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Somatostatin Receptor Subtype 1 Selective Activation in Human Growth Hormone (GH)- and Prolactin (PRL)-Secreting Pituitary Adenomas: Effects on Cell Viability, GH, and PRL Secretion

Section of Endocrinology (M.C.Z., D.P., F.T., M.R.A., A.M., E.C.d.U.), Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, 44100 Ferrara, Italy; Division of Neurosurgery (R.P.), Hospital of Ferrara, 44100 Ferrara, Italy; Division of Neurosurgery (M.S.), Hospital of Padova, 35100 Padova, Italy; and Biomeasure Inc./Beaufour-IPSEN (M.D.C.), Milford, Massachusetts 01757-3650

Address all correspondence and requests for reprints to: Ettore C. degli Uberti, M.D., Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}dns.unife.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Somatostatin (SRIF) analogs interacting with SRIF receptor subtype (SSTR) 2 and SSTR5 are known to reduce secretion in GH-secreting pituitary adenomas. We investigated the effects of SRIF and a SSTR1 selective agonist, BIM-23926, on GH and prolactin (PRL) secretion and cell viability in primary cultures deriving from 15 GH- and PRL-secreting adenomas expressing SSTR1. Quantitative RT-PCR showed SSTR1 mRNA mean levels of 6 ± 2.2 x 10⁴ molecules/µg reverse-transcribed total RNA. SSTR2 and SSTR5 were frequently expressed (93.3%), on the contrary of SSTR3 (53.3%) and SSTR4 (6.7%). GH secretion was significantly reduced by SRIF and BIM-23926 (45 ± 8.6% and 32 ± 18.1% inhibition, respectively) as well as PRL secretion (16.1 ± 4% and 19.7 ± 3.5% inhibition, respectively). After treatment with SRIF and BIM-23926, cell viability was significantly reduced by 17.5 ± 5% and 20 ± 3.9%, respectively. SSTR1 mRNA levels correlated with the degree of GH and PRL secretion inhibition. These results demonstrate that SSTR1 selective activation inhibits hormone secretion and cell viability in GH- and PRL-secreting adenomas in vitro and suggest that SRIF analogs with affinity for SSTR1 may be useful to control hormone hypersecretion and reduce neoplastic growth of pituitary adenomas.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
SOMATOSTATIN (SRIF) TRANSDUCES its inhibitory signals on hormone secretion and cell proliferation in many tissues through five transmembrane G-coupled SRIF receptor subtypes (SSTR 1 to 5) (1). SSTR subtypes 1, 2, 3, and 5 are expressed in normal and neoplastic human pituitary somatotroph cells, and a large body of evidence indicates that SRIF analogs, mainly interacting with SSTR2 and SSTR5, can reduce GH and prolactin (PRL) secretion in GH- and in PRL-secreting pituitary adenomas, both in vivo (2) and in vitro (3, 4). Furthermore, an inhibitory effect of compounds preferentially interacting with SSTR2 and SSTR5 has recently been demonstrated on the proliferation of GH-secreting pituitary adenoma cells (5). Although the involvement of SSTR2 and SSTR5 in mediating the inhibitory action of SRIF and its analogs on pituitary function and proliferation are being investigated, the effect of SSTR1 selective activation in human GH-secreting adenomas has so far not been explored. We therefore investigated SSTR (1 to 5) expression in 15 GH- and PRL-secreting adenomas by RT-PCR and quantitatively evaluated SSTR1 mRNA expression. We then tested, in primary cultures derived from SSTR1-expressing tumors, the effects of SRIF and a SSTR1 selective agonist, BIM-23926, on GH and PRL secretion and on cell viability.

