GH Preparations Research Articles

Activation of growth hormone short loop negative feedback delays puberty in the female rat.

Implantation of GH in the median eminence of the hypothalamus of 23-day-old female rats tonically inhibited serum GH levels throughout prepubertal development (days 23-36) and depressed GH diurnal pulsatile release. Puberty was significantly delayed in GH-deficient animals, a delay associated with a blunted in vitro ovarian steroidal responsiveness to gonadotropins (particularly estradiol) and a marked decrease in prepubertal uterine weight, as evaluated 4, 7, and 13 days after GH implantation. The prepubertal body weight increase was also depressed. Neither ovarian weight nor serum levels of LH, FSH, PRL, or TSH were consistently altered by the GH implant. In addition, evaluation of pulsatile PRL release in 33-day-old rats revealed no difference between control and GH-deficient animals. Ovarian LH receptor content was lower in GH-implanted rats than in controls, suggesting that a decrease in LH receptors may be one of the mechanisms by which a chronic decrease in serum gH depressed the prepubertal ovarian estradiol and, to a lesser extent, the progesterone response to gonadotropins. A direct ovarian site of action for GH was indicated by the results of experiments in which GH was administered to immature hypophysectomized estrogen-treated rats. The in vitro ovarian progesterone response of the GH-treated animals to both hCG and human FSH was distinctly increased by prior in vivo GH treatment. This effect of GH was not reproduced either by the in vivo administration of LH at a dose calculated by RIA to be contaminating the GH preparation or by FSH at a dose that induced a marked increase in aromatase activity in the same ovaries. GH treatment of hypophysectomized rats failed to affect either aromatase activity or hCG-induced estradiol release, indicating that GH does not directly facilitate the production of estradiol by the ovary. The fact that intact rats with GH implants did not actually show a decrease in the ovarian estradiol response to hCG but, rather, failed to show an increased response at the same time as control animals strongly suggests that ovarian maturation was delayed in GH-deficient rats. It is suggested that, in addition to PRL, GH may also play a role in the process of prepubertal reproductive development by enhancing the steroidal response of the ovary to gonadotropins.

Specific binding of growth hormone by rat adipocytes.

Human GH (hGH) binding by isolated epididymal fat cells was studied in nonhypophysectomized Wistar rats. Total binding of [125I]hGH was temperature and time dependent; nonspecific binding was maximal within 30 min of incubation at both 24 and 37 C. Equilibrium binding of [125I]hGH by small adipocytes (mean volume, 40–70 pl) from young lean rats (150– 220 g BW) at 24 C was reached at 90 min and remained stable for at least 3 h. At 2 h, nonspecific binding was 30% of the total radioactivity bound. Specific binding was demonstrable and directly related (r = 0.998; P < 0.001) to the number of cells present in the flasks from 0.2 to 2.4 × 106 fat cells/ml. The amounts of hGH specifically bound to small fat cells increased as the hGH concentration in the medium increased from 10 to 100 ng/ml, and remained at a constant level with further increments in medium hGH concentration. Several nonprimate GH preparations suppressed [125I]hGH binding to adipocytes, but did so to a lesser extent than biologically equivalen...

High incidence of anti-GH antibodies in subjects treated with the GH clinical preparation available in Italy

We have characterized the GH clinical preparation available in Italy (Grorm, Serono) by gel chromatography and immunoreactivity. Only 50-60% of this preparation can be identified with the active GH monomeric form. Both higher and lower molecular weight compounds are present as well. Then, using a radioimmunoassay technique, we looked for anti-GH antibodies in 21 subjects under Grorm treatment and found them in 6 paitents (28.6%). Two subjects having anti-GH antibodies with high affinity had a lower growth rate. These data suggest that anti-GH antibodies measurement is clinically useful in patients treated with this GH preparation. A more purified preparation should be used when a slowing down of the growth rate is observed in the presence of anti-GH antibodies.

Relative effects of the somatomedins, multiplication-stimulating activity, and growth hormone on myoblasts and myotubes in culture.

