Localization of 5-HT3 receptor binding sites in human dorsal vagal complex

European Journal of Pharmacology,

127

174 (1989) 127-130

Elsevier UP 20521

Short communication

Localization of 5HT, receptor binding sites in human dorsal vagal complex D. John

M. Reynolds,

Ronald A. Leslie, David G. Grahame-Smith

and Jennifer

M. Harvey

M. R. C. Clinical Pharmacology Unit and University Department of CIinical Pharmacology, and Oxford University, Bet-chum Centre for Applied Neuropsychobiology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K.

Received 10 October 1989, accepted 24 October 1989

Binding of the 5-HT, receptor ligand [3H]BRL 43694 was investigated in the human medulla oblongata using in vitro autoradiography. High levels of saturable, displaceable binding (B,,,,, 1.88 pmol/mg protein, K, 1.21 nM) were seen in the dorsal vagal complex but in no other medullary region. The results provide evidence for the existence of 5-HT, receptor binding sites in a brain region involved in the control of vomiting in man. 5-HT, receptors;

Emesis; Autoradiography;

1. Introduction In the last few years 5-HT, receptor antagonists have emerged as effective treatments for vomiting caused by cancer chemotherapy (Miner and Sanger, 1986) both in experimental animals and in humans (Andrews et al., 1988). Although it is clear that peripheral 5-HT, receptors may mediate this effect (Andrews et al., 1988) recent evidence indicates that the central nervous system also contains 5-HT, receptor binding sites. This has been demonstrated in a variety of species using a number of different radiolabelled ligands (Kilpatrick et al., 1988; Waeber et al., 1989; Barnes et al., 1988; Nelson and Thomas, 1989; Peroutka and Hamik, 1988). The highest density of such binding sites has been reported to be in the nucleus tractus solitarii (NTS) and area postrema (AP) in the medulla oblongata (Waeber et al., 1989; Kilpatrick et al., 1988). These brainstem areas receive visceral sensory information via the vagus nerve and are

Medulla oblongata

part of the dorsal vagal complex (DVC) or the caudal brainstem that is involved in the control of the emetic response (Leslie, 1985). Medullary binding of the 5-HT, receptor antagonist BRL 43694 is markedly reduced following vagotomy in the rat (Pratt and Bowery, 1989) and in the ferret (Reynolds et al., 1989) suggesting that these binding sites are located on vagal afferent terminals. Thus far most of the published information on DVC binding of 5-HT, receptor ligands has come from work on animal tissues such as the rat or the ferret, a commonly used model for studies of cytotoxic drug- and radiation-induced vomiting (Andrews et al., 1988). Waeber et al. (1989) have used [3H]ICS 205-930 to label 5-HT, receptor binding sites in the human brain. We have used autoradiography to identify and characterize the binding of another tritiated 5-HT, receptor antagonist, BRL 43694 (granisetron), in the human medulla oblongata.

2. Materials and methods Correspondence to: D.J.M. Reynolds, M.R.C. Clinical Pharmacology Unit, University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K. 0014-2999/89/$03.50

Human medulla oblongata from three patients (two female, one male, ages 72-76) who had died

0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

128 f r o m n o n - n e u r o l o g i c a l disease was o b t a i n e d at post m o r t e m within 37 h of death. Serial frozen sections were cut at 12 /am and m o u n t e d on gelatinized glass slides and stored at - 7 0 ° C until use. Sections were i n c u b a t e d with varying conc e n t r a t i o n s of [ 3 H ] B R L 43694 (0.11-5.8 n M ) in 50 m M H E P E S buffer, p H 7.4 at r o o m t e m p e r a t u r e for 45 min. T h e specific activity of the r a d i o l i g a n d was 21.2 C i / m m o l . A d j a c e n t sections were similarly i n c u b a t e d with the a d d i t i o n of 100 /~M m e t o c l o p r a m i d e to define non-specific binding. F o l l o w i n g i n c u b a t i o n all sections were washed twice for 10 s each in 50 m M H E P E S buffer at 4 ° C followed by a rapid wash in distilled water at 4 ° C . Sections were air-dried and e x p o s e d to A m e r s h a m H y p e r f i l m for 2-8 weeks along with brain paste s t a n d a r d s to allow s u b s e q u e n t q u a n t i tative d e n s i t o m e t r i c analysis. F o l l o w i n g exposure

