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Evidence for a dual function of oxytocin in the control of growth hormone secretion in rats
Regulatory Peptides 69 (1997) 1–5
Evidence for a dual function of oxytocin in the control of growth hormone secretion in rats a, a *, M.D. Anna-Lena Hulting b , M.D. Kerstin Uvnas-Moberg ¨ ¨ Eva Bjorkstrand a
Department of Physiology and Pharmacology, Division of Pharmacology, Karolinska Institute, Stockholm, Sweden b Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden Received 24 June 1996; revised 27 September 1996; accepted 17 October 1996
Abstract The aim of the present study was to investigate the role of oxytocin (Oxy) in the control of growth hormone (GH) release. Oxy was administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) to male rats. The animals were decapitated and trunk blood was collected at 30 and 120 min after Oxy administration. GH levels were analyzed by radioimmunoassay. Oxy (100 mg, s.c) increased plasma levels of GH significantly 30 min after administration. Oxy (2 ng, i.c.v.) caused a significant rise of GH after 120 min. This effect was completely abolished by previous administration of the Oxy antagonist 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin. When 5 mg of Oxy were given i.c.v. or 1 mg s.c., an inhibition of GH secretion was seen after 120 min. This effect was also abolished by the Oxy antagonist. Thus Oxy may influence GH in opposite directions depending on the doses given. 1997 Elsevier Science B.V. All rights reserved Keywords: Oxytocin; Oxytocin antagonist; Growth hormone; Somatostatin; Rats
1. Introduction Oxytocin (Oxy) has been shown to influence the secretion of several pituitary hormones. It stimulates prolactin secretion —an effect which seems to be exerted directly on the prolactin-producing cells [1]. Oxytocinergic neurons from the paraventricular nucleus (PVN) that project to the median eminence allow Oxy to be released into the hypophysial portal system [2]. Thus, high amounts of Oxy may reach the anterior pituitary by this route and thereby influence hormonal release [3]. Oxy also influences growth hormone (GH) secretion —although data as to the effect of Oxy are conflicting. Lumpkin et al. [1] reported an increase of plasma levels of GH in response to intracerebroventricular (i.c.v.) injections of Oxy (1 mg) in conscious male rats, but failed to demonstrate effects in vitro. Franci et al. [4], on the other
*Corresponding author. Telefax. 1 0046 8 332237 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-0115( 96 )02101-5
hand, found evidence for an inhibitory effect of Oxy on GH release, since antiserum against Oxy applied into the third ventricle increased GH levels. The aim of the present study was to explore the effects on GH release in conscious rats of a wide range of doses of Oxy applied i.c.v. or subcutaneously (s.c.).
2. Materials and methods
2.1. Animals Male Sprague–Dawley rats (B & K Universal, Sollentuna, Sweden) weighing 200–350 g were used in this study. The animals were housed in air-conditioned and temperature-controlled rooms (228C) illuminated between 7.00 and 19.00 h and had free access to food (Lactamin, Vadstena, Sweden) and water.
2
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
2.2. Surgery
2.6. Somatostatin
Before surgery, the animals were anaesthetized with chloral hydrate (Apoteksbolaget, Stockholm, Sweden) 400 mg kg 21 body weight intraperitoneally. The skull was uncovered and a stainless-steel guide cannula (21G) was stereotaxically fixed to the skull by means of stainless-steel screws and acrylic dental cement. The coordinates were 1.00 mm posterior and 1.30 mm lateral to the bregma. The guide reached, but did not penetrate the dura mater. The animals were allowed to recover for one week before the experiments. At the end of each experiment, the site of injection into the lateral ventricle was confirmed by injection of 2 ml of toluidine blue and the brain was removed, frozen and sectioned.
Before determination of somatostatin, the peptide was separated from plasma proteins using Sep-Pak C 18 cartridges (Water Assoc., Milford, MA, USA). Somatostatin RIA was then performed as described by Efendic et al. [6]. The limit of detection was 2 pmol l 21 . The intra- and inter-assay coefficients of variation were 10% and 9%, respectively.
