(Hypertension. 1996;28:1026-1033.)
© 1996 American Heart Association, Inc.
Articles |
Markers of Adenosine Removal in Normotensive and Hypertensive Rat Nervous Tissue
the Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Correspondence to Margie Castillo-Melendez, Department of Pharmacology, Monash University, Clayton, Victoria 3168, Australia. E-mail casmel@monash.med.edu.au.
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Abstract |
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Adenosine mechanisms are altered in brain stem nuclei associated with cardiovascular control in spontaneously hypertensive rats (SHR). Therefore, in the present study we used a number of techniques to compare the binding of the adenosine transport inhibitor [3H]nitrobenzylthioinosine ([3H]NBMPR) as well as adenosine deaminase immunoreactivity (ADA-IR) in brain stems and nodose ganglia of SHR and age-matched normotensive Donryu rats (DRY). Saturation binding revealed a single class of [3H]NBMPR binding sites in the dorsal brain stem of both strains, with Kd and Bmax values of 65±9 pmol/L and 282±31 fmol/mg protein, respectively, in SHR and 129±2 pmol/L and 217±23 fmol/mg protein in DRY. The Kd for [3H]NBMPR was significantly lower in SHR than in DRY. In competition assays, NBMPR, dilazep, dipyridamole, and adenosine displaced [3H]NBMPR binding, with Kd values of 0.21±0.04, 57.16±16.20, 1340±100, and 87 000±12 500 nmol/L, respectively, in DRY and 0.17±0.04, 28.24±3.60, 621±100, and 32 000±6820 in SHR. Kd values for all displacers were lower in SHR; however, only values for dipyridamole and adenosine reached statistical significance. Autoradiography of adenosine transport sites with [3H]NBMPR revealed that unilateral nodose ganglionectomy reduced [3H]NBMPR binding on the denervated side of the nucleus tractus solitarius by 20.6±1.1% in DRY and 18.7±2.3% in SHR. The density of [3H]NBMPR binding in nodose ganglia was significantly lower in SHR (0.99±0.06 Bq/mm2) than in DRY (1.25±0.08). Immunohistochemical studies demonstrated ADA-IR in the dorsal vagal complex, associated with both nerve cells and fibers. Measurement of ADA-IR in the dorsal vagal complex with an 125I-labeled secondary antibody revealed a significantly higher level of ADA-IR in SHR (122%) than in DRY. In the nodose ganglia, ADA-IR was associated with a population of vagal perikarya. The present study helps provide a molecular explanation for the previously reported impaired cardiovascular responses to intra–nucleus tractus solitarius microinjection of adenosine in hypertensive rats.
Key Words: adenosine • thioinosine • autoradiography • immunohistochemistry • adenosine deaminase • rats, inbred SHR • solitary nucleus
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Introduction |
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The NTS, located in the medulla oblongata, is the major relay nucleus for the central processing of cardiovascular control.1 Both anatomic2 3 4 5 6 and functional6 7 8 9 studies have demonstrated that in the central nervous system, the site of first termination of baroreceptor afferent fibers is in the NTS.
