Synthetic Human Growth Hormone-releasing Research Articles

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.

Solution structure of human growth hormone‐releasing factor fragment (1–29) by CD: Characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.

The secretion of human growth hormone stimulated by human growth hormone releasing factor following severe cranio-cerebral trauma

Patients suffering from severe cranio-cerebral trauma show alterations of the secretory patterns of thyroid stimulating hormone (TSH) and human growth hormone (HGH) which may be of prognostic significance. We studied 10 patients following severe brain injury and prospectively compared a new synthetic human growth hormone releasing factor (HGHRF) test with the thyrotropin releasing hormone (TRH) test. On admission, all patients had a Glasgow Coma Scale score of 3 or 4. All patients had a low T3 syndrome. In the patients who died the TSH response after stimulation with TRH was also absent. In the patients who survived a significant TSH increase was observed (p less than 0.05). In comparison to the patients who died those who survived showed a significant (p less than 0.001) HGH increase after HGHRF stimulation. This test might be useful as an additional tool in establishing early prognosis in patients with severe brain injury.

Plasma Growth Hormone, Insulin-Like Growth Factor-I, and Milk Production Responses to Exogenous Human Growth Hormone-Releasing Factor Analogs in Dairy Cows.

Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5 ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmunoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3 ng/ml at about 5 h from 1.4-1.7 ng/ml at 0 h (just before injection), and returned to the level before injection after 10-12 h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6 h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4 ng/ml at 20 h from 43.7-46.4 ng/ml at 0 h. The IGF-I concentration at 24 h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2 h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline injected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows.

Effects of growth hormone-releasing hormone on the secretion of islet hormones and on glucose homeostasis in lean and genetically obese-diabetic (ob/ob) mice and normal rats

The effect of synthetic human growth hormone-releasing hormone(1-40) (hGHRH-40) on the function of the endocrine pancreas and on glucose homeostasis in lean and genetically obese-diabetic (ob/ob) mice and normal rats has been examined. The addition of 1 mumol hGHRH-40/1 to incubated islets from normal lean mice increased insulin release by 90 and 37% at 5.6 and 16.7 mmol glucose/l respectively. Lower concentrations of hGHRH-40 did not affect insulin release. hGHRH-40 (1 mumol/l) increased pancreatic polypeptide release by 50% at 5.6 mmol glucose/l. A range of concentrations of hGHRH-40 (1 nmol/l-1 mumol/l) reduced glucagon release by 42-73% at 5.6 mmol glucose/l, and by 38-70% at 16.7 mmol glucose/l. Somatostatin release was increased (eightfold) by 1 mumol hGHRH-40/1 at 5.6 mmol glucose/1, but at 1 nmol hGHRH-40/1 somatostatin release was reduced (by greater than 50%). At 16.7 mmol glucose/litre 0.01-1 mumol hGHRH-40/1 increased somatostatin release (three- to fourfold), but 1 nmol hGHRH-40/1 produced a reduction of 50%. In vivo, administration of hGHRH-40 (50 micrograms/kg body weight i.p.) to fasted lean and ob/ob mice did not alter basal plasma concentrations of glucose and insulin, or the glucose and insulin responses to a concomitant i.p. glucose challenge. Intravenous injection of hGHRH-40 (20 micrograms/kg body weight) to anaesthetized rats increased plasma concentrations of insulin in the hepatic portal vein. A lower dose of hGHRH-40 (0.2 micrograms/kg) was ineffective, and neither dose of hGHRH-40 altered plasma glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Growth Hormone-Releasing Hormone on Human Peripheral Blood Leukocyte Chemotaxis and Migration in Normal Subjects

The effect of synthetic human growth hormone-releasing hormone (GHRH) on chemotactic response and migration inhibition of human peripheral blood leukocytes has been studied. In the assay performed by using modified Nelson methods, significant inhibition of chemotactic response was observed at 10(-6)M-10(-8) M concentrations of GHRH. There is a strong negative correlation (r = -0.519; p less than 0.001) between the chemotactic response of peripheral blood leukocytes and the concentration of GHRH. In contrast, GHRH tested at the same concentration range was not active in the migration inhibition assay. This finding provides additional evidence for the neuroimmunomodulatory action of GHRH.