2.3. Experiments
3. Results
On the day of experiment, Oxy or the Oxy antagonist 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin (Ferring, Malmo¨ Sweden) dissolved in 5 ml of saline, or saline as a control was slowly injected into the lateral ventricle over a period of 1 min. Injections were performed using a 27G stainless-steel injection needle inserted into the guide cannula. The injection needle was connected to a 10 ml Hamilton syringe via a polyethylene tube. The injection needles reached 3.8 mm below the dura mater, with the needle-tip in the lateral ventricle. In a second group of animals, 0.3 ml of Oxy was injected s.c. This system of administration is less stressful to the animals than intravenous injections. Control animals received the corresponding volume of saline i.c.v. or s.c.
3.1. Intracerebroventricular administration
2.4. Blood samples At the end of each experiment, the rats were decapitated and trunk blood was collected in chilled plastic tubes containing 10 I.U. ml 21 heparin (Kabi, Stockholm, Sweden) and 500 KIE ml 21 aprotinin (Bayer, Stockholm, Sweden). The samples were centrifuged immediately and plasma was removed and frozen.
2.5. Radioimmunoassay ( RIA) 2.5.1. GH Rat GH levels were measured by the radioimmunoassay ´ et al. [5]. Highly purified rat method described by Eden GH (NIDDK) with a potency of 2.0 I.U. mg 21 was used as a reference. Plasma GH concentrations were expressed as ng ml 21 in terms of the rGH RP-2 standard. The limit of detection was 0.31 ng ml 21 . Intra- and inter-assay coefficients of variation in the RIA were 11% and 14%, respectively.
2.7. Statistical calculations Data are presented as mean values 6S.D. based on four to seven observations per group. Differences between groups were evaluated with the Mann–Whitney U-test.
Oxy (2 ng) tended to increase GH levels after 30 min and caused a significant increase 120 min after administration of the drug. This stimulatory effect on GH secretion was completely abolished by pretreatment with an Oxy antagonist (1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin) (Fig. 1). No effect on plasma levels of somatostatin was seen (Fig. 1). A higher dose of Oxy, 200 ng, caused a significant GH increment after 30 min and significantly decreased plasma levels of somatostatin (Fig. 2). In contrast, when a very high dose of Oxy (5 mg) was given, plasma GH was unchanged at 30 min but decreased after 120 min (Fig. 3). No effect on plasma levels of somatostatin was seen (Fig. 3).
3.2. Subcutaneous administration When Oxy was given s.c., 10 mg Oxy tended to, and 100 mg significantly stimulated GH release after 30 min. A stimulatory effect of Oxy could not be obtained by using a higher dosage of Oxy. In fact, injection of 1 mg kg 21 of Oxy inhibited GH release at 120 min. This effect was abolished by pretreatment with the Oxy antagonist (Fig. 4).
4. Discussion In the present study we found evidence for a stimulatory as well as an inhibitory effect on GH secretion by Oxy. The stimulatory and the inhibitory effects of Oxy on GH release were observed in different dose ranges and at different times. The GH release-promoting effect of Oxy given i.c.v. was obtained in the ng range and the inhibitory effect in the mg range. Interestingly, a similar difference was observed following s.c. injections of Oxy, although the doses used were 1000-fold higher. It is possible that
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
3
Fig. 2. Plasma levels of GH (upper panel A) and somatostatin (lower panel B) 30 min after administration of 200 ng oxytocin i.c.v. in the lateral ventricle. Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05 and *** p , 0.001. Fig. 1. Effects of oxytocin and / or an oxytocin antagonist administered i.c.v. in the lateral ventricle on plasma levels of GH (upper panel A) and somatostatin (lower panel B). Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
