Suppression of ovulation in the rat by an orally active antagonist of luteinizing hormone-releasing hormone.

A synthetic antagonist of luteinizing hormone-releasing hormone blocked ovulation in rats in a dose-dependent manner when given by gavage on the afternoon of proestrus. Ovulation was delayed for at least 1 day in all animals given 2 milligrams of antogonist and in some of the animals treated with 1 or 0.5 milligram. Oral administration of 2 milligrams also blocked the preovulatory surge of luteinizing hormone. This demonstration that antagonists of luteinizing hormone-releasing hormone can have oral antiovulatory activity clearly enhances their therapeutic potential.

Corticotropin-releasing factor stimulates secretion of melanocyte-stimulating hormone from the rat pituitary.

Administration of synthetic ovine corticotropin-releasing factor led to rapid, parallel increases in adrenocorticotropin and alpha-melanocyte-stimulating hormone concentrations in rat plasma. Prior treatment with dexamethasone almost completely blocked the adrenocorticotropin response but not the increase in melanocyte-stimulating hormone. These data demonstrate that corticotropin-releasing factor is a potent stimulator not only of adrenocorticotropin secretion from the corticotrophs of the anterior pituitary gland but also of peptide secretion from the intermediate lobe. Such data suggest that melanocyte-stimulating hormone and beta-endorphin play a role in the physiological response to stress.

Comparison of the distribution of dynorphin systems and enkephalin systems in brain.

A study of the anatomical distribution of the endogenous opioid dynorphin in rat brain showed that the peptide is localized in a widespread system with multiple cell groups and projections. This network is revealed by the use of multiple antiserums against dynorphin and can be distinguished from the system containing methionine-enkephalin and leucine-enkephalin, which is mapped by the use of antiserums against the enkephalins and biosynthetically related peptides in the adrenal. It thus appears that the brain contains at least three separate opioid neuronal networks: an enkephalin family with components similar to those found in the adrenal, a beta-endorphin family, and a dynorphin family.

Nonopiate effects of dynorphin and des-Tyr-dynorphin.

Intracerebroventricular administration of dynorphin produced potent and long-lasting effects on motor function and the electroencephalogram in rats. In addition, local iontophoretic or pressure ejection of dynorphin consistently inhibited hippocampal unit activity. None of these effects were significantly affected by naloxone even at high doses. Moreover, a fragment of dynorphin that failed to displace any of a number of tritiated narcotics from rat brain homogenates produced similar effects on these physiological measures in vivo. On the basis of a variety of criteria for "opiate action," the results suggest that a second biologically active site within the dynorphin sequence is capable of quite potent but nonopiate effects.

Corticotropin-releasing factor stimulates accumulation of adenosine 3', 5'-monophosphate in rat pituitary corticotrophs.

The presence of synthetic ovine corticotropin-releasing factor leads to a rapid and marked stimulation of adenosine 3', 5'-monophosphate accumulation in an enriched population of rat pituitary corticotrophs in primary culture. The increase, observed as early as 60 seconds after the addition of corticotropin-releasing factor, suggests that changes in the intracellular concentration of the cyclic nucleotide coincide with or precede the secretion of adrenocorticotropic hormone in response to corticotropin-releasing factor.

An analog of enkephalin having prolonged opiate-like effects in vivo.

Intraventricular administration of the enkephalin analog, [D-Ala2]-metenkephalin, induces profound and long-lasting analgesia, as well as other opiate-like behavioral effects in the rat. This analgesia was highly dose dependent, of much greater magnitude, and about 30 times longer lasting than that produced by the naturally occurring peptide, methionine-enkephalin. The behavioral effects of the [D-Ala2] analog could be completely reversed by the opiate antagonist, naloxone, suggesting that these effects were mediated by opiate receptors. Systemic administration of naloxone alone resulted in a significant increase in pain sensitivity. These findings support the view that endogenous opiate systems may play an important role in modulating pain sensitivity.

Blockage of ovulation in rats by inhibitory analogs of luteinizing hormones-releasing hormone.

An antagonist of luteinizing hormone-releasing hormone (LH-RH). [D-Phe2-Phe3-D-Phe6]-LH-RH (Phe, phenylalanine), suppressed luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in male rats in response to LH-RH for at least 4 hours. Three subcutaneous injection of 1 milligram of this antagonist into rats during proestrus completely suppressed ovulation, while a single injection of 1.5 milligrams per rat inhibited 95.3 percent of the preovulatory surge of LH, 84.2 percent of the FSH surge, and suppressed ovulation by 86.4 percent.

Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide, inhibits glucagon secretion via somatostatin (receptor subtype 2) in the perfused rat pancreas.

