Differential release of vasopressin and oxytocin in response to abdominal vagal afferent stimulation or apomorphine in the ferret

Brain Research, 438 {1988) 193-198 Elsevier

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Differential release of vasopressin and oxytocin in response to abdominal vagal afferent stimulation or apomorphine in the ferret J. Hawthorn l, P.L.R. Andrews 1, V.T.Y. Ang 2 and J.S. Jenkins 2 Departments of t Physiology and "Medicine I!, St. George's Hospitai Medical School, London ( U. K. (Accepted 23 June 1987)

Key words: Vasopressin; Oxytocin; Vagal afferent; Nausea; Ferret

The aim of this study was to investigate whether direct afferent stimulation of the abdominal vagus could promote release of the neurohypophyseal hormones. The nucleus of the solitary tract is the major recipient of vagal afferent informatioa, and this region of the brainstem may also be activated by stimulation of the area postrema. For this reason apomorphine, a D 2 dopaminergic agonist which acts on the area postrema, and can evoke: ;-asopressin secretion in man, was also investigated for its effect on vasopres~n and oxytocin release. Our results show that vasopressin, but not oxytocin is released in vast amounts in response to electrical afferent stimulation of the abdominal vagus. Administration of apomorphine also evoked a massive vasopressin release with less marked effects on oxytocin. The possible functional implications of these results are discussed especially in the context of nausea and vomiting.

INTRODUCTION The release of vasopresai~ rna~ be affected by a variety of stimuli acting via vagal afferents associated with the cardiovascular system, e.g. baroreceptors or cardiac volume receptors 4-9. This cardiovascular information is integrated in the nucleus of the tractus solitarius (NTS) 7 which can influence the hypothaiamic paraventricular nuclei (PVN) (a major source of vasopressin and oxytocin) by several pathways z6. The NTS is also the major site of termination of vagal afferents innervating the abdominal viscera ma~ and therefore the possibility exists that abdominal vagal afferents may also be able to influence vasopressin release via the outputs of the NTS to the PVN. The major aim of this study was to investigate whether abdominal vagu.l afferent stimulation could evoke the release of vasopressin and oxytocin. Neuroanatomical studies ~2 have demonstrated a

subpopulation of abdominal vagal afferents projecting to the area postrema (AP) and as the A P itself sends projections to the NTS it is possible that the PVN could be influenced by abdominal vagal afferents acting via the A P rather than the NTS directly. This possibility of an A P - N T S - P V N pathway was tested by using the dopamine D 2 agonist apomorphine to activate the A P 29. MATERIALS AND METHODS

Surgery Albino or fitch ferrets (Mustela putorius furo L; 500-1100 g) of either sex were used. The animals were anaesthetized by an i.p. injection of urethane (1.5 g/kg) and cannulae (Portex 5FG) were inserted into the carotid artery and external jugular vein. The arterial cannula was connected via a 3-way tap to a pressure transducer (Druck, Neurolog NL 108) to allow monitoring of blood pressure and sampling of ar-

Correspondence: J. Hawthorn, Department of Physiology, St. George's Hospital Medical School, Cranmer Terrace, London SWI7 ORE, U.K. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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terial blood. For the vagal stimulation study the abdominal vagi were exposed via a ventral midline incision. The dorsal abdominal vagus was ligated and sectioned just caudal to the gastro-oesophageal junction and located on bipolar platinum electrodes for stimulation. The ventral vagus in the ferret, as in other laboratory species, is the major route taken by the vagal fibres supplying the liver 14. As osmoreceptors with afferent axons in the vagus are present in the liver and portal vein 22 and we wished to avoid changes in vasopressin which were due primarily to activation of these osmoreceptors it was decided to confine the study to the effects of stimulation of the dorsal abdominal vagus. The abdomen was not opened in the apomorphine study. After surgery all animals were left for 40-50 min and t h e , a basal blood sample was taken. A second basal blood sample was taken after 25 min and vagal stimulation or apomorphine was given 5 rain later. The vagus was stimulated continuously for 5 min at 3 or 30 Hz at a stimulus strength previously shown to be supramaximal for unmyelinated fibres in this species (25 V, 0.5 ms). Apomorphine (Sigma) was given as an i.v. bo'us of 100/~g/kg in 0.5-1.0 ml of 154 mM NaCI.