Oxy exerted its effects via central mechanisms also following s.c. administration, since 0.1% of a dose of Oxy given s.c. passes the blood–brain barrier [7]. In line with the present findings, Oxy has been shown to exert different behavioral as well as physiological effects depending on dose. Some effects such as increased sexual behavior, anxiolytic-like effects and activation of vagal nerve activity leading to bradycardia, increased HCl secretion and insulin release are induced by ng amounts of Oxy given i.c.v. In contrast, mg amounts of Oxy, administered i.c.v., are needed to induce effects on food intake, sedation and elevation of pain threshold (for a review see [8]). A possible pathway for the Oxy effects described in the present paper is the portal blood system, since Oxy neurons project not only to the posterior pituitary [9] but also to other hypothalamic sites including the median eminence [2]. By this route, Oxy may influence GH secretion either directly on the somatotroph cells or by interfering with the hypothalamic neurons that control GH secretion, e.g. the GHRH and somatostatin-containing
neurons. The stimulatory effect on GH secretion observed was obtained with very low doses of Oxy. In a previous study we have observed that ng-amounts of Oxy given i.c.v. inhibit circulating levels of somatostatin. This inhibitory effect on somatostatin is found to be abolished by pretreatment with atropine [10], suggesting that this somatostatin derives from cholinergically innervated somatostatin-producing cells in the gastrointestinal tract. If hypothalamic somatostatin is inhibited by ng-amounts of Oxy in a similar way, the stimulatory effect of Oxy on GH secretion may be explained. In a recent study we showed that Oxy in concentrations of 10 210 –2 3 10 26 M lowered Oxy secretion from dispersed anterior pituitary cells [11]. This inhibition was completely restored by addition of the Oxy antagonist used in the present study. Oxy also inhibited GHRH-induced GH release and the maximal magnitude of this response was similar to that caused by somatostatin. Considering this, Oxy might inhibit GH release via a direct action on Oxy receptors located on the GH cells. However, the inhibition of GH secretion seen in the present study in response to high doses of Oxy given i.c.v. or s.c, was not seen until 120 min after injection. Therefore the possibility that this inhibitory effect is indirect cannot be excluded.
4
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
The fact that the inhibitory effect caused by 1 mg kg 21 of Oxy given s.c. was reversed by the Oxy antagonist indicates that oxytocinergic mechanisms are involved. Lactation is associated with enhanced Oxy secretion both in the CNS and in the circulation [7]. In addition, suckling in lactating rats is followed by a depletion of pituitary concentration of GH after 30 and 180 min of suckling [12,13] and is clearly associated with increased GH secretion [14]. This effect might correspond to our data that show a stimulatory effect of Oxy on GH secretion after 30 and 120 min. It has been known for two decades that the GH secretion in male rats is markedly pulsatile [15]. In this study we have relatively low GH levels with a relatively small spread between the samples. We cannot completely explain this but one reason could be that the experiments were always performed at the same time of the day, two hours after the light was turned on, although we cannot exclude a stress factor during blood sampling. Finally, it should be noted that obese people, known to have a blunted GH response to certain stimuli, e.g. insulininduced hypoglycemia, also have high Oxy levels [16]. It remains to be established whether there is a causal relationship between these two findings. In conclusion GH is affected in two opposite ways by Oxy. In response to low doses of Oxy (ng, i.c.v.) GH is released and in response to high doses (mg, i.c.v.) GH release is inhibited. The mechanisms behind the stimulatory and the inhibitory actions of Oxy on GH secretion remain to be clarified and the functional importance to be established. Fig. 3. Effect of oxytocin administered i.c.v. in the lateral ventricle on plasma levels of GH (upper panel A) and somatostatin (lower panel B). Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
