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infusion of saline or different 5-HT antagonists. Results: Restraint stress (5min), hypotensive hemorrhage or dehydration for 24h increased AVP secretion fivefold ...
European Journal of Endocrinology (2002) 147 815–824

ISSN 0804-4643

EXPERIMENTAL STUDY

Serotonergic involvement in stress-induced vasopressin and oxytocin secretion Henrik Jørgensen1, Ulrich Knigge1,2, Andreas Kjær1,3 and Jørgen Warberg1 1 Department of Medical Physiology, The Panum Institute, 2 Department of Surgery C and 3 Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

(Correspondence should be addressed to Henrik Jørgensen, Department of Medical Physiology, The Panum Institute 12.3.21, University of Copenhagen, Blegdamsvej 3, DK-2200N, Denmark; Email: [email protected])

Abstract Objective: To investigate the involvement of serotonin (5-hydroxytryptamine – 5-HT) receptors in mediation of stress-induced arginine vasopressin (AVP) and oxytocin (OT) secretion in male rats. Design: Experiments on laboratory rats with control groups. Methods: Different stress paradigms were applied after pretreatment with intracerebroventricular infusion of saline or different 5-HT antagonists. Results: Restraint stress (5 min), hypotensive hemorrhage or dehydration for 24 h increased AVP secretion fivefold and OT secretion threefold. Swim stress for 3 min had no effect on AVP secretion, but increased OT secretion threefold. Ether vapor or hypoglycemia had no effect on AVP or OT secretion. The restraint stress-induced AVP response was inhibited by pretreatment with the 5-HT2Aþ2C antagonists ketanserin (KET) and LY-53857 (LY) and the 5-HT3þ4 antagonist ICS205930 (ICS), whereas the 5-HT1A antagonist WAY-100635 (WAY) had no effect. The OT response to restraint stress was inhibited by WAY, KET and LY but not by ICS. KET and LY inhibited OT response to dehydration, and LY inhibited OT response to hemorrhage. Neither of the antagonists affected AVP responses to dehydration or hemorrhage, nor the swim stress-induced OT response. Conclusion: 5-HT2A, 5-HT2C and possibly 5-HT3 and 5-HT4 receptors, but not 5-HT1A receptors, are involved in the restraint stress-induced AVP secretion. 5-HT does not seem to be involved in the dehydration- or hemorrhage-induced AVP response. The restraint stress-induced OT response seems to be mediated via 5-HT1A, 5-HT2A and 5-HT2C receptors. The dehydration and hemorrhage-induced OT responses are at least mediated by the 5-HT2A and 5-HT2C receptors. The 5-HT3 and 5-HT4 receptors are not involved in stress-induced OT secretion. European Journal of Endocrinology 147 815–824

Introduction Arginine-vasopressin (AVP) and oxytocin (OT) are synthesized in the magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN). Following axonal transport, AVP and OT are released from nerve terminals in the neurohypophysis upon stimulation. Various types of psychological or physical stressors have been shown to stimulate the hypothalamo –neurohypophysial system and to increase the secretion of AVP and OT (1 – 3). However, the relative release of AVP and OT depends on the specific stressor applied (4 – 6). Two decades ago it was considered that OT but not AVP was a stress hormone in the rat (7, 8). This has since been modified, but controversies about the AVP response to different stressors still exist. It has been proposed that primary physical stressors such as hypovolemia, hemorrhage,

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hypoglycemia or exercise increase AVP secretion (9, 10), whereas predominant psychological stressors such as restraint stress, ether-vapor stress or forced swimming have minor or no effect (6, 11 –13). However, recent studies indicate that this is not necessarily true, since repeated psychological stress was found to increase the concentration of AVP in the PVN (14) or the median eminence (15) or gene expression of AVP in the PVN (16). Together, these findings indicate that both types of stress activate the hypothalamo – neurohypophysial system, which may not necessarily be reflected in elevated peripheral hormone levels (17, 18). Several neurotransmitters seem to be involved in the mediation of the stress-induced release of AVP and OT, since administration of acetylcholinergic, histaminergic and aminergic receptor antagonists and administration of GABA and opiate receptor agonists inhibit the

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neurohypophysial hormone response to various stressors (19 –23). In addition, serotonin (5-hydroxytryptamine – 5-HT) seems to be involved in the mediation of the basal as well as the stress-induced secretion of the neurohypophysial hormones (24, 25). The effect of 5-HT on the secretion of the neurohypophysial hormones is exerted via centrally located receptors, of which primarily 5-HT2C receptors are known to be involved in the release of AVP, and 5-HT1A and 5-HT2 receptors in OT release (26 –28). Recently, we reported that the 5-HT4 receptor might also be involved in the 5-HT-induced secretion of AVP and OT, whereas the 5-HT3 receptor seems to be of minor importance (29). We have previously found that 5-HT1, 5-HT2 and 5-HT3 receptors are involved in stress-induced release of prolactin (30) and that 5-HT1, 5-HT2 and possibly the 5-HT4 receptors seem to be involved in the stressinduced adrenocorticotropin (ACTH) secretion (31). Therefore, the aim of the present study was to investigate (1) which types of stress affect AVP and OT secretion, (2) whether the serotonergic system participates in the mediation of the release of the neurohypophysial hormones in response to different stress paradigms, and (3) which 5-HT receptors are involved in this response.

reduce or eliminate all unnecessary stress or pain to the animals.

