Marie C. Gelato

Growth Hormone-Releasing Factor

We have previously described in detail the clinical and laboratory findings of a patient who harbored a pancreatic tumor secreting a growth hormone releasing factor (GRF).(1) This 40-amino-acid-containing peptide (hpGRF-40) was extracted, isolated, sequenced, and a synthetic replicate produced by the Peptide Biology Laboratory at the Salk Institute.(2,3) A 44-amino-acid peptide (hpGRF-44) was identified from another pancreatic tumor and subsequently synthesized by the Laboratories for Neuroendocrinology at the Salk Institute.(4) One year later convincing immunological, biochemical, and genetic information appears to have confirmed that human hypothalamic growth hormone-releasing hormone (GHRH) is identical to human pancreatic tumor GRF(s) and has approximately 70% homology with rat hypothalamic GHRH.(5–8) In this chapter we will review our first 11 months of clinical experience with GRF and compare our results to those reported by other groups.

The effects of prolactin on rat ovarian function

Hyperprolactinemia has been associated with several reproductive disorders. To investigate whether hyperprolactinemia directly affects rat ovarian function, we examined the ovarian histopathology and the activities of the four ovarian enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17-hydroxylase (17-OH), 17,20-desmolase (17,20-D) and aromatase in hyperprolactinemic rats and controls. Hypophysectomized, gonadotropin-treated Fisher rats were made hyperprolactinemic by isografting pituitary glands under the kidney capsule. The control animals received skeletal muscle. The ovaries were resected, pooled according to prolactin levels and microsomal enzyme activities were measured from each pool. Prolactin (PRL) levels were 344 +/- 23 ng/ml in the hyperprolactinemic rats and 18 +/- 5 ng/ml in the controls (p less than 0.001). Estradiol concentrations were 609 +/- 47 pg/ml in the hyperprolactinemic animals and 56 +/- 13 pg/ml in the controls (p less than 0.001). Ovarian and uterine weights were significantly higher in the hyperprolactinemic rats (p less than 0.02). Ovarian histopathology demonstrated benign polycystic transformation in the hyperprolactinemic animals. Hyperprolactinemia had no effect on 3 beta-HSD, but was associated with significant decreases in the 17-OH, 17,20-D and aromatase activities when compared to controls (p less than 0.001). We conclude that prolactin has a direct effect on rat ovarian function which appears to be independent of changes in gonadotropin secretion.

Sex Steroids Increase Spontaneous Growth Hormone Secretion in Short Children

Spontaneous growth hormone (GH) secretion and GH responses to standard provocative tests and to growth hormone releasing hormone (GHRH) were evaluated in twenty-one children with idiopathic short stature. Eight children (two female, six male) were studied after brief sex steroid administration (estradiol or testosterone), whereas thirteen children (seven female, six male) were studied under baseline conditions without sex steroid treatment. Children were prepubertal or in early puberty, with no endocrine abnormalities. Children who were given sex steroids showed a greater frequency of growth hormone pulses during the day (6.0 ± 0.3 [SEM] versus 3.2 ± 0.3, ρ < 0.001) and a higher mean 24-hour growth hormone level (5.4 ± 0.8 vs. 3.6 ± 0.5 ng/ml, ρ < 0.02) than children who were untreated. GH peak responses to arginine and to L-dopa were higher in treated children (27.3± 5.5 vs. 9.8 ± 1.9 ng/ml, and 11.4 ± 3.3 vs. 5.7 ± 2.2 ng/ml, respectively). In contrast, responses to insulin and to GHRH were not different between the two groups. We conclude that sex steroids increase both spontaneous and stimulated GH secretion in children with idiopathic short stature, and that sex steroid induced increases in GH secretion may contribute to the pubertal growth spurt.

Production of monoclonal antibodies against human growth hormone releasing hormone and their use in an enzyme-linked immunosorbent assay (ELISA)☆

Two murine monoclonal antibodies (mAbs) specific for human growth hormone releasing hormone (GHRH-44-NH2) were produced from a fusion of spleen cells from a BALB/c mouse immunized with GHRH-conjugated BSA with SP 2/0 myeloma cells. The antibodies were of the IgG1 kappa, and IgG2b-kappa isotypes. The binding of both antibodies to GHRH-coated plates was inhibited by a 30-44 amino acid fragment but not by a 1-26 fragment. Thus, both antibodies are directed against the carboxy terminus of the peptide. Furthermore, both antibodies bind to the same epitope on the 30-44 amino acid portion since they cross-inhibit each others binding to intact GHRH. Using these mAbs, a direct binding GHRH enzyme-linked immunosorbent assay (ELISA) was developed which had a least detectable dose of 30 pg. The availability of these antibodies and their use in ELISA methodology permits consistent and specific detection of GHRH in a non-isotope assay. They should prove of value in screening acromegalic patients for ectopic sources of GHRH secretion and in studies of ontogenic analysis of GHRH production.

