Presence of functional vasopressin V1 receptors in rat vagal afferent neurones

Neuroscience Letters, 145 (1992) 79-82 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Presence of functional vasopressin VI receptors in rat vagal afferent neurones X. G a o a, P.A. PhiUips a, R . E . W i d d o p b, D . T r i n d e r a, B. J a r r o t t b a n d C.I. J o h n s t o n a QDepartment of Medicine and bClinical Pharmacology and Therapeutics Unit, University of Melbourne, Austin Hospital, Heidelberg, Vic. (Australia)

(Received 29 January 1992; Revised version received 24 June 1992; Accepted 25 June 1992) Key words: Arginine vasopressin; Brain; Nodose ganglion; Nucleus tractus solitarii; Receptor; Vagus

The nucleus of the solitary tract (NTS) is one of the brain regions by which arginine vasopressin (AVP) influences blood pressure. This series of experiments in adult male rats was designed to determine whether the AVP binding sites which have been demonstrated in the NTS by in vitro autoradiography might be presynaptic on vagal afferents from the nodose ganglion; whether the AVP binding sites on vagal afferent neurones are functional receptors; and whether vagal transport of AVP receptors to other organs also occurs. High affinity binding sites (using the selective V~ antagonist radioligand [~25I][d(CH2)5Sar7]AVPand in vitro autoradiography) with characteristics of V~ receptors in the medial subnucleus of the NTS were reduced by 40% ipsilateral to nodose ganglionectomy.The nodose ganglion itself also contained high affinityV~AVP binding sites that localised over cell bodies of vagal sensory neurones. That these binding sites were functional receptors was apparent when low concentrations of AVP but not oxytocin were found to depolarise the isolated nodose ganglion utilizing the 'silicone grease gap' technique. Furthermore, the actions of AVP were antagonised by low concentrations of a selectiveVt receptor antagonist. However,there was no accumulation of AVP binding sites adjacent to either the proximal or distal vagal ligations suggestingthat peripheral vagal transport of AVP receptors may not occur. Therefore these results are consistent with functional AVP V~receptors occurring in the nodose ganglion. These receptors may occur on central terminals of vagal sensory neurones in the medial subnucleus of the NTS, but there was no evidence for peripheral transport of AVP V~ receptors.

Arginine vasopressin (AVP) influences blood pressure in m a n y ways. Circulating AVP acts as a direct vasoconstrictor and also can sensitise the baroreflex [4, 11]. In the brain, circulating AVP m a y act on the area postrema (which is outside the b l o o d - b r a i n barrier) to influence cardiovascular regulation [4]. Inside the b l o o d - b r a i n barrier, AVP m a y act as a neurotransmitter or neuromodulator in several regions thereby influencing blood pressure [11]. The nucleus of the solitary tract (NTS) m a y be one of these regions. Micro-injections of AVP into the N T S alters blood pressure probably via V1 AVP receptors [8]. The N T S also receives vasopressinergic innervation from the hypothalamic paraventricular nucleus [12] and contains high affinity binding sites for [3H]AVP and radiolabelled V~ antagonists [9, 10, 13, 14]. Similarly, another vasoactive neuropeptide, angiotensin II, m a y influence blood pressure via the N T S [2, 10]. Micro-injections of angiotensin II in this region increase blood pressure [2, 11] and the N T S contains high affinity binding sites for radiolabelled A I I antagonists [1, 7]. These All receptors Correspondence: P.A. Phillips, Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Vic. 3084, Australia. Fax: (61)(3)457 5485.

seem to be presynaptic in the N T S on vagal sensory afferents since nodose ganglionectomy reduces A l l receptor density in the N T S [7]. Similarly, ligation of the distal vagus demonstrates that A l l receptors are transported peripherally [1] in a similar manner to other receptors such as muscarinic [18], N-methyl-D-aspartate [3], opioid [16] and cholecystokinin [17] receptors. Furthermore, A l l is potent in depolarising the isolated rat nodose ganglion and this depolarisation is blocked by the specific A l l antagonist saralasin [15]. The aim of this series of studies was to determine whether the AVP binding sites seen in the N T S might be presynaptic arising in vagal sensory neurones in the nodose ganglion; whether these binding sites are functional receptors; and whether vagal transport of AVP receptors peripherally also occurs. Some of these results have been reported previously in a preliminary communication associated with a scientific meeting [10]. In the first series of experiments male S p r a g u e - D a w ley rats (200-300 g, n=4) were anaesthetized with methohexitone (32 mg/kg)/amylobarbitone (60 mg/kg) and the left or right nodose ganglion excised and processed for in vitro autoradiographic determination of AVP binding site density (see below). The contralateral

