Somatostatin analogs.

Somatostatin is a hypothalamic peptide hormone that inhibits the secretion of growth hormone, glucagon, insulin, gastrin and secretin, and also plays a role in neural transmission. Because of its wide range of possible clinical applications hundreds of somatostatin analogs have been synthesized and bioassayed to date. This review gives a historical perspective, summarizing approximately 30 years of research on somatostatin. The main focus is on the structure-activity relationships and conformational studies of the last generation of somatostatin agonists and their selectivity for five somatostatin receptor subtypes. Achievements in the synthesis of nonpeptide somatostatin analogs, as well as the first somatostatin antagonists, are also discussed. Finally, the use of a cyclic somatostatin scaffold to design ligands for other G-protein-coupled receptors, such as opioid and melanocortin receptors, is mentioned.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

The therapeutic problem of proliferative diabetic retinopathy: targeting somatostatin receptors.

Clinical management of proliferative diabetic retinopathy has changed very little in the last 5 decades, relying primarily on laser ablation of the retinal vasculature. Several lines of clinical and experimental evidence suggest that somatostatin analogues may be efficacious in inhibiting neovascularization associated with proliferative retinopathy but the mechanism of action for these compounds is unclear. Inhibition of growth hormone secretion and the subsequent suppression of insulin-like growth factor 1 (IGF-1) production by somatostatin has been suggested as the mechanism of action, however, in vitro studies suggest that somatostatin analogues suppress endothelial cell growth through a direct, somatostatin receptor-mediated inhibition of pro-survival signaling pathways. The advent of a new generation of modified peptide and peptidomimetic somatostatin analogues has allowed investigators to more carefully define the receptor subtypes responsible for somatostatin-induced endothelial cell death and may eventually lead to the clinical development of somatostatin analogues that can reduce endothelial cell proliferation, independent of suppression of circulating hormone levels.     

Cyclic hexa- and pentapeptide somatostatin analogues with reduced gastric inhibitory activity

The gastric inhibitory activity of cyclic hexa- and pentapeptide analogues of somatostatin was investigated in conscious cats with gastric fistulae. Gastric acid and pepsin secretions were stimulated by pentagastrin. Cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe) showed no inhibition of acid secretion at molar doses up to 50-times the ID50 for somatostatin. This peptide inhibited pepsin secretion at the highest dose (50 micrograms kg-1 hr-1), and its potency is approximately 0.005 compared with somatostatin (1.0). Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) inhibited acid (approximately 50%) and pepsin (approximately 85%) secretions, but the inhibition was not dose-related being similar with doses of 10 to 50 micrograms kg-1 hr-1. The cyclic pentapeptide, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr), was inactive in the dose range studied, with a potency less than 0.01. Cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] has been described as a somatostatin antagonist with respect to inhibition of growth hormone, insulin and glucagon release in rats [2]. Up to 60-fold molar excesses of this peptide failed to antagonise the inhibitory activity of somatostatin in the stomach. The results demonstrate that residues outside the central 6-11 region of somatostatin are very important for its gastric activity. The lack of gastric antagonistic activity of the pentapeptide antagonist indicates that these residues are likely to be involved in receptor recognition/binding.     

Localization of hormone secreting pathways in the brain by immunohistochemistry and light microscopy: a review.

Immunocytochemical techniques are now being used to localize hypothalamic neurosecretory hormones and related peptides in the mammalian brain. The data are probably incomplete, due primarily to false negative results. A number of previous assumptions concerning these pathways have been confirmed while other unexpected results were obtained. As expected, vasopressin and oxytocin and their associated proteins, neurophysins, were found in the magnocellular cell bodies of the hypothalamus and in their axonal projections to the neural lobe of the pituitary. Gonadotropin-releasing hormone (Gn-RH), somatostatin, and thyrotropin-releasing hormone (TRH) were located in what appears to be parvicellular nerve terminals on portal capillaries. Gn-RH has been found in perikarya in the arcuate nucleus, which is considered a source of fibers to the portal capillary bed. An extensive network of cell bodies and fibers in the preoptic area was also found to contain Gn-RH, and others in the periventricular nucleus in the anterior hypothalamus reacted with antiserum to somatostatin. Unexpected was considerable evidence that vasopressin is secreted directly into hypophyseal portal blood. This hormone and its neurophysin were also found in parvicellular neurons in the suprachiasmatic nucleus of rodents. All the hormones were found in fibers in the organum vasculosum of the lamina terminalis and in the posterior pituitary gland.     

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.     

Growth hormone stimulates hypothalamic somatostatin.