Acknowledgments ¨ Sweden for the oxytocin and We thank Ferring, Malmo, the oxytocin antagonist used in this study. This study was supported by grants from the Swedish Medical Council (B94-04X-05207-17B 1 B96-19x-11245-02B
References
Fig. 4. Effect of 10–1000 mg kg 21 oxytocin and / or an oxytocin antagonist administered s.c. on plasma levels of GH. Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
[1] Lumpkin, M.D., Samson, W.K. and McCann, S.M., Hypothalamic and pituitary sites of action of oxytocin to alter prolactin secretion in the rat, Endocrinology, 112 (1983) 111–115. [2] Vandesande, F., Dierickx, K. and Demey, J., The origin of the vasopressinergic and oxytocinergic fibres of the external region of the median eminence of the rat hypophysis, Cell Tiss. Res., 180 (1977) 443–452. [3] Gibbs, D.M., High concentrations of oxytocin in hypophysial portal plasma, Endocrinology, 114 (1984) 1216–1218. [4] Franci, C.R., Anselmo-Franci, J.A., Kozlowski, G.P. and McCann, S.M., Actions of endogenous vasopressin and oxytocin on anterior pituitary hormone secretion, Neuroendocrinology, 57 (1993) 693– 699. ´ S., Albertsson-Wikland, K. and Isaksson, O., Plasma levels of [5] Eden, growth hormone in female rats of different ages, Acta Endocrinol. (Copenh), 88 (1978) 676–690. ´ A. and Uvnas-Wallensten, ¨ [6] Efendic, S., Enzmann, F., Nylen K.,
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
[7]
[8] [9]
[10]
Sulphonurea (Glibenamide) enhances somatostatin and inhibits glucagon release induced by arginine, Acta Physiol. Scand., 108 (1980) 231–233. Kendrick, K., Keverne, E., Baldwin, B. and Sharman, D., Cerebrospinal fluid levels of acethylcholinesterase, monoamines and oxytocin during labour, parturition, vagocervical stimulation, lamb separation and suckling in sheep, Neuroendocrinology, 44 (1986) 149–156. Richard, P., Moos, F. and Freund-Mercier, M.-J., Central effects of oxytocin, Physiol. Rev., 71 (1991) 331–370. Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration: organisation of the paraventricular and supraoptic nuclei, Ann. Rev. Neurosci., 6 (1983) 269–324. ¨ ¨ Bjorkstrand, E., Ahlenius, S., Smedh, U. and Uvnas-Moberg, K., The oxytocin receptor antagonist 1-deamino-2-D-Tyr-(OEt)-4-Thr-8Orn-oxytocin inhibits effects of the 5-HT1A agonist 8-OH-DPAT on insulin, cholecystokinin and somatostatin levels, Regulatory Peptides, 63 (1996) 47–52.
5
¨ ¨ [11] Hulting, A.L., Grenback, E., Pineda, R., Coya, R., Uvnas-Moberg, ¨ K., Meister, B. and Hokfelt, T., Effect of oxytocin on growth hormone release in vitro, Regulatory Peptides, 67 (1996) 69–73. [12] Grosvenor, C.E., Krulich, L. and McCann, S.M., Depletion of pituitary concentration of growth hormone as a result of suckling in the lactating rat, Endocrinology, 82 (1968) 617–619. [13] Sar, M. and Meites, J., Effects of suckling on pituitary release of prolactin, GH, and TSH in postpartum lactating rats, Neuroendocrinology, 4 (1969) 25–31. [14] Saunders, A., Terry, L.C., Audet, J. Brazeau, P. and Martin, J.B., Dynamic studies of growth hormone and prolactin secretion in the female rat, Neuroendocrinology, 21 (1976) 193–203. [15] Tannenbaum, G.S. and Martin, J.B., Evidence for an endogenous ultraradian rythm governing growth horrmone secretion in the rat, Endocrinology, 98 (1976) 562–570. ¨ L., Backman, L., Matthiesen, A.S. and Uvnas¨ [16] Stock, S., Granstrom, Moberg, K., Elevated plasma levels of oxytocin in obese subjects before and after gastric banding, Inter. J. Obes., 13 (1989) 213–222.
