Growth hormone-releasing hormone: structure, gene expression and molecular heterogeneity.
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Amino Acid Sequence↗
Biomedical subjects
T R Downs
Publications and source records attributed to T R Downs.
Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia.
To elucidate the role of growth hormone (GH)-releasing hormone (GRH) and somatostatin (SRIH) in the regulation of the growth hormone (GH) secretory pattern, we collected portal blood from five unanesthetized ovariectomized ewes for repeated measurements of GRH and SRIH simultaneous with those of peripheral GH. Hormones were measured at 10-min intervals for 5.5 h and their interrelationships analyzed. Mean portal GRH was 20.4 +/- 6.7 (SD) pg/ml and the estimated overall secretion rate was 13 pg/min. GRH secretion was pulsatile with peaks of 25-40 pg/ml and a mean pulse interval of 71 min. Mean portal SRIH was 72 +/- 33 pg/ml and the estimated overall secretion rate was 32 pg/min. SRIH secretion was also pulsatile with peaks of 65-160 pg/ml and a mean pulse interval of 54 min. The GH pulse interval was 62 min. A significant association was present between GRH and GH secretory peaks though not between GRH and SRIH or SRIH and GH. Insulin hypoglycemia resulted in a rapid and brief stimulation of SRIH secretion followed by a decline in GH levels. No effect was observed on GRH secretion until 90 min, when a slight increase occurred. The results suggest (a) the presence of an independent neural rhythmicity of GRH and SRIH secretion with a primary role of GRH in determining pulsatile GRH secretion, and (b) that the inhibitory effects of insulin hypoglycemia on GH in this species are attributable to a combination of enhanced SRIH secretion and possibly other factors, though without significant inhibition of GRH.
Animals↗
Growth hormone and prolactin secretion in cultured somatomammotroph cells.
A somatomammotropic cell line (P0) derived from adult rat pituitaries has been maintained in culture for 2 yr. Secretion of GH and PRL by this cell line has been studied in response to hypophysiotropic peptides known to affect the release of both hormones as well as agents that affect second messenger systems in an attempt to characterize the stimulus-secretion mechanisms used by these cells. GH and PRL release during short term (4 h) incubations of P0 cells and primary cultures of dispersed rat pituitary cells was initially measured in response to GRF, TRH, vasoactive intestinal peptide (VIP), and SRIF. In P0 cells, the minimal effective dose of each of the hypophysiotropic peptides was comparable with respect to GH and PRL secretion. The effects of TRH and VIP were similar to those in freshly dispersed cells with respect to PRL release, whereas those of GRF and SRIF were less potent with respect to GH release. The stimulation of GH and PRL release in P0 cells by adenylate cyclase-related agents ((Bu)2 cAMP and forskolin) was comparable to that for GH secretion in mature somatotrophs but much greater than that of PRL release in mature lactotrophs. Stimulation of GH and PRL release in P0 cells by protein kinase C-related agents (diacylglycerol and phorbol ester) was also similar to that observed for GH release from mature pituitary cells, whereas minimal or undetectable effects were observed on PRL release from mature cells. The results indicate that the P0 somatomammotropic cell line possesses receptors, second messenger systems, and secretory characteristics of both somatotrophs and lactotrophs, although where differences exist, there is more resemblance to somatotrophs. They also demonstrate that the responses to each of the agents studied are bihormonal and appear to be regulated by a common mechanism.
Animals↗
Tissue distribution and molecular heterogeneity of human growth hormone-releasing factor in the transgenic mouse.
Transgenic mice expressing the human growth hormone-releasing factor (hGRF) gene linked to the metallothionein promoter exhibit high circulating levels of hGRF and GH and increased growth. We have described the distribution of GRF immunoreactivity (GRF-IR) in various tissues and characterized its molecular heterogeneity using gel filtration and high performance liquid chromatography (HPLC) and two separate RIAs that recognized mid-molecule and carboxyl-terminal epitopes of hGRF. The highest levels of GRF-IR were in the pituitary, followed by the pancreas. Intermediate levels were present in hypothalamus and liver, and lower levels in visceral organs, heart, and gonads. The pituitary and brain revealed evidence of the two mature hormone forms [hGRF(1-44)-NH2 and hGRF(1-40)-OH and in addition a more hydrophobic form that is believed to represent the hGRF precursor (proGRF) on the basis of its estimated molecular size (approximately 9,000) and selective recognition by the mid-molecule RIA. The profiles of GRF in pancreas and gut were similar except that only minimal amounts of hGRF(1-40)-OH were detected. In contrast, neither mature hormonal form was present in the liver and plasma contained primarily hGRF(3-44)-NH2, the major circulating metabolite of hGRF. The results provide evidence for variable processing of the hGRF precursor that is tissue specific and indicate that several extrahypothalamic tissues possess the necessary complement of enzymes to generate the mature hormonal forms.