Responses of muscle cells in culture to somatomedins A and B (SM-A and SM-B), Temin's multiplication-stimulating activity (MSA), and a number of GH preparations from various species have been determined at both the myoblast and myotube stages of differentiation. Proliferation and amino acid (AIB) uptake by myoblasts as well as AIB uptake by myotubes were equally responsive to SM-A and MSA, demonstrating that MSA serves as a useful surrogate for SM-A in studies on muscle cells. SM-B exhibited no activity in either system, and it did not enhance the actions of SM-A. A number of GH preparations were inactive in muscle cell cultures from various sources, but bovine GH stimulated AIB uptake in myotubes. Both the magnitude of the response and the sensitivity of myotubes to bovine GH increased with time after differentiation of the cells in vitro, but conditions could not be found to demonstrate an effect of GH at or near physiological levels of the hormone. On the basis of our observations and published studies on the effects of GH on isolated muscles, we suggest that the early growth of muscle is stimulated primarily by the somatomedins, and that a direct response to GH develops relatively late after the differentiation of skeletal muscle.

Identification and Characterization of High and Low Affinity Binding Sites for Growth Hormone in Rabbit Liver*

The specific binding of rat (r) and rabbit (rb) [l25I]iodo-GH was studied in liver membrane preparations isolated from male, virgin female, late pregnant, and midlactating rabbits. Scatchard plots of dose-response displacement curves were consistently nonlinear and suggested the existence of at least two classes of binding sites for both GHs. The average binding affinity constants (Ka) for rGH on the low affinity (1.7 × 108 M-1) and high affinity sites(s) (1.0 × 1010 M-1) were similar to those for the rbGH. Displacement of [125I]iodo-rGH with preparations of GH, PRL, and placental lactogen (PL) from different species demonstrated that the potencies and slopes of the dose-response curves varied considerably, indicating that the displacement activities of many hormones differ at the two sites. Neither the potencies nor the slopes of the dose-response curves correlated with the growth-promoting activities of the various hormones. On the bases of parallelism and potency, rbGH, equine GH, and bovine (b) PRL we...

On the Isolation of Human Pituitary Hormones*

Six human pituitary hormones can be obtained from a single batch of human pituitary glands by the procedure described. Starting from an acetone powder of the glands, the hormones are segregated into three fractions; one containing GH and PRL, a second containing the glycoprotein hormones, and the third fraction containing smaller peptides including ACTH and MSH. Clinical grade GH is extracted from the residue at alkaline pH, followed by a series of ammonium sulfate and pH fractionations. Further chromatography on G-100 yields a highly purified GH preparation. FSH is purified by chromatography on carboxymethyl cellulose at pH 5.4 followed by gel filtration on G-100 and fractionation on sulfopropyl Sephadex C-50 at pH 5.4. LH and TSH are separated by DEAE-cellulose chromatography at pH 9.5. The GH and glycoprotein hormones are recovered in a highly purified form and good yields. The GH is obtained in an active form which is easily soluble and well suited for clinical use. A large part of the PRL is also saved during the extraction procedure.

Distribution of Binding Sites for Ovine Placental Lactogen in the Sheep*

Specific binding sites for ovine placental lactogen (oPL) have been detected in the 100,000 × g pellet obtained from tissues derived from non-pregnant and pregnant ewes as well as from fetal tissues. Specific binding of ovine [125I]iodo-PL (percentage per milligram of protein) was found in non-pregnant ewe liver (28%), adipose tissue (20%), ovary (10%), corpus luteum (6%), uterus (5%), and fetal liver (5%). The specific binding of oPL was less than 3% in the following maternal tissues: mammary gland, adrenal, kidney, spleen, pancreas, lung, heart, skeletal muscle brain (cortex), and uterus near term (130–135 days’ gestation). Less than 3% specific binding was found also in several fetal tissues including adipose tissue, adrenal, kidney, spleen, pancreas, lung, heart, skeletal muscle, brain (cortex), and placenta near term (both fetal cotyledons and maternal caruncles). The binding of oPL to maternal liver, adipose tissue, ovary, corpus luteum, and fetal liver was inhibited by ovine GH but not by ovine PRL or ovine LH. In the radioreceptor assay for oPL using liver (100,000 × g pellets) from the ewe, ovine [125I]iodo-PL was inhibited by several GH preparations and by human placental lactogen. PRL preparations from several species as well as other pituitary hormones did not compete for the binding sites. Conversely, oPL only slightly inhibited the binding of ovine [125I]iodo-PRL in the radioreceptor assay for oPRL using pregnant ewe uterus. These observations suggest that oPL may act more like a GH than a PRL, and hence may mediate important metabolic effects during the greater part of pregnancy in the ewe. (Endocrinology102: 632, 1978)

