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Radioligand binding and autoradiographic visualization of adenosine transport sites in human inferior vagal ganglia and their axonal transport along rat vagal afferent neurons
Journal of the
ELSEVIER
Autonomic Nervous System Journal of the Autonomic Nervous System 57 (1996) 36-42
Radioligand binding and autoradiographic visualization of adenosine transport sites in human inferior vagal ganglia and their axonal transport along rat vagal afferent neurons Margie Castillo-Mel6ndez *, Bevyn Jarrott, Andrew J. Lawrence Department of Pharmacology, Monash University, WellingtonRoad, Clayton, Victoria 3168, Australia Received 17 May 1995; revision received 2 September 1995; accepted 4 September 1995
Abstract
The present study has employed membrane-binding studies and in vitro autoradiography to demonstrate the presence of adenosine transport sites in human inferior vagal ganglia using [3H]nitrobenzylthioinosine ([3H]NBMPR), a potent inhibitor of adenosine transport. In addition, [3H]NBMPR was used to determine whether adenosine transport sites are subject to axonal transport along the rat vagus nerve. Binding of [3H]NBMPR to human inferior vagal ganglia membranes was saturable and reversible. Saturation experiments revealed a single class of high affinity-binding sites with a K d of 93.73 + 23.13 pM and Bm~, of 413.50+ 50.40 fmol/mg protein. In displacement experiments, the adenosine transport inhibitor dipyridamole was the most potent displacer of [3H]NBMPR binding (K i m 42.7 + 28.0 nM). Adenosine itself was able to fully displace [3H]NBMPR binding with a K i of 115.0 _ 34.0 /zM. The A l//A2a adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) was able to fully displace [3H]NBMPR binding in only one experiment at a concentration of 100/zM, yielding an affinity 1000-fold higher than its affinity for adenosine receptors. All competition curves obtained from displacement experiments displayed monophasic profiles, indicating the presence of a single class of [3H]NBMPR binding sites. Incubation of human inferior vagal ganglia sections with [3H]NBMPR (0.7 nM) revealed dense binding which appeared to be consistent with the distribution of neuronal cell bodies in this tissue. Following unilateral ligation of the vagus nerve in the rat, acccumulation of [3H]NBMPR binding sites occurred both proximal and distal to the vagal ligatures. These results suggest that [3H]NBMPR binds with high affinity to a single class of adenosine transport sites, and that these sites are present on vagal afferent neurons in the human and undergo bidirectional axonal transport along the rat vagus nerve. Keywords: Adenosine; Transport site; Sensory ganglion; Axonal flow; Nitrobenzylthioinosine; Autoradiography
1. Introduction The purine nucleoside adenosine has been defined as an endogenous neuromodulatory substance in the central nervous system [8]. The physiological actions of adenosine are believed to be mediated primarily by interactions with extracellular adenosine receptors coupled to adenylate cyclase systems [21]. Adenosine is the endogenous ligand at Pl purinoeceptors termed A l, A 2 [31], A 3 [28] and A 4 [6]. The A 2 adenosine receptor has been further subdivided into m2a and A2b subtypes [10]. Recently, there has been a growing body of evidence, suggesting a possible role for
* Corresponding author. Tel.: (61-3) 9905-4855; Fax: (1-613) 99055851. 0165-1838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 65- 1 8 3 8 ( 9 5 ) 0 0 0 9 8 - 4
adenosine in the processing of baroreceptor information at the central terminals of baro-afferents within the nucleus tractus solitarius (NTS) in the dorsal medulla oblongata. Initial support came from studies which indicated that microinjection of adenosine antagonists into the NTS was capable of inhibiting baroreceptor reflex activity within this nucleus [25]. In addition, studies by Barraco and colleagues [1] demonstrated that intra-NTS microinjection of adenosine agonists led to different cardiovascular response pattems depending on the adenosine receptor subtype activated. Furthermore, microdialysis studies in the rabbit have shown that adenosine administration into the NTS leads to increases in local extracellular levels of glutamate [26], the favoured candidate neurotransmitter of primary baroreceptor afferent neurons [5,17,29]. More recently, microdialysis experiments in the rat have demon-
M. Castillo-MeMndez et al./ Journal of the Autonomic Nervous System 57 (1996) 36-42
strated that activation of adenosine A2a receptors in the NTS results in increased extracellular glutamate levels within this nucleus [4]. The rapid transport of adenosine from the extracellular fluid back into the cells and subsequent catabolism by adenosine kinase and deaminase are believed to be the most important mechanisms by which the physiological effects of this nucleoside are terminated [27]. The presence of a high density of adenosine transport sites in the NTS [2] and a high-affinity adenosine transport system in synaptosomes prepared from dorsal brain stem [16] have been demonstrated. The presence of adenosine transport sites in the inferior vagal (nodose) ganglion, where the cell bodies of aortic baro-afferent neurons are located [12], has not yet been reported. Therefore, the present study has employed radioligand-binding studies using membrane preparations of human inferior vagal ganglia to characterize the binding properties of the potent adenosine transport inhibitor [SH]nitrobenzylthioinosine ([3H]NBMPR) [2]. In addition, in vitro autoradiography studies were carried out using [3H]NBMPR to visualize adenosine transport sites on sections of human inferior vagal ganglia. Evidence has indicated that receptors for various neurotransmitters and neuromodulators are synthesized in vagal afferent cell bodies and then axonally transported along the nerve to the terminals [15]. Thus, unilateral vagal ligation in the rat has been employed to determine whether adenosine transport sites are subject to such axonal transport mechanisms along the vagus nerve.
2. Materials and methods
2.1. Materials [3H]NBMPR (19.5 Ci/mmol) was obtained from NEN Dupont, NBMPR was from Research Biochemicals Incorpotated (RBI), tritium sensitive film (Hyperfilm) and tritium microscales were from Amersham. Adenosine, dipyridamole and 5'-(N-ethylcarboxamido)-adenosine (NECA) were all from Sigma. EcoLite scintillant was obtained from ICN. All other reagents were either analytical or laboratory grade from various suppliers.