AIMS/HYPOTHESIS: The glucose-lowering effect of glucagon-like peptide-1 (GLP-1) is based not only upon its potent insulinotropic actions but also on its ability to restrain glucagon secretion. Surprisingly, the closely related glucose-dependent insulinotropic peptide (GIP) stimulates glucagon release. We examined whether the islet hormone somatostatin, which strongly inhibits glucagon secretion, is involved in this divergent behaviour. METHODS: At 1.5 mmol/l glucose and therefore minimal insulin secretion, the glucagon, insulin and somatostatin responses to 20 mmol/l glucose, GLP-1, GIP and somatostatin were studied in the presence of a high-affinity monoclonal somatostatin antibody and of a highly specific somatostatin receptor subtype 2 (SSTR2) antagonist (PRL-2903) in the isolated perfused rat pancreas. RESULTS: In control experiments, GLP-1 at 1 and 10 nmol/l reduced glucagon secretion significantly to 59.0 +/- 6.3% (p < 0.004; n = 5; SSTR2 series; each vs pre-infusion level) and to 48.0 +/- 2.6% (p < 0.001; n = 6; somatostatin antibody series) respectively. During somatostatin antibody administration, GLP-1 still inhibited glucagon secretion significantly, but the effect was less pronounced than in control experiments (p < 0.018). Co-infusion of the SSTR2 antagonist completely abolished the GLP-1-induced suppression of glucagon secretion. In contrast, neither the GIP-induced stimulation of glucagon release nor its inhibition by 20 mmol/l glucose was altered by somatostatin antibody or SSTR2 antagonist administration. CONCLUSIONS/INTERPRETATION: We conclude that GLP-1 is capable of inhibiting glucagon secretion even in the absence of secretory products from the beta cell. It is highly likely that this is mediated via somatostatin interacting with SSTR2 on rat alpha cells. In contrast, GIP and glucose seem to influence the alpha cell independently of somatostatin secretion.

Effect of growth hormone-release inhibiting hormone on hormones stimulating exocrine pancreatic secretion.

The nature and extent of growth hormone-release inhibiting hormone (GH-RIH, somatostatin)-induced inhibition of pancreatic secretion of bicarbonate and protein, an index of enzyme secretion, were studied by administration of exogenous secretin or cholecystokinin (CCK) and of a number of stimulants for endogenous release of these hormones in fasted pancreatic fistula dogs with and without an infusion of GH-RIH. The results of this study show that GH-RIH inhibits the pancreatic fluid and bicarbonate secretion induced by duodenal acidification and exogenous secretion. The kinetic analysis shows that the interaction between GH-RIH and secretin affecting pancreatic bicarbonate secretion possesses the characteristics of competitive inhibition. GH-RIH does not change the pancreatic protein response to exogenous CCK, but profoundly inhibits pancreatic response to a variety of the endogenous stimulants of CCK release, including duodenal perfusion of sodium oleate, amino acid mixture, or feeding of a peptone meal. We conclude that GH-RIH is a very potent inhibitor of the endogenous release of CCK from the intestinal mucosa and inhibits competitively the action of secretin but not CCK on the exocrine pancreatic secretion.

Somatostatin receptor subtype specificity in human fetal pituitary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone, and prolactin regulation.

Somatostatin (SRIF), a hypothalamic inhibitor of pituitary growth hormone (GH) and thyroid-stimulating hormone (TSH) secretion, binds to five distinct receptor (SSTR) subtypes. We therefore tested SSTR subtype-specific SRIF analogs in primary human fetal pituitary cultures (23-25-wk gestation) to elucidate their role in regulating human pituitary function. Using reverse transcription-PCR, mRNA expression of SSTR2 and SSTR5 were detected in fetal pituitary by 25 wk. SRIF analog affinities were determined by membrane radioligand binding in cells stably expressing the human SSTR forms. GH secretion was suppressed equally (40-60%, P < 0.005) by analogs preferential for either SSTR2 (IC50 for receptor binding affinity, 0.19-0.42 nM) or SSTR5 (IC50, 0.37 nM), and compounds with enhanced affinity for SSTR2 were more potent (EC50 for GH suppression, 0.05-0.09 nM) than Lanreotide (EC50, 2.30 nM) and SRIF (EC50, 0.19 nM). Similarly, analogs with high affinity for SSTR2 or SSTR5 decreased TSH secretion (30-40%, P < 0.005). However, prolactin was effectively inhibited only by compounds preferentially bound to SSTR2 (20-30%, P < 0.05). Luteinizing hormone was modestly decreased (15-20%) by SSTR2- or SSTR5-specific analogs. An SSTR5-specific analog also exclusively inhibited GH in acromegalic tumor cells. Thus, SRIF regulation of GH and TSH in primary human fetal pituitary cells is mediated by both SSTR2 and SSTR5, both of which are abundantly expressed by 25 wk. In contrast, suppression of prolactin is mediated mainly by SSTR2. These results indicate that SSTR5 is critical for physiologic regulation of GH and TSH. SRIF analogs with selective affinity for this receptor may therefore be more effective in the treatment of hormone-secreting pituitary adenomas.

