid exposure. TG at 10 µM induced Ca2+ mobilization because of Ca2+ release from intracellular stores (Fig. 8A). Vehicle alone (Me2SO at 0.1%) did not affect [Ca2+]i (data not shown). After treatment with vehicle, both C12 and bombesin induced Ca2+ mobilization. Intracellular [Ca2+]i returned nearly to basal values 15 min after TG treatment, at which time cells were exposed to 500 µM C12 or 10 nM bombesin in the continued presence of TG. Bombesin at 10 nM, which had been shown to induce Ca2+ release from the store only (Figs. 2 and 3), failed to induce any further increase in [Ca2+]i after TG treatment (Fig. 8A), confirming that Ca2+ stores were depleted completely by TG treatment. However, C12 was still effective in inducing a further rise in [Ca2+]i after TG treatment. This contrasted with the inability of C12 to cause any additional increase in [Ca2+]i in the absence of extracellular Ca2+ (Fig. 2). Fig. 8B shows the area under the curve for the fluorescence ratio changes during 2 min exposure to C12 or bombesin after TG or vehicle treatment. The bombesin-induced Ca2+ response was abolished by TG treatment (p = 0.0027), but the C12-induced Ca2+ response was unchanged (p = 0.9152).



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  		FIG. 8.
Changes in [Ca2+]i in response to C12 or bombesin after TG treatment in the presence of extracellular Ca2+ in STC-1 cells. In the presence of extracellular Ca2+, STC-1 cells were exposed to 10 µM TG or vehicle (0.1% dimethyl sulfoxide (DMSO)) for 15 min followed by exposure to 500 µM C12 (open circles) or 10 nM bombesin (BBS; no symbols) for 2 min. A, values are mean fluorescence ratio ± S.E. of 10–30 cells obtained from a single experiment. Data are representative of at least four separate experiments. B, values are mean area under curve (AUC) of changes in fluorescence ratio induced by 500 µM C12 or 10 nM bombesin exposure for 2 min after TG or vehicle treatment (n = 4, 7, 4, and 5 for Me2SO + C12, TG + C12, Me2SO + bombesin, and TG + bombesin, respectively). N.S., not significantly different between Me2SO and TG treatment. *, significantly different between Me2SO and TG treatment by Student's unpaired t test, p < 0.05.

 
C12 Release Ca2+ from the Store After Permeabilization, and in the Absence of Extracellular Ca2+ in Several Cell Types—To investigate whether the effect of C12 on [Ca2+]i is specific to this particular CCK-producing enteroendocrine cell, several additional cell lines were exposed to C12 in the presence or absence of extracellular Ca2+, and also exposed to C12 after magfura loading and SLO permeabilization (Table I). In the presence of extracellular Ca2+, only STC-1 cells responded to C12. On the other hand, in the absence of extracellular Ca2+ in intact cells, and in SLO-permeabilized cells, all cell lines showed some release of stored Ca2+ by 500 µM C12. However, responses in STC-1 cells remained greater than in all the other cell lines studied.


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  		TABLE I
Changes in fluorescent ratio (340 / 380 nm) by 500 µM C12 exposure in intact cells in the presence or absence of extracellular Ca2+, or in permeabilized cells

Values are the mean fluorescent ratio changes (340/380 nm) in 30-133 cells from two to seven separate experiments. The intracellular Ca2+ level was measured in fura-2-loaded intact cells in the presence or absence of extracellular Ca2+ (Ca2+o), and in magfura-2-loaded cells permeabilized by SLO. In permeabilized cells, values decreased the fluorescent ratio of C12 in 3 min.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The cellular mechanisms by which fatty acids induce chain length-specific responses in enteroendocrine cells are still uncharacterized. The present study has demonstrated that fatty acids can act directly on endoplasmic reticulum Ca2+ stores in a manner that retains the key chain length specificity. Nonetheless, fatty acids clearly also induce Ca2+ entry from extracellular sources. Importantly, the [Ca2+]i kinetics observed for each site of action of fatty acid are distinctive, in keeping with the coexistence of at least two discrete sensing mechanisms. The evidence supports both of the outlined possibilities.