to H y p e r f i l m , all sections were stained with cresyl violet to aid a n a t o m i c a l localization of binding. W i t h reference to a c a l i b r a t i o n curve constructed f r o m the b r ai n paste standards, densitometric analysis was p e r f o r m e d using a c o m p u t e r based i m ag e analysis system ( K o n t r o n I B A S 2) on a m i n i m u m of four sections for each c o n c e n t r a t i o n of ligand per subject. Values for b i n d i n g in the presence of m e t o c l o p r a m i d e were s u b t r a c t e d f r o m the values o b t a i n e d in a d j a c e n t sections i n c u b a t e d in the absence of displacing d r u g to give specific binding.

3. Results High c o n c e n t r a t i o n s of [3 H ] B R L 43694 specific b i n d i n g were f o u n d in the N T S of the h u m a n

B

~

L D

. . . . . . . . . . . . . . . . . . . . . . . . . . .

Fig. 1. (A) Negative image of an autoradiogram generated over a tissue section of human caudal brainstem, showing total binding of [3H]BRL 43694 (see Methods). Note the distinct localization of heavy binding to the dorsomedial region of the nucleus tractus solitarii; the area of heavy binding on the right side is outlined and shown in higher magnification in 1C. Bar = 3 mm. (B) Autoradiogram generated over a section taken from the same region as that illustrated (A), showing non-specific binding in the presence of 100 ~M metoclopramide; bar = 3 mm. (C) Higher magnification of outlined region of autoradiogram in (A). The largest region of heavy binding appears to be in the most dorsomedial region of the nucleus tractus solitarii, just subjacent to the area postrema (refer to diagram 1D which illustrates the region of the autoradiogram indicated by dashed lines). Bar = 1 ram. (D) Line diagram of outlined area of (C). AP = area postrema, TS = solitary tract, X = dorsal motor nucleus of the vagus, IV = fourth ventricle, GN = gracile nucleus.

129

brainstem, particularly in dorsomedial regions of the nucleus (fig. 1). Non-specific binding, as determined by displacement of the radioligand with unlabelled metoclopramide, was low, accounting for less than 5% of total binding levels. No displaceable binding was observed in any of the other brainstem regions examined (e.g. hypoglossal nucleus, dorsal motor nucleus of the vagus or the nucleus of the spinal tract of the trigeminal nerve); data not shown. Binding was seen in the inferior olivary nucleus (fig. 1A) but it was not displaceable with 100 PM metoclopramide (fig. 1B). Autoradiograms of total and non-specific binding of [3H]BRL 43694 are shown in fig. 1. Displaceable binding is clearly seen in the NTS and appears to extend into the AP which is located subjacent to the ependymal lining of the floor of the caudal part of the fourth ventricle (fig. 1). Similar appearances were found in all three brains examined. Saturation analysis was performed with varying concentrations of [ 3H]BRL 43694 and fig. 2 shows a Scatchard transformation of the mean saturation data from the three brains which gives a K, of 1.21 nM and a B,,, of 1.88 pmol/mg protein.

BOUND/FREE

1500

1000

500

O_

BOUND

4. Discussion

Fig. 2. Scatchard transformation of the mean saturation analysis data obtained by autoradiography from three human brains. [‘H]BRL 43694 bound (fmol/mg protein) vs. bound/free. K, = 1.21 nM, B,, = 1.88 pmol/mg protein.