Drugs The following 5-HT receptor antagonists were used: the 5-HT1A receptor antagonist WAY-100635, N-tertbutyl-3-(4-(2-methoxyphenyl) piperazine-1-yl)-2-phenylpropionamide (WAY), gift from Lundbeck Inc., Valby, Denmark; the 5-HT2A(þ2C) receptor antagonist ketanserin, 3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]2,4(1H,3H)-quinazolinedione tartrate (KET), RBI, Natick, MA, USA; the 5-HT2C(þ2A) receptor antagonist LY-53857, 6-methyl-1-(-methyl ethyl)-ergoline-8bcarboxylic acid 2-hydroxy-1-methyl propyl ester maleate (LY), RBI; and the 5-HT3þ4 receptor antagonist ICS-205930, endo-8-methyl-8-axabiocylol [3.2.1]oct3-ol indol-3-yl-carboxylate hydrochloride (ICS; tropisetrone), RBI. The receptor affinities of the antagonists are given in Table 1. Insulin was purchased from Actrapid, Novo Nordisk, Bagsværd Denmark. All drugs were dissolved in saline and adjusted to pH 6.8 –7.5, except for KET, which was dissolved in 0.9% saline acidified with 0.05% HCl and adjusted to pH 6.8 with 0.05 mol/l NaOH.

Experimental procedures

Materials and methods Animal procedures Male Wistar rats (250–325 g), bred at the Panum Institute, were used in all experiments. The rats were housed under controlled temperature ð22^1 8CÞ and humidity (80%), in cages of four animals before and one per cage after surgery. A cycle of 12 h light: 12 h darkness with lights on at 0600 was used. The rats had free access to laboratory chow and tap water, when not dehydrated. The experimental protocol was in accordance with, and accepted by, the European Communities Council directive of 24 November 1986 and the Danish Ministry of Justice, Board for Animal Experimental Research. The animal experiments endeavored to reduce the overall number of animals and to

General procedures For intracerebroventricular (i.c.v.) administration of 5-HT antagonists, a permanent stainless steel cannula was implanted in the right lateral cerebral ventricle through a drilled hole in the skull. The coordinates were: P ¼ 0:0; L ¼ 1:5; H ¼ 24:0 relative to bregma. The cannula was closed with a platinum obturator. Compounds were infused intracerebroventricularly over a period of 2.5 min in a volume of 5 ml. All operations were performed during pentobarbital anesthesia (66 mg/kg i.p.) 4– 5 days prior to the experiments. On the day of the experiments the animals were brought to the laboratory and allowed to de-stress in their cage 3 h prior to injections. After 90 min i.v. catheters and i.c.v. cannulas were extended by polypropylene plastic tubes permitting infusion of test substances without disturbing the rats. The antagonists

Table 1 The 5-HT receptor antagonists used with the respective receptor affinities; values in pKA or pKi. Shaded areas indicate the primary type of receptor specificity. A pKi above 7.0 is considered to indicate specificity for that receptor. Numbers in parentheses are reference numbers.

WAY (64) 5-HT1A KET 5-HT2A+2C LY 5-HT2A+2C ICS 5-HT3+4

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5-HT1A

5-HT1B

5-HT2A

5-HT2C

5-HT3

8.9

,7.0

,7

5-HT5

5-HT7





5.9 (65)

5.7 (65)

8.7 (66)

7.2 (66)

,4 (67)



4.8

6.7

6.4 (68)

5.5 (68)

7.7 (66)

8.3 (66)









5.3 (68)

4.6 (68)

8.5 (67)

6.2 (67)

,7

5-HT4 ,7

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were infused intracerebroventricularly at 215 min, unless otherwise stated. The time for administration and the doses of antagonists were chosen on the basis of previous experiments, with the antagonists administered both intraperitoneally and intracerebroventricularly (30, 32, 33). In all experiments, blood samples were obtained by decapitation of rats at 0 min, which was scheduled between 1000 and 1200 h. Blood samples were collected in chilled polyethylene tubes containing 50 ml aprotinin (Trasylol, 20 000 kIU/ml; Bayer, Leverkusen, Germany) and 100 ml 0.5 mol/l EDTA and were kept on ice until centrifuged at 4 8C. The plasma was separated into polyethylene tubes and stored at 2 20 8C until hormone analysis could be carried out. Experiment 1. Effect of stress on AVP or OT secretion Rats were exposed to one of the following seven different stressors. Restraint stress was performed by fixing the animal on its back in a supine position for 5 min. Cold-swim stress was performed by allowing the rat to swim in cold (4 8C), deep water in a plastic bowl for 3 min followed by a 2-min period to allow it to shake the skin dry. Dehydration was performed for 24 h starting at noon. The length of dehydration chosen has previously been shown to cause significant AVP release (20) and did not lead to any visible discomfort for the animals. The Danish Government Animal Research Control Committee, Ministry of Justice, has approved dehydration for up to 48 h as ethically acceptable. Hemorrhage stress was performed by withdrawal of 3.0 ml venous blood (approximately 20% of the blood volume) from 2 10 to 2 8 min. Ether stress was performed by placing the rat in an ether-vapor filled glass beaker for 5 min from 2 5 to 0 min. Hypoglycemia stress was achieved by i.p. injection of 3 IU/kg rapidworking insulin preparation at 2 45 min. The rats were decapitated at 0 min. Each experiment consisted of eight rats. Experiment 2. Effect of 5-HT antagonists on restraint stress-induced AVP or OT secretion Rats were exposed to restraint stress from 2 5 to 0 min after pretreatment with either saline or one of the following 5-HT antagonists (i.c.v. infusion at 2 15 min): the 5-HT1A antagonist WAY (10 nmol); the 5HT2Aþ2C antagonist KET (50 nmol); the 5-HT2Cþ2A antagonist LY (50 nmol); or the 5-HT3þ4 antagonist ICS (10 nmol). The seven rats in each group were decapitated at 0 min. Experiment 3. Effect of 5-HT antagonists on dehydration-induced AVP or OT secretion Rats were exposed to 24 h of dehydration. One group of rats was continuously infused intracerebroventricularly with 5-HT antagonists via a microosmotic pump (Alzet, Cupertino, CA, USA; infusion rate 2 ml/h equalizing 3 nmol/h), placed intraperitoneally during the

