SRIH Receptor Research Articles

Sulfadiazine Partially Protects the Rat Temporal Cortex from Amyloid Beta Peptide (25–35)-Induced Alterations of the Somatostatinergic System

The aim of this study was to elucidate whether sulfadiazine, shown to improve cognitive capacity in the elderly, can prevent amyloid beta peptide (Aβ) (25–35)-induced impairment of the somatostatinergic system previously reported by our group in rat temporal cortex. Male Wistar rats were thus treated with sulfadiazine (160 mg/kg) or vehicle, via a gastric cannula, twice on the day prior to Aβ(25–35) treatment. On the following day and during 14 days, Aβ(25–35) was administered intracerebroventricularly (i.c.v.) via an osmotic minipump connected to a cannula implanted in the right lateral ventricle (300 pmol/day). Sulfadiazine (80 mg/kg) or vehicle was administered once again the last 2 days of the Aβ(25–35) infusion. All animals were sacrificed by decapitation 24 h after the last sulfadiazine dose. The findings obtained reveal that sulfadiazine partially prevents the decrease in somatostatin (SRIH)-like immunoreactivity content in the temporal cortex of rats infused with Aβ(25–35) during 14 days. In addition, sulfadiazine blocks the Aβ(25–35)-induced reduction in the SRIH receptor density and in SRIH receptor subtype 2 expression. Sulfadiazine treatment also restored the inhibitory effect of SRIH on basal adenylyl cyclase activity back to control values. Altogether, the results suggest that sulfadiazine might have beneficial effects in the early treatment of Alzheimer’s disease.

Control of pituitary adenoma cell proliferation by somatostatin analogs, dopamine agonists and novel chimeric compounds

The antisecretory effects of somatostatin (SRIH) and its analogs are widely recognized and provide the basis for treatment of hormonal hypersecretion in patients with pituitary adenomas, especially in the settings of acromegaly. Dopamine (DA) agonists have also been used for medical treatment of prolactin and/or GH hypersecretion, and recent evidence points to an even greater antisecretory effect for a chimeric molecule, having high affinity for both SRIH and DA receptors. Evidence for an antiproliferative effect of these compounds has also been provided. This review focuses on the antiproliferative effects of SRIH and its analogs, of DA and chimeric compounds on pituitary adenomas, and on the clinical consequences on tumor volume of pharmacological treatment of pituitary adenomas with these drugs.

Role of Complex Cyclin D1/Cdk4 in Somatostatin Subtype 2 Receptor-Mediated Inhibition of Cell Proliferation of a Medullary Thyroid Carcinoma Cell Line in Vitro

Somatostatin (SRIH) inhibits cell proliferation by interacting with five distinct SRIH receptor subtypes (SSTRs) activating several pathways in many tissues. We previously demonstrated that SRIH, by activating Src homology-2-containing protein, inhibits cell proliferation of the human medullary thyroid carcinoma cell line, TT, which expresses all SSTRs. However, the effects of SRIH on cell cycle proteins have not been investigated so far. We therefore evaluated the effects of SRIH and a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on cyclin D1 and its associated kinases. Our data show that SRIH and the selective SSTR2 agonist, BIM-23120, reduce cell proliferation and DNA synthesis as well as induce a delay of the cell cycle in G(2)/M phase. Moreover, treatment with both SRIH and BIM-23120 decreases cyclin D1 levels, with a parallel increase in phosphocyclin D1 levels, suggesting protein degradation. Moreover, our data show an increase in glycogen synthase kinase-3beta activity, which triggers phosphorylation-dependent cyclin D1 degradation. Indeed, we observed a reduction in cyclin D1 protein half-life under treatment with SRIH or the SSTR2 selective agonist. A reduction in cdk4 protein levels is also observed with a parallel reduction in Rb phosphorylation levels at Ser-780. Our data indicate that the subtype 2 receptor-mediated antiproliferative effect of SRIH on TT cell proliferation may be exerted through a decrease in cyclin D1 levels.

