The use of pigs as an animal model to evaluate the efficacy, potency and specificity of two growth hormone releasing factor analogues.

In 1982, Guillemin et al reported the isolation of the human (h) growth hormone (GH) releasing factor (GRF) from a pancreatic tumour in an acromegalic patient. Since then, work to develop potent GRF analogues has been widespread and the rat has been the main animal model used. The aim of the present study was to compare the efficacy, potency and specificity of two GRF analogues with those of the native GRF(1-29)NH(2)using pig (p) as the animal model. Two analogues, Al ([His(1), D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) and A2 ([D-Ala(2), Ala(8,9,15,17), D-Arg(29)] hGRF(1-29)NH(2)) were compared with the h or pGRF(1-29)NH(2). Five studies were designed using 28-48 kg BW growing barrows. Results showed that the two GRF analogues were more potent than the native GRF molecule, were highly specific, were active for long periods of time and were able to induce changes in body composition similar to those reported with GH or other GRF analogues. Because of the similarity between swine and human species with respect to the amino acid sequence of GRF and to the physiology, secretion and effects of GH, it can be proposed that the pig could be used as a pre-clinical animal model to study and test new GRF molecules over short and long periods of time.

Serum insulin-like growth factor binding protein-3 in the hypophosphatemic mouse: decreased activity and abnormal modulation by dietary phosphate.

The hypophosphatemic mouse, the murine homologue of X-linked hypophosphatemia, is characterized by renal defects in phosphate reabsorption and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) production and by an osteoblast dysfunction. In view of the potential importance of insulin-like growth factors (IGFs) in the regulation of these processes and the role of IGF-binding proteins (IGFBPs) as modulators of IGF action, we asked whether Hyp mice have alterations in IGFs or IGFBPs. Using specific radioimmunoassays and Western ligand blot analysis, we evaluated serum levels of IGFs (IGF-1 and IGF-II) and IGFBPs, respectively, in normal and Hyp mice. We also examined the effect of dietary phosphatase on these parameters. Serum levels of IGF-1 and IGF-II in Hyp mice were not significantly different from those in normal mice, but IGFBP-3 levels were significantly lower (70% of normal, p < 0.05) in the mutant strain. The other IGFBP species appear unchanged. Phosphate supplementation normalized serum phosphate levels in Hyp mice and elicited a significant decrease in serum IGF-I levels (23%, p < 0.05) and a further deduction in IGFBP-3 (22%, p < 0.02). Phosphate deprivation induced hypophosphatemia IGF-II. The present results indicate that the low serum IGFBP-3 activity in Hyp mice is not related to hypophosphatemia per se. Based on the documented effects of parathyroid hormone (PTH) on IGF-I and IGFBP-3, we propose that the secondary hyperparathyroidism displayed by Hyp mice and its exacerbation by phosphate supplementation may contribute to low IGFBP-3 levels in control Hyp mice and to the decreases in serum IGF-I and IGFBP-3 in phosphate-supplemented Hyp mice.

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

Dynamics of growth hormone responsiveness to growth hormone releasing factor in aging rats: peripheral and central influences.

The in vivo and in vitro dynamics of somatotroph responsiveness to rGRF (1-29) NH2 (rat growth hormone releasing factor) were evaluated in 2-, 4-, 8-, 12-, and 20-month-old male rats. In vivo, using pentobarbital-anesthetized animals, we observed that the rGH (rat growth hormone) responsiveness to 0.4 and 1.6 micrograms/kg rGRF started to decline at the higher dose in 12-month-old rats and was completely blunted at both rGRF doses in 20-month-old animals. In vitro, using freshly dispersed perifused pituitary cells, we also documented a decrease of rGRF-induced rGH secretion in 12- and 20-month-old rats. Moreover, as the animals aged, the rGRF-induced rGH secretion was differentially affected by the inhibiting action of somatostatin (p less than 0.001), suggesting a loss of pituitary sensitivity to somatostatin in the presence of a high concentration of rGRF. The pituitary rGH content increased until rats reached 12 months of age, but was diminished in 20-month-old rats. In contrast, the pituitary somatostatin content increased twofold in 20-month-old rats as compared with younger rats. The hypothalamic somatostatin content was highest in 8-month-old rats and only slightly diminished in 20-month-old animals. Finally, plasma insulin-like growth factor I concentrations were highest in 8-month-old rats and lowest in 20-month-old animals. Altogether, these results indicate that the physiological loss of somatotroph responsiveness associated with the process of aging starts around 12 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosteroids: regulatory mechanisms in male rat brain during heterosexual exposure.

