DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 9(2):115-123,1992
NEUROENDOCRINE REGULATION OF GROWTH HORMONE SECRETION IN SHEEP.V. GROWTH HORMONE RELEASING FACTOR AND THYROTROPHIN RELEASING HORMONE G.S.G. Spencer, W.M. Aitken, S.C. Hodgkinson and J.J. Bass Growth Physiology, MAFTech, Ruakura Agricultural Centre, Private Bag, Hamilton, New Zealand Received June 27, 1991
ABSTRACT The effects of intravenous (IV) and intracerebroventricular (ICV) administration of either bovine growth hormone releasing hormone (GRF) or thyrotrophin releasing hormone (TRH) on plasma growth hormone (GH) and glucose levels have been examined in sheep. Intravenous GRF 1-29NH 2 at 3 and 30/ag stimulated an increase in GH levels in a dosedependent fashion; administration of GRF into a lateral cerebral ventricle, however, produced a smaller GH response which was similar at these two doses. Evaluation of somatostatin levels in petrosal sinus blood (which collects pituitary effluent blood) showed that ICV administration of GRF stimulated a release of somatostatin into the blood. Furthermore, concurrent administration of GRF and a potent anti-somatostatin serum ICV resulted in a much enhanced release of GH which was similar to that obtained with a comparable dose of GRF given IV. TRH (as another putative GH-secretagogue) was also administered both IV and ICV. When given IV, 200 pg (but not 100 lag) TRH produced an elevation in GH levels. By contrast, when 5 tag TRH was given ICV there was a decrease in circulating GH levels, but no change in plasma somatostatin concentrations. These results indicate that the smaller GH response to ICV- compared with IV-administered GRF is due to the release of somatostatin within the brain. In addition, it would seem that TRH is not a physiological GH-secretagogue in sheep. INTRODUCTION An enormous number and variety of factors are known to influence the release of GH; this may be a reflection of the multi-faceted nature of growth. It is believed that most of the factors regulating GH secretion act through either, or both, of two hypothalamic hormones: GH-releasing factor (GRF) and somatostatin. These hypothalamic hypophysiotropic peptides are, in turn, regulated by adjacent hypothalamic neurones which respond to stimulation by biogenic amines acting as neurotransmitters of higher central nervous system messages. Another hypothalamic peptide, thyrotrophin releasing hormone (TRH), is also a putative GH-secretagogue. In avian species the stimulatory effect of TRH on GH release is clear (1), but the results from other species are equivocal and contradictory (2). In sheep, TRH has been reported to stimulate GH release from pituitaries in culture (3,4) but most reports indicate that intravenous administration of TRH to sheep has no effect on GH release (5,6). A clear appreciation of the endogenous control exerted by hypothalamic peptides over GH release is of both practical and academic interest. To date, the rat has provided the main animal model for in vivo examination of neuroendocrine mechanisms, but there are fewer data on farm animal species. In the present paper we report the effect of both intravenous and intracerebroventricular administration of bovine GRF and of TRH on GH and glucose levels in sheep, and examine the interaction between endogenous somatostatin and exogenous GRF in regulating GH release. Copyright © 1992 Butterworth-Heinemann
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MATERIALS AND METHODS Coopworth sheep (30-44 kg liveweight) were individually housed indoors in adjacent pens and fed a complete pelleted diet. After adaptation to these conditions the animals were placed on a maintenance ration of 450g/d (approximately 1.5% body weight). This amount has previously proved sufficient to elevate basal GH levels slightly without producing malnutrition, stress or any other effects that may interfere with the study (7,8). On the days of study, the sheep were blood sampled via jugular vein catheters at 15 min intervals for 1 hr prior to treatment and for 90 min following various doses of solutions of GRF or TRH administered either intravenously (IV) or directly into the CSF intracerebroventricularly (ICV). Blood samples were collected into heparinised tubes, centrifuged and the plasma stored at -20 C. TREATMENTS I. Intravenous GRF: Eleven sheep were given GRF (bovine growth hormone releasing factor 1-29 amide; Sigma Chemical Co., St Louis, MO) intravenously in 100 ~1 saline and flushed in with 10 ml saline. Seven sheep received 30 lag GRF (approximately 1 ktg/kg; 0.3 nmols/kg); four sheep received 3 lag (0.03 nmols/kg) GRF. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. H. Intracerebroventricular GRF: Sheep fitted with both jugular vein catheters and with indwelling intracerebroventricular cannulae positioned in a lateral ventricle as described previously (7) were studied. Eight sheep received 300 ng GRF in 100 lal saline directly into a lateral ventricle; five sheep received 3 lag, seven received 30 tag in 100 lal saline, and four received 100 lal saline. