Muscarinic receptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10-day treatment with growth hormone (GH) (1 mg/kg body weight day) and somatostatin (SRIF) (0.25 mg/kg body weight day) subcutaneously on the activity of muscarinic (M) receptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using (3H)quinuclidinyl benzylate [(3H)QNB] as radioligand. 2. The administration of GH and SRIF significantly decreased the M-receptor binding affinity in the hypothalamus. 3. In the pituitary the M-receptor affinity was increased after both GH and SRIF treatment. 4. In the hypothalamus and the pituitary the binding capacity of muscarinic receptors was unchanged. 5. In the cerebral cortex the chronical GH injection induced an increase in the number of antagonist binding sites and a decrease of their affinity, while the similar SRIF treatment led to an increase of the binding affinity without any change of M-receptor capacity. 6. These results indicate that GH and SRIF selectively and region-specifically modulate muscarinic receptor binding affinity and capacity and provide new insight into the feedback regulatory mechanisms of GH secretion.     

Somatostatin analog SMS 201995 inhibits proliferation in human leukemia T-cell line: relevance of the adenylyl cyclase stimulation

Octreotide SMS 201995 is a stable somatostatin (SRIF) analog with potent antiproliferative actions in numerous cell types including normal T lymphocytes. It is currently used in the clinical treatment of different malignancies. However, the possible beneficial actions of octreotide in T-cell leukemia have not been addressed before, although these cells express SRIF receptors. For instance, human leukemia Jurkat T cells have been shown to express a single SRIF receptor isotype: sst3 that can be pharmacologically targeted by octreotide. In this study, we therefore studied SMS 201995 effects on in vitro [(3)H-CH3]thymidine incorporation in Jurkat T cells. Our data show that octreotide inhibits the proliferation of Jurkat cells both in the absence and in the presence of mitogens. By contrast, SRIF28, an endogenous SRIF analog sharing with SMS 201995 an almost identical affinity for somatostatin sst3 receptors, increases [(3)H-CH3]thymidine uptake in both mitogen-activated and nonactivated cells. To assess the mechanisms of the opposite actions of these two analogs on leukemia T-cell proliferation, we next studied their effects on adenylyl cyclase activity in whole Jurkat cells. At least in the presence of mitogens, SMS 201995 significantly enhances the adenylyl cyclase activity whereas SRIF28 inhibits it. Taken together these data are in accordance with the current hypothesis according to which increase and decrease in cAMP production are required to allow the inhibition and stimulation of T-cell proliferation, respectively. They also point to a potential therapeutic benefit of SMS 201995 in the management of human T-cell leukemia.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Analogues of Somatostatin Bind Selectively to Brain Somatostatin Receptor Subtypes

Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.     

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

SSTR2 mediates the somatostatin-induced increase in intracellular Ca(2+) concentration and insulin secretion in the presence of arginine vasopressin in clonal beta-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca(2+)](i) and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca(2+)](i) and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca(2+)](i) and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca(2+)](i) or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca(2+)](i) and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.     

Beta-adrenoceptor activity change after prolonged treatment with growth hormone and somatostatin.

1. The effect of 10 days treatment with growth hormone (GH) (1 mg/kg body wt/day) and somatostatin (SRIF) (0.25 mg/kg body wt/day) subcutaneously on the activity of beta-adrenoceptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using [3H]dihydroalprenolol ([3H]DHA) as radioligand. 2. The administration of GH significantly increased the beta-adrenoceptor binding affinity and the administration of SRIF decreased the beta-adrenoceptor binding capacity in the hypothalamus. 3. In the pituitary the beta-adrenoceptor binding affinity was significantly decreased after both hormonal applications. 4. In the cerebral cortex the beta-adrenoceptor binding affinity was significantly decreased after the GH treatment and increased after the SRIF treatment. 5. The present study provides direct evidence for GH and SRIF effects on the activity of rat beta-adrenoceptors and supports the view about the involvement of beta-adrenergic mechanisms in the neurotransmitter regulation of GH secretion in the rat.     

