Rat Growth Hormone-releasing Factor Research Articles

Long-Term Alterations in Growth Hormone and Insulin Secretion after Temporary Dietary Protein Restriction in Early Life in the Rat

Protein malnutrition early in life stunts subsequent physical growth in both humans and rats, but the mechanism(s) is unknown. To test the hypothesis that temporary early life dietary protein restriction produces long-term alterations in the growth hormone (GH) neuroendocrine axis, we examined the effects of 3 wk of exposure to dietary protein restriction in male rats postweaning (3-6 wk of age) on spontaneous and GH-releasing factor (GRF)-stimulated GH secretion at 12 wk of age. In comparison to rats weaned onto a normal diet (23% protein), rats weaned onto a low (4%) protein diet failed to catch up growth when transferred to the normal diet between 6 and 12 wk of age. Spontaneous 6-h GH secretory profiles of adult rats fed the low protein diet early in life showed a 41% reduction in mean GH peak amplitude and a significant suppression in overall mean 6-h plasma GH concentrations (37.5 +/- 4.5 versus 56.9 +/- 5.9 micrograms/L; p < 0.02). The magnitude of the GH response to 1 microgram of rat GRF(1-29)NH2 i.v. challenge was augmented during the GH trough period in these rats (165.7 +/- 30.4 versus 43.9 +/- 17.6 micrograms/L; p < 0.01). Although basal plasma IGF-I levels and glucose tolerance of protein-deprived rats were normal at 12 wk of age, the insulin response to ip glucose was severely blunted [insulin integrated area under the curve: 303.0 +/- 32.7 versus 778.3 +/- 105.0 pmol/L/75 min; p < 0.01]. These results demonstrate that temporary protein malnutrition early in life 1) blunts spontaneous pulsatile GH secretion, 2) augments GH responsiveness to GRF challenge, and 3) reduces the insulin secretory response to glucose in adulthood. Our findings suggest that dietary protein in early life is an important determinant for CNS control of GH secretion as well as for the development of pancreatic beta-cell sensitivity to glucose. Such alterations in the GH neuroendocrine axis, together with the subnormal insulin secretion, likely contribute to the lack of catch-up growth in this model.

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor does not disturb the course of sexual maturation and fertility in the female rat.

The importance of normal GH secretion for the onset of sexual maturation is a subject of controversy. Also, the need to achieve a minimal body size or body fat content has been postulated to be of importance for determining the timing of the onset of puberty. To evaluate the importance of GH secretion on the onset of sexual maturation in the female rat, GH deprivation has been induced by treating prepubertal rats with antirat GRF serum to passively immunize these animals against GRF. Chronic administration of anti-GRF serum produced in all series an impressive reduction in growth rate (from 5 to 2 g/day), resulting in a body weight averaging 50-60% the normal value at 50 days of life. Despite this deficit in growth, sexual maturation, as established by vaginal opening and first estrous cycles, occurred at the normal age in three of four series of rats; in one series, however, sexual maturation was delayed by 4 days, but thereafter, all parameters indicated that the gonadotropic axis was normally activated. In one series, fertility was tested at 59 days of age in females with a body weight corresponding to 51% of the control weight; these females conceived and delivered a reduced number of pups (9.4 +/- 0.7 instead of 14.2 +/- 0.8 in control dams), but the pups were of normal size. In a second experimental approach, the effect of GH deprivation was evaluated in a model of late sexual maturation obtained by severe food restriction followed by a switch to ad libitum feeding. Severe food restriction initiated at approximately 28 days, when the body weight was 75 g, drastically reduced the growth rate and completely prevented sexual maturation. A switch to ad libitum feeding at 50 days provoked an important compensatory growth and the occurrence of sexual maturation 4 days later. Passive immunization against GRF during this recovery phase did reduce the growth rate, but did not delay sexual maturation. Plasma insulin-like growth factor-I (IGF-I) secretion was very low in food-restricted rats and in each situation with induced GH deprivation. During food restriction, plasma IGF-binding protein-3 (IGFBP-3) and to a lesser extent IGFBP-1 were decreased, and IGFBP-2 was increased; after switching to ad libitum feeding, plasma levels of IGFBP-2 normalized, but levels of IGFBP-1 and IGFBP-3 remained low in the face of normalized plasma IGF-I levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor does not disturb the course of sexual maturation and fertility in the female rat

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor does not disturb the course of sexual maturation and fertility in the female rat