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Fig. 1. In vitro autoradiographic demonstration of (A) reduction of [t25I]dSAVP binding to the medial subnucleus of the NTS (MNTS) ipsilateral to nodose ganglionectomy but not to the lateral subnucleus of the NTS (LNTS), inferior olive (10) or area postrema (AP); (B) no reduction in binding in bilaterally sham operated animals; (C) total binding of [~2~I]dSAVPto the nodose ganglion; (D) non-specific binding to the nodose ganglion; (E), no accumulation of radioligand binding adjacent to proximal or distal ligatures around vagus distal to nodose ganglion (arrows indicate sites of ligation) as compared with nonspecific binding (F) and sham ligated vagus (G); and non-specific binding of a sham ligated vagus (H). The patchy high intensity non-specific binding in both ligated total and non-specific autoradiographs of vagi represents non-specific binding to regenerative connective tissue.

receptor binding the excised tissues were immediately frozen in isopentane at - 3 0 ° C and 20 p m frozen sections prepared as described previously [9]. The ligand used, [1-(fl-mercapto-fl,fl-cyclopentamethylene propionic acid), sarcosine7,argS]vasopressin ([125I]dSAVP), is a selective V~ receptor antagonist radioligand with little crossreactivity to oxytocin receptors or other types o f AVP receptor [6]. Non-specific binding was determined in parallel incubations containing 1 pmol/l unlabelled dSAVP. The autoradiographic images were quantitated in terms o f 125I counts b o u n d per unit area (d/min per m m 2) with a computerised image analysis system (Imaging Research Inc., Ontario) using radioactivity standards to obtain computer-generated standard curves. Specific binding was calculated by subtracting non-specific binding in the regions o f interest from total binding. Specific binding densities in the medial and lateral subnuclei o f the NTS, the inferior olive and area postrema ipsilateral and contralateral to nodose ganglionectomy were then compared by paired t-test. Specific binding was observed in the rostral, medial and lateral subnuclei o f the NTS, area postrema, inferior olive and caudal subnucleus o f the spinal trigeminal tract as well as in the nodose ganglion (Fig. 1). There was no binding in the dorsal m o t o r nucleus o f the vagus. Binding was reduced by 40% in the medial subnucleus o f the NTS (P < 0.05) ipsilateral to nodose ganglionect o m y c o m p a r e d with the contralateral side (Fig. 1 and Table I). However, there was no reduction in binding in the rostral NTS, lateral subnucleus o f the NTS, inferior olive or area postrema (Fig. 1). Bilaterally sham-operated rats had no alteration in binding in any o f the regions (Fig. 1). Also there was no reduction in binding to the medial subnucleus o f the N T S contralateral to nodose ganglionectomy c o m p a r e d to control rats suggesting no extensive crossing o f afferent fibres carrying AVP binding sites. These results are consistent with the presTABLE 1 [~25I]dSAVP dpm/mm 2 BOUND TO DIFFERENT BRAIN REGIONS AS DETERMINED BY COMPUTERIZED DENSITOMETRY Nucleus of the solitary tract Medial L a t e r a l Rostral subnucleus subnucleus

ganglion was exposed but not excised. In a separate control g r o u p o f rats both ganglia were exposed but not excised (n = 4). Seven days later the experimental and control rats were decapitated and the hindbrains rem o v e d and processed for in vitro autoradiography. F o r in vitro autoradiographic determination o f AVP

Inferior olive

Sideof 30.6+3.8* 50.5±5.8 31.9_+15.1 77.7_+15.8 ganglionectomy Side contralateral 50.5-~6.0 53.9_+6.2 35.0_+10.2 84.4+18.1 to ganglionectomy *P<0.05.

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-LOG PEPTIDE CONC (M) Fig. 2. A: concentration-response curves for depolarisation of the rat isolated nodose ganglion by either vasopressin (AVP) or oxytocin (OXY) at 37°C. Each point is the mean + S.E,M. of 6 tissues. In another two experiments, the AVP concentration-response curves were repeated before and during superfusion with a 1 nM concentration of the selective V~ receptor antagonist [d(CH2)s,argS]AVP(,-*). B: depolarisation of the rat isolated nodose ganglion by vasopressin (AVP) before (,--o) and during superfusion with a 1 nM concentration of the selective V1 receptor antagonist [1-fl-mercapto-fl,fl-cyclopentamethylene propionic acid), O-Me-Tyr2,argS]vasopressin (ANT, *-,). Each point is the mean of two experiments.

ence of AVP V~ receptors on the central branches of vagal afferents projecting from the nodose ganglion to the medial subnucleus of the NTS. In the second series of experiments the aim was to determine whether the binding sites demonstrated in the nodose ganglion represent functional AVP receptors.