Collection of blood samples and assays As this is the first time, to our knowledge, that vasopressin and oxytocin levels have been reported for the ferret we subjected samples of plasma to highperformance liquid chromatography (HPLC) to verify the authenticity of the hormones and also used a bioassay to demonstrate that the vasopressin was true antidiuretic hormone. Blood samples (1 ml) were usually taken at 2, 5, 15, 30 and 60 min after the stimulus. After each blood sample was withdrawn an equivalent volume of dextran or haemaccel solution at 37 °C was replaced to maintain blood volume. Blood was collected into heparirJzed syringes and centrifuged immediately in a microfuge (Eppendoff). The plasma was removed, an aliquot reserved for measurement of osmolarity and the rest was frozen in cardice and stored at -20 °C until extraction and assay for vasopressin (pAVP) and oxytocin (pOXT). Osmolarity was measured using a vapour pressure osmometer (Wescor Inc). Details of the radioimmunoassays and HPLC conditions are as published previously l°.

Bioassay was performed by administration of extracted ferret plasma in a diuresing rat preparation. Control animals for the vagal stimula£ioil experiments were subjected to laparotomy and mobilization of the vagus but no stimulus was applied. The controls for apomorphine administration ieceived an equivalent volume of 154 mM NaCl. Blood pressure and plasma osmolarity were measured before and after either stimulus tc evaluate these parameters as possible contributory ~stimuli to vasopressin release. RESULTS

Vasopressin Ferret plasma contained vasopressin which diluted in parallel to standard AVP in our radioimmunoassay, and eluted as a single peak in the same position as standard peptide from HPLC. A pooled sample of ferret plasma extracted on sep-pak was biologically active in the diuresing rat (Fig. 1). In 3 conscious ferrets dehydration for 24 h caused a rise in plasma vasopressin from 31 + 1 to 79 + 1 pg/ml. Concomitant plasma osmolarity values were 328 + 7 and 366 _+ 11 mOsm/kg. Control animals with (vagal stimulation study) and without (aponlorphine study) laparotomy showed no differences in plasma vasopressin levels and are grouped together as controls.

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195

Vagal stimulation. Stimulation of the dorsal vagus at 3 Hz produced a marked elevation of pAVP from 61 + 9 pg/ml (n = 4) before stimulation, to 360 + 92 pg/ml at 2 rain after the onset of the stimulus, rising further to 468 + 63 pg/ml at 5 min (Fig. 2a). These stimulated values were significantly different from control values at equivalent time points; at 2 min control values were 94 + 27 pg/mi (P < 0.05) and at 5 min 91 + 33 pg/ml (P < 0.01). Stimulation at 30 Hz produced a similar pattern of changes in plasma vasopressin, however the response was enhanced, compared to stimulation at 3 Hz. Peptide levels rose from 109 + 28 pg/ml (n = 6) before stimulation to 472 + 140 pg/ml at 2 rain after the onset of stimulation (P < 0.05 compared to controls) rising further 521 _+ 142 pg/ml at 5 min (Fig. 2a) (P < 0.05 compared to controls). There was no significant difference between the levels of peptide in the groups

receiving vagai stimulatio~ whether the frequency was 3 or 30 Hz. Control animals did show slightly elevated plasma vasopressin levels by 60 min (168 + 78 pg/ml) (n = 5). We assume that this was a response to withdrawal of blood samples even though t[v~ ~lood was replaced immediately by an equal volume of dextran solution or haemaccel.

Apomorphine. Apomorphine also produced a rapid release of vasopressin, giving plasma levels of peptide almost as great as those observed with the lower level of vagai stimulation (Fig. 2b). The pattern of release, however, differed in that maximal plasma values were obtained at 2 min; 342 +_ 69 pg/ml (n = 5, P < 0.01 compared with controls). At 5 min the plasma level was 337 + 74 pg/ml (P < 0.05 compared with controls). By 15 min the plasma levels were not significantly different from controls.

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Oxytocin levels were unchanged by vagal stimulation showing that the effect on vasopressin was quite specific. Plasma oxytocin values 43.6 + 7.9 pg/ml (n = 6) before vagal stimulation. After 30 Hz stii.mlation values were 44.0 + 6.0 pg/ml at 2 rain and 56.3 + 19.9 pg/ml at 5 rain. Oxytocin values were only measured in two of the animals receiving stimulation at 3 Hz and these did not rise (48.5 pg/mi before stim-

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Fig. 2. a: plasma vasopressin levels in response to stimulation of the abdominal vagus in the ferret, q~----O, 30-Hz stimulation; • - - - @, 3-Hz stimulation; • . . . . . O, control. The stimulus was applied at time zero. Values are mean +_ S.E.M. • P < 0.05 **P < 0.0!. b: plasma vasopressin in response to a bolus i.v. injection of apomorphine (100pg/kg). O O, apomorphine, • . . . . . O, control. Apomorphine was given at time zero. Results are mean ";-_:S.E.M. *P < 0.05.