Evidence for a dual function of oxytocin in the control of growth hormone secretion in rats a, a *, M.D. Anna-Lena Hulting b , M.D. Kerstin Uvnas-Moberg ¨ ¨ Eva Bjorkstrand a
Department of Physiology and Pharmacology, Division of Pharmacology, Karolinska Institute, Stockholm, Sweden b Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden Received 24 June 1996; revised 27 September 1996; accepted 17 October 1996
Abstract The aim of the present study was to investigate the role of oxytocin (Oxy) in the control of growth hormone (GH) release. Oxy was administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) to male rats. The animals were decapitated and trunk blood was collected at 30 and 120 min after Oxy administration. GH levels were analyzed by radioimmunoassay. Oxy (100 mg, s.c) increased plasma levels of GH significantly 30 min after administration. Oxy (2 ng, i.c.v.) caused a significant rise of GH after 120 min. This effect was completely abolished by previous administration of the Oxy antagonist 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin. When 5 mg of Oxy were given i.c.v. or 1 mg s.c., an inhibition of GH secretion was seen after 120 min. This effect was also abolished by the Oxy antagonist. Thus Oxy may influence GH in opposite directions depending on the doses given. 1997 Elsevier Science B.V. All rights reserved Keywords: Oxytocin; Oxytocin antagonist; Growth hormone; Somatostatin; Rats
1. Introduction Oxytocin (Oxy) has been shown to influence the secretion of several pituitary hormones. It stimulates prolactin secretion —an effect which seems to be exerted directly on the prolactin-producing cells [1]. Oxytocinergic neurons from the paraventricular nucleus (PVN) that project to the median eminence allow Oxy to be released into the hypophysial portal system [2]. Thus, high amounts of Oxy may reach the anterior pituitary by this route and thereby influence hormonal release [3]. Oxy also influences growth hormone (GH) secretion —although data as to the effect of Oxy are conflicting. Lumpkin et al. [1] reported an increase of plasma levels of GH in response to intracerebroventricular (i.c.v.) injections of Oxy (1 mg) in conscious male rats, but failed to demonstrate effects in vitro. Franci et al. [4], on the other
*Corresponding author. Telefax. 1 0046 8 332237 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-0115( 96 )02101-5
hand, found evidence for an inhibitory effect of Oxy on GH release, since antiserum against Oxy applied into the third ventricle increased GH levels. The aim of the present study was to explore the effects on GH release in conscious rats of a wide range of doses of Oxy applied i.c.v. or subcutaneously (s.c.).
2. Materials and methods
2.1. Animals Male Sprague–Dawley rats (B & K Universal, Sollentuna, Sweden) weighing 200–350 g were used in this study. The animals were housed in air-conditioned and temperature-controlled rooms (228C) illuminated between 7.00 and 19.00 h and had free access to food (Lactamin, Vadstena, Sweden) and water.
2
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
2.2. Surgery
2.6. Somatostatin
Before surgery, the animals were anaesthetized with chloral hydrate (Apoteksbolaget, Stockholm, Sweden) 400 mg kg 21 body weight intraperitoneally. The skull was uncovered and a stainless-steel guide cannula (21G) was stereotaxically fixed to the skull by means of stainless-steel screws and acrylic dental cement. The coordinates were 1.00 mm posterior and 1.30 mm lateral to the bregma. The guide reached, but did not penetrate the dura mater. The animals were allowed to recover for one week before the experiments. At the end of each experiment, the site of injection into the lateral ventricle was confirmed by injection of 2 ml of toluidine blue and the brain was removed, frozen and sectioned.
Before determination of somatostatin, the peptide was separated from plasma proteins using Sep-Pak C 18 cartridges (Water Assoc., Milford, MA, USA). Somatostatin RIA was then performed as described by Efendic et al. [6]. The limit of detection was 2 pmol l 21 . The intra- and inter-assay coefficients of variation were 10% and 9%, respectively.
2.3. Experiments
3. Results
On the day of experiment, Oxy or the Oxy antagonist 1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin (Ferring, Malmo¨ Sweden) dissolved in 5 ml of saline, or saline as a control was slowly injected into the lateral ventricle over a period of 1 min. Injections were performed using a 27G stainless-steel injection needle inserted into the guide cannula. The injection needle was connected to a 10 ml Hamilton syringe via a polyethylene tube. The injection needles reached 3.8 mm below the dura mater, with the needle-tip in the lateral ventricle. In a second group of animals, 0.3 ml of Oxy was injected s.c. This system of administration is less stressful to the animals than intravenous injections. Control animals received the corresponding volume of saline i.c.v. or s.c.
3.1. Intracerebroventricular administration
2.4. Blood samples At the end of each experiment, the rats were decapitated and trunk blood was collected in chilled plastic tubes containing 10 I.U. ml 21 heparin (Kabi, Stockholm, Sweden) and 500 KIE ml 21 aprotinin (Bayer, Stockholm, Sweden). The samples were centrifuged immediately and plasma was removed and frozen.