Somatostatin receptors

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors.This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst₁-sst₅), one of which is represented by two splice variants (sst_2A and sst_2B). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst₂ and sst₅ receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.     

A transplantable phosphorylation probe for direct assessment of G protein-coupled receptor activation

The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst(2) receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst(3) and sst(5) receptors but was only a partial agonist at the sst(2) receptor. Octreotide exhibited potent agonistic properties at the sst(2) receptor but produced very little sst(5) receptor activation. Like octreotide, somatoprim was a full agonist at the sst(2) receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.     

Two amino acids, located in transmembrane domains VI and VII, determine the selectivity of the peptide agonist SMS 201-995 for the SSTR2 somatostatin receptor.

Human somatostatin receptor subtypes (SSTR1-5) bind their natural ligands SRIF-14 and SRIF-28 with high affinity. By contrast, short synthetic SRIF analogues such as SMS 201-995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub-nanomolar affinity only for the receptor subtype SSTR2. To understand the molecular nature of selective peptide agonist binding to somatostatin receptors we have now, by site-directed mutagenesis, identified amino acids mediating SMS 201-995 specificity for SSTR2. Sequentially, amino acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201-995, were exchanged for the corresponding SSTR2 residues. After three consecutive steps, in which eight amino acids were exchanged, a SSTR1 mutant receptor with high affinity for SMS 201-995 was obtained. Receptor mutants with different combinations of these eight amino acids were then constructed. A single Ser305 to Phe mutation in TM VII increased the affinity of SSTR1 for SMS 201-995 nearly 100-fold. When this mutation was combined with an exchange of Gln291 to Asn in TM VI, almost full susceptibility to SMS 201-995 was obtained. Thus, it is concluded that the specificity of SMS 201-995 for SSTR2 is mainly defined by these two amino acids in transmembrane domains VI and VII. Using the conjugate gradient method we have, by analogy to the well established structure of bacteriorhodopsin, built a model for SRIF receptor-ligand interactions that explains the importance of Gln291 and Ser305 for the selectivity of agonists.     

Somatostatin receptors

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst(1)-sst(5)), one of which is represented by two splice variants (sst(2A) and sst(2B)). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst(2) and sst(5) receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.     

Differential distribution of somatostatin sst2 receptor splice variants in rat gastric mucosa

The tetradecapeptide somatostatin (SRIF) has an inhibitory action on acid secretion in the stomach. It has been suggested that somatostatin may act directly on parietal cells as well as indirectly via histamine-producing cells. A family of high affinity membrane-bound receptors, which are termed sst1-sst5 receptors, mediates the physiological effects of somatostatin. On the basis of functional studies it has been suggested that somatostatin may mediate its actions in the stomach by activation of a somatostatin sst2 receptor type. Two splice variants of the rat sst2 receptor exist, sst2(a) and sst2(b), which differ in length and composition of their intracellular carboxy termini. To date, little information is available on the distribution of the somatostatin sst2(b) receptor in any peripheral tissue. Here we show for the first time the localisation of this receptor isoform in the rat oxyntic mucosa, where the receptor protein was found to be present in parietal cells. This is in contrast to sst2(a) receptor, which was localised to enterochromaffin-like cells and nerve fibres. The differential localisation of the receptor isoforms to two key cell types, parietal cells and enterochromaffin-like cells, may explain how somatostatin inhibits acid secretion by more than one mechanism.     

111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptor-positive tumors: synthesis, radiolabeling and in vitro validation.