Pregnenolone (delta 5-P) and dehydroepiandrosterone (DHEA) were measured in the limbic system of young adult male rats (M) exposed for several days to the scent of cycling females but in absence of sexual contacts (M/F), and compared to levels obtained in males similarly exposed to other males (M/M). delta 5-P was highest in the olfactory bulbs of M/M, as compared to other regions of the limbic system. It decreased greater than 50% in M/F olfactory bulbs, but was identical in the olfactory tubercles, the amygdalas and the hypothalamus of M/M and M/F, as well as in the plasma, the adrenals and the spleen (taken as a representative non-endocrine organ). In comparison with M/M levels, DHEA selectively increased in the hypothalamus of M/F. These results demonstrate very different steroid concentrations in different regions of the brain, and they reveal their selective and eventually opposite changes upon heterosexual exposure. Therefore, they suggest regulatory mechanisms specific to various parts of the brain which are not directly related to the hormonal levels in the blood, and which could be part of the response to still undefined signals emitted by animals of the other sex.

Growth hormone-releasing factor from ovine and caprine hypothalamus: isolation, sequence analysis and total synthesis.

Peptides with high intrinsic growth hormone releasing activity (growth hormone-releasing factor, GRF) were isolated from 2100 ovine and 2600 caprine (goat) hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration and reverse phase HPLC. Structural characterization of the 44 amino acid ovine peptide by gas-liquid phase sequencing and peptide mapping established its primary structure as Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser -Tyr-Arg-Lys-Ile-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met -Asn -Arg-Gln-Gln-GLy-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-NH2. Caprine GRF was found to possess the same sequence except for the replacement of the isoleucine residue in position 13 with valine and thus is identical to bovine GRF.

Rat hypothalamic growth hormone-releasing factor: isolation, sequence analysis and total synthesis.

Growth hormone-releasing factor (GRF) was isolated from acid extracts of approximately 35,000 rat hypothalami by means of immunoaffinity chromatography, gel filtration and two steps of reverse-phase HPLC. Amino acid analysis, gas-liquid phases sequencing and peptide mapping established that rat GRF is a 43 amino acid peptide with the amino acid sequence His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly- Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly- Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH, confirming the primary structure reported earlier (Spiess et al Nature 303, 532 (1983).

Isolation, primary structure, and synthesis of human hypothalamic somatocrinin: growth hormone-releasing factor.

The hypophysiotropic peptide, growth hormone-releasing factor (GRF), was isolated from human hypothalamic-hypophysial tissues by means of acid extraction, immunoaffinity chromatography, gel filtration, and two steps of reverse-phase high-performance liquid chromatography. Amino acid sequence determination using a gas-phase sequencer and reverse-phase liquid chromatography of the native peptide and its synthetic replicates showed its primary structure to be as follows: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln -Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser -Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH2, which is identical to that of the GRF recently isolated and characterized from a human pancreatic tumor that had caused acromegaly. Human hypothalamic GRF shows major homologies (93%, 89%, and 86%, respectively) when its primary structure is compared to that of the hypothalamic GRF from the porcine, bovine, caprine, and ovine species.

Isolation and characterization of the bovine hypothalamic growth hormone releasing factor.

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 500 bovine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analysis. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala -Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln -Gly-Ala-Lys-Val-Arg-Leu-NH2 using approximately 2 nmol of material.

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor.

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.

Human hypothalamic growth hormone releasing factor (GRF): evidence for two forms identical to tumor derived GRF-44-NH2 and GRF-40.

Human hypothalamic growth hormone-releasing factor (GRF) was purified by gel filtration and reverse-phase HPLC. Bioassay and two radioimmunoassays of different specificity revealed the presence of two major forms of GRF-activity which coelute with human pancreas GRFs, hpGRF-44-NH2 and hpGRF-40 previously characterized in pancreas tumors. The bioactive material coeluting with hpGRF-44-NH2 is recognized by two antibodies which are directed against the amidated COOH-terminal sequence and the central portion of the GRF-44 peptide. The bioactive GRF which coelutes with hpGRF-40 reacts only with the antibody directed against the central portion of hpGRF. These data strongly suggest that the human hypothalamus contains the same major forms of GRF that were identified in pancreas tumors responsible for acromegaly in the absence of a pituitary tumor.

Topographical study of the neurons containing hpGRF immunoreactivity in monkey hypothalamus.

Hypothalamic neurons producing growth hormone-releasing factor (GRF) have been characterized by immunohistochemistry in monkey hypothalamus, using an antiserum raised against hpGRF1-40, a peptide with GRF activity isolated from a human pancreatic tumor. Cell bodies with hpGRF immunoreactivity were found in arcuate and ventromedial nuclei. From these neurons, bundles of fibers innervate median eminence and appear to terminate in contact with portal vessels. In addition to median eminence, hpGRF immunoreactive fibers were found mostly in the anterior hypothalamus and the arcuate and ventromedial nuclei where they give perineuronal endings. These results correlate with earlier physiological data on hypothalamic control of growth hormone secretion and suggest that GRF is also involved in interneuronal relationships related or unrelated to neurohumoral control of pituitary secretions.

Regulation of neurotensin secretion in a mammalian C cell line: effect of dexamethasone.

