cis-Unsaturated Free Fatty Acids Block Growth ... - Semantic Scholar

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 1 Printed in U.S.A.

cis-Unsaturated Free Fatty Acids Block Growth Hormone and Prolactin Secretion in ThyrotropinReleasing Hormone-Stimulated GH3 Cells by Perturbing the Function of Plasma Membrane Integral Proteins* ´ REZ, XESU ´ S CASABIELL, JESU ´ S P. CAMIN ˜ A, JOSE ´ L. ZUGAZA, FRANCISCO R. PE AND FELIPE F. CASANUEVA Cellular Endocrinology Laboratory, Department of Medicine, Compostela University School of Medicine and Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain ABSTRACT In vivo FFA block basal and stimulated GH secretion and have been implicated in the pathogenesis of the altered GH secretion present in obesity and Cushing’s syndrome. Although a direct action on the somatotroph cell has been postulated, the FFA mechanism of action is unknown. The main biological target for FFA action is the cellular membrane, and it has been shown that these metabolites can block the activity of a number of plasma membrane pumps, channels, and receptor systems. In the present work, it was observed using different types of pituitary cells (GH3, GH4C1, and rat pituitary primary cultures) that cis-unsaturated fatty acids, such as oleic, 1) do not perturb TRH binding or the homologous desensitization of the TRH receptor;

2) inhibit TRH-induced inositol 1,4,5-trisphosphate/diacylglycerol generation, probably by a direct perturbation of phospholipase C; 3) reduce the TRH-induced intracellular Ca21 redistribution and the ensuing changes in membrane potential; 4) completely inhibit the [Ca21]i rise due to the TRH-induced opening of voltage-gated Ca21 channels; and 5) abolish the TRH-induced Ca21 efflux through plasma membrane Ca21 pumps. These results suggest that cis-unsaturated FFA such as oleic acid selectively perturb the function of integral membrane proteins such as enzymes, channels, and pumps without perturbing the binding of ligands to receptors. (Endocrinology 138: 264 –272, 1997)

A

and Ga11 proteins (9). PLC, in turn, mediates the hydrolysis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2), generating inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) (10). DAG will cause the activation of protein kinase C (11) and the ensuing phosphorylation of several intracellular substrates, whereas Ins(1,4,5)P3 will lead to a rapid and transient release of Ca21 from intracellular stores, followed by a more persistent phase of calcium influx from the extracellular medium (12). This biphasic Ca21 signal triggers stimulated secretion of both GH and PRL in GH3 cells (13). In the present work, the biological effects of FFA have been studied in TRH-stimulated GH3 cells in an attempt to determine the intracellular target where these metabolites perturb signal transduction. The results presented here show that after incorporation of the fatty acid into the plasma membrane, it blocks TRH-induced hormone secretion by altering the functioning of some plasma membrane integral proteins implicated in the signaling pathway.

CLASSIC endocrinological feedback loop exists between FFA and GH secretion. In fact, GH has lipolytic properties on adipose tissue, and, in turn, plasma FFA reduction stimulates both spontaneous and stimulated GH secretion (1, 2). It is surprising that FFA are able to suppress both basal GH levels and GH secretion elicited by all known stimuli including GH-releasing hormone and GH-releasing peptide-6 (3). Although a partial involvement of somatostatin secretion has been postulated (4), unambiguous data exist showing that FFA block GH secretion by operating directly at the somatotroph cell (3, 5). This inhibitory action of FFA on somatotroph GH secretion is rapid (within minutes), dose and time dependent, and heavily dependent on the structure of the FFA tested (6). However, the precise mechanisms of FFA action as well as the points where they disrupt the intrasomatotroph signaling system are at present unknown. The GH3 cell line is a well characterized and widely used model to study GH secretion in vitro. This cell line expresses 50,000 –100,000 copies/cell of the TRH receptor, a protein that belongs to the family of G protein-coupled receptors characterized by seven transmembrane domains (7). TRH binds to its receptor with an apparent Kd of 10 nm (8), eliciting a rapid increase in phospholipase C (PLC) b-activity via Gaq

