Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).     

Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Influence of chronic treatment with the growth hormone secretagogue Ipamorelin,in young female rats: somatotroph response in vitro

Growth hormone (GH) is secreted in the anterior pituitary gland by the somatotroph cells. Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the composition of the somatotroph cell population and on somatotroph GH content, an in vitro analysis was performed of the percentage of somatotroph cells (% of total), the ratio of different GH cell types (strongly/weakly-staining) and individual GH content, in pituitary cell cultures obtained from young female rats receiving Ipamorelin over 21 days (Ipamorelin group) and the effects were compared with those of GHRH (GHRH group) or saline (saline group). The ultrastructure of somatotroph cells did not change, but the volume density of secretion granules was increased (P<0.05) by previous in vivo Ipamorelin or GHRH treatment. In 3-day basal pituitary cell monolayer cultures, the percentage of somatotroph cells showed no modifications between groups, nor was there any change in the ratio of strongly/weakly immunostaining GH cells. In the Ipamorelin group alone, in vitro treatment with Ipamorelin (10(-8) M), or GHRP 6 (10(-8) M), or GHRH (10(-8) M) for 4 hours, increased the percentage of somatotroph cells, without modifying the ratio of strongly/weakly immunostained GH cells. Basal intracellular GH content in somatotroph cells over 4 hours was lower in the Ipamorelin group and the GHRH group than in the saline group. Only in the Ipamorelin group did Ipamorelin (10(-8) M), GHRP 6 (10(-8) M) and GHRH (10(-8) M) prompt increased intracellular GH content. These data suggest that, at least in the young female rat, the GHS Ipamorelin is able to exert a dynamic control effect on the somatotroph population and on GH hormone content.     

Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation

The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.     

Growth hormone secretagogues in critical illness

Alterations within the somatotropic axis occurring during the course of critical illness follow a biphasic pattern. The initial stress response consists of activated growth hormone (GH) release whereas circulating levels of GH-dependent insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 fall and IGFBP-1 concentrations rise. In contrast, in the chronic intensive care-dependent phase of severe illness, pulsatile GH secretion substantially decreases whereas the non-pulsatile fraction remains relatively elevated, resulting in an abnormally flat GH secretory pattern and low-normal mean nocturnal GH serum concentrations. Specifically the reduced amount of GH released in pulses is found to be related to low circulating levels of IGF-I, IGFBP-3 and acid-labile subunit (ALS), which suggests that a relative hyposomatotropism may participate in the pathogenesis of the wasting syndrome distinctively in the chronic phase of critical illness. The relative hyposomatotropism seems at least in part of hypothalamic origin since the whole somatotropic axis has been found to be very responsive to continuous infusion of GH releasing peptide (GHRP), administered alone or in combination with GH releasing hormone (GHRH), as evidenced by reactivated pulsatile GH secretion followed by substantial increases in circulating levels of IGF-I, IGFBP-3 and ALS. GHRH alone, however, is unable to exert the same effect, which may point to an underlying reduced availability of the endogenous ligand for the GHRP receptor. The presence of considerable responsiveness to restored endogenous pulsatile GH secretion using GHRPs not only further delineates the distinct pathophysiological paradigm of the chronic phase of critical illness, as opposed to the acute phase, which is thought to be primarily a condition of GH resistance, but may also have important therapeutic consequences. Recent data revealed that this novel strategy evokes metabolic improvement related to the balanced endocrine responses. Whether GH secretagogues also enhance clinical recovery of protracted critically ill patients remains to be elucidated.     

Bioactivity of growth hormone releasing hormone (1-29) analogues after SC injection in man

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.     

Evaluation of the growth-hormone-releasing hormone test in short normal and growth-hormone-deficient children

Growth-hormone-releasing hormone (GHRH) tests were performed once [GHRH(1-29)NH2, 1 microgram/kg] or on 2 consecutive days [GHRH(1-44)NH2, 1 and 2 microgram/kg administered in random order] in 27 children with idiopathic, isolated growth hormone (GH)-deficiency and in 49 short normal children, all clinically prepubertal. No differences in GH release were found between the tests performed on the 1st and 2nd day or according to GHRH dose or sex, both in GH-deficient and control children. 80% of GH-deficient and 87% of control children responded (GH peak greater than 10 ng/ml) to GHRH(1-29)NH2, and 65% of GH-deficient and all control children to GHRH(1-44)NH2. No differences in GH release were found between GH-deficient GHRH responders and control children. 17% of GH-deficient and 10% of control children responded only to one of the two tests performed on 2 consecutive days; the lack of responsiveness was unrelated to GHRH dose and sequence of GHRH administration (1st or 2nd day). The GHRH test does not seem to be a reproducible test for the evaluation of GH release, nor is it useful to differentiate GH-deficient GHRH responders from short normal children.     

Nonpeptidyl somatostatin agonists demonstrate that sst2 and sst5 inhibit stimulated growth hormone secretion from rat anterior pituitary cells.

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat.

Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution conformation of Substance P antagonists-[D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP by CD, NMR and MD simulations

Substance P (SP) is an important neuropeptide involved in pain transmission and induction of inflammation. Its antagonists are being extensively investigated for their non-narcotic analgesic and anti-inflammatory activity. With a view towards better understanding the structural requirements of these analogs for efficient interaction with the SP receptor, the conformation of three SP antagonists [D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP has been studied by CD, NMR and molecular dynamics (MD) simulations. All three peptides exhibit a high dependence of structure on the solvent. The molecules tend to adopt beta-turns in solvents like DMSO and H2O and form helices in a hydrophobic environment. A direct relation between the helix forming potential of these antagonists with their receptor binding potency has been observed.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

Effect of the bombesin receptor blockers [Leu13, psi CH2NH-Leu14]bombesin and N-pivaloyl GRP(20-25) alkylamide (L 686,095-001C002) on basal and neuromedin C-stimulated PRL and GH release in pituitary cell aggregates.