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anesthesia for the i.c.v. cannula. On the morning of intervention the pumps were filled with either saline or one of the following 5-HT antagonists: KET (5-HT2Aþ2C), LY (5-HT2Cþ2A) or ICS (5-HT3þ4), and connected to the i.c.v. cannula. Euhydrated rats served as the control. At the end of the 24-h period, blood samples were obtained as described above. There were eight rats in each treatment group. Experiment 4. Effect of 5-HT antagonists on hemorrhage-induced AVP or OT secretion Four to five days prior to the experiment a silicone catheter was implanted in the right jugular vein as previously described (33), and an i.c.v. cannula was implanted as described above. Rats were pretreated intracerebroventricularly with saline, LY (50 nmol) or ICS (10 nmol) at 2 20 min. At 2 10 min, 3.0 ml of blood was drawn from the vein catheter over a period of 2 min. The rats (six in each group) were decapitated at 0 min. Experiment 5. Effect of 5-HT antagonists on AVP or OT secretion induced by cold-swim stress Groups of seven or eight rats were exposed to cold-swim stress for 3 min from 2 5 to 2 2 min and decapitated at 0 min, after pretreatment (intracerebroventricularly at 2 15 min) with saline or the following 5-HT antagonist: the 5-HT1A antagonist WAY (10 nmol); the 5-HT2Aþ2C antagonist KET (50 nmol); the 5-HT2Cþ2A antagonist LY (50 nmol); or the 5-HT3þ4 antagonist ICS (10 nmol).

Hormone analysis AVP and OT were analyzed by RIA in extracted plasma by means of C18 Sep-Pak cartridges as previously described (20). The least detectable quantity for AVP and OT was 0.1 –0.3 pmol/l plasma and 4 –6 pmol/l respectively. Intra- and interassay coefficients were 10 and 15% respectively for both assays.

Osmolality analysis Measurement of plasma osmolality was performed on plasma samples in triplicate by freezing-point depression (Advanced Microosmometer 3MO, Advanced Instruments Inc., Needham Heights, MA, USA) by a previously described method (34).

Statistical tests Results are presented as the mean ^ S.E.M. and evaluated by ANOVA followed by Newman – Keul’s test for multiple comparisons when appropriate. The level of significance was set at P , 0:05: www.eje.org

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Results Experiment 1. Effect of stress on AVP or OT secretion Five minutes of restraint stress, 24 h of dehydration and 3.0 ml hemorrhage increased plasma AVP level six-, five- and fourfold respectively (P , 0:01; Fig. 1a), whereas swim, ether, endotoxin or hypoglycemia stress had no effect (Fig. 1a). Restraint, cold-swim, dehydration and hemorrhage stress increased plasma OT level 3.5-, 2-, 3- and 2-fold respectively (P , 0:01; P , 0:05; P , 0:01 and P , 0:05 respectively, Fig. 1b), whereas ether or hypoglycemia stress had no effect (Fig. 1b).

Experiment 2. Effect of 5-HT antagonists on restraint stress-induced AVP or OT secretion The restraint stress-induced AVP response was inhibited almost 70% by pretreatment with the 5-HT2Aþ2C antagonist KET, the 5-HT2Cþ2A antagonist LY and the 5-HT3þ4 antagonist ICS (P , 0:01; Fig. 2a), whereas

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the 5-HT1A antagonist WAY had no effect (Fig. 2a). WAY inhibited the OT response to restraint stress almost 80% (P , 0:01; Fig. 2b). KET and LY reduced the OT response to restraint stress about 60% (P , 0:01; Fig. 2b), whereas ICS had no significant effect.

Experiment 3. Effect of 5-HT antagonists on dehydration-induced AVP or OT secretion Twenty-four hours of dehydration increased plasma osmolality 4% from 288^1:2 mosmol=l to 300^ 1:2 mosmol=l ðP , 0:05Þ; AVP secretion twofold ðP , 0:05Þ; and OT secretion almost fourfold ðP , 0:05Þ (Fig. 3a and 3b). Infusion of 5-HT antagonists had no effect on the dehydration induced AVP response (Fig. 3a). However, continuous i.c.v. infusion of the 5-HT2Cþ2A antagonist LY or the 5-HT2A antagonist KET inhibited the OT response 75% (P , 0:01; Fig. 3b), whereas the 5-HT3þ4 antagonist ICS had no effect.

