Hypothalamic Growth Hormone-releasing Factor Research Articles

Specific depletion of immunoreactive growth hormone-releasing factor by monosodium glutamate in rat median eminence.

A potent and specific growth hormone-releasing factor (GRF) was recently isolated and characterized from a human islet cell tumour of the pancreas that caused acromegaly. Antibodies raised against the synthetic replicate of this peptide have allowed the immunohistochemical identification of GRF-producing neurones within the primate central nervous system. Such neurones are found mainly in the arcuate nucleus in human and monkey hypothalamus, suggesting that this nucleus is a primary source of GRF. We have further investigated this hypothesis by studying the anatomical organization of GRF neurones in rat hypothalamus, using an antibody raised against the recently characterized rat hypothalamic GRF in normal animals and in animals neonatally treated with monosodium glutamate (MSG), a treatment which results in the selective destruction of arcuate nucleus neurones. We present here the results which show that GRF-producing neurones are located mainly in the arcuate nucleus of rats. MSG treatment results in the complete loss of GRF-immunoreactive cell bodies within this nucleus and provokes a selective disappearance of GRF-immunoreactive fibres in the median eminence. These results show that the arcuate nucleus is the origin of the GRF-containing fibres that project to the median eminence and establish the MSG-treated rat as an in vivo model for studying growth hormone secretion in the absence of neurohumoral GRF.

Isolation and characterization of the bovine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 500 bovine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analysis. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr Ala-Asp-Ala-Ile- Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg- Leu-NH 2 using approximately 2 nmol of material.

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp- Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH 2 using approximately 6 nmol of material.

Invivo biological potency of rat and human growth hormone-releasing factor and fragments of human growth hormone-releasing factor

The biological potency of the synthetic replicates of three peptides isolated from a human pancreatic tumor with growth hormone releasing activity and rat hypothalamic growth hormone-releasing factor was evaluated in conscious freely-moving rats and anesthetized rats. All 4 peptides are equipotent on a molar basis in their ability to stimulate GH secretion. Studies with synthetic fragments of the human derived material indicated that the amino-terminal amino acid is required for activity. Deletion of as many as 13 amino acids from the carboxy-terminal failed to decrease GH-releasing activity; however, deletion of 16 amino acids resulted in a significant decrease and deletion of 20 amino acids resulted in complete loss of bioactivity.

Human hypothalamic growth hormone releasing factor (GRF): Evidence for two forms identical to tumor derived GRF-44-NH 2 and GRF-40

Human hypothalamic growth hormone-releasing factor (GRF) was purified by gel filtration and reverse-phase HPLC. Bioassay and two radioimmunoassays of different specificity revealed the presence of two major forms of GRF-activity which coelute with human pancreas GRFs, hpGRF-44-NH 2 and hpGRF-40 previously characterized in pancreas tumors. The bioactive material coeluting with hpGRF-44-NH 2 is recognized by two antibodies which are directed against the amidated COOH-terminal sequence and the central portion of the GRF-44 peptide. The bioactive GRF which coelutes with hpGRF-40 reacts only with the antibody directed against the central portion of hpGRF. These data strongly suggest that the human hypothalamus contains the same major forms of GRF that were identified in pancreas tumors responsible for acromegaly in the absence of a pituitary tumor.

Human growth hormone releasing factor and somatostatin from two pancreatic tumors: isolation and characterization.

Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior.

Immunohistochemical detection of growth hormone-releasing factor in brain.

The concept of a hypothalamic neurohumoral control for anterior pituitary secretion postulates the existence of a growth hormone-releasing factor (GRF) of neuronal origin that stimulates the pituitary gland to release growth hormone (GH). Such a compound has not yet been isolated and characterized from the brain, although there is extensive physiological and biochemical evidence for its existence (reviewed in ref. 2). However, a 44-amino-acid amidated peptide having the physiological properties of GRF as well as chemical similarities was recently isolated from a human pancreatic tumour that had caused acromegaly. Two shorter biologically active fragments of 40 and 37 residues were also isolated. The synthetic replicates of these human pancreas GRF (hpGRF) peptides specifically stimulate GH release in vitro and in vivo. Assuming similarity or identity between the putative hypothalamic GRF and the tumour-derived hpGRF, we have used immunohistochemistry to search for hpGRF-like immunoreactivity in the brain. We report here that antisera against the hpGRF1-40 peptide specifically stain neuronal cell bodies in the arcuate nucleus of the primate hypothalamus, with fibres projecting to the median eminence and ending in contact with portal vessels. This topography is characteristic of a neuronal system elaborating a releasing factor. These results provide evidence that hypothalamic GRF is very similar, if not identical, to hpGRF.

