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(Stroke. 1995;26:1627-1633.)
© 1995 American Heart Association, Inc.

Articles

Adenosine Modulates N-Methyl-D-Aspartate–Stimulated Hippocampal Nitric Oxide Production In Vivo

Anish Bhardwaj, MD; Frances J. Northington, MD; Raymond C. Koehler, PhD; Theodore Stiefel; Daniel F. Hanley, MD Richard J. Traystman, PhD

From the Departments of Neurology (A.B., T.S., D.F.H.), Anesthesiology/Critical Care Medicine (A.B., R.C.K., D.F.H., R.J.T.), and Pediatrics (F.J.N.), The Johns Hopkins Medical Institutions, Baltimore, Md.

Correspondence to Anish Bhardwaj, MD, Neurocritical Care Division, The Johns Hopkins Hospital, Meyer 8-140, 600 N Wolfe St, Baltimore, MD 21287-7840.

	Abstract

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Background and Purpose Adenosine acts presynaptically to inhibit release of excitatory amino acids (EAAs) and is thus considered to be neuroprotective. Because EAA-stimulated synthesis of nitric oxide (NO) may play an important role in long-term potentiation and excitotoxic-mediated injury, we tested the hypotheses that adenosine agonists attenuate basal and EAA-induced NO production in the hippocampus in vivo and that adenosine A₁ receptors mediate this response.

Methods Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of adult Sprague-Dawley rats under pentobarbital anesthesia. Probes were perfused for 5 hours with artificial cerebrospinal fluid containing 3 µmol/L [¹⁴C]L-arginine. Recovery of [¹⁴C]L-citrulline in the effluent was used as a marker of NO production. In 10 groups of rats, time-dependent increases in [¹⁴C]L-citrulline recovery were compared between right- and left-sided probes perfused with various combinations of N-methyl-D-aspartate (NMDA), adenosine agonists, adenosine antagonists, and the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME).

Results Recovery of [¹⁴C]L-citrulline during perfusion with artificial cerebrospinal fluid progressively increased to 141±27 fmol/min (±SEM) over 5 hours. Contralateral perfusion with 1 mmol/L NMDA augmented [¹⁴C]L-citrulline recovery to 317±62 fmol/min. Perfusion of 1 mmol/L L-NAME with NMDA inhibited [¹⁴C]L-citrulline recovery compared with NMDA alone. Perfusion with 0.1 mmol/L 2-chloroadenosine attenuated basal as well as NMDA-enhanced [¹⁴C]L-citrulline recovery. This action of 2-chloroadenosine was reversed by infusion of 0.1 mmol/L 8-cyclopentyl-1,3-dipropylxanthine, a specific A₁ receptor antagonist. Infusion of 0.1 mmol/L (2S)-N⁶-[2-endo-norboryl]adenosine, a specific A₁ receptor agonist, also attenuated the 0.1 mmol/L and 1 mmol/L NMDA-enhanced [¹⁴C]L-citrulline recovery.

Conclusions Using an indirect method of assessing NO production in vivo, these data are consistent with in vitro results showing that NMDA receptor stimulation enhances NO production. Furthermore, we conclude that stimulation of A₁ receptors can attenuate the basal as well as NMDA-induced production of NO. Because NMDA receptor stimulation amplifies glutamate release, our data are consistent with presynaptic A₁ receptor–mediated inhibition of EAA release and consequent downregulation of NO production.

Key Words: adenosine • hippocampus • N-methyl-D-aspartate • nitric oxide • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nitric oxide plays a multifaceted role in the brain as a neurotransmitter^{1 2} and a regulator of cerebral blood flow.^{1 3} Sites of NO production include neurons, vascular endothelium, perivascular neurons, and possibly astrocytes.³ If present in abnormally high concentrations, NO may exert neurotoxic effects.^{4 5 6} Experiments in primary neuronal cultures implicate NO as a mediator of glutaminergic neurotoxicity acting via NMDA receptors.⁵ Excessive stimulation of the NMDA receptors allows influx of ionized calcium into neurons, which may stimulate NOS.⁷ Inhibition of NOS in neuronal culture can ameliorate glutamate toxicity.⁵ It is well established that the hippocampus is one of the regions most vulnerable to ischemia and possesses prominent glutamatergic receptors.^{8 9} Furthermore, stimulation of NMDA receptors stimulates glutamate release,¹⁰ and this amplifying effect was attenuated by NOS inhibition.¹¹