Animals↗
Effects of thyroid hormone deficiency and replacement on rat hypothalamic growth hormone (GH)-releasing hormone gene expression in vivo are mediated by GH.
The role of thyroid hormone and GH in the regulation of hypothalamic GH-releasing hormone (GRH) gene expression in the rat was examined after the induction of thyroid hormone deficiency by thyroidectomy. Thyroidectomy resulted in a time-dependent decrease in hypothalamic GRH content, which was significant by 2 weeks postoperatively, and a reduction in pituitary GH content to 1% of the control level by 4 weeks. In contrast, GRH secretion by incubated hypothalami under both basal and K(+)-stimulated conditions was increased after thyroidectomy. Hypothalamic GRH mRNA levels also exhibited a time-dependent increase, which was significant at 1 week and maximal by 2 weeks after thyroidectomy. Administration of antirat GH serum to thyroidectomized rats resulted in a further increase in GRH mRNA levels. T4 treatment of thyroidectomized rats for 5 days, which also partially restored pituitary GH content, lowered the elevated GRH mRNA levels. However, comparable effects on GRH mRNA levels were observed by rat GH treatment alone. These results suggest that the changes in hypothalamic GRH gene expression after thyroidectomy in the rat are due to the GH deficiency caused by thyroidectomy, rather than a direct effect of thyroid hormone on the hypothalamus, since the changes were reversible by GH alone despite persistent thyroid hormone deficiency. In addition, they further support the role of GH as a physiological negative feedback regulator of GRH gene expression.
Animals↗
Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.
The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step cleavage by a dipeptidylpeptidase (DPP) rather than sequential aminopeptidase cleavages. Conversion to GRH(3-44)-NH2 was blocked by diprotin A, a DPP type IV (DPP IV) competitive inhibitor. D-Amino acid substitution at either position 1 or 2 also prevented hydrolysis, characteristic of DPP IV. Analysis of endogenous plasma GRH immunoreactivity from a human GRH transgenic pig revealed that the major peak coeluted with GRH(3-44)-NH2. Native GRH exhibited trypsin-like degradation at the 11-12 position but cleavage at the 12-13 site occurred only with GRH(1-32)-NH2 and GRH(1-29)-NH2. Formation of these metabolites was independent of prior DPP IV hydrolysis but was greatly reduced by trypsin inhibitors. Evaluation of plasma stability of potential GRH super analogues, designed to resist degradation by these enzymes, confirmed that GRH degradation in plasma occurs primarily by DPP IV, and to a lesser extent by trypsin-like enzyme(s).
Amino Acid Sequence↗
Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.
We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat.
Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.
Animals↗
Differential effects of somatostatin (SRIH) and a SRIH analog, SMS 201-995, on the secretion of growth hormone and thyroid-stimulating hormone in man.