2.5. Radioimmunoassay ( RIA) 2.5.1. GH Rat GH levels were measured by the radioimmunoassay ´ et al. [5]. Highly purified rat method described by Eden GH (NIDDK) with a potency of 2.0 I.U. mg 21 was used as a reference. Plasma GH concentrations were expressed as ng ml 21 in terms of the rGH RP-2 standard. The limit of detection was 0.31 ng ml 21 . Intra- and inter-assay coefficients of variation in the RIA were 11% and 14%, respectively.
2.7. Statistical calculations Data are presented as mean values 6S.D. based on four to seven observations per group. Differences between groups were evaluated with the Mann–Whitney U-test.
Oxy (2 ng) tended to increase GH levels after 30 min and caused a significant increase 120 min after administration of the drug. This stimulatory effect on GH secretion was completely abolished by pretreatment with an Oxy antagonist (1-deamino-2-D-Tyr-(OEt)-4-Thr-8-Orn-oxytocin) (Fig. 1). No effect on plasma levels of somatostatin was seen (Fig. 1). A higher dose of Oxy, 200 ng, caused a significant GH increment after 30 min and significantly decreased plasma levels of somatostatin (Fig. 2). In contrast, when a very high dose of Oxy (5 mg) was given, plasma GH was unchanged at 30 min but decreased after 120 min (Fig. 3). No effect on plasma levels of somatostatin was seen (Fig. 3).
3.2. Subcutaneous administration When Oxy was given s.c., 10 mg Oxy tended to, and 100 mg significantly stimulated GH release after 30 min. A stimulatory effect of Oxy could not be obtained by using a higher dosage of Oxy. In fact, injection of 1 mg kg 21 of Oxy inhibited GH release at 120 min. This effect was abolished by pretreatment with the Oxy antagonist (Fig. 4).
4. Discussion In the present study we found evidence for a stimulatory as well as an inhibitory effect on GH secretion by Oxy. The stimulatory and the inhibitory effects of Oxy on GH release were observed in different dose ranges and at different times. The GH release-promoting effect of Oxy given i.c.v. was obtained in the ng range and the inhibitory effect in the mg range. Interestingly, a similar difference was observed following s.c. injections of Oxy, although the doses used were 1000-fold higher. It is possible that
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
3
Fig. 2. Plasma levels of GH (upper panel A) and somatostatin (lower panel B) 30 min after administration of 200 ng oxytocin i.c.v. in the lateral ventricle. Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05 and *** p , 0.001. Fig. 1. Effects of oxytocin and / or an oxytocin antagonist administered i.c.v. in the lateral ventricle on plasma levels of GH (upper panel A) and somatostatin (lower panel B). Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
Oxy exerted its effects via central mechanisms also following s.c. administration, since 0.1% of a dose of Oxy given s.c. passes the blood–brain barrier [7]. In line with the present findings, Oxy has been shown to exert different behavioral as well as physiological effects depending on dose. Some effects such as increased sexual behavior, anxiolytic-like effects and activation of vagal nerve activity leading to bradycardia, increased HCl secretion and insulin release are induced by ng amounts of Oxy given i.c.v. In contrast, mg amounts of Oxy, administered i.c.v., are needed to induce effects on food intake, sedation and elevation of pain threshold (for a review see [8]). A possible pathway for the Oxy effects described in the present paper is the portal blood system, since Oxy neurons project not only to the posterior pituitary [9] but also to other hypothalamic sites including the median eminence [2]. By this route, Oxy may influence GH secretion either directly on the somatotroph cells or by interfering with the hypothalamic neurons that control GH secretion, e.g. the GHRH and somatostatin-containing
neurons. The stimulatory effect on GH secretion observed was obtained with very low doses of Oxy. In a previous study we have observed that ng-amounts of Oxy given i.c.v. inhibit circulating levels of somatostatin. This inhibitory effect on somatostatin is found to be abolished by pretreatment with atropine [10], suggesting that this somatostatin derives from cholinergically innervated somatostatin-producing cells in the gastrointestinal tract. If hypothalamic somatostatin is inhibited by ng-amounts of Oxy in a similar way, the stimulatory effect of Oxy on GH secretion may be explained. In a recent study we showed that Oxy in concentrations of 10 210 –2 3 10 26 M lowered Oxy secretion from dispersed anterior pituitary cells [11]. This inhibition was completely restored by addition of the Oxy antagonist used in the present study. Oxy also inhibited GHRH-induced GH release and the maximal magnitude of this response was similar to that caused by somatostatin. Considering this, Oxy might inhibit GH release via a direct action on Oxy receptors located on the GH cells. However, the inhibition of GH secretion seen in the present study in response to high doses of Oxy given i.c.v. or s.c, was not seen until 120 min after injection. Therefore the possibility that this inhibitory effect is indirect cannot be excluded.