Adenosine exerts ischemic neuroprotection not only by promoting vasodilation but probably also by its neuromodulatory actions on presynaptic, postsynaptic, and possibly extrasynaptic A₁ and A₂ receptors.^{12 13 14} Presynaptically adenosine inhibits release of neurotransmitters.^{15 16 17} Postulated mechanisms for this action include inhibition of adenylate cyclase, opening of K⁺ channels, and reduced flux through Ca²⁺ channels.¹⁵ The A₁ receptors exhibit a heterogeneous distribution within the brain.¹⁸ In the hippocampus, A₁ receptors are prominent on dendrites.¹⁹ Adenosine has been shown to modulate the release of glutamate from synaptosomes and to suppress ischemia-evoked excitatory amino acid release in hippocampus, striatum, and cortex.^{20 21 22 23} Adenosine also blocks Ca²⁺ spikes in hippocampal slices.²⁴ Conversely, glutamate enhances the release of adenosine in rat cortical slices.²⁵ Furthermore, stimulation of NMDA receptors on hippocampal interneurons may cause adenosine release, which then presynaptically inhibits glutamate release from adjacent neurons.²⁶ Thus, adenosine appears to exert negative feedback on glutamate release, which in turn may reduce the increase in NO production normally associated with stimulation of NMDA and non-NMDA receptors.²⁷

Direct measurement of NO in brain in vivo has proved difficult because of the lability of the gas. Some investigators have used a NO-sensitive porphyrinic microsensor²⁸ or a microdialysis hemoglobin trapping technique.²⁹ NO is formed from arginine by NOS, which oxidizes a guanidino-nitrogen of arginine, releasing NO and citrulline.¹ Activity of NOS in vitro can be measured by the conversion of radiolabeled arginine to citrulline.¹ We have adapted this technique to the in vivo setting by infusing [¹⁴C]L-arginine into a microdialysis probe and measuring recovery of [¹⁴C]L-citrulline in the dialysis effluent.³⁰ Perfusion of lamb cortex with NMDA increased citrulline recovery.³⁰ Because both basal and NMDA-induced increase in citrulline recovery were inhibited by the NOS inhibitor L-NAME, in vivo [¹⁴C]L-citrulline recovery appears to provide an indirect marker of NO production.

Using this technique, we tested the hypotheses that (1) stimulation of NMDA receptors increases NO production in rat hippocampus in vivo and (2) stimulation of adenosine A₁ receptors inhibits both basal and NMDA-induced NO production.

	Materials and Methods

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The experimental protocol was approved by the institutional animal care and use committee of the Johns Hopkins University and conforms to the National Institutes of Health guidelines for the care and use of animals in research. Adult male Sprague-Dawley rats (300 to 450 g) were anesthetized with intraperitoneal pentobarbital (30 mg/kg). Additional pentobarbital was given during the course of the experiment in response to "leg-pinch" stimulus (15 mg/kg per hour). Rats were tracheotomized and mechanically ventilated with a mixture of oxygen and nitrogen to maintain arterial pH of 7.35 to 7.40, PCO₂ of 35 mm Hg, and PO₂ of 150 mm Hg. Cannulas were inserted into the tail artery to continuously monitor arterial blood pressure, heart rate, and arterial blood gases. Temperature was monitored with a rectal probe and maintained at 37°C to 38°C with a warming blanket. Care was taken to avoid direct heating of the head, which would alter the normal thermal gradients between the brain and the core.

The rat's head was placed in a Kopf stereotaxic frame for placement of microdialysis cannulas into the CA3 region of the hippocampus (0.3 mm anterior and 4.5 mm lateral to lambda; depth of 8 mm from the dura).³¹ A 2x2-mm area of the skull was removed with a variable-speed drill. A thin layer of bone was left intact and removed with forceps under microscopic observation to minimize trauma to the cortex. Cannulas were advanced to predetermined coordinates with a micromanipulator and were fixed in position with dental cement. The animal was then removed from the stereotaxic apparatus and allowed a 60-minute postsurgical equilibration period before the experiment was begun.

Microdialysis
Dialysis probes used in these studies were made according to the description of Johnson and Justice³² and as modified by Van Wylen et al³³ and others.³⁴ The probes consisted of a single, hollow dialysis fiber, one end of which was sealed with epoxy. The dialysis membrane was 300 µm in diameter and had a molecular mass cutoff of 5 kD. Two hollow silica perfusion tubes were inserted into the dialysis fiber so that their ends were 5 mm apart. The distance between the tips constitutes the effective dialyzing area of the cannula.^{30 34} The cannulas were perfused at 1 µL/min. The millimolar concentration of artificial CSF was as follows: NaCl 131.8, NaHCO₃ 24.6, CaCl₂ 2.0, KCl 3.0, MgCl₂ 0.65, urea 6.7, and dextrose 3.7. The CSF was filtered, warmed to 37°C, and bubbled with 95% N₂ and 5% CO₂ until O₂ and CO₂ tensions were similar to those of CSF and brain tissue.^{30 34}