13.9 pg/ml 10 rain later. Animals receiving apomorphine (n = 4) had basal values at 57.5 + 6.4 pg/ml at the -5-min time point which rose to 70.1 + 16.9 pg/mi at 2 rain and 128.1 + 54.8 pg/ml at 5 rain; however, there was an enormous variation in response, and this increase was not statistically significam.

Plasma osmolarity and blood pressure. Plasma osmolarity was relatively high in both the conscious (328 + 7 mOsm/kg) and anaesthetized (322.5 + 3.9 and 328 + 2.6 mOsm/kg) animals (apomorphine and vagal stimulation groups, respectively). Following apomorphine or vagal stimulation plasma osmolarities of 323.3 + 3.9 and 326.5 + 7.3 mOsm/kg, respectively, were measured. Apomorphine caused a reduction in systolic blood pressure from 127 + 3 mm

196 Hg to 81 + 3 mm Hg whereas vagai stimulation elevated blood pressure from 124 + 3 mm Hg to 179 + 14 mm Hg with 3 Hz stimulation and from 130 .+, mm Hg to 163 + 10 mm Hg with 30 Hz stimulation. DISCUSSION Vasopressin levels have been measured in the ferret (Mustelaputorius furo L.) and found to be somewhat higher in both conscious and anaesthetized animals than in other species. The reason for this is not clear but may be related to their low fluid intake (combined with dry pelleted food) and high normal plasma osmolarity (>300 mOsm/kg). Relevant to this is evidence suggesting that the ferret is a domesticated form of a desert-dwelling mustelid 19. As this was the first time we have measured AVP in this species the hormone was characterized by testing for the ability to dilute in parallel to standard vasopressin in radioimmunoassay and separation on HPLC as well as monitoring the hormone response to water deprivation and assessing the hormone in a bioassay. After extraction by octadecyl silica (Sep-pak, Waters) cartridges the material in ferret plasma behaved identically to pure hormone in the radioimmunoassay and only one peak was eluted from the HPLC column which reacted with our antibody. Thus, the expected rise in hormone levels seen after fluid deprivation and its activity in the diuresing rat preparation demonstrate that we are measuring true antidiuretic hormone. The results presented here demonstrate for the first time that electrical stimulation of abdominal vagal afferents in the ferret (a carnivore) evokes a prompt and massive increase in plasma vasopressin, but not oxytoc!n. The plasma levels reached were considerably greater than those produced by fluid deprivation, and also they occurred in the face of a substantial increase in blood pressure; a stimulus which would normally produce a marked reduction in vasopressin secretion. Similar large and specific increases in vasopressin were produced by apomorphine. This was coincident with a decrease in blood pressure which is sufficient to release vasopressin in other species46, and must therefore be treated with caution. However a previous report has shown elevated plasma levels of vasopressin in humans given apo-

morphine, independent of any known stimulus to vasopressin secretion including changes in blood r,~e_~ure ~blood volume or intrathoracic pressure 22. Neuroaaatomical studies in the ferret have demonstrated that the NTS is the major site of termination of vagal afferents 18. Vagal afferents also project to the area pastrema in the ferret and electrical stimulation of abdominal vagal afferents, comparable to that used in the present study increased the uptake of [3H]2-deoxyglucose in this area, as did apomorphine 3. No studies of the outputs of the NTS have been undertaken in this species but in other species (rat, monkey and man) several pathways from the NTS to the PVN have been demonstrated. Thus the NTS projects to the parabrachial nucleus and the A~ and A 5 catecholamine cell groups 13. Noradrenergic projections arise from the parabrachial nucleus and terminate in the parvocellular PVN and a noradrenergic pathway to the magnocellular region of the PVN arises from the A1 cell group 25. Furthermore long ascending projections from the NTS directly terminate in the paraventricular nucleus or the limbic system 17'2°. Therefore visceral information may be relayed direct to the paraventricular nucleus, by several different pathways. Substances acting at the area postrema, such as apomorphine, could also activate this pathway as afferent fibres from the AP project to the NTS and there is also a projection from the AP to the parabrachial nucleus 15. At present we can only speculate about the function of vasopressin release by abdominal vagal afferents or apomorphine, but several pieces of evidence suggest that it may be concerned with nausea or the emetic reflex. It has been known for some time that nausea is associated with very high levels of plasma vasopressin far in excess of those required for maximum antidiuresis 22 and which can override stimuli such as alcohol or water loading which would otherwise inhibit vasopressin release 6"22. Patients receiving chemotherapy experience nausea and elevated vasopressin levels independent of changes in plasma osmolarity or blood pressure 8. In addition nausea is a common symptom in patients given an infusion of vasopressin and is often followed by retching 27. Following apomorphine administration to humans elevated vasopressin and nausea were inseparably related 22 and subjects re-