Somatostatin receptor subtypes mediate contractility on human colonic smooth muscle cells.

This study demonstrates the expression of functional somatostatin receptor (sstr) subtypes in human circular and longitudinal colonic smooth muscle cells (SMC). Native somatostatin (SS) and sstr subtype-specific analogues were used to characterize the sstr subtypes present in both cell types by contraction/relaxation studies. Qualitative and quantitative mRNA analysis and immunohistochemistry of sstr subtypes were also carried out. sstr subtype 2 mRNA was expressed in circular SMC, and various levels of subtypes 1, 2 and 3 mRNA were expressed in longitudinal colonic SMC. Native SS and each subtype-specific analogue exerted a modest, but significant, contraction, although inhibition of carbachol-induced contraction (relaxation) was the main effect on SMC from both layers. CH-288, a sstr subtype 1-specific analogue, and octreotide, a sstr subtype 2-specific analogue, were the most effective relaxant analogues on longitudinal and circular SMC, respectively. sstr subtypes display a distinct expression pattern on human colonic SMC; on circular SMC, subtype 2 is the only sstr, whereas sstr subtypes 1, 2 and 3 are expressed on human SMC isolated from the longitudinal layer. The contractile effects of SS are mediated through sstr subtype 2 and sstr subtype 1 on circular and longitudinal human colonic SMC, respectively.

Treatment of R-3327 prostate tumors with a somatostatin analogue (somatuline) as adjuvant therapy following surgical castration.

This study addresses, in an animal tumor model, the clinical problem of "escape from castration inhibition." Somatuline (BIM-23014C), an octapeptide analogue of somatostatin with enhanced potency and longer duration of biological activity was administered as a therapeutic agent, over a period of 90 and 197 days, to male Copenhagen rats bearing syngeneic Dunning R-3327-H prostate tumors. Androgen sensitivity was confirmed by the response of tumors to castration and by the significant inhibition of tumor growth in intact animals by treatment with a luteinizing hormone-releasing hormone antagonist (BIM-21009). Inhibition of tumor growth resulting from castration persisted for 102 days, after which progressive regrowth occurred, indicating an escape from castration inhibition. When Somatuline treatment was initiated as an adjuvant therapy 5 days after castration, the rate of tumor regrowth during escape was significantly retarded. During the period of 197 days postcastration, tumors in the vehicle-treated, intact controls grew to an average diameter of 38.6 +/- 7.6 mm and tumors in vehicle-treated castrate controls grew to an average diameter of 23.3 +/- 4.1 mm (60% test/control). Treatment with the luteinizing hormone-releasing hormone antagonist induced no significant additional tumor inhibitory effects in castrated animals which developed tumors having an average diameter of 30.2 +/- 8.2 mm (78% test/control). Treatment of tumors in castrate animals with Somatuline, on the other hand, induced a significant (P less than 0.01) tumor-inhibitory effect that was greater than that produced by castration alone, developing an average tumor diameter of only 14.3 +/- 2.6 mm, (37% test/control). A growth inhibitory effect was also inducible in animals having tumors that had already escaped castration inhibition. The relative nontoxicity of a somatostatin analogue such as Somatuline suggests that chronic or maintenance therapy of slow-growing prostate cancers may be both feasible and acceptable in a clinical setting.

Response of human lung tumor xenografts to treatment with a somatostatin analogue (Somatuline).

Four human small cell lung carcinomas, NCI-H69, NCI-N417, NCI-H345, LX-1, and a non-small cell lung carcinoma, H-165, implanted s.c. as tumor xenografts in athymic nude mice, were treated with Somatuline (BIM-23014C), an endocrinologically potent octapeptide analogue of somatostatin. All tumors responded, although in varying degrees, with percentage of test/control values ranging from 3 to 88. Somatuline administered as a perilesional infusion effectively inhibited xenograft growth inducing prolonged remissions. When treatment was terminated, some tumors regrew, suggesting antimitogenic activity rather than cytocidal. Absence of observable systemic or local toxicity during prolonged treatment would support this conclusion and suggest the feasibility of long term maintenance therapy with a resultant extended survival.

Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Bombesin stimulates adhesion, spreading, lamellipodia formation, and proliferation in the human colon carcinoma Isreco1 cell line.

