Neuroendocrine regulation of growth hormone secretion in sheep. VII. Effects of GABA

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Regulatory Peptides 52 (1994) 181-186

Neuroendocrine regulation of growth hormone secretion in sheep. VII. Effects of GABA G.S.G. Spencer*, C.J. Berry, J.J. Bass Growth Physiology, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, New Zealand

Received 7 February 94; revised version received 10 April 1994; accepted 28 April 1994 Abstract The effects of intravenous (i.v.) or intracerebroventricular (i.c.v.) administration of 7 aminobutyric acid (GABA) on plasma growth hormone (GH) concentrations have been examined in sheep. Intravenous administration of GABA resulted in a rapid, significant (P< 0.001) increase in plasma GH. Administration of 10 mg of GABA i.c.v, produced a significant (P< 0.001) increase in GH release. By contrast, 100 mg given i.c.v, was inhibitory and resulted in a decrease (P< 0.05) in plasma GH levels. Concurrent administration of somatostatin (0.5 #g/min i.v. over 1 h) did not alter the plasma GH response to 10 mg GABA given i.c.v. These data are consistent with the concept of dual sites of action for GABA in regulating GH release in sheep, but the exact mechanism through which this effect is mediated remains unclear. Key words: Growth hormone; GABA; Sheep; Somatostatin

1. Introduction Release of G H is controlled from within the brain by a wide range of factors. There are many reports on neuro-endocrine control of G H release in man and rats [ 1], but relatively little is known about neurophysiological regulation of G H in sheep. Among the substances which have been reported to affect G H release in other species is the neurotransmitter 7 aminobutyric acid (GABA). Both inhibitory and stimulatory effects of G A B A on G H release have been reported in rat and man [2-6], * Corresponding author. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 01 67-01 1 5 ( 9 4 ) 0 0 0 4 1 - U

although the majority of evidence suggests that G A B A potentiates G H release. There is some evidence to suggest that this pathway for G H release is via the release of G R F [7] although there may also be direct pituitary effects. Immunohistological studies suggest that GABAergic mechanisms may also be involved in somatostatin release [8] although other studies have found no evidence to support this view [9]. To date few data have been presented on the effects of G A B A on G H release in sheep. This paper reports the effects of both central and peripheral administration of G A B A on plasma G H levels in sheep.

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Materials

G.S.G. Spencer et al. / Regulator), Peptides 52 (1994) 181-186 and methods

Castrated male sheep of about 40 kg liveweight were housed individually indoors and were fed a complete pelleted diet at a maintenance level (1.5 ~o body weight). The sheep were fitted with indwelling jugular vein catheters and with steel intracerebroventricular (i.c.v.) cannulae placed in a lateral ventricle while under anaesthesia (for details of the procedure see [10]). After surgery the sheep were placed in individual pens and allowed at least 4 days to recover. The sheep were fed at 08.00 h and the sampling started at 09.00 h. 2.1. Intravenous administration of GABA Blood samples were taken through the jugular vein catheter during a pretreatment sampling period of one hour for measurement of basal G H levels. Following this period, 800 mg of G A B A (Sigma, St. Louis, MO) was given intravenously to six sheep and flushed in with 20 ml saline. Controls received 20 ml of saline alone. Blood samples were collected from the jugular vein catheter at 15 min intervals after treatment. 2.2. Intracerebroventricular administrat&n of GABA

Blood samples were collected into heparinized tubes on ice, centrifuged and the plasma stored at - 2 0 ° C until assayed for o G H by specific radioimmunoassay [ 10]. Data were log-transformed to normalise distribution and the data were then analysed in two-ways: by one-way A N O V A comparing the mean pretreatment G H levels for each animal with the mean G H level in the hour after treatment, and by comparison with saline controls.

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Results

Intravenous administration of 800 mg G A B A resulted in a rapid significant ( P < 0.001) increase (from

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After a one hour pretreatment sampling, 22 sheep were given G A B A i.c.v, at various doses: 1 mg (n = 6), 10 mg (n= 6) or 100 mg (n= 6), and four controls received a similar volume (100/~1) of saline i.c.v. Blood samples were collected from the jugular vein catheter at 15 min intervals after treatment. A further six sheep also received an i.c.v, injection of 10 mg GABA, but concurrently received an intravenous infusion of 0.5 #g/kg of somatostatin (Sigma) over 1 h. Control sheep for this experiment received a similar somatostatin infusion but without concurrent administration of GABA. In this study the sheep were not fed until after the experiment, and blood samples were collected at 10, 20, 30, 45 and 60 min after injection of GABA.

