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INSERM U-159, Centre Paul Broca, 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. J. Epelbaum, INSERM U-159, 2ter rue d’Alésia, 75014 Paris, France. E-mail: epelbaum{at}broca.inserm.fr
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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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Five different somatostatin receptor subtypes (sst1 to sst5) have recently been cloned and characterized in various species, including human, rat and mouse (for review, see Refs. 6 and 7). However, the functional significance of these multiple isoforms has not yet been clarified. The anterior pituitary is one of the few tissues in which transcripts of all five subtypes have been detected (8). In situ hybridization studies demonstrated that discrete pituitary cell types could express several isoforms of the somatostatin receptors, but whether the profile of sst receptor expression is cell specific is still debated. High levels of sst4 and sst5 receptor messenger RNA (mRNA) have been reported in somatotropes, whereas thyrotropes may predominantly express sst2 mRNA (9). According to another study, sst5 and sst2 transcripts are preferentially expressed in somatotropes and thyrotropes compared with the other cell types; sst5 mRNA is more broadly expressed than sst2 (10). Finally, it was reported recently that in the 7315b rat prolactinoma model, sst2 and sst3 expression is primarily dependent upon the presence of estrogens (11).
Although no fully selective agonist has been developed to date for the sst receptors, several synthetic compounds were reported to exhibit a relative specificity for discrete rat subtypes when transfected in various cell lines. For example, octreotide (OCT) was reported to be more active on sst2, whereas BIM-23052, BIM-23056, and BIM-23268 exhibited a higher potency for sst3 and sst5 (12, 13, 14).
In the present study, we investigated whether the same sst receptor subtypes were involved in somatostatin inhibition of GH and PRL secretion and of cAMP accumulation in the female rat pituitary by comparing the potency ratios of SRIF and its agonists on these parameters. We also used a recently described method for measuring sst mRNA levels by quantitative RT-PCR (15) to determine whether basal and E2-induced pituitary levels of all five sst mRNA subtypes are correlated to the hormonal responses obtained under both conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH and PRL release
After removal of the culture medium, the monolayers were rinsed
with DMEM and exposed to forskolin (1 µM) alone or in
combination with different concentrations of SRIF or somatostatin
receptor agonists at 37 C for 3 h in DMEM-HEPES (15
mM; pH 7.4)-buffered medium containing BSA (0.1%). At the
end of the treatment, media were collected, aliquoted, and stored
frozen for hormone assay. GH and PRL levels in the medium were measured
by competitive enzyme immunoassays using rat GH (rGH) or rat PRL (rPRL)
coupled to acetylcholinesterase as tracers and highly specific
polyclonal immune sera obtained from goats for rGH and from rabbits for
rPRL (18). Dose-response curves established using NIAMDD RP-2 rGH or
RP-3 rPRL as standards displayed ED50 values of 3.3 and 2.5
ng/ml, respectively. OCT and
AcNH-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2
were gifts from Novartis (Basel, Switzerland), and BIM-23052,
BIM-23056, and BIM-23268 were obtained from Biomeasure (Milford,
MA).
Intracellular [3H]cAMP accumulation
After removal of the culture medium, cells were labeled with
[3H]adenine (2 µCi/ml) in serum-free
DMEM-HEPES-buffered medium at 37 C for 2 h. The labeling medium
was aspirated, and cells were incubated with different test substances
diluted in DMEM-HEPES containing 1 mM
3-isobutyl-1-methylxanthine (Sigma). After 2 h, the supernatant
was eliminated, and the reaction was terminated by the addition of
ice-cold 5% trichloroacetic acid (Sigma) containing 1 mM
cAMP (Boehringer Mannheim) and 1 mM ATP (Boehringer
Mannheim). Isolation of [3H]cAMP was performed by
sequential chromatography on Dowex (Pharmacia, Uppsala, Sweden) and
alumina columns according to the technique described by Salomon
et al. (19). Radioactivity was evaluated by liquid
scintillation counting (LKB-Pharmacia), and the data were expressed as
[3H]cAMP/[3H]ATP ratios, which permits
estimates of adenylate cyclase activity by the conversion of
[3H]ATP to [3H]cAMP.
Assay of sst mRNAs levels by quantitative RT-PCR
mRNA levels for each somatostatin receptor isoform were
quantified by means of the competitive RT-PCR assay described by
Viollet et al. (15). The principle of the method is to
coamplify an internal standard together with the mRNA species of
interest in each assay. The internal standard consists of an artificial
RNA containing the sequences required for specific amplification of
each sst. Coamplification was permitted by using pairs of RT-PCR
primers consisting of a 5'-primer specific for a given sst1 to sst5
mRNA and a 3'-primer common to all five species.