Selective Activation of Somatostatin Receptor Subtypes Differentially Modulates Secretion and Viability in Human Medullary Thyroid Carcinoma Primary Cultures: Potential Clinical Perspectives

Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells. We previously demonstrated that somatostatin (SRIH) reduces cell growth in the human MTC cell line, TT, which expresses all SRIH receptor (SSTR) subtypes and responds differently to selective SSTR agonists. To clarify the possible effects of SRIH analogs on hormone secretion and proliferation in MTC primary cultures, we evaluated SSTR expression and assessed the in vitro effects on calcitonin (CT) and chromogranin A secretion as well as cell viability of SRIH analogs interacting with SSTR1, SSTR2, and SSTR5. Thirty-five patients affected by MTC were recruited from 2003 to 2005. After total thyroidectomy, the samples were examined for CT, chromogranin A, and SSTR expression by RT-PCR. Primary cultures were developed and tested with SRIH analogs interacting with SSTR1, SSTR2, and SSTR5. We selected 18 MTC tumor samples, expressing SSTR1, SSTR2, and SSTR5. Two different groups were identified according to CT secretion inhibition by the clinically available SRIH analog, lanreotide. In the responder group, CT secretion was reduced by compounds interacting with SSTR1, SSTR2, and SSTR5, whereas cell viability was not affected. On the other hand, in the nonresponder group, CT secretion was reduced by the SSTR1 selective agonist, whereas cell viability was inhibited by SSTR2 selective agonists. Our data suggest that SRIH analogs might be useful in medical therapy of MTC because they could have antiproliferative effects despite the lack of antisecretory activity and vice versa.

Role of Glucocorticoids in the Regulation of Pituitary Somatostatin Receptor Subtype (sst1–sst5) mRNA Levels: Evidence for Direct and Somatostatin-Mediated Effects

Glucocorticoids can differentially regulate somatostatin (SRIH) receptor subtype expression depending on the duration of treatment, dose used and tissue type examined. In order to determine if glucocorticoids are critical regulators of pituitary SRIH receptor synthesis in vivo, we examined the effect of adrenalectomy (ADX), with and without dexamethasone (DEX; 200 µg/day for 8 days) treatment, on the relative expression levels of the SRIH receptor subtypes, sst1–sst5, by multiplex RT-PCR. ADX increased pituitary sst2 mRNA levels, but did not significantly alter mRNA levels of the other SRIH receptor subtypes. These findings indicate that pituitary sst2 synthesis is normally under inhibitory control of endogenous glucocorticoids. High-dose DEX resulted in a decrease in sst1–sst4 mRNA and an increase in sst5 mRNA, independent of adrenal status. DEX also decreased sst2, sst3 and sst4 mRNA levels and increased sst5 mRNA levels by short-term in vitro application (10 nM, 4 h) in primary rat pituitary cell cultures, indicating DEX regulation of sst2–sst5 in vivo is at least in part due to a direct action at the level of the pituitary. However, the inhibitory actions of DEX on sst1 mRNA levels observed in vivo were not consistently replicated in vitro. In order to determine if the somatotrope population of the pituitary would display a similar response to DEX, fluorescent-activated cell sorting was used to obtain somatotrope-enriched cultures (>95% growth hormone immunopositive cells). DEX treatment (10 nM, 4 h) of somatotropes decreased sst2 and sst3, but did not alter sst5 mRNA levels. These results indicate that the effects of DEX on sst5 mRNA levels observed in unsorted pituitary cell cultures might be due to changes in sst5 expression in pituitary cell types other than somatotropes. Since excess glucocorticoids are thought to enhance SRIH tone, we also tested if ligand activation of SRIH receptor subtypes in vitro could mimic any of the actions of DEX on SRIH receptor mRNA levels observed in vivo. To this end, unsorted pituitary cell cultures and somatotrope-enriched cultures were treated with SRIH (1 and 100 nM) for 4 h. SRIH increased sst3 and sst5 mRNA levels, in both culture systems. These results suggest that the DEX-induced increase in endogenous SRIH tone may contribute to enhanced sst5 mRNA levels observed in vivo. However, the stimulatory actions of SRIH on sst3 mRNA levels observed in vitro might be overridden by direct inhibitory actions of DEX.

Identification of somatostatin receptors controlling growth hormone and thyrotropin secretion in the chicken using receptor subtype-specific agonists.