Growing evidence implicates the purine nucleoside adenosine in the processing of baroreceptor information within this brain stem nucleus. The potential of adenosine to act as a neuromodulatory substance at barosensitive neurons was suggested by findings that microinjection of adenosine receptor antagonists into the NTS inhibited the baroreceptor heart rate reflex in normotensive rats.10 In addition, intra-NTS administration of adenosine analogues has been shown to result in either a pressor response mediated via the activation of adenosine A1 receptors or a depressor response mediated by A2a receptors.11 Furthermore, the depressor action of adenosine in the rat NTS can be selectively blocked by antagonists of both adenosine and glutamate receptors, and in the rabbit, microdialysis studies have shown that intra-NTS administration of adenosine leads to increased local extracellular levels of glutamate,12 the proposed neurotransmitter at baroreceptor afferent terminals.13 14 In support of functional interactions between adenosine and glutamate in the NTS, studies by Tao and Abdel-Rahman15 have demonstrated that both cardiovascular and neuronal responses to microinjection of adenosine in the caudal NTS of the rat mimic the effects of intra-NTS glutamate, and more recently, experiments in our laboratory have shown increased neuronal glutamate release in the rat NTS following activation of A2a receptors in this nucleus.16
As for classic neurotransmitters and neuromodulators, inactivation of extracellular adenosine occurs largely by uptake mechanisms consisting of rapid transport across the cell membrane followed by deamination to inosine by ADA or phosphorylation to 5'-AMP by adenosine kinase.17 Thus, putative ligands for adenosine transporters such as [3H]NBMPR18 19 20 21 and [3H]dipyridamole22 as well as ADA23 24 25 have been used as markers for identification of purinergic neural systems. Autoradiographic studies with [3H]NBMPR have demonstrated that the highest density of adenosine transport sites in the rat brain is in the NTS and that such a high density of these sites suggests the involvement of adenosine in the central regulation of cardiovascular function.18 In addition, the presence of a high-affinity transport system for adenosine in rat dorsal brain stem synaptosomes has been reported.26 With the use of immunohistological techniques, the presence of ADA in restricted regions of the rat brain has been investigated,25 27 and previous studies of ADA immunoreactivity in dorsal root ganglion neurons have suggested ADA to be a marker for the utilization of purines by primary afferent neurons.24
Alterations in purinergic mechanisms in brain stem nuclei associated with cardiovascular control in the SHR have been reported. The affinity of central A1 receptors has been shown to be decreased in the SHR model.28 Furthermore, a recent microinjection study demonstrated attenuated cardiovascular responses to intra-NTS administration of adenosine in SHR compared with normotensive rats.29 In view of these findings, it is possible that mechanisms involved in the uptake (transport and metabolism) of adenosine in the NTS may be altered in hypertension; therefore, in the present study, we compared the binding characteristics of the adenosine transport inhibitor [3H]NBMPR and ADA-IR in brain stems and nodose ganglia of SHR and age-matched normotensive DRY.
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All experiments were carried out in accordance with the Prevention of Cruelty to Animals Act 1986 under the guidelines of the Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia.
Before experiments were begun, systolic pressure readings were obtained in conscious SHR and DRY by the tail-cuff method. Blood pressures ranged between 70 and 120 mm Hg in DRY and 140 and 200 in SHR. All rats used in the present study were 16- to 18-week-old males.
Materials
[3H]NBMPR (19.5 Ci/mmol) was obtained from NEN-DuPont; S-(4-nitrobenzyl)-6-thioinosine was from Research Biochemicals Inc; dilazep, dipyridamole, and adenosine were from Sigma Chemical Co; tritium-sensitive film (Hyperfilm) and tritium microscales were from Amersham; and D-19 developer was from Kodak. Diaminobenzidine, stable peroxidase buffer, and immunoperoxidase suppressor were from Pierce Chemicals; NGS was from Sigma Immunochemicals; and streptavidin-conjugated horseradish peroxidase and biotinylated secondary anti-rabbit IgG were from Silenius Australia. The rabbit anti-ADA serum was a gift from Prof J.D. Geiger. The goat anti-rabbit 125I-IgG was from NEN-DuPont; x-ray film was from Kodak; and 14C microscales were from American Radiolabeled Chemicals Inc. All other reagents were either analytical or laboratory grade from various suppliers.