Total RNA was extracted and purified from control and E2-treated cells via the guanidium/CsCl gradient method (20). One microgram of total RNA was mixed with serial dilutions of the synthetic RNA standards. The mixture was denatured and reverse transcribed in a 20-µl volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 625 µM of each deoxy-NTP (Boehringer Mannheim), 100 µM random hexamers (Pharmacia), 20 U RNasin (Promega Biotech, Madison, WI), and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, NY). Samples were incubated for 90 min at 37 C, followed by 10 min at 70 C and chilled on ice. Two types of controls were performed. For the absence of genomic DNA contamination during the preparation of RNA samples, aliquots of each total RNA sample were submitted to the RT reaction in the absence of reverse transcriptase. Additionally, the RT reaction was performed in the absence of RNA sample.
Amplification conditions were the same for all receptors. One tenth of the RT sample was amplified in a 50-µl volume in 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM of each deoxy-NTP (Boehringer Mannheim), 25 pmol of each sense and reverse primer, 0.5 x 106 cpm 5'-end 32P-labeled reverse primer, and 1.5 U Taq polymerase (Promega Biotech, Madison, WI). The amplification included an initial denaturation step at 94 C for 30 sec, 29–32 cycles (denaturation at 94 C for 30 sec, annealing at 60 C for 1 min, elongation at 72 C for 30 sec), and a final step at 72 C for 10 min. Amplification was performed in an automatic thermocycler (Hybaid, Teddington, UK). In each experiment, a control including the PCR mixture with no complementary DNA template was added to check possible external contamination.
A 5-µl aliquot of each PCR reaction was electrophoresed in a 5% or 8% polyacrylamide gel (Bio-Rad, Hercules, CA). In the case of sst1 detection, amplification products were digested for 1 h at 37 C with XbaI restriction enzyme (Boehringer Mannheim) before migration to distinguish both standard and target PCR products. After migration, the gel was dried and exposed to X-Omat autoradiographic films (Eastman Kodak, Rochester, NY). Autoradiogram bands corresponding to the amplified products were cut and counted in a ß-scintillation counter. The amount of radioactivity recovered from the excised gel was plotted as the internal standard RNA/target mRNA ratio against the initial internal standard RNA amount. Linear regression of the curve was calculated. The number of target molecules was obtained by extrapolating the number of internal standard RNA molecules corresponding to an isomolar ratio of both species. Results were expressed as molecules per µg total RNA.
Statistical analysis
Each experiment was repeated at least three times using
triplicate or quadruplicate independent culture dishes per experimental
condition. Data obtained from independent experiments were normalized
and pooled as indicated in the figure legends. The IC50 of
dose-response analyses were calculated using the GraphPad Prism
program. Data were submitted to ANOVA and parametric F tests. A
two-tailed t test was used for RT-PCR data analysis. Results
are expressed as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, upper
panel). Addition of 1 nM E2 to
the culture for 96 h induced a moderate decrease
(P = 0.09) in basal and a significant decrease
(P < 0.001) in forskolin-stimulated GH secretion with
respect to those recorded in estrogen-depleted cultures, but did not
affect the hormonal response to SRIF agonists. Analysis of
dose-response curves obtained on E2-treated
cells (Fig. 2, upper panel)
allowed them to be ranked in a SRIF = OCT >> BIM-23052 >
BIM-23268 decreasing order of potency, with similar maximal inhibition
for all four analogs (Table 1).
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, lower panel). In contrast, an inhibitory response
to SRIF, OCT, and BIM-23052 was detected after pretreatment of the
cells with the steroid.
Dose-response plots (Fig. 2, lower panel) suggest that in
steroid-supplemented medium, the order of potency of the agonists on
PRL and GH release is identical. However, the potencies of SRIF, OCT,
and BIM-23052 were 1 order of magnitude higher for GH than for PRL
inhibition. BIM-23268, which was weakly active on GH secretion,
appeared inactive on PRL secretion. Although maximal SRIF, OCT, and
BIM-23052 inhibition of forskolin-stimulated GH secretion reached
values lower than basal levels, this was not the case for inhibition of
PRL secretion.
BIM-23056 did not have any effect by itself on the release of either
hormone under all conditions tested (Figs. 1 and 2). As the analog has
been proposed as a SRIF antagonist on the human recombinant sst5
receptor (21), we tested its action in the presence of SRIF (0.01
µM) on GH or PRL release. BIM-23056 (0.1
µM) weakly potentiated the inhibitory effect of SRIF on
PRL release in E2-treated cultures only (Table 2). Another somatostatin analog,
AcNH-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2,
which was reported to have antagonistic properties on the sst2 receptor
(compound 4 in Ref.22), was not effective as such on SRIF inhibition
of GH and PRL release under our experimental conditions (Table 2).
However, it weakly potentiated the inhibitory effect of SRIF on GH
release in E2-treated cultures.