Perifusion of rat anterior pituitary cell aggregates, cultured in estrogen-supplemented serum-free medium with 1 nM of the bombesin (BBN)-like peptide, neuromedin C (NMC), significantly stimulates GH and PRL release. This effect is dose-dependently inhibited by the BBN receptor blocker L 686,095-001C002 [an N-pivaloyl-gastrin-releasing-peptide(20-25) alkylamide]. The IC50 was 0.20 nM in the case of the GH response and 0.16 nM in the case of the PRL response. The antagonist has no effect on basal PRL or GH release. [Leu13, psi CH2NH-Leu14]BBN (psi BBN) displays an IC50 of 0.41 microM for inhibiting the GH response and 0.36 microM for inhibiting the PRL response to NMC. At a concentration of 0.5 microM or 5 microM, however, the latter antagonist stimulates PRL and GH release when perifused alone. This stimulatory effect is dose dependent, augments when aggregates are cultured in 1 nM E2 (as is the case for NMC) and is abolished by 2 nM L 686,095-001C002. It is concluded that L 686,095-001C002 is a potent and pure antagonist of pituitary BBN receptors mediating PRL and GH release, whereas psi BBN is a relatively weak antagonist with considerable partial agonist activity.     

Interactions of substance P antagonists with serotonin in the mouse spinal cord

Administered intrathecally (IT) to mice, the putative substance P antagonist [D-Pro2,D-Trp7,9-substance P (DPDT) blocked substance P- and serotonin-induced reciprocal hindlimb scratching with ID50 values of 4.6 (2.9-6.9) and 3.0 (1.9-4.8) micrograms, respectively. The duration of this antagonistic effect was 90-120 min. In contrast, DPDT did not block bombesin-, somatostatin-, glycine- or glutamate-induced scratching. These data indicate that DPDT is an effective antagonist of serotonin-induced behaviors in the mouse spinal cord. Phenoxybenzamine (IT) also blocked substance P- and serotonin-induced scratching. Its onset of action was more rapid for serotonin than for DPDT implying differences in agonist-induced receptor activation. Methysergide (IT) blocked serotonin-induced scratching [ID50 = 0.7 (0.3-1.5) micrograms], but not substance P-induced scratching. Similar to DPDT, [D-Arg1,D-Trp7,9,Leu11]-substance P, [des-Arg1,D-Pro2, D-Trp7,9]-substance P(2-11) and [D-Pro4,D-Trp7,9]-substance P(4-11) blocked substance P and serotonin-induced scratching. In contrast, [D-Pro2,D-Phe7,D-Trp9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P(4-11) blocked only substance P-induced scratching. Thus, some, but not all putative substance P antagonists may also be behavioral antagonists of serotonin in the mouse spinal cord.     

Evaluation of the biological potency of new agmatine analogs of growth hormone-releasing hormone in the bovine.

Four new growth hormone-releasing hormone (GHRH) analogs with C-terminal agmatine were compared with the parent human GHRH(1-29)NH2 fragment to assess their abilities to increase serum concentrations of growth hormone (GH) in the bovine. The four analogs were: [D-Ala2, Nle27] GHRH(1-28)Agm (JG-73); [desNH2-Tyr1, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-51); [desNH2-Tyr1, Ala15, D-Lys21, Nle27] GHRH(1-28)Agm (MZ-2-75); and [desNH2-Tyr1, D-Lys12,21, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-87). The special characteristic of all four GHRH analogs is that arginine was replaced by agmatine (Agm) in Position 29. Five pregnant Holstein cows received these peptides subcutaneously at the following doses: 0.0156, 0.0625, 0.25, 1, and 4 micrograms/kg body wt. Each cow received each analog-dose combination according to a 5 x 5 Greco-Latin square design repeated for the 5-week treatment. Each cow also received saline vehicle only at the end of the 5-week treatment. Blood samples were collected from 30 min before until 360 min after treatment injection. Total area under the GH response curves for the 6-hr sampling period for each dose of each GHRH analog was compared. There was a linear dose-dependent GH release in response to hGHRH(1-29)NH2 and its four GHRH(1-28)Agm analogs. At the dose of 0.25 micrograms/kg, two GHRH analogs, JG-73 and MZ-2-75, stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). No differences were seen at the two lowest doses, 0.0625 and 0.156 micrograms/kg. When both total area under the GH response curves and GH peak amplitudes for each treatment were averaged for all doses, JG-73 and MZ-2-75 stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). In summary, three GHRH(1-28)Agm analogs, JG-73, MZ-2-75, and MZ-2-51, were found to be 11.8, 11.3, and 6.5 times more potent, respectively, on a weight basis, than hGHRH(1-29)NH2 in stimulating the release of GH in cows.     

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.     

Reduction induced by substance P of the initial accumulation of myo(3H)inositol in acinar cells of the parotid gland in rats

The stimulation of rat parotid acinar cells by substance P(SP) resulted in a significant reduction in the initial accumulation of cytosolic myo[3H]inositol and in the initial labelling of phosphoinositides. The SP-induced reduction was concentration-dependent, the EC50 of SP was 5.8 +/- 2.5 nM. Spantide, [D-Arg1, D-Trp7,9, Leu11]SP, a SP antagonist, used at a concentration of 10(-5) M, competitively shifted the dose-response curve of SP. The pharmacological analysis of the effects of several tachykinins and analogues, suggests the implication of NK1 receptors (specific receptor of SP).