C. Dieguez

GROWTH HORMONE NEUROREGULATION AND ITS ALTERATIONS IN DISEASE STATES

12-392. Neuroregulation of GH secretion 131 BIRGE, C.A., Pwm, G.T., MARIZ, I.K. & DAUGHADAY, W.H. (1967) Radioimmunoassayable growth hormone in the rat pituitary gland: effects of age, sex and hormonal state. Endocrinology, 81, 195-204. BLACKLAY, A., GROSSMAN, A., Ross, R.J.M., SAVAGE, M.O., DAVIES, P.S.W., PLOWMAN, P.N., COY, D.H. & BESSER, G.M. (1986) Cranial irradiation for cerebral and nasopharyngeal tumours in children: evidence for the production of a hypothalamic defect in growth hormone release. Journalof Endocrinology, lOS,25-29. BLOCH, B., LING, N.. BENOIT, R., WEHRENBERG, W.B. & GUILLEMIN, R. (1984a) Specific depletion of immunoreactive growth hormone-releasing factor by monosodium glutamate in rat median eminence. Nature, 307,272-273. BLOCH, B., GAILLARD, R.C., BRAZEAU, P., LIN, H.D. & LING, N. (1984b) Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus. Regulatory Pepti&s,8,21-31. BORKENSTEIN, M. (1986) The effects of intranasal insufflation of growth hormone releasing factor analogue GRF (1-29) NHz on growth hormone secretion in children with short stature. Acta Endocrinologica (Suppl.), 279, 135-138. BOWERS, C.Y., MOMANY. F A., REYNOLDS, G.A. & HONG, A. (1984) On the in vitro and in viuo activity of a new synthetic hexapeptide that acts on the pituitary to specifically releasegrowth hormone. Endocrinology, 114, BRAMNERT, M. & H ~ W L T , B. (1987) Naloxoneenhances the increase in plasma growth hormone induced by aladrenergic stimulation in healthy males. Acra Endocrinologica, 114, 308-313. B-N, J.L., CWVEQUIN, M.C., FELLMAN, D. & BUGNON C. (1984) Ontogeny of the neuroglandular system revealed with hpGRF 44 antibodies in human hypothalamus. Neuroendocrinology. 39.68-73. BRUHN. T.O., MASON, R.T. & VALE W.W. (1985) Presence of growth hormone-releasing factor-like immunoreactivity in rat duodenum. Endocrinology, 117, 1710-1712. CABEZAS-CERRATO, J., PENALVA, ., VIDAL, P., IGLESIAS, M.. LADO, J. & ARAUXO, D. (1987) Pituitary response ofnormal subjects to fast sequential stimulus by the four hypothalamic releasing hormones beforeand after activation of GABA synapses by sodium valproate. 3rd Meeting of the European Neuroendocrinc. Associarion. London, Abstract 67. CAMPBELL, P.J.. BOLLI. G.B.. CRYER, P.E. & GERICH, J.E. (1985) Pathogenesis of the dawn phenomenon in patients with insulin dependent diabetes mellitus. New England Journal of Medicine, 312, 1473-1479. CASANUEVA. F.F.. B ~ I . R., CELLA S.G., MULLER, E.E. dr MANTEGAZZA, P. (1983) Effects of agonists and antagonists of cholinergic neurotransmission on growth hormone release in the dog. Acfa Endocrinologicu. CASANUEVA, F.F., VILLANUEVA, L., CABRANFS, J.A., CABEZAECERRATO. J. & FERNANDEZ-CRUZ, A. (1984a) Cholinergic mediation of growth hormone secretion elicited by arginine, clonidine and physical exercise in man. Journal of Clinical Endocrinology und Metabolism, 59, 526530. CASANUEVA, F.A.. VILLANUEVA, L., F’ENALVA, . CABEZAS-CERRATO, J. (1984b) Depending on the stimulus. central serotoninergic activation by fenfluramine blocks or does not alter growth hormone secretion in man, Neurwn&crinology, 30, 302-308. CASANWA. F.F., VILWNUEVA, L.. DIEGUEZ, C., CABRANES, J.A.. DIAZ. Y.. SZOKE, B., SCANLON, M.F.. SCHALLY A.F. & FERNANDEZ-CRUZ, A. (1986) Atropine blockade of growth hormone (GH)-releasing hormone-induced GH secretion in man is not exerted a t pituitary level. Journal of Clinical Endocrinology and Metabolism, 62, 186491. CASANUEVA, F.F., VILLANUEVA, L., DIEGUEZ, C., Duz, Y., CABRANES, J.A.. SZOKE, B., SCANLON. M.F., SCHALLY A.V. 8t FERNANDEZ-CRUZ, A. (1987) Free fatty acids (FFA) blockade of GHRH-induced GH secretion in man. Journal of Clinical Endocrinology and Meiabolism, 65,634840. CAVAGNINI, F. I ”! , C., DI LANDRO. A., TENCONI, L., MARASCHINI, C. & GIRorn, G. (1977) Effects of a GABA derivative baclofen, on growth hormone and prolactin secretion in man. Journal of Clinical Endocrinology and Metabolism, 45, 579-584. CEDA, G.P. &HOFFMAN, A.R. (1985)Growth hormone-releasing factor densensitisation in rat anterior pituitary cells in virro. Endocrinology, 116, 1334-1340. CELLA, S.G., MORGESE, M., MANTEGAZA, P. & MULLER, E.E. (1984) Inhibitory action of the a,-adrenergic receptor on growth hormone secretion in the dog. Endocrinology, 114,24062408. CHATELAIN, B., DAVID, M. & FUNCOIS, R. (1985) Effect of acute intravenous growth hormone-releasing factor on plasma prolactin in short children and patients with growth hormone deficiency. Hormone Research, 22, 46-51. CHIODINI, P.G., LlUZZl, A., DALLABONZANA. D., Oppizzi, G. & VERDE, G. (1985) Changes in growth hormone 1537-1 545.