The absence of an age-related change in the pituitary response to growth hormone-releasing factor in rats.

The capability of the anterior pituitary gland to secrete growth hormone (GH) in response to an intravenous injection of growth hormone-releasing factor, hpGRF-44, was evaluated in male rats ranging from 22 days to 24 months of age. The increase in GH concentrations following a submaximal dose of hpGRF-44 (0.5 microgram/kg) was similar among the different aged rats suggesting there is no age-related change in pituitary sensitivity to hpGRF-44. Likewise, the response to a maximal dose of this peptide (25 micrograms/kg) was not different in young and old animals indicating there is no decrease in the readily releasable pool of GH. Thus, the age-related changes in GH secretion observed in rats do not appear to be due to a change in the pituitary response to the hypothalamic growth hormone-releasing factor.

A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor.

Rats were passively immunized with an antiserum against somatostatin and a monoclonal antibody against rat hypothalamic growth hormone-releasing factor (rGRF-mAb) which does not recognize the biologically active 1-40 amino acid fragment of hpGRF-44 (hpGRF-40). Using this paradigm we have observed that the pituitary possesses a resilient capacity to release growth hormone (GH) following repeated injections of hpGRF-40 and that this response follows a dose-dependent relationship.

Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro.

Purified (rat) hypothalamic growth hormone releasing factor (GRF), native human GRF isolated from an islet cell tumor of the pancreas that had caused acromegaly, and the synthetic replicates of the human material are potent secretagogues of immunoreactive growth hormone (GH) by primary cultures of rat pituitary cells. Native or synthetic peptides give identical dose-response curves, with identical slopes and identical maximal effects. The median effective dose of the tumor-derived GRF is 15 x 10(-12) M. The effect of hypothalamic GRF or of a synthetic replicate of tumor-derived GRF is immediate, being demonstrable in less than or equal to 30 sec after contact in a pituitary cell perifusion system. The effect of hypothalamic GRF or of tumor-derived GRF is highly specific for stimulating release of immunoreactive growth hormone; there is no demonstrable concomitant effect on the secretion of other pituitary hormones. Somatostatin-28 and somatostatin-14 inhibit the release of growth hormone produced by hypothalamic GRF or tumor-derived GRF in typical noncompetitive antagonism. On the basis of the results reported here, hypothalamic GRF and tumor-derived GRF are qualitatively indistinguishable in their ability to stimulate the secretion of immunoreactive growth hormone in vitro. The name "somatocrinin" is proposed to replace the acronym GRF.

Monoclonal antibodies to hypothalamic growth hormone-releasing factor with picomoles of antigen.

Monoclonal antibodies specific for rat hypothalamic growth hormone-releasing factor (rGRF) have been produced by in vitro immunization of mouse spleen cells with less than 1 nanomole of rGRF in a partially purified preparation. Hybridoma supernatants were screened for anti-rGRF activity by use of a pituitary culture assay system that can detect growth hormone-releasing factor in the femtomole range. Such highly sensitive in vitro techniques permit the use of picomole quantities of an antigen in partially purified preparations for the isolation of monoclonal antibodies, which can in turn be used in biological studies and in immunochemical procedures for large-scale purification and isolation of that antigen.

Paradoxical elevation of growth hormone by intraventricular somatostatin: possible ultrashort-loop feedback.

Somatostatin, the growth hormone-inhibiting factor, when microinjected into the third ventricle of the rat brain, paradoxically induced the release of growth hormone. A pituitary site of action having been ruled out, this result supports the concept that exogenous somatostatin within the hypothalamus acts either to suppress the release of somatostatin from somatostatin-containing neurons, possibly via an ultrashort-loop feedback mechanism, or to augment release of hypothalamic growth hormone-releasing factor, thereby inducing a release of growth hormone. Injection of somatostatin into the third ventricle also decreased plasma concentrations of luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, probably by inhibiting the release of luteinizing hormone-releasing factor and thyrotropin-releasing factor.

Effect of a bovine hypothalamic factor on the release and biosythesis of growth hormone.