	Materials and Methods

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Human pituitary adenomas

We examined 15 pituitary macroadenomas (mean maximal diameter 1.87 ± 0.75 cm; mean volume 3.7 ± 0.9 cm³) from acromegalic patients, six males and nine females, aged 44 ± 3.9 yr (median, 46 yr), with serum GH of 23.4 ± 2.8 µg/liter (not suppressing to <1 µg/liter during an oral glucose tolerance test) and IGF-I levels of 812 ± 63.4 µg/liter. Serum PRL levels were 32.6 ± 8.52 µg/liter. Eight patients had previously been treated with SRIF analogs (lanreotide or octreotide) and two with dopaminergic drugs (cabergoline). All treatments were stopped 2 months before surgery. All patients underwent transsphenoidal surgery, and immunohistochemical examination for anterior pituitary hormones was performed on all specimens. Of the 15 tumors, nine stained only for GH, and six were positive for both GH and PRL. Human total RNA from normal pituitary was used as a control for RT-PCR reactions (Analytical Biological Services Inc., Wilmington, DE).

Tissue collection and primary culture

Tissue samples were collected in accordance with the guidelines of the local committee on human research. A fragment was immediately frozen in liquid nitrogen under ribonuclease (RNase)-free conditions at the time of surgery and stored at -80 C until RNA isolation was performed. A portion of the fresh tissue was washed in high-glucose DMEM (EuroClone Ltd., Torquay, UK) supplemented with 0.3% BSA, 2 mM glutamine, and penicillin/streptomycin (EuroClone Ltd.) and then minced and enzymatically dissociated using 0.35% collagenase (Sigma, Milano, Italy) and 1% trypsin (EuroClone Ltd.) at 37 C for 60 min, as previously described (3). Cells were resuspended in high-glucose DMEM with 10% fetal bovine serum and antibiotics (EuroClone Ltd.), seeded in 96-well culture plates (2 x 10⁴ cells/well; 80 wells/tumor) and incubated at 37 C in a humidified atmosphere of 5% CO₂ and 95% air. Fibroblast contamination was excluded by treatment with cis4-hydroxy-L-proline and morphological examination of the cultured cells (5). Medium was then changed to serum-free high-glucose DMEM containing 0.2% BSA, 120 nM transferrin, 5 U/liter insulin, 2 mM glutamine, and antibiotics. Cells were then treated with SRIF or a SSTR1 selective agonist (BIM-23926) for evaluation of hormone secretion in conditioned medium, which was collected and stored at -20 C for later hormone measurement. Medium was then replaced and cells were treated with SRIF or BIM-23926 for cell viability experiments.

Isolation of RNA and RT-PCR

To demonstrate the pituitary origin of the samples, RT-PCR analysis for GH expression was performed in each specimen, and further expression analysis for PRL and SSTR1–5 was performed only in GH-expressing tissues. Frozen tissues were disrupted using a dismembrator (B. Braun Biotech International, Milano, Italy), and total RNA from the pulverized tumors was extracted with TRIzol reagent (Invitrogen, Milano, Italy), according to the manufacturer’s protocol. Total RNA was dissolved in diethylpyrocarbonate-treated water to a concentration greater than 1 µg/µl and stored at -80 C until use. To prevent DNA contamination, RNA was treated with RNase-free deoxyribonuclease (Promega Corp., Milano, Italy). Using a first-strand cDNA synthesis kit (SuperScript preamplification system for first-strand cDNA synthesis, Invitrogen), 1 µg total RNA was reverse transcribed with random hexamers according to the manufacturer’s protocol. Reverse transcription (RT) reactions were performed by using the GeneAmp 9700 PCR system (Applera, Monza, Italy) as previously described (6). A negative control for each sample was performed by running the reaction without adding the reverse transcriptase enzyme (RT-). The cDNA (1 µl RT reaction, corresponding to 50 ng reverse-transcribed total RNA) was then amplified by PCR in 50 µl with 1 U Taq DNA polymerase (Promega Corp.) in the conditions recommended by the suppliers. PCR conditions and oligonucleotide primers for amplification are listed in Table 1. Glyceraldehyde phosphate dehydrogenase amplification was performed as control for RT reaction. PCR products were run on a 2% agarose gel, visualized by ethidium bromide staining, and analyzed with the Fluor-S Multi Imager (Bio-Rad Laboratories, Inc., Milano, Italy). To confirm the correct identification of RT-PCR products, their specificity was verified after gel purification by Quiaex II (QIAGEN, Valencia, CA) by restriction enzyme digestion and direct sequencing (data not shown).