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cleared of connective tissue and homogenized in 8 ml of 0.32 M sucrose using an Ultra-Turrax homogenizer. The homogenate was centrifuged at 1000 × g for 10 min. at 4°C and the resulting supematant was recentrifuged at 30000 × g for 30 min. Following centrifugation, the resulting pellet was resuspended in 25 vols. (w/v) of Tris. HC1 buffer (50 mM; pH 7.4) and centrifuged again at 30 000 × g for 20 min. The final pellet was resuspended in 50 vols. (w/v) of Tris. HC1 buffer. Protein concentration was determined as described by Lowry et al. [22] using bovine serum albumin as standard (0.25-4.0 mg/ml).
2.2.2. Binding assays All binding assays were carried out in duplicate, and were commenced by addition of 0.1 ml of membrane preparation, representing 36-56 /.tg of protein in a final volume of 0.5 ml. Saturation experiments were performed by incubating the membranes with nine different concentrations of [3H]NBMPR ranging from 0.025 to 6.4 nM (n = 3). Displacement experiments (n = 3 per displacer) consisted of incubation of the membranes with 0.3 nM [3H]NBMPR and eight different concentrations of either dipyridamole (1 nM-3 /xM), adenosine (3 /zM-10 mM) or NECA (30 nM-100 /zM). Non-specific binding was determined in the presence of 5 /xM unlabelled NBMPR. Incubations for both saturation and displacement assays were for 30 min at room temperature, and were terminated by rapid vacuum filtration through Whatman glass microfibre filters (GF/B) with 4 × 4 ml ice-cold Tris. HC1 (50 mM; pH 7.4) rinses using a Brandel cell harvester. Bound radioactivity was measured by scintillation counting of /3-emission (efficiency approx. 50%). 2.2.3. Analysis of [ZH]NBMPR binding The maximal number of [3H]NBMPR-binding sites (Bm~x) and apparent affinity of the ligand for the binding site (K d) were determined by Scatchard analysis from the saturation binding data. The affinities of the displacers for the [3H]NBMPR-binding sites (K i) were determined from the displacement curves obtained from the competition binding data. All parameters were obtained using the EBDA/LIGAND computer software [24]. 2.3. Preparation of the tissue slices
2.2. Membrane-binding studies 2.2.1. Preparation of the membranes Inferior vagal ganglia were obtained from cadavers at the Victorian Institute of Forensic Pathology. Donors were of either sex and had no previous history of neurological disease. The age range of the donors was between 25 and 70 years of age and in all cases the delay between death and autopsy was less than 48 h. Ethical permission for the study was granted by the Ethics and Integrity in Research Committees of both Monash University and the Victorian Institute of Forensic Pathology. The tissue samples were
2.3.1. Human studies Inferior vagal ganglia from human subjects were frozen over liquid nitrogen and sections were cryostat cut (10 /zm), thaw-mounted on to gelatin/chrome-alum-coated slides and stored at - 80°C until further processed. 2.3.2. Rat studies Male Wistar-Kyoto rats (280-350 g) were anaesthetized with sodium methohexitone (60 mg/kg, i.p.) and the left nodose ganglion and vagus nerve were exposed. Two tight ligations (5 mm apart) were applied to the nerve approx.
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M. Castillo-Mel£ndez et al./ Journal of the Autoru~mic Nervous System 57 (1996) 36-42
10 mm peripheral to the nodose ganglion using 6 / 0 silk thread (n = 5). In a similar manner, corresponding sham ligations were performed on a separate group of rats by placing two loosely fitting ligatures around the vagus nerve (n = 4). After 24 h, rats were re-anaesthetized with sodium pentobarbitone (60 mg/kg, i.p.) and the ligated portion of the vagus nerve and nodose ganglion were excised and the ligatures removed. Tissue samples were then placed in embedding compound (OCT) and frozen over supercooled isopentane. Longitudinal 10-/~m sections were cut in a cryostat, thaw-mounted on to gelatin/chrome-alum coated microscope slides and stored at - 8 0 ° C until used.
2.4. In vitro autoradiography The autoradiographic experiments were carried out using a previously described protocol [2]. Briefly, tissue sections were wai'med to room temperature prior to incubation with 0.7 nM [3H]NBMPR (in 50 mM Tris-HCI buffer, pH 7.4) for 30 min. Non-specific binding was determined on adjacent sections by addition of an excess of NBMPR (20 /xM final concentration) to the incubation media. Following the incubation period, sections were washed (3 X 5 min) in ice-cold 50 mM Tris- HCI buffer (pH 7.4) and rinsed twice in distilled water. The slides were then dried under a stream of cool air and apposed to tritium sensitive film for 8 weeks, in the presence of tritium microscales.
2.5. Image analysis Densitometric quantification of the developed films was carried out using an MCID M4 image analysis system (Imaging Research). The binding over whole sections of human inferior vagal ganglia (n = 4; 4 sections/specimen), rat ligated vagus nerve with nodose ganglion (n = 5; 3-8 sections/animal) and sham ligated vagus nerve and nodose ganglion (n = 4; 5 - 8 sections/animal) was quantified by comparing the optical density of the autoradiograms to the standard tritium microscales. Sections of both human inferior vagal ganglia and rat vagus nerve and nodose ganglia were stained with 0.1% thionin and coverslipped for histological assessment, using an Olympus BH-2 photomicroscope.