Partial purification of growth hormone (GH)-releasing factor (GRF) by acid extraction followed by gel filtration on Sephadex G-25 has been attained from bovine hypothalami. When rat pituitaries were incubated in 2 ml Krebs Ringer-bicarbonate-glucose (KRBG) medium, a stimulatory effect of the GRF fraction on immunoreactive GH (IR-GH) release was observed, while that of the factor neither on GH synthesis nor release of the synthesized GH was demonstrated. Stimulation of the GH release was exerted maximally within 30 min of incubation. Cycloheximide and actinomycin D, at a concentration which inhibited protein and RNA synthesis to less than 5 and 20% of the control, respectively, were without effect on the stimulatory action of the factor on GH release. On the other hand, stimulation of GH synthesis was observed under incubation in 0.3 ml medium with the factor and enhancing effect of the factor on the IR-GH release was undetectable. These results suggest that stimulation of the release and synthesis of GH mediated by the hypothalamic GRF fraction is under influence of the pool size of incubation media.

Immediate release of prolactin and biphasic effects on growth hormone release following electrical stimulation of the median eminence.

The median eminence (ME) region of the hypothalamus was electrically stimulated through permanently implanted electrodes in 1 unanesthetized sheep. Plasma concentrations of GH and prolactin (PRL) were measured at 10-min intervals before, during and after 30-min periods of electrical stimulation or sham procedures. The onset of ME stimulation decreased plasma GH in all 17 cases in which prestimulation GH levels were 1.0 ng/ml or higher. In all 27 cases of ME stimulation, plasma GH never increased during the 30-min stimulation period. However, spontaneous GH increases were noted during sham procedures in 9 of 31 cases. The termination of ME stimulation was followed within 10--20 min by markedly increased GH levels in 24 of 27 cases. All 4 ewes responded to electrical stimulation with comparable biphasic effects on GH release even though electrode locations varied slightly. These results indicate localization and stimulation-induced release of endogenous neurohormones in the ME region with activities inhibiting the release of GH (GHIF). The secondary increase in plasma GH following the end of ME stimulation indicates a reversal of conditions unfavorable to GH release. This reversal may represent a rebound from the inhibitory action of GHIF or the post-inhibitory action of a hypothalamic GH-releasing factor or both. In 3 ewes with electrodes implanted in the region of the anterior ME, the onset of stimulation increased plasma PRL within 10--20 min in 17 out of 20 cases. The electrode in the fourth animal was located in the posterior ME, and when all cases were considered, stimulation through this electrode did not significantly increase or decrease plasma PRL. However, in 5 out of 7 cases stimulation of the posterior ME decreased plasma PRL. In summary, these results indicate the presence in the anterior ME of andogenous neurohormones with PRL-releasing activity (PRF). Conclusions about the posterior ME are equivocal, but this region may contain a PRL-release inhibiting compound in combination with PRF.

Serial plasma growth hormone concentration during selective removal of pituitary tumors in acromegaly.

✓ The pituitary function of two patients with acromegaly was monitored before, during, and after selective transnasal pituitary adenomectomy. The purpose of the study was to verify the efficacy of this technique, and to see if there was normal function in the residual pituitary tissue. Following surgery, both patients showed clinical improvement with no evidence of significant anterior or posterior pituitary dysfunction. Concentrations of plasma growth hormone obtained serially during surgery were elevated prior to tumor removal but fell precipitously after adenomectomy. The half-life for immunoreactive growth hormone in these patients was less than 35 minutes. We conclude that selective transnasal pituitary adenomectomy is an effective treatment for pituitary tumors. Furthermore, our data suggest that the pituitary tumor in these patients did not arise because of excessive stimulation by hypothalamic growth hormone-releasing factor.

Inhibition and Stimulation of Rat Growth Hormone Secretionin vitroBy Hypothalamic Extracts

An extract of ovine hypothalami, partially purified on Sephadex, inhibited the synthesis and release of GH in vitro by rat anterior pituitaries incubated sequentially in the presence of 14C-leucine and then 3H-leucine with the inhibitory hypothalamic factor (GIF). Effects on GH synthesis and on the release of both newly synthesized and stored GH were determined using an immunoprecipitation technique. GIF decreased GH synthesis and decreased release of newly synthesized GH while not affecting release of older, stored GH. This effect contrasted with that of a hypothalamic GH releasing factor (GRF) which increased the release of newly synthesized GH while not affecting the rate of GH synthesis or the release of older GH. GIF inhibition was reversible since glands preincubated in its presence later responded characteristically to GRF. GIF reduced the release of both newly synthesized and stored GH produced by 21mM K+. Prolactin synthesis and release were not affected by GIF or GRF. The findings support the concept of two pools of pituitary GH. Each pool, distinguishable according to time of synthesis, was differentially affected by GIF and GRF with both hypothalamic factors exerting primary effects upon the newly synthesized pool.