To perform quantitative PCR (QPCR) for human SSTR1 mRNA, we used primers and probe for SSTR1 designed using Primer Express software (PE Applied Biosystems, Monza, Italy), based on the sequence data available on GenBank (accession no. NM_001049). The forward primer was 5'-GAACGGGACCTTGAGCGAG-3'; the reverse primer was 5'-GGCACACCACGGAGTAGATGA-3'; the probe had the following sequence: 5'-AGGGCAGCGCCATCCTGATCTCTT-3', and the size of the amplicon was 68 bp.

A TaqMan probe (PE Applied Biosystems) labeled with a fluorescent dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethylrhodamine) was used. For amplification of the housekeeping gene, the predeveloped TaqMan assay reagents for the human 18S rRNA (20x) were used (PE Applied Biosystem).

QPCR amplification was carried out in triplicate in 50-µl reaction volumes consisting of 1x TaqMan universal PCR master mix (PE Applied Biosystems), 0.05 µM forward primer, 0.3 µM reverse primer, and 0.2 µM probe. Two microliters of cDNA (corresponding to 100 ng reverse-transcribed total RNA) and 2 µl RT- were amplified according to the following thermal profile: 50 C for 2 min, 95 C for 10 min and 45 cycles of 95 C for 15 sec, 59 C for 1 min. All the QPCR reactions were performed, recorded, and analyzed using the ABI 7700 Prism sequence detection system (PE Applied Biosystems).

Absolute quantitation of mRNA copy number in the samples was carried out following the standard curve methods (separate tubes) (user bulletin #2 ABI PRISM 7700 sequence detection system, PE Applied Biosystems). Serial dilutions of the single-stranded SSTR1 sense oligonucleotide amplicon (from 10⁹ to 10² molecules) were carried out in triplicate. The log copy numbers of unknown samples were calculated from the regression line according to the equation: logN = (Ct - q)/m, where Ct is the threshold cycle, q is the y-intercept, and m is the slope of the standard curve line. Slopes for all assays reported were -3.3 ± 0.3.

All samples were carried out in triplicate (100 ng reverse-transcribed total RNA per well) and repeated at least twice. For each sample one point of 18S rRNA was loaded to evaluate the retrotranscription efficiency in the same plate and PCR conditions. No template control and RT- controls were run in each experiment.

SRIF and SSTR1 selective agonist

SRIF (Stilamin 250) was purchased from Serono Pharma (Roma, Italy). The SRIF analog selective for SSTR1, BIM-23926, was provided by Biomeasure Incorporated (Milford, MA). The human SSTR subtype specificity (IC₅₀) of SRIF and BIM-23926, listed in Table 2, was determined by radioligand-binding assay using membrane preparations of CHO-K1 cells stably transfected with human SSTR1–4 genes or SSTR5 cDNA, and the biological activity of the SSTR1 selective agonist was evaluated as previously described (7).

To explore the effects of SRIF and the SSTR1 selective agonist on primary pituitary cultures, human GH and PRL levels were measured by immunoradiometric assay (IRMA) with reagents supplied by Nichols Institute Diagnostics (San Juan Capistrano, CA). The limit of detection for GH was 0.05 µg/liter, with intra- and interassay variation coefficients of 3.3% and 6.1%, respectively. The limit of detection for PRL was 0.47 µg/liter, with intra- and interassay variation coefficients of 6.4% and 5.9%, respectively. Hormone assays were performed in duplicate after appropriate sample dilutions of conditioned medium from treated cells in eight replicates for each pituitary adenoma.

Cell viability

The effect of SRIF and the SSTR1 selective agonist on cell viability of pituitary adenomas in vitro was assessed by the CellTiter 96 Aq_ueous nonradioactive cell proliferation assay (Promega Corp.), as previously described (7), after incubation in medium without or with SRIF or BIM-23926.

