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A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r=0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n=24) and 14% (n=9) respectively. Minimal detectable amounts of rGH were 0,6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Involvement of endogenous corticotropin releasing hormone (CRH) in the regulation of spontaneous growth hormone (GH) secretion was investigated. A CRH antagonist, α helical CRH 9–41, was intracerebroventricularly infused for 36 h at a rate of 1 μg/0·5 μl/h to freely moving, cannulated adult male rats. Serial blood samples were drawn every 20 min for the last 8 hours of α helical CRH 9–41 infusion. The treatment induced a marked increase in GH peak amplitude without affecting either trough levels or numbers of peaks. In parallel, levels of growth hormone releasing hormone (GHRH) mRNA in the arcuate nucleus, but not of somatotropin release inhibiting hormone (SRIH) mRNA in the periventricular and arcuate nuclei, were increased. These data suggest that, in addition to its action in the stress-induced inhibition of GH secretion through regulation of periventricular SRIH neurons, CRH can also act as a modulator of endogenous GH secretion through regulation of arcuate GHRH neurons. Whether the modulatory effects of CRH on GHRH neurons are direct or indirect remains to be established. Journal of Endocrinology (1997) 152, 431–436

To test the hypothesis of the involvement of centrally expressed rat growth hormone receptors (rGH-R) in the ultradian rhythmicity of pituitary GH secretion, adult male rats were submitted to a 60 hr intracerebroventricular infusion of an antisense (AS) oligodeoxynucleotide (ODN) complementary to the sequence of rGH-R mRNA. Eight hour (10 A.M.–6 P.M.) GH secretory profiles, obtained from freely moving male rats infused with 2.0 nmol/hr of rGH-R AS, revealed a marked increase in GH peak amplitude (150 ± 12 vs 101 ± 10 ng/ml), trough levels (16.2 ± 3.0 vs 5.4 ± 1.4 ng/ml), and number of peaks (2.9 ± 0.3 vs 1.8 ± 0.2). No change was observed in rats treated with an ODN complementary to the prolactin receptor mRNA sequence (2.0 nmol/hr). Infusion of increasing ODN concentrations resulted in a dose-dependent stimulation of GH release. In parallel, somatogenic binding sites in the choroid plexus were decreased by 40%, and levels of rGH-R mRNA were increased in the periventricular nucleus (PeV) but unchanged in the arcuate nucleus (ARC). Levels of somatostatin mRNA, in the PeV but not in the ARC, were lowered by the treatment. Levels of GH-releasing hormone mRNA in the ARC were not affected. These data suggest that GH negative feedback results from a direct effect on central GH receptors and a subsequent activation of hypophysiotropic somatostatin neurons located in the anterior periventricular hypothalamus.

Noradrenergic neurons of the locus coeruleus (LC) negatively regulate the endogenous rhythmicity of growth hormone (GH) secretion. These neurons express high concentrations of receptors for somatostatin (SRIH) and galanin (GAL), two neuropeptides which can affect electrical activity of LC neurons and also centrally modulate plasma GH levels. We thus investigated whether somatostatin and galanin receptors located in the LC are involved in GH regulation. Pulsatile patterns of endogenous GH secretion were monitored after unilateral infusion of the peptides into the lateral ventricle (ICV) or into the LC, after lesion of contralateral LC neurons by 6 hydroxydopamine. Neither unilateral LC lesions nor administration of saline affected GH release. When administered ICV, both SRIH (5 micrograms/microliter/15 min) and GAL (1 microgram/microliter/15 min) resulted in a marked increase in GH secretion. Infusion of SRIH into the LC induced a significant but weaker stimulation of plasma GH as compared to ICV injections. In contrast, infusion of GAL into the LC was ineffective. These results indicate that somatostatin can exhibit direct effects on noradrenergic neurons of the LC involved in GH regulation, whereas central effects of galanin on the hormone are mediated by distinct structures.