Hypothalamic Growth Hormone Releasing Factor: Release and Synthesis After Exposure to a High Ambient Temperature

SummaryThis study was designed to investigate temporal effects of environmental heat stress on the release and synthesis of hypothalamic GHRF in rats. The experiments consisted of five treatment groups; one group of normals at 25° ± 1° and four groups, each exposed to 35.5° ± 0.5° for 1, 4, 96, and 240 hr, respectively. GHRF activity was measured by in vivo depletion of pituitary GH content in immature rats. Vasopressor and antidiuretic activities of crude milk median eminence extracts were abolished by treating the extracts with thioglycollic acid. Exposures for 1, 4, and 96 hr significantly (p < 0.01) elevated GHRF activity as compared to the control which returned to a normal level by 240 hr. No significant difference existed in GHRF activity for two consecutive periods of exposures, indicating a continuous synthesis of GHRF. Enhanced GHRF activity on heat exposure has been discussed in light of the increased endogenous turnover of hypothalamic norepinephrine on heat exposure and the enhanced firing ra...

A dialyzable heat stable factor in acromegalic plasma associated with increased in vitro growth hormone release.

A number of reports in the recent literature have suggested the possibility of persistent hypothalamic control in acromegaly (1-3), and further that a primary pathogenetic defect may be characterized by excessive secretion of hypothalamic growth hormone releasing factor. In a previous communication from this laboratory we have shown greater in vitro release of monkey pituitary growth hormone in the presence of acromegalic plasma than in the presence of normal plasma (4). It is the purpose of this report to further characterize this observation. Methods Intact rhesus monkey pituitary glands were obtained and handled in a manner previously described (4). The incubation mixture of 10 ml included lactated ringers 7.5 ml, containing 50 μg/ml neomycin, and a glucose concentration of 100 mg/100 ml, to which 2.5 ml aliquots of plasma or dialysates of plasma with added albumin were added for study purposes. All incubations were carried out in a Dubnoff shaker at 37°, aliquots for immunoreactive growth hormone were obtained at 30 sec after the start of an incubation, during and at the conclusion of two hour incubation periods. Growth hormone was assayed using minor modifications of a solid phase radioimmunoassay (5), with highly specific rabbit anti-growth hormone antibodies, produced in our laboratory. The standard is purified human growth hormone, NIH HS-1147. Three types of experiments were carried out. In one, whole pituitaries were incubated for 2 hr periods, first either in the presence of normal or acromegalic plasma, washed and transferred to a different plasma and then finally back to the original medium. Three intact glands were started first in acromegalic plasma, transferred to normal plasma and back for the final 2 hr of incubation to acromegalic palsma. Two pituitary glands were incubated in reverse order.

In vitro release of monkey pituitary growth hormone by acromegalic plasma.

ABSTRACT Several recent reports have proposed that acromegaly may be caused by excessive release of hypothalamic growth hormone releasing factor. In an effort to gain additional support for this hypothesis, evidence for the presence of growth hormone releasing substances in the peripheral blood of acromegalics was sought. Monkey pituitary glands incubated in the presence of acromegalic plasma demonstrated a 4- to 5-fold greater release of growth hormone than obtained from pituitaries in the presence of normal plasma. Although this may reflect raised levels of a circulating hypothalamic growth hormone releasing factor in acromegalic plasma, other factors may be responsible.

Regulation of growth and plasma growth hormone in a Laron's dwarf

Laron identified a group of children with severe growth failure and high levels of immunoreactive human growth hormone (IR-HGH). To investigate why these patients often have elevated HGH, we studied a 7½ year old Saudia Arabian boy with a height of 85 cm (HA 2yrs) and basal IR-HGH levels of 32 to 84 ng/ml (normal—less than 5 nanagrams per ml). Fasting blood sugars were often in the hypoglycemic range, and hyperglycemia induced by glucose infusion did not suppress his high IR-HGH. The serum IR-HGH was increased markedly by arginine infusion. High dose HGH treatment (7.5 mg q 12 hrs × 9 doses) did not modify his IR-HGH response 1 hour after arginine (increment over control was 111 and 121 ng/ml before and after treatment). Plasma sulfation factor (SF) was low and did not rise with treatment, as it does in the usual HGH deficient patient. Chronic adminsitration of HGH (2.5 mg × per week) produced a modest growth acceleration of 2.6 cm over the 5 months period compared to an expected response of 6.5 to 12 cm per year, in patients lacking HGH. These studies suggest that the persistently elevated IR-HGH is due to either a lack of SF to suppress the hypothalamic growth hormone releasing factor or that the hypothalamic HGH receptors share the same defects as those receptors responsible for the initiation of SF synthesis. Regardless of the interpretation, the IR-HGH in these patients does not appear to be under the hypothalamic control system demonstrable in normal individuals.