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. Three sheep were fitted with jugular and ICV cannulae and also with petrosal sinus cannulae ~to allow collection of effluent blood from the pituitary (9). In these sheep, blood samples were collected additionally at 5, 10 and 20 min after GRF administration (30 lag) and somatostatin levels were measured in plasma at -10, -5, 0, 5, 10, 15, 20 and 30 min. III. Intracerebroventricular GRF and anti-somatostatin serum: Six sheep were sampled at 15 min intervals for 1 hr and then were given 30 ~tg GRF ICV as above, together with 250 lal of a concentrated potent, specific, anti-somatostatin serum raised in sheep (10). Seven sheep were given 250 IJ of the anti-somatostatin serum alone. The amount of antiserum given was capable of binding 118 Jag of somatostatin in vitro and has been shown to be able to elicit an elevation in plasma GH when given either intravenously (11,12,13) or intracerebrally (8). Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. IV. Thyrotrophin releasing hormone: Six sheep were given 100 lag of TRH (Sigma) and five received 200 lag TRH in 20 ml solution by IV injection. Four further sheep were given 1 lag TRH (in 100 lxl) ICV and four received 5 lag by ICV injection in a similar volume. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations and samples were also taken for somatostatin as in (2) above. Glucose was measured using an automated glucose oxidase system (Beckman Glucose Analyzer 2, Palo Alto, USA), and plasma GH levels were measured using a specific, sensitive, homologous radioimmunoassay (7), with intra- and inter-assay variations of 2.1% and 7%, respectively in these studies. Plasma samples for measurement of somatostatin were collected into chilled tubes containing EDTA and 500 KIU of Trasylol (Aprotinin; Sigma), kept on ice for a minimum time and processed at
Vol. 9(2):115-123,1992
NEUROENDOCRINE REGULATION OF GROWTH HORMONE SECRETION IN SHEEP.V. GROWTH HORMONE RELEASING FACTOR AND THYROTROPHIN RELEASING HORMONE G.S.G. Spencer, W.M. Aitken, S.C. Hodgkinson and J.J. Bass Growth Physiology, MAFTech, Ruakura Agricultural Centre, Private Bag, Hamilton, New Zealand Received June 27, 1991
ABSTRACT The effects of intravenous (IV) and intracerebroventricular (ICV) administration of either bovine growth hormone releasing hormone (GRF) or thyrotrophin releasing hormone (TRH) on plasma growth hormone (GH) and glucose levels have been examined in sheep. Intravenous GRF 1-29NH 2 at 3 and 30/ag stimulated an increase in GH levels in a dosedependent fashion; administration of GRF into a lateral cerebral ventricle, however, produced a smaller GH response which was similar at these two doses. Evaluation of somatostatin levels in petrosal sinus blood (which collects pituitary effluent blood) showed that ICV administration of GRF stimulated a release of somatostatin into the blood. Furthermore, concurrent administration of GRF and a potent anti-somatostatin serum ICV resulted in a much enhanced release of GH which was similar to that obtained with a comparable dose of GRF given IV. TRH (as another putative GH-secretagogue) was also administered both IV and ICV. When given IV, 200 pg (but not 100 lag) TRH produced an elevation in GH levels. By contrast, when 5 tag TRH was given ICV there was a decrease in circulating GH levels, but no change in plasma somatostatin concentrations. These results indicate that the smaller GH response to ICV- compared with IV-administered GRF is due to the release of somatostatin within the brain. In addition, it would seem that TRH is not a physiological GH-secretagogue in sheep. INTRODUCTION An enormous number and variety of factors are known to influence the release of GH; this may be a reflection of the multi-faceted nature of growth. It is believed that most of the factors regulating GH secretion act through either, or both, of two hypothalamic hormones: GH-releasing factor (GRF) and somatostatin. These hypothalamic hypophysiotropic peptides are, in turn, regulated by adjacent hypothalamic neurones which respond to stimulation by biogenic amines acting as neurotransmitters of higher central nervous system messages. Another hypothalamic peptide, thyrotrophin releasing hormone (TRH), is also a putative GH-secretagogue. In avian species the stimulatory effect of TRH on GH release is clear (1), but the results from other species are equivocal and contradictory (2). In sheep, TRH has been reported to stimulate GH release from pituitaries in culture (3,4) but most reports indicate that intravenous administration of TRH to sheep has no effect on GH release (5,6). A clear appreciation of the endogenous control exerted by hypothalamic peptides over GH release is of both practical and academic interest. To date, the rat has provided the main animal model for in vivo examination of neuroendocrine mechanisms, but there are fewer data on farm animal species. In the present paper we report the effect of both intravenous and intracerebroventricular administration of bovine GRF and of TRH on GH and glucose levels in sheep, and examine the interaction between endogenous somatostatin and exogenous GRF in regulating GH release. Copyright © 1992 Butterworth-Heinemann
115
0739-7240/92/$3.00
116
SPENCER ET AL.
