Paul Roos

Characterization of putative growth hormone receptors in human choroid plexus

The ability to bind iodine-labelled human growth hormone ([125I]hGH) was measured in different parts of the human brain. The choroid plexus contained the highest amount of binding sites (receptors) and was therefore selected for further studies. The binding between [125I]hGH and the receptor was saturable, of high affinity (Ka = 0.63 nM-1) and pH- as well as time-dependent. After solubilization with Triton X-100 the receptors retained their hormone-binding properties and eluted in the high molecular weight range (greater than 500,000) upon molecular sieve chromatography. Analysis by an affinity cross-linking technique indicated a hormone-binding unit of molecular weight 51,000. The molecular characteristics of the identified binding sites are discussed in comparison to those of growth hormone receptors of human and animal origin.

Age-related reduction of human growth hormone-binding sites in the human brain

Previous studies have shown that alterations in various neuroendocrine functions occur with increasing age. We here report a study of growth hormone (GH)-binding sites in different areas of post-mortem human brains collected from individual males and females of different age. The results indicate that there exists a significant negative correlation between the density of GH-binding sites and increasing age. This phenomenon was observed in both sexes in brain areas such as choroid plexus, hippocampus, hypothalamus, pituitary and putamen but not in e.g. thalamus. In all tissues (except for choroid plexus), the GH binding was significantly higher in those originating from females than those from males. This discrepancy was found likely to be associated with the affinity of GH to lactogenic rather than to somatogenic sites as no pronounced sex difference in binding was observed in the presence of excessive amounts of human prolactin. Data also indicate that the putative GH receptors in the various brain regions differ with regard to binding constants and to the estimated molecular size of the hormone-binding units. The loss of GH receptors in brain of elderly people may have consequences in several physiological courses. The decrease in GH binding at hypothalamic and pituitary levels may be of importance for the mechanisms behind the release or secretion of the hormone.

Preparation of human growth hormone by gel filtration

Abstract 1. (1) A simple method for preparation of human pituitary growth hormone has been described. It involves extraction of homogenized glands, ammonium sulfate fractionation and gel filtration on Sephadex G-100. All procedures were performed in solutions of near neutral pH. 2. (2) The active product yielded a single peak when refiltrated in columns packed with Sephadex G-100, G-75, and G-50, respectively, and a single sedimentation boundary in the ultracentrifuge. 3. (3) The yield human pituitary growth hormone was about 5 mg/g frozen whole pituitaries and the material contained negligible amounts of follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone and thyrotropic hormone. 4. (4) Subsequent zone electrophoresis in agarose suspensions revealed the presence in the active material of more than one molecular species with growth-stimulating activity. No significant difference in specific activity in these components has been established.

Pregnancies following treatment with human gonadotropins: With special reference to the problem of multiple births☆☆☆

Abstract During a 4 year period about 100 women with primary or secondary amenorrhea were treated for sterility with human pituitary FSH and HCG. About 90 per cent ovulated once or several times, and 50 per cent became pregnant. So far 43 have been delivered, 20 women of a single infant, 14 of twins, and 9 of triplets or more fetuses. Thus, the incidence of multiple births was the same as that of single births. Repeated pregnancies were induced in 10 patients. The rate of abortion was high in those women who conceived triplets or more while it was negligible in those with single pregnancies. No congenital malformations were observed. To avoid multiple births the daily dose of human pituitary FSH, the pattern of FSH administration and the endogenous gonadotropin production must be taken into careful consideration with each new patient. This kind of treatment is, therefore, best confined to special centers with a fully equipped gynecologic endocrine unit.

Characterization of growth hormone binding sites in rat brain

The binding of 125I‐labelled rat growth hormone (GH) to different areas in the brain was studied in male Sprague‐Dawley rats. A high density of GH binding was found in the choroid plexus, hypothalamus, hippocampus, pituitary and spinal cord, whereas a lower binding density was observed in the cortex. Binding of the hormone to the various brain regions was age dependent. Binding was also dependent on time, pH and protein concentration. The binding affinity of the labelled hormone to choroid plexus was 4.3 per nmol/l and the binding capacity was 33.4 nmol/mg protein. The corresponding figures for binding of 125I‐labelled GH to hypothalamus were 5.6 per nmol/1 and 21.6 nmol/mg protein. By sodium dodecyl sulphate electrophoresis of the cross‐linked hormone‐receptor complexes, molecular weights of 60,000 and 61,000 were determined for the binding units in the choroid plexus and hypothalamus, respectively. It was further indicated that the binding unit for rat GH was distinct from that for prolactin.