Figure 1 Effect of different stressors on plasma level of AVP (a) or OT (b). Restraint stress (RS; 5 min), dehydration (Dehy; 24 h), hypotensive hemorrhage (Hemo; 3.0 ml at 210 min), cold swim (Swim; 3 min in 4 8C cold water), ether stress (Ether; 5 min) or hypoglycemia (Hypo; i.p. 3 IU insulin at 245 min). Rats were decapitated at 0 min. Data represent means ^ S.E.M. of eight rats. ## P , 0:01 and # P , 0:05 compared with control rats.

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Figure 2 Effect of i.c.v. pretreatment at 215 min with saline, the 5-HT1A antagonist WAY (10 nmol), the 5-HT2Aþ2C antagonists KET or LY (50 nmol), or the 5-HT3þ4 antagonist ICS (10 nmol) on restraint stress-induced AVP (a) and OT (b) response. Rats were decapitated at 0 min. Data represent means ^ S.E.M. of seven or eight rats; ## P , 0:01 compared with control; *P , 0:05 and **P , 0:01 compared with saline-treated restraint stressed rats.

Experiment 4. Effect of 5-HT antagonists on hemorrhage-induced AVP or OT secretion

Discussion

Withdrawal of 3.0 ml of blood from the jugular vein increased AVP and OT secretion seven- and twofold respectively (P , 0:05; Fig. 4a and P , 0:01; Fig. 4b, respectively). Pretreatment with the 5-HT antagonist had no effect on hemorrhage-induced AVP secretion. OT secretion was inhibited 50% by LY, while ICS had no effect (P , 0:05; Fig. 4b).

The present experiments show that physical as well as psychological stress paradigms stimulate the secretion of the neurohypophysial hormones AVP and OT, since

Experiment 5. Effect of 5-HT antagonists on AVP or OT secretion induced by cold-swim stress Three minutes of cold-swim stress had no effect on AVP secretion, but increased OT secretion twofold (Table 2, Fig. 1). None of the 5-HT antagonists affected this response (Table 2).

Table 2 Effect of i.c.v. infusion of different 5-HT antagonists on the AVP and OT response to 3 min of cold-swim stress from 25 to 22 min, and decapitation at 0 min. Values are in pmol/l expressed as means of seven or eight rats ^ S.E.M. *P , 0:05 versus control. Stress

Antagonist

AVP

OT

Control Swim Swim Swim Swim Swim

Saline Saline WAY (5-HT1A) KET (5-HT2A) LY (5-HT2C+2A) ICS (5-HT3+4)

1.9^0.2 1.5^0.2 1.5^0.1 1.9^0.2 1.6^0.1 2.4^0.2

11^1.0 19^2.1* 23^2.3* 21^1.7* 26^1.5* 25^3*

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Figure 3 Effect of continuous i.c.v. infusion (0.3 nmol/h) of saline, KET or LY (5-HT2A+2C) or ICS (5-HT3+4) during a 24-h dehydration period on the AVP (a) and OT (b) response. Rats were decapitated at the end of the dehydration period (0 h). Data represent means ^S.E.M. of eight rats; ## P , 0:01 compared with control; **P , 0:01 compared with saline-treated dehydrated rats.

dehydration and hemorrhage (physical stressors), as well as restraint stress (psychological stressor) increased the secretion of both hormones. However, some of the applied stress paradigms, such as ether and hypoglycemia, did not affect the secretion of either of the two hormones. The stress response is probably an interaction between the neuroendocrine system, the sympathetic nervous system and the target organs, resulting in activation of different neuronal pathways and release of specific hormones (35). Stress in itself releases various neurotransmitters, which may have either stimulating (catecholamines, 5-HT, histamine) or inhibiting (GABA) effect on hypothalamic and pituitary hormone release (19). The secretion of the two neurohypophysial hormones seem to be differentially affected by stress, since coldswim stress, which is considered to be combined physical and psychological stressor, stimulated OT secretion, but not AVP secretion. The independent regulation of www.eje.org

the neurohypophysial hormones illustrates the flexibility of the hypothalamo – neurohypophysial system, which is suitable to achieve a maximal overall positive effect on the target organs under different physiological conditions, such as bleeding, labor, hypovolemia, etc. (5). A heterogeneous hormone response in respect to AVP and OT has also been found after immobilization, forced swimming, ether stress, hemorrhage, endotoxin and osmotic loading (4 –6, 36). The present experiments show that involvement of 5-HT and the effect of 5-HT receptor blockade on the AVP or OT responses to different stress paradigms are not general, but seem to be as important as the involvement of other neurotransmitters, such as norepinephrine and histamine (20, 37, 38). The present finding of an increased level of plasma AVP after 5 min of restraint stress is in accordance with two studies (5, 7), but in contrast to the a previous finding, probably due to a short period of stress (1 min) in that

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Figure 4 Effect of i.c.v. pretreatment at 220 min with saline, LY (5-HT2Aþ2C; 50 nmol) or ICS (5-HT3þ4; 10 nmol) on the hemorrhage (3.0 ml venous blood at 210 min) induced AVP (a) and OT (b) response. Rats were decapitated at 0 min. Data represent means ^ S.E.M. of six rats; # P , 0:05 and ##P , 0:01 compared with control; *P , 0:05 compared with saline-treated hemorrhage-stressed rats.