Therefore, both nodose ganglia with attached vagal trunks from male Sprague-Dawley rats were placed in a twin-chambered perspex bath to record peptide-induced extracellular depolarisation of the nodose gangion as described previously [15]. Each compartment was superfused continuously with Krebs' solution at 37°C and concentration-response curves were determined by applying peptides to separate ganglia as described previously [15]. For studies using the V~ receptor antagonist [l-(fl-mercapto-fl,fl-cyclo-pentamethylene propionic acid), O-Me-Tyr2,argS]vasopressin (dTMAVP) the nodose ganglion was superfused with a 1 nM concentration for 20-30 min before a second AVP concentration-response curve was constructed. It was found that low nanomolar concentrations of AVP depolarised the isolated nodose ganglion preparation (Fig. 2). The structurally similar neuropeptide oxytocin was approximately 500 times less potent than AVP (Fig. 2) and the depolarising action of AVP was potently antagonised by superfusion with the specific V~ receptor antagonist dTMAVP (Fig. 2). These findings suggest that the actions of AVP are initiated after selective stimulation of the V~ subclass of AVP receptors. This conclusion is supported by the nodose ganglion autoradiographic findings that the high affinity binding site has the characteristics of a V~ receptor. In the final series of experiments the aim was to determine whether there was peripheral transport of AVP receptors from the nodose ganglion. Male Sprague-Dawley rats (200-250 g, n = 8) were anaesthetized with methohexitone (32 mg/kg)/ambylobarbitone (60 mg/kg) and the right nodose ganglion was exposed. Two 6/0 silk ligatures were then applied to the peripheral vagus approximately 5 mm apart, the most distal being about 10 mm distal to the nodose ganglion. The ligatures were pulled tight thus ligating the vagus and the wound was closed. In a separate group of rats (n=7) control ligations were made in the same way exept that the ligatures were not pulled tight. After 24 h the rats were re-anaesthetized and the vagus nerve was exposed, the sutures were carefully dissected from the ligated nerve and the nerve trunk was excised and processed for in vitro autoradiography of AVP receptors. Binding site densities in the appropriate control rats and in the vagus 2 mm proximal to the proximal ligature and 2 mm distal to the distal ligature were compared with the appropriate control animals by unpaired t-test. There was no evidence of accumulation of AVP binding sites adjacent to either the proximal or distal vagal ligations (Fig. 1) suggesting that peripheral transport of AVP receptors does not occur. Similarly, AVP receptors have not been identified in structures such as the heart or gut [9] from which vagal

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sensory afferents arise, providing further support for this lack of transport. This is in contrast to the findings for angiotensin II [1], muscarinic [18], N-methyl-D-aspartate [3], opioid [16] and cholecystokinin [17] receptors which seem to be transported peripherally in the vagus nerve using similar methodology to that used here. The ligated vagus has proven a convenient model for the demonstration of such axonal transport of neurotransmitters and neuroreceptors. However although specific V~ AVP receptor binding sites were found in the NTS and nodose ganglion, and binding sites in the medial subnucleus of the NTS were reduced following unilateral nodose ganglionectomy, suggesting central transport of AVP receptors, there was no evidence for peripheral vagal transport of AVP receptors. This was surprising given the pseudobipolar nature of these neurones. One possibility for the lack of demonstration of peripheral transport of AVP receptors might be that they are transported by slow axonal flow and so a longer ligation time might have been necessary. However, in other similar studies, AII receptor accumulation occurred adjacent to ligatures within 24 h and was easily demonstrable [1]. If axonal flow of AVP receptors distally was to occur it would be reasonable to hypothesise that it would occur in the same way that angiotensin II receptors were transported. Since we did not demonstrate AVP receptors in the dorsal motor nucleus of the vagus it suggests that the AVP receptors are not on motor fibers and that they arise in the nodose ganglion and are transported centrally to the medial subnucleus of the vagus. Similarly since there was no reduction of binding in the area postrema (despite the area postrema receiving sensory input from the vagus) it would seem that the receptors in this region which lacks a blood-brain barrier, may be postsynaptic perhaps mediating the inhibitory effects of circulating AVP on the baroreflex [4]. In conclusion, this study has identified high affinity binding sites with characteristics of AVP Vj receptors both on the central terminals of vagal sensory nerves in the medial subnucleus of the NTS and in the cell bodies of the nodose ganglion of rats. Furthermore, AVP was a potent depolarising agent on the isolated nodose ganglion and by inference [5] on the AVP receptors at the central terminals of vagal afferents in the NTS. Thus, AVP could alter baroreflex function centrally through actions on these receptors on vagal afferent nerves. This research was supported by the N&HMRC of Australia. We thank Ms. Elena Krstew for her excellent technical assistance. 1 Allen, A.M., Lewis, S,J., Verberne, A.J.M. and Mendelsohn,

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