Subtypes Sst Research Articles

Somatostatin Receptor Imaging In Vivo by Planar Scintigraphy Facilitates the Diagnosis of Canine Insulinomas

Somatostatin receptors expressed by insulinomas in 5 dogs were imaged in vivo by means of indium In 111 pentetreotide (OctreoScan) scintigraphy. The diagnosis in each dog was supported by the presence of hypoglycemia ( 20 μIU/mL), and histopathologic review of neoplastic tissue. All insulinomas expressed high-affinity somatostatin receptors of subtype sst2, as shown by receptor autoradiography in vitro using 1 2 5 I-[tyrosine 3 ]-octreotide and 1 2 5 I-[leucine 8 , D-tryptophan 2 2 , tyrosine 2 5 ]-somatostatin-28 with an sst2 subtype-selective analogue. Scintigrams were obtained at 1, 4, 12, and 24 hours after the IV administration of 74-222 MBq of OctreoScan to each patient. Abnormal foci of activity were 1st observed from I hour after administration of the radioligand in dog 3, to 24 hours after its administration in dog 4; in dogs I and 2, abnormal foci of activity were visible from 12 hours. Dog 5 showed a questionable abnormal focus of activity at 12 hours, but not at 24 hours. Scintigraphy enabled accurate prediction of the anatomical location of the primary tumor in 1 of 4 dogs, but was unable to differentiate a right- from a left- pancreatic lobe tumor, or vice versa, in 3 dogs; the 5th dog had equivocal results. 1 1 1 In-pentetreotide scintigraphy is a useful diagnostic adjunct to the clinical evaluation of the insulinoma patient, but is unable to localize the tumor in some cases.

Somatostatin Receptor Imaging In Vivo by Planar Scintigraphy Facilitates the Diagnosis of Canine Insulinomas

Somatostatin receptors expressed by insulinomas in 5 dogs were imaged in vivo by means of indium in 111 pentetreotide (OctreoScan) scintigraphy. The diagnosis in each dog was supported by the presence of hypoglycemia (<60 mg/dL), hyperinsulinemia (>20 microU/mL), and histopathologic review of neoplastic tissue. All insulinomas expressed high-affinity somatostatin receptors of subtype sst2, as shown by receptor autoradiography in vitro using 125I-[tyrosine3]-octreotide and 125I-[leucine8, Dtryptophan22, tyrosine25]-somatostatin-28 with an sst2 subtype-selective analogue. Scintigrams were obtained at 1, 4, 12, and 24 hours after the i.v. administration of 74-222 MBq of OctreoScan to each patient. Abnormal foci of activity were 1st observed from 1 hour after administration of the radioligand in dog 3, to 24 hours after its administration in dog 4; in dogs 1 and 2, abnormal foci of activity were visible from 12 hours. Dog 5 showed a questionable abnormal focus of activity at 12 hours, but not at 24 hours. Scintigraphy enabled accurate prediction of the anatomical location of the primary tumor in 1 of 4 dogs, but was unable to differentiate a right- from a left-pancreatic lobe tumor, or vice versa, in 3 dogs; the 5th dog had equivocal results. 111In-pentetreotide scintigraphy is a useful diagnostic adjunct to the clinical evaluation of the insulinoma patient, but is unable to localize the tumor in some cases.

Somatostatin receptor subtype 1 and subtype 2 mRNA expression is regulated by nutritional state in rainbow trout ( Oncorhynchus mykiss)

Somatostatin receptors (sst) mediate the numerous physiological actions (e.g., aspects of growth, development, and metabolism) of the somatostatin family of peptides. In this study, we used rainbow trout ( Oncorhynchus mykiss) to establish the pattern of sst subtype 1A, 1B, and 2 mRNA expression in selected tissues (optic tectum of brain, endocrine pancreas, and liver) and to evaluate nutritional regulation of sst expression. Quantitative real-time reverse transcription-PCR, sensitive to less than 100 copies, revealed that sst1s and sst2 was differentially expressed, both in terms of distribution among the tissues of study and in terms of relative abundance within a particular tissue. Under normal physiological (fed) conditions, sst1B levels were two times greater than those of sst1A in all tissues examined and levels of sst2 were 2–5 times greater those of sst1B, except in optic tectum, in which sst1B and sst2 mRNA levels appeared equal. Nutritional state modulated the pattern of sst1 and sst2 mRNAs expression. Fasting for 2 or 6 weeks reduced the expression of sst mRNAs in optic tectum; whereas, fasting increased the expression of sst mRNAs in both pancreas and liver. Re-feeding animals for 2 weeks following a 4-week fast restored mRNA levels to near those in tissues from animals which were fed continuously. These findings indicate that the pattern of sst expression in optic tectum, pancreas, and liver is regulated by nutritional state.