3. Results
3.1. Human studies 3.1.1. Membrane-binding studies
Using the current protocol, [3H]NBMPR bound to human inferior vagal ganglia membranes in a saturable manner. Scatchard analysis of saturation experiments yielded a Bma x of 413.50 ___50.40 fmol/mg protein and a K d of
120-
100:
8060-
,~ O ~9
40200 -9
-
-7
-
-5
-4
-3
-2
Displacer (log M) Fig. I. Displacement of [3H]NBMPR (0.3 nM) binding to membranes of human inferior vagal ganglia by dipyridamole ( • ) , adenosine ( 0 ) and NECA ([2). Experiments were conducted in duplicate using and eight concentrations of each displacer. The data illustrated are mean + SEM values from 3 separate experiments per displacer for dipyridamole and adenosine. The competition curve for NECA is from an individual experiment. In two other cases, NECA was unable to fully inhibit the binding of [3H]NBMPR.
93.73 + 23.13 pM. Within the [3H]NBMPR concentration range studied, the data best described a one-site interaction as indicated by the obtained Hill coefficient of 1.00 + 0.05 (n = 3). In displacement binding assays, the specific binding of [3H]NBMPR was found to represent 93.25 _+ 0.49% of the total binding (n = 9). The results from displacement studies for adenosine, dipyridamole and NECA are illustrated in Fig. I. As can be seen, both adenosine and the adenosine transport inhibitor dipyridamole were able to fully displace [3H]NBMPR binding with K i values of 115.00 + 34.00 /xM and 42.70 _+ 28.00 nM, respectively. Despite the apparently low Hill slopes of 0.66 _+ 0.06 for adenosine and 0.85 _+ 0.12 for dipyridamole, ligand analysis of the data indicated a single site fit for both competitors. The non-selective adenosine receptor agonist NECA was unable to fully displace [3H]NBMPR binding in two experiments, however, in a third experiment, NECA inhibited the binding by 90% at a concentration of 100 /xM.
3.1.2. Autoradiography studies Multiple slide mounted sections of human inferior vagal ganglia obtained from four subjects were analysed, two from the right and two from the left vagus. [3H]NBMPR (0.7 nM) extensively bound to human inferior vagal ganglia sections (Fig. 2A). Microscopic examination revealed that the distribution pattern of [3H]NBMPR binding was consistent with the localization of neuronal cell bodies in this tissue, as seen in an adjacent thionin-stained section (Fig. 2C). Densitometric quantitation of, the autoradiograms indicated that total binding of [3H]NBMPR represented 64.3 + 2.0 d p m / m m 2 (n = 16 sections). A preliminary experiment where inferior vagal ganglia sections were wiped off the slides and counted in a r-emission scintilla-
M. Castillo-Mel~ndez et al. / Journal of the Autonomic Nervous System 57 (1996) 36-42
tion counter revealed that, in the presence of NBMPR (20 /xM) specific binding of [3H]NBMPR represented 95% of the total binding. Indeed, sections representing non-specific binding did not produce a strong enough signal on film for quantification (Fig. 2B). 3.2. Rat studies
Fig. 3A represents a photomicrograph of a thioninstained section of rat nodose ganglion with the vagal trunk, demonstrating the effect of surgical ligation of the vagus nerve. In the sham-ligated vagus nerve of the rat, [3H]NBMPR binding was almost completely associated
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with the nodose ganglion (Fig. 3B). In the ligated vagus nerve, [3H]NBMPR binding occurred not only on the nodose ganglion but also was present in the vagal trunk, both proximal and distal to the ligatures (Fig. 3C). Image analysis of the autoradiograms indicated a binding density of 17.3 +__0.8 and 18.0 _ 1.1 d p m / m m 2 for [3H]NBMPR on whole sections of sham and ligated nodose-vagal sampies, respectively. Binding of [3H]NBMPR on both sham and ligated vagus nerve sections was completely abolished in the presence of NBMPR (20 /zM) and the images obtained were too faint to be quantified.
4. Discussion
Fig. 2. Photomicrograph of human inferior vagal ganglia sections. (.~) Total [3H]NBMPR (0.7 nM) binding on a section of inferior vagai ganglia. Well defined, punctate binding can be clearly seen. Scale bar = 1.5 mm. (B) A section of human inferior vagal ganglia showing the absence of binding in the presence of 20 p,M unlabelled NBMPR. Scale bar = 1.5 rnm. (C) Light-field photomicrograph of an adjacent tissue section to A, stained with 0.1% thionin, demonstrating alternate bands of neuronal cell bodies and nerve fibres. Scale bar = 1.2 mm.
The present investigation has characterized the binding properties of the widely used adenosine transport inhibitor [3H]NBMPR in human inferior vagal ganglia membrane preparations. The binding of [3H]NBMPR to these membranes was saturable, reversible and specific, and of high affinity to a single population of binding sites. Displacement experiments revealed that out of the compounds tested, dipyridamole was the most potent inhibitor of [3H]NBMPR binding. Adenosine was also able to fully inhibit [3H]NBMPR binding, whereas the A I / A E a adenosine receptor agonist NECA produced 0, 50 and 90% inhibition of binding at a concentration of 100 /xM in the three experiments carried out. In the assay where almost total inhibition was achieved, NECA was found to display a micromolar potency. The affinity of this agonist for A J A 2 a adenosine receptors has been reported to be less than 10 nM [3], suggesting that the sites being labelled by [3H]NBMPR are not likely to represent an adenosine receptor. The ability of NECA to fully displace [3H]N-BMPR binding could be attributed to NECA being a close structural analogue of adenosine and therefore, at high concentrations, NECA may display low affinity for the adenosine transport site. The present study has also visualised the presence of a high density of [3H]NBMPR binding sites on sections of inferior vagal ganglia of humans using in vitro autoradiography. Furthermore, the distribution of binding sites was punctate, paralleling the cytoarchitecture of the ganglia, indicating the possibility that human vagal afferent neurons possess adenosine transport sites. The distribution of [3H]NBMPR binding is similar to the recent visualization of dopamine D 2 binding sites on this ganglion, where binding appeared to be restricted to neuronal cell bodies [18]. This observed pattern of [3H]NBMPR binding to sections of inferior vagal ganglia indicates the likelihood of a proportion of these binding sites being associated with vagal perikarya. An absolute confirmation at the light microscopic level is not possible due to the use of low specific activity tritium as radioligand rendering the use of nuclear emulsion unfeasible.