Growth hormone (GH) responses to GH-releasing hormone in depression: Correlation with GH release following clonidine

Twenty subjects (10 patients with major depressive disorder and 10 controls matched for age, gender, and ovarian status) received 1 μg/kg synthetic human growth hormone-releasing hormone (GHRH)-44 amide as an i.v. bolus dose. Compared to controls, depressed patients showed a significant attenuation of net growth hormone (GH) responses to GHRH associated with normal basal GH concentrations. The blunted GH responses occurred in the face of significantly higher somatomedin C (Sm-C) concentrations. Comparison of GH responses after GHRH with GH output following the α 2-agonist clonidine (CLON) revealed a significant positive correlation. The concordance between GH responses after specific challenges at different levels of the GHRH-GH-somatomedin axis indicates the integrity of the hypothalamic-pituitary-somatotropic system in depression and supports the view that altered GH secretory patterns in depression may primarily be due to a suprapituitary disturbance.

Injection of Synthetic Human Growth Hormone-Releasing Factors in Dairy Cows. 1. Effect on Feed Intake and Milk Yield and Composition

Eighteen multiparous Holstein cows in the second half of their lactation were used to determine the effect of human growth hormone-releasing factor (1–44)NH2 and a fragment of growth hormone-releasing factor (1–29)NH2 on lactational performance and feed intake. Saline, the 44-amino acid peptide or the 29-amino acid fragment, at the same dose per injection (.2 nmol·kg−1) was injected intravenously at 4-h intervals for 10 d. Average milk yield, milk composition, feed intake, and feed efficiency were compared for the second half of each 10-d preinjection, injection, and post-injection period. Injections of the 44-amino acid peptide and the 29-amino acid fragment increased milk yield 18.6 and 14.6%, respectively. Feed intake was not changed, but feed efficiency was increased 23.9 and 18.8% over control following 44-amino acid peptide and the 29-amino acid fragment injection, respectively. The lactational response was not different between the two peptides for any of the variables measured. This study demonstrates the feasibility of using a growth hormone-releasing factor fragment as an alternative method of elevating milk yield in cattle via somatotropins.

Hypothalamic hypopituitarism following cranial irradiation for nasopharyngeal carcinoma.

Eight patients, one male and seven females, with no pre-existing hypothalamic-pituitary disease, who developed symptoms of hypopituitarism following cranial irradiation for nasopharyngeal carcinoma were studied 5 years or more after radiotherapy. All were GH deficient. Four of the patients with no GH response during insulin tolerance tests (ITT) showed increased GH in response to synthetic human growth hormone releasing factor (GRF-44). Four patients had impaired cortisol responses to ITT, and gradual but diminished cortisol responses to ovine corticotrophin releasing factor (CRF-41). There was no significant difference between mean peak increments in response to ITT and those in response to CRF-41. TSH responses to TRH were delayed in five and absent in two patients; four of these had low free T4 index. Prolactin was raised in all seven women and increased further in response to TRH. Two patients had impaired gonadotrophin responses to LHRH. None of the patients had clinical or biochemical evidence of diabetes insipidus. These data suggest that post-irradiation hypopituitarism in these patients results from radiation damage to the hypothalamus leading to varying degrees of deficiency of the hypothalamic releasing or inhibitory factors.

Characteristics of the desensitization of growth hormone and cyclic AMP responses to growth hormone-releasing factor and prostaglandin E 2 in rat anterior pituitary cells in culture

Upon first exposure, synthetic human growth hormone-releasing factor (GRF) and prostaglandin E 2 (PGE 2) cause a rapid and marked stimulation of cyclic AMP accumulation and GH release in rat adenohypophysial cells in primary culture. However, a marked attenuation of these responses occurs following previous incubation with the 2 compounds. A 50% desensitization of the cyclic AMP and GH responses is observed after 100 and 150 min of preincubation with 300 nM GRF, respectively. After a prior exposure to 3 μM PGE 2, a 50% maximal decrease of the cyclic AMP and GH responsiveness to a subsequent 3 h incubation with PGE 2 is obtained at 90 and 120 min, respectively. Following preincubation with GRF, a loss of responsiveness of the cyclic AMP and GH responses is also observed after heterologous stimulation with PGE 2. A similar heterologous desensitization to the action of GRF is observed following pretreatment with PGE 2. The desensitizing action of GRF on the cyclic AMP and GH responses is obtained at respective IC 50 values of 2 and 7 nM for both the homologous and heterologous responses. The sensitivity of the desensitizing effect of GRF (7 nM) is thus identical to that of its stimulatory action on GH release (6.2 nM). The desensitization to GRF, in analogy to that to PGE 2, is mainly due to a decrease in the maximal action of GRF. Although GH cell content is decreased by previous exposure to GRF and/or PGE 2, the ability of forskolin, cholera toxin, 8-bromo 3',5'-adenosine cyclic monophosphate and 3-isobutyl-1-methylxanthine to stimulate GH release remains unchanged in cells pretreated with these compounds, thus indicating that the loss of responsiveness to GRF and PGE 2 is not due to a depletion of the releasable pool of GH. On the other hand, nifedipine, a potent calcium channel antagonist, completely abolishes the stimulatory effect of GRF on GH release while not affecting basal and GRF- or PGE 2-induced cyclic AMP accumulation. Preincubation with nifedipine has no influence on the desensitizing effect of GRF or PGE 2 on either the cyclic AMP or GH responses to the same stimuli. In addition to showing the cross-desensitization by GRF and PGE 2, the present results strongly suggest that the desensitization does not result from a depletion of the GH releasable pool but most likely results from a down-regulation and/or an impairment of coupling of a component of the adenylate cyclase system independent from calcium uptake.