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA.

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.

Somatostatin receptors in pituitary function, diagnosis and therapy.

Recent studies have demonstrated that human pituitary adenomas express multiple somatostatin receptor (sst) subtypes. The expression of sst subtypes in human pituitary adenomas is highly variable. This variability in sst subtype expression may explain the variable responsiveness of patients with pituitary adenomas to medical treatment with the sst2-preferring SS-analogs octreotide and lanreotide. In human GH-secreting pituitary adenomas, both sst2 and sst5 are involved in the regulation of GH secretion. In prolactinomas, sst5 receptors are the key receptors in regulating responsiveness to SS. The low abundance of sst2 in prolactinomas explains the lack of efficacy of octreotide in lowering the elevated PRL levels in prolactinoma patients. Octreotide and lanreotide successfully suppress TSH levels, including normalization of thyroid hormone levels in the large majority of patients with TSH-secreting pituitary adenomas, probably due to the high level of sst2, expression in the adenomas. Although sst2 receptors are expressed by a significant proportion of gonadotroph and clinically non-functioning pituitary adenomas, the overall efficacy of sst2-preferring SS-analogs seems low in this type of patients. Finally, corticotroph adenomas may express multiple sst subtypes as well. Octapeptide SS-analogs do not lower circulating ACTH levels in patients with untreated pituitary-dependent Cushing's disease, whereas SS and the SS-analog octreotide suppress pathological ACTH release in some patients with Nelson's syndrome. This review discusses the expression and potential role of sst subtypes in the different types of human pituitary adenomas.

Somatostatin Receptors and Their Interest in Diagnostic Pathology

Since the discovery of somatostatin (SS) and of its interactions with a family of specific somatostatin receptors (sst), a wide body of evidence has been reported on its biological activities. Those activities include inhibition of hormone secretion, neuromodulatory properties in the central nervous system, cell growth control, and induction of apoptosis. At the same time, the distribution of sst has been analyzed in both normal and pathological tissues and sst subtype selective SS-analogs, able to mimic most SS functions, have been developed. The results have been fundamental insights into sst physiology and potent clinical implications in a variety of neoplastic and non neoplastic diseases. Neuroendocrine tumors have been particular targets of investigation. Alternative methods have been validated and are available to analyze the presence and functionality of sst at the level of either mRNA or protein. These methods include RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, autoradiography, and in vivo scintigraphy. Tissue localization techniques are now accessible to many pathology laboratories worldwide and the role of the pathologist in typing the different sst present in a given sample is becoming more and more crucial. This is particularly, but not exclusively, the case in the field of neuroendocrine oncology, where sst typing may affect the clinical management of patients with sst-positive tumors.

Somatostatin receptors 2 and 5 are the major somatostatin receptors in insulinomas: an in vivo and in vitro study.

Somatostatin (SRIF) receptors (sst) are present on normal pancreatic endocrine beta-cells. However, the use of SRIF analogs in the scintigraphic imaging of insulinomas and in the medical management of these tumors seems to be restricted to a subgroup of patients. The aim of this study was to determine the prevalence of sst expression in vitro and characterize sst subtype binding in insulinomas and its correlation with in vivo sst receptor scintigraphy (SRS). In vitro studies were performed on 27 insulinomas from 25 patients: 22 with benign and three with malignant tumors. Semiquantitative RT-PCR of sst mRNAs was performed for 20 of these insulinomas. Sst2 and sst5 were expressed in 70%, sst1 in 50%, and sst3 and sst4 subtypes only in 15-20% of the tumors. (125)I-Tyr(0)DTrp(8)SRIF(14) binding was assessed by quantitative autoradiography in 18 insulinomas, and competition experiments were performed with SRIF(14) and L797-591, L779-976, L796-778, L803-087, L817-818, selective agonists of the five sst subtypes, and BIM23244, a selective agonist of sst2 and sst5. Significant specific binding was observed in 72% of the insulinomas. Displacement experiments with ligands of higher affinity for each of the sst receptors revealed significant binding with the sst2 and sst5 ligands in 72%, sst3 in 44%, sst1 in 44%, and sst4 in 28% of cases. All insulinomas displaying sst2 binding were also sst5 sensitive. However, the ratio of sst5/sst2 displacement was variable and only equal to that for SRIF(14) in experiments with the sst2/sst5 agonist BIM23244. SRS was performed 10 times in nine patients; it detected 60% of the tumors, including metastases of a malignant insulinoma. All the tumors detected by SRS displayed high levels of (125)I-Tyr(0)DTrp(8)SRIF(14) binding. The mechanisms underlying the loss of expression of sst2/sst5 in a third of insulinomas remains to be determined, but this loss of expression may be involved in beta-cell dysfunction.