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M. Castillo-Meldndez et al./ Journal of the Autoru)mic Nervous System 57 (1996) 36-42
In addition, the study has demonstrated for the first time the presence of a high density of binding sites for [3H]NBMPR over sections of rat nodose ganglia. Furthermore, the accumulation of [3H]NBMPR binding in the rat vagus nerve following vagal ligation demonstrates that adenosine transport sites are subject to axonal transport along the vagus nerve in this species, and thus a similar process would be expected in humans, The ligated rat vagus nerve has been established as a suitable model for the demonstration of axonal transport of neuroreceptors and transmitters [15]. Accumulation of [3H]NBMPR binding in tigated vagus nerve sections was observed on both the proximal and distal sides of the vagal ligatures suggesting bidirectional transport of adenosine transport sites. Axonal transport along the rat vagus nerve has been previously demonstrated using this surgical procedure for opioid receptors [32], muscarinic receptors [13], glutamate receptors [20], vasopressin V 1 receptors [9], cholecystokinin receptors [23] and /3-adrenoceptors [19]. In addition, the existence of orthograde axoplasmic transport has been demonstrated for various transmitters, enzymes, labelled proteins and peptides along the axons of different nerve fibres [14]. However, the present study represents the first demonstration of axoplasmic transport of adenosine transport sites. The accumulation of adenosine transport sites both proximal and distal to the ligatures indicate that these transport sites undergo anterograde axonal transport from the cell bodies towards the peripheral vagal synaptic terminals, as well as retrograde transport from these terminals back towards the perikarya. This is consistent with find-
ings indicating that opiate receptors in peripheral vagal terminals detach themselves from their synaptic terminals and are axonally transported back towards their cell bodies [14]. As the process of axoplasmic flow is clearly occurring on the peripheral side of the nodose ganglion, one would expect a similar pattern of transport towards central vagal terminals in the brain stem. Thus, adenosine transport sites synthesized in the cell bodies of the nodose ganglion could also flow towards central vagal nerve endings in the brain stem by axoplasmic transport mechanisms and then by the same process, be transported back towards their perikarya for reprocessing. The terminals of vagal afferent neurons have been located in the NTS, the area postrema and the dorsal motor nucleus of the vagus [1 1]; therefore, it is possible that adenosine transport sites present in these brain stem nuclei are, at least in part, located presynaptically on vagal afferent neurons. Although the rate of the axoplasmic transport was not determined, the accumulation of adenosine transport sites adjacent to the ligatures occurred only 24 h after unilateral vagal ligation. These findings suggest that the transport of these sites along the vagus nerve involves a rapid process. Previous findings have demonstrated that both anterograde and retrograde transport are fast processes, attaining velocities of 200 to 400 m m / d a y and 100 to 200 m m / d a y , respectively. The motor proteins involved in this process are likely to be kinesin and dynein which have both been associated with axonal transport mechanisms [7]. The present study has demonstrated the presence of a single class of saturable, high-affinity adenosine transport sites on human inferior vagal ganglia membrane prepara-
Fig. 3. (A) Histologicalphotomicrographof rat nodose ganglion with ligated vagal trunk, stained with 0.1% thionin. Arrowheads indicate the position of the ligatures on the distal vagal trunk. Scale bar = 178 ~m. (B) Autoradiogramdemonstrating total [3HINBMPR(0.7 nM) binding on rat nodose ganglia and vagus nerve in a sham-ligatedanimal. Scale bar = 550 p.m. (C) Total [3H]NBMPR(0.7 nM) binding on rat nodose ganglion with ligated vagal trunk. The arrows point to the position of the ligatures where accumulationof [3H]NBMPR binding can be observedboth proximaland distal to the ligation sites. Scale bar = 550/.tm. NG, nodose ganglion; V, vagus nerve.
M. Castillo-Mel~ndez et al. / Journal of the Autonomic Nervous System 57 (1996) 36-42
tions. In addition, in vitro autoradiogaphic studies have demonstrated that adenosine transport sites are associated with human vagal afferent neurons and that these transport sites undergo bidirectional axonal flow along the rat vagus nerve. These results provide further evidence implicating adenosine as a potential neuromodulator of vagal afferent neurons. The significance of the presence of such high densities of adenosine transport sites in the central terminals [2] and soma of vagal afferent neurons with respect to the putative neuromodulatory role of adenosine in baroreceptor reflex activity is yet to be elucidated. Although the present study has not distinguished between baroreceptor cell bodies and other vagal sensory perikarya, it is likely that at least a portion of adenosine transport sites in the inferior (nodose) vagal ganglion are associated with central projections of baroreceptor afferent neurons. This hypothesis is based on previous neuropharmacological and neurochemical studies indicating a potent effect of adenosine on barosensitive neurons in the NTS [1,4,26,30]. Furthermore, central vagal afferent terminals in the medial NTS (region containing predominantly baroreceptor terminals) are known to contain adenosine A 2a receptors [4]. Therefore, it is likely that endogenous adenosine is found in physiologically relevant concentrations within this region of the NTS and thus an efficient adenosine transport system would be expected to exist in the medial NTS. In accordance with this requirement, autoradiographic studies have documented that the NTS contains the highest density of central adenosine transport sites as labelled by [3H]NBMPR
[21. In conclusion, the present study has demonstrated the presence of high affinity adenosine transport sites associated with vagal afferent neurons of humans and rats. Furthermore, these transport sites are subject to bidirectional axoplasmic transport and thus their presence on vagal afferent terminals within the NTS would be expected.
Acknowledgements
This work was supported by a project grant from the National Health and Medical Research Council of Australia of which A.J.L. is a senior research officer. The authors would like to thank Dianne Watkins for her assistance.