Materials and Methods Chemicals Fura-2/AM and bis-oxonol were obtained from Molecular Probes (Eugene, OR). BSA, thapsigargin, and TRH were purchased from Sigma Chemical Co. (St. Louis, MO). Myo-[2-N-3H]inositol was obtained from New England Nuclear (Boston, MA). All other chemicals were reagent grade from Sigma. The Partisil SAX HPLC column was obtained from Whatman (Clifton, NJ). FFA were purchased from Sigma at the highest purity available and checked by TLC in our laboratory. They were then stored at 220 C as 100- to 1000-fold concentrated ethanolic solutions under an inert atmosphere and administered to cells under continuous

Received July 24, 1996. Address all correspondence and requests for reprints to: Dr. F. F. Casanueva, P.O. Box 563, 15780 Santiago de Compostela, Spain. E-mail: [email protected]. * This work was supported by grants from Fondo de Investigacion Sanitaria, Spanish Ministry of Health (94/0213-E), and Fundacioń Espanõla contra el Cańcer.

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FFA BLOCK TRH-INDUCED SIGNAL TRANSDUCTION stirring. All controls received an equivalent amount of ethanol (0.1–1%, vol/vol), which had no effect on the measured responses.

Cell cultures GH3 pituitary cancer cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% (vol/vol) horse serum (HS) and 2.5% (vol/vol) FCS (both from Life Technologies, Grand Island, NY). GH4C1 cells were grown in DMEM plus 10% FCS. Media were supplemented with penicillin G (sodium salt; 100 U/ml) and streptomycin sulfate (100 mg/ml; both from Sigma), and cells were grown under a humidified atmosphere of 95% air-5% CO2 at 37 C. Cells were passaged once a week, and fresh medium was added on alternate days.

Secretion assays Cumulative PRL and GH secretion was determined by RIA essentially as previously described (14, 15). Briefly, GH3 cells were subcultured into 24-wells plates with DMEM-10% HS-2.5% FCS. After 3 days, monolayers were washed with Krebs-Ringer HEPES (KRH) of the following composition: 125 mm NaCl, 5 mm KCl, 1.2 m MgSO4, 2 mm CaCl2, 2 mm KH2PO4, 6 mm glucose, and 25 mm HEPES, pH 7.4; they were then preincubated for 5 or 10 min at 37 C in 2 ml serum-free DMEM supplemented with oleic acid (50 mm) or vehicle (1% ethanol in all plates). TRH (100 nm, final concentration) or an equivalent amount of KRH was added to the plates, and incubation resumed for the indicated time. In some assays oleic acid was added after TRH. Media were collected 1 h after the addition of TRH and stored at 280 C until assayed by RIA. RIA determinations were performed with materials generously provided by the National Pituitary Hormone Distribution Program (NIDDK, Bethesda, MD).

Measurement of the intracellular free Ca21 concentration, ([Ca21]i) GH3 cells were maintained in DMEM-15% HS-2.5% FCS for 4 days after confluence. They were then washed twice with Ham’s medium (122 mm CaCl2, 1 mm NaH2PO4, 3 mm KCl, and 30 mm HEPES) and treated at 37 C with the same buffer containing trypsin (0.05%, wt/vol) and EDTA (0.9 mm). Detachment of the cells from the dish was complete within 1 min. After centrifugation at 300 3 g for 5 min, cells were resuspended in KRH and loaded with 3 mm fura-2/AM for 30 min at 37 C under gentle continuous shaking. Cell suspensions were then diluted 1:4 with KRH and maintained at room temperature for 15 min. For fluorimetric measurements, 1 ml cell suspension was centrifuged, and the pellet was resuspended in 2 ml warm KRH. Cells were then placed in a cuvette positioned in a holder, thermostatically controlled at 37 6 1 C, and the fluorescence signal was measured under continuous stirring in a Perkin-Elmer LS-5B fluorimeter adjusted to lex 5 345 nm and lem 5 490 nm. Absolute [Ca21]i values were calculated using the formula: [Ca21]i 5 Kd (F 2 Fmin)/Fmax 2 F), where F is the fluorescence at the unknown [Ca21]i, Fmax is the fluorescence after the addition of 0.02% (vol/vol) Triton X-100 and 4 mm CaCl2, and Fmin is the fluorescence after Ca21 in the solution is chelated with 10 mm EGTA and 4 mm Tris, as previously described (16). The Kd was 225 nm for fura-2 (17).