An immunological approach to the study of protein conformational heterogeneity: its application to growth hormone.

The study of multiple forms of a hormone due to changes in its secondary or tertiary structure (conformational heterogeneity) has been restricted to methods of physical chemistry which require relatively large quantities of purified preparations and has precluded such studies on circulating hormones. The present report describes an immunological approach to the study of GH conformational heterogeneity based on the simultaneous use of two homologous RIAs: one which recognizes porcine (p) GH in its native conformation (pGH RIA) and a second (pB RIA) for a 55-residue S-aminoethylated cyanogen bromide fragment (pB) of pGH (corresponding to residues 126-180 of human GH). In the pGH RIA, pB was nonreactive, whereas pGH displacement of [125I]iodo-pB from anti-pB serum required about 10(3) M excess and was nonparallel to pB. Treatment of pGH with urea plus mercaptoethanol (ME), which would be expected to affect the conformation of the GH molecule, increased its immunoreactivity 68-fold in the pB RIA and its dilution curve became parallel to that of pB. Similar treatment of porcine pituitary extract increased its immunoreactivity 97-fold in pB RIA with a corresponding slope change. In contrast, urea plus ME decreased pGH and pituitary extract immunoreactivity by 84% and 92%, respectively, in the pGH RIA. The pB RIA/pGH RIA ratio of immunoreactivity for pGH and for pituitary extract increased 795-fold and 569-fold, respectively, after combined treatment, whereas lesser increases were observed after urea or ME treatment alone. In conditions where alterations in GH conformation were suspected (highly purified, aged human GH preparations which were no longer satisfactory as radioiodinated tracers for radioimmunoassay), calculation of the pB RIA/hGH RIA ratio of immunoreactivity indicated increased values. Based on these results, a model is proposed in which unfolding a native hormone, with concomitant exposure of the antigenic determinant of pB, hidden within pGH, leads in increased immunoreactivity in the pB RIA and decreased immunoreactivity in the pGH RIA. In this model, the pB RIA/pGH RIA ratio of immunoreactivity would thus serve as an index of the state of hormonal unfolding.

Lactogenic activity of mammalian growth hormones in vitro.

SummaryGrowth hormone preparations from several mammalian species were investigated for their ability to initiate secretion in organ cultures of prelactating mammary tissue from strain C3H and A mice. All the growth hormones studies showed some degree of activity in C3H mammary tissues. Bovine, ovine and porcine GH were 20-50% as active as ovine prolactin, although the prolactin content of the preparations was shown to be low. Simian and human GH, whose prolactin-like activity in other systems is well-known, showed activity equal to or greater than ovine prolactin in C3H mammary tissues. On the other hand, a single human GH preparation (one of three studied) was the only GH to show significant activity in strain A mammary tissues. These results suggest an intrinsic lactogenic activity of GH, particularly well-marked in the case of human GH, whose expression in mouse mammary gland cultures is in part determined by the genetic background of the tissues.

PITUITARY GROWTH HORMONE AND INTERMEDIARY METABOLISM

Several significant concepts have emerged from the impressive mass of information on the basic principles of GH function. Before 1955 no distinctive differences in growth hormone effect on various animals had been noted. Now it has been demonstrated that growth hormone derived from certain animals fails to show definite results and, in some instances, has no activity in specimens from other species. In general, growth hormone obtained from the more advanced species will be active in the same or in genetically lower groups; the reverse fails to occur (47) and, in particular, GH preparations from other animals have been found inactive in man.