The effects of octreotide, a long-acting somatostatin agonist selective of the sstr2/sstr3/sstr5 receptor subtypes, on ectopic GH secretion and tumor growth were investigated in Wistar-Furth female rats implanted with GH secreting (GC) cells which express mostly somatostatin receptors of the sstr1 and sstr2 subtypes. Octreotide dose dependently inhibited thymidine incorporation (-57%) and GH secretion (-41%) from GC cells in culture. In vivo, 6 weeks after GC cell implantation, plasma GH, IGF-1 and insulin levels were highly elevated. Cluster analysis of GH secretory dynamics revealed that GH secretion was less pulsatile in GC-implanted than in control animals. Furthermore, in GC-implanted animals, passive immunization either with SRIH or GHRH antisera, did not affect GH plasma levels. Three weeks after GC cell implantation, when tumors became palpable, octreotide (1 micrograms/h/kg BW) or saline was infused constantly for three weeks by osmotic minipumps. In octreotide treated rats, GH, IGF-1 and insulin levels were not different from sham-implanted animals and tumors weight were reduced by 80%. High affinity somatostatin binding sites were found in equivalent amounts on tumors from octreotide-treated or saline-treated animals. These findings indicate that GH secretion in GC-rats is mainly derived from the tumors and independent of hypothalamic control and that octreotide reduces both GH secretion and tumor growth. We conclude that the GC-implanted rat represents a good animal model to test the antisecretory and antitrophic properties of somatostatin analogs in vivo.

In the present work we investigated a possible role of an alpha 1-noradrenergic (NA) pathway involving the hypothalamic paraventricular nucleus (PVN) in the central regulation of growth hormone (GH) release. A week after bilateral electrolytic lesions of the PVN, pulsatile GH-secretory patterns were monitored in unanesthetized, freely moving control or lesioned male rats. While the pulsatility of GH secretion was maintained, the amplitude of the pulses and the area under the curve during an 8-hour sampling period were twice as high in PVN-lesioned than in control rats. Trough levels of GH were similar in the two groups. Inactivation of PVN alpha 1-receptors by local infusion of an alpha 1-NA antagonist, prazosin (50 ng/rat), also induced an increase in GH release. In control animals, intravenous injection of the alpha 1-NA agonist methoxamine (0.02 mg/100 g body weight) elicited a decrease in GH release but was ineffective when administered to PVN-lesioned rats. These data show that alpha 1-NA receptors, mediating GH inhibition, are located in the PVN. In light of the analogous effects observed herein on PVN-lesioned animals and, previously, after locus coeruleus (LC) lesions it is suggested that GH inhibition by the LC is relayed by the PVN via a local alpha 1-receptor population.

Although the role of serotonin (5HT) in the regulation of anterior pituitary hormone secretion is well documented, the involvement of specific 5HT receptor subtypes in this action is not yet fully elucidated. In the present work we attempted to determine the neuroendocrine role of the 5HT1A receptor subtype. This was chosen mainly because highly selective pharmacological tools are available for that subtype. 8-Hydroxy-2-(di-n-propylamino)tetralin (8OHDPAT) and ipsapirone were injected iv in conscious, freely moving male rats cannulated in the jugular vein. For the sake of comparison, a 5HT receptor agonist with preference for the 5HT1B and 5HT1C receptor subtypes 1-(m-trifluoromethyl-phenyl)piperazine (TFMPP) was also administered. Plasma PRL, ACTH, and beta-endorphin levels increased in a dose-dependent manner after 8-OHDPAT (0.01-1 mg/kg) or ipsapirone (0.5-7.5 mg/kg) injection. Maximal effects were obtained between 7-15 min. Only the highest dose of 1-(m-trifluoromethyl-phenyl)piperazine (5 mg/kg) resulted in the same response. In contrast, none of the drugs used affected plasma GH, TSH, or LH levels at any dose tested. The results indicate that 5HT1A receptors are involved in the regulation of PRL as well as ACTH and beta-endorphin secretion. 8OHDPAT was almost 40 times more potent than ipsapirone. The maximal effects of the two drugs on PRL release were comparable. In contrast, ipsapirone behaved as a partial agonist only on ACTH and beta-endorphin secretion, thus suggesting that different neuronal targets are involved in the stimulation of the three hormones by 5HT.