Estimation of NO production was performed using modifications of the assay described by Bredt et al.¹ Arginine is converted to equimolar concentrations of citrulline and NO by the action of NOS.¹ During continuous infusion of CSF containing 3 µmol/L [¹⁴C]L-arginine, 60-µL effluent dialysate samples were collected over 1-hour periods and assayed for [¹⁴C]L-citrulline content. Samples were diluted with 200 µL of water and were poured over 0.5 mL resin AG-50Wx8 (Na⁺ form, pH 7.0) 400-mesh columns. Columns were washed with 2 mL buffer containing 30 mmol/L HEPES (pH 5.2) and 3 mmol/L EDTA and 1 mL of water. To determine resin efficiency of arginine trapping, 60 µL of artificial CSF containing 3 µmol/L [¹⁴C]L-arginine, not used for dialysis, was diluted in 200 µL of water, poured over a column, and washed as above. Radioactivity of the flow-through was quantified by liquid scintillation spectroscopy. Flow-through radioactivity of the nondialyzed CSF was assumed to be arginine, on the presumption that no conversion of arginine to citrulline occurred in the CSF that was not exposed to any source of enzyme activity. Specific activity was corrected for counting efficiency and background activity and was expressed as femtomoles per minute of perfusion. As an internal control, 100 µL of CSF not used for dialysis was directly assayed for activity to ensure that consistent concentrations of [¹⁴C]L-arginine were added to the CSF.

Experimental Groups
Rats were divided into 10 treatment groups to receive perfusates containing various combinations of NMDA, L-NAME, 2-CADO, the A₁ receptor agonist S(-)-ENBA, and the A₁ antagonist DPCPX in artificial CSF with 3 µmol/L [¹⁴C]L-arginine. Within each group, the particular drug combination was randomly assigned to either right or left hippocampus, and the effluent concentrations of [¹⁴C]L-citrulline were compared on a paired basis. The experimental groups were as follows: (1) control CSF versus 1 mmol/L NMDA (n=8); (2) control CSF versus 0.1 mmol/L 2-CADO (n=8); (3) 1 mmol/L NMDA versus 1 mmol/L NMDA plus 0.01 mmol/L 2-CADO (n=8); (4) 1 mmol/L NMDA versus 1 mmol/L NMDA plus 0.1 mmol/L 2-CADO (n=8); (5) 1 mmol/L NMDA plus 0.1 mmol/L 2-CADO versus 1 mmol/L NMDA plus 2-CADO plus 0.1 mmol/L DPCPX (n=8); (6) 0.1 mmol/L NMDA versus 0.1 mmol/L NMDA plus 0.1 mmol/L S(-)-ENBA (n=8); (7) 1 mmol/L NMDA versus 1 mmol/L NMDA plus 0.1 mmol/L S(-)-ENBA (n=7); (8) 1 mmol/L NMDA versus 1 mmol/L NMDA plus 1 mmol/L L-NAME (n=8); (9) 1 mmol/L NMDA versus 1 mmol/L NMDA plus 1 mmol/L D-NAME (n=6); and (10) control CSF versus control CSF (n=8).

Perfusions of both [¹⁴C]L-arginine and drugs began together at the start of perfusion (1 hour after probe insertion). After 5 hours of perfusion, rats were killed with an intravenous injection of potassium chloride while still anesthetized, and dialysis probes were perfused with methylene blue. Animals were decapitated, and the brains were removed and stored in 4% paraformaldehyde for 48 hours. Brains were then dissected to visualize the probe tracts. Recovery of [¹⁴C]L-citrulline was measured in vitro on 14 probes before implantation in seven rats and again after removal of the probes at the end of the experiment.

Immunohistochemical Studies
Naive rats were given intravenous heparin and were perfused through the ascending aorta with 4% paraformaldehyde and 1% acrolein. Brains were removed and frozen en bloc at -80°C until processing. Immunohistochemical experiments were performed on 40-µm floating sections of tissue. The tissue was processed using a peroxidase/antiperoxidase detection method with 3,3'-diaminobenzidine (DAB) as the chromogen. Incubation with or without the primary antibody was carried out at 4°C for 48 hours on a rocker plate. Nonspecific binding was inhibited by preincubating tissues with 4% goat serum. A well-characterized antineuronal NOS antibody is a polyclonal rabbit antibody that recognizes a 155-kD protein.³⁵

Materials
[¹⁴C]L-Arginine (317 mCi/mmol) was obtained from Dupont-NEN Products; L-NAME, 2-CADO, and NMDA were obtained from Sigma Chemical Co. S(-)-ENBA and DPCPX were obtained from Research Biochemical International.

Statistical Analysis
Within each group, the effluent citrulline data were analyzed by two-way ANOVA where the two treatments delivered to the two hippocampi were one within-subject factor, and the 5-hourly collections were a second within-subject factor. If the overall effect of treatment or the treatment multiplied by time interaction was significant, comparisons of mean values between the two treatments at individual time points were made by Newman-Keuls multiple-range test. A value of P<.05 was considered significant. Within each group, time-dependent changes in hemodynamic and blood gas data were determined with one-way repeated-measures ANOVA. Data are presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using a well-characterized antineuronal NOS antibody, we demonstrated the presence of NOS-containing neurons in the CA3 region of the hippocampus (Fig 1). NOS-positive neurons were also identified in the dentate gyrus of the hippocampus.