MATERIALS AND METHODS Coopworth sheep (30-44 kg liveweight) were individually housed indoors in adjacent pens and fed a complete pelleted diet. After adaptation to these conditions the animals were placed on a maintenance ration of 450g/d (approximately 1.5% body weight). This amount has previously proved sufficient to elevate basal GH levels slightly without producing malnutrition, stress or any other effects that may interfere with the study (7,8). On the days of study, the sheep were blood sampled via jugular vein catheters at 15 min intervals for 1 hr prior to treatment and for 90 min following various doses of solutions of GRF or TRH administered either intravenously (IV) or directly into the CSF intracerebroventricularly (ICV). Blood samples were collected into heparinised tubes, centrifuged and the plasma stored at -20 C. TREATMENTS I. Intravenous GRF: Eleven sheep were given GRF (bovine growth hormone releasing factor 1-29 amide; Sigma Chemical Co., St Louis, MO) intravenously in 100 ~1 saline and flushed in with 10 ml saline. Seven sheep received 30 lag GRF (approximately 1 ktg/kg; 0.3 nmols/kg); four sheep received 3 lag (0.03 nmols/kg) GRF. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. H. Intracerebroventricular GRF: Sheep fitted with both jugular vein catheters and with indwelling intracerebroventricular cannulae positioned in a lateral ventricle as described previously (7) were studied. Eight sheep received 300 ng GRF in 100 lal saline directly into a lateral ventricle; five sheep received 3 lag, seven received 30 tag in 100 lal saline, and four received 100 lal saline. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. Three sheep were fitted with jugular and ICV cannulae and also with petrosal sinus cannulae ~to allow collection of effluent blood from the pituitary (9). In these sheep, blood samples were collected additionally at 5, 10 and 20 min after GRF administration (30 lag) and somatostatin levels were measured in plasma at -10, -5, 0, 5, 10, 15, 20 and 30 min. III. Intracerebroventricular GRF and anti-somatostatin serum: Six sheep were sampled at 15 min intervals for 1 hr and then were given 30 ~tg GRF ICV as above, together with 250 lal of a concentrated potent, specific, anti-somatostatin serum raised in sheep (10). Seven sheep were given 250 IJ of the anti-somatostatin serum alone. The amount of antiserum given was capable of binding 118 Jag of somatostatin in vitro and has been shown to be able to elicit an elevation in plasma GH when given either intravenously (11,12,13) or intracerebrally (8). Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations. IV. Thyrotrophin releasing hormone: Six sheep were given 100 lag of TRH (Sigma) and five received 200 lag TRH in 20 ml solution by IV injection. Four further sheep were given 1 lag TRH (in 100 lxl) ICV and four received 5 lag by ICV injection in a similar volume. Blood samples were collected at 15 min intervals for measurement of GH and glucose concentrations and samples were also taken for somatostatin as in (2) above. Glucose was measured using an automated glucose oxidase system (Beckman Glucose Analyzer 2, Palo Alto, USA), and plasma GH levels were measured using a specific, sensitive, homologous radioimmunoassay (7), with intra- and inter-assay variations of 2.1% and 7%, respectively in these studies. Plasma samples for measurement of somatostatin were collected into chilled tubes containing EDTA and 500 KIU of Trasylol (Aprotinin; Sigma), kept on ice for a minimum time and processed at
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