Stimulation and inhibition of food intake in sheep by centrally-administered hypothalamic releasing factors

Short-term effects of hypothalamic releasing factors on feeding behavior and digestive motility patterns were assessed in hay-fed sheep trained to eat more than half the total amount eaten over 8 h within the first 3 h after food presentation. Thyrotropin-releasing hormone (TRH) given intracerebroventricularly (ICV, 30 ng/kg) or intravenously at higher doses (IV, 3 μg/kg) reduced food consumption by 20 p. cent. The ICV or IV TRH-induced reduction was associated with behavioral excitation and stimulation of antroduodenal motor activity without changes in water intake. The ovine corticotropin releasing factor (oCRF 41) decreased food and water intake by 30–50 % when administered ICV (60 ng/kg) but was not active when given systemically at doses up to 6 μg/kg. The synthetic human growth hormone releasing factor (hGRF 44) administered centrally (60 ng/kg) increased the amount of food intake and the antral motor activity without behavioral excitation. The results indicate a centrally-mediated facilitation of food intake by GRF and its inhibition by CRF which also affect water consumption. The presence of digestive motor effects suggests that extrapituitary pathways may be involved, as for TRH, in the action of both GRF and CRF.

Response of growth hormone release to human growth hormone-releasing factor and its analogs in the bovine.

Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH2 analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH2 in female dairy calves. Four- and 12-month-old calves were injected intravenously with 0.25 microgram of hGRF-44-NH2 or its analogs per kg of bw. Blood samples were collected before, and during 180 min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180 min after injection of hGRF-44-NH2 and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF(1-26)-NH2 was significantly lower than that of hGRF-44-NH2 (P less than 0.05). On the other hand, hGRF(1-29)-NH2 possessed similar potency to hGRF-44-NH2. [D-Tyr1]-hGRF-44-NH2 showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH2. Also, [D-Ala2]-hGRF(1-29)-NH2 exhibited prolonged GH-releasing activity, and its potency was 2.5 (P less than 0.05) and twice (P less than 0.05) as great as that of hGRF-44-NH2 and hGRF(1-29)-NH2, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala2]-hGRF(1-29)-NH2 has longer and greater activity, on a dose basis, than hGRF-44-NH2 in the calves.

Synthetic human growth hormone releasing factor 1-44 (GRF) stimulates glucose transport in isolated rat adipocytes.

Synthetic human growth hormone releasing factor 1-44 (GRF) stimulates glucose transport in isolated rat adipocytes.

ChemInform Abstract: STUDIES ON PEPTIDES. CXXV. 3‐(3‐P‐METHOXYBENZYLTHIOPROPIONYL)THIAZOLIDINE‐2‐THIONE AND ITS ANALOGS AS REAGENTS FOR THE INTRODUCTION OF THE MERCAPTO GROUP INTO PEPTIDES AND PROTEINS

A new SH-introducing reagent, 3-(3-p-methoxybenzylthiopropionyl) thiazolidine-2-thione (MTPTT), was prepared by condensation of 3-p-methoxybenzylthiopropionic acid and thiazolidine-2-thione using DCC. Reaction of MTPTT with peptides can be monitored by following the disappearance of the pale yellow color of the reagent. As an example, synthetic human growth hormone-releasing factor (hGRF-40) was treated with MTPTT, followed by 1M trifluoromethanesulfonic acid-thioanisole/trifluoroacetic acid. The resulting HS-CH2CH2-CO-(hGRF-40) was conjugated with bovine serum albumin (BSA) by using a newly introduced heterobifunctional conjugating reagent, 2, 4-dinitrophenyl p-(β-nitrovinyl) benzoate for antigen preparation. Four other similar SH-introducing reagents were also prepared.

