Neurohypophysial peptides in cerebrospinal fluid: an update.
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Animals↗
Biomedical subjects
I C Robinson
Publications and source records attributed to I C Robinson.
Serial blood microsampling for neurohypophysial hormones.
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Animals↗
Systemic administration of growth hormone-releasing peptide activates hypothalamic arcuate neurons.
The synthetic hexapeptide growth hormone-releasing peptide selectively releases growth hormone in many species including man. Growth hormone-releasing peptide directly stimulates growth hormone release by an action at the level of the pituitary, at a different receptor site to that for the endogenous 44-amino acid peptide, growth hormone-releasing hormone, and when administered with growth hormone-releasing hormone has a synergistic effect. In addition to this pituitary action, we have suggested that the potent in vivo growth hormone-releasing activity of growth hormone-releasing peptide reflects a hypothalamic action and growth hormone-releasing peptide binding sites have been reported to be present in the hypothalamus. We have now found more direct evidence for a hypothalamic action of growth hormone-releasing peptide in two ways. First, we have found that a sub-population of hypothalamic neurons show strongly increased fos expression in response to systemic growth hormone-releasing peptide administration. Fos is the protein product of the immediate early gene, c-fos, which is induced in many neuronal systems following their activation. Second, extracellular recordings from putative growth hormone-releasing hormone neurons in the arcuate nucleus showed that growth hormone-releasing peptide also stimulates the firing of neurons in this area.
Animals↗
Growth hormone (GH) receptors, GH binding protein and GH: an autoregulatory system?
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Animals↗
Growth hormone binding protein in the rat: effects of gonadal steroids.
In normal rats, females have higher circulating GH-binding protein (GHBP) levels than males, whereas in the GH-deficient dwarf (Dw) rat, there is no sexual dimorphism in plasma GHBP, suggesting that GH secretion may be involved in this difference. In order to study the relationship between gonadal steroids and GH on GHBP and GH receptor regulation, the levels of plasma GHBP, hepatic bovine GH, and human GH (hGH) binding as well as GHBP and GH receptor messenger RNA (mRNA) have now been studied in normal, Dw, hypophysectomized (Hx), or ovariectomized (Ovx) rats, subjected to different GH and gonadal steroid exposure. In normal male rats, estradiol (E2, 12.5-25 micrograms/day for 1 or 2 weeks) markedly increased plasma GHBP and hepatic hGH, and bGH binding. These effects of E2 were diminished in Dw rats, absent in Hx rats, but restored in Hx rats given exogenous hGH. Plasma GHBP rose in female rats given E2, and fell in females given the anti-estrogen tamoxifen. Ovx animals had lower plasma GHBP and hepatic GH binding which was reversed by E2, but not testosterone treatment. Continuous hGH infusions in Ovx rats restored hepatic GH binding, and increased plasma GHBP. In Dw males, hGH increased plasma GHBP and hepatic GH binding, whereas testosterone had no effect on GHBP or GH receptors and did not affect their up-regulation by hGH. Hepatic levels of GHBP-, and GH receptor mRNA transcripts showed the same trends in response to steroid or GH treatment, but the differences were rarely significant, except in Ovx animals which had higher GHBP mRNA transcripts after GH or E2 treatment. Thus E2 and GH increase both plasma GHBP and hepatic GH receptor binding. GH up-regulates GHBP in the absence of E2, whereas E2 treatment does not raise GHBP in the absence of GH. Whereas some of the effects of estrogen could be mediated via alterations in GH secretion, estrogen may also directly influence GHBP production at the liver, but only in the presence of GH.
Animals↗
Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system.
The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).
Animals↗
Growth hormone receptor regulation in growth hormone-deficient dwarf rats.
In the rat, many actions of GH depend upon the sexually dimorphic pattern of exposure to GH. Hepatic human GH (hGH) receptor binding differs between the sexes and is sensitive to GH deficiency, but this has mostly been studied in acutely hypophysectomized rats, which lack all pituitary hormones. We have used a strain of GH-deficient dwarf (Dw) rats to determine whether chronic GH deficiency alters the normal developmental pattern and sexually dimorphic expression of hepatic GH receptors. Adult female Dw rats had lower levels of 125I-labelled hGH binding (reflecting predominantly lactogenic receptors) than their normal counterparts whereas there was no difference between adult Dw and normal males; binding capacity increased from 25 days of age, becoming sexually dimorphic from 40 days to adulthood in both strains (% specific binding/mg protein: normal males 1.6 +/- 0.3, normal females 13.2 +/- 1.1, Dw males 2.1 +/- 0.4, Dw females 10.0 +/- 0.6). In contrast, hepatic 125I-labelled bovine GH (bGH) binding (somatogenic receptors) was much lower, and similar in both Dw and normal animals. A sex difference in 125I-labelled bGH binding was only seen in adult animals, and was considerably less marked in Dw rats compared with normal animals (normal males 1.3 +/- 0.1, normal females 2.5 +/- 0.2, Dw males 1.9 +/- 0.2, Dw females 2.4 +/- 0.2%/mg protein). Continuous hGH infusion stimulated growth in female Dw rats, and raised somatogenic and lactogenic GH binding (3.2 +/- 0.4 and 19.6 +/- 2.5%/mg protein) compared with sham-infused controls (2.4 +/- 0.2 and 7.9 +/- 0.6%/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)
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Isolation of an insulin-like growth factor II cDNA from guinea pig liver: expression and developmental regulation.