Statistical analysis

Results of hormone assays and cell viability experiments are expressed as the mean ± SE. A preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), paired or unpaired t test was used to evaluate individual differences between means. To measure the strength of association between pairs of variables without specifying dependencies, Spearman order correlations were run. P less than 0.05 was considered significant in all tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Somatostatin receptor mRNA expression

All tissue samples were subjected to RT-PCR and all adenomas expressed GH. The expression of PRL and SSTR1–5 and absolute levels of SSTR1 mRNA were therefore investigated in each sample. The mean ± SE level of SSTR1 mRNA was 6 ± 2.2 x 10⁴ molecules/microgram of reverse-transcribed total RNA (median, 4.47 x 10⁴ molecules/microgram of reverse-transcribed total RNA), which is comparable to that found in normal pituitary (5.4 ± 1.02 x 10⁴ molecules/microgram of reverse-transcribed total RNA). SSTR1 mRNA levels did not correlate to patients’ preoperative GH or PRL plasma levels, immunohistochemical findings, age, or sex. Among the 15 SSTR1-expressing adenomas, all expressed PRL, and SSTR2, as well as SSTR5, was expressed in 14 adenomas (93.3%). SSTR3 expression was detected in eight adenomas (53.3%), and SSTR4 mRNA expression was found in only one adenoma (6.7%). Table 3 shows patient characteristics and SSTR1 mRNA levels, which ranged from 0.1–26 x 10⁴ molecules/microgram of reverse-transcribed total RNA.

To investigate the effects of SRIF and SSTR1 selective agonist on hormone secretion, GH and PRL levels were measured in conditioned media collected from primary pituitary cultures derived from SSTR1 expressing adenomas. As indicated in Table 3, all adenomas secreted measurable amounts of both GH and PRL in the culture medium.

Dose-response studies were performed to evaluate GH and PRL secretion from pituitary cultures incubated with either SRIF or BIM-23926 at concentrations ranging from 10^-12 to 10^-6 M for 6 h (Fig. 1). Both SRIF and BIM-23926 significantly reduced GH (Fig. 1, top) and PRL secretion (Fig. 1, bottom) at concentrations ranging from 10^-10 to 10^-6 M. SRIF and BIM-23296 at 10^-8 and 10^-6 M maximally suppressed the secretion of both GH (EC₅₀ = 7 pmol/liter and EC₅₀ = 3 pmol/liter, respectively) and PRL (EC₅₀ = 5.7 pmol/liter and EC₅₀ = 4.6 pmol/liter, respectively). Therefore, the concentration of 10^-8 M was chosen for subsequent experiments as the lowest dose with the maximal efficacy.

View larger version (27K): [in this window] [in a new window]   Figure 1. Dose-response experiments for evaluating the effects of SRIF and BIM-23926 on GH (top) and PRL secretion (bottom). Pituitary cells were incubated in 96-well plates for 6 h in a culture medium supplemented with either SRIF or BIM-23296 at concentrations ranging from 10-12 to 10-6 M. Control cells were treated with vehicle solution. GH and PRL levels in conditioned media from each primary culture were measured by IRMA. Data from primary cultures derived from five GH- and PRL-secreting adenomas were evaluated independently with eight replicates and expressed as the mean ± SE percentage GH and PRL secretion inhibition vs. untreated control cells. *, P < 0.05; and **, P < 0.01 vs. untreated control cells; +, P < 0.05 vs. samples treated with 10-10 M SRIF or BIM-23296.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of SRIF and SSTR1 selective agonist on GH and PRL secretion. Primary pituitary cultures derived from 15 GH- and PRL-secreting adenomas expressing SSTR1 were treated with either SRIF or BIM-23926 at 10-8 M for 6 h. GH and PRL levels were assessed by IRMA in the conditioned medium from eight replicates for each adenoma. Results are expressed as the mean ± SE percentage GH and PRL suppression in treated cells vs. untreated control cells. *, P < 0.05 vs. untreated control cells.

To determine the effects of SRIF and the SSTR1 selective agonist on cell viability of SSTR1-expressing adenomas, we assessed cell number in pituitary primary cultures after treatment with SRIF or BIM-23926.