Cholinergic muscarinic receptor blockade with pirenzepine abolishes slow wave sleep-related growth hormone release in normal adult males

Cholinergic pathways play an important role in the regulation of GH secretion from the anterior pituitary gland, and in this study we have investigated whether cholinergic muscarinic receptor blockade with pirenzepine displayed any inhibitory action on slow wave sleep‐related GH release in normal subjects. Six adult males (ages 24–37 years) were studied in a randomized order and fasted from 1800 h on each study day. All subjects showed episodes of slow wave sleep on each occasion and this was followed by peaks of GH release when placebo alone was administered (range of GH peaks 4–50 mU/l). In contrast, pirenzepine treatment (100 mg p.o. at 2200 and 2400 h) completely abolished nocturnal GH release in each individual without altering the occurrence of slow wave sleep itself. These data demonstrate clearly that cholinergic muscarinic receptor blockade completely abolishes slow wave sleep‐related GH release in normal adult subjects. Because of the striking effects it is reasonable to conclude that acetylcholine plays an important stimulatory role in mediating slow wave sleep‐related GH release. This finding may have investigational and therapeutic applications in young patients with Type 1 diabetes mellitus since GH is implicated in some acute metabolic and chronic microvascular complications of this disease.

GROWTH HORMONE (GH) RESPONSES TO ARGININE AND L‐DOPA ALONE AND AFTER GHRH PRETREATMENT

In order to investigate the mechanisms by which arginine and L‐dopa cause GH release in humans we measured the GH response to GHRH 1–44 (200 μg i.v.), arginine (30 g i.v. over 30 min) and L‐dopa (500 mg orally) administered alone and 120 minutes following pretreatment with GHRH 1–44 (200 μg i.v.) in normal male subjects. Prior GHRH administration abolished the GH response to subsequent GHRH. Arginine infusion induced a rise in GH levels maximal at 45 min. Following GHRH pretreatment the GH response to arginine was enhanced, with peak values of 19.3 ± 6.4 vs 53.3 ± 16.5 mU/l (mean + SEM) respectively (P < 0.02). L‐dopa alone induced a rise in GH levels maximal at 90 min (17.6 ± 7.4 mU/l, mean ± SEM) but this rise was abolished by pretreatment with GHRH.