Measurement of membrane potential (Vm) Membrane potential changes were monitored with the slow response fluorescent dye bis-oxonol (18). For these experiments, cell suspensions were prepared as described for [Ca21]i measurements. After washing once with KRH, cells from four dishes were resuspended in 10 ml KRH, and 750 ml were washed again by centrifugation at 104 3 g. The resuspended pellet was transferred to the fluorimeter cuvette, and 100 nmbis-oxonol was added from a 1000-fold concentration solution in dimethylsulfoxide. The cells were allowed to equilibrate with the dye for 10 min before the addition of the test substances. Downward or upward deflections of the fluorescence signal represent hyper- or depolarizations, respectively. Data were expressed as the percent decrease from basal fluorescence values.

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Measurement of inositol phosphate (InsP) generation InsP levels were determined as previously described (16). Briefly, GH3 cells were plated in 35-mm diameter dishes and grown until 70 – 80% confluent in complete DMEM. Labeling with myo-[2-N-3H]inositol was performed by incubating cell monolayers with the radioisotope (10 mCi/ml) in a 1:4 mixture of DMEM and Eagle’s Basal Medium (inositolfree) plus 10% dialyzed (Mr cut-off 5 12,000) FCS (final inositol concentration, ;10 mm). Twenty-four hours later, the incubation medium was removed, and the monolayers were washed three times with cold KRH. Cells were preincubated for 5 min at 37 C in KRH and incubated for 5 min at 37 C with shaking (120 rpm) in 2 ml prewarmed KRH with or without 50 mm oleic acid. After incubation, the medium was removed, and 1 ml prewarmed KRH, with or without the fatty acid, supplemented with TRH (100 nm) was added to the dishes, At the stated times, the media were aspirated, and the reactions were stopped by the addition of 2 ml ice-cold 10% (vol/vol) trichloroacetic acid. Acid-soluble radioactivity was extracted on ice for 30 min with occasional rocking. The entire supernatant was transferred to glass tubes and washed five times with 1 vol diethyl ether. Final extracts were diluted to 2.5 ml with ultrapure water and injected on an anion exchange HPLC column [Partisil 10 SAX (Whatman, Clifton, NJ); 250 3 4.6 mm]. InsP were resolved by a stepwise gradient of ammonium formiate at 1 ml/min and detected by liquid scintillation counting after mixing 0.4-ml fractions with 5.5 ml scintillation fluid (16). Experiments were performed in triplicate, and the results are expressed as the mean 6 sd of peak areas obtained by the trapezoidal method. The identities of Ins(1,4,5)P3 and Ins(1,3,4)P3 peaks were determined by comparison with tritiated standards.

Primary rat pituitary cell cultures Pituitary glands from Sprague-Dawley rats (150 –200 g) were obtained and processed essentially as previously described (19). Briefly, cells were dispersed by enzymatic digestion in Earle’s Balanced Salts Solution plus 1% FCS containing collagenase (0.4%), deoxyribonuclease (0.01%), dispase (0.2%), and hyaluronidase (0.1%) and cultured in 100-mm petri dishes (Ca21 experiments) or 24-well multiwells (secretion experiments) in a mixture of Ham’s F-12-DMEM-BGjb (6:3:1) (20) supplemented with BSA (2 g/liter), HEPES (2.4 g/liter), hydrocortisone (143 mg/liter), insulin-like growth factor I (10 ng/liter), T3 (0.4 mg/liter), glucagon (10 ng/liter), epidermal growth factor (EGF; 0.1 mg/liter), fibroblast growth factor (0.2 mg/liter), transferrin (10 mg/liter), NaHCO3 (1.8 g/liter), FCS (2.5%, vol/vol), and antibiotics.

Statistical analysis Statistical significance was calculated by the nonparametric MannWhitney test.

Results

TRH binds to specific receptors in GH3 cells that elicit a variety of intracellular responses after activation, among them a transient rise in [Ca21]i (13). The calcium signal elicited by the activation of TRH receptors in GH3 cells (Fig. 1A) can be subdivided into three main phases: 1) a first phase caused by a fast and transient rise of the [Ca21]i by redistribution of the ion from intracellular stores (Fig. 1A, a), which was suppressed by preincubation of the cells with thapsigargin (Fig. 1B), a drug able to block the sarco/endoplasmic reticulum Ca21 pumps (21); 2) a second, more sustained, phase (Fig. 1A, c) caused by the influx of Ca21 from the extracellular medium, which was abolished by depletion of extracellular Ca21 (Fig. 1C); this phase is probably due to the opening of different Ca21 channels at the plasma membrane (13, 22, 23), as shown by the fact that the L-type channel blocker nimodipine only partially inhibited this calcium influx (Fig. 1D); 3) between the first and second phases of the [Ca21]i rise, a minor calcium efflux current occurred that was