study (4). The stimulating effect of restraint stress on plasma OT is in agreement with our own and others previous findings (4, 39, 40). Therefore, it seems that both AVP and OT are stimulated by restraint stress. A single prolonged period of restraint stress (30 – 150 min) has been found to increase AVP mRNA in the medial parvocellular part but not in the magnocellular part of the PVN (41, 42). However, an increased synthesis of AVP in the parvocellular PVN does not inevitably induce an increased plasma level of AVP in the peripheral circulation, since AVP produced in the parvocellular part of the PVN is released primarily into the pituitary portal circulation. The pattern emerging from the previous and the present findings indicate that single ultra short-term restraint stress (1 – 2 min) performed by mechanical fixation or restraint in a plastic glass box, does not seem to be sufficient to increase either AVP mRNA in the hypothalamic PVN or AVP in peripheral plasma (36, 43),

whereas a 3– 5-min period of manual restraint, as used in this experiment, is sufficient to increase peripheral plasma levels of AVP (5, 7). Serotonergic antagonists with affinity for 5-HT2, 5-HT3 and 5-HT4 receptors inhibit the AVP response to restraint stress, whereas 5-HT1A and 5-HT2 antagonists reduce the OT response to this stressor. There are no available comparable data, but in unstressed rats, the AVP response to serotonergic stimulation is mediated via 5-HT2A and 5-HT2C receptors (26, 28, 44, 45), while 5-HT1A and 5-HT2 receptors are involved in the 5-HT-induced OT response via the PVN (46 –49). We found that dehydration is a potent physiological stimulus for both AVP and OT secretion, which confirms previous findings (9, 50). An involvement of the serotonergic system in AVP response to dehydration is possible but does not seem likely, since none of the antagonists (5-HT2A, 5-HT2C, 5-HT3þ4) used in the present experiments had any effect on this response. An www.eje.org

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involvement of other receptors cannot be excluded. The possibility that the receptor blockade was insufficient is limited, since we used continuous i.c.v. infusion of compounds via an osmotic pump. In addition, using an identical experimental design, we previously found that the AVP response to dehydration was inhibited by histamine receptor antagonists (20). Furthermore, an increase in AVP secretion, induced by osmotic challenge with 500 mmol NaCl/l, was not affected by either the 5-HT2 antagonist ritanserin or the 5-HT2Aþ2C agonist DOI (51). Hypotensive hemorrhage-induced by acute depletion of approximately 20% of the total blood volume potently increased AVP and OT in peripheral plasma in our study. This is in accordance with previous findings, which showed that a 15 – 30% acute reduction in blood volume increased AVP (9, 52 – 54), and OT concentration in peripheral (5, 50, 55) and pituitary portal plasma (56), and increased AVP content and mRNA in the hypothalamus (52, 57, 58). The OT response to hemorrhage seems to be mediated via 5-HT2 receptors, since the response was inhibited by pretreatment with the 5-HT2Cþ2A antagonist LY. The AVP response also tended to be inhibited, but this was not significant. Cold-swim stress stimulated OT but not AVP secretion, which is in agreement with previous observations (6, 59). In the present experiments, rats were exposed to deep cold water (4 8C for 2 min). The increase in OT secretion could be due either to the immersion in cold water or the swimming exercise, or both. Exposure to cold environment (4 8C for 30 min) has previously been found to increase OT levels (5), and single or repeated forced swim stress (20 8C for 10 min) increased both AVP and OT in microdialysates from both the SON and the PVN, but only OT in peripheral plasma (59). The OT response induced by coldswim stress does not seem to involve the investigated 5-HT receptors. Five minutes of ether stress did not affect AVP or OT secretion, which conflicts with previous findings both of ourselves and of others (4, 5, 36, 39). A reason for the missing response to ether stress may be the stresstime schedule, since it was found that 1 min of ether stress increased AVP or OT secretion twofold, measured 1 min after the end of the stress period, whereas the hormone response disappeared 4 min after the stress period was discontinued (4). In accordance with most of the previous studies (5, 39), we found no effect on insulin-induced hypoglycemia stress on AVP or OT secretion. The present findings demonstrate that 5-HT is differentially involved in the stress-induced secretion of AVP and OT. One explanation might be that various stress paradigms activate cortical, limbic and hypothalamic areas differently, and these areas in addition have a heterogeneous distribution of 5-HT neurons. 5-HT nerve fibers are localized differently within the hypothalamus. www.eje.org

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Although relatively few in the PVN, they are more abundant in the parvocellular than in the magnocellular part and are primarily projecting to OT neurons, whereas they are very few and scattered at AVP neurons in the PVN (60, 61). The few 5-HT fibers in the SON are found to be in close contact with AVP neurons, but sparse at the OT neurons (62). The involvement of different serotonergic receptors in the mediation of the stress-induced AVP and OT responses is found to be differentiated for, for example, blockade of 5-HT1A receptors inhibited restraint stressinduced OT, but not AVP secretion. This finding is in accordance with previous findings of a different involvement of 5-HT receptors in AVP and OT secretion (28, 63). The reason for this dissimilarity may be absence of 5-HT1A receptors on AVP neurons. The present study has not investigated the specific localization of the interaction between the 5-HT system and the neurohypophysial system. Further studies, for example, with specific anatomical lesions, are required to elucidate this. In conclusion, we found that various stress paradigms affect AVP and OT secretion differently and that the serotonergic system is involved differently in these responses. The restraint stress-induced AVP and OT responses are at least mediated through 5-HT2Aþ2C and 5-HT4 receptors, and via 5-HT1A and 5-HT2Aþ2C receptors respectively. The dehydrationand hemorrhage-induced OT response is in part mediated through the 5-HT2Aþ2C receptors, whereas the AVP responses to these stressors do not seem to involve the investigated 5-HT receptors. Swim stress increased only OT secretion, but the investigated 5-HT receptors are not involved in this response. Ether and hypoglycemia stress had no effect on AVP or OT secretion.