EFFECT OF THYROXINE REPLACEMENT THERAPY ON PLASMA INSULIN‐LIKE GROWTH FACTOR 1 LEVELS AND GROWTH HORMONE RESPONSES TO GROWTH HORMONE RELEASING FACTOR IN HYPOTHYROID PATIENTS

The aim of this study was to evaluate the effect of T4 replacement therapy on plasma insulin‐like growth factor 1 (IGF‐1) levels in patients with primary hypothyroidism to see whether recovery of pituitary GH responsiveness to GRF was associated with increased plasma IGF‐1 levels. IGF‐1 levels and GH responses to GRF (1 μg/kg) were measured in 21 patients with primary hypothyroidism before and after T4 replacement therapy. T4 increased plasma IGF‐1 levels (57.2 ± 4.4 vs 75.9 ± 8‐8 ng/ml, mean ± SEM, P<0.05) and GH responses to GRF as assessed both by peak GH levels (9 ± 1.5 ng/ml before treatment vs 16.7 ± 3 ng/ml after treatment, mean ± SEM, P <0.05) and area under curve (496 ± 92 before treatment vs 896 ± 161 after treatment, mean‐± SEM, P < 0.05). Linear regression analysis showed a positive correlation between free T3 and IGF‐1 levels after treatment (r= 0‐37, P <0.05) and a negative relationship between plasma IGF‐1 levels before treatment and a IGF following T4 replacement therapy (r= 0.45, P < 0.025). However, no correlation was found between plasma IGF‐1 levels and GH responses to GRF, suggesting that GH responses to GRF are of no predictive value in relation to the recovery of plasma’ IGF‐1 levels following T4 replacement therapy in hypothyroid patients.

INFLUENCE OF DOPAMINERGIC, ADRENERGIC AND CHOLINERGIC BLOCKADE AND TRH ADMINISTRATION ON GH RESPONSES TO GRF 1–29

In order to establish the influence of dopaminergic, á‐adrenergic and cholinergic pathways on GRF‐mediated GH release we have studied the GH responses to GRF 1–29 (100 or 50 μg as i.v. bolus) alone and in combination with metoclopramide (MCP, 10 mg, i.v.), thymoxamine (THYM, 210 μg/min, 150 min infusion), and atropine (1.2 mg, i.v.). We have also investigated any possible interaction between TRH and GRF in view of the reported inhibitory effects of TRH infusion on stimulated GH release. Dopaminergic and á‐adrenergic blockade with MCP and THYM respectively, did not have any effect on the GH responses to GRF. This lack of effect strongly suggests that any action which these neurotransmitters may exert on GH secretion is not at a pituitary level. TRH did not modify the GH response to GRF suggesting that the inhibitory effect on stimulted GH secretion is exerted at a hypothalamic level. In contrast, GH responses to GRF were significantly reduced by prior administration of atropine. These data support the view that cholinergic pathways play an important role in the regulation of GH secretion and such control may be exerted at both hypothalamic and pituitary levels.

ALPHA‐2‐ADRENERGIC PATHWAYS RELEASE GROWTH HORMONE VIA A NON‐GRF‐DEPENDENT MECHANISM IN NORMAL HUMAN SUBJECTS

Administration of a supramaximal dose of GRF 1‐44 (200 μg, i.v.) to normal human volunteers increased GH levels while a further bolus of GRF (200 μg i.v.) given 2 hours later failed to increase plasma GH levels. In contrast, alphaadrenergic receptor agonism with either propranolol‐adrenaline infusion or clonidine increased plasma GH levels at a time when GH responses to this supramaximal dose of GRF were absent. This indicates that alpha‐adrenergic pathways stimulate GH secretion through a non‐GRF‐dependent mechanism in normal human subjects.