Somatostatin (SRIH) functions as an endocrine mediator in processes such as growth, immune resistance and reproduction. Five SRIH receptors (sstr1-5) have been identified in mammals, where they are expressed in both the brain and peripheral tIssues. To study the specific function of each receptor subtype, specific agonists (ag1-5) have been synthesized. The high degree of homology between mammalian and avian SRIH receptors suggests that these agonists might also be used in chickens. In this paper we describe two in vitro protocols (static incubation and perifusion system) to identify the SRIH receptors controlling the secretion of GH and TSH from the chicken pituitary. We found that basal GH or TSH secretion were never affected when SRIH or an agonist (1 microM) were added. SRIH diminished the GH as well as the TSH response to TSH-releasing hormone (TRH; 100 nM) in both systems. Our results have indicated that the SRIH actions at the level of the pituitary are regulated through specific receptor subtypes. In both the static and flow incubations, ag2 lowered the GH response to TRH, whereas stimulated TSH release was diminished by both ag2 and ag5. Ag3 and ag4 tended to increase rather than decrease the responsiveness of both pituitary cell types to TRH in perifusion studies. Our data have indicated that SRIH inhibits chicken pituitary function through sstr2 and sstr5. Only sstr2 seems to be involved in the control of chicken GH release, whereas both sstr2 and sstr5 inhibit induced GH secretion in mammals. The possible stimulatory action of ag3 and ag4 may point towards a species-specific function of sstr3 and sstr4.

Somatostatin Receptor Subtypes 2 and 5 Inhibit Corticotropin-Releasing Hormone-Stimulated Adrenocorticotropin Secretion from AtT-20 Cells

Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst₁–sst₅) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with ¹²⁵I-SRIH-14 and ¹²⁵I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst₂ (L-779,976; compound 2) or sst₁/sst₅) (L-817,818; compound 5) receptor subtypes potently displaced ¹²⁵I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst₁ (L-779,591; compound 1), sst₃ (L-796,778; compound 3) or sst₄ (L-803,087; compound 4) subtypes were inactive. Tyr¹¹-SRIH-14, compound 2 (sst₂) or compound 5 (sst₅) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst₂ and sst₅ agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst₂ and sst₅ receptor subtypes, but not sst₁, sst₃ or sst₄, inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope.

Somatostatin, but not somatostatin receptor subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and gene expression in the human medullary thyroid carcinoma cell line, TT.

Somatostatin (SRIH) analogs are commonly used to treat symptoms in medullary thyroid carcinoma (MTC), that expresses SRIH receptors (SSTR1 to SSTR5), as does the human MTC cell line TT. The aim of this work was to evaluate whether SRIH, SSTR2 and SSTR5-selective agonists influence calcitonin (CT) secretion and gene expression in the TT cell line. CT secretion was evaluated by chemiluminescence, and gene expression was analyzed by Northern blot. TT cell line proliferation was also assessed by [(3)H] thymidine ([(3)H]thy) incorporation and viable cell number count. SRIH significantly (p < 0.05) reduced [(3)H]thy incorporation (approx. 50 %), viable cell number (approx. 20 %), CT secretion (-30 %) and CT gene expression (approx. 2-fold). Exposure to the SSTR2-selective agonist, BIM-23 120, and to the SSTR5-selective agonist, BIM-23 206, did not modify CT secretion and mRNA levels in TT cells. Thus, SRIH inhibits DNA synthesis, cell proliferation, CT secretion and CT gene expression in the TT cell line, while SSTR2 and 5 selective agonists, although influencing DNA synthesis and cell proliferation, do not modify CT gene expression, suggesting that SRIH may influence gene expression acting through SSTRs other than subtypes 2 and 5. Furthermore, these findings may explain the erratic response of MTC patients in terms of CT plasma levels to treatment with SRIH analogs, like octreotide and lanreotide, which interact mainly with SSTR2 and 5.

Cortistatin-14 inhibits cell proliferation of human thyroid carcinoma cell lines of both follicular and parafollicular origin