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ELSEVIER
Autonomic Nervous System Journal of the Autonomic Nervous System 57 (1996) 36-42
Radioligand binding and autoradiographic visualization of adenosine transport sites in human inferior vagal ganglia and their axonal transport along rat vagal afferent neurons Margie Castillo-Mel6ndez *, Bevyn Jarrott, Andrew J. Lawrence Department of Pharmacology, Monash University, WellingtonRoad, Clayton, Victoria 3168, Australia Received 17 May 1995; revision received 2 September 1995; accepted 4 September 1995
Abstract
The present study has employed membrane-binding studies and in vitro autoradiography to demonstrate the presence of adenosine transport sites in human inferior vagal ganglia using [3H]nitrobenzylthioinosine ([3H]NBMPR), a potent inhibitor of adenosine transport. In addition, [3H]NBMPR was used to determine whether adenosine transport sites are subject to axonal transport along the rat vagus nerve. Binding of [3H]NBMPR to human inferior vagal ganglia membranes was saturable and reversible. Saturation experiments revealed a single class of high affinity-binding sites with a K d of 93.73 + 23.13 pM and Bm~, of 413.50+ 50.40 fmol/mg protein. In displacement experiments, the adenosine transport inhibitor dipyridamole was the most potent displacer of [3H]NBMPR binding (K i m 42.7 + 28.0 nM). Adenosine itself was able to fully displace [3H]NBMPR binding with a K i of 115.0 _ 34.0 /zM. The A l//A2a adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA) was able to fully displace [3H]NBMPR binding in only one experiment at a concentration of 100/zM, yielding an affinity 1000-fold higher than its affinity for adenosine receptors. All competition curves obtained from displacement experiments displayed monophasic profiles, indicating the presence of a single class of [3H]NBMPR binding sites. Incubation of human inferior vagal ganglia sections with [3H]NBMPR (0.7 nM) revealed dense binding which appeared to be consistent with the distribution of neuronal cell bodies in this tissue. Following unilateral ligation of the vagus nerve in the rat, acccumulation of [3H]NBMPR binding sites occurred both proximal and distal to the vagal ligatures. These results suggest that [3H]NBMPR binds with high affinity to a single class of adenosine transport sites, and that these sites are present on vagal afferent neurons in the human and undergo bidirectional axonal transport along the rat vagus nerve. Keywords: Adenosine; Transport site; Sensory ganglion; Axonal flow; Nitrobenzylthioinosine; Autoradiography
1. Introduction The purine nucleoside adenosine has been defined as an endogenous neuromodulatory substance in the central nervous system [8]. The physiological actions of adenosine are believed to be mediated primarily by interactions with extracellular adenosine receptors coupled to adenylate cyclase systems [21]. Adenosine is the endogenous ligand at Pl purinoeceptors termed A l, A 2 [31], A 3 [28] and A 4 [6]. The A 2 adenosine receptor has been further subdivided into m2a and A2b subtypes [10]. Recently, there has been a growing body of evidence, suggesting a possible role for
* Corresponding author. Tel.: (61-3) 9905-4855; Fax: (1-613) 99055851. 0165-1838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 01 65- 1 8 3 8 ( 9 5 ) 0 0 0 9 8 - 4
adenosine in the processing of baroreceptor information at the central terminals of baro-afferents within the nucleus tractus solitarius (NTS) in the dorsal medulla oblongata. Initial support came from studies which indicated that microinjection of adenosine antagonists into the NTS was capable of inhibiting baroreceptor reflex activity within this nucleus [25]. In addition, studies by Barraco and colleagues [1] demonstrated that intra-NTS microinjection of adenosine agonists led to different cardiovascular response pattems depending on the adenosine receptor subtype activated. Furthermore, microdialysis studies in the rabbit have shown that adenosine administration into the NTS leads to increases in local extracellular levels of glutamate [26], the favoured candidate neurotransmitter of primary baroreceptor afferent neurons [5,17,29]. More recently, microdialysis experiments in the rat have demon-
M. Castillo-MeMndez et al./ Journal of the Autonomic Nervous System 57 (1996) 36-42
strated that activation of adenosine A2a receptors in the NTS results in increased extracellular glutamate levels within this nucleus [4]. The rapid transport of adenosine from the extracellular fluid back into the cells and subsequent catabolism by adenosine kinase and deaminase are believed to be the most important mechanisms by which the physiological effects of this nucleoside are terminated [27]. The presence of a high density of adenosine transport sites in the NTS [2] and a high-affinity adenosine transport system in synaptosomes prepared from dorsal brain stem [16] have been demonstrated. The presence of adenosine transport sites in the inferior vagal (nodose) ganglion, where the cell bodies of aortic baro-afferent neurons are located [12], has not yet been reported. Therefore, the present study has employed radioligand-binding studies using membrane preparations of human inferior vagal ganglia to characterize the binding properties of the potent adenosine transport inhibitor [SH]nitrobenzylthioinosine ([3H]NBMPR) [2]. In addition, in vitro autoradiography studies were carried out using [3H]NBMPR to visualize adenosine transport sites on sections of human inferior vagal ganglia. Evidence has indicated that receptors for various neurotransmitters and neuromodulators are synthesized in vagal afferent cell bodies and then axonally transported along the nerve to the terminals [15]. Thus, unilateral vagal ligation in the rat has been employed to determine whether adenosine transport sites are subject to such axonal transport mechanisms along the vagus nerve.
2. Materials and methods
2.1. Materials [3H]NBMPR (19.5 Ci/mmol) was obtained from NEN Dupont, NBMPR was from Research Biochemicals Incorpotated (RBI), tritium sensitive film (Hyperfilm) and tritium microscales were from Amersham. Adenosine, dipyridamole and 5'-(N-ethylcarboxamido)-adenosine (NECA) were all from Sigma. EcoLite scintillant was obtained from ICN. All other reagents were either analytical or laboratory grade from various suppliers.