Molecular cloning of cDNA encoding the precursor for hamster hypothalamic growth hormone-releasing factor

The structure of rat and mouse growth hormone-releasing factor (GRF) peptide and precursor shows considerable divergence from that of the human counterpart and also within rodents themselves. To study such structural divergence in another rodent, we cloned a cDNA encoding the GRF precursor from golden hamster. The hamster GRF (haGRF) cDNA clone had an open-reading frame that predicts a haGRF precursor protein with 107 amino acids. The haGRF precursor bore greater overall homology (82%) to the human than the same rodent homologue (58-64%) and contained two processing sites identical to the human sequence that would generate mature haGRF peptide. Furthermore, the haGRF peptide, like human but unlike rat or mouse GRF, consisted of 44 amino acids and also had greater homology to the human (89%) than the rodent sequence (64-75%), conserving a Tyr residue at the N-terminus and an amidated Leu residue at the C-terminus. Thus, both haGRF precursor and peptide are structurally more related to those of human than of other rodents, suggesting that rodent GRF precursor diverged from the human sequence at differential rates within the species.

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences

Native human GRF(1–44)-NH 2 (hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala 2 and Asp 3. The hGRF44 is also subject to chemical rearrangement (Asn 8 → Asp 8, β-Asp 8 via aminosuccinimide formation) and oxidation [Met 27 → Met(O) 27] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop long-acting GRF analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Ala 15 substitution (for Gly 15), previously demonstrated to enhance receptor binding affinity, would be predicted to improve GRF analogue potency. Substitution of [His 1,Val 2]-(from the mouse GRF sequence) for [Tyr 1,Ala 2]-(human sequence) in [Ala 15,Leu 27]hGRF(1–32)-OH analogues completely inhibited (24-h incubation) DPP-IV cleavage and greatly increased plasma stability in vitro. Additional substitution of Thr 8 (mouse GRF sequence), Ser 8 (rat GRF sequence), or Gln 8 (not naturally occurring) for Asn 8 (human GRF sequence) resulted in analogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resistant hGRF(1–32)-OH analogues, containing His 1, Val 2, Thr/Ser/Gln 8, Ala 15, and Leu 27 replacements, were then bioassayed for growth hormone (GH)-releasing activity in vitro (rat pituitary cell culture) and in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1–32)-OH analogues. In vitro, these analogues were approximately threefold more potent than hGRF44, whereas in vivo they were eleven- to thirteenfold more potent. As the in vitro results reflect only receptor affinity and signal transduction, the increment in potency observed in vivo is likely due to the increased biological half-life of these analogues (i.e., the result of decreased enzymatic and chemical decomposition such that more bioactive peptide is available per unit time).

Timing of exposure to somatostatin relative to growth hormone-releasing factor dictates the rat anterior pituitary cell growth hormone response.

The temporal interactions of rat GH-releasing factor (GRF) and somatostatin (SRIF) on the secretion of GH from perifused rat anterior pituitary cells have been studied. SRIF and GRF were employed at concentrations in a range close to levels reported in the hypophysial circulation of the rat. GH secretion was inhibited by pulses of 1 nmol SRIF/l (6 min) or 0.3 nmol SRIF/l (6 or 20 min). No rebound GH stimulation was observed. Exposure of cells to 6-min pulses of 0.3 nmol rat GRF/l repeated three times resulted in rapid stimulatory responses which became attenuated. Concomitant exposure to 0.3 nmol SRIF/l during a GRF pulse resulted in transient inhibition followed by a delayed and enhanced GH response, measured as the area under the curve but not peak height, whereas continuation of SRIF after a GRF pulse abolished the GH response. Exposure to 0.3 nmol SRIF/l prior to but not during a GRF pulse did not delay or inhibit the GH response. The area under the curve was increased under these conditions because of the lowered baseline in cells perifused with SRIF. SRIF alters somatotroph responsivity to equimolar concentrations of rat GRF and under different temporal conditions and can inhibit, enhance or delay GH secretion.