Cortistatin (CST-14, Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys]-Lys-NH2), a neuropeptide member of the SRIH family, binds to all 5 SRIH receptor (sst) subtypes, but also possesses a significant binding affinity to GH secretagogue receptors (GHS-R), which have been reported to mediate the antiproliferative activity of GHS on thyroid cancer cells. The effect of CST-14 on cell proliferation was studied in 3 different human thyroid carcinoma cell lines of follicular origin (N-PAP, WRO, ARO) and in one thyroid medullary carcinoma cell line (TT). CST-14 1 pM determined a significant inhibition of cell proliferation in TT, N-PAP and WRO cells and this effect was dose-dependent and more pronounced than that displayed by SRIH-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH) treatment. To a minor extent, CST-14, but not SRIH-14, also temporary inhibited ARO cell proliferation. By immunofluorescence, sst2, sst3 and sst5 have been demonstrated in TT cells, whereas types 3 and 5 only were expressed in N-PAP and WRO cells, and no sst subtype was found in ARO cells. The presence of both GHS-Rla and lb mRNA has been studied and demonstrated in the TT medullary carcinoma cell line, whereas follicular derived cell lines were already known to express GHS binding sites. Addition of EP-80874 (D-Mrp-c[D-Cyspyridilalanyl3-D-Trp-Lys-Val-Cys]-Mrp-NH2), a synthetic peptide that binds to SRIH and GHS-R, completely abolished the antiproliferative effects of CST-14 or SRIH-14 on sst/GHS-R positive thyroid carcinoma cell lines (WRO, N-PAP and TT). EP-80874 was also able to antagonize the inhibitory activity of CST-14 on the growth of cells (ARO) expressing GHS-R but not sst. Taken together, these data firstly demonstrate that EP-80874 has a mixed SRIH/CST antagonist activity and suggest that the oncostatic effect of CST-14 on thyroid cancer cells could be mediated by both sst and/or GHS-R.

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l μg/5 μl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.

Homologous Upregulation of sst2 Somatostatin Receptor Expression in the Rat Arcuate Nucleus in vivo

In vitro studies using various cell systems have provided conflicting results regarding homologous regulation of somatostatin (SRIH) receptors, and information on whether SRIH regulates the expression of its own receptors in vivo is lacking. In the present study we examined, by in situ hybridization, the effects of pretreatment with the sst2-preferring SRIH analog, octreotide, in vivo, on mRNA levels of two SRIH receptor subtypes, sst1 and sst2, in rat brain and pituitary. ¹²⁵I-[DTrp⁸]-SRIH binding was also measured in these regions. Three hours after the iv injection of 50 µg octreotide to conscious adult male rats, there was a 46% increase (p < 0.01) in the labeling density of sst2 mRNA-expressing cells in the hypothalamic arcuate nucleus compared to normal saline-pretreated controls, but not in any of the other brain regions examined. Computer-assisted image analysis revealed that 3 h exposure to octreotide significantly (p < 0.01) augmented both the number and labeling density of sst2 mRNA-expressing cells in the arcuate nucleus, compared to those in saline-treated controls. By contrast, within the anterior pituitary gland, in vivo exposure to octreotide did not affect the expression of sst2 mRNA. No changes in sst1 mRNA-expressing cells were observed after octreotide treatment in any of the regions measured, indicating that the observed effects were homologous, i.e. specific of the receptor subtype stimulated. Octreotide pretreatment was also without effect on the density of ¹²⁵I-[DTrp⁸]-SRIH binding in either the arcuate nucleus or pituitary. These results demonstrate, for the first time, that SRIH preexposure in vivo upregulates the expression of a subtype of its own receptors, sst2, within the central nervous system. They further suggest that pretreatment with SRIH in vivo does not cause sst2 receptor desensitization in arcuate nucleus and pituitary. Such homologous regulatory mechanisms may play an important role in the neuroendocrine control of growth hormone (GH) secretion by the arcuate nucleus.

Somatostatin Inhibits Alpha-2-Adrenergic-Induced Secretion of Growth Hormone-Releasing Hormone

The purpose of this experiment was to determine the role of growth hormone-releasing hormone (GHRH) and somatostatin (SRIH) neurons in mediating α₂-adrenergic receptor-induced stimulation of growth hormone (GH) secretion in cattle. Our first objective was to determine if stimulation of α₂-adrenergic receptors increases activity of GHRH neurons in the arcuate nucleus (ARC) and/or decreases activity of SRIH neurons in periventricular (PeVN) and ARC nuclei. Clonidine (an α₂-adrenergic agonist) or vehicle (saline) were injected i.v. into steers and dual-label immunohistochemistry was performed to quantify the number of GHRH and SRIH neurons expressing Fos and Fos-related antigens (Fos/FRA) as markers of neuronal activity. Clonidine increased concentrations of GH in serum and decreased activity of SRIH neurons in the PeVN, but not in the ARC. Clonidine did not alter activity of GHRH neurons in the ARC. Our second objective was to determine if clonidine decreases secretion of SRIH from perifused slices of hypothalami, which contain perikarya and terminals of GHRH and SRIH neurons, and from explants of hypophysial stalk alone, which contain only terminals of GHRH and SRIH neurons. Clonidine failed to alter release of GHRH or SRIH from hypothalamic slices, but stimulated release of GHRH from explants of hypophysial stalk. Blockade of SRIH receptors enabled clonidine to stimulate release of GHRH from slices of hypothalami, but also stimulated release of SRIH. These results suggest that α₂-adrenergic-induced secretion of GH occurs via a dual mechanism involving inhibition of SRIH neurons in the PeVN and direct stimulation of GHRH release from axon terminals in the median eminence.