We established in culture two colony clones of rMTC 44-2 cells, rMTC 44-2B and 44-2C which secrete substantially greater quantities of neurotensin (NT) than the parent cell line. We describe here the effects of the synthetic glucocorticoid, dexamethasone, on NT and cAMP release. Medium and intracellular levels of NT and cAMP were measured by specific RIAs. Long-term release experiments were performed in Dulbecco's Modified Eagle's Medium supplemented with 15% horse serum (DMEM). Short-term release experiments were performed in Krebs-Ringer-bicarbonate-glucose buffer (KRBG) supplemented with 1.0 mm Ca2+. Dexamethasone stimulated NT release and increased intracellular NT levels. The ED50 values for stimulation of NT release following 24 or 48 h incubation of cells in DMEM with dexamethasone were 5 X 10(-9) and 7 X 10(-9) M, respectively. Dexamethasone markedly enhanced intracellular levels of NT in rMTC 44-2 cells while it decreased cell growth. Cells pretreated with dexamethasone for 48 h released greater amounts of NT in response to Ca2+ (1.0 mM) with or without K+ (50 mM) or NE (10(-6) M) following a 10 min incubation with these substances in KRBG. This experimental paradigm was also used to measure the efflux of cAMP following a brief (10 min) exposure of cells to NE. We conclude that the rMTC 44-2B and 44-2C cells are useful tools for studying the effects of dexamethasone on the regulation of cell growth, as well as the secretion of NT and cAMP.

Regulation of neurotensin secretion in mammalian C cell lines: potassium and norepinephrine affect the rapid release of the peptide.

Calcitonin (CT) and neurotensin (NT) secreting cell lines (6-23,44-2) were established from a transplantable rat medullary thyroid carcinoma (rMTC). The 44-2 line was used to obtain two colony clones 44-2 B and C secreting 20-30-fold greater NT than CT. These cells were used to study the regulation of Ca2+-modulated NT secretion and to ascertain the role of K+ and NE in the rapid release of NT. Medium NT was measured by a specific RIA with the antiserum N-1-11. Secretion experiments were in replicate 35 mm dishes in Krebs-Ringer-bicarbonate buffer supplemented with 90 mg% glucose (KRBG). Ca2+ (0.5-4.0 mM) stimulated NT release in a dose-dependent manner with an ED50 of 2.0 mM. Ca2+ was required for NT release induced by the Ca2+ ionophore, ionomycin, also K+ (50 mM) and norepinephrine (NE). To determine the mode of NT release in the presence of control (1.0 mM Ca2+) or experimental conditions (Ca2+ 1.0 mM plus 10(-6) NE and/or 50 mM K+), 44-2 cells were incubated in KRBG using an experimental paradigm wherein medium was changed at 10-min intervals, and NT release was quantitated. NE stimulated release of NT by these cells and the amount of NT released with each repetitive pulse of NE remained constant throughout the experiment. K+ (50 mM) elicited a rapid release of NT in the first 0-10 min incubation and the amount of NT released into the buffer was greater than that measured with NE; however, in these experiments, the response to K+ declined progressively and reached basal values at 20-30 min. Our results show neither pulse stimulation nor continuous incubation of cells with NE affected the response of the cells to a subsequent challenge with K+. These results suggest the presence of differentially stimulated, releasable pools of NT in these cells. We conclude that these newly established 44-2 B and C cells provide a useful model to study the regulation of NT release.

Presence of somatostatin-28-(1-12) in hypothalamus and pancreas.

Acid extracts from rat pancreas and hypothalamus were analyzed for the presence of the antigenic determinant corresponding to the NH2 terminus of somatostatin-28 (SS28), using an antiserum directed against amino acids 1 to less than or equal to 11 of the SS28 molecule. On gel permeation chromatography the majority of the immunoreactive material from each tissue extract eluted in one zone compatible with a peptide of 1250 daltons. Purification of this immunoreactive material by reverse-phase HPLC and cation-exchange chromatography yielded two immunoreactive peptides from each tissue extract. The amino acid compositions of both peptides in pancreas and hypothalamus correspond to the fragment 1-12 of SS28. The more hydrophobic peptide from each tissue coeluted with synthetic SS28-(1-12) on HPLC, while the other one coeluted with synthetic SS28-(1-12)-amide. We conclude that the prosomatostatin fragment Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-OH is present in both rat hypothalamus and rat pancreas.

Isolation and chemical characterization of somatostatin-28 from rat hypothalamus.

A peptide with somatostatin-like immuno- and bioactivity has been isolated from an aqueous extract of 96 800 rat hypothalamic by means of immunoaffinity chromatography, gel filtration and reverse-phase high-performance liquid chromatography (HPLC). Chemical characterization by amino acid analysis, tryptic peptide mapping and retention behavior in two HPLC system showed that the peptide was indistinguishable from somatostatin-28 previously characterized from several species. This evidence suggests that rat hypothalamic somatostatin-28 is identical in structure to porcine and ovine somatostatin-28.