In the presence of extracellular Ca2+, C12 forms insoluble aggregates that must be dispersed by sonication. The time elapsed after sonication affects solubility, and re-aggregation of fatty acid occurs, which in turn affects the cellular responses to C12 (10). This has led to the suggestion that fatty acid effects may in part be exerted by the extracellular aggregates themselves (10, 11). Removing Ca2+ from the buffer renders C12 far more soluble and stable in its physicochemical state, yet results in [Ca2+]i responses that are rapid, reversible, and more reproducible than in the presence of extracellular Ca2+. Ca2+-free conditions permit detailed and separate study of the intracellular Ca2+ releasing mechanism, under more constant and controlled physicochemical conditions. The responses showed the same chain length dependence as previously described in vivo (8), indicating that the same basic sensing mechanisms were probably involved.

The entry of extracellular Ca2+ induced by C12 has previously been shown to be via L-type Ca2+ channels (9, 12). In the absence of extracellular Ca2+, C12 mobilized Ca2+ from intracellular stores, as confirmed by prior depletion of intracellular Ca2+ stores using thapsigargin (10 µM). This observation contrasts with data we have reported in a previous study, but the discrepancy can be explained by use of a lower concentration of thapsigargin (1 µM) or by the shorter incubation time (7 min) employed in the earlier study (12). Indeed, Fig. 6B shows that emptying of Ca2+ stores by thapsigargin treatment in STC-1 cells is a slow process. In the present study, the inclusion of bombesin as a positive control confirmed store emptying by thapsigargin, and therefore supports the current interpretation.

To probe the transduction mechanisms by which C12 acts to mobilize Ca2+ from intracellular stores, we initially employed specific blockers for several known cellular signaling pathways linked to Ca2+ mobilization. Where appropriate, the bombesin pathway was used as a positive control. These data were particularly important in excluding a role for IP3. All the blockers tested were ineffective. Although clearly many other transcytosolic signaling pathways could be involved, these data tend to rule out several obvious candidates.

Our subsequent data using permeabilized cells largely overcome the theoretical limitations associated with purely negative results, and with the need for suitable controls for every putative pathway, because in this model system the cytoplasm is replaced with an artificial intracellular buffer. This technique has been previously applied to study intracellular Ca2+ stores or exocytosis in several cell types including gastric epithelial cells (24), pancreatic acinar cells (13, 25), and platelets (26). We optimized and validated this method for use in STC-1 cells. These manipulations indicate that permeabilized STC-1 cells remain physiologically intact in being capable of accumulating Ca2+ into intracellular stores by an energy-dependent process, then releasing it in response to C12. Crucially, the specificity of response to fatty acid chain length remained identical to that seen in intact cells. It was important to exclude a nonspecific detergent effect of C12 causing leakage of all ER contents. This was confirmed by monitoring the 380 nm excitation data alone, in addition to the 340/380 ratio. A reduction in 380 nm intensity on C12 exposure would have been expected if leakage of dye were responsible for the fall in 340/380 fluorescence ratio. However, the 380 nm intensity actually rose after exposure to C12 (Fig. 5B). Indeed, magfura-2 is a small molecule (Mr 722), so its retention in the ER demonstrates retained membrane selectivity, and the observations following C12 can be ascribed to Ca2+ flux, rather than general membrane permeabilization with redistribution of Ca2+ dye.

Permeabilization was monitored in real time on the fluorescence microscope to ensure that cytoplasmic magfura-2 was lost while washing repeatedly with the intracellular buffer. Hence cytosolic signaling molecules such as phospholipases, protein kinases, IP3, and cAMP must be lost together with cytosolic magfura-2. Generation of new signaling molecules at the plasma membrane that could diffuse into the intracellular buffer cannot be totally excluded, but it seems unlikely that such cascades could be reconstituted quickly enough to explain the rapid time course demonstrated in response to C12. Moreover, if restoration of washed out signaling molecules occurred by regeneration in situ, bombesin would be expected to evoke a [Ca2+]i response in permeabilized cells, but this was not the case. Recently, there has been a resurgence of interest in the possibility that mitochondrial Ca2+ is involved in intracellular signaling (27, 28). However, Ca2+ release was unaffected in STC-1 cells in the presence of mitochondrial Ca2+ uptake inhibitors (Fig. 5E), suggesting that C12 releases Ca2+ from the ER Ca2+ store and not from mitochondria.