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FIG. 1. TRH-induced Ca21 signal in GH3 cells. [Ca21]i levels were continuously monitored in GH3 cell suspensions with the Ca21-sensitive dye fura-2, as described in Materials and Methods. The effect of TRH (100 nM) on [Ca21]i tracings was evaluated in control cells (A), cells preincubated for 30 min in the presence of the sarco/endoplasmic reticulum Ca21 pump inhibitor thapsigargin (B; 50 nM), cells incubated under nominal Ca21-free conditions (C), and cells preincubated for 2 min with 1 mM nimodipine to block L-type Ca21 channels (D). In control cells, the Ca21 signal can be subdivided in three phases: 1) a sharp rise to peak values, followed by 2) a fast return to basal levels and 3) a slow and sustained phase of Ca21 influx.

FIG. 2. Effect of oleic acid on TRH-induced [Ca21]i and Vm increases in GH3 cells. Cell suspensions were loaded with fura-2 to measure intracellular Ca21 levels (A and B) or with bis-oxonol to measure membrane potential (C and D), as described in the text. Fluorescence changes in response to TRH (100 nM) were continuously monitored in both controls (A and C) and cells preincubated with 50 mM oleic acid for 5 min (B and D). Downward and upward deflections of the fluorescence tracing in C and D reflect hyper- and depolarizations, respectively, and are expressed in arbitrary fluorescence units. All controls received the same amount of vehicle (0.1% ethanol, final concentration), which had no effect on the studied responses.

responsible for the inflexion between the first and the second peak (intermediate phase; Fig. 1A, b) (24). To understand the mechanisms by which cis-unsaturated FFA, like oleic acid, can abolish GH secretion both in vivo and in vitro, GH3 cells were exposed to oleic acid for some minutes before TRH administration. As Fig. 2, A and B shows, 50 mm oleic acid, administered 5 min previously, exerted a profound inhibition of the TRH-induced [Ca21]i signal in GH3 cells. The first [Ca21]i rise, caused by Ca21 redistribution, was inhibited by about 50%, and the second [Ca21]i rise, caused by Ca21 influx, was completely suppressed. When using the fluorescent probe bis-oxonol to assess membrane potential in GH3 cells, the administration of 100 nm TRH caused a rapid and transient hyperpolarization, followed by a slower depolarization (Fig. 2C). The initial hyperpolarization was due to the extrusion of intracellular K1 mediated by the previous [Ca21]i rise, whereas the subsequent depolarization may well be due to the entrance of extracellular Ca21 (25). When the cells were pretreated with 50 mm oleic acid, both the hyperpolarization and the subsequent depolarization were markedly reduced (Fig. 2D). The inhibitory effect of oleic acid on the TRH-mediated [Ca21]i signal was both dose and time dependent. As Fig. 3A shows, a dose of 50 mm oleic acid (a dose within the physiological range), administered 5 min previously, was able to reduce the TRH-mediated [Ca21]i rise by 50%. The time course of oleic acid (50 mm) action showed that the first phase of the TRH-mediated [Ca21]i signal was inhibited by about 50% in 5 min and was virtually suppressed after 10 min of

cell exposure to the FFA (Fig. 3B). The second phase was far more sensitive to the inhibitory effect of oleic acid, being inhibited by 50% in just 1 min and completely blocked when oleic acid was added to cells 5 min before TRH. This dissociation in oleic acid actions may reflect a rapid partition of the FFA on the plasma membrane, followed by a slower redistribution to the intracellular membranes. To understand whether the observed inhibitory actions of oleic acid on TRH-mediated early intracellular signals may be due to an impairment in the binding of TRH to its cognate receptor, an indirect approach was undertaken based on two facts. First, the administration of a saturating dose of 100 nm TRH desensitizes the TRH receptor to a second TRH administration (Fig. 4A). Second, the inhibitory action of oleic acid on TRH action (Fig. 4B) is rapidly eliminated by washing out the FFA in the extracellular milieu (Fig. 4C), clearly showing that no actions described here for oleic acid could be attributed to toxic effects. Then, when 50 mm oleic acid was administered to GH3 cells 5 min before 100 nm TRH, and cells were washed (twice, 10 min total) to eliminate the FFA, a second dose of TRH was completely ineffective (Fig. 4D). These results clearly demonstrate that oleic acid was not preventing either the binding of TRH or the desensitization of TRH receptor. As the inhibitory effect of FFA on the transmembrane signaling of the TRH receptor cannot be explained by a perturbation of ligand binding, the generation of InsP in response to TRH was assessed. No effect of oleic acid was observed on TRH-mediated generation of total InsP (data not