Dose-response studies, performed by incubating the cells with SRIF or BIM-23296 at concentrations ranging from 10^-12 to 10^-6 M for 48 h, indicated that cell viability was maximally inhibited by treatment with 10^-8 M SRIF or BIM-23926 (EC₅₀ = 5.7 pmol/liter and EC₅₀ = 4.6 pmol/liter, respectively) (Fig. 3). Time course evaluation, performed by treating the cells with 10^-8 M SRIF or BIM-23926 for 24, 48, and 72 h, showed that treatment with 10^-8 M SRIF or BIM-23926 resulted in maximal inhibition of cell viability after 48 h. Treatment for 72 h was less effective owing to spontaneous detachment of both control and treated cells. In keeping with these results, each adenoma was treated with 10^-8 M SRIF or BIM-23926 for 48 h. We found that SRIF and BIM-23926 significantly (P < 0.05) reduced cell viability (17.5 ± 5%, and 20 ± 3.9% inhibition vs. control, respectively). The difference in the reduction of cell viability observed after treatment with SRIF or BIM-23926 was not statistically significant.

View larger version (17K): [in this window] [in a new window]   Figure 3. Dose-response experiments for evaluating the effects of SRIF and BIM-23926 on cell viability. Pituitary cells were incubated in 96-well plates for 48 h in a culture medium supplemented with either SRIF or BIM-23296 at concentrations ranging from 10-12 to 10-6 M. Control cells were treated with vehicle solution. Cell viability of each primary culture was measured as absorbance at 490 nm. Data from primary cultures derived from five GH- and PRL-secreting pituitary adenomas were evaluated independently in eight replicates and were expressed as the mean ± SE percentage cell viability inhibition vs. untreated control cells. *, P < 0.05; and **, P < 0.01 vs. untreated control cells. +, P < 0.05 vs. samples treated with 10-10 M SRIF or BIM-23296.

The degree of GH secretion inhibition by both SRIF and BIM-23926 in vitro was significantly correlated (P < 0.001) with the level of SSTR1 mRNA expression (Table 4); no statistical difference could be found between the slopes of the two regression lines. Similarly, the extent of PRL secretion inhibition by both SRIF and BIM-23926 was significantly (P < 0.01) correlated with the level of SSTR1 mRNA expression. No correlation was found between the degree of cell viability inhibition induced by either SRIF or BIM-23926 and SSTR1 mRNA expression levels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The role of specific SSTR subtypes in regulating pituitary hormone secretion has been extensively investigated, focusing mainly on SSTR2 and 5, which are involved in mediating the actions of the currently available SRIF analogs, used for treatment of acromegaly (8). Both SSTR2 and SSTR5 have been implicated in mediating the suppression of GH secretion induced by SRIF and its analogs in somatotroph cells, and inhibition of PRL secretion in pure PRL- and in mixed GH/PRL-secreting adenomas seems to be mediated mainly by SSTR5 (3, 9, 10). The role of SSTR2 activation in the control of pituitary function has recently been further highlighted by the finding that a hybrid molecule, BIM-23A387, which binds with high affinity and selectivity to both SSTR2 and the dopamine receptor subtype 2 (DR2), reduces both GH and PRL secretion by pituitary adenomas with enhanced potency (11). The observed potentiation of the inhibitory effect is not explained on the basis of binding affinities, suggesting that a ligand-induced dimerization process between SSTR2 and DR2 may occur. Indeed, recent evidence has shown that, upon ligand binding, G protein-coupled receptors can interact with each other forming homo- and heterodimers, such as SSTR5/DR2 or SSTR5/SSTR1 heterodimers, with enhanced functional activity. However, SSTR1 appears to remain monomeric on ligand activation, even if it can be recruited in heterodimer formation by an activated SSTR5 (12). Therefore, the significant reduction in hormone secretion and cell survival induced by the selective SSTR1 agonist shown in the present study may be achieved through the specific activation of SSTR1 alone, independently of other SSTRs in the tissue. Furthermore, SSTR1 selective agonist treatment affects both hormone secretion and cell survival. This observation differs from what has been previously observed with SSTR2 and SSTR5 selective agonists, whose inhibitory effects on secretion appear to be independent of those on proliferation (5). The inhibitory effects of SRIF on cell viability and GH secretion in pituitary adenomas shown here are in line with previously reported data (4, 5, 10). However, our data seem to indicate that SRIF reduces PRL secretion to a lesser extent than previously reported (4).