How do fatty acids induce Ca2+ release from the endoplasmic reticular stores? Perhaps there is a specific fatty acid receptor awaiting characterization, expressed in the ER membrane and working in parallel to the IP3 or ryanodine receptors. Another possibility is that fatty acids may directly act on Ca2+ channels or pumps on the store membrane. Alternatively, fatty acids incorporated into the ER membrane may modify a biophysical membrane property to rapidly enhance efflux of Ca2+. Answering these fundamental questions is an important aim for future studies.

In contrast to Ca2+ release evoked by IP3, the Ca2+ response to C12 in permeabilized cells was irreversible. This is most likely because of loss of cytoplasmic fatty acid shuttling (or buffering) proteins that are responsible for the rapid permeation of C12 throughout the intact cell, or perhaps reflects an inability to wash out fatty acid that enters the ER membrane in these modified conditions. The loss of counter-regulatory systems is an inevitable drawback of cellular permeabilization.

Comparison with several other cell types showed that intact STC-1 cells are clearly specialized and sensitive as fatty acid sensors. However, other cells become responsive to C12 when the fatty acid is rendered more available (i.e. presented in Ca2+-free medium or following permeabilization). This suggests that if adequate C12 enters any cell type, it can act on the Ca2+ store. It is likely that, in the absence of extracellular Ca2+, soluble C12 fatty acids are readily able to cross the plasma membrane, because of their hydrophobic properties and relatively small size. The fatty acid can then induce Ca2+ release via an intracellular site of action. However, only STC-1 cells responded to the less soluble fatty acids presented in the presence of extracellular Ca2+. Fatty acids have been shown to rapidly permeate STC-1 cells (12), so a theoretical component of the intact STC-1 cell fatty acid sensing mechanism may be a high affinity uptake system.

Taken together with our previous data, the current results strongly suggest the involvement of two pathways, one initiated at the plasma membrane to trigger influx of extracellular Ca2+ and another operated by fatty acid arriving at the endoplasmic reticulum store to trigger Ca2+ release. This duality may explain the small discrepancies in chain length dependence of fatty acid stimulation: C8 and C10 had a small effect on the Ca2+ response in the presence of extracellular Ca2+ (9, 12), but they had no direct effect on Ca2+ release from the store as shown in the present study (Figs. 1 and 6). The specificity of the STC-1 cell as a lipid sensor is likely to be explained by a combination of one or more specialized cell surface detection systems, and high avidity fatty acid uptake that allows rapid access to deeper compartments.

In vivo, it is likely that fatty acids in the gut lumen after a meal co-exist as a mixture of aggregate and soluble states, and that EEC are able to respond to both fatty acid states. Therefore, both pathways may be biologically important, because the small intestinal epithelium is the only organ that will ordinarily be exposed to such high concentrations of free fatty acids. In all other biological compartments, free fatty acids are transported mainly bound to protein, or repackaged in esterified form to circulate with lipoproteins.

In conclusion, medium chain fatty acid that releases CCK (C12, but not C8 or C10) induces intracellular Ca2+ release from ER stores in EEC. It also induces Ca2+ entry from the extracellular medium to maintain a high intracellular [Ca2+]i, via a different sensing mechanism. Cell surface receptors or EEC-specific transport systems may be involved in the two mechanisms.


   FOOTNOTES
 
* This work was supported by the Digestive Disorders Foundation, UK, and the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 44-161-206-4362; Fax: 44-161-206-1495; E-mail: john.mclaughlin{at}man.ac.uk.

1 The abbreviations used are: EEC, enteroendocrine cells; CCK, cholecystokinin; 2-APB, 2-aminoethyldiphenyl borate; SLO, streptolysin-O; ER, endoplasmic reticulum; TG, thapsigargin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. C. M. Townsend, Jr. for providing BON cells, and Dr. P. Padfield for SLO and expert advice on its use.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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