4
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
The fact that the inhibitory effect caused by 1 mg kg 21 of Oxy given s.c. was reversed by the Oxy antagonist indicates that oxytocinergic mechanisms are involved. Lactation is associated with enhanced Oxy secretion both in the CNS and in the circulation [7]. In addition, suckling in lactating rats is followed by a depletion of pituitary concentration of GH after 30 and 180 min of suckling [12,13] and is clearly associated with increased GH secretion [14]. This effect might correspond to our data that show a stimulatory effect of Oxy on GH secretion after 30 and 120 min. It has been known for two decades that the GH secretion in male rats is markedly pulsatile [15]. In this study we have relatively low GH levels with a relatively small spread between the samples. We cannot completely explain this but one reason could be that the experiments were always performed at the same time of the day, two hours after the light was turned on, although we cannot exclude a stress factor during blood sampling. Finally, it should be noted that obese people, known to have a blunted GH response to certain stimuli, e.g. insulininduced hypoglycemia, also have high Oxy levels [16]. It remains to be established whether there is a causal relationship between these two findings. In conclusion GH is affected in two opposite ways by Oxy. In response to low doses of Oxy (ng, i.c.v.) GH is released and in response to high doses (mg, i.c.v.) GH release is inhibited. The mechanisms behind the stimulatory and the inhibitory actions of Oxy on GH secretion remain to be clarified and the functional importance to be established. Fig. 3. Effect of oxytocin administered i.c.v. in the lateral ventricle on plasma levels of GH (upper panel A) and somatostatin (lower panel B). Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
Acknowledgments ¨ Sweden for the oxytocin and We thank Ferring, Malmo, the oxytocin antagonist used in this study. This study was supported by grants from the Swedish Medical Council (B94-04X-05207-17B 1 B96-19x-11245-02B
References
Fig. 4. Effect of 10–1000 mg kg 21 oxytocin and / or an oxytocin antagonist administered s.c. on plasma levels of GH. Data are presented as means 6S.D. Statistical evaluation was performed by means of the Mann–Whitney U-test. * p , 0.05.
[1] Lumpkin, M.D., Samson, W.K. and McCann, S.M., Hypothalamic and pituitary sites of action of oxytocin to alter prolactin secretion in the rat, Endocrinology, 112 (1983) 111–115. [2] Vandesande, F., Dierickx, K. and Demey, J., The origin of the vasopressinergic and oxytocinergic fibres of the external region of the median eminence of the rat hypophysis, Cell Tiss. Res., 180 (1977) 443–452. [3] Gibbs, D.M., High concentrations of oxytocin in hypophysial portal plasma, Endocrinology, 114 (1984) 1216–1218. [4] Franci, C.R., Anselmo-Franci, J.A., Kozlowski, G.P. and McCann, S.M., Actions of endogenous vasopressin and oxytocin on anterior pituitary hormone secretion, Neuroendocrinology, 57 (1993) 693– 699. ´ S., Albertsson-Wikland, K. and Isaksson, O., Plasma levels of [5] Eden, growth hormone in female rats of different ages, Acta Endocrinol. (Copenh), 88 (1978) 676–690. ´ A. and Uvnas-Wallensten, ¨ [6] Efendic, S., Enzmann, F., Nylen K.,
¨ E. Bjorkstrand et al. / Regulatory Peptides 69 (1997) 1 – 5
[7]
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