Inhibitory Role of the Somatostatin Receptor SST2 on the Intracrine-regulated Cell Proliferation Induced by the 210-Amino Acid Fibroblast Growth Factor-2 Isoform: IMPLICATION OF JAK2

The fibroblast growth factor (FGF)-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to grow in low serum concentrations through still unknown mechanisms called intracrine regulations. Different peptide hormones and cytokines have been found to be nuclearized through NLS and to induce cell proliferation. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. Pancreatic cells AR4-2J were stably transfected to express selectively the HMW FGF-2. We demonstrated that activation of the somatostatin receptor subtype SST2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates Galphai/JAK2/SHP-1. The Galphai inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-immunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with SST2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the following evidence: 1) the intracrine-induced proliferation can be reversed by extracellular acting polypeptides; 2) SST2 inhibitory signaling may involve the JAK2/SHP-1 pathway.

Somatostatin receptors in gastroentero-pancreatic neuroendocrine tumours.

Five somatostatin receptor (sst) subtype genes, sst(1), sst(2), sst(3), sst(4) and sst(5), have been cloned and characterised. The five sst subtypes all bind natural somatostatin-14 and somatostatin-28 with high affinity. Endocrine pancreatic and endocrine digestive tract tumours also express multiple sst subtypes, but sst(2) predominance is generally found. However, there is considerable variation in sst subtype expression between the different tumour types and among tumours of the same type. The predominant expression of sst(2) receptors on pancreatic endocrine or carcinoid tumours is essential for the control of hormonal hypersecretion by the octapeptide somatostatin analogues such as octreotide and lanreotide. Somatostatin and its octapeptide analogues are also able to inhibit proliferation of normal and tumour cells. The high density of sst(2) or sst(5) on pancreatic endocrine or carcinoid tumours further allows the use of radiolabelled somatostatin analogues for in vivo visualisation. The predominant expression of sst(2) receptors in these tumours and the efficiency of sst(2) receptors to undergo agonist-induced internalisation is also essential for the application of radiolabelled octapeptide somatostatin analogues. Currently, [(111)In-DTPA(0)]octreotide, [(90)Y-DOTA(0),Tyr(3)]octreotide, [(177)Lu-DOTA(0)Tyr(3)]octreotate, [(111)In-DOTA(0)]lanreotide and [(90)Y-DOTA(0)]lanreotide can be used for this purpose.

Somatostatin receptor genes are expressed in lymphocytes from retroorbital tissues in Graves' disease.

The radiolabeled somatostatin (SST) analog octreotide accumulates within the orbits of active Graves' ophthalmopathy (GO), and octreotide and lanreotide have been proposed to treat this disorder. It is still unclear which retroorbital cells the SST analogs target. Lymphocytic infiltration of retroorbital tissues is a peculiarity of GO, and labeled octreotide could accumulate at specific sites on retroorbital-activated lymphocytes. The accumulation of radiolabeled analogs is due to the interaction with specific cell surface SST receptors. Five subtypes of somatostatin receptors (SST1-5), member of the G protein-coupled, seven-transmembrane superfamily, are described. It still unknown which SST subtype is expressed in retroorbital activated lymphocytes. The aim of this study was to evaluate the expression of SST1-5 genes in lymphocytes recovered from retroorbital tissues obtained from patients with GO undergoing orbital decompression. Cultured phytohemagglutinin-stimulated lymphocytes from retroorbital blood samples, drawn during orbital surgery in five patients with GO and in two control patients without autoimmune or thyroid diseases and without orbital inflammatory conditions, were also studied. RT-PCR of total RNA extracted from lymphocytes was performed using primers for SST1-5 and, as internal control, for glyceraldehyde-3-phosphate dehydrogenase. All SSTs transcripts were found in lymphocytes both from GO retroorbital tissues and blood samples. The levels of expression of SST1, -2, and -4 mRNA were higher than those of the SST3 and -5 transcripts. In the lymphocytes from control subjects, the SST subtypes with high affinity for octreotide were barely found. The presence, even if at different concentrations, of all SST1-5 receptors in retroorbital lymphocytes from GO shows that they are targeted by SST analogs and could explain the effects described in GO patients treated with SST analogs.