Proteolytic degradation of rat growth hormone-releasing factor(1–29) amide in rat pituitary and hypothalamus

The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1–29)NH 2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1–29)NH 2 (10 μM) was incubated (0–120 min, 37°C) in the presence of a pituitary (237 ± 51μg protein/ml) or hypothalamus homogenate (576 ± 27μg protein/ml). Using analytical high pressure liquid chromatograpy (HPLC), apparent half-lives of (22 ± 3min and25 ± 4min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys 21-Leu 22 (trypsin-like cleavage site), Leu 14-Gly 15 and Tyr 10-Arg 11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1–21)OH while TPCK blocked the cleavage of Leu 14-Gly 15. The low affinity of fragment (1–21)NH 2 for pituitary GRF binding sites suggests that hydrolysis of the Lys 21-Leu 22 bond inactivates rGRF(1–29)NH 2 in this target tissue.

Somatostatin reduces transcription of the growth hormone gene in rats.

To examine whether somatostatin (SS) exerts influences on the steady state levels of GH-releasing factor (GRF), the effect of SS on GH gene transcription was examined in rats. This approach was used because it has been shown that GRF stimulates GH gene transcription independent of GH release, and SS does not inhibit basal or GRF-stimulated GH gene transcription. Therefore, it is assumed that an effect of SS on GH gene transcription would be mediated by a change in GRF levels. Adult female Sprague-Dawley rats were provided with right atrial cannulae. Studies were performed using unanesthetized rats. Pituitary GH gene transcription was measured by transcription assay. An iv administration of antiserum to rat GRF 150 min previously significantly decreased GH gene transcription compared with that in control rats given normal goat serum. A continuous infusion of SS (300 micrograms/kg.h) via the cannula for 150 min significantly decreased GH gene transcription compared with that in control rats receiving 0.9% NaCl. When GRF (3 micrograms/kg.h) was given simultaneously with SS (300 micrograms/kg.h), GH gene transcription increased significantly compared with that in rats receiving SS infusion alone. After the withdrawal of SS infusion, GH gene transcription rapidly and significantly increased. The data suggest that SS reduces the steady state levels of GRF.

Simultaneous solubilization of high-affinity receptors for VIP and glucagon and of a low-affinity binding protein for VIP, shown to be identical to calmodulin

Anion-exchange chromatography of solubilized pig liver cell membranes on DEAE-Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP ( K d 1.5 ± 0.6 nM and B max 1.3 ± 0.4 pmol mg ). The order of potency for VIP-related peptides to inhibit [ 125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP-γ-S inhibited [ 125I]vip binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [ 125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [ 125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon ( K d 0.08 ± 0.03 nM and B max 0.31 ± 0.01 pmol mg ). A low-affinity VIP binding protein (IC 50 0.7 μM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10–28) > secretin > PHI. This rGRF-preferring protein (18 kDa) was purified and had a partial amino-acid sequence identical to that of calmodulin. Its [ 125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co-solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP-binding calmodulin.

Growth hormone-releasing factor regulation by somatostatin, growth hormone and insulin-like growth factor I in fetal rat hypothalamic-brain stem cell cocultures.

Information about growth hormone-releasing factor (GRF) regulation by somatostatin, GH and IGF-I is scarce and controversial. This could be due to the in vivo interactions among these signals and the lack of models for individualizing the action of one of them from the others upon GRF regulation. The aim of the present work was to study GRF regulation by these signals, using primary fetal rat hypothalamic-brain stem cell cocultures. Coculturing of these two cytotypes increases hypothalamic immunoreactive rat GRF (IR-rGRF) content in cells by 45% and in media by 36%. The effect of SS on GRF in cocultures was examined by using a multiple approach: (1) depleting endogenous SS by adding 1 mM cysteamine (CSH); (2) blocking endogenous SS by incubation with SS antiserum, and (3) incubating with synthetic SS14 at different concentrations and exposure periods. 1 mM CSH depleted IR-SS content (pg/plate, mean +/- SE) in cells (CSH-treated: 68 +/- 8 vs. control: 322 +/- 10, p < 0.01) and media (CSH-treated: 211 +/- 15 vs. control: 880 +/- 70; p < 0.01). In the CSH-induced SS-depleted cultures, a slight reduction in the IR-rGRF content in cells was observed (CSH-treated: 93.5 +/- 4.5 vs. control: 111 +/- 6; p < 0.05), with no effect on media content. When SS antiserum was added to plates, there was a slight reduction in the IR-rGRF content in cells and media, but it was not significantly different from the controls. However, SS14 (10(-10)-10(-8) M) could not modify IR-rGRF content in media and cells. The GH effect on IR-rGRF was studied in the absence of CSH and in CSH-induced SS-depleted cultures. GH (5 microM, 24 h) decreased (52%) the IR-rGRF content in media (GH-treated: 28.7 +/- 4.6 vs. control: 60.2 +/- 7; p < 0.01) without causing changes in cell content. In SS-depleted cultures, the inhibitory action of GH on media IR-rGRF was greater (62% decrease) (GH-treated: not detected, control 56 +/- 10; p < 0.01) and also affected IR-rGRF cell content (GH-treated: 64.3 +/- 7.3 vs. control: 160 +/- 9.6; p < 0.01). In the same experiments, GH increased IR-SS content in cells (GH-treated: 31.8 +/- 4.6 vs. control 20.9 +/- 0.5; p < 0.01) and in media (GH-treated: 413 +/- 7 vs. control: 286 +/- 9; p < 0.01). 1 mM CSH again depleted IR-SS content and abolished the GH stimulatory effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.