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). Treatment with
E2 resulted in a 2-fold increase in the amplitude
of the responses, to 75 ± 1%, 76 ± 2%, and 62 ±
1%, respectively. BIM-23056 was ineffective under control conditions
(0.5 ± 1% activation), but induced a moderate, but significant,
inhibition of cAMP accumulation in cells exposed to
E2.
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). No
difference in PCR efficiency occurred between wild-type and standard
mRNA, as shown by the parallelism of both amplification curves
illustrated for sst2 (Fig. 4, inset). Highest mRNA levels in control cells correspond to
sst2 and sst3 subtypes (31% and 34% of total ssts, respectively).
sst1 mRNA was moderately expressed (19% of the total ssts), and sst4
and sst5 mRNAs were weakly expressed (9% and 6%, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether E2-induced PRL sensitivity to SRIF involves selective up-regulation of discrete somatostatin receptor subtypes, we compared the pattern of expression of pituitary sst receptor mRNA, measured by quantitative RT-PCR, with the efficacy of SRIF and of relatively selective SRIF analogs (OCT, BIM-23052, BIM-23056, and BIM-23268) in inhibiting GH and PRL secretion and cAMP accumulation (for the first three analogs) in anterior pituitary cells. We used a phenol red-free defined culture medium, because this frequently used pH indicator exhibits a weak estrogenic activity and by itself is able to stimulate cell content and release of PRL (23, 24).
Under the present experimental conditions, exposure of cells to E2 did not affect somatostatin inhibition of forskolin-stimulated GH release, in agreement with previous data obtained on female Sprague-Dawley pituitary cells grown for 72 h in the presence of 0.1 µM E2 (25). The fact that E2 did not affect the amplitude of the GH response suggests that receptor numbers on somatotropes are already maximal under basal conditions. At the same time E2-induced sensitivity of lactotropes resulted in a significant inhibition of forskolin-stimulated PRL secretion by SRIF, OCT, and BIM-23052, but not by BIM-23056 or BIM-23268.
Using quantitative RT-PCR assay we determined the absolute amounts of somatostatin receptor mRNAs expressed in pituitary cultures. The RT-PCR quantitation was always achieved within the range of parallel amplification of synthetic standard and sample mRNAs. Under these conditions, all five known sst subtypes were expressed after 4 days in culture. Major forms of mRNAs coding for SRIF receptors corresponded to isoforms sst2 and sst3, whereas mRNAs coding for the other subtypes were present in low amounts only. In addition, our data indicate that sst5 represents a minor component only of total pituitary sst receptor mRNA, although this subtype appears more strongly expressed in the pituitary than in most other tissues such as the brain or the gastrointestinal tract. Our results are in agreement with those of Bruno et al., who reported a similar pattern of expression in adult male rat Sprague-Dawley pituitaries using a semiquantitative hybridization/nuclease protection method (8). In situ hybridization studies have also shown that all sst subtypes seem to be present in all pituitary cell types and do not exhibit clear-cut colocalization with given subtypes (9). In another study, sst2 and sst5 transcripts were found to be more abundant in somatotropes and thyrotropes than in the other cell types (10). sst3 mRNA-containing cells were also frequently observed in the intermediate lobe of the pituitary, and sst2 and sst3 mRNAs were found in scattered cells of the neural lobe (9).
Treatment of the cells with estradiol for 4 days in culture resulted in selective overexpression of sst2 and sst3 subtypes. This observation agrees with the report by Visser-Wisselaar et al. (11, 26) that addition of E2 for 7 or 14 days in vitro induces the expression of sst2 and sst3 receptor subtypes in the 7315b rat prolactinoma cells, whereas sc administration of estradiol in vivo to 7315b tumor-bearing rats provokes sst2 mRNA expression only. In the GH4C1 cell line, 24-h treatment with estrogen was reported to increase not only sst2 and sst3, but also sst1 expression (27). Other conclusions were drawn concerning the gonadal steroid regulation of somatostatin receptor expression. In situ hybridization studies suggested that pituitary sst2 mRNA levels were unaffected by gonadal steroids in Sprague-Dawley adult rats (28). In contrast, in the same study, pituitary sst1 and sst3 mRNA expression appeared sexually dimorphic, with higher levels of sst1 in the female and higher levels of sst3 in the male.
As far as the other receptor subtypes are concerned, we observed lower levels of sst1 receptor mRNAs after estradiol treatment, whereas the decrease in the expression of sst4 and sst5 receptor mRNAs did not reach statistical significance. This suggests the existence of a differential regulation of sst5 with respect to that of sst2 and sst3. Such differential regulation of SRIF receptor subtypes has also been reported under different experimental conditions, in particular between sst1 and sst3, on the one hand, and sst5, on the other, in the pituitaries of food-deprived rats (29).