We compared the ability of SRIH and SRIH analog, SMS 201-995 (SMS), to inhibit stimulated GH and TSH secretion in men who received 120-min iv infusions of saline, SRIH (5, 50, and 500 micrograms/h), and SMS (3, 30, and 300 ng/kg.h) together with a bolus iv injection of GHRH (1 microgram/kg) and TRH (500 micrograms). Integrated GH secretion during the 60 min after GHRH plus TRH injection was decreased compared to that after saline by (mean +/- SE) 32 +/- 14% (P = 0.059), 78 +/- 5% (P less than 0.001), and 88 +/- 3% (P less than 0.001) during the 5, 50, and 500 micrograms/h SRIH infusions, and by 13 +/- 7% (P = NS), 50 +/- 15% (P less than 0.05), and 80 +/- 6% (P less than 0.001) during the 3, 30, and 300 ng/kg.h SMS infusions. In contrast, integrated TSH secretion was unaltered during the 5 micrograms/h SRIH and 3 ng/kg.h SMS infusions; it decreased by only 43 +/- 5% (P less than 0.001) and 66 +/- 4% (P less than 0.001) during the 50 and 500 micrograms/h SRIH infusions and by 33 +/- 8% (P less than 0.05) and 50 +/- 3% (P less than 0.001) during the 30 and 300 ng/kg.h SMS infusions. Analysis of the dose-response curves indicated approximately 10- and 5-fold greater potencies of SRIH and SMS, respectively, in inhibiting GH as compared to TSH secretion. These results quantify the effect of SRIH as an inhibitor of GH secretion and suggest that if SRIH has a physiological role in the inhibition of TSH secretion in man, it is limited to conditions associated with marked suppression of GH.
Adult↗
Comparison of the sensitivity of growth hormone secretion to somatostatin in vivo and in vitro in acromegaly.
Somatostatin (SRIH) sensitivity in acromegaly was evaluated in vivo by comparing the inhibition of GHRH (1 microgram/kg, iv)-stimulated GH secretion in eight acromegalic and six normal subjects. A SRIH infusion (50 micrograms/h) that inhibited the mean plasma GH response to GHRH by 74 +/- 5% (+/- SE) in normal subjects had no significant effect in the acromegalic patients. However, when two acromegalic patients in whom SRIH had no suppressive effect were excluded from the analysis, the effect of SRIH in the other six (82 +/- 7%) was comparable to that in the normal subjects. Within the acromegalic group, the percent suppression of basal and GHRH-stimulated GH secretion was inversely correlated with both basal plasma GH (r = -0.751; P = 0.03 and r = -0.727; P = 0.04, respectively) and insulin-like growth factor I (r = -0.800; P = 0.02 and r = -0.727; P = 0.04, respectively) concentrations. The in vitro sensitivity to SRIH was studied in pituitary adenomas from five of the acromegalic patients in 3- to 4-day monolayer cultures of dispersed cells. The SRIH IC50 values were lowest in the tumors (8.6-44 pmol/L) from the three patients who had in vivo SRIH sensitivity (suppression of GHRH-stimulated GH secretion) comparable to that in the normal subjects. The IC50 values were higher in the tumors (150 and 21,000 pmol/L) from the two patients that were least responsive to SRIH in vivo. These results indicate that there is considerable variability of SRIH sensitivity in patients with acromegaly. Although the role of this defect in the pathogenesis of acromegaly is uncertain, it may be an important determinant in the degree of elevation of plasma GH levels.
Acromegaly↗
Feedback regulation of growth hormone (GH)-releasing hormone gene expression by GH in rat hypothalamus.
The role of the pituitary, and in particular, of GH in GH-releasing hormone (GRH) gene expression was studied in hypophysectomized rats with and without GH treatment. Hypothalamic GRH mRNA was 6-fold greater in hypophysectomized than in control rats. Increased levels of GRH mRNA were observed at 3 days and the maximal increase was noted at 7 days, postoperatively. Administration of GH to hypox rats partially reversed the increase in GRH mRNA, suggesting a negative feedback regulation by GH of GRH gene activity at the transcript accumulation level. The overall regulation of GRH gene expression, however, appears more complex since GRH mRNA levels and GRH content exhibited discordant changes after both hypophysectomy and GH treatment, suggesting that factors other than GH are required for efficient translation of GRH mRNA.
Animals↗
Intravenous medroxalol stimulates prolactin secretion in normal and hypertensive subjects.
This study evaluated the effects of medroxalol on prolactin secretion. Twelve normal subjects received medroxalol, 1 mg/kg, intravenously and on a separate occasion, 5% dextrose in water. Integrated prolactin secretion during the 3 hours after medroxalol injection was significantly increased as compared with dextrose (P less than 0.001). Intravenous administration of medroxalol, 2 mg/kg, to 10 hypertensive subjects resulted in significant elevation of mean prolactin levels above basal levels at all time intervals measured from 30 to 240 minutes after injection. Oral medroxalol administration to 11 hypertensive subjects for up to 15 months did not alter mean prolactin levels. Medroxalol neither stimulated prolactin release nor decreased dopamine suppression of prolactin release from pituitary cell cultures. In conclusion, intravenous medroxalol stimulates prolactin secretion in both normal and hypertensive subjects. This effect is not likely mediated by a direct action of the drug on the pituitary but rather by an effect either within the central nervous system or of a drug metabolite.