Insulin-like growth factor II (IGF-II) cDNA was isolated from adult guinea pig liver by polymerase chain reaction (PCR) screening. A cDNA sequence was obtained corresponding to part of the preproIGF-II, including the signal peptide, the mature IGF-II and 37 amino acids of the acid carboxy-terminal E-domain. Amino acid sequence prediction, based on the cDNA clone, showed that mature guinea pig IGF-II has a high homology with both human and rat IGF-II, 100 and 94% identity, respectively. Levels of IGF-II mRNA in guinea pigs of different ages were analyzed by solution hybridization/RNase protection assay using part of the isolated IGF-II cDNA as a probe. There is a marked developmental regulation of IGF-II after birth. IGF-II mRNA levels were high in fetal livers, and decreased 15- to 30-fold in adults. As in man, but in contrast to rats, adult guinea pigs have significant levels of IGF-II mRNA in the liver. In fetal guinea pigs, the expression of IGF-II mRNA was 5-, 2- and 70-fold lower in kidney, skeletal muscle and brain cortex, respectively, than in liver. IGF-II mRNA levels in kidney and skeletal muscle of fetal guinea pigs were 5- and 4-fold higher, respectively, compared with adults. Similar sizes of IGF-II mRNA transcripts could be observed on Northern blots in newborn rats and in fetal guinea pigs. Our conclusions are that the mature IGF-II peptide in the guinea pig is 100% identical to the mature peptide in the human.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Growth hormone (GH) binding protein and GH interactions in vivo in the guinea pig.
GH binding proteins (GHBPs) are present in the blood of several species and by complexing with circulating GH may alter its clearance and distribution. The actions of human GHBP (hGHBP) have hitherto been studied indirectly via their effects on the clearance of hGH. In the present experiments, recombinant preparations of hGHBP and a shorter variant lacking exon 3 (delta 3hGHBP) were tested in vivo, either alone or after preincubation with recombinant hGH, recombinant 20K methionyl-hGH, or rat GH. Multiple serial blood sampling was performed in conscious chronically cannulated guinea pigs and both GHBP and GH variants monitored in the same samples by specific RIAs, unaffected by endogenous activities in this species. hGHBP, delta 3hGHBP, hGH, and 20K met-hGH (10-40 micrograms) were all cleared rapidly when injected alone (t1/2 = 11-20 min). However, when the same amounts of hGHBP and hGH were incubated together for 60 min and then injected, the clearance of both proteins was greatly prolonged (t1/2 hGHBP = 75-94 min, hGH = 80-137 min). Similar results were obtained for hGH complexed with delta 3hGHBP, and over a range of GH:GHBP ratios from 0.3:1 to 4:1. The effect was specific for 22K hGH; neither rat GH nor 20K met-hGH altered hGHBP clearance, nor were their clearances slowed by preincubation with hGHBP. Rapid complex formation also could be demonstrated in vivo. Injections of hGH 30 min after delta 3hGHBP, or vice versa, altered the established plasma disappearance curve of hGHBP, and hGH clearance was slowed in parallel. During a continuous infusion of hGH to steady state, injections of delta 3hGHBP increased plateau hGH concentrations by forming a delta 3hGHBP/hGH complex in the circulation. These experiments show that the conscious chronically cannulated guinea pig provides a useful model in which the in vivo interaction between hGHBP and hGH can be studied dynamically. The results imply that the location, extent, and rate of complex formation between GHBP and GH may be an important determinant of the passage of GH between the intra- and extravascular compartments, which could affect the pattern of tissue exposure to GH.
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Growth hormone (GH)-binding protein in normal and GH-deficient dwarf rats.