An antiproliferative effect resulting from SSTR1 activation has previously been demonstrated in SSTR1-transfected CHO-K1 cells (13) and the human leukemia cell lines HL60 and BALL-1 (14). Furthermore, we recently provided evidence that SSTR1 selective agonists inhibit cell proliferation, calcitonin secretion, and gene expression in a cell line derived from a human medullary thyroid carcinoma (7), suggesting that these SSTR1 selective agonists may have a wide range of action on neuroendocrine cells.

Our data suggest that SRIF analogs with enhanced affinity for SSTR1 may have great potentiality as pharmacological tools for the treatment of pituitary adenomas, not only to reduce hypersecretion but also to control neoplastic growth. Previous studies have shown that SSTR1 is highly expressed in pure prolactinomas (9, 15) but poorly expressed in pure GH-secreting adenomas (4). On the contrary, we found that SSTR1 mRNA was expressed at various level in all the examined adenomas.

Moreover, we have shown that SSTR1 mRNA correlates with the extent of hormone secretion inhibition induced by both SRIF and the SSTR1 selective agonist, suggesting that the antisecretory effects of SRIF may also be mediated by SSTR1 activation and SSTR1 selective agonists may be very useful in controlling hormone hypersecretion. Our findings are in contrast with a previously reported study, showing the lack of effect of a nonpeptidic SSTR1 selective agonist (L-797,591) on GH secretion in normal rat pituitary cultures (16). Such a different effect can be explained by the evidence that SSTR1 is the least expressed SSTR subtype in normal rat somatotrophs, occurring in only 5 ± 1.2% of the cells (17). Moreover, Rohrer et al. (16) describe the effect of a SSTR1 selective agonist on normal rat pituitary primary cultures, but our experiments are performed with primary cultures from human pituitary adenomas. Furthermore, it has recently been shown that SRIF inhibits GH secretion in rat pituitary tumor cells through activation of both SSTR1 and SSTR2 (18). Therefore, the modulatory role of SSTR1 on GH secretion by rat pituitary may be different between normal and neoplastic tissue. On the other hand, there is evidence showing the involvement of SSTR1 in modulating basal GH secretion at pituitary level in mice (19), suggesting a species specificity of the control of pituitary GH secretion by SSTRs.

There is evidence that almost all human tumors express SSTR1 mRNA (20), which led us to hypothesize that compounds selectively targeting SSTR1 may represent a novel tool for the control of neoplastic growth. However, in our study, the inhibition of cell viability induced by SRIF treatment did not correlate with SSTR1 mRNA levels, suggesting that SRIF transduces its effects through interaction with additional SSTR subtypes. On the other hand, the fact that BIM-23926 effects also did not correlate with SSTR1 mRNA levels may suggest that even low levels of SSTR1 expression may warrant an antiproliferative activity of compounds binding to this receptor. Therefore, the availability of stable SRIF analogs that bind with high affinity to SSTR1, whether selectively or together with other SSTR subtypes, may indeed open a new frontier in the treatment of GH- and PRL-secreting adenomas. It has recently been highlighted that a universal SSTR agonist with a broader binding profile for SSTRs, similar to native SRIF, might be very powerful (21), in keeping with the marked overlap of intracellular pathways triggered by differential SSTR activation and with the finding that cells typically express multiple SSTR subtypes. Such a new SRIF analog with a unique binding pattern (SSTR1, 2, 3, and 5) has recently been identified and shown to be very effective in reducing GH and IGF-I secretion in animals (22).

In conclusion, our results show that SSTR1 may be very important in mediating SRIF actions at pituitary level, inducing us to consider this receptor a good target for new therapies attempting to control growth and hormonal secretion of pituitary tumors.

	Footnotes

 
This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (60%, 2001; MIUR 2002067251-003), Fondazione Cassa di Risparmio di Ferrara, IPSEN S.p.A. (Milano, Italy), and the Associazione Ferrarese dell’Ipertensione Arteriosa.

Abbreviations: IRMA, Immunoradiometric assay; PRL, prolactin; QPCR, quantitative PCR; RT, reverse transcription; SRIF, somatostatin; SSTR, SRIF receptor subtype.

Received November 20, 2002.

Accepted March 10, 2003.

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