Repetitive injections of growth hormone releasing hormone (GHRH1-44) to normal volunteers and patients with growth hormone deficiency

The present study was designed to answer the following three questions: Is there any difference between the growth hormone (GH) response to i.v. injections of GHRH 1-44 by a slowly injecting hormone pump or to a s. s. or rapid i. v. injection by syringe? Do nocturnal injections of GHRH 1-44 i. v. elicit different GH levels than during daytime? Can repetitive administration of GHRH 1-44 in patient with GH deficiency induce a physiological GH pattern and thereby normalize the condition resulting from a hypothalamic defect? A rapid i. v. bolus injection of 50 micrograms GHRH 1-44 by syringe with an injection time of one second elicited in the same subject at the same time of the day a twofold greater response than a slowly injecting (60 seconds) hormone pump. In six male adult volunteers each GHRH i. v. bolus was followed by a GH secretory pulse. The GH response at night (area under the curve and peak plasma GH levels) was significantly greater than at daytime (P less than 0.05) and greater than the GH pulses measured during a spontaneous nocturnal profile (P less than 0.05). Out of six GH deficient young adult patients who had been receiving extractive GH until two years prior to the study, three responded much like the controls, the other three patients-those who lacked any spontaneous nocturnal GH peaks-had markedly lower GH levels after GHRH (P less than 0.05). However, there was a clear-cut GH release after GHRH injection in each patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bisanthraquinone glycosides of Hypericum perforatum with binding inhibition to CRH-1 receptors

Four new bisanthraquinone glycosides, S-(+)-skyrin-6-O-beta-glucopyranoside (1), R-(-)-skyrin-6-O-beta-glucopyranoside (2), S-(+)-skyrin-6-O-beta-xylopyranoside (3) and S-(+)-skyrin-6-O-beta-alpha-arabinofuranoside (4), have been isolated from an ethanol-water (1:1, v/v) dry extract of the aerial parts of Hypericum perforatum L. The structures were elucidated by spectroscopic methods, mainly NMR and mass spectrometry. Circular dichroism was used to determine their axial stereochemistry revealing 1 and 2 to be atropisomers. 1 and 2 inhibited [125I]sauvagine binding to corticotropin releasing hormone (CRH-1) receptors.     

Failure to confirm consistent stimulation of growth hormone by diazepam

Diazepam has been reported to influence pituitary hormone secretion, with several studies claiming that diazepam provokes growth hormone release. Normal volunteers were therefore examined for anterior pituitary responsiveness to 10 mg diazepam i.v. and p.o. The drug had no significant effect on the secretion of prolactin or thyrotropin when compared to control saline injections in all subjects. Growth hormone response was variable; serum growth hormone increased significantly in only 4 of 10 patients after i.v. diazepam and in only 1 of 7 subjects tested with oral diazepam. Serum cortisol rose in only 1 subject, precluding a stress-related explanation for the increase in growth hormone. We conclude that diazepam inconsistently stimulates growth hormone secretion and should not be relied upon as a test of growth hormone reserve.     

A 1H-NMR study of the solution conformation of secretin. Resonance assignment and secondary structure

The solution conformation of the 27 residue polypeptide hormone secretin has been investigated by 1H-NMR spectroscopy under conditions where it adopts a fully ordered structure as judged by circular dichroism spectroscopy, namely in an aqueous solution of 40% (v/v) trifluoroethanol. Using a combination of two-dimensional NMR techniques the 1H-NMR spectrum of secretin is completely assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. It is shown that under these conditions secretin adopts a conformation consisting of an N-terminal irregular strand (residues 1-6) followed by two helices (residues 7-13 and 17-25) connected by a 'half-turn' (residues 14-16); the last two residues (26 and 27) are again irregular. This conformation is shown to be very similar to that of glucagon in perdeuterated dodecylphosphocholine micelles and to that of the active 1-29 fragment of growth hormone releasing factor in 30% (v/v) trifluoroethanol:     

Restoration of microcephalic cerebrum with hypomyelination in the growth hormone-deficient mouse (lit): Stimulatory effects of GH restricted to the first 20 days of postnatal life