Membrane Binding Studies
Male SHR and DRY weighing between 290 and 310 g were killed by decapitation. Their brain stems were removed, cut 3 mm on either side of the obex, and hemisected; the dorsal portion was homogenized in 3 mL of 0.32 mol/L sucrose with a polytetrafluoroethylene-glass homogenizer. The homogenate was centrifuged at 1000g for 10 minutes at 4°C, and the resulting supernatant was centrifuged at 30 000g for 30 minutes. After centrifugation, the resulting pellet was resuspended in 25 vol (wt/vol) of 50 mmol/L Tris-HCl buffer (pH 7.4) and centrifuged at 30 000g for 20 minutes. The final pellet was resuspended in 50 vol (wt/vol) Tris-HCl buffer. Protein concentration was determined as described by Lowry and colleagues30 with the use of bovine serum albumin standards (0.25 to 4.00 mg/mL). All binding assays were carried out in duplicate and were begun by addition of 0.1 mL crude membrane preparation representing 83±4 µg membrane protein in a final volume of 0.5 mL. Saturation experiments were performed by incubation of membranes with nine different concentrations of [3H]NBMPR ranging from 0.0125 to 3.2 nmol/L. Displacement experiments consisted of incubation of membranes with 0.3 nmol/L [3H]NBMPR and different concentrations of either unlabeled NBMPR (0.03 to 100 nmol/L), dipyridamole (1 nmol/L to 30 µmol/L), adenosine (1 µmol/L to 10 mmol/L), or dilazep (2 nmol/L to 3 µmol/L). Nonspecific binding was determined in the presence of 5 µmol/L unlabeled NBMPR. Incubations for both saturation and displacement assays were for 30 minutes at room temperature and were terminated by rapid vacuum filtration through glass microfiber filters (Whatman GF/B) followed by washing four times with 4 mL ice-cold Tris-HCl buffer (50 mmol/L) through a Brandel cell harvester (Biomedical Research & Development Laboratories Inc). Bound radioactivity was measured by scintillation counting of ß-emission (efficiency, ~45%).
The maximal number of [3H]NBMPR binding sites (Bmax) and apparent affinity of the binding (Kd) were determined by Scatchard analysis of the saturation binding data. The affinities of the displacers for the [3H]NBMPR binding sites (Kd values) were determined from the displacement curves obtained from the competition binding data. All parameters were obtained with the iterative computer program RADLIG 40 (an updated version of EBDA/LIGAND).31 Statistical evaluation of the kinetic parameters was performed with unpaired t tests.
In Vitro Autoradiography
Male DRY and SHR weighing between 260 and 305 g underwent left nodose ganglionectomy. In brief, rats were anesthetized with sodium methohexital (60 mg/kg IP) and placed on their backs. A midline incision was made in the neck, and the left nodose ganglion was exposed and excised (n=4 per strain). The operation involved the removal of a section containing the left nodose ganglion as well as trunks of the vagus, superior laryngeal, and inferior pharyngeal nerves at the respective points of connection with the ganglion. Corresponding sham control operations were performed on both DRY (n=5) and SHR (n=3) in which the left nodose ganglion was exposed but not excised. After a 2-week recovery period, the rats were reanesthetized with sodium pentobarbital (60 mg/kg IP), and the remaining nodose ganglia were removed. The rats were then killed by decapitation and their brain stems removed. The tissue samples collected from these rats were immediately frozen over liquid nitrogen and stored at -80°C until further processing. Systolic arterial pressure readings for both SHR and DRY were obtained before surgery and after the recovery period.
The autoradiographic procedure involved the incubation of slide-mounted tissue sections with [3H]NBMPR (721.5 GBq/mmol) and was carried out according to a published protocol.18 Briefly, cryostat-cut 10-µm sections of brain stem (bregma -14.3 mm to bregma -12.8 mm)32 and nodose ganglia were thaw-mounted onto gelatin/chrome alum–coated microscope slides and stored at -80°C. Tissue sections were allowed to warm to room temperature and were then incubated with [3H]NBMPR (0.7 nmol/L) in 50 mmol/L Tris-HCl (pH 7.4) for 30 minutes. Nonspecific binding was determined by addition of 20 µmol/L NBMPR to the incubation medium. After the 30-minute incubation period, the slides were placed in racks and washed in ice-cold Tris-HCl (3x5 minutes), rinsed twice in distilled water, and dried under a stream of cold air. The slides were then apposed to tritium-sensitive film in light-proof cassettes in the presence of tritium microscales. After 8 weeks, the films were developed and quantified with an MCID M4 image-analysis system (Imaging Research Inc) by comparing the optical density of the autoradiograms with that of the microscale standards. Statistical analysis was carried out by comparing the denervated (left) with the intact (right) side of the NTS with the use of a paired Student's t test. Unpaired t tests were used for comparison of the density of [3H]NBMPR binding on brain stem and nodose ganglia sections between strains.