Hypothalamic somatostatin concentration is increased in normal rats treated with growth hormone. Somatostatin is reduced in the median eminence of hypophysectomized rats but restored to normal following growth hormone administration. These results suggest that growth hormone exerts a positive feedback effect on hypothalamic somatostatin mechanism by which it may regulate its own secretion.     

Peptidase inactivation of hypothalamic releasing hormones.

With the structural characterization of the hypothalamic hormones, luteinizing hormone-releasing hormone (LH-RH), thyrotrophin-releasing (TRH), melanocyte-stimulating hormone release-inhibiting hormine (MIH), and growth hormone release-inhibiting hormone, (GH-RIH or somatostatin), it has been possible to investigate their enzymic inactivation by peptidases which are present at various sites in the body. Enzymes may play an important part in the control of polypeptide hormone levels and the peptidases acting on these four hypothalamic hormones may regulate the amount of TRH, LH-RH, MIH and somatostatin released from the hypothalamus, or their action at the level of the pituitary and their removal from the circulation. By studying the peptidase enzymes, further information may be obtained on the physiological mechanisms controlling the secretion and actions of hypothalamic hormones, as well as on the design of analogues with increased or competitive activity.     

New medical approaches in pituitary adenomas

Recently, the medical approach to patients with secreting and clinically non-functioning pituitary adenomas has received great impulse thanks to the availability of new, selective and long-lasting compounds with dopaminergic activity, such as cabergoline, and of somatostatin analogues provided in slow-release formulations, such as lanreotide and octreotide long acting release (LAR). In particular, the use of cabergoline has induced control of hyperprolactinaemia and tumour shrinkage in the great majority of patients with micro- and macroprolactinomas. Cabergoline treatment restores fertility both in women and men, and partially improves osteoporosis, one of the major complications of hyperprolactinaemia. In acromegaly, disease control (growth hormone [GH] <2.5-1.0 microg/l as a fasting or glucose-suppressed value, respectively, together with age-normalised insulin-like growth factor [IGF]-I) is achievable in more than half of patients receiving treatment with lanreotide or octreotide-LAR. Improvement in cardiomyopathy, sleep apnoea and arthropathy has been reported during GH/IGF-I suppression after pharmacotherapy. A synthetic GH analogue, B2036-PEG, that antagonises endogenous GH binding to its receptor-binding sites and a GH-releasing hormone antagonist that blocks the effect of this releasing factor on the hypothalamus and pituitary are presently under investigation in acromegaly. Preliminary studies have clearly demonstrated the effectiveness of the GH receptor antagonist in suppressing IGF-I levels in acromegalic patients previously unresponsive to somatostatin analogues. Beneficial effects of subcutaneous octreotide and lanreotide have also been reported in adenomas secreting thyroid-stimulating hormone, while the results of treatment with dopamine agonists or somatostatin analogues remain disappointing in patients with clinically non-functioning adenomas. In these patients the possibility of visualising in vivo the expression of D(2) receptors using specific radiotracers such as (123)I-methoxybenzamide has allowed selection of patients likely to respond to cabergoline. Scant effects of pharmacotherapy have also been reported in patients with adenomas secreting adrenocorticotropic hormone. However, some preliminary data suggest a potential use of cabergoline in combination with ketoconazole, or alone, in selected cases of Cushing's disease or Nelson's syndrome.     

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

The somatostatin receptor on isolated pancreatic acinar cell plasma membranes. Identification of subunit structure and direct regulation by cholecystokinin

Somatostatin binding to its receptors on rat pancreatic acinar membranes was characterized with [125I-Tyr1]somatostatin. Binding at 24 degrees C was rapid reaching a maximum after 60 min and was reversible upon the addition of 1 microM unlabeled ligand. Scatchard analysis revealed a single class of binding sites, with a Kd of 0.32 +/- 0.03 nM and a binding capacity of 600 +/- 54 fmol/mg of protein. Specificity for the somatostatin was demonstrated with the inhibition of labeled hormone binding by somatostatin analogs in proportion to their biological activities. When [125I-Tyr1]somatostatin was cross-linked to its receptors with the photoreactive cross-linker n-hydroxysuccinimidyl-4-azidobenzoate, the hormone was associated with Mr = 90,000 protein. Similar mobilities of the radioactive band were observed in the presence and absence of dithiothreitol. In contrast to other unrelated peptides, cholecystokinin (CCK) and its analogs directly reduced [125I-Tyr1] somatostatin binding to isolated membranes. The effect of CCK was one-half-maximal at 3 nM and maximal at 100 nM. In the presence of 3 nM CCK8, the binding capacity for somatostatin was decreased to 237 +/- 39 fmol/mg of protein without a significant change in affinity. Dibutyryl cyclic GMP, a CCK receptor antagonist, blocked this action of CCK8 indicating that the CCK receptor mediated the decrease in [125-Tyr1]somatostatin binding. In contrast cerebral cortex membranes, which also possess a somatostatin receptor, were not regulated by CCK. These results indicate, therefore, that 1) purified pancreatic acinar plasma membranes contain specific receptors for somatostatin, 2) the receptor has an apparent Mr of about 90,000, and 3) the binding of somatostatin to its receptor on pancreatic plasma membranes is regulated by CCK analogs acting via the CCK receptor.     