EFFECT OF ORAL ADMINISTRATION OF MELATONIN ON GH RESPONSES TO GRF 1–44 IN NORMAL SUBJECTS

In order to investigate the role of melatonin on the neuroregulation of GH secretion, eight healthy male volunteers each underwent four separate tests in random order separated by at least 1 week. Following oral administration of melatonin (500 mg at – 60 min and at – 30 min) plasma GH levels were higher than after placebo at 45 min (mean + SEM 2.9 ± 0.8 vs 0.9 ± 0.4 ng/ml, P<0‐01) and 60 min (mean ± SEM 2.9 ± 0.4 vs 0.8 ± 0.1 ng/ml, P<005). Likewise, after prior administration of melatonin, GH responses to GRF 1‐44 (1 μg/kg i.v. at 0 min) were greater than placebo plus GRF at 15 min (mean ± SEM 22‐4 ± 6‐1 ng/ml vs 11.3 ± 2.3 ng/ml, P <005), 45 min (mean ± SEM 26.2 ± 5.3 ng/ml vs 13.3 ± 2.5 ng/ml, P < 0.01) and 60 min (mean ± SEM, 24.7 ± 7.4 ng/ml vs 11.1 ± 2.5 ng/ml, P <0.05). In contrast we did not observe any effect of either 10−9M, 10−7M or 10−5M melatonin on in‐vitro basal GH release and GH responses to 10−8M GRF by rat anterior pituitary cells in monolayer culture. These data suggest that melatonin plays a facilitatory role in the neuroregulation of GH secretion, probably by acting at the hypothalamic level.

ADDITIVE EFFECTS OF GROWTH HORMONE RELEASING FACTOR AND INSULIN HYPOGLYCAEMIA ON GROWTH HORMONE RELEASE IN MAN

We have measured GH and PRL changes following separate and combined administration of insulin and GH releasing factor (GRF) in six normal males. Peak GH responses to separate administration of insulin and GRF were comparable (71 4 ± 10.2 vs 70.1 ± 27.7 mU/1; mean ± SEM). However, the peak GH response following combined administration was significantly higher (120.8 ± 29.7, P < 0.05) as was the total GH released as calculated by measuring the area under the curve (P <005). In contrast the PRL response to hypoglycaemia was not altered by the combined administration of insulin and GRF. This effect was not due to any direct action of hypoglycaemia or insulin at pituitary level since basal and 10−8 M GRF stimulated GH release from rat anterior pituitary cells in vitro was not influenced by varying glucose and insulin levels. Our findings support the hypothesis that GRF and insulin‐induced hypoglycaemia release GH via different pathways which are, at least in part, additive.

CHOLINERGIC MUSCARINIC RECEPTOR BLOCKADE WITH PIRENZEPINE ABOLISHES SLOW WAVE SLEEP‐RELATED GROWTH HORMONE RELEASE IN YOUNG PATIENTS WITH INSULIN‐DEPENDENT DIABETES MELLITUS

Cholinergic receptor blockade has been shown to abolish GH secretion in a variety of physiological and pharmacological situations in normal subjects. We have investigated the effect of pirenzepine on nocturnal GH secretion in young adult patients with Type I insulin‐dependent diabetes mellitus. Five patients (three male, two female; aged 20‐27 years) were studied in a randomized order on two days separated by at least 1 week. All patients showed episodes of slow wave sleep on each occasion and this was followed by peaks of GH release when placebo alone was administered (range of GH peaks 6–115 mU/1). In contrast, cholinergic muscarinic receptor blockade with pirenzepine (100 mg orally at 2200 and 2400 h) completely abolished nocturnal GH release in each individual without altering the occurrence of slow wave sleep itself. Mean plasma glucose levels at each sampling time between each study did not differ significantly. The ability to abolish nocturnal GH secretion may be important in the field of diabetes, since excess GH secretion is implicated in several acute metabolic and chronic microvascular complications of the disease.

THE EFFECTS OF CHOLINERGIC BLOCKADE ON THE GROWTH HORMONE AND PROLACTIN RESPONSE TO INSULIN HYPOGLYCAEMIA

The effect of cholinergic blockade on growth hormone (GH) and prolactin (PRL) secretion during insulin‐induced hypoglycaemia was assessed in six normal male volunteers (mean age 23, age range 21–25). Each subject underwent two insulin tolerance tests with and without atropine. GH responses were significantly lower 45 min after insulin administration with atropine (17.5 · 2.5 mU/l (mean · SEM) than with placebo (37.6 · 3.6 mU/l, P < 0.0006). In contrast PRL responses were higher (P < 0.01) at 45 and 90 min after insulin during treatment with atropine. These data demonstrate that cholinergic mechanisms are involved in stimulatory and inhibitory pathways in the medication of the respective GH and PRL responses to insulin induced hypoglycaemia in man.