There are GH-binding proteins (GHBPs) present in the blood of many species, and these correspond to the extracellular GH-binding domain of the GH receptor. In the rat, GHBP arises by alternative splicing of the GH receptor mRNA, but little is known of the physiological role of circulating GHBP, or its relationship with episodic GH secretion. We have developed a sensitive radioimmunoassay based on recombinant GHBP, and have measured rat GHBP levels in small samples of plasma from normal and GH-deficient dwarf rats. In normal adult rats, GHBP levels were two- to threefold higher in females than in males (16.6 +/- 0.8 vs 6.4 +/- 0.4 micrograms/l, P < 0.001), but this sex difference was not seen in dwarf rats. A continuous infusion of human GH in dwarf males raised plasma GHBP to 23.5 +/- 3.5 micrograms/l compared with 6.7 +/- 0.5 micrograms/l in sham-infused animals, whereas suppression of GH by continuous infusion of a long-acting somatostatin analogue in female dwarf rats had no effect on GHBP. In anaesthetized rats, large changes in plasma GH caused by i.v. administration of rat GH, somatostatin or GH-releasing factor did not affect GHBP acutely. Both GH and GHBP were also measured in serial blood samples from conscious normal and dwarf rats. A sexually dimorphic GH secretory pattern was observed in both strains. Males showed peaks and troughs of GH every 3 h varying over a 100-fold range, whereas females exhibited more continuous GH secretion. Despite the large fluctuations in endogenous GH, GHBP levels remained relatively constant in individual normal or dwarf males, as well as in females of both strains, and there was no significant correlation between GH and GHBP either in individual rats or as a group. Our results suggest that GHBP is GH-dependent in the longer term, and that the higher GHBP levels in female rats require their continuous GH secretory pattern. However, plasma GHBP levels remain stable and are not affected by acute changes in endogenous or exogenous GH.
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The interaction of growth hormone releasing hormone and somatostatin in the generation of a GH pulse in man.
OBJECTIVE: To study the regulation of the growth hormone (GH) response to growth hormone releasing hormone (GHRH) in the presence or absence of somatostatin pretreatment. DESIGN: Seven healthy adult male volunteers of normal height and weight and aged between 19 and 29 years underwent four separate studies (each containing three cycles in one day) in random order. The studies were separated from each other by at least a week. On day 1, three consecutive cycles (between 0800 and 2000 hours) consisted each of a saline infusion for 3 hours which was stopped prior to a bolus injection of saline and followed by 60 minutes of more intensive blood sampling. On day 2, the bolus injections were of GHRH given after saline infusion. On days 3 and 4 somatostatin infusions were administered instead of saline over the 3-hour periods followed by bolus injections of saline or GHRH respectively. In all studies, samples were collected for the measurement of serum GH concentration at 15-minute intervals from time 0 to 180 minutes and then at 5-minute intervals for a further 60 minutes, this cycle being repeated three times. MEASUREMENTS: Serum GH concentrations were analysed by serial array averaging. RESULTS: Prompt release of GH was observed in response to GHRH given against a saline background (day 2, cycle 1) (mean at 60 minutes 49.2 +/- 14.7 mU/l) but the responses observed during the second and third cycles were attenuated (mean at 60 minutes 17.2 +/- 4.0 mU/l; P = 0.025). GH release between somatostatin infusions (somatostatin withdrawal; day 3) occurred twice as often as that observed during saline infusions (62% day 3: 29% day 1). The response, although qualitatively similar to that induced by GHRH, was reduced in amplitude and the time of onset variable (5-45 minutes). On day 4, the administration of GHRH as a bolus injection combined with somatostatin withdrawal led to consistent and repeatable GH responses (mean at 60 minutes, cycle 1, 39.7 +/- 10.8 mU/l; cycles 2 and 3, 37.4 +/- 9.4 mU/l) which were similar to those observed with GHRH alone (day 2, cycle 1) (mean 39.7 +/- 10.8 mU/l) (P = NS). CONCLUSIONS: These data suggest that endogenous somatostatin secretion is important in determining the ability of the somatotroph to respond to repeated growth hormone releasing hormone stimulation and that for regular GH pulse generation a close interplay between growth hormone releasing hormone and somatostatin is required.
Adult↗
The growth hormone secretory pattern: a response to neuroendocrine signals.
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Animals↗
Corticotrophin-releasing factor-41, vasopressin and oxytocin release into hypophysial portal blood in the rat: effects of electrical stimulation of the hypothalamus, amygdala and hippocampus.