Somatostatin receptor-positive human tumors can be detected using radioiodinated analogues of somatostatin, both in vitro and in vivo. [123I-Tyr3]-octreotide has been successfully used in the visualization of somatostatin receptor-positive tumors by gamma camera scintigraphy, but this radiopharmaceutical has some major drawbacks, which can be overcome with other radionuclides such as 111In. As starting material for a potentially convenient radiopharmaceutical, a diethylenetriaminopentaacetic acid (DTPA) conjugated derivative of octreotide (SMS 201-995) was prepared. This peptide, [DTPA-D-Phe1]-octreotide (SDZ 215-811) binds more than 95% of added 111In in an easy, single-step labeling procedure without necessity of further purification. The specific somatostatin-like biologic effect of these analogues was proven by the inhibition of growth hormone secretion by cultured rat pituitary cells in a dose-dependent fashion by octreotide, [DTPA-D-Phe1]-octreotide and non-radioactive [115In-DTPA-D-Phe1]-octreotide. The binding of [111In-DTPA-D-Phe1]-octreotide to rat brain cortex membranes proved to be displaced similarly by natural somatostatin as well as by octreotide, suggesting specific binding of [111In-DTPA-D-Phe1]-octreotide to somatostatin receptors. The binding of the indium-labeled compound showed a somewhat lower affinity when compared with the iodinated [Tyr3]-octreotide, but indium-labeled [DTPA-D-Phe1]-octreotide still binds with nanomolar affinity. In conjunction with in vivo studies, these results suggest that [111In-DTPA-D-Phe1]-octreotide is a promising radiopharmaceutical for scintigraphic imaging of somatostatin receptor-positive tumors.     

Somatostatin receptors in the gastrointestinal tract in health and disease.

The multiple actions of somatostatin are mediated by specific membrane-bound receptors present in all somatostatin target tissues, such as brain, pituitary, pancreas, and gastrointestinal tract. Three different types of tissues in the human gastrointestinal tract express somatostatin receptors: (1) the gastrointestinal mucosa, (2) the peripheral nervous system, and (3) the gut-associated lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogs. Somatostatin receptors are also expressed in pathological states, particularly in neuroendocrine tumors of the gastrointestinal tract. Ninety percent of the carcinoids and a majority of islet-cell carcinomas, including their metastases, usually have a high density of somatostatin receptors. Only 10 percent of the colorectal carcinomas and none of the exocrine pancreatic carcinomas, however, contain somatostatin receptors. The somatostatin receptors in tumors are identified with in vitro binding methods or with in vivo imaging techniques; the latter allow the precise localization of the tumors and their metastases in the patients. Since somatostatin receptors in gastroenteropancreatic tumors are functional, their identification can be used to assess the therapeutic efficacy of octreotide to inhibit excessive hormone release in the patients.     

Inhibition of basal insulin secretion induces insulin resistance in normal man: evidence for a tonic effect of insulin on carbohydrate metabolism

Insulin resistance has been demonstrated both in insulin deficiency and insulin excess in man and in animals. This study was carried out in normal man to evaluate the role of insulinopenia in the pathogenesis of insulin resistance. Insulin suppression was obtained by 4 h somatostatin (SRIF) infusion. Insulin receptors on circulating monocytes were evaluated before and after SRIF infusion; an insulin tolerance test (ITT) was performed after SRIF, saline or SRIF and replacing basal insulin secretion. Insulin binding to circulating monocytes did not change after 4 h insulinopenia (2.19 +/- 0.30 vs. 2.35 +/- 0.80%), while insulin sensitivity appeared decreased after SRIF (KITT = 0.97 +/- 0.13) as compared with saline (KITT = 3.30 +/- 0.42), and this effect was prevented by insulin (KITT = 2.46 +/- 0.38). A relationship was detected between KITT and plasma insulin concentration before ITT (r = 0.85, p less than 0.01), suggesting that insulin deficiency is the main cause of the phenomenon observed. The present data suggest that basal insulin concentration plays an essential role in the control of insulin sensitivity. If insulin binding on monocytes mimics the behavior of major insulin target tissues, it is possible that the impaired insulin action after 4 h of insulin deficiency is related to a post binding effect.     