Somatostatin receptors and signaling cascades coupled to them

The peptide hormone somatostatin controlling functions of CNS and peripheral organs and tissues realizes its regulatory effects via five types of somatostatin receptors (SomR) coupled to heterotrimeric G-proteins. Targets of the hormone action are the enzymes generating second messengers (adenylyl cyclase, phospholipase C, phosphatidylinositol-3-kinase), phosphotyrosine phosphatases, ion channels. The review summarizes and analyzes literature data and results of our studies on molecular mechanisms of transduction of the somatostatin signal into the cell, selectivity of interaction of SomR with heterotrimeric G-proteins and intracellular effectors as well as on effect of SomR oligomerization on their functional activity.     

Physiology of the ECL cells.

The enterochromaffin-like (ECL) cells of the oxyntic mucosa (fundus) of the stomach produce, store and secrete histamine, chromogranin A-derived peptides such as pancreastatin, and an unanticipated but as yet unidentified peptide hormone. The cells are stimulated by gastrin and pituitary adenylate cyclase activating peptide and suppressed by somatostatin and galanin. Choline esters and histamine seem to be without effect on ECL cell secretion. The existence of a gastrin-ECL cell axis not only explains how gastrin stimulates acid secretion but also may help to explore the functional significance of the ECL cells with respect to the nature and bioactivity of its peptide hormone. From the results of studies of gastrectomized/fundectomized and gastrin-treated rats, it has been speculated that the anticipated ECL-cell peptide hormone acts on bone metabolism.     

Carboxypeptidase E in rat antropyloric mucosa: distribution in progenitor and mature endocrine cell types

Processing of most gut hormones involves cleavage between dibasic amino acids followed by carboxypeptidase-catalyzed removal of the COOH-terminal basic residue, resulting in peptides with a COOH-terminal glycine. Such peptides may subsequently be converted to amidated peptides or can be directly secreted. It is believed that carboxypeptidase E (CPE) is involved in gut hormone processing but its presence in gut endocrine cells has never been studied. We have analyzed the distribution of CPE in the antropyloric mucosa of rat stomach and report that gastrin cells and progenitor gastrin-somatostatin (G/D) cells express CPE while mature somatostatin cells and the majority of serotonin cells fail to express CPE. These data indicate that immature G/D cells are able to process gastrin to glycine-extended forms and that CPE-mediated processing is not a characteristic of mature somatostatin and serotonin cells.     

The interrelationship of somatostatin and guanylate cyclase activity

Summary Somatostatin has been shown to inhibit the release of various polypeptide hormones including insulin, glucagon, gastrin, thyroid stimulating hormone, and growth hormone. The mechanism by which somatostatin inhibits the release of these various polypeptide hormones has not been fully eluciadated. It has been reported that somatostatin increases the level of the second messenger cyclic GMP in rat brain and in the anterior pituitary gland. The present investigation was designed to determine if these responses seen in the anterior pituitary gland and brain were due to activation of guanylate cyclase GTP-pyrophosphate lyase (cyclizing), E.C.4.6.1.2., the enzyme that catalyzes the formation of cyclic GMP. Somatostatin at a concentration of 2 pm enhanced guanylate cyclase activity two-fold in rat cerebrum and anterior pituitary gland. This enhancement of guanylate cyclase activity was also seen in rat liver, pancreas, stomach, and small intestine at the same concentration of somatostatin. Increasing the concentration of somatostatin to 20 m, caused a marked inhibition of guanylate cyclase activity in all these tissues. Dose-response curves done on gastric guanylate cyclase activity revealed that over a concentration range of 2 pm to 0.2 m, somatostatin had a stimulatory effect on guanylate cyclase activity while at concentrations above 10 m somatostatin was inhibitory to guanylate cyclase activity. The biphasic pattern of enhancement of guanylate cyclase activity at lower concentrations of somatostatin and inhibition at higher concentrations may help to explain some of the discrepancies seen with previous investigations with somatostatin, hormone release, and cyclic nucleotide metabolism.     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.