Immunohistochemistry
Male SHR and DRY (270 to 300 g) were anesthetized with sodium pentobarbital (60 mg/kg IP) and transcardially perfused with 100 mL of 0.05 mol/L PBS (pH 7.4) followed by 500 mL of Zamboni's fixative (4% paraformaldehyde, 0.2% picric acid in 0.16 mol/L PBS; pH 6.9). The brain stem and nodose ganglia were removed, post-fixed overnight in Zamboni's solution, and cryoprotected with sucrose solution (10% sucrose in 0.1 mol/L phosphate, pH 7.4) for 24 hours at 4°C. The brain stems and nodose ganglia collected were then frozen over liquid nitrogen, stored at -80°C, and subsequently cryostat cut at 20 and 12 µm, respectively, and mounted onto gelatin/chrome alum–coated microscope slides. The slides were incubated with a primary antibody to ADA (1/500)33 containing 0.3% Triton X-100 and 1% NGS for 48 hours at 4°C. Then the slides were washed in PBS (3x15 minutes, pH 7.4) and incubated with 0.5% immunopure peroxidase suppressor in PBS (15 minutes) followed by three 15-minute washes in PBS (pH 7.4). After this, slides were incubated with a biotinylated secondary antibody (1/200) in PBS containing 1% NGS and 0.3% Triton X-100 for 3 hours at room temperature and washed again in PBS (3x15 minutes). Next, the slides were incubated for 1 hour at room temperature with streptavidin-conjugated horseradish peroxidase (1/100) in PBS containing 1% NGS and 0.3% Triton X-100 and were washed (3x15 minutes) in Tris-HCl buffer (50 mmol/L, pH 7.4). Finally, the slides were incubated with a metal-enhanced diaminobenzidine kit (Pierce) for 20 minutes at room temperature and dehydrated in serial alcohol dilutions (75%, 95%, and 100%), cleared, and coverslipped. A separate group of slides was incubated with an iodinated secondary antibody for visualization of ADA-IR by autoradiography. After incubation with the primary antibody to ADA, the slides were washed (3x5 minutes) in PBS (pH 7.4) in the presence of 0.1% NGS. The slides were then incubated with 125I-IgG (1/750) in PBS containing 0.1% NGS, followed by four 10-minute washes in PBS (pH 7.4) and two 30-second washes in distilled water. The slides were dried under a cold stream of air and apposed to x-ray film for 2 days in a light-proof cassette in the presence of 14C microscales. The autoradiograms were quantified with an MCID M4 image analyzer. An unpaired t test was used for determination of statistical significance in ADA-IR between SHR and DRY.
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Membrane Binding
All binding constants in the present study were obtained by data analysis with RADLIG 40. This program uses a multi-iterative process to account for the phenomenon of ligand depletion. For the same reason, all affinity constants for the competing ligands are expressed as Kd rather than Ki values.
Specific binding of [3H]NBMPR to dorsal brain stem membrane preparations was saturable and found to routinely represent between 90% and 95% of the total [3H]NBMPR binding in both SHR and DRY. Scatchard analysis of the saturation binding data revealed monophasic profiles in both strains, indicating the presence of a single population of [3H]NBMPR binding sites in the dorsal brain stem. Statistical evaluation of the data revealed a significant difference in the apparent affinity of [3H]NBMPR binding, where the Kd value was significantly lower in SHR than in DRY (unpaired t test, P<.01), but there was no difference in the [3H]NBMPR Bmax values between strains (Fig 1
). A correlation coefficient of -.2 was obtained for both SHR and DRY when blood pressure readings were correlated to Kd values (data not shown).
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), nonspecific (
), and specific (
) binding; mean±SEM of five separate experiments per rat strain. Data were plotted with the nonlinear regression computer program GraphPad PRISM. C and D, Corresponding Scatchard transformations of A and B, respectively. Experiments were conducted in duplicate with nine concentrations of [3H]NBMPR. Kd and Bmax values obtained from data analyses with the RADLIG 40 computer software were 65±9 pmol/L and 282±31 fmol/mg protein, respectively, in SHR (n=5) and 129±2 pmol/L and 217±23 fmol/mg protein in DRY (n=5). A significant difference in Kd values was found between strains (unpaired t test, P<.05).