We administered bovine growth hormone to the Little (lit), a promising model of isolated growth hormone deficiency, during the first and second 20 days after birth. Positive results were obtained only when bovine growth hormone was given during the first 20 days of postnatal life. We observed a distinct increase in cerebral weight, DNA content, and 2, 3-cyclic nucleotide 3-phosphohydrolase activity. The latter administration of bovine growth hormone was ineffective. These data prove that growth hormone has an independent action on cerebral development, apart from the complementary or synergistic action of thyroid hormones, and that the administration of exogenous growth hormone led to increased myelinogenesis through its stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

Genetic engineering of single-chain gonadotropins and hormone-receptor fusion proteins

The gonadotropin hormone family is distinguished by its heterodimeric structure in which the members share a common alpha subunit and a hormone-specific beta subunit. Since assembly of the heterodimer is often the rate-limiting step in production of functional hormone, single-chain hormones have been engineered by genetically linking the two subunits. The single-chain hormone can in turn be fused to its receptor to produce a functional single-chain hormone-receptor complex. These fusion constructs offer a valuable new approach in structure-function studies and in the generation of hormone analogs. In this article we describe the experimental design for the generation of single-chain human chorionic gonadotropin and single-chain hormone-receptor fusion complex and strategies for the expression of these fusion proteins.     

TRH dissociates the aggressivity of vegetative and motor phenomena induced by carbachol in the cat

In these experiments interaction of thyrotropin releasing hormone (TRH) and carbachol injected into the cerebral ventricles of unanaesthetized cats has been investigated. Intracerebroventricular (i.c.v.) carbachol as well as i.c.v. TRH produced emotional behaviour, autonomic and motor phenomena. The most impressive feature of i.c.v. carbachol was the aggressive behaviour, whereas that of i.c.v. TRH the autonomic changes. In cats treated with i.c.v. TRH, the aggressive behaviour, but the autonomic and motor changes of i.c.v. carbachol was potentiated. Since there is evidence that carbachol acts mainly on muscarinic M-2 receptors, the potentiation by TRH of aggressive behaviour, but not the autonomic and motor changes induced by carbachol could indicate heterogeneity of central muscarinic M-2 receptors.     

The isolation and identification of cyasterone from Ajuga chia (labiatae)

Cyasterone, an insect molting hormone, has been isolated from Ajuga chia and identified by means of chromatographic (column, TLC) and spectroscopic (u.v., i.r., NMR, m.s.) methods.     

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.     

Glucagon action upon plasma post-heparin lipolytic and monoglyceride hydrolase activities. Studies involving administration of exogenous hormone during suppression of endogenous hormone secretion with somatostatin

It has been previously demonstrated that glucagon increased plasma post-heparin lipolytic activity (PHLA) in normal rats, but this was not the case in alloxan diabetic rats. The present work was designed to determine if the administration of exogenous glucagon (0.2 mg i.v.) during suppression of endogenous hormone secretion with somatostatin modifies the plasma post-heparin lipolytic activity in normal rats and the action of such hormone upon monoglyceride hydrolase (MGH) activity. It was found that exogenous glucagon significatively increased PHLA and MGH activity in normal rats after 18-24 hours of starvation. However, both enzymatic activities were not influenced by exogenous glucagon when they were measured during somatostatin administration. Therefore it is believed that the enhancement of these activities observed when somatostatin was not simultaneously given was due to the insulin secretion that follows the glucagon injection.     

Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...     

Impact of euglycaemia and hyperglycaemia on the release of growth hormone and prolactin in type II-diabetics

The influence of different blood glucose concentrations on the arginine (30 g/30 min i.v.) and TRH (400 micrograms i.v.) induced release of growth hormone and prolactin was studied in six male type II-diabetic patients. Blood glucose concentrations were clamped at euglycaemic (4-5 mmol/l) or hyperglycaemic (12-18 mmol/l) levels by means of an automated glucose-controlled insulin infusion system. The response of growth hormone to arginine, and irregular spikes in growth hormone concentrations following TRH seen in the euglycaemic state were suppressed during hyperglycaemia. The suppression of the arginine-induced release of growth hormone by hyperglycaemia was observed both with and without concomitant administration of exogenous insulin. The rise in serum prolactin concentrations in response to arginine was unaffected by hyperglycaemia, whereas the TRH-induced release of prolactin was suppressed. Since arginine induces the release of growth hormone and prolactin via the hypothalamus, while TRH acts at the pituitary level, the glycaemic state appears to exert a modulatory effect on the secretion of growth hormone and prolactin in type II-diabetics at both locations.     

Interaction of substance P and its N- and C-terminal fragments with Ca2+: implications for hormone action.

