Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

Abstract

Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor )NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OHJ were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund's adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster in-jection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioinmunoassays for these peptides. By using antibodies with an apparent K. of 3.3x 10-12 (human) and 7.7 x 10-11 (rat), the sensitivity of these assays in both human and rat was found to be <1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOHterminal region of the molecule, as shown by its crossreactivity with various hpGRF analogues: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(140)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 12,5-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 ± 0.2 for the human and 11.1 ± 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2 to 16 weeks and a correlation between increasing body weight and GRF content. These radioimmunoassays winl serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.Since 1959 (1), it has been known that the hypothalamus is involved in the control of growth hormone (GH) secretion. Experimental and clinical evidence has since indicated that the hypothalamus performs a dual function in the regulation of GH secretion from the anterior pituitary (2-4). Inhibition of GH release is accomplished by an inhibitory peptide, somatostatin (SRIF), which in 1972 was isolated from ovine hypothalami, purified, and characterized as a tetradecapeptide (5). In contrast, as suggested by physiological evidence (2-4) and GH-releasing activity in hypothalamic extracts (6-8), stimulation of GH release is accomplished by a stimulatory peptide, GH-releasing factor (GRF). Until 1982, howev-er, the isolation and characterization of GRF remained elusive. The difficulty in its characterization had been due to the minute quantities of the peptide in hypothalamic tissue, the large quantities of SRIF that interfere in the bioassay, and the fact that the methodology used for peptide sequencing and purification has only recently become available (9-12).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2970Many investigators have reported the presenc...

Sexual and Developmental Differences in Peptides Regulating Growth Hormone Secretion in the Rat

“…The present data verifies an abstract that reports higher hypothalamic rGRF-IR content in adult male versus female rats, and agrees with previously reported levels of this peptide [3,6,32]. Although nonsignificant sex differences in whole hypothalamic rGRF-IR content have been reported, the magni tude of these differences (35%) compares with the present study [19], We analyzed the median eminence separately in the juve nile-development study to isolate this important nerve terminal bed.…”

Immunocytochemical localization of growth hormone and growth hormone‐releasing hormone immunoreactivity in the brain and pituitary of the little brown bat

“…Identification of GHRH-immunoreactive cell bodies in this nucleus was unexpected, as it has not been reported for other mammals. Consistent observations of immunoreactive perikarya and fibers in the neocortex were also surprising, as only one previous study refers to immunocytochemical localization of GHRH in rat cerebral cortex (Audhya et al, 1985). In little brown bats, Laemle and Cotter (1988a) recently reported VIPlike immunoreactivity in suprachiasmatic neurons as well as in neuronal elements in the cerebral cortex.…”

Role of Locally Produced Growth Hormone-Releasing Factor in Somatostatin Regulation by Fetal Rat Brain Cells in Culture

“…Although immunohistochemical and RIA studies showed that the highest SS concentrations are found in the hypothalamus [2,3], this peptide is wide spread throughout the central [4] and peripheral [5] ner vous systems, where it has been suggested that SS may have a role as a neurotransmitter or neuromodulator, thus integrating neuroendocrine and behavioral responses [6]. Similarly, the highest GRF concentrations are located in the median eminence and arcuate nucleus [7], but detect able levels of immunoreactive GRF (IR-GRF) have also been identified in extrahypothalamic brain areas, with functions yet to be defined [8], Ultrastructural [9,10] and biochemical [11,12] evi dence strongly suggest that SS and GRF may interact within the hypothalamus to modulate hypophysial GH secretion, but their regulatory interactions have not been adequately delineated. Furthermore, because GRF and SS are both synthesized and released by extrahypothala mic neurons, a possible functional interaction related to neuromodulation may exist between these neuropeptides in the cortex.…”