Acknowledgements This study was supported by grants from the Danish Medical Research Council; NOVO’s Foundation; The Lundbeck Foundation; Nordic Insulin Foundation Committee; The Danish Hospital Foundation for Medical Research, Regions of Copenhagen, The Faroe Islands and Greenland; and the Danish Medical Association Research Fund. We thank Elsa Larsen and Jytte Oxbøll for skilled technical assistance. The materials for RIA of AVP and OT were kindly provided by the National Hormone and Pituitary Program of the NIDDK.

References 1 Udelsman R & Chrousos GP. Hormonal responses to surgical stress. Advances in Experimental Medical Biology 1988 245 265–272.

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2 Van de Kar LD, Richardson Morton KD & Rittenhouse PA. Stress: neuroendocrine and pharmacological mechanisms. Methods and Achievements in Experimental Pathology 1991 14 133– 173. 3 Chrousos GP & Gold PW. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 1992 267 1244–1252. 4 Hashimoto K, Murakami K, Takao T, Makino S, Sugawara M & Ota Z. Effect of acute ether or restraint stress on plasma corticotropin-releasing hormone, vasopressin and oxytocin levels in the rat. Acta Medicina Okayama 1989 43 161–167. 5 Kasting NW. Simultaneous and independent release of vasopressin and oxytocin in the rat. Canadian Journal of Physiology and Pharmacology 1988 66 22 –26. 6 Lang RE, Heil JW, Ganten D, Hermann K, Unger T & Rascher W. Oxytocin unlike vasopressin is a stress hormone in the rat. Neuroendocrinology 1983 37 314–316. 7 Husain MK, Manger WM, Rock TW, Weiss RJ & Frantz AG. Vasopressin release due to manual restraint in the rat: role of body compression and comparison with other stressful stimuli. Endocrinology 1979 104 641 –644. 8 Landgraf R, Malkinson TJ, Veale WL, Lederis K & Pittman QJ. Vasopressin and oxytocin in rat brain in response to prostaglandin fever. American Journal of Physiology 1990 259 R1056–R1062. 9 Fyhrquist F, Tikkanen I & Linkola J. Plasma vasopressin concentration and renin in the rat: effect of hydration and hemorrhage. Acta Physiologica Scandinavia 1981 113 507 –510. 10 Baylis PH & Robertson GL. Rat vasopressin response to insulininduced hypoglycemia. Endocrinology 1980 107 1975–1979. 11 Yagi K & Onaka T. Suppressive vasopressin response to emotional stress: the neuroactive substance that may be involved. Annals of the New York Academy of Sciences 1993 689 685–688. 12 Keil LC & Severs WB. Reduction in plasma vasopressin levels of dehydrated rats following acute stress. Endocrinology 1977 100 30 –38. 13 Gibbs DM. Vasopressin and oxytocin: hypothalamic modulators of the stress response: a review. Psychoneuroendocrinology 1986 11 131– 139. 14 Wotjak CT, Kubota M, Liebsch G, Montkowski A, Holsboer F, Neumann I et al. Release of vasopressin within the rat paraventricular nucleus in response to emotional stress: a novel mechanism of regulating adrenocorticotropic hormone secretion? Journal of Neuroscience 1996 16 7725–7732. 15 de Goeij DC, Dijkstra H & Tilders FJ. Chronic psychosocial stress enhances vasopressin, but not corticotropin-releasing factor, in the external zone of the median eminence of male rats: relationship to subordinate status. Endocrinology 1992 131 847–853. 16 Ma XM, Lightman SL & Aguilera G. Vasopressin and corticotropin-releasing hormone gene responses to novel stress in rats adapted to repeated restraint. Endocrinology 1999 140 3623–3632. 17 Engelmann M, Ebner K, Landgraf R, Holsboer F & Wotjak CT. Emotional stress triggers intrahypothalamic but not peripheral release of oxytocin in male rats. Journal of Neuroendocrinology 1999 11 867– 872. 18 Engelmann M, Wotjak CT, Ebner K & Landgraf R. Behavioural impact of intraseptally released vasopressin and oxytocin in rats. Experimental Physiology 2000 85 125S–130S. 19 Van de Kar LD & Blair ML. Forebrain pathways mediating stressinduced hormone secretion. Frontiers of Neuroendocrinology 1999 20 1– 48. 20 Kjaer A, Knigge U, Rouleau A, Garbarg M & Warberg J. Dehydration-induced release of vasopressin involves activation of hypothalamic histaminergic neurons. Endocrinology 1994 135 675– 681. 21 Stratakis CA & Chrousos GP. Neuroendocrinology and pathophysiology of the stress system. Annals of the New York Academy of Sciences 1995 771 1–18. 22 Renaud LP & Bourque CW. Neurophysiology and neuropharmacology of hypothalamic magnocellular neurons secreting