Radioautographic determinations of 125I-[Tyr0,D-Trp8]somatostatin-14 (125I-SRIF) binding sites were performed on frozen serial sections of the locus coeruleus (LC) of control rats and of rats subjected to either bilateral microinjections of 6 hydroxydopamine (6-OHDA) into the LC or unilateral microinjection into the ascending noradrenergic bundles. These experiments were performed in order to determine whether 125I-SRIF binding was localized to noradrenergic-containing cells and in which regions the cells which contain the binding sites are projecting. The extent of the lesions was assessed by measuring norepinephrine (NE) levels in the hippocampus (88% decrease as compared to sham-operated animals) for bilateral LC lesions and in the frontal cortex (87% reduction vs. contralateral side) for unilateral bundle lesions. In control rats, 125I-SRIF binding sites were restricted to the boundaries of the LC and followed closely the distribution of tyrosine hydroxylase-labeled cells. Three weeks after bilateral injections of 6-OHDA, 125I-SRIF binding decreased by 79% in all regions of the LC. In contrast, unilateral destruction of the ascending noradrenergic bundles resulted in a moderate decrease only in the middle part of the LC with a more important effect in the dorsal (55%) than in the ventral (24%) portion of the nucleus. These data demonstrate that: 1) most SRIF receptors in the LC are located in the vicinity of NE-containing cell bodies and 2) NE-containing cells bearing SRIF receptors project to the forebrain as well as to other terminal areas located more caudally in the brain. These data suggest a general role for SRIF in the control of the multiple functions of the LC.

The pulsatile pattern of GH secretion slowly develop in the postnatal period concomitantly with the dual network of GHRH and somatostatin (SRIH) hypothalamic neurons. We investigated whether an early postnatal treatment with a long acting SRIH analogue, octreotide, could affect maturation and subsequent operation of those networks in the adult rat. Octreotide administration (5 mu g/rat SC) every other day during the first 10 days of life resulted in growth retardation in the adult. In parallel, the amplitude of plasma GH secretory episodes in free moving unanesthetized animals was markedly reduced. The numbers of arcuate GHRH mRNA-containing and periventricular SRIH-mRNA containing neurons were not affected by the treatment. GHRH mRNA levels per neuron however was decreased by 30%, and median eminence GHRH stores by 50%. SRIH expression in the arcuate nucleus was also diminished, as was the number of 125I-SRIH labeled neurons in that nucleus. The effects of octreotide were compared to the hyposomatotropinemia induced by administration of monosodium glutamate (MSG), every other day during the first 10 days of life. Growth retardation and inhibition of GH secretory episodes in adult rats neonatally treated with MSG were slightly more pronounced than after octreotide. In contrast to octreotide, MSG induced a massive loss of GHRH neurons and a concomitant decrease in 125I-SRIH binding. Somatostatin did not protect GHRH neurons against the neurotoxic action of MSG since octreotide treatment did not further affect any of the parameters impaired by MSG. In conclusion, these experiments demonstrate that neonatally injected octreotide cannot counteract the toxic effect of MSG on arcuate neurons. However, a neonatal treatment with the SRIH agonist affects permanently growth rate and GH pulsatility. This effect is mediated in the hypothalamus by permanently impairing the neural networks that control GH secretion.