In competition experiments, [3H]NBMPR binding to dorsal brain stem membranes was fully displaced by adenosine and adenosine transport inhibitors such as NBMPR, dilazep, and dipyridamole in both SHR and DRY (Fig 2). All of the inhibition constants (Kd) obtained in the SHR were found to be lower than Kd values obtained for the DRY; however, only dipyridamole and adenosine reached statistical significance, indicating an increased affinity of these ligands for [3H]NBMPR binding sites in the hypertensive group (Table 1). Although the mean Kd for NBMPR in competition experiments in SHR appears higher than that obtained for [3H]NBMPR in saturation assays, the ranges of values from individual experiments overlap (83 to 300 pmol/L in competition experiments and 42 to 88 in saturation experiments). Computer analysis of the data revealed monophasic competition curves for all of the displacers, suggesting a homogeneity of transport sites labeled by [3H]NBMPR (Fig 2). The Hill coefficient (nH) values obtained for dipyridamole in DRY and for NBMPR in SHR were found to be significantly different from 1 (unpaired t test, P<.05; Table 1).
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), dilazep (
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In Vitro Autoradiography
Autoradiographic studies on brain stem sections revealed a high density of [3H]NBMPR binding in the NTS and inferior olive in both SHR and DRY (Fig 3). Quantification of the autoradiograms revealed that in the ganglionectomized group, the density of [3H]NBMPR binding was significantly reduced on the denervated side of the NTS (left) by 20.6±1.1% in DRY (n=22 sections, P<.05) and 18.7±2.3% in SHR (n=30 sections, P<.05; Fig 3B and 3F). No significant differences were found in the density of [3H]NBMPR binding in the NTS between strains (1.65±0.07 Bq/mm2 for DRY and 1.72±0.08 for SHR); however, in the nodose ganglia, the density of [3H]NBMPR binding was found to be significantly lower (unpaired t test, P<.05; Fig 3D and 3H) in SHR (0.99±0.06 Bq/mm2, n=42 sections) than in DRY (1.25±0.08, n=41 sections). The autoradiograms obtained for nonspecific binding for both brain stem and nodose ganglia were too faint to be quantified (Fig 3C and 3G).
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Immunohistochemistry
The distribution of ADA in the brain stem and nodose ganglia of DRY and SHR was demonstrated by immunoreactivity. ADA-IR was concentrated in the dorsal vagal complex comprising the area postrema, the NTS, and the dorsal motor nucleus of the vagus (DMX, Fig 4A and 4D). Both nerve cells and nerve fibers in these brain stem nuclei were found to be immunopositive for ADA in both strains; however, in the DMX and the more rostral parts of the NTS, ADA-IR was predominantly associated with nerve fibers (Fig 4B and 4E). In the nodose ganglia, ADA-IR was found in a subpopulation of cell bodies in both strains (Fig 4C and 4F). Control sections, in which the slides were incubated in the absence of the primary antibody, showed no staining. Quantification of the autoradiograms revealed 125I–ADA-IR densities of 0.84±0.01 and 0.69±0.01 Bq/mm2 in the dorsal vagal complex of SHR and DRY, respectively; in the nodose ganglia, 125I–ADA-IR densities were 0.45±0.09 Bq/mm2 for SHR and 0.37±0.04 for DRY. Statistical evaluation revealed that the density of 125I–ADA-IR was significantly higher in the dorsal vagal complex of SHR (122%) than in that of DRY (unpaired t test, P<.05) but was not significantly different in the nodose ganglia between strains.
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Discussion |
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The SHR has been a useful model for the study of human hypertension34 ; for comparison with the SHR, we have used the genetically related DRY inbred strain35 36 as normotensive controls. Considering recent findings that demonstrate attenuated cardiovascular responses to adenosine in the NTS of SHR,29 we used a number of techniques in the present study to determine whether adenosine uptake mechanisms (transport and metabolism) in the NTS are altered in the hypertensive state.