Adult↗
Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat.
The possible regulation of hypothalamic GH-releasing factor (GRF) by GH was studied in untreated and GH-treated hypophysectomized rats by measurement of rat hypothalamic GRF content and release in vitro with a specific GRF RIA. Two weeks after hypophysectomy, animals not receiving hormone replacement showed a marked reduction in hypothalamic GRF content (to 28% of sham-operated control values; P less than 0.001). Replacement therapy with T4, cortisone, and testosterone for 9 days did not correct the decrease in hypothalamic GRF content in hypophysectomized rats, though the addition of GH therapy partially restored GRF levels (to 55% of control values; P less than 0.001). GRF release from incubated mediobasal hypothalamic fragments of hypophysectomized rats was decreased both basally and in response to 30 mM K+. This defect was completely corrected by prior GH treatment. The results suggest an impairment of GRF synthesis and release in the presence of GH deficiency.
Animals↗
Receptor-associated resistance to growth hormone-releasing factor in dwarf "little" mice.
Anterior pituitaries from the dwarf mouse strain "little" did not release growth hormone or accumulate adenosine 3',5'-monophosphate (cyclic AMP) in response to human and rat growth hormone-releasing factor (GRF). Dibutyryl cyclic AMP, as well as the adenylate cyclase stimulators forskolin and cholera toxin, markedly stimulated growth hormone (GH) release. The basis of the GH deficiency in the little mouse may therefore be a defect in an early stage of GRF-stimulated GH release related either to receptor binding or to the function of the hormone-receptor complex.
Animals↗
Measurement of growth hormone-releasing factor.
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Animals↗
Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism.
The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH.
Animals↗
Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.
The effect of plasma on degradation of human growth hormone-releasing hormone (GRH) was examined in vitro and in vivo using high performance liquid chromatography (HPLC), radioimmunoassay (RIA), and bioassay. When GRH(1-44)-NH2 was incubated with human plasma, the t1/2 of total GRH immunoreactivity was 63 min (RIA). However, HPLC revealed a more rapid disappearance (t1/2, 17 min) of GRH(1-44)-NH2 that was associated with the appearance of a less hydrophobic but relatively stable peptide that was fully immunoreactive. Sequence analysis indicated its structure to be GRH(3-44)-NH2. Identity was also confirmed by co-elution of purified and synthetic peptides on HPLC. Biologic activity of GRH(3-44)-NH2 was less than 10(-3) that of GRH(1-44)-NH2. After intravenous injection of GRH(1-44)-NH2 in normal subjects, a plasma immunoreactive peak with HPLC retention comparable to GRH(3-44)-NH2 was detected within 1 min and the t1/2 of GRH(1-44)-NH2 (HPLC) was 6.8 min. The results provide evidence for GRH inactivation by a plasma dipeptidylaminopeptidase that could limit its effect on the pituitary.
Adult↗
Identification of a putative GH-releasing factor (GRF) batch as predominantly ovine CRF with a small quantity of human GRF.
The existence of discordant results regarding the effects of intracerebroventricular (icv) administration of GRF on GH secretion prompted a reexamination of the central actions of GRF and a detailed chemical characterization of the peptide designated as hpGRF-44-NH2. The icv injection of 10 micrograms rat (r) GRF to freely-moving rats caused an acute stimulation of GH release, whereas 10 micrograms of the putative hpGRF peptide icv continued to suppress spontaneous GH secretion. Through a series of biochemical and immunologic studies we demonstrate that peptide hpGRF-44-NH2, code number 92-81-5G-41-47, is predominantly ovine (o) CRF and also contains a small amount (3-5%) human (h) GRF-44-NH2. We conclude that the major effect of icv administered GRF, at high doses, is to stimulate GH release and that the central actions previously attributed to the hpGRF peptide are, in fact, due to the oCRF component of this compound.
Amino Acids↗