Differential Inhibition of Growth Hormone Secretion by Analogs Selective for Somatostatin Receptor Subtypes 2 and 5 in Human Growth-Hormone-Secreting Adenoma Cells in vitro

Somatostatin (SRIH), a cyclic tetradecapeptide hormone originally isolated from mammalian hypothalamus, is a potent suppressor of pituitary growth hormone (GH) secretion. SRIH acts through a family of G-protein-coupled membrane receptors containing seven transmembrane domains. Five genes encoding distinct SRIH receptor (SSTR) subtypes have so far been cloned in human and other species and termed SSTR1–5. In human somatotrophe pituitary adenomas GH secretion is controlled by both SSTR2 and SSTR5. However, in clinical practice only somatostatin analogs selective for SSTR2 (octreotide and lanreotide) are available. This may explain why clinical and in vitro responses to these analogs in acromegaly are only partial. In this study, we investigated the inhibitory effect of two new SRIH analogs with high selectivity for SSTR2 (NC-4-28B) and SSTR5 (BIM-23268) and compared it to that of native somatostatin (SRIH-14) on a large number of GH-secreting adenomas obtained by transphenoidal neurosurgery. Tissues from 16 adenomas were enzymatically dispersed and plated in 24-well dishes at 50,000 cells/well. After 3 days, groups of three wells were incubated for 4 h with medium alone, SRIH-14 or analogs NC-4-28B or BIM-23268, at the concentrations of 0.01, 0.1 and 1 µM. Our results show that 9 out of 16 adenomas were responsive (GH suppression: 20–40% vs. control, p < 0.05) to SRIH. In this group only 4 adenomas showed similar responses to both selective analogs, with 2 nonresponders (expression of other SRIH receptor subtypes) and 2 responders (concomitant expression of SSTR2 and SSTR5) to both analogs. GH release was selectively inhibited by NC-4-28B in 3 adenomas and by BIM-23268 in the remaining 2 adenomas, suggesting predominant expression of SSTR2 and SSTR5, respectively. SRIH failed to inhibit GH release in 7 adenomas (43%). Interestingly, in that group a better inhibitory effect was obtained with BIM-23268 (5 out of 7 adenomas) than with NC-4-28B, suggesting expression of a few SSTR5 receptors only, or of both SSTR2 and SSTR5, respectively. We conclude that the availability of somatostatin analogs selective for SSTR5 will enhance the treatment potency and spectrum in acromegaly.

Effects of aging and dietary restriction on mRNA levels of receptors for growth hormone-releasing hormone and somatostatin in the rat pituitary.

Aging impairs and dietary restriction may modulate pituitary response to growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SRIH) for GH secretion. Using the semiquantitative reverse-transcription polymerase chain reaction method, we analyzed the mRNA levels of the GHRH receptor (grfr) and SRIH receptor subtype 2 (sstr2) and subtype 5 (sstr5) in anterior pituitaries of male rats fed ad libitum or 30% dietary restricted. Aging reduced the mRNA levels of these receptors in a slightly different manner. The levels of grfr progressively decreased between 6 and 24 months, whereas those of sstr2 and sstr5 declined after 16 months. Dietary restriction did not diminish the aging-dependent changes, although it slightly augmented the levels of grfr, but not sstr2 and sstr5. The present results suggest that the aging-dependent impairment in pituitary response for GH secretion could result mostly from a decline in grfr rather than relative increase of sstrs. Although DR could slightly enhance the pituitary sensitivity to GHRH, the antiaging action may be minor at the level of gene expression.