FIG. 3. Time and dose dependence of the inhibition of TRH-induced [Ca21]i signal by oleic acid. GH3 cells were preincubated for 5 min with increasing doses of oleic acid (A) or with a fixed dose of the fatty acid (50 mM) for increasing time periods (B). TRH-induced [Ca21]i signals were evaluated by fluorimetry with fura-2, as described in Materials and Methods, and expressed (mean 6 SD of four independent experiments) as a percentage of the control value. In B, the effects of oleic acid were analyzed separately for the first and second phases of the Ca21 signal.

shown), probably due to the high turnover of inositol lipids in GH3 cells. When InsP isomers were determined individually by strong anion exchange (SAX)-HPLC, Ins(1,4,5)P3 rose after 100 nm TRH in vehicle-treated GH3 cells with a time course that closely matched the first phase of the [Ca21]i response (Fig. 5). Pretreatment of the cells with 50 mm oleic acid 5 min before TRH led to a parallel inhibition of both Ins(1,4,5)P3 and [Ca21]i rises. This result taken together with the evidence that FFA did not perturb TRH binding to its receptor points toward the activation of PLC as the biochemical target of cis-unsaturated fatty acids. In the absence of extracellular Ca21, oleic acid exerted a more pronounced inhibition of the TRH-induced [Ca21]i response (Fig. 6, A–C). This fact together with the observation

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FIG. 4. Oleic acid does not prevent TRH binding and receptor desensitization. To assess the effect of oleic acid on the binding of TRH to its receptor, an indirect approach was undertaken, based on the fact that TRH administration leads to cell desensitization to a second TRH dose (A). The inhibitory action of oleic acid pretreatment on the TRHinduced Ca21 rise (B) was completely prevented when cells were washed out after oleic acid addition (C). When cells treated with oleic acid and then TRH were washed out, a second TRH dose was ineffective (D), thus showing that the first TRH dose has bound to its receptors. The washing process itself did not have any effect on the binding of TRH to its receptors (E). All experimental groups were washed twice by centrifugation (10-min total washing time), as indicated.

that FFA exerted a more pronounced inhibition on the second phase of the Ca21 response to TRH than on the first phase (Fig. 2) suggests that a perturbation in the activity of PLC may well not be the only action of oleic acid. A direct perturbation on the plasma membrane Ca21 channels that mediate Ca21 entry was studied. As Fig. 6D shows, the stimulation of GH3 cells by TRH opened Ca21 channels at the plasma membrane. This is indicated by the facilitated entry of Mn21 from the extracellular medium into the cell, with the subsequent quenching by Mn21 of the fura-2 fluorescence (26). When GH3 cells were preincubated with oleic acid (50 mm, 5 min previously) and then exposed to the same dose of TRH, the quenching was even slower than that in control cells, which suggests a profound inhibition of Ca21 channel opening. Recently, a Ca21 efflux current has been described in TRH-

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FIG. 5. Effect of oleic acid on TRH-induced generation of Ins(1,4,5)P3. GH3 cells were loaded with fura-2 for high speed [Ca21]i measurements (upper panel) or labeled with myo-[2-N-3H]inositol for determination of InsP3 isomers by strong anion exchange (SAX)-HPLC (lower panel). The time course of the inhibition of the TRH-induced Ca21 rise by oleic acid was paralleled by the inhibition of Ins(1,4,5)P3 generation (in the lower panel, data are expressed as the mean 6 SD of quadruplicate samples).

