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Cellular Endocrinology Laboratory, Department of Medicine, Compostela University School of Medicine and Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Dr. F. F. Casanueva, P.O. Box 563, 15780 Santiago de Compostela, Spain. E-mail: meffcasa{at}uscmail.usc.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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) ß-activity via
G
q and G11 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
Ca2+ from intracellular stores, followed by a more
persistent phase of calcium influx from the extracellular medium (12).
This biphasic Ca2+ 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg/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 µM) 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 -80 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 Ca2+concentration,
([Ca2+]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 x g for 5 min, cells were
resuspended in KRH and loaded with 3 µM 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 ± 1 C, and the fluorescence signal was measured under
continuous stirring in a Perkin-Elmer LS-5B fluorimeter adjusted to
ex = 345 nm and em = 490 nm.
Absolute [Ca2+]i values were calculated using the formula: [Ca2+]i = Kd (F - Fmin)/Fmax - F), where F is the fluorescence at the unknown [Ca2+]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 Ca2+ 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
[Ca2+]i measurements. After washing once
with KRH, cells from four dishes were resuspended in 10 ml KRH, and 750
µl were washed again by centrifugation at 104 x
g. The resuspended pellet was transferred to the fluorimeter
cuvette, and 100 nM-bis-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.
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 µCi/ml) in a 1:4 mixture of DMEM
and Eagle’s Basal Medium (inositol-free) plus 10% dialyzed
(Mr cut-off = 12,000) FCS (final inositol
concentration, 
10 µM). 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 µM 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 x 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 ± 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 (Ca2+ 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 µg/liter), insulin-like growth
factor I (10 ng/liter), T3 (0.4 µg/liter),
glucagon (10 ng/liter), epidermal growth factor (EGF; 0.1 µg/liter),
fibroblast growth factor (0.2 µg/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
Mann-Whitney test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) can be subdivided into
three main phases: 1) a first phase caused by a fast and transient rise
of the [Ca2+]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 Ca2+ pumps (21); 2) a
second, more sustained, phase (Fig. 1A, c) caused by the influx of
Ca2+ from the extracellular medium, which was abolished by
depletion of extracellular Ca2+ (Fig. 1C); this phase is
probably due to the opening of different Ca2+ 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
[Ca2+]i rise, a minor calcium efflux current
occurred that was responsible for the inflexion between the first and
the second peak (intermediate phase; Fig. 1A, b) (24).
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, A
and B shows, 50 µM oleic acid, administered 5 min
previously, exerted a profound inhibition of the TRH-induced
[Ca2+]i signal in GH3 cells. The
first [Ca2+]i rise, caused by
Ca2+ redistribution, was inhibited by about 50%, and the
second [Ca2+]i rise, caused by
Ca2+ influx, was completely suppressed.
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). The initial
hyperpolarization was due to the extrusion of intracellular
K+ mediated by the previous
[Ca2+]i rise, whereas the subsequent
depolarization may well be due to the entrance of extracellular
Ca2+ (25). When the cells were pretreated with 50
µM oleic acid, both the hyperpolarization and the
subsequent depolarization were markedly reduced (Fig. 2D).
The inhibitory effect of oleic acid on the TRH-mediated
[Ca2+]i signal was both dose and time
dependent. As Fig. 3A shows, a dose of 50
µM oleic acid (a dose within the physiological range),
administered 5 min previously, was able to reduce the TRH-mediated
[Ca2+]i rise by 50%. The time course of
oleic acid (50 µM) action showed that the first phase of
the TRH-mediated [Ca2+]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.
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). 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 µM
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.
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).
Pretreatment of the cells with 50 µM oleic acid 5 min
before TRH led to a parallel inhibition of both
Ins(1, 4, 5)P3 and [Ca2+]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.
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, A–C). This fact together with the observation that FFA exerted a more
pronounced inhibition on the second phase of the Ca2+
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
Ca2+ channels that mediate Ca2+ entry was
studied. As Fig. 6D shows, the stimulation of GH3 cells by
TRH opened Ca2+ channels at the plasma membrane. This is
indicated by the facilitated entry of Mn2+ from the
extracellular medium into the cell, with the subsequent quenching by
Mn2+ of the fura-2 fluorescence (26). When GH3
cells were preincubated with oleic acid (50 µM, 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 Ca2+ channel opening.
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, b (intermediate
phase), and normally it is difficult to see because of its location
between two increases of [Ca2+]i. This efflux
may also be the instrument of the rapid return toward basal values of
[Ca2+]i after TRH stimulation in
GH3 cells deprived of extracellular Ca2+ (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 Ca2+ channel, but is probably due to a pump that
extrudes the ion out of the cell. In fact, this Ca2+ efflux
is partially blocked by La3+ ions, which inhibit plasma
membrane Ca2+ adenosine triphosphatases (27) (Fig. 7B).
This TRH-mediated outward Ca2+ current became more evident
in GH3 cells in which previous calcium entry has been
induced by K+ depolarization (Fig. 7C). Interestingly
enough, when a Ca2+ entry had been induced by
K+ depolarization, oleic acid was able to completely block
the TRH-induced Ca2+ efflux (Fig. 7D) in just 1 min,
indicating that even this uncharacterized membrane pump, which expels
Ca2+ against the gradient, may be perturbed by FFA.
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). Similar results were
found when the secretion of PRL was analyzed (Fig. 8B).
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). The inhibitory action on
both the first and the second phase of TRH-induced
[Ca2+]i rise was observed only for oleic acid
(C18
9-cis), and both caprylic
acid (C8) and estearic acid (C18) were devoid
of action. It is most relevant that elaidic acid
(C189-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).
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). 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 [Ca2+]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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 PLC
either by direct inhibition of the
enzyme or by disrupting the interaction between the activated receptor
and PLC (16, 30, 31, 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 Ca2+ signal has two main components, as revealed by pharmacological and electrophysiological experiments: 1) a rapid, but transient, phase of Ca2+ redistribution from intracellular stores, and 2) a slower, but more sustained, phase of Ca2+ 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; Ca2+ 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 Ca2+ stores to the second messengers that trigger the Ca2+ 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 Ca2+ 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, 31, 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 Ca2+ signal. The influx phase, dependent on the voltage-gated opening of L-type Ca2+ 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 Ca2+ channels by oleic acid using the Mn2+-quenching technique, resulting in a Ca2+ signal very similar to the tracing observed in control cells when TRH was administered in nominally Ca2+-free medium. In addition, the TRH-induced Ca2+ efflux current recently described by Nelson et al. (24) and originated by the activation of a Ca2+ 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 Ca2+ 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 obtained for both the
Ca2+ 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, 31, 32, 33, 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 trans-unsaturated 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 TRH-mediated hormone secretion, perturbing the normal functioning of at least three integral membrane proteins: PLC, a channel responsible for Ca2+ entrance, and a Ca2+-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 Ca2+ fluxes in the cell.
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Acknowledgments |
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Footnotes |
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Received July 24, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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