Inhibitory effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells: Involvement of pertussis toxin-sensitive G-proteins

The effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells of the rat medullary thyroid carcinoma cell line rMTC 6–23 was investigated. Intracellular cAMP accumulation as well as calcitonin secretion could be dose-dependently stimulated by rat growth hormone releasing factor (rGRF). The long-acting somatostatin analogue octreotide inhibited rGRF-stimulated cAMP accumulation and calcitonin secretion dose dependently but failed to block 8-bromo-cAMP-stimulated calcitonin secretion. The inhibitory effect of octreotide on rGRF-induced calcitonin secretion was partially abolished by pretreating the cells with pertussis toxin. The octreotide effect was not due to changes in the degradation of cAMP, as it was similarily seen in the presence of isobutylmethylxanthine. Thus we conclude that pertussis toxin-sensitive G-proteins are involved in the cAMP-mediated regulation of calcitonin secretion in C-cells.

Catabolism of rat growth hormone-releasing factor(1–29) amide in rat serum and liver

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1–29)NH 2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1–29)NH 2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1–29)NH 2 ( 30 μM) was incubated (0–120 min, 37°C) in serum ( 49 ± 8 mg prot./ml ). The rGRF(1–29)NH 2 ( 10 μM) was also incubated (0–120 min, 37°C) with liver homogenate ( 200 ± 6 μ g prot./ml ). Time course studies of rGRF(1–29)NH 2 disappearance showed apparent half-lives of 18 ± 4 min and 13 ± 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1–20)OH and (3–20)OH. A small amount of rGRF(12–29)NH 2, coeluting with rGRF(3–20)OH, was also found by sequencing. In the liver, rGRF(1–18)OH, (3–18)OH, and (1–10)OH were identified. The peptide bond Ala 2-Asp 3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg 11-Arg 12 and Arg 20-Lys 21 (trypsin-like cleavage site) in the serum, and Tyr 10-Arg 11 and Tyr 18-Ala 19 (chymotrypsin-like cleavage site) in the liver. The enzyme responsible for the cleavage of Ala 2-Asp 3 was sensitive to diprotin A, while those responsible for the cleavage of Tyr 18-Ala 19 and Arg 20-Lys 21 were sensitive to leupeptin. The low affinity of fragments (1–20)NH 2 and (12–29)NH 2 for pituitary GRF binding sites suggests that hydrolysis of Arg 11-Arg 12, Tyr 18-Ala 19, and Arg 20-Lys 21 bonds inactivates rGRF(1–29)NH 2 in this target tissue.

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Effect of Antiserum to Rat Growth Hormone (GH)- Releasing Factor on Physiological GH Secretion in the Female Rat

To further study the physiological role of GH-releasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH secretion in the normal female rat. Two groups of six conscious female rats were passively immunized with either nonimmune rabbit serum (NRS) or antirat GRF serum via a chronic indwelling atrial catheter. The secretory profiles of GH were observed by collecting blood samples at 15-min intervals for 1 h before and 4 h after administration. The NRS-treated rats showed a characteristic female pattern of spontaneous GH secretion. GH pulses were of low amplitude (mean +/- SEM, 26.8 +/- 2.4 ng/ml) and occurred irregularly at a frequency of 4.2 +/- 0.2/5 h, while interpeak through levels of GH were relatively high, with nadir values of 8.6 +/- 0.7 ng/ml. Synthetic rat GRF, given iv at a dose of 1 microgram/kg BW after the last blood sampling, stimulated GH release to a peak level of 153 +/- 37 ng/ml in the control rats. Administration of GRF antiserum caused a profound suppression of both pulse and trough components of GH secretion. This effect occurred rapidly, within 15 min after injection of antiserum, and GH secretion decreased uniformly to very low levels (3.4 +/- 0.1 ng/ml), with little or no fluctuation throughout the observation period. GRF antiserum also abolished the synthetic rat GRF-induced GH release, indicating sufficient potency of immunoneutralization. These results demonstrate that both GH pulses and troughs are dependent upon hypothalamic GRF in normal female rats, thereby substantiating earlier observations in male rats which demonstrated the physiological role of GRF in GH secretion.