Somatostatin receptor gene expression in neuroblastoma

Somatostatin receptor expression is a favorable prognostic factor in human neuroblastoma. Somatostatin receptors have been demonstrated in vitro by pharmacologic analysis of tumor tissue and in vivo by diagnostic radioreceptor scintigraphy. However, which receptor subtypes (sst(1), sst(2), sst(3), sst(4), and sst(5)) are expressed in these tumors has not yet been delineated. We used RT-PCR to analyze expression of the five somatostatin receptor genes in 32 neuroblastoma tumor specimens. All 32 tumor specimens expressed mRNA for c-abl and sst(1); sst(2) mRNA was detected in 27/32 samples and somatostatin mRNA was detected in 30/32 tumor specimens. The remaining receptor subtypes, sst(3), sst(4), and sst(5) were variably expressed. Receptor protein for sst(1) and sst(2) was visualized in tumor neuroblasts as well as in endothelial cells of tumor vessels using immunostaining with specific anti-receptor antibodies. The effect of high expression of somatostatin receptors on cell proliferation was examined in SKNSH neuroblastoma cells transfected with sst(1) and sst(2). SS(14) binding to wild-type SKNSH cells was undetectable; but the native peptide bound with high affinity to the SKNSH/sst(1) and SKNSH/sst(2) neuroblastoma cell lines. Pharmacologic analysis of binding with two long-acting analogues, CH275 and octreotide, confirmed selective expression of sst(1) and sst(2) in stably transfected SKNSH cells. Formation of neuroblastoma xenograft tumors in nude mice was significantly delayed for both SKNSH/sst(1) (P<0.001) and SKNSH/sst(2) (P<0.05) cells compared to wild-type SKNSH. We conclude that: (1) Somatostatin receptors, sst(1) and sst(2), are expressed in the majority of neuroblastomas at diagnosis; and (2) upregulation of functional sst(1) or sst(2) in neuroblastoma cell lines suppresses tumorigenicity in a xenograft model. These observations suggest that somatostatin receptors may be a useful therapeutic target in neuroblastoma.     

Beta-adrenoceptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10 days treatment with growth hormone (GH) (l mg/kg body wt/day) and somatostatin (SRIF) (0.25 mg/kg body wt/day) subcutaneously on the activity of β-adrenoceptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using [³H]dihydroalprenolol ([³H]DHA) as radioligand.2. The administration of GH significantly increased the β-adrenoceptor binding affinity and the administration of SRIF decreased the β-adrenoceptor binding capacity in the hypothalamus.3. In the pituitary the β-adrenoceptor binding affinity was significantly decreased after both hormonal applications.4. In the cerebral cortex the β-adrenoceptor binding affinity was significantly decreased after the GH treatment and increased after the SRIF treatment.5. The present study provides direct evidence for GH and SRIF effects on the activity of rat β-adrenoceptors and supports the view about the involvement of β-adrenergic mechanisms in the neurotransmitter regulation of GH secretion in the rat.     

A Study of Somatostatin Receptors in Amygdaloid-Kindled Rat Brain

Abstract: Kindling is an animal model of epilepsy. Previously somatostatin (SRIF) was implicated in seizure activity in the brain. Recently we reported a significant increase in brain SRIF content in the temporal cortices and cortices of kindled rats. Since the interaction between the neurotransmitter and the receptor eventually is responsible for the biological response, the present study was undertaken to examine evidence for the participation of SRIF receptor in the kindled state. In this study we present a procedure for detection of SRIF receptors using radiolabeled (D-Tyr⁸)-SRIF as a tracer. The present study indicates that in kindled rats there are no differences in the total number or affinity of the binding sites in the temporal cortex and a slight increase in the total number of binding sites in the cortex when compared with controls. These results, in view of our other observations, suggest that in kindled rat brain there may be an increased release of SRIF but no down-regulation of SRIF receptors in temporal cortex and cortex. There appears to be a significant decrease in the number of SRIF receptors in kindled hippocampus. The mechanism by which this occurs remains unclear.