Effect of dopamine, bombesin and cysteamine hydrochloride on plasma growth hormone response to synthetic growth hormone-releasing factor in rats

In urethane anesthetized rats, an intracerebroventricular (icv) injection of 2 μg bombesin 5 min prior to the administration of synthetic human growth hormone-releasing factor (GRF) (1 μg/kg, iv) inhibited plasma growth hormone (GH) response, while cysteamine hydrochloride (90 mg/kg, sc) administered 150 min beforehand depleted immunoreactive somatostatin content in the pituitary-stalk median eminence and consequently potentiated the response to GRF. Under the same experimental conditions, central injection of 1.89 μg (10 −8M) dopamine hydrochloride or iv administration of L-DOPA (10 mg/kg) did not influence the subsequent plasma GH response to GRF. Results suggest indirectly that bombesin and cysteamine, but not dopamine, predominantly modulate somatostatin release from the hypothalamus.

Plasma GH and somatomedin C responses to single and repeated administrations of human growth hormone releasing factor (hGRF).

Plasma growth hormone (GH) and somatomedin C responses to single and repeated administrations of synthetic human growth hormone releasing factor (hGRF-44) were studied in 29 patients with GH deficiency. hGRF-44 administered by single iv bolus (10 micrograms/kg), repeated iv boluses (50 or 100 micrograms, once a day), repeated iv infusions (2.5 micrograms/min for 90 min, once a day), and/or repeated im injections (100 micrograms, twice a day) for three to six consecutive days. Ten of 16 patients had plasma GH responses to a single iv bolus injection, i.e., their plasma GH increased above 5 ng/ml or twice the basal level. However none of them showed a plasma somatomedin C increase. To repeated iv bolus injections and repeated iv infusions of hGRF-44, 7 of 17 patients showed plasma GH responses, however in none of 7 patients did somatomedin C increase. Plasma somatomedin C did not increase after repeated im administrations of hGRF-44 for 5 days. Plasma somatomedin C increase was observed in two patients, from 0.32 to 0.54 U/ml and from 0.16 to 0.46 U/ml, in response to repeated iv boluses and repeated iv infusions, respectively. These results suggest that hGRF-44 stimulates plasma GH secretion in some patients with GH deficiency, however the increases are not enough to stimulate somatomedin generation.

Plasma growth hormone response to human growth hormone releasing factor in rats administered with chlorpromazine and antiserum against somatostatin. Effects of hypo- and hyperthyroidism.

The effect of hypo- and hyperthyroidism on the plasma growth hormone (GH) response to synthetic human growth hormone releasing factor (GRF) was determined in conscious, freely moving rats pretreated with chlorpromazine and antiserum against somatostatin. Chlorpromazine plus somatostatin antiserum pretreated rats gave consistent response to GRF which was not observed in untreated rats. Chlorpromazine alone has no effect on GH secretion induced by GRF in rat pituitary monolayer culture. In rats made hypothyroid by thyroidectomy, both basal and peak plasma GH responses to a small (0.25 microgram/kg bw) and a moderate dose of GRF (1 microgram/kg bw) were significantly reduced as compared to controls. In rats made hyperthyroid by the administration of thyroxine, basal and peak plasma GH responses to a small but not to a moderate dose of GRF were significantly reduced as compared to controls. A reduced plasma GH response to a small dose of GRF was observed 8 days after the cessation of thyroxine administration. The pituitary GH reserve was markedly reduced in hypothyroid but not in hyperthyroid rats as compared to their respective controls. These results indicate that plasma GH response to GRF is reduced both in hypo- and hyperthyroidism. The mechanism involved in the phenomenon appears to be different between the two conditions.

Studies on peptides. CXXV. 3-(3-p-Methoxybenzylthiopropionyl)thiazolidine-2-thione and its analogs as reagents for the introduction of the mercapto group into peptides and proteins.

A new SH-introducing reagent, 3-(3-p-methoxybenzylthiopropionyl) thiazolidine-2-thione (MTPTT), was prepared by condensation of 3-p-methoxybenzylthiopropionic acid and thiazolidine-2-thione using DCC. Reaction of MTPTT with peptides can be monitored by following the disappearance of the pale yellow color of the reagent. As an example, synthetic human growth hormone-releasing factor (hGRF-40) was treated with MTPTT, followed by 1M trifluoromethanesulfonic acid-thioanisole/trifluoroacetic acid. The resulting HS-CH2CH2-CO-(hGRF-40) was conjugated with bovine serum albumin (BSA) by using a newly introduced heterobifunctional conjugating reagent, 2, 4-dinitrophenyl p-(β-nitrovinyl) benzoate for antigen preparation. Four other similar SH-introducing reagents were also prepared.