stimulated GH3 cells (24). It may be responsible for the inflexion between the first and the second peak of [Ca21]i shown in Fig. 1A, b (intermediate phase), and normally it is difficult to see because of its location between two increases of [Ca21]i. This efflux may also be the instrument of the rapid return toward basal values of [Ca21]i after TRH stimulation in GH3 cells deprived of extracellular Ca21 (Fig. 7A). As this flow occurs against the chemical gradient of the ion, it is unlikely to be the result of the opening of any cell membrane Ca21 channel, but is probably due to a pump that extrudes the ion out of the cell. In fact, this Ca21 efflux is partially blocked by La31 ions, which inhibit plasma membrane Ca21 adenosine triphosphatases (27) (Fig. 7B). This TRH-mediated outward Ca21 current became more evident in GH3 cells in which previous calcium entry has been induced by K1 depolarization (Fig. 7C). Interestingly enough, when a Ca21 entry had been induced by K1 depolarization, oleic acid was able to completely block the TRH-induced Ca21 efflux (Fig. 7D) in just 1 min, indicating that even this uncharacterized membrane pump, which expels Ca21 against the gradient, may be perturbed by FFA. To determine whether the perturbations caused by oleic acid on TRH-mediated early intracellular events were reflected in hormonal secretion in GH3 cells, similar experiments were undertaken measuring either GH or PRL. Preincubation of GH3 cells for 5 min with 50 mm oleic acid had

FIG. 6. Effect of oleic acid on TRH-induced Ca21 influx. The inhibition of the TRH-induced [Ca21]i signal by oleic acid (50 mM) was evaluated in both the absence (A and C) and presence (B) of extracellular Ca21. In D, the rate of influx through plasma membrane Ca21 channels was evaluated by the Mn21-quenching technique, using fura-2-loaded cells in the presence of high (500 mM) extracellular Mn21. Mn21 ions can flow through Ca21 channels in the plasma membrane, quenching the fluorescence of the intracellular fura-2. The rate of quenching can then be used as an index to monitor the number of Ca21 channels opened at a given moment. Oleic acid pretreatment resulted in a functional inactivation of plasma membrane Ca21 channels (D), causing a Ca21 tracing in response to TRH in complete medium analog to the signal obtained for control cells in Ca21-free medium (A and B).

no effect by itself, but was able to significantly suppress (P , 0.05 at 10 and 15 min; P , 0.01 at 30 min) the GH response after the administration of TRH (Fig. 8A). Similar results


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FIG. 7. Effect of oleic acid on TRH-induced Ca21 efflux in GH3 cells. [Ca21]i levels in response to TRH were monitored under nominal Ca21-free conditions (A and B) and in complete medium (C and D). When Ca21 influx was suppressed by the elimination of extracellular Ca21, [Ca21]i returned rapidly to basal levels after TRH (100 nM) stimulation (A). This process was partly blocked by La31 ions, which are known to inhibit plasma membrane Ca21 adenosine triphosphatases (B). When a maximal Ca21 flux was produced by KCl (50 mM) depolarization, the administration of TRH (100 nM) caused a transient downward deflection of the fluorescence tracing due to the activation of a Ca21 efflux current (C). This current was blocked when oleic acid (50 mM) was administered 1 min before TRH (D). Data are from representative experiments and are expressed in arbitrary fluorescence units.

were found when the secretion of PRL was analyzed (Fig. 8B). Next we tested whether the effects observed were nonspecific or, on the contrary, related to the spatial conformation of the FFA used. GH3 cells were preincubated for 5 min with different FFA at 50 mm (Fig. 9). The inhibitory action on both the first and the second phase of TRH-induced [Ca21]i rise was observed only for oleic acid (C18D9-cis), and both caprylic acid (C8) and estearic acid (C18) were devoid of action. It is most relevant that elaidic acid (C18D9-trans), an isomeric form of oleic acid, with the same chain length and with a double bond in the same position, was completely ineffective. The difference in spatial structure (oleic acid is a cis-monounsaturated and elaidic is a trans-monounsaturated FFA) may well be the explanation for these findings (6, 16, 28). The inhibitory actions of oleic acid on the TRH-generated intracellular calcium rise, was not a peculiarity of the tumoral line GH3. In fact, a similar inhibition was observed when GH4C1 cells were preincubated with 50 mm oleic acid for 5 min before TRH (Fig. 10). The oleic acid-mediated inhibition of TRH stimulation of early intracellular signals was more evident in the normal dispersed rat pituitary cells. The inhibitory action of oleic acid on the [Ca21]i rise was paralleled by the inhibition of either GH and PRL secretion in both GH4C1 cells and rat primary pituitary cells (Fig. 11, A and B). These facts suggest that the observed actions of oleic acid are a generalized phenomenon and not a peculiar finding of any special cell line.