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.     

Structure-activity relationships on hyperglycemia by representatives of the adipokinetic/hyperglycemic hormone family in Blaberus discoidalis cockroaches

Summary Several members of the adipokinetic/hyperglycemic neurohormone family from several different invertebrate species have been prepared by solid-phase peptide synthesis and assayed by a modified in vivo hyperglycemic bioassay in Blaberus discoidalis cockroaches. The hypertrehalosemic hormone (HrTH) is the endogenous hypertrehalosemic factor for B. discoidalis and was the most potent peptide in the assay. The more divergent the sequence of a family member from Blaberus HrTH, the less potent was the bioanalog. Manduca adipokinetic hormone is the most divergent peptide of the family and was totally inactive in the bioassay. Locusta adipokinetic hormone I had reduced maximum activity in the assay, which suggests that Ser⁵ is an important residue for the transduction of the hyperglycemic response. The direct relation between bioanalog similarity to Blaberus HrTH sequence and potency suggests that the hormone and target cell receptor for HrTH have evolved to maintain an optimal fit.Abbreviations AKH adipokinetic hormone - HrGH hyperglycemic hormone - HrTH hypertrehalosemic hormone - RPCH red pigmentconcentrating hormone - CAH cardioacceleratory hormone. Hormone abbreviations are according to the convention of Gäde and Raina (1989) except that the genus names are not abbreviated     

Insulin-like growth factors (IGFs) as autocrine/paracrine regulators of granulosa cell differentiation and growth: studies with a neutralizing monoclonal antibody to IGF-I

Evidence that granulosa cells secrete and respond to insulin-like growth factors (IGFs) suggests, but does not prove, the importance of IGFs as intraovarian regulators. To further assess the role of these peptides in ovarian function, a neutralizing monoclonal antibody to IGF-I was employed to block the actions of IGFs in porcine follicular fluid and in granulosa cell-conditioned medium. In one series of experiments, granulosa cells from immature porcine follicles were cultured in medium containing porcine follicular fluid that had been charcoal-treated to remove steroids. As noted before, fluid from large follicles (LFF) stimulated progesterone production in a dose-dependent manner. The stimulatory effect of LFF (30% v/v) could be inhibited by greater than 50% by the anti-IGF monoclonal antibody. This inhibitory action was specific for the anti-IGF antibody and could be overcome by the addition of excess exogenous IGFs. In another series of experiments, granulosa cells were made dependent on endogenously produced IGFs by culturing them in a serum-free medium without exogenous growth factors. The effects of follicle-stimulating hormone (FSH), estradiol (E2), growth hormone (GH), and combinations thereof on progesterone production were inhibited by approximately 50% by the anti-IGF antibody. The effects of IGFs on indices of cell growth (judged by the criterion of being inhibited by the anti-IGF antibody) were less dramatic. A modest 18% increase in cell number was observed with FSH and E2 treatment in serum-free medium; this effect was virtually abolished by the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.     

Splenic Leukocytes Traffic to the Thyroid and Produce a Novel TSHβ Isoform during Acute Listeria monocytogenes Infection in Mice

The thyroid stimulating hormone beta-subunit (TSHβ) with TSHα form a glycoprotein hormone that is produced by the anterior pituitary in the hypothalamus-pituitary-thyroid (HPT) axis. Although TSHβ has been known for many years to be made by cells of the immune system, the role of immune system TSH has remained unclear. Recent studies demonstrated that cells of the immune system produce a novel splice variant isoform of TSHβ (TSHβv), but little if any native TSHβ. Here, we show that within three days of systemic infection of mice with Listeria monocytogenes, splenic leukocytes synthesized elevated levels of TSHβv. This was accompanied by an influx of CD14⁺, Ly6C⁺, Ly6G⁺ cells into the thyroid of infected mice, and increased levels of intrathyroidal TSHβv gene expression. Adoptive transfer of carboxyfluorescein succinimidyl ester (CFSE)-labeled splenic leukocytes from infected mice into non-infected mice migrated into the thyroid as early as forty-eight hours post-cell transfer, whereas CFSE-labeled cells from non-infected mice failed to traffic to the thyroid. These findings demonstrate for the first time that during bacterial infection peripheral leukocytes produce elevated levels of TSHβv, and that spleen cells traffic to the thyroid where they produce TSHβv intrathyroidally.