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes.

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.

Somatostatin inhibits glucagon-like peptide-1-induced insulin secretion and proliferation of RINm5F insulinoma cells.

Glucagon-like peptide-1 [GLP-1; formerly GLP-1(7-36)amide] and somatostatin (SS) are two postprandially or paracrine released peptide hormones that regulate insulin secretion from pancreatic islets. Using the rat insulinoma cell line RINm5F as a model, we investigated the effects of both peptides alone and in combination on insulin release, proliferation, and intracellular signal transduction. In addition, we determined the SS receptor subtypes expressed and involved by reverse transcription-polymerase chain reaction and use of selective SS agonists. GLP-1 stimulated insulin release, cell proliferation, intracellular cAMP accumulation and activation of the transcription factor cAMP-response element binding protein (CREB) which all could be reduced to basal values by co-incubation with SS. Incubation with SS alone did not affect basal levels. RINm5F cells express the somatostatin receptor (sst) subtypes sst1 and sst2 as well as traces of sst3. In accordance, the sst1- or sst2-selective non-peptide agonists L-797591 or L-054522 and peptide agonist octreotide (SMS 201995; sst2, sst3, and sst5 selective) potently inhibited GLP-1-induced insulin secretion whereas the sst3-selective agonist L-796778 showed little effect. Moreover, the sst1- and sst2-selective agonists slightly reduced also basal insulin release. The experiments show that GLP-1 and SS are perfect opponents for regulating pancreatic beta-cell insulin secretion.

Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice.

We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled.

SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile.

The aim of the present study was to identify a small, metabolically stable somatotropin release inhibiting factor (SRIF) analog with a more universal binding profile similar to that of natural somatostatin, resulting in improved pharmacological properties and hence new therapeutic uses. A rational drug design approach was followed by synthesizing alanine-substituted SRIF-14 analogs to determine the importance of single amino acids in SRIF-14 for SRIF receptor subtype binding. The incorporation of structural elements of SRIF-14 in a stable cyclohexapeptide template in the form of modified unnatural amino acids resulted in the identification of the novel cyclohexapeptide SOM230. SOM230 binds with high affinity to SRIF receptor subtypes sst1, sst2, sst3 and sst5 and displays a 30- to 40-fold higher affinity for sst1 and sst5 than Sandostatin (octreotide; SMS 201-995) or Somatuline (BIM 23014). In vitro, SOM230 effectively inhibited the growth hormone releasing hormone (GHRH)-induced growth hormone (GH) release in primary cultures of rat pituitary cells with an IC(50) of 0.4+/-0.1 nmol/l (n=5). In vivo, SOM230 also potently suppressed GH secretion in rats. The ED(50) values determined at 1 h and 6 h post injection of SOM230 indicated its very long duration of action in vivo. This property was also reflected in pharmacokinetic studies comparing plasma levels of SMS 201-995 and SOM230 after subcutaneous application. Whereas SMS 201-995 had a terminal elimination half life of 2 h, this was markedly prolonged in SOM230-treated animals (t(1/2)=23 h). Furthermore, in rats SOM230 demonstrated a much higher efficacy in lowering plasma insulin-like growth factor-I (IGF-I) levels compared with SMS 201-995. The infusion of 10 microg/kg/h of SOM230 using subcutaneously implanted minipumps decreased plasma IGF-I levels far more effectively than SMS 201-995. After 126 days of continuous infusion of SOM230 plasma IGF-I levels were decreased by 75% of placebo-treated control animals. For comparison SMS 201-995, when used under the same experimental conditions, resulted in only a 28% reduction of plasma IGF-I levels, indicating a much higher efficacy for SOM230 in this animal model. It is important to note that the inhibitory effect of SOM230 was relatively selective for GH and IGF-I in that insulin and glucagon secretion was inhibited only at higher doses of SOM230. This lack of potent inhibition of insulin and glucagon release was also reflected in the lack of effect on plasma glucose levels. Even after high dose treatment over 126 days no obvious adverse side effects were noticed, including changes in plasma glucose levels. We have identified a novel short synthetic SRIF peptidomimetic, which exhibits high affinity binding to four of the five human SRIF receptor subtypes and has potent, long lasting inhibitory effects on GH and IGF-I release. Therefore SOM230 is a promising development candidate for effective GH and IGF-I inhibition and is currently under evaluation in phase 1 clinical trials.