37
cleared of connective tissue and homogenized in 8 ml of 0.32 M sucrose using an Ultra-Turrax homogenizer. The homogenate was centrifuged at 1000 × g for 10 min. at 4°C and the resulting supematant was recentrifuged at 30000 × g for 30 min. Following centrifugation, the resulting pellet was resuspended in 25 vols. (w/v) of Tris. HC1 buffer (50 mM; pH 7.4) and centrifuged again at 30 000 × g for 20 min. The final pellet was resuspended in 50 vols. (w/v) of Tris. HC1 buffer. Protein concentration was determined as described by Lowry et al. [22] using bovine serum albumin as standard (0.25-4.0 mg/ml).
2.2.2. Binding assays All binding assays were carried out in duplicate, and were commenced by addition of 0.1 ml of membrane preparation, representing 36-56 /.tg of protein in a final volume of 0.5 ml. Saturation experiments were performed by incubating the membranes with nine different concentrations of [3H]NBMPR ranging from 0.025 to 6.4 nM (n = 3). Displacement experiments (n = 3 per displacer) consisted of incubation of the membranes with 0.3 nM [3H]NBMPR and eight different concentrations of either dipyridamole (1 nM-3 /xM), adenosine (3 /zM-10 mM) or NECA (30 nM-100 /zM). Non-specific binding was determined in the presence of 5 /xM unlabelled NBMPR. Incubations for both saturation and displacement assays were for 30 min at room temperature, and were terminated by rapid vacuum filtration through Whatman glass microfibre filters (GF/B) with 4 × 4 ml ice-cold Tris. HC1 (50 mM; pH 7.4) rinses using a Brandel cell harvester. Bound radioactivity was measured by scintillation counting of /3-emission (efficiency approx. 50%). 2.2.3. Analysis of [ZH]NBMPR binding The maximal number of [3H]NBMPR-binding sites (Bm~x) and apparent affinity of the ligand for the binding site (K d) were determined by Scatchard analysis from the saturation binding data. The affinities of the displacers for the [3H]NBMPR-binding sites (K i) were determined from the displacement curves obtained from the competition binding data. All parameters were obtained using the EBDA/LIGAND computer software [24]. 2.3. Preparation of the tissue slices
2.2. Membrane-binding studies 2.2.1. Preparation of the membranes Inferior vagal ganglia were obtained from cadavers at the Victorian Institute of Forensic Pathology. Donors were of either sex and had no previous history of neurological disease. The age range of the donors was between 25 and 70 years of age and in all cases the delay between death and autopsy was less than 48 h. Ethical permission for the study was granted by the Ethics and Integrity in Research Committees of both Monash University and the Victorian Institute of Forensic Pathology. The tissue samples were
2.3.1. Human studies Inferior vagal ganglia from human subjects were frozen over liquid nitrogen and sections were cryostat cut (10 /zm), thaw-mounted on to gelatin/chrome-alum-coated slides and stored at - 80°C until further processed. 2.3.2. Rat studies Male Wistar-Kyoto rats (280-350 g) were anaesthetized with sodium methohexitone (60 mg/kg, i.p.) and the left nodose ganglion and vagus nerve were exposed. Two tight ligations (5 mm apart) were applied to the nerve approx.
38
M. Castillo-Mel£ndez et al./ Journal of the Autoru~mic Nervous System 57 (1996) 36-42
10 mm peripheral to the nodose ganglion using 6 / 0 silk thread (n = 5). In a similar manner, corresponding sham ligations were performed on a separate group of rats by placing two loosely fitting ligatures around the vagus nerve (n = 4). After 24 h, rats were re-anaesthetized with sodium pentobarbitone (60 mg/kg, i.p.) and the ligated portion of the vagus nerve and nodose ganglion were excised and the ligatures removed. Tissue samples were then placed in embedding compound (OCT) and frozen over supercooled isopentane. Longitudinal 10-/~m sections were cut in a cryostat, thaw-mounted on to gelatin/chrome-alum coated microscope slides and stored at - 8 0 ° C until used.
2.4. In vitro autoradiography The autoradiographic experiments were carried out using a previously described protocol [2]. Briefly, tissue sections were wai'med to room temperature prior to incubation with 0.7 nM [3H]NBMPR (in 50 mM Tris-HCI buffer, pH 7.4) for 30 min. Non-specific binding was determined on adjacent sections by addition of an excess of NBMPR (20 /xM final concentration) to the incubation media. Following the incubation period, sections were washed (3 X 5 min) in ice-cold 50 mM Tris- HCI buffer (pH 7.4) and rinsed twice in distilled water. The slides were then dried under a stream of cool air and apposed to tritium sensitive film for 8 weeks, in the presence of tritium microscales.
2.5. Image analysis Densitometric quantification of the developed films was carried out using an MCID M4 image analysis system (Imaging Research). The binding over whole sections of human inferior vagal ganglia (n = 4; 4 sections/specimen), rat ligated vagus nerve with nodose ganglion (n = 5; 3-8 sections/animal) and sham ligated vagus nerve and nodose ganglion (n = 4; 5 - 8 sections/animal) was quantified by comparing the optical density of the autoradiograms to the standard tritium microscales. Sections of both human inferior vagal ganglia and rat vagus nerve and nodose ganglia were stained with 0.1% thionin and coverslipped for histological assessment, using an Olympus BH-2 photomicroscope.