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Fig. 1. Plasma growth hormone (oGH) concentration in sheep before and after intravenous administration of: (upper panel) 800 mg GABA (n = 6) or (lowe panel) saline (n = 3) at time 0. Values are means + S.E.M.; **P<0.01; ***P
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1.65 + 0.02 to 8.02 + 1.4 ng/ml) in plasma G H concentrations (Fig. 1, upper panel), while saline infusion had no effect (Fig. 1, lower panel). The difference between saline treatment and G A B A was significant ( P < 0.01). Intracerebroventricular administration of 10 mg

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of G A B A also resulted in a significant (P<0.001) increase in G H , with peak G H levels (7.4 + 1.2 ng/ ml) being reached 30 mins after injection (Fig. 2B). A lower i.c.v, dose of 1 mg G A B A had no significant effect compared with saline controls, but when inter-animal variation was removed by comparing each animal with its own control values, there was a small significant effect (Fig. 2A). Following 100 mg G A B A i.c.v., there was a small decrease (from 3.2+0.42 to 2.4+0.43 ng/ml) in G H levels (Fig. 2C) which only achieved statistical significance when compared with individual basal levels. Administration of saline i.c.v, did not affect plasma G H (Fig. 2D). Concurrent infusion of somatostatin did not inhibit the G H secretion stimulated by 10 mg of G A B A given i.c.v. (Fig. 3) although infusion of somatostatin alone increased circulating plasma somatostatin concentrations more than 10-fold (from 54 + 10.4 pg/ml to 684 + t41 pg/ml). In line with previously published experiments [11 ], infusion of somatostatin at such a dose failed to have any effect on basal plasma G H concentrations (Fig. 3).

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Time ( min ) Fig. 2. Plasma growth hormone (oGH) concentration in sheep before i.c.v, administration of: either GABA at (A) 1 mg (n = 6); (B) 10 mg (n = 6); (C) 100 mg (n = 6), or (D) saline (n = 4), at time 0. Values are means+ S.E.M.; *P<0.05; **P<0.01 compared with mean pre-treatment values for each individual sheep.

The data presented here show that GABAergic mechanisms may be involved in regulating G H release in the sheep. The doses of G A B A used were chosen to relate (on a per kg body weight basis) to doses used in rats. The stimulatory effect on G H release of intracerebroventricularly administered G A B A at lower doses is similar to that reported in experiments with rats [ 5 ]. In addition, the inhibitory effect at higher doses is also consistent with the speculation [12,13] that G A B A has dual sites of action of opposite sign and may have both stimulatory and inhibitory actions. The mechanisms through which G A B A may alter G H release are still unclear. It has been suggested [7] that the effect of G A B A on G H release is mediated by G R F , but others have indicated that the

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Fig. 3. Plasma growth hormone (oGH) concentration in sheep before (black bars) and during intravenous infusion of somatostatin (0.5 #g/kg) over 60 min following i.c.v, administration of 10 mg GABA (hatched bars) or saline (dotted bars) at time 0. Values are means_+ S.E.M.; *P<0.05; **P<0.01 values are significantly different compared with mean pre-treatment values for each individual sheep.

effect of G A B A is independent of G R F [ 14,15]. In addition, evidence has also been presented that both G R F [16] and S R I F [8] secretion may be affected by GABA. These conflicting results are also be consistent with the hypothesis of a dual action of GABA. The inability of somatostatin infusion (other than at gross pharmacological levels) to inhibit basal G H release in normal subjects is now well established [11,17-20]. However, the lack of effect of concurrent somatostatin infusion on the GABA-stimulated G H release in these studies suggests that the G H stimulating action of G A B A is not brought about through decreasing endogenous SRIF. This finding, together with the conflicting results concerning the involvement of G R F , and the potent effects of G A B A given peripherally, may indicate that there may also be a direct action of G A B A at the pituitary level. The only reports of the effects of G A B A on G H

release in sheep [21 ] showed that neither the G A B A agonist muscimol, nor the antagonist picrotoxin, had any effect on G H release in neonatal or adult sheep; this lack of effect may have been due to the use of an inappropriate dose. However, the difference between that observation [21] and those report in the present paper may relate to sex. It has been suggested that there is a sex difference in the growth hormone response to GABAergic stimulation in humans; female subjects failed to show a G H response to the G A B A agonist baclofen while males responded with elevated plasma G H levels [3]. In the present study we have found a GH-stimulatory effect in castrated male sheep while Gluckman [21] could find no effect of G A B A in ewes. In apparent contrast to these results, it has also been reported that the G A B A agonist muscimol inhibits G H release in the ovine fetus [21]. Such inhibition of G H release is the opposite of the major

G.S.G. Spencer et al. / Regulatory Peptides 52 (1994) 181-186

effect f o u n d in t h e p r e s e n t s t u d y u s i n g p o s t n a t a l sheep. H o w e v e r , this m a y reflect different sensitivity to G A B A e r g i c s t i m u l a t i o n at different stages o f development, and GABAergic neurones have a u n i q u e influence d u r i n g t h e late g e s t a t i o n a n d early n e o n a t a l p e r i o d s [22]. F u r t h e r m o r e , the m e c h a n i s m s regulating G H r e l e a s e are n o t fully d e v e l o p e d in the fetus.

Acknowledgements W e are grateful to T. W a t s o n a n d M. H o l m e s for c a r e o f the a n i m a l s , S. J o h n s t o n a n d R. L a s e n b y for e x p e r i m e n t a l a s s i s t a n c e , a n d to Dr. N e i l C o x for statistical a d v i c e a n d analysis o f the d a t a .

[ 8]

[ 9]

[10]

[ 11 ]

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