Morphometric and Functional Changes of Rat Pituitary Somatotropes and Lactotropes after Central Administration of Somatostatin

This study examined effects of intracerebroventricularly (i.c.v.) administered somatostatin (SRIH-14 and SRIH-28) on growth and function of pituitary somatotropes (GH cells) and lactotropes (PRL cells). Male rats received three i.c.v. injections (1 µg/5 µl) of SRIH-14 or SRIH-28 every second day. Blood samples were collected for hormone assays and pituitaries were removed for histological and morphometric evaluation 5 days after the last i.c.v. treatment. Compared to control animals, SRIH treatment decreased (p < 0.05) pituitary weight and all morphometric measurements obtained in GH and PRL cells. Concentrations of serum growth hormone (GH) and prolactin (PRL) in both SRIH-treated groups were also lower (p < 0.05) than in control rats. These findings suggest that centrally administered somatostatin is specifically involved in the control of growth and secretory activity of GH and PRL cells. Thus, pharmacological manipulation of SRIH receptors reached from cerebrospinal fluid may alter systemic effects of GH and PRL.

Long-Term Inhibitory Effects of Somatostatin and Insulin-Like Growth Factor 1 on Growth Hormone Release by Serum-Free Primary Culture of Pituitary Cells from European Eel (Anguilla anguilla)

To investigate the ability of hypothalamic and peripheral factors to directly regulate growth hormone (GH) release in a primitive teleost, the European eel (Anguilla anguilla L.), we used primary cultures of dispersed pituitary cells. When cultured for 12 days in a serum-free medium, pituitary cells continuously released large amounts of GH, which exceeded the initial cellular content. Somatotropin-release inhibiting hormone (SRIH-14) dose-dependently inhibited GH release (EC₅₀ 0.75 nM) up to a maximal inhibitory effect of 95%. No desensitization of somatotropes to SRIH was observed over the 12 days of culture. Use of receptor subtype-selective SRIH agonists suggests the existence on eel somatotropes of SRIH receptor(s) related to the mammalian sst₂/sst₃/sst₅ class rather than to the sst₁/sst₄ class. Insulin-like growth factor 1 (IGF1) dose-dependently inhibited GH release (EC₅₀ 0.03 nM) up to a maximal inhibitory effect of 85%, without desensitization. IGF1 and IGF2 were equipotent in inhibiting GH release, whereas insulin was 1,000 times less active, suggesting the implication of a receptor related to the mammalian IGF type 1 receptor. These results indicate that eel somatotropes are active in vitro without any specific additional factors, and suggest the existence of a dominant inhibitory control of GH release in vivo. Two potential candidates for this chronic negative regulation are a neurohormone, SRIH and a circulating factor, IGF1. These data underline the early evolutionary origin of the molecular and functional SRIH-GH-IGF1 neuroendocrine axis in vertebrates.

Different expression patterns of somatostatin receptor subtypes in cultured epithelial cells from human normal prostate and prostate cancer.

The transcripts of five SRIH receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) were investigated by RT-PCR in epithelial cells (EC) and stromal cells (SC) from primary cultures of five normal human prostates and six prostate cancers. Primary cultures of prostate EC were established in serum-free keratynocyte medium with 5% FCS, epidermal growth factor, and bovine pituitary extract; SC were cultured in MEM with 10% FCS. Total RNA was extracted from EC and SC using a modified guanidine thiocyanate method. RT-PCR was performed after deoxyribonuclease treatment, using SSTR1-, SSTR2-, SSTR3-, SSTR4-, and SSTR5-specific-primers and adding glyceraldehyde-3-phosphate dehydrogenase-specific primers as internal control. A PCR product of the expected size of 334 bp, corresponding to SSTR1, was expressed only in EC from prostate cancer, whereas the expected 461-bp product of SSTR2 was found only in EC from normal prostate. SSTR3 messenger RNA was undetectable in normal and cancer EC, whereas SSTR4 and SSTR5 were present in both cell types. SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 messenger RNAs were not expressed in SC from both normal and cancer prostates. The RT-PCR method clearly demonstrated SSTRs' expression in the human prostate EC in vitro with differences between normal and tumoral samples. Our results may explain the ineffectiveness of some SSTR2 selective SRIH analogues in the treatment of prostate cancer and suggest that the absence of SSTR2 could represent a growth advantage in prostate cancer.

Somatostatin receptor subtype expression in human thyroid and thyroid carcinoma cell lines.