FIG. 8. Effect of oleic acid on TRH-induced secretion of GH and PRL in GH3 cells. Cultures were grown in 24-well multiwells as described in Materials and Methods. Then, the time course of GH (A) and PRL (B) secretion was determined by RIA in control cells (solid squares), cells preincubated with 50 mM oleic acid for 5 min (open squares), cells stimulated with 100 nM TRH (solid circles), and cells preincubated with oleic acid for 5 min before the administration of TRH (open circles). Data are expressed as the mean 6 SD (n 5 4). Similar results were obtained for three independent experiments. *, P , 0.05.

Discussion

Nonsterified fatty acids are circulating metabolites whose levels in plasma oscilate widely in response to nutritional status and in certain pathological situations. Elevated plasma FFA levels have been associated with a plethora of biological effects, but it is not known whether their elevation in situations such as hypoxia or cetoacidosis plays a significant role in the pathophysiology of these clinical conditions. FFA have attracted considerable attention in recent years because they inhibit basal GH secretion and, in particular, block GH release elicited by all stimuli studied to date (3). Elevated plasma FFA levels have been implicated in the pathogenesis of this impaired GH secretion in obesity and Cushing’s syndrome, especially as recent reports show that FFA impinge

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FIG. 9. Effect of different FFAs on TRH-induced [Ca21]i. Fluorescence changes in response to TRH addition (100 nM) were observed in fura-2-loaded cells in the absence (control) or presence of different FFAs (5-min preincubation; 50 mM). The effects over the first and second phases of [Ca21]i response were analyzed separately. Data are expressed as the mean 6 SD (three independent triplicate experiments). *, P , 0.05 vs. control.

FIG. 11. Effect of oleic acid on TRH-induced GH and PRL secretion in different cell types. GH3, GH4C1, and rat pituitary cell suspensions were grown in 24-well plates as described for secretion experiments. After preincubation with either vehicle or 50 mM oleic acid for 5 min, cells were stimulated or not with TRH (100 nM). Then, GH (A) and PRL (B) were measured after 1 h. Data are expressed as the mean 6 SD (n 5 4). *, P , 0.005 vs. respective TRH values.

FIG. 10. Effect of oleic acid on the [Ca21]i signal of different cell types. GH3, GH4C1, and rat pituitary cell suspensions were loaded with fura-2, and [Ca21]i in response to TRH (100 nM) was monitored as described in Materials and Methods in both control cells and cells preincubated with 50 mM oleic acid for 5 min. Data are expressed as the mean 6 SD (n 5 4). *, P , 0.05 vs. respective TRH values.

directly on the somatotroph cell to exert their inhibitory action (5, 6, 29), a fact only observed for the physiological inhibitor of GH secretion, somatostatin. However, the precise mechanism of FFA action and the targets affected by them in the intracellular machinery of the somatotroph cell are far from being understood.

The inhibition of GH secretion by FFA could be due, in principle, to a disruption of the stimulus-secretion coupling, to a direct blockade of the secretory process, or to a combination of both mechanisms. We have previously shown in a fibroblast cell line transfected with the human EGF receptor that cis-unsaturated fatty acids, such as oleic acid, are able to block EGF-mediated generation of early ionic signals while leaving unaffected signal transduction by the tyrosine kinase pathway. It was postulated that oleic acid acts at the EGF receptor, blocking the activity of PLCg either by direct inhibition of the enzyme or by disrupting the interaction between the activated receptor and PLC (16, 30 –32). In the present work, we have studied the action of oleic acid on TRH-activated transmembrane signaling in GH3 cells. Pretreatment of cells with oleic acid was able to block the calcium signal elicited by a saturating dose of TRH. This Ca21 signal has two main components, as revealed by pharma-