The role of the paraventricular nuclei (PVN), amygdala and hippocampus in the control of the hypothalamic-pituitary-adrenal axis has been studied by determining the effect of electrical stimulation of the PVN, amygdala and hippocampus on the release of corticotrophin-releasing hormone (CRF-41) and arginine vasopressin (AVP) into hypophysial portal blood and ACTH and corticosterone into peripheral blood. Adult female Wistar rats were anaesthetized with sodium pentobarbitone and stimulation was carried out through previously implanted bipolar, glass-insulated platinum electrodes. Hypophysial portal blood was collected 30 min before and 30 min during the application of the stimulus which consisted of trains (30 s on and 30 s off) of biphasic rectangular pulses with a frequency of 50 Hz, pulse width 1 ms and amplitude 1 mA. Bilateral stimulation of the PVN increased while unilateral stimulation of the amygdala decreased the release of CRF-41 into hypophysial portal blood. The threefold increase in release of CRF-41 induced by PVN stimulation correlated with a marked increase in peripheral plasma concentrations of ACTH and corticosterone. Stimulation of the hippocampus had no significant effect on CRF-41 release, and stimulation of each of the three brain regions had no effect on AVP release into portal blood. These findings were extended in a second study to compare the effects of unilateral bipolar electrical stimulation of the PVN and of the supraoptic nucleus (SON) on the release of CRF-41, AVP and oxytocin. This study was carried out on adult male rats, anaesthetized with sodium pentobarbitone, in which the stimulus was applied through previously implanted concentric stainless-steel electrodes.(ABSTRACT TRUNCATED AT 250 WORDS)
Amygdala↗
The episodic secretory pattern of growth hormone regulates liver carbonic anhydrase III. Studies in normal and mutant growth-hormone-deficient dwarf rats.
Carbonic anhydrase III (CAIII) occurs in male rat liver at concentrations twenty times those in the female, and is sensitive to the pattern of growth hormone (GH) release. Males release GH episodically and have high concentrations of CAIII; females produce GH in a more continuous fashion and have lower CAIII levels. In normal female rats, the endogenous GH secretory pattern was masculinized, either by regular injections of GH-releasing factor (GRF) or by intermittent infusions of somatostatin (90 min on/90 min off). Both treatments induced regular GH pulses and stimulated growth, but only intermittent somatostatin infusions raised CAIII levels (controls, 1.5 +/- 0.5; somatostatin-treated, 9.0 +/- 2.9 micrograms/mg; means +/- S.D.). GRF pulses (4 micrograms every 4 h) did not however raise CAIII levels (controls 1.8 +/- 0.5; GRF-treated 1.4 +/- 0.4 micrograms/mg). Surprisingly, hepatic CAIII is also sexually dimorphic (males, 18.8 +/- 3; females, 2.22 +/- 0.4 micrograms/mg) in a GH-deficient dwarf rat strain which has low plasma GH levels without 3-hourly GH peaks. Intermittent somatostatin infusions in female dwarf rats partially masculinized hepatic CAIII, an effect reduced by co-infusion with GRF. This CAIII response was not secondary to growth induction, since neither somatostatin nor GRF stimulated growth in dwarf rats, and pulses of exogenous GH stimulated growth in female dwarfs without masculinizing CAIII levels. Furthermore, continuous GH infusion in male dwarf rats partially feminized hepatic CAIII levels (to 9.1 +/- 2.4 micrograms/mg), whereas infusions of insulin-like growth factor-1, which induced the same body weight gain, did not affect hepatic CAIII (20.8 +/- 6 micrograms/mg). These results show that hepatic CAIII expression is highly sensitive to the endogenous GH secretory pattern, independent of growth. They also implicate the low basal GH levels between pulses, rather than the peak GH levels, as the primary determinant of the sexually dimorphic hepatic CAIII expression in the rat.
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Changes in a proposed new neuroendocrine marker of oestrogen receptor function in postpartum women.
We describe a novel neuroendocrine test which reflects a central response to activation of oestrogen receptors. This is achieved by measurement of plasma levels of oestrogen-stimulated neurophysin (ESN) following an oestrogen challenge. In normal women the ESN response to ethinyl oestradiol is dose-dependent. This response is attenuated in normal women during the first postpartum month, although it is unchanged in patients with anorexia nervosa, in spite of their similar concurrent hypo-oestrogenic state. The altered puerperal response may result from the acute oestrogen withdrawal which occurs at delivery. The time course of the altered ESN response coincides with the period of maximum risk for puerperal psychosis. The ESN response to oestrogen provides a novel neuroendocrine measure to test the relevance of changes in central oestrogen receptor responsiveness in the pathogenesis of puerperal psychosis.
Adolescent↗
Somatostatin and its physiological significance in regulating the episodic secretion of growth hormone in the rat.