The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells.

Isolation and structure of several peptides from porcine hypothalami.

Ten peptides were isolated from porcine hypothalami and structurally elucidated. These included four dipeptides Arg-Phe, Phe-Tyr, Val-Trp, and Tyr-Phe; a tripeptide Lys-Phe-Tyr; two tetrapeptides Gly-Lys-Val-Asn and Phe-Glu-His-Glu, a nonapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; a decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe and a hexadecapeptide Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asn-Leu. The tetrapeptide Gly-Lys-Val-Asn, the nonapeptide, the decapeptide and the hexadecapeptide most probably represent artifact fragments of alpha- and beta-chains of porcine hemoglobin. The natural or synthetic Gly-Lys-Val-Asn and Phe-Glu-His-Glu had some growth hormone releasing activity while Val-Trp, Tyr-Phe and Lys-Phe-Tyr had slight prolactin releasing activity. The biological activities of other peptides have not been determined yet.

Progress in the development of potent bombesin receptor antagonists.

Bombesin and the mammalian-related peptides gastrin-releasing peptide (GRP), GRP and neuromedin B have been shown to have numerous actions in the CNS, gastrointestinal tract and on growth. However, the role of the peptides in various physiological processes has remained unclear because of the lack of potent antagonists. Recent in vitro studies have described four different classes of bombesin receptor antagonist, some of which are active in the nanomolar range and in vivo. Robert Jensen and David Coy describe recent insights into peptide structural determinants of biological activity. Evidence from structure-function studies have resulted in identification of some analogues that function as potent antagonists in all systems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist.

Reversal of somatostatin inhibition of insulin and glucagon secretion.

These studies were designed to elucidate the mechanism of inhibitory action of somatostatin (SRIF) on glucagon (IRG) and insulin (IRI) secretion. Studies were carried out in the unrecirculated isolated rat pancreas perfusion with arginine 19.2 mM and glucose 5.5 mM as stimulus primarily for IRG but also IRI secretion. The effects of excess Ca++ (15.2 mEq./L.) and excess K+ (12.8 mEq./L.) on IRG, IRI, and the SRIF-inhibited pancreas were studied. Ca++ excess in five perfusions strikingly stimulated IRG secretion (+92 per cent) but only stabilized IRI secretion compared with control perfusions. K+ excess (in seven perfusions) markedly inhibited IRG secretion (-39 per cent) while stimulating IRI secretion (+16 per cent). Restoration of normal concentrations of K+ resulted in a rebound of IRG to levels 120 per cent that of controls. SRIF, at concentrations from 0.1-20 ng./ml., produced inhibition of both IRG and IRI. In 11 perfusions, with SRIF at 10 ng./ml., IRG decreased more than IRI (-75.2 per cent IRG and -46.9 per cent IRI). In five perfusions, addition of Ca++ (15.2 mEq./L.) 10 minutes after SRIF was started resulted in a reversal of IRG inhibition to 69.4 per cent and IRI to 73.2 per cent of the arginine controls. The reversal by Ca++ of SRIF effect on IRG was greater at higher concentrations of Ca++, suggesting some form of competition. In four perfusions, excess K+ reversed SRIF-induced IRI inhibition to 79.6 per cent that of controls but had no effect on IRG inhibition. Studies in vitro with isolated islets revealed that SRIF (2 mug./ml.) inhibited 45Ca uptake of islets as did epinephrine (10(-5) M). It was concluded that SRIF-induced inhibition of hormone release appears related to an action on Ca++ uptake.

Characterization of a putative cellular receptor for HIV-1 transmembrane glycoprotein using synthetic peptides.

The transmembrane glycoprotein (gp41 or TM) of HIV-1 contains limited sequence similarity to TM of some immunosuppressive animal retroviruses. A specific HIV-1 TM sequence, denoted CS3, inhibits T-cell activation in vitro and antibody specific to CS3 has been linked to the absence of disease. CS3, when conjugated to human serum albumin (HSA) and labeled with fluorescein, binds specifically to CD4+ cell lines. Cross-linking of CS3-HSA to its binding activity on the CD4+ cell line RH9 reveals a putative subunit size of approximately 44 kD. Incubation of RH9 cells with CS3-HSA prior to addition of HIV-1 prevented HIV-1-mediated cell lysis and inhibited infection. These results suggest that the CS3 region of TM plays an important role in the pathogenesis of the AIDS virus, HIV-1.

Endogenous opiates: through 1978.

The discovery of receptors in the brain for opiates and the structure of the endogenous peptides for these receptors has led to an explosion of interest in this field. The present review is the first of an annual series. It summarizes many of the highlights of research with opiate peptides published with a date of 1978 or earlier.