Somatostatin (SRIH) analogs can suppress the proliferation of human differentiated thyroid carcinoma cell lines that express SRIH receptors (SSTRs) demonstrated by radioligand binding analysis. Five distinct human SSTR subtypes (hSSTR1-5) that bind native SRIH exhibit diverse affinities to a wide range of SRIH analogs. Reverse transcriptase-PCR amplification of ribonucleic acids (RNAs) obtained from normal thyroid tissues and nine human thyroid carcinoma cell lines, grown as monolayer cultures and xenograft tumors in nude mice, were used to discriminate expression of SSTR subtype messenger RNAs (mRNAs). The cell lines were derived from a follicular adenoma (KAK-1), two follicular carcinomas (MRO-87 and WRO-82), two papillary carcinomas (NPA87 and KAT-10), and four anaplastic thyroid carcinomas (DRO-90, ARO-81, KAT-4, and KAT-18). Most thyroid cancer cell line monolayers and xenografts expressed SSTR3 and SSTR5 mRNAs. SSTR1 expression was more varied between monolayers and xenografts, whereas SSTR2 mRNA was only faintly detectable at the most extreme resolution. SSTR4 mRNA was faintly positive in only one anaplastic carcinoma xenograft. Normal thyroid also expressed SSTR3 and SSTR5 mRNAs, with only faint expression of SSTR1 and SSTR2 mRNAs (in one of five and three of five samples, respectively). SSTR mRNA expression was dependent upon in vitro culture conditions, as xenograft SSTR mRNA expression tended to decrease compared to that in each respective monolayer culture. Characterization of SSTR subtype expression in human thyroid carcinomas may permit targeting of specific SRIH analogs to inhibit proliferation of differentiated and anaplastic thyroid carcinomas in patients.

Somatostatin receptors in human prostate and prostate cancer.

Benign as well as malignant human prostatic tissues were evaluated for their content of somatostatin (SRIH) receptors (SRIH-R). In vitro receptor autoradiography techniques on cryostat sections were performed using 125I-labeled [Tyr3]octreotide as well as 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28 as radioligands. SRIH-R were identified in all normal and hyperplastic prostates in the smooth muscles of the stroma, whereas the glands did not express the receptors. Muscular nodules were strongly receptor positive as well. The receptors were of high affinity (Kd = 0.4 nmol/L) and high specificity for biologically active SRIH analogs; high affinity for SRIH-14, SRIH-28, and octreotide was detected, suggesting the presence of the SSTR2 receptor subtype. In situ hybridization studies confirmed the presence of SSTR2 messenger ribonucleic acid in these tissues. Primary prostate cancers did not have SRIH-R identified with 125I-labeled [Tyr3]octreotide. However, they were expressing SRIH-R identified with 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28, with a high affinity for SRIH-14 and SRIH-28, but low affinity for octreotide. The receptors were located on tumoral cells. In situ hybridization studies revealed a preferential expression of SSTR1. Primary human prostate cancers, therefore, express a different SRIH-R subtype than benign prostate tissue. Several veins and the ganglion cells from the prostatic plexus in the surroundings of the tumors were expressing SRIH-R with high affinity for octreotide as well. These data suggest that the human prostate as well as prostate cancers may be targets for SRIH therapy; however, SRIH analogs with different selectivities for SRIH-R subtypes are required in each case.

Molecular and pharmacological characterization of somatostatin receptor subtypes in adrenal, extraadrenal, and malignant pheochromocytomas.

SRIH receptors were quantified by radioautography in 33 pheochromocytomas and 5 normal adrenals. Binding was evenly distributed over the tumors, whereas it was more intense in adrenal medulla than cortex. Binding levels were significantly higher in tumoral than in normal tissue, but did not differ among tumors. At 100 nmol/L, SRIH-14 and octreotide (or BIM23014 in cross-linking experiments to a 57-kilodalton component) comparably displaced SRIH binding, BIM23042 and BIM23052 were less potent, and BIM23056 was inefficient. In increasing doses, the rank order of potency was SRIH-14 > SRIH-28 > octreotide > BIM23052 >> BIM23042 >> > BIM23056. All five species of SRIH receptor (SSTR1-5) messenger ribonucleic acids (mRNAs) were measurable in pheochromocytomas and normal adrenals, SSTR2 and SSTR4 mRNA were the most expressed moieties. The proportion of SSTR5 mRNA species was higher in normal adrenals (21%) than in pheochromocytomas (6%). In the presence of guanylylimidodiphosphate, SRIH binding was reduced by 83%. However, SRIH did not alter basal or forskolin-stimulated adenylyl cyclase activity. Taken together, these pharmacological and molecular data indicate that SRIH binding on pheochromocytomas depends on a mixed population of receptors, mainly of the SSTR2 and SSTR4 subtypes, efficiently coupled to G proteins, but not to adenylyl cyclase inhibition.