In Sprague-Dawley rats, Freund-adjuvant-induced arthritis (AIA) results in an increase in the amplitude of ultradian growth-hormone (GH)-secretory episodes without modification of their frequency. This is most apparent at the time of maximal inflammation, i.e. 14-21 days after inoculation of the adjuvant. GH responsiveness to a maximal dose of clonidine (10 micrograms/100 g body weight, BW), a secretagogue known to act at the hypothalamic level, is comparable in AIA and control rats. In contrast, GH response to a maximal dose of GH-releasing hormone (GHRH, 1 microgram/100 g BW), a peptide acting directly on pituitary somatotropes, is greater in AIA than in control rats. Furthermore AIA affects significantly neither hypothalamic somatostatin and GHRH mRNA levels nor pituitary GH content. In adult rats treated neonatally with monosodium glutamate (MSG), a neurotoxin which destroys the majority of GHRH neurons of the arcuate nucleus and reduces considerably plasma GH levels, clinical symptoms observed 14 days after inoculation of the Freund adjuvant are more marked than in AIA. The MSG-treated rats exhibit in particular a significantly higher increase in hindpaw diameter. Pulsatile administration of GH (40 micrograms/day/rat, with successive periods of 2 h of GH and 4 h of mineral oil) restoring the endogenous GH-secretory pattern throughout the 15-day period of arthritis development prevents hindpaw diameter increase. These results indicate that the impact of AIA on GH regulation occurs at the pituitary but not the hypothalamic level and suggest that increased GH secretion observed in AIA rats is an adaptive mechanism involved in the regulation of the inflammatory process.

In vitro and in vivo release of pituitary hormones were studied in the presence of (hydroxyproline9)LHRH ((Hyp)LHRH), a newly characterized endogenous molecular form of LHRH. Results were compared to those obtained with LHRH itself. (Hyp)LHRH, as LHRH, stimulated both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a homothetic manner. The hydroxylated compound was, however, 24 times (in vitro) and 5 times (in vivo) less potent than LHRH. The lower activity of (Hyp)LHRH than of LHRH in the in vitro assay correlated well with a 28-fold lesser potency in a binding test using pituitary membrane preparations. The higher relative potency and the prolonged effect of (Hyp)LHRH in the in vivo test were related to a lesser susceptibility of the hydroxylated form to proteolytic degradation. Effects of LHRH and of (Hyp)LHRH were not additive, both peptides were equally able to desensitize gonadotrophs to a subsequent challenge by the other. Taken together, these observations suggest that both forms of LHRH act at the same receptor site. The lesser affinity of the hydroxylated compound is compensated to a certain extent by its higher resistance to enzymatic degradation. It is concluded that in spite of its lesser potency, (Hyp)LHRH may participate in the regulation of gonadotropins.

In several diseases chronic pain is associated with long-lasting pathophysiological responses which differ strongly from those observed in acute situations. When persisting, acute pain often results in physical and psychological stress which may in turn aggravate the initial pathological state. In the present work we examined the secretory patterns of pituitary hormones related to acute stress (growth hormone (GH), prolactin (PRL) and β-endorphin (β-END)) in rats during the phase of Freund adjuvant-induced arthritis (AIA, a model used for chronic pain studies) when chronic pain is maximum (14 and 21 days, postinoculation (PI)). Using radio-immunoassay hormones were measured in plasma samples taken every 30 min for 7 h in free-moving rats 14 and 21 days after Freund adjuvant or vehicle injection and in control animals. The total amount of GH secretion was higher at 14 and 21 days PI in AIA rats as compared to vehicle-treated and control animals, and the pulsatility of GH secretory pattern was not modified by AIA. PRL and β-END secretion were not significantly different in arthritic rats as compared to controls. These results show that GH, PRL and β-END responses induced by acute stress are not observed during the AIA phase when chronic pain is maximum. Thus, in our experimental conditions, β-END and PRL do not seem to be good plasma markers of chronic pain. The increase of GH secretion, at a stage of the illness when chronic pain is well developed, raises the questions of (1) the possible involvement of peptides, such as substance P or cytokines, released during chronic inflammatory painful syndromes in the regulation of pulsatile GH secretion, and (2) the possible involvement of a decrease in noradrenergic inhibitory inputs from the locus coeruleus exerted on GH secretion. The role of GH as a plasma marker of some aspects of chronic pain is a possibility that cannot be ruled out.