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.

Adenylyl cyclase stimulation by VIP in rat seminal vesicle membranes

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in membranes from rat seminal vesicle. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 μM. The stimulation of adenylyl cyclase by VIP was time and temperature dependent. The response was linear with time up to 15 min at 30°C. Half-maximal adenylyl cyclase activation (in the presence of 10 μM GTP) was achieved at 3.0 nM VIP. The enzyme activity increased about 150% with respect to basal values at the maximal VIP concentration tested (1 μM). The relative potency of peptides upon stimulation of adenylyl cyclase activity was: VIP > helodermin > peptide histidine isoleucinamide > rat growth hormone-releasing factor. Other agents like GTP (0.1 mM), GppNHp (0.1 mM), forskolin (0.1 mM) and sodium fluoride (10 mM) increased the adenylyl cyclase activity 1.8-, 4.4-, 6.7- and 2.4-fold, respectively. Taken together, the presence of VIP in nerve terminals innervating the seminal vesicle of rats and the existence of VIP receptors coupled to adenylyl cyclase strongly suggest a physiological role for this neuropeptide in the modulation of seminal vesicle cell function.

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50–60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20–240 min after the start of the perifusion and was stimulated by depolarization induced by high K + concentration. The release of GRF was inhibited by somatostatin at concentrations of 10 −11 to 10 −8 M with maximum inhibition to 52.5% of the basal release at a concentration of 10 −9 M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.

Disappearance half-life times of exogenous and growth hormone-releasing factor-stimulated endogenous growth hormone in normal rats

This study was performed to determine the disappearance half-life times of endogenous and exogenous rat GH in conscious normal rats and to compare these with the decay characteristics of GH at the end of spontaneous normal bursts. The endogenous half-life was determined in five rats by giving an i.v. injection of rat GH-releasing factor followed after 10 min by an i.v. injection of long-acting somatostatin analogue (octreotide) and taking blood samples for 85 min. The half-lives (mean +/- S.E.M.) were 3.4 +/- 0.4 min and 13.2 +/- 1.1 min for the first and second exponential respectively as determined by bi-exponential analysis. The exogenous GH half-life was determined in ten rats by giving i.v. octreotide followed after 10 min by i.v. rat GH and sampling for 85 min. The half-lives of exogenous GH were 3.3 +/- 0.2 min and 17.5 +/- 1.4 min by bi-exponential analysis and there was no significant difference between the half-lives of endogenous and exogenous GH. The half-life of the decline of GH levels at the end of spontaneous bursts in nine rats was 14.4 +/- 0.9 min, not different from the half-life of endogenous GH, the secretion of which was terminated by octreotide. This suggests that the end of spontaneous GH bursts is marked by sudden cessation of GH release and may provide an indication of the rapidity of change in the levels of the underlying hypothalamic hormones which control GH release.

A comparison of the biological activities of authentic rat GRF(1–43)OH with the analogue rat GRF(1–29)NH2

The purpose of this study was to characterize the biological activity of the synthetic rat growth hormone releasing factor analogue rGRF(1-29)NH2 and to compare its action on growth hormone (GH) release to that of authentic rGRF(1-43)OH. We first compared the concentration-response characteristics of the two peptides in static incubation, and then examined the reversibility and repeatability of the GH response in a perifusion system. Authentic rGRF(1-43)OH was significantly more potent in static incubation (EC50 = 3 x 10(-11) M) than the analogue (5 x 10(-11) M), whereas the reverse held true in perifusion. The shapes of the GH responses were similar for both peptides in the perifusion system. However, while the GH response to authentic rGRF was repeatable, the prior administration of rGRF(1-29)NH2 significantly reduced (greater than 50%) the GH response to the subsequent administration of either rGRF(1-29)NH2 or rGRF(1-43)OH. Thus authentic rGRF and the synthetic fragment may have different actions at the level of the GRF receptor or at a postreceptor (second messenger) step.