In this study we have used the 5-HT, receptor antagonist [3H]BRL 43694 to localize 5-HT, receptor binding sites in the human medulla oblongata. High levels of saturable and displaceable binding were demonstrated in the dorsomedial NTS with much lower levels of binding in the AP. In a recent report, Waeber et al. (1989) demonstrated binding of the 5-HT, receptor antagonist, [3H]ICS 205-930 in the human brain. They reported that the highest levels of binding occurred in the AP in the caudal medulla oblongata, with somewhat lower levels in the dorsal motor nucleus of the vagus nerve and much lower levels in the NTS. We have performed a careful histological analysis of the distribution of binding of [ 3H]BRL 43694 in the human medulla and have found that the greatest amount of binding is immediately

subjacent to the AP in the dorsomedial subdivision of the NTS. The dorsomedial subdivision of the NTS has been shown to receive the largest concentration of gastric vagal afferent terminals in several animal species with far fewer vagal afferent fibres terminating in the AP (Leslie, 1985). Consistent with the results of the binding study reported here, are the reports that binding of [3H]BRL 43694 is greatly decreased in the NTS following de-afferentation of the vagus nerve in the rat (Pratt and Bowery, 1989) and in the ferret (Reynolds et al., 1989). Furthermore, the distinction between the AP and the NTS is important from a physiological viewpoint as the AP has a very weak blood-brain diffusion barrier, unlike the more conventional blood-brain barrier in the NTS.

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We saw no binding of [3H]BRL 43694 in the spinal trigeminal nucleus, which contrasts with that reported for [3H]ICS 205-930 binding in this brain region by Waeber et al. (1989). The presence of a binding site for [3H]ICS 205-930 in the spinal trigeminal nucleus, which is not recognized by [3H]BRL 43694, suggests that separate classes of binding sites might be labelled by these two ligands.

Acknowledgements [3H]BRL 43694 and metoclopramide hydrochloride were generously provided by Beechams Pharmaceuticals. We are grateful to Dr. B. McDonald, Nuffield Dept. of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford, for supplying the human brain tissue.

References Andrews, P.L.R., W.G. Rapeport and G.J. Sanger, 1988, Neuropharmacology of emesis induced by anti-cancer therapy, Trends Pharmacol. Sci. 9, 334. Barnes, N.M., B. Costall, J.W. Ironside and R.J. Naylor, 1988, Identification of 5-HT3 recognition sites in human brain tissue using [3H]zacopride, J. Pharm. Pharmacol. 40, 668.

Kilpatrick, G.J., B.J. Jones and M.B. Tyers, 1988, The distribution of specific binding of the 5-HT3 receptor ligand [3H]GR65630 in rat brain using quantitative autoradiography, Neurosci. Lett. 94, 156. Leslie, R.A., 1985, Neuroactive substances in the dorsal vagal complex of the medulla oblongata; nucleus of the tractus solitarius, area postrema and dorsal motor nucleus of the vagus, Neurochem. Int. 7, 191. Miner, W.D. and G.J. Sanger, 1986, Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism, Br. J. Pharmacol. 88, 497. Nelson, D.R. and D.R. Thomas, 1989, [3H]BRL 43694 (granisetron), a specific ligand for 5-HT3 binding sites in rat brain cortical membranes, Biochem. Pharmacol. 38, 1693. Peroutka, S.J. and A. Hamik, 1988, [3H]Quipazine labels 5-HT3 recognition sites in rat cortical membranes, European J. Pharmacol. 148, 297. Pratt, G.D. and N.G. Bowery, 1989, The 5-HT3 receptor ligand, [3H]BRL 43694, binds to presynaptic sites in the nucleus tractus solitarius of the rat, Br. J. Pharmacol. 97, 414P. Reynolds, D.J.M., P.L.R. Andrews, R.A. Leslie, J.M. Harvey, P.M. Grasby and D.G. Grahame-Smith, 1989, Bilateral abdominal vagotomy abolishes bindign of [3H]BRL 43694 in ferret dorsovagal complex, Br. J. Pharmacol. 98, 692P. Waeber, C., D. Hoyer and J.M. Palacios, 1989, 5-Hydroxytryptamine3 receptors in the human brain: autoradiographic visualization using [3H]ICS 205-930, Neuroscience 31, 393.