As the turnover of subtype-specific mRNAs is not known, any extrapolation of our data to corresponding receptor proteins should be interpreted with caution. To evaluate functional receptors, we thus analyzed the pharmacology of GH and PRL release in response to OCT, BIM-23052, BIM-23056, and BIM-23268, four subtype-selective SRIF analogs.
Involvement of sst2 receptors in somatostatin inhibition of GH secretion was already indicated by previous data for rat (30, 31), sheep (31), and fetal human (14) pituitary cultures, whereas sst5 appeared involved in the last two species only. Our data indicate that E2 pretreatment does not modify the pharmacology of SRIF inhibition of GH secretion, as only sst2 active agonists (SRIF itself and OCT) displayed high potency compared with sst3 and sst5 preferential agonists (BIM-23052 and BIM-23268). A recently reported sst2-selective antagonist (22) did not display antagonistic properties, but weakly potentiated the actions of SRIF on GH secretion. It might be recalled that at the dose that we used, it slightly inhibited cAMP accumulation from GH4C1 cells (22).
Our pharmacological results suggest that inhibition of lactotropes by SRIF is also preferentially mediated by sst2. After exposure to E2, OCT, and BIM-23052, but not BIM-23268 and BIM-23056, induced a dose-dependent inhibition of PRL release with IC50 values 1 order of magnitude higher than those corresponding to GH inhibition. This rank of potency for SRIF agonist is in keeping with 1) the 20 times lower membrane binding affinity of OCT in cells transfected with rat sst3 compared with cells transfected with rat sst2 (32) (Meyerhof, W., personal communication), 2) the 25- to 40-fold higher affinity of BIM-23052 (13) and BIM-23268 (Taylor, J. E., personal communication) for rat sst3 and sst5 than for rat sst2 in transfected cells, and 3) the absence of effects of the weak sst5 agonist BIM-23056 (13). These data are strongly suggestive that sst2 is the major receptor controlling GH and PRL inhibition. On the other hand, however, increased efficacy of OCT and BIM-23052 to inhibit cAMP accumulation after treatment with E2 may result from a combined action of sst2 and sst3. Finally, the failure of a putative antagonist (22) to show the same effect in our system suggests that data obtained from transfected cells cannot be easily extrapolated to normal pituitary cells.
Lower potency of all three active SRIF analogs on PRL than on GH secretion cannot be explained by differential receptor involvement, because their potency ratio was found homothetic in both cases. Alternative splicing of the sst2 isoform (33), which was reported to exhibit different coupling properties (34), may be one of the possible mechanisms responsible for the lower potency of analogs in lactotropes.
Participation of the rat sst5 isoform in GH inhibition or in E2-induced PRL inhibition by SRIF appears of minor importance, as BIM-23056, an analog with highest (although rather weak) affinity for rat sst5 (13), was ineffective as either an agonist or antagonist despite reports (21) that the analog acted as a potent antagonist (0.1 µM) in CHO-K1 cells transfected with the human recombinant sst5 receptor. In fact, BIM-23056 was only able to slightly potentiate the effect of SRIF on PRL secretion. In addition, sst5 mRNA appeared more down-regulated than up-regulated by exposure to estradiol. Nevertheless, inhibition of cAMP accumulation by BIM-23056 after pretreatment of cells with estradiol suggests that the agonist may also be recognized by cell types other than lactotropes and somatotropes. On the other hand, this observation is also consistent with recurrent doubts about the extent of direct cAMP involvement in the regulation of hormone secretion.
In summary, our results indicate that estrogens selectively increase pituitary mRNA levels corresponding to sst2 and sst3 receptor subtypes in the female rat. In addition, they confirm that sst2 is the most relevant somatostatin receptor for rat GH inhibition regardless of the steroid environment. However, there appears to be species differences in the receptors involved, as sst5 may also play a significant role in the regulation by SRIF of sheep (31) and human fetal (14) GH secretion. sst2 is also the major receptor responsible for rat PRL inhibition after priming of lactotropes with estrogens as also found in human fetal pituitary cells (14). Although participation of the sst3 subtype cannot be formally excluded, our pharmacological data suggest that its role in GH and PRL control is relatively minor. The pituitary functions of lowly expressed sst1 and sst4 receptor transcripts remain to be established.
Received September 18, 1997.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Basal hormone release; 
,
forskolin (1 µM)-stimulated hormone release; |og,
forskolin- plus 0.1 µM SRIF-stimulated hormone release;
, forskolin- plus 0.1 µM OCT-stimulated hormone
release; 
, forskolin- plus 0.1 µM BIM-23052-stimulated
hormone release; 
forskolin- plus 0.1 µM
BIM-23056-stimulated hormone release. Values are the mean ±
SEM of 6–10 independent determinations. a,
P < 0.001 vs. forskolin-stimulated
release.
, octreotide; 
,
BIM-23052; 
, BIM-23056; *, BIM-23268.