The effects of dopamine on GH release were investigated both in vivo in freely moving intact rats and in rats with a mediobasal hypothalamic lesion, and in vitro in a perifusion system using dispersed male rat pituitary cells kept in primary culture. In vivo, dopamine (5 mg/kg body weight) induced a rapid and very transient increase in plasma GH levels in lesioned but not in intact rats. This increase was markedly inhibited by a prior injection of the D1 antagonist SCH 23390 (0·5 mg/kg) but not of the D2 antagonist domperidone (0·5 mg/kg). The D, agonist SKF 38393 induced a dose-dependent stimulation of GH release in lesioned rats, and the effect obtained with a dose of 5 mg/kg was abolished by pretreatment with SCH 23390 (0·5 mg/kg). In vitro, dopamine (0·1 μmol/l) and SKF 38393 (0·1 μmol/l) provoked a rapid and reversible release of GH from superfused rat pituitary cells; this effect was markedly inhibited by simultaneous superfusion of SCH 23390 (1 μmol/l). These findings indicate that dopamine can stimulate basal GH release at the pituitary level and that this stimulation is mediated by D1 but not by D2 receptors. They also support the hypothesis that unidentified hypothalamic neurohormones may modulate this effect. Journal of Endocrinology (1990) 127, 191–196

Effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) secretion were investigated in vivo (on intact or mediobasal hypothalamic lesioned rats tested under either anesthesia or free moving conditions) as well as in vitro (in incubation or perifusion systems of anterior pituitary tissue). The peptide induced a rapid, dose-dependent increase of plasma GH levels in free moving animals bearing an extensive lesion of the mediobasal hypothalamus including the median eminence. Under comparable conditions, TRH was ineffective in intact animals. After chloral hydrate anesthesia a GH response to TRH was recorded in both groups, but lesioned rats exhibited a better responsiveness to all doses tested. In vitro TRH increased GH release from incubated or perifused pituitaries sampled from both intact and lesioned rats in a transient and concentration-dependent manner. A similar effect was obtained with the (3 Me His2) analogue of TRH. These findings indicate that TRH can affect GH secretion at the pituitary level under specific experimental conditions and support the hypothesis that either peripheral hormones or other, still unidentified hypothalamic neurohormones may modulate this effect.

1. Diets deficient in phosphorus have been shown to preserve the renal function of uraemic rats, but the role of concomitant alterations in the intake of other nutrients caused by such diets was not elucidated. 2. Five groups of uraemic rats were compared. Three (A, B, C) received (ad libitum) diets deficient (0·03%), low (0·2%) or normal (0·5%) in P. Two groups (D, E) received restricted amounts of 0·2% and 0·5% P diets by pair-feeding with group A rats. 3. Phosphorus deficient diet resulted in marked anorexia, growth arrest, low P and high calcium plasma levels and severe rickets, but renal function became stabilized. Groups B and C had both higher total food intake and weight gain but a significantly higher mortality rate. After 36 weeks, mortality was 90% in these groups, and was 19%, 28% and 44% in groups A, D and E rats respectively, despite a higher P consumption in group E than in group B rats. 4. Normal P diet was associated with severe renal calcium deposits and high calcium heart content, which were related to the degree of hyperparathyroidism evaluated by bone histology. 5. Preservation of renal function was mainly mediated by reduction in some other components of the diet and not by P restriction itself. Normal P diet with restricted dietary allotments had little adverse effect on survival, and its influence on renal histology is not easy to explain. 6. Restriction in P sufficient to cause moderate hypophosphataemia, disappearance of phosphaturia and to prevent hyperparathyroidism without causing anorexia and other severe side effects, had little or no beneficial effect on renal deterioration.