3. Results
3.1. Human studies 3.1.1. Membrane-binding studies
Using the current protocol, [3H]NBMPR bound to human inferior vagal ganglia membranes in a saturable manner. Scatchard analysis of saturation experiments yielded a Bma x of 413.50 ___50.40 fmol/mg protein and a K d of
120-
100:
8060-
,~ O ~9
40200 -9
-
-7
-
-5
-4
-3
-2
Displacer (log M) Fig. I. Displacement of [3H]NBMPR (0.3 nM) binding to membranes of human inferior vagal ganglia by dipyridamole ( • ) , adenosine ( 0 ) and NECA ([2). Experiments were conducted in duplicate using and eight concentrations of each displacer. The data illustrated are mean + SEM values from 3 separate experiments per displacer for dipyridamole and adenosine. The competition curve for NECA is from an individual experiment. In two other cases, NECA was unable to fully inhibit the binding of [3H]NBMPR.
93.73 + 23.13 pM. Within the [3H]NBMPR concentration range studied, the data best described a one-site interaction as indicated by the obtained Hill coefficient of 1.00 + 0.05 (n = 3). In displacement binding assays, the specific binding of [3H]NBMPR was found to represent 93.25 _+ 0.49% of the total binding (n = 9). The results from displacement studies for adenosine, dipyridamole and NECA are illustrated in Fig. I. As can be seen, both adenosine and the adenosine transport inhibitor dipyridamole were able to fully displace [3H]NBMPR binding with K i values of 115.00 + 34.00 /xM and 42.70 _+ 28.00 nM, respectively. Despite the apparently low Hill slopes of 0.66 _+ 0.06 for adenosine and 0.85 _+ 0.12 for dipyridamole, ligand analysis of the data indicated a single site fit for both competitors. The non-selective adenosine receptor agonist NECA was unable to fully displace [3H]NBMPR binding in two experiments, however, in a third experiment, NECA inhibited the binding by 90% at a concentration of 100 /xM.
3.1.2. Autoradiography studies Multiple slide mounted sections of human inferior vagal ganglia obtained from four subjects were analysed, two from the right and two from the left vagus. [3H]NBMPR (0.7 nM) extensively bound to human inferior vagal ganglia sections (Fig. 2A). Microscopic examination revealed that the distribution pattern of [3H]NBMPR binding was consistent with the localization of neuronal cell bodies in this tissue, as seen in an adjacent thionin-stained section (Fig. 2C). Densitometric quantitation of, the autoradiograms indicated that total binding of [3H]NBMPR represented 64.3 + 2.0 d p m / m m 2 (n = 16 sections). A preliminary experiment where inferior vagal ganglia sections were wiped off the slides and counted in a r-emission scintilla-
M. Castillo-Mel~ndez et al. / Journal of the Autonomic Nervous System 57 (1996) 36-42
tion counter revealed that, in the presence of NBMPR (20 /xM) specific binding of [3H]NBMPR represented 95% of the total binding. Indeed, sections representing non-specific binding did not produce a strong enough signal on film for quantification (Fig. 2B). 3.2. Rat studies
Fig. 3A represents a photomicrograph of a thioninstained section of rat nodose ganglion with the vagal trunk, demonstrating the effect of surgical ligation of the vagus nerve. In the sham-ligated vagus nerve of the rat, [3H]NBMPR binding was almost completely associated
39
with the nodose ganglion (Fig. 3B). In the ligated vagus nerve, [3H]NBMPR binding occurred not only on the nodose ganglion but also was present in the vagal trunk, both proximal and distal to the ligatures (Fig. 3C). Image analysis of the autoradiograms indicated a binding density of 17.3 +__0.8 and 18.0 _ 1.1 d p m / m m 2 for [3H]NBMPR on whole sections of sham and ligated nodose-vagal sampies, respectively. Binding of [3H]NBMPR on both sham and ligated vagus nerve sections was completely abolished in the presence of NBMPR (20 /zM) and the images obtained were too faint to be quantified.
4. Discussion
Fig. 2. Photomicrograph of human inferior vagal ganglia sections. (.~) Total [3H]NBMPR (0.7 nM) binding on a section of inferior vagai ganglia. Well defined, punctate binding can be clearly seen. Scale bar = 1.5 mm. (B) A section of human inferior vagal ganglia showing the absence of binding in the presence of 20 p,M unlabelled NBMPR. Scale bar = 1.5 rnm. (C) Light-field photomicrograph of an adjacent tissue section to A, stained with 0.1% thionin, demonstrating alternate bands of neuronal cell bodies and nerve fibres. Scale bar = 1.2 mm.
The present investigation has characterized the binding properties of the widely used adenosine transport inhibitor [3H]NBMPR in human inferior vagal ganglia membrane preparations. The binding of [3H]NBMPR to these membranes was saturable, reversible and specific, and of high affinity to a single population of binding sites. Displacement experiments revealed that out of the compounds tested, dipyridamole was the most potent inhibitor of [3H]NBMPR binding. Adenosine was also able to fully inhibit [3H]NBMPR binding, whereas the A I / A E a adenosine receptor agonist NECA produced 0, 50 and 90% inhibition of binding at a concentration of 100 /xM in the three experiments carried out. In the assay where almost total inhibition was achieved, NECA was found to display a micromolar potency. The affinity of this agonist for A J A 2 a adenosine receptors has been reported to be less than 10 nM [3], suggesting that the sites being labelled by [3H]NBMPR are not likely to represent an adenosine receptor. The ability of NECA to fully displace [3H]N-BMPR binding could be attributed to NECA being a close structural analogue of adenosine and therefore, at high concentrations, NECA may display low affinity for the adenosine transport site. The present study has also visualised the presence of a high density of [3H]NBMPR binding sites on sections of inferior vagal ganglia of humans using in vitro autoradiography. Furthermore, the distribution of binding sites was punctate, paralleling the cytoarchitecture of the ganglia, indicating the possibility that human vagal afferent neurons possess adenosine transport sites. The distribution of [3H]NBMPR binding is similar to the recent visualization of dopamine D 2 binding sites on this ganglion, where binding appeared to be restricted to neuronal cell bodies [18]. This observed pattern of [3H]NBMPR binding to sections of inferior vagal ganglia indicates the likelihood of a proportion of these binding sites being associated with vagal perikarya. An absolute confirmation at the light microscopic level is not possible due to the use of low specific activity tritium as radioligand rendering the use of nuclear emulsion unfeasible.