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24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

823

vasopressin and oxytocin. Progress in Neurobiology 1991 36 131–169. Reichlin S. Neuroendocrinology. In Textbook of Endocrinology, pp 165–248. Eds JD Wilson, DW Foster, HM Kronenberg & PR Larsen. Philadelphia: W B Saunders, 1998. Iovino M & Steardo L. Effect of substances influencing brain serotonergic transmission on plasma vasopressin levels in the rat. European Journal of Pharmacology 1985 113 99– 103. Van de Kar LD. Neuroendocrine pharmacology of serotonergic neurons. Annual Review of Pharmacology and Toxicology 1991 31 289–320. Li Q, Brownfield MS, Battaglia G, Cabrera TM, Levy AD, Rittenhouse PA et al. Long-term treatment with the antidepressants fluoxetine and desipramine potentiates endocrine responses to the serotonin agonists 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) and (þ 2)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI). Journal of Pharmacology and Experimental Therapeutics 1993 266 836–844. Uvnas MK, Hillegaart V, Alster P & Ahlenius S. Effects of 5-HT agonists, selective for different receptor subtypes, on oxytocin, CCK, gastrin and somatostatin plasma levels in the rat. Neuropharmacology 1996 35 1635 –1640. Bagdy G, Sved AF, Murphy DL & Szemeredi K. Pharmacological characterization of serotonin receptor subtypes involved in vasopressin and plasma renin activity responses to serotonin agonists. European Journal of Pharmacology 1992 210 285–289. Jorgensen H, Knigge U, Kjaer A & Warberg J. Stress-induced neurohypophysial hormone secretion is partly mediated by serotonergic neurons. In International Union of Pharmacology IV, Fourth IUPHAR sattelite meeting on serotonin, Rotterdam, pp 112, 1998. Jorgensen H, Knigge U & Warberg J. Effect of selective serotonin receptor agonists on prolactin secretion in male rats. Neuroendocrinology 1993 57 401–407. Jorgensen H, Knigge U, Kjaer A, Vadsholt T & Warberg J. Serotonergic involvement in stress-induced ACTH release. Brain Research 1998 811 10–20. Jorgensen H, Knigge U & Warberg J. Effect of serotonin 5-HT1, 5HT2, and 5-HT3 receptor antagonists on the prolactin response to restraint and ether stress. Neuroendocrinology 1992 56 371– 377. Jorgensen H, Knigge U, Kjaer A & Warberg J. Adrenocorticotropic hormone secretion in rats induced by stimulation with serotonergic compounds. Journal of Neuroendocrinology 1999 11 283– 290. Kjœr A, Knigge U, Jørgensen H & Warberg J. Dehydration-induced vasopressin secretion in humans: involvement of the histaminergic system. American Journal of Physiology and Endocrinology and Metabolism 2000 279 E1305–E1310. Bohus B, Benus RF, Fokkema DS, Koolhass JM, Nyakas C, van Oortmerssen GA et al. Neuroendocrine states and behavioral and physiological stress responses. In Progress in Brain Research, pp 57 –70. Eds ER De Kloet, VM Wiegant & D de Wied. Amsterdam: Elsevier, 1987. Gibbs DM. Dissociation of oxytocin, vasopressin and corticotropin secretion during different types of stress. Life Sciences 1984 35 487–491. Onaka T. Catecholaminergic mechanisms underlying neurohypophysial hormone responses to unconditioned or conditioned aversive stimuli in rats. Experimental Physiology 2000 85 101S–110S. Itoi K, Helmreich DL, Lopez-Figueroa MO & Watson SJ. Differential regulation of corticotropin-releasing hormone and vasopressin gene transcription in the hypothalamus by norepinephrine. Journal of Neuroscience 1999 19 5464–5472. Kjaer A, Knigge U & Warberg J. Involvement of oxytocin in histamine- and stress-induced ACTH and prolactin secretion. Neuroendocrinology 1995 61 704–713. Jezova D, Michajlovskij N, Kvetnansky R & Makara GB. Paraventricular and supraoptic nuclei of the hypothalamus are not equally important for oxytocin release during stress. Neuroendocrinology 1993 57 776–781.