The administration of gamma-hydroxybutyrate (GHB) induced a consistent secretory episode of growth hormone (GH) in the morning followed by basal levels of secretion of GH for several hours. The measurement of endogenous noradrenaline, dopamine and serotonin (5-HT) following infusion of GHB showed that dopamine concentrations were significantly increased in the striatum; at the level of the hypothalamus, however, no significant differences were observed between control and GHB-treated animals. The data reported in this study are consistent with the interpretation that the neurotransmitter regulation of GH release and the modulation of hypothalamic gluco-receptor systems are not fundamentally different in rodents and primates. Clonidine, an α-adrenergic agonist, enhanced the peak of GH observed in the morning and caused a rapid increment of GH during the period when it was normally at basal levels. Under the same experimental conditions, dopamine agonists, apomorphine and levodopa, had no effect on GH secretion. The inhibition of catecholamine synthesis by α-methyl-p-tyrosine blocked the secretory episode of GH following administration of GHB and after insulin hypoglycaemia whereas the GH rise induced by clonidine was unchanged. The inhibition of 5-HT synthesis by p-chlorophenylalanine also suppressed the secretory episode of GH seen in the morning and the release of GH induced by hypoglycaemia; both being partly restored in animals pretreated with 5-hydroxytryptamine.

Effects of vasoactive intestinal peptide (VIP) on growth hormone (GH) secretion were investigated both in vivo on freely moving, intact or mediobasal hypothalamic lesioned rats, and in vitro in incubation or superfusion systems of anterior pituitary tissue. In vivo, IV injection of VIP induced a rapid but transient increase in plasma GH levels in all animals and in vitro, VIP stimulated GH release from incubated or superfused rat pituitaries in a concentration dependent manner. This increase was potentiated by forskolin, a potent activator of adenylate cyclase. These findings indicate that VIP exerts a direct stimulating action on somatotrophs and that the effect seems to imply a coupling of VIP receptors with cAMP production.

The β-adrenergic agonist, isoprenaline, and antagonist, propranolol, had no effect on the delayed basal secretion of GH consistently observed in rats treated with the narco-analgesic gamma-hydroxybutyrate. Under the same experimental conditions, GH release was distinctly stimulated by infusion of the α-adrenergic agonist, clonidine, and by morphine; both responses were dose-dependent. The effects of β-adrenergic agonists and antagonists on these GH responses were as follows: in rats pretreated with isoprenaline the GH release induced by clonidine and morphine was abolished whereas it was enhanced in rats pretreated with propranolol. These data confirmed and extended previous reports from this laboratory on the inhibitory role of β-adrenergic receptors on GH regulation.

In order to determine the central or peripheral origin of the starvation-induced modifications of growth hormone (GH) and thyroid-stimulating hormone (TSH) secretions, the effects of starvation were studied in freely moving male rats with hypothalamo-hypophyseal disconnection. Five days after the disconnection GH secretion exhibited lower maximal values and higher trough levels and ultradian pulsatile secretion was lost as compared to controls. TSH levels were also decreased. The lesion did not modify pituitary somatostatin (SRIF) receptors as assessed by 125I-Tyr-O-D-Trp-8-SRIF binding or inhibition of adenylate cyclase activity. On the other hand, the growth hormone releasing factor (GRF) capacity to stimulate adenylate cyclase was strongly reduced by the lesion without modification of the affinity. Exposure to 72 h food deprivation decreased GH pulses and TSH levels in control rats but did not modify GH secretory profiles or TSH levels of lesioned rats. Plasma glucose and insulin levels were equally decreased after fasting in control and lesioned rats. Altogether, our results demonstrate that starvation-induced modifications of GH and TSH secretions are of central origin while glucose and insulin changes are peripherally triggered. They suggest that the hypothalamus is the only source of SRIF implicated in this effect.