40
M. Castillo-Meldndez et al./ Journal of the Autoru)mic Nervous System 57 (1996) 36-42
In addition, the study has demonstrated for the first time the presence of a high density of binding sites for [3H]NBMPR over sections of rat nodose ganglia. Furthermore, the accumulation of [3H]NBMPR binding in the rat vagus nerve following vagal ligation demonstrates that adenosine transport sites are subject to axonal transport along the vagus nerve in this species, and thus a similar process would be expected in humans, The ligated rat vagus nerve has been established as a suitable model for the demonstration of axonal transport of neuroreceptors and transmitters [15]. Accumulation of [3H]NBMPR binding in tigated vagus nerve sections was observed on both the proximal and distal sides of the vagal ligatures suggesting bidirectional transport of adenosine transport sites. Axonal transport along the rat vagus nerve has been previously demonstrated using this surgical procedure for opioid receptors [32], muscarinic receptors [13], glutamate receptors [20], vasopressin V 1 receptors [9], cholecystokinin receptors [23] and /3-adrenoceptors [19]. In addition, the existence of orthograde axoplasmic transport has been demonstrated for various transmitters, enzymes, labelled proteins and peptides along the axons of different nerve fibres [14]. However, the present study represents the first demonstration of axoplasmic transport of adenosine transport sites. The accumulation of adenosine transport sites both proximal and distal to the ligatures indicate that these transport sites undergo anterograde axonal transport from the cell bodies towards the peripheral vagal synaptic terminals, as well as retrograde transport from these terminals back towards the perikarya. This is consistent with find-
ings indicating that opiate receptors in peripheral vagal terminals detach themselves from their synaptic terminals and are axonally transported back towards their cell bodies [14]. As the process of axoplasmic flow is clearly occurring on the peripheral side of the nodose ganglion, one would expect a similar pattern of transport towards central vagal terminals in the brain stem. Thus, adenosine transport sites synthesized in the cell bodies of the nodose ganglion could also flow towards central vagal nerve endings in the brain stem by axoplasmic transport mechanisms and then by the same process, be transported back towards their perikarya for reprocessing. The terminals of vagal afferent neurons have been located in the NTS, the area postrema and the dorsal motor nucleus of the vagus [1 1]; therefore, it is possible that adenosine transport sites present in these brain stem nuclei are, at least in part, located presynaptically on vagal afferent neurons. Although the rate of the axoplasmic transport was not determined, the accumulation of adenosine transport sites adjacent to the ligatures occurred only 24 h after unilateral vagal ligation. These findings suggest that the transport of these sites along the vagus nerve involves a rapid process. Previous findings have demonstrated that both anterograde and retrograde transport are fast processes, attaining velocities of 200 to 400 m m / d a y and 100 to 200 m m / d a y , respectively. The motor proteins involved in this process are likely to be kinesin and dynein which have both been associated with axonal transport mechanisms [7]. The present study has demonstrated the presence of a single class of saturable, high-affinity adenosine transport sites on human inferior vagal ganglia membrane prepara-
Fig. 3. (A) Histologicalphotomicrographof rat nodose ganglion with ligated vagal trunk, stained with 0.1% thionin. Arrowheads indicate the position of the ligatures on the distal vagal trunk. Scale bar = 178 ~m. (B) Autoradiogramdemonstrating total [3HINBMPR(0.7 nM) binding on rat nodose ganglia and vagus nerve in a sham-ligatedanimal. Scale bar = 550 p.m. (C) Total [3H]NBMPR(0.7 nM) binding on rat nodose ganglion with ligated vagal trunk. The arrows point to the position of the ligatures where accumulationof [3H]NBMPR binding can be observedboth proximaland distal to the ligation sites. Scale bar = 550/.tm. NG, nodose ganglion; V, vagus nerve.
M. Castillo-Mel~ndez et al. / Journal of the Autonomic Nervous System 57 (1996) 36-42
tions. In addition, in vitro autoradiogaphic studies have demonstrated that adenosine transport sites are associated with human vagal afferent neurons and that these transport sites undergo bidirectional axonal flow along the rat vagus nerve. These results provide further evidence implicating adenosine as a potential neuromodulator of vagal afferent neurons. The significance of the presence of such high densities of adenosine transport sites in the central terminals [2] and soma of vagal afferent neurons with respect to the putative neuromodulatory role of adenosine in baroreceptor reflex activity is yet to be elucidated. Although the present study has not distinguished between baroreceptor cell bodies and other vagal sensory perikarya, it is likely that at least a portion of adenosine transport sites in the inferior (nodose) vagal ganglion are associated with central projections of baroreceptor afferent neurons. This hypothesis is based on previous neuropharmacological and neurochemical studies indicating a potent effect of adenosine on barosensitive neurons in the NTS [1,4,26,30]. Furthermore, central vagal afferent terminals in the medial NTS (region containing predominantly baroreceptor terminals) are known to contain adenosine A 2a receptors [4]. Therefore, it is likely that endogenous adenosine is found in physiologically relevant concentrations within this region of the NTS and thus an efficient adenosine transport system would be expected to exist in the medial NTS. In accordance with this requirement, autoradiographic studies have documented that the NTS contains the highest density of central adenosine transport sites as labelled by [3H]NBMPR
[21. In conclusion, the present study has demonstrated the presence of high affinity adenosine transport sites associated with vagal afferent neurons of humans and rats. Furthermore, these transport sites are subject to bidirectional axoplasmic transport and thus their presence on vagal afferent terminals within the NTS would be expected.
Acknowledgements
This work was supported by a project grant from the National Health and Medical Research Council of Australia of which A.J.L. is a senior research officer. The authors would like to thank Dianne Watkins for her assistance.
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