[3H]NBMPR has been shown to bind to a nucleoside transporter in both nonneuronal20 and neuronal21 37 tissues. [3H]NBMPR binding to dorsal brain stem membrane preparations from both SHR and DRY was saturable, specific, and of high affinity, indicating that [3H]NBMPR was labeling a site that appears to be a nucleoside transporter. The Kd value for [3H]NBMPR obtained in SHR was found to be significantly lower than that of the DRY group, whereas no significant changes were observed in Bmax values between strains. For rat brain, Kd values ranging from 0.0538 to 0.1821 nmol/L have been reported with [3H]NBMPR. The Kd values obtained for both DRY (0.125 nmol/L) and SHR (0.065 nmol/L) clearly fall within this range; however, the Kd value obtained in SHR is at the lower end of this range, suggesting a high affinity of [3H]NBMPR for adenosine transport sites in the dorsal brain stem of SHR. On the other hand, Bmax values have been previously shown to range between 11339 and 27840 fmol/mg protein. The values obtained in our experiments were 217 and 282 fmol/mg protein for DRY and SHR, respectively; such a high number of [3H]NBMPR binding sites in the dorsal brain stem are consistent with autoradiographic studies that have demonstrated that the highest density of adenosine transport sites labeled with [3H]NBMPR is in the NTS.18 These results suggest that the apparent affinity of [3H]NBMPR for adenosine transport sites is increased in SHR compared with DRY, although the number of these sites is not altered; however, the possible presence of an NBMPR-insensitive transporter should be considered. At least six different nucleoside transporters have been identified on the basis of Na+ dependency, substrate selectivity, and sensitivity to inhibition by NBMPR.38 Of the two recognized Na+-independent transporters, termed es and ei, only the es type is sensitive to inhibition by NBMPR. Furthermore, only one of the four Na+-dependent nucleoside transporters is NBMPR sensitive (cs),41 whereas the three remaining transporters, termed N1 (cif), N2 (cit), and N3 (cib), based on substrate selectivity are all insensitive to inhibition by NBMPR.38 In the dorsal brain stem, however, we have previously shown that 90% of the adenosine transport system is Na+ dependent and that only 25% of the total transport capacity could be inhibited by NBMPR.26 Together, these findings would indicate that only a proportion of adenosine transport sites are labeled with [3H]NBMPR; nevertheless, this ligand remains the best available tool for labeling of adenosine transport sites in rat brain.
In competition experiments, [3H]NBMPR binding was fully displaced by the adenosine transport inhibitors NBMPR, dilazep, and dipyridamole as well as by adenosine in both SHR and DRY. All of the Kd values obtained for the inhibitors in the SHR were found to be lower than those of the DRY, suggesting an increased sensitivity for the displacers, including adenosine itself, in the hypertensive state. This finding may reflect either a conformational change of the transporter binding site or the absence of an endogenous inhibitory factor in SHR. In both strains, displacers yielded monophasic displacement curves, indicating a single class of binding site. However, NBMPR in SHR and dipyridamole in DRY had nH values significantly different from 1, suggesting [3H]NBMPR binding site heterogeneity. Alternatively, shallow Hill slopes may be explained by nonspecific binding of the displacers.
The surgical procedure of unilateral nodose ganglionectomy has been used extensively for determination of the location of receptor sites on vagal afferent neurons.16 42 43 44 In the present study, autoradiography with [3H]NBMPR revealed that in brain stem sections obtained from both SHR and DRY, the binding density of [3H]NBMPR was reduced by 18.7% and 20.6%, respectively, on the denervated side of the NTS, suggesting that a proportion of these sites are located presynaptically on vagal afferent neurons. In agreement with this, previous studies in our laboratory have characterized bidirectional axonal transport of [3H]NBMPR binding sites along the rat vagus nerve.45 However, the physiological relevance of this observation requires further investigation. [3H]NBMPR binding was also demonstrated on nodose ganglia sections from both rat strains, where the density of [3H]NBMPR binding sites was lower in SHR than in DRY. It is possible that the concentration of [3H]NBMPR (0.7 nmol/L) used in these experiments may not completely saturate the binding sites, therefore resulting in the observed differences. The presence of such a high density of [3H]NBMPR binding sites in the soma and central terminals of vagal afferents further suggests a neuromodulatory role for adenosine in the NTS.
