Effects of dopamine on immunoreactive growth hormone-releasing factor and somatostatin secretion from rat hypothalamic slices perifused in vitro.

The effects of dopamine (DA) on the release of GRF and somatostatin (SRIF) from the hypothalami of adult male rats were examined in an in vitro perifusion system using horizontal hypothalamic slices, 400 micron thick, including the median eminence and arcuate nuclei. When hypothalamic slices from five animals were perfused in a chamber with artificial cerebrospinal fluid (ACSF) at a flow rate of 100 microliters/min under a gaseous phase of 95% O2 and 5% CO2 at 37 C, rat (r) GRF- and SRIF-like immunoreactivities (-LI) were constantly detected in 30-min perifusates at least until 240 min of perifusion, and during the perifusion with 60 mM K+, the concentrations of rGRF-LI and SRIF-LI were increased 2.1 and 3.2 times, respectively, over basal values. Under the perifusion with ACSF containing normal goat gamma-globulin, the addition of 10(-8) M DA resulted in a significant increase in SRIF-LI from 8.2 +/- 0.3 to 14.3 +/- 1.5 pg/hypothalamus.30 min, but conversely, it caused a slight but significant decrease in rGRF-LI from 4.5 +/- 0.9 to 2.0 +/- 0.3 pg/hypothalamus.30 min. On the other hand, 10(-8) and 10(-6) M DA significantly stimulated rGRF-LI release from hypothalamic slices perifused with ACSF containing anti-SRIF goat gamma-globulin. These findings suggest that DA is a secretagogue for both SRIF and rGRF in the hypothalamus, but the rGRF-stimulating effect of DA is masked unless the action of endogenous SRIF is attenuated.

Galanin stimulates immunoreactive growth hormone-releasing factor secretion from rat hypothalamic slices perifused in vitro.

The effect of galanin (GAL) on the release of GH-releasing factor (GRF) and somatostatin (SRIF) was examined in an in vitro perifusion system of rat hypothalamic slices. GAL at doses of 10(-7) and 10(-6)M stimulated the release of immunoreactive GRF while it failed to affect SRIF release. Therefore, in vivo stimulation of GH release by GAL may be explained in part by the GRF-releasing effect of this peptide.

GHRH treatment: studies in an animal model.

This study examined the effects of chronic deletion of circulating growth hormone-releasing (GHRH) and/or somatostatin (SRIF) on normal growing male rats, as well as the effects of exogenous GHRH (1-29)NH2 and/or SMS 201-995 administration on the growth of rats with hypothalamic ablation. Passive immunization with anti-rat GHRH goat gamma-globulin (GHRH-Ab) for 3 weeks caused a marked decrease in the levels of pituitary GH mRNA and severe growth failure. Treatment with anti-SRIF goat gamma-globulin (SRIF-Ab) for 3 weeks produced a more modest decrease in GH mRNA levels in the pituitary and a slight but significant inhibition of normal somatic growth. Hypothalamic ablation produced a marked decrease in the level of mRNA in the pituitary. Chronic continuous administration of GHRH (1-29)NH2 stimulated pituitary GH synthesis, elevated serum levels of insulin-like growth factor I and increased body weight gain in rats with hypothalamic ablation treated with replacement doses of cortisone, testosterone and L-thyroxine. Combined treatment with GHRH (1-29)NH2 and SMS 201-995 appeared to promote the effect of GHRH on pituitary GH release and somatic growth in these animals. The results suggest that continuous administration of GHRH will be useful in the treatment of children with growth retardation resulting from hypothalamic disorders. In children with combined GHRH and somatostatin deficiencies, the addition of somatostatin to a GHRH treatment regimen may produce better results.

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6).

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.

Molecular genetics of growth hormone-releasing factors.