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.

Rat growth hormone and hypothalamic catecholamine nerve termianl systems. Evidence for rapid and discrete reductions in dopamine and noradrenaline levels and turnover in the median eminenece of the hypophysectomized male rat

Rat growth hormone (rGH) (100 micrograms/kg) produced 2-4 h after its i.v. injection a rapid reduction of catecholamine stores and turnover in the subependymal layer and in the medial and lateral palisade zone of the median eminence. It is suggested that rGH may inhibit its own secretion partly via reduction of DA synthesis and release in the median eminence leading to increased somatostatin release and partly via reduced noradrenaline synthesis and turnover in the median eminence leading to reduced secretion of a growth hormone releasing factor.

Isolation of five active thyrotropin components from human pituitary gland

A procedure is described for the isolation of human pituitary thyroid-stimulating hormone (thyrotropin). The starting material was a side-fraction provided by the earlier developed process for the purification of growth hormone from whole frozen pituitaries. This fraction was further purified by successive chromatography on Bio-Gel P-150, Bio-Gel HT hydroxyapatite, and SP-Sephadex C-50. The resulting preparation was obtained in yields of 10 mg/kg of pituitary tissue and had a thyrotropin potency of 11 units human Research Standard A/mg as measured by a specific radioimmunoassay. Contamination by other pituitary hormone activities was low. In the ultracentrifuge a single sedimenting boundary was registered with an s20,w value of 2.7 S. The molecular weight as determined by sedimentation-equilibrium experiments was 34 000 in phosphate buffer, pH 7.0, and 17 700 in 1 M propionic acid. This thyrotropin preparation was, however, electrophoretically heterogenous. Following preparative polyacrylamide gel electrophoresis five different components associated with thyrotropin activity were isolated. Isolation on a preparative scale of electrophoretically homogeneous human thyrotropin has not earlier been reported. One of the thyrotropin components was characterized with respect to molecular weight and amino acid composition. The data were consistent with a molecular weight of 33 000 from sedimentation-equilibrium analysis at pH 7 and with 268 amino acid residues per molecule.

Human pituitary luteinizing hormone: Isolation and characterization of four glycoproteins with luteinizing activity

Two major and two minor components of human luteinizing hormone (lutropin) were isolated from whole frozen pituitaries by a procedure involving extraction of homogenized pituitaries, (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, Sephadex G-100, and SE-Sephadex C-50 and electrophoresis in polyacrylamide gel. The isolation procedure was monitored by both bioassays and radioimmunoassays. Contamination of the final products by other pituitary hormone activities was very low. The four lutropin components were all homogeneous by polyacrylamide gel electrophoresis (a sieving medium) and by free zone electrophoresis (a non-sieving medium). No heterogeneity was observed when the components were studied in the ultracentrifuge by sedimentation-equilibrium technique. The molecular weights of the components were in the range of 34 000-40 000. Sedimentation velocity experiments with the two major components revealed in each case one boundary with S20,W values of 3.2 S and 3.5 S. Further evidence for the homogeneity of the components was the observation of only one precipitin line for each component upon immunodiffusion against a rabbit anti-human lutropin serum. Amino acid and carbohydrate analyses indicated close similarity among the four components. From the analysis data the molecular weights of the components were calculated to be 31 000-33 000.

Vascular permeability to growth hormone in the rat central nervous system after focal spinal cord injury. Influence of a new anti-oxidant H 290/51 and age

Vascular permeability to the growth hormone (GH) across the blood-brain barrier (BBB) is unknown. This investigation was undertaken to examine vascular permeability to 125I-labelled rat growth hormone (rGH) in the central nervous system (CNS) of normal animals. Since age and spinal cord injury influences the metabolism of GH, these factors were also included. No statistically significant difference was seen regarding rGH permeability between young (aged 19-21 weeks) and old (age 38-42 weeks) animals. A focal trauma to the cord, produced by an incision into the right dorsal horn of the T10-11 segments in young animals, increased rGH permeability in several spinal cord segments at 0.5-5.0 h after injury. This permeability increase progressed over time. Similar trauma to old rats resulted in a significantly less increase in rGH permeability in the spinal cord 5 h after the trauma. This indicates that trauma-induced increased permeability of rGH is age-dependent. Pretreatment of normal young animals with a new antioxidant (H 290/51) did not influence the rGH permeability. However the drug prevented the trauma-induced increase of rGH permeability at 5 h after injury. This indicates that inhibition of lipid peroxidation has some protective effect on trauma-induced increase in rGH permeability.