197 ceiving apomorphine at subemetic do~es, or who did not experience nausea at doses which are usually emetogenic, did not show elevated pAVP showing that the rise in hormone is related to the nausea not the apomorphine z5. Plasma oxytocin levels in response to apomorphine were double basal values but extremely variable and not statistically significant. A previous study has reported a rise in plasma oxytocin in response to apomorphine 2s, but vasopressin levels here were not elevated. This discrepancy could be due to species variations as these authors used rats, which lack an emetic response 29. Vagal stimulation at levels comparable to those used in this study will promote retching and vomiting in the ferret 2. Electrophysiological studies have identified vagal afferents that fire in response to a range of substaaces (e.g. NaCI, KCI, glucose) when they are applied to the upper gut, and these substances are known to cause nausea and vomiting in man. The synchronous electrical stimulation of all the fibres in the abdominal vagus used here may be similar to the massive vagal afferent discharge evoked chemically and result in an emetic response. It has been implied that vasopressin release is under tonic inhibitory control by vagal afferents supplying the cardiovascular baro- and chemoreceptors. Interruptien of afferent and efferent vagal pathways by section or cold blockade causes a large, but transient, release of vasopressin 5"24. If tonic inhibition is important then cervical vagotomy would be expected to cause a sustained elevation of AVP, which did not occur 16. Our results show that stimulation of the central end of the abdominal vagus actually causes a release of vasopressin and previous studies may actually be

REFERENCES 1 Andrews, P.L.R., Vagal afferent innereation of the gastrointestinal tract, Progr. Brain Research, 67 (1986) 65-86. 2 Andrews, P.L.R., Bingham, S. and Davis, C.J., Retching evoked by stimulation of ak~dominal vagal afferents in the anaesthetised ferret, J. Phys;.e! (London), 358 (1984) 103P. 3 Andrews, P.L.R., Davis, C.J., Grahame-Smith, D.G. and Leslie~ R.A., Increase in [3H]2-deoxyglucose uptake in the ferret area postrema produced by apomorphine administration or electrical stimulation of the abdominal vagus, 3. Physiol. (London), 382 (1986) 187P. 4 Baylis, P.H., Posterior pituitary function in health and disease, Clin. Endo. Metab., 12 (1983) 747-770.

demonstrating the effect of a massive vagal injury discharge which could occur on sectioning. Elevated plasma vasopressin is obviously part of the emetic response; however, whether vasopressin is in itself a mediator of nausea is a matter for speculation. As decerebrate ferrets will retch in response to vagal afferent stimulation it is unlikely that a massive release of vasopressin is required to evoke the retching and vomiting phases of the emetic response (Andrews, unpublished observations). Vagal influences on vasopressin secretion are important and vet) rcomplex as any consideration of the effects of blood volume or pressure on vasopressin secretion will show. Furthermore sodium, and osmosensitive vagal afferents have been found in several regions of the gut including the liver ~ and these latter afferents also project to the NTS and parabrachial nucleus 2~. Thus the regulation of vasopressin secretion in normal physiological circumstances may involve the vagal-solitary tract nucleus-hypothalamus pathway and emesis may represent a massive stimulation of this pathway. In conclusion, this study has demonstrated that vagal afferent stimulation can evoke vasopressin secretion but the functional significance of this pathway awaits further studies. ACKNOWLEDGEMENTS We are most grateful to Dr. H.S. Chowdrey for performing the bioassay of ferret plasma. We are also grateful to the Wellcome Trust for financial support.

5 Bishop, V.S., Thames, M.D. and Schmid, P.G., Effects of bilateral vagal cold block on vasopressin in conscious dogs, Am. J. Physiol., 246 (1~84) R566-569. 6 Coutinho, E.M., Oxytocin and antidiuretic effects of nausea in women, Am. J. Obst. Gynaecol., 105 (1969) 127-131. 7 Donoghue, S., Garcia, M., Jordan, D. and Spyer, K.M., The brain stem projections of pulmonary stretch afferents in cats and rabbits, J. Physiol. fLondon). 322 (1982) 353-364. 8 Fisher, R.D., Rentschler, R.E., Nelson, J.C., Godfrey~ T.E. and Wilbur, D.W., Elevation of plasma antidiuretic hormone (ADH) associated with chemotherapy-induced emesis in man, Cancer Trcat. Rep., 66 (1982) 25-29. 9 Gauer, O.H. and Henry, J.P., Circulatory basis of volume

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