www.eje.org

824

H Jørgensen and others

41 Aubry JM, Bartanusz V, Jezova D, Belin D & Kiss JZ. Single stress induces long-lasting elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurones, but repeated stress is required to modify AVP immunoreactivity. Journal of Neuroendocrinology 1999 11 377 –384. 42 Herman JP. In situ hybridization analysis of vasopressin gene transcription in the paraventricular and supraoptic nuclei of the rat: regulation by stress and glucocorticoids. Journal of Comparative Neurology 1995 363 15–27. 43 Makino S, Smith MA & Gold PW. Increased expression of corticotropin-releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus during repeated stress: association with reduction in glucocorticoid receptor mRNA levels. Endocrinology 1995 136 3299–3309. 44 Brownfield MS, Greathouse J, Lorens SA, Armstrong J, Urban JH & Van de Kar LD. Neuropharmacological characterization of serotoninergic stimulation of vasopressin secretion in conscious rats. Neuroendocrinology 1988 47 277– 283. 45 Bagdy G, Calogero AE, Szemeredi K, Gomez MT, Murphy DL, Chrousos GP et al. Beta-endorphin responses to different serotonin agonists: involvement of corticotropin-releasing hormone, vasopressin and direct pituitary action. Brain Research 1990 537 227 –232. 46 Van de Kar LD, Rittenhouse PA, Li Q, Levy AD & Brownfield MS. Hypothalamic paraventricular, but not supraoptic neurons, mediate the serotonergic stimulation of oxytocin secretion. Brain Research Bulletin 1995 36 45–50. 47 Van de Kar LD, Javed A, Zhang Y, Serres F, Raap DK & Gray TS. 5-HT2A receptors stimulate ACTH, corticosterone, oxytocin, renin, and prolactin release and activate hypothalamic CRF and oxytocin-expressing cells. Journal of Neuroscience 2001 21 3572–3579. 48 Bagdy G. Role of the hypothalamic paraventricular nucleus in 5-HT1A, 5-HT2A and 5-HT2C receptor-mediated oxytocin, prolactin and ACTH/corticosterone responses. Behavioural Brain Research 1996 73 277–280. 49 Li Q, Brownfield MS, Levy AD, Battaglia G, Cabrera TM & Van de Kar LD. Attenuation of hormone responses to the 5-HT1A agonist ipsapirone by long-term treatment with fluoxetine, but not desipramine, in male rats. Biological Psychiatry 1994 36 300–308. 50 Summy-Long JY, Miller DS, Rosella-Dampman LM, Hartman RD & Emmert SE. A functional role for opioid peptides in the differential secretion of vasopressin and oxytocin. Brain Research 1984 309 362 –366. 51 Faull CM, Charlton JA, Butler TJ & Baylis PH. The effect of acute pharmacological manipulation of central serotonin neurotransmission on osmoregulated secretion of arginine vasopressin in the rat. Journal of Endocrinology 1993 139 77–87. 52 Shoji M, Kimura T, Kawarabayasi Y, Ota K, Inoue M, Yamamoto T et al. Effects of acute hypotensive hemorrhage on arginine vasopressin gene transcription in the rat brain. Neuroendocrinology 1993 58 630 –636. 53 Burnard DM, Pittman QJ & Veale WL. Increased motor disturbances in response to arginine vasopressin following hemorrhage or hypertonic saline: evidence for central AVP release in rats. Brain Research 1983 273 59 –65. 54 Sapru HN, Krieger AJ & Haldar J. Vasopressin release in the spontaneously hypertensive rat. Research Communications in Chemical Pathology and Pharmacology 1977 16 195 –198.

www.eje.org

EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 147

55 Dunning BE, Verbalis JG & Fawcett CP. Evidence for participation of the neurohypophysial hormones in the hyperglucagonemic response to hemorrhage in the rat. Neuroendocrinology 1985 41 385– 389. 56 Plotsky PM, Bruhn TO & Vale W. Evidence for multifactor regulation of the adrenocorticotropin secretory response to hemodynamic stimuli. Endocrinology 1985 116 633–639. 57 Ota M, Crofton JT & Share L. Hemorrhage-induced vasopressin release in the paraventricular nucleus measured by in vivo microdialysis. Brain Research 1994 658 49–54. 58 Demotes-Mainard J, Chauveau J, Rodriguez F, Vincent JD & Poulain DA. Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation in rats. Brain Research 1986 381 314–321. 59 Wotjak CT, Ganster J, Kohl G, Holsboer F, Landgraf R & Engelmann M. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 1998 85 1209–1222. 60 Sawchenko PE, Swanson LW, Steinbusch HW & Verhofstad AA. The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat. Brain Research 1983 277 355–360. 61 Sawchenko PE & Swanson LW. Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. Journal of Comparative Neurology 1982 205 260–272. 62 Ferris CF, Melloni JR, Koppel G, Perry KW, Fuller RW & Delville Y. Vasopressin/serotonin interactions in the anterior hypothalamus control aggressive behavior in golden hamsters. Journal of Neuroscience 1997 17 4331–4340. 63 Li Q, Levy AD, Cabrera TM, Brownfield MS, Battaglia G & Van de Kar LD. Long-term fluoxetine, but not desipramine, inhibits the ACTH and oxytocin responses to the 5-HT1A agonist, 8-OHDPAT, in male rats. Brain Research 1993 630 148–156. 64 Fletcher A, Forster EA, Bill DJ, Brown G, Cliffe IA, Hartley JE et al. Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist. Behavioural Brain Research 1996 73 337–353. 65 Hoyer D. 5-Hydroxytryptamine receptors and effector coupling mechanisms in peripheral tissues. In Peripheral Actions of 5-HT, pp 72–99. Ed. J Fozard. Oxford: Oxford University Press, 1989. 66 Schreiber R, Brocco M, Audinot V, Gobert A, Veiga S & Millan MJ. (1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane)-induced headtwitches in the rat are mediated by 5-hydroxytryptamine (5HT) 2A receptors: modulation by novel 5-HT2A/2C antagonists, D1 antagonists and 5-HT1A agonists. Journal of Pharmacology and Experimental Therapeutics 1995 273 101 –112. 67 Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ et al. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacology Reviews 1994 46 157–203. 68 Hoyer D. Functional correlates of serotonin 5-HT1 recognition sites. Journal of Receptor Research 1988 8 59– 81.

Received 6 March 2002 Accepted 22 July 2002