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.

Early cerebral activities of the environmental estrogen bisphenol A appear to act via the somatostatin receptor subtype sst(2).

Recently, considerable interest has been aroused by the specific actions of bisphenol A (BPA). The present investigation represents a first study dealing with the interaction of BPA with the biologically more active somatostatin receptor subtype (sst(2)) in the rat limbic circuit. After treating pregnant female Sprague-Dawley rats with two doses (400 microg/kg/day; 40 microg/kg/day) of BPA, the binding activity of the above receptor subtype was evaluated in some limbic regions of the offspring. The higher dose proved to be the more effective one, as demonstrated by the elevated affinity of sst(2) with its specific radioligand, [(125)I]-Tyr(0)somatostatin-14. The most dramatic effects of BPA on sst(2) levels occurred at the low-affinity states of such a subtype in some telencephalic limbic areas of postnatal rats (10 days of age; postnatal day [PND] 10). These included lower (p < 0.05) sst(2) levels in the gyrus dentate of the hippocampus and basomedial nucleus of the amygdala; significantly higher (p < 0.01) levels were observed only for the high-affinity states of the periventricular nucleus of the hypothalamus. A similar trend was maintained in PND 23 rats with the exception of much lower levels of the high-affinity sst(2) receptor subtype in the amygdala nucleus and ventromedial hypothalamic nucleus. However, greater changes produced by this environmental estrogen were reported when the binding activity of sst(2) was checked in the presence of the two more important selective agonists (zolpidem and Ro 15-4513) specific for the alpha-containing Gamma-aminobutyric acid (GABA) type A receptor complex. In this case, an even greater potentiating effect (p < 0.001) was mainly obtained for the low-affinity sst(2) receptor subtype in PND 10 animals, with the exception of the high-affinity type in the ventromedial hypothalamic nucleus and gyrus dentate. These results support the contention that an sst(2) subtype alpha-containing GABA type A receptor system might represent an important neuromediating station capable of promoting estrogenlike mechanisms of BPA, especially during the early developmental phases.

Somatostatin and somatostatin analogues: diagnostic and therapeutic uses.

Somatostatin and its octapeptide analogues exert their effects through interaction with somatostatin receptor (sst) subtypes 1 through 5 (sst 1-5 ). Somatostatin binds with high affinity to all sst subtypes, whereas the currently commercially available octapeptide analogues bind only with a high affinity to sst 2 and sst 5. Pituitary tumors, endocrine pancreatic tumors, and carcinoid tumors express multiple sst subtypes, but sst 2 predominance is found in 90% of carcinoids and 80% of endocrine pancreatic tumors. Sst 2 and sst 5 predominance is found in growth hormone-secreting pituitary tumors. In patients harboring sst 2 - or sst 5 -positive neuroendocrine tumors, clinical symptomatology can be controlled by the chronic administration of one of the currently commercially available octapeptide somatostatin analogues. Tumors and metastases that bear sst 2 or sst 5 can be visualized in vivo after injection of radiolabeled octapeptide analogues. Radiolabeled octapeptide analogues can also be used for radiotherapy of sst 2 - and sst 5 -positive advanced or metastatic neuroendocrine tumors.