ADA-IR has been previously reported in cell bodies of dorsal root ganglia of the rat, suggesting that ADA is a marker for the use of purines by primary afferent neurons.24 To our knowledge, there are no previous visualizations of ADA-IR in the rat dorsal vagal complex; however, ADA activity has been demonstrated in the rat medulla.23 In the present study, ADA-IR was found to be associated with both perikarya and nerve fibers in the area postrema, DMX, and NTS. In the NTS, particularly in the more rostral sections examined (corresponding to the medial subnucleus), dense fiber networks were found to be immunopositive for ADA in both strains. Furthermore, in both SHR and DRY, ADA-IR was observed in a subpopulation of cell bodies in the nodose ganglia. The presence of dense ADA-IR in neuronal cell bodies of the nodose ganglion as well as in axons and terminals in the dorsal vagal complex (area postrema, DMX, and NTS) of both SHR and DRY is consistent with a neuromodulatory role for adenosine in vagal afferent fibers. It has been demonstrated that brain regions containing perikarya and ADA-immunopositive fibers have the highest ADA activity.23
As the results obtained with diaminobenzidine indicated that there was a higher density of ADA-IR in the dorsal vagal complex of SHR than in that of DRY, we used an iodinated secondary antibody to allow quantification of 125I–ADA-IR in brain stem and nodose ganglia. Our results revealed that the density of 125I–ADA-IR was significantly higher in the dorsal vagal complex of SHR than in that of DRY, whereas no significant differences in 125I–ADA-IR densities between strains were observed in the nodose ganglia. These findings suggest that the differences observed in ADA-IR density in the dorsal vagal complex observed between strains were possibly due to a postsynaptic source of ADA.
The present study has demonstrated that the binding characteristics of [3H]NBMPR and ADA-IR in brain stems and nodose ganglia of SHR and DRY differ. In addition, [3H]NBMPR binding sites and ADA-IR essentially show a parallel topographic distribution profile at the level of the medulla oblongata examined in both strains. This is consistent with previous findings demonstrating that structures immunoreactive for ADA in rat brain closely correspond to those exhibiting a high density of binding sites for [3H]NBMPR.25 It appears that in the SHR, adenosine uptake mechanisms could be more effective, as indicated both by the increased sensitivity of adenosine transport sites for adenosine transport blockers as well as to adenosine itself in the dorsal brain stem and by the increased ADA-IR levels observed in the dorsal vagal complex. These results help provide a molecular basis for the observed functional differences found between normotensive and hypertensive rats, in which an attenuated pressor response to adenosine in the area postrema and NTS of SHR has been reported.29 It is also possible that the differences found in the present study between SHR and DRY were due to a general strain difference; however, these two rat strains are known to be genetically homogeneous.35 36 In both SHR and DRY, a correlation coefficient of -.2 was obtained, indicating a lack of correlation between blood pressure and apparent affinity values for [3H]NBMPR in both strains; however, given that the n values available in saturation experiments (n=5) are small and that the tail-cuff method is inherently inaccurate, it is impossible to draw definite conclusions. The presence of uptake mechanisms for adenosine in the soma and terminals of vagal afferent neurons provides further evidence implicating this purine nucleoside as a potential neuromodulator of neuronal activity in the medulla. Whether the observed molecular differences in purinergic mechanisms within this brain stem nuclei are contributing to the development and/or maintenance of hypertension remains to be elucidated.
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Acknowledgments |
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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 are extremely grateful to Prof Jonathon D. Geiger from the University of Manitoba for kindly providing the primary antibody against ADA.
Received May 20, 1996;
first decision July 1, 1996; first decision July 30, 1996;
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References |
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