ACCELERATION OF LINEAR GROWTH AFTER REPEATED DOSES OF GROWTH HORMONE-RELEASING FACTOR (GRF)

GRFs are potent stimulators of GH secretion in man. We administered 1 μg/kg GRF 1-44 NH2 to 15 children with documented GH deficiency, ages 4-20 years. 12/15 had a measurable GH response. Following discontinuation of GH therapy for at least 2 months, 2 responders and 1 nonresponder then received 1 μg/kg GRF IV q 3 h for 10-12 d. Short-term growth was assessed by the lower leg measurement technique of Valk et al. (Growth 47:53, 1983). GH was measured q 20 min for 12 h on the 1st, and 5th or 7th days of multiple-dose therapy and for the first 12 hours off GRF. Somatomedin-C (SmC) levels were drawn daily. Baseline GH levels were <0.7 ng/ml. In the 2 responders, mean peak GH response to GRF increased, SmC rose, and lower leg growth velocity (GV) accelerated (Table). GH levels returned to baseline after GRF was stopped. The third patient had no hormonal or growth response.Thus most children with GH deficiency respond to GRF, and multiple doses of GRF can accelerate short-term linear growth. GRF may form the basis for an alternative treatment of GH deficiency.

Repeated Stimulation with Growth Hormone-Releasing Hormone Can Induce a Growth Hormone Response in Initially Unresponsive Growth Hormone Deficient Patients

More than eighty per cent of growth hormone deficient (GHD) children respond to a 1 μξ/Kg intravenous bolus of growth hormone releasing hormone (GHRH-[1-44] NH2) with an increase in growth hormone (GH) levels. To determine whether the remaining patients have a pituitary deficit or whether the lack of response is secondary to chronically unstimulated somatotrophs, we administered repeated pulsatile doses of GHRH, either 1 μ-g/Kg every 3 h, or 3 μ-g/Kg sc q 1.5 h at night, to seven GHD patients who had failed to respond to an initial dose of GHRH. A l/xg/Kg iv bolus of GHRH was given at the beginning and the end of the pulsatile stimulation period and the responses

The growth hormone response to growth hormone-releasing hormone stimulation in infantile protein-calorie malnutrition

Abstract Growth hormone kinetics in infantile malnutrition are characterized by high basal values and decreased responses to various provocative stimuli. The mechanism of this alteration has not been explained. To determine whether altered sensitivity to growth hormone releasing hormone (GHRH) plays a role in this phenomenon, we perfomed GHRH testing in 19 infants with primary malnutrition of grades II and III before and after 6 weeks of nutritional rehabilitation. GHRH (1–44) NH2 was administered intravenously by bolus injection at a dose of 1 mcg/kg. Mean baseline GH maximum elevation above baseline (ΔGH), and area under the Gh response curve (Σ GH) were measured. The injections stimulated a rapid rise in GH which reached a maximum at 15–30 mins after GHRH. There were no significant differences in the magnitude or time couse of the responses between infants in the malnourished and recovering states (Δ GH 29±7 vs. 34±5 ng ml:Σ GH 3379±571 vs. 3459±397 ng. min/ml respectively), and responses were similar to thoses of normal prepuberal children. We conclude that altered sensitivity of pituitary to GHRH appears not to underlie the alterations of growth hormone dynamics observed in malnutrion.

Direct effect of the luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH on rat testicular steroidogenesis.

The luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH (LHRHa) inhibits rat testicular testosterone secretion. To determine whether LHRHa decreases serum testosterone concentrations solely by inhibiting gonadotropin secretion or, in addition, by influencing directly testicular testosterone biosynthesis, we examined the effects of LHRHa on the activities of 5 key testicular steroidogenic enzymes. Thirty hypophysectomized, hOG treated rats were given either LHRHa (1 micrograms sc/day) or saline during 7 days. The LHRHa treated animals exhibited a significant decrease of serum testosterone when compared to the control group (498 +/- 37 ng/dl vs 2044 +/- 105 ng/dl, mean +/- SEM, P less than 0.001). 17-Hydroxyprogesterone serum levels were also decreased in the LHRHa treated rats (61 +/- 6 ng/dl vs 93 +/- 7 ng/dl, P less than 0.005), while serum progesterone levels were similar in both groups of animals. These changes in steroid concentrations were associated with decreases in the microsomal enzyme activities of 17-hydroxylase (37 +/- 9 vs 654 +/- 41 pmol/mg protein/min, P less than 0.001), 17,20-desmolase (103 +/- 9 vs 522 +/- 47 pmol/mg protein/min, P less than 0.001), 3 beta-hydroxysteroid dehydrogenase (1.7 +/- 0.02 vs 4.1 +/- 0.1 nmol/mg protein/min, P less than 0.001), aromatase (95 +/- 7 vs 228 +/- 6 pmol/mg protein/min, P less than 0.001) and 17-ketosteroid reductase (167 +/- 9 vs 290 +/- 18 pmol/mg protein/min, P less than 0.01) in the LHRHa treated animals. These findings indicate that LHRHa can inhibit directly rat testicular testosterone biosynthesis.