cological and electrophysiological experiments: 1) a rapid, but transient, phase of Ca21 redistribution from intracellular stores, and 2) a slower, but more sustained, phase of Ca21 influx from the extracellular medium. Both components were blocked by oleic acid in a dose-dependent way, although to a different extent and with different time courses; Ca21 influx was more sensitive to inhibition by FFA. This inhibition could be explained by a perturbation of TRH binding to its receptor induced by oleic acid. However, both TRH binding and TRH-induced desensitization of the TRH receptor took place in the presence of oleic acid. Another possibility could be the induction of some kind of resistance of the intracellular Ca21 stores to the second messengers that trigger the Ca21 response in activated cells, namely Ins(1,4,5)P3 and DAG. However, the generation of Ins(1,4,5)P3 and, by inference, DAG was blocked by oleic acid with a time course that closely matched that of the Ca21 signal blockade. Thus, oleic acid did not inhibit TRH binding or TRH receptor activation, but inhibited Ins(1,4,5)P3 generation, pointing toward a perturbation in PLC activation or an interference in the interaction of PLC with phosphatidylinositol(4,5)bisphosphate PtdIns(4,5)P2 as the point of action for oleic acid. This fact has been observed in other cell types and with quite different receptor systems (16, 30 –32). These results agree with the established view that the main target for the biological actions of FFA are cellular membranes. In fact, the interaction of oleic acid with the plasma membrane of GH3 cells is very rapid (within seconds), in sharp contrast with the longer time (minutes) required for interaction of the fatty acid with intracellular components (data not shown). These data fit well with the observed time courses for the inhibition of the different components of the Ca21 signal. The influx phase, dependent on the voltage-gated opening of L-type Ca21 channels located externally on the plasma membrane, was inhibited faster and at lower doses than the redistribution phase, which has a more complex activation process, implicating enzymes located internally. In fact, we observed a direct blockade of plasma membrane Ca21 channels by oleic acid using the Mn21-quenching technique, resulting in a Ca21 signal very similar to the tracing observed in control cells when TRH was administered in nominally Ca21-free medium. In addition, the TRH-induced Ca21 efflux current recently described by Nelson et al. (24) and originated by the activation of a Ca21 extrusion pump located at the plasma membrane was also completely blocked by oleic acid. The inhibitory action of oleic acid on TRH-mediated early signals in GH3 cells was paralleled by the inhibition of both GH and PRL secretion, a fact that supports the view that Ca21 is instrumental for hormone release in those cells. Despite these prominent effects of oleic acid on membrane components responsible for the generation of transmembrane signals, we cannot entirely rule out a role for more distal effects of fatty acids, such as the perturbation of the fusion of secretory granules with the plasma membrane. In fact, whereas the inhibition of ionic transmembrane signals was not complete (;50%), GH secretion was usually blocked. A considerable amount of work will be required to clarify this possibility. Also, when the experiments were repeated in a different clone of somatotroph cells (GH4C1) or directly on pituitary dispersed cells, essentially the same results were

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obtained for both the Ca21 signal and the secretion of GH and PRL, suggesting that the effects of oleic acid were specific and not due to a feature of the cell line studied. A large body of evidence suggests that the biological actions of FFA are exerted mainly by their effects on biological membranes and the ensuing perturbation of the integral proteins residing there (28, 33). After their incorporation into lipid bilayers, FFA cause a biophysical perturbation at the core of the lipid bilayer, modifying its fluidity (34), causing a disruption of lipid-lipid and lipid-protein interactions. As a result, many biological functions were affected, including cell to substrate adhesion, surface receptor capping, and transmembrane signaling (16, 30 –34). FFA have been classified in two groups: type A FFA, which are cis unsaturated, and type B FFA, which are saturated or trans-unsaturated molecules (28). Type A FFA molecules, as oleic acid, have an angular structure due to the spatial conformation of the cis double bond, which prevents their regular packing when inserted into lipid bilayers, acting as potent functional disruptors of biological membranes. Type B FFA molecules, on the other hand, display a linear structure that can pack into biological membranes causing little or no distortion at this level, determining a significantly smaller biological potency. In fact, when we tested the abilities of various FFA to block calcium fluxes in TRH-stimulated cells, saturated and transunsaturated FFA were completely devoid of action, in sharp contrast with the marked inhibition caused by the same dose of a cis-unsaturated molecule such as oleic acid. This was particularly evident for elaidic acid, the trans isomer of oleic acid. These results suggest that oleic acid disrupts TRHmediated hormone secretion, perturbing the normal functioning of at least three integral membrane proteins: PLC, a channel responsible for Ca21 entrance, and a Ca21-extruding pump. In conclusion, in TRH-stimulated GH-secreting cell lines and rat somatotrophs, oleic acid was able to inhibit GH and PRL secretion by a time- and dose-dependent specific mechanism of action. Oleic acid exerted its action by multipoint activity, inhibiting the effectiveness of PLC as well as some channels and/or pumps that mediated Ca21 fluxes in the cell. Acknowledgments We thank Ms. M. Lage and Ms. V. Pin˜eiro for their excellent technical assistance.

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