Identification and characterization of a type five-like somatostatin receptor in goldfish pituitary

In this study, a somatostatin receptor (Sst) cDNA was cloned and sequenced from goldfish pituitary. The cDNA encodes a 390-amino acid type five-like Sst (designated as gfSst 5). The amino acid sequence of the receptor has slightly higher homology to mammalian Sst 5′, compared with other mammalian Sst subtypes and recently identified fish Sst 1, Sst 2 and Sst 3. In CCL39-SRE-Luci cells stably expressing the cloned receptor, agonist radioligand [ 125I]LTT-SRIF 28′, a mammalian SRIF 28 analog, bound to a homogenous population of receptors with high affinity (nM K d). Competition binding studies showed that all three natural goldfish SRIF ligands, SRIF 14, [Pro 2]SRIF 14, and goldfish SRIF 28 (gfSRIF 28), and LTT-SRIF 28 bind the cloned gfSst 5 with high affinity and significantly stimulate [ 35S]GTPγS binding, with SRIF 28 peptides showing higher affinity in receptor binding and potency in [ 35S]GTPγS binding compared with SRIF 14 peptides. The receptor gene is highly and predominately expressed in pituitary; lower levels of the receptor mRNA were also detected in different brain regions by reverse transcriptor-polymerase chain reaction (RT-PCR) followed by Southern blot analysis.

Expression of somatostatin receptor types 2, 3 and 5 in biopsies and surgical specimens of human lung tumours. Correlation with preoperative octreotide scintigraphy.

The increasingly popular use of somatostatin analogs in clinical practice for both diagnostic and therapeutic purposes prompted extensive investigations on somatostatin receptor (sst) expression in human tumors by autoradiography, nucleic acid analysis and, recently, immunohistochemistry (IHC). The currently employed radiotracer for scintigraphy (Octreoscan) is octreotide, a somatostatin analog having a high affinity for sst types 2, 3, and 5. In this study on 25 patients, we compared sst 2, 3, and 5 expression in surgical and biopsy specimens of lung tumors, as revealed by immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR), with the octreoscan outcome (which was positive in 20/25 cases). By IHC, the tumors mainly expressed sst2 (17/25, 68%) at the cell membrane level, while sst 3 and 5 were detected in a fraction of cases (24% and 20%, respectively). Comparing RT-PCR and IHC data, a correlation was found in 83.3% of cases, while octreoscan findings and sst expression were correlated in 22/25 cases (88%). In addition, cytological and biopsy specimens expressed the same sst type found in the corresponding surgical sample, thus indicating that a cell membrane sst immunoreactivity in a biopsy reliably predicts the tumor-receptor profile before its resection. Finally, sst expression was not restricted to neuroendocrine lung tumors, but was also a feature of some non-neuroendocrine carcinomas, although to a lesser extent. The occasional expression of sst subtypes in intratumoral lymphocytes, endothelia and necrotic areas is an additional feature to be considered in the interpretation of Octreoscan findings, since the in vivo procedure does not allow to define the sst cellular distribution. IHC can therefore be usefully coupled to radionuclear investigations to better characterize the sst cellular location and subtype in lung tumors.

Immunohistochemical detection of somatostatin receptor types 1-5 in medullary carcinoma of the thyroid.

We have analysed the distribution of the five somatostatin receptors (sst1-5) by immunohistochemistry in a large retrospective series of 51 medullary carcinoma of the thyroid (MCT) specimens and correlated the pattern of sst expression with expression of somatostatin (SRIF) peptide, tumour pathology and clinical outcome. Immunohistochemistry was performed with rabbit polyclonal antipeptide antibodies directed against the extracellular domains or cytoplasmic tail of human (h) sst1-5. SRIF immunoreactivity was investigated in parallel paraffin sections. Eighty-five percent of the tumours were positive for one or more sst, localized to both tumour cells as well as surrounding peritumoural structures, especially blood vessels. Forty-nine percent of the tumours were positive for sst1, 43% for sst2, 47% for sst3, 4% for sst4, and 57% for sst5. Fifty-one percent of tumours expressed one or two sst subtypes; 33% were positive for three or more sst isoforms. All five sst receptors were detected in only two cases. Tumours expressing octreotide sensitive subtypes (sst2,3,5) accounted for 75% of the series. 50% of the tumours co-expressed SRIF suggesting tumour cell regulation by endogenous SRIF via paracrine/autocrine circuits. There was no correlation between sst1-5 expression and age, sex, tumour size or stage, histological type or clinical outcome. Simultaneous analysis of primary tumour and lymph node metastases revealed a similar pattern of sst immunoreactivity indicating that sst expression is not modified in the course of disease progression. With the exception of sst4, medullary carcinoma of the thyroid display a rich but heterogeneous expression of sst subtypes. Immunohistochemical typing of sst receptor expression using specific antireceptor antibodies represents an ideal approach for characterizing sst subtype expression in medullary carcinoma of the thyroid for optimizing receptor targeted diagnosis and therapy with somatostatin analogs.