To elucidate the effects of GRF on GH synthesis and of the GH-IGF-I axis on hypothalamic GRF synthesis, we measured the mRNA levels of hypothalamic GRF and pituitary GH in pubertal male rats treated for 3 weeks with antirat GRF gamma-globulin (GRF-ab), antisomatostatin gamma-globulin (SRIF-ab) or both. Immunoneutralization of circulating endogenous GRF resulted in a marked decrease in serum IGF-I and pituitary GH mRNA levels in Northern blot analysis, whereas it significantly increased GRF mRNA levels in the arcuate nucleus in both Northern blot and in situ hybridization analysis. Immunoneutralization of circulating endogenous SRIF failed to affect GRF mRNA levels but caused a slight reduction in pituitary GH mRNA. Then, we examined the effect of systemic replacement with rat GH or IGF-I for 1 week on GRF mRNA levels in the hypothalamus of rats treated with GRF-ab for 2 weeks. Replacement with either rat GH or IGF-I significantly suppressed the increased hypothalamic GRF mRNA levels. These data indicate that endogenous GRF is essential in normal synthesis of pituitary GH and that both GH and IGF-I negatively regulate the synthesis of hypothalamic GRF.

Intranasal administration of His-D-Trp-Ala-Trp-D-Phe-LysNH2 (growth hormone releasing peptide) increased plasma growth hormone and insulin-like growth factor-I levels in normal men.

The effects of intranasal and iv administration of His-D-Trp-Ala-Trp-D-Phe-LysNH2 (GHRP) on plasma GH, PRL, LH, FSH, TSH, cortisol, insulin, IGF-I as well as GHRH-like immunoreactivity (LI) levels were examined in 6 healthy male subjects. An iv bolus injection of GHRP(1 micrograms/kg BW) caused a remarkable increase in plasma GH levels with a mean (+/- SE) peak of 54.9 +/- 4.2-micrograms/L. In addition an intranasal administration of GHRP resulted in a significant, dose-related increase in plasma GH with peaks of 39.6 +/- 15.3 micrograms/L at a dose of 30 micrograms/kg BW, 14.1 +/- 5.0 micrograms/L at 15 micrograms/kg BW and 7.5 +/- 5.7 micrograms/L at 5 microgram/kg BW. Plasma PRL and cortisol levels were slightly but significantly increased after iv administration of GHRP, whereas GHRP totally failed to affect plasma TSH, LH, FSH, insulin, blood sugar and GHRH-LI levels. Seven consecutive, intranasal administrations of 15 micrograms/kg BW GHRP every 8h were well tolerated in all subjects examined. During this treatment, GH responsiveness to GHRP was not attenuated by desensitization and plasma IGF-I was increased from 94.5 +/- 5.8 micrograms/L before GHRP to 125.8 +/- 6.0 micrograms/L after repeated GHRP administration. These findings indicate that intranasal administration of GHRP stimulates GH secretion and consequently enhances IGF-I production in normal subjects. If GHRP is demonstrated to be beneficial in the treatment of some patients with GH deficiency, the intranasal route of administration may be more useful than the painful injection because a prolonged period is required for the treatment.

Genotype-phenotype correlation of patients with multiple endocrine neoplasia type 2 in Japan.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN 2 is caused predominantly by germ-line mutations of the RET proto-oncogene. This study aimed to clarify the genotype-phenotype correlation in MEN 2 patients in Japan in order to modify the clinical management according to the genotype. METHODS: Constitutive DNA of 64 MEN 2 patients (48 kindreds) were searched for mutations at exons 10, 11, 13, 14 and 16 of the RET proto-oncogene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing and restriction enzyme digestion. The clinical characteristics of the patients were obtained from a previous nationwide questionnaire survey. RESULTS: Overall, 62 (96.9%) out of 64 patients had a germ-line point mutation at the hot spots. MTC and pheochromocytoma occurred equally in every genotype except C630S. Specific genotype had a correlation between tumor size and age at the operation for MTC or extent of MTC, i.e. C618S developed late onset type of MTC as compared with that of C634R, C634Y and M918T. Small MTC in C634R may be less aggressive than those in C634Y and M918T. CONCLUSIONS: DNA testing has good clinical implications for the management of patients with MEN 2 and the timing and operative procedures of thyroidectomy can be modified according to the genotype.