Somatostatin (SS) is a powerful inhibitor of growth hormone (GH) secretion both in vitro and in vivo, and is generally regarded as having a negative influence on GH secretion and growth. In the conscious rat, the GH secretory pattern, the response to repeated injections of GH-releasing hormone (GHRH), and the feedback mechanism by which GH regulates its own release are all sexually dimorphic and probably reflect a sexually dimorphic pattern of SS release. Prolonged infusions of SS initially block GHRH-induced GH release but responses begin to break through with time. Withdrawal of SS induces a rebound release of GH in vivo, largely dependent on the release of endogenous GHRH. Prolonged exposure to intermittent infusions of SS in normal female rats produces a paradoxical growth response by inducing regular peaks of GH secretion whereas continuous infusions of SS do not. This response is GH-dependent, and is not due to other effects of SS since intermittent infusions of SS do not increase growth in dwarf rats which are deficient in pituitary GH. The sex differences in GH secretion are also reflected in the expression of several GH-dependent liver enzymes, one of which, carbonic anhydrase III, responds to manipulations of the endogenous secretory pattern by SS in both normal and dwarf rats, and appears to be sensitive to differences in basal, rather than peak, GH levels.
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Effect of food withdrawal and insulin on growth hormone secretion in the guinea pig.
The guinea pig is unusual in that its postnatal growth appears to be independent of GH even though its pituitary gland produces a GH molecule. The effects of fasting on the GH secretory pattern and the GH responses to insulin, GH-releasing factor (GRF), and somatostatin (SS) during fasting have now been studied by automatic microsampling of blood in chronically cannulated normal guinea pigs. Withdrawal of food in both male and female guinea pigs changed the GH secretory pattern dramatically. The normal episodic GH secretory pattern [large GH peaks occurring at 3.6 +/- 0.4-h intervals over a low (approximately 0.5-1.5 ng/ml) baseline secretion] was altered to a pattern of more continuous GH output, characterized by a 10-fold elevated baseline secretion (5-15 ng/ml) with no large secretory episodes or troughs. Glucose injections (three injections of 600 mg, iv, at hourly intervals) in fasted guinea pigs lowered their elevated blood GH levels significantly (from 9.1 +/- 1.1 to 6.5 +/- 0.9 ng/ml). Insulin injections (1, 2, or 6 U, iv) inhibited spontaneous GH pulses in normally fed animals, but had little effect on the high continuous GH tone during fasting. The elevated GH secretion in fasted animals could be inhibited by continuous infusion of SS or a single iv injection of a long-acting SS analog. The secretion of GH during fasting could be further increased, either by injections of GRF (two injections of 2 micrograms, iv, 90 min apart), producing peak levels of 102 +/- 16 and 68 +/- 21 ng/ml (above a baseline output of 8.8 +/- 2.2 ng/ml), or by a continuous iv infusion of GRF (12 micrograms/h). Because the GH secretory pattern in the guinea pig is so sensitive to nutrition and insulin, this species may provide an interesting model in which to study selectively the metabolic, as opposed to growth-promoting, actions and regulation of GH.
Animals↗
Growth hormone secretion in the guinea-pig.
The guinea-pig is unusual in that it continues to grow at a normal rate after hypophysectomy. Although its pituitary gland appears to contain a GH, this has not been isolated or characterized, and nothing is known about its secretion or physiological control. We have identified guinea-pig GH, established a sensitive heterologous radioimmunoassay and adapted our automatic blood microsampling method to study spontaneous GH secretion in this species. In male guinea-pigs, GH is released in an episodic pattern, reminiscent of the rat. Large multicomponent pulses of GH secretion occur every 3-4 h between periods of low or undetectable GH release, whereas most females showed a more uniform pulsatile pattern with pulses every 1-2 h. GH was released in response to GH-releasing factor (GRF) injections (2, 10 or 20 micrograms [Nle27]-GRF(1-29)NH2) in a dose-dependent fashion, and i.v. infusion of somatostatin (50 micrograms/h) blocked spontaneous GH pulses, eliciting a rebound release (from 2.0 +/- 0.8 (S.E.M.) to 36 +/- 17 micrograms/l 30 min after stopping the infusion). Infusions of a GH-releasing hexapeptide (100 or 400 micrograms/h for 4 h) also released GH. These results provide the first description of the pattern of GH release in the guinea-pig, and suggest that the striking episodic pattern is controlled by the same hypothalamic peptides that regulate GH in other species. Since the guinea-pig grows well in the absence of GH, this species may use GH for its metabolic, rather than growth-promoting actions. The guinea-pig may well prove a useful model, now that methods are available for studying its endogenous GH secretion.
Amino Acid Sequence↗