View larger version (139K): [in this window] [in a new window]   Figure 1. Photomicrograph demonstrating positive immunocytochemical staining for neuronal NOS neurons in rat hippocampus. A, Low magnification (x10); B, high magnification (x100) of CA3.

In group 1, perfusion with [¹⁴C]L-arginine in artificial CSF resulted in time-dependent increases in [¹⁴C]L-citrulline in the effluent. These time-dependent increases represent the time required for diffusion of labeled arginine into the tissue and the time required for diffusion of labeled citrulline back to the probe. In the side perfused with 1 mmol/L NMDA, recovery of labeled citrulline was increased from the third through the fifth hour of perfusion compared with the control side (Fig 2A).

View larger version (22K): [in this window] [in a new window]   Figure 2. Line graphs depict [14C]L-citrulline recovery in dialysates from probes perfused with artificial CSF on one side and 1 mmol/L NMDA on the contralateral side (A) and with control CSF on one side and 0.1 mmol/L 2-CADO on the contralateral side (B). Values are mean±SEM (n=8 rats); *P<.05 between treatments.

In group 2, perfusion with 0.1 mmol/L 2-CADO reduced citrulline recovery from the second to the fifth hour compared with perfusion with artificial CSF alone (Fig 2B).

In group 3, combined perfusion of 0.01 mmol/L 2-CADO with 1 mmol/L NMDA did not reduce citrulline recovery compared with 1 mmol/L NMDA alone (data not shown). However, increasing the dose of 2-CADO to 0.1 mmol/L (group 4) attenuated the increase in citrulline recovery during the fourth and fifth hours compared with perfusion with 1 mmol/L NMDA alone (Fig 3A). This effect was reversed by the A₁ antagonist DPCPX in group 5 (Fig 3B). Citrulline recovery during perfusion with 0.1 mmol/L DPCPX plus 0.1 mmol/L 2-CADO plus 1 mmol/L NMDA was greater than citrulline recovery during perfusion with 0.1 mmol/L 2-CADO plus 1 mmol/L NMDA. This effect was significant from the second through fifth hours of perfusion.

View larger version (25K): [in this window] [in a new window]   Figure 3. Line graphs depict [14C]L-citrulline recovery in dialysates from probes perfused with 1 mmol/L NMDA on one side and 1 mmol/L NMDA plus 0.1 mmol/L 2-CADO on the contralateral side (A) and with 1 mmol/L NMDA on one side and 1 mmol/L NMDA plus 0.1 mmol/L 2-CADO plus 0.1 mmol/L DPCPX on the contralateral side (B). Values are mean±SEM (n=8 rats); *P<.05 between treatments.

With a lower dose of NMDA (0.1 mmol/L) and a more specific A₁ agonist, S(-)-ENBA (group 6), citrulline recovery was reduced from the second through fifth hours when compared with citrulline recovery during 0.1 mmol/L NMDA perfusion (Fig 4A). When the dose of NMDA was increased to 1 mmol/L in group 7, 0.1 mmol/L S(-)-ENBA attenuated citrulline recovery from the second through fourth hours but not during the fifth hour (Fig 4B).

View larger version (23K): [in this window] [in a new window]   Figure 4. Line graphs depict [14C]L-citrulline recovery in dialysates from probes perfused with 0.1 mmol/L NMDA on one side and 0.1 mmol/L NMDA plus 0.1 mmol/L S(-)-ENBA on the contralateral side (n=8 rats) (A) and with 1 mmol/L NMDA on one side and 1 mmol/L NMDA plus 0.1 mmol/L S(-)-ENBA on the contralateral side (n=7 rats) (B). Values are mean±SEM; *P<.05 between treatments.

In group 8, citrulline recovery did not increase over time on the side perfused with 1 mmol/L L-NAME plus 1 mmol/L NMDA. Additionally, citrulline recovery during L-NAME plus NMDA was less than on the side perfused with 1 mmol/L NMDA from the second to the fifth hours (Fig 5A). In contrast, citrulline recovery during perfusion with 1 mmol/L NMDA plus 1 mmol/L D-NAME was not less than on the side perfused with 1 mmol/L NMDA (Fig 5B).

View larger version (23K): [in this window] [in a new window]   Figure 5. Line graphs depict [14C]L-citrulline recovery in dialysates from probes perfused with 1 mmol/L NMDA on one side and 1 mmol/L NMDA plus 1 mmol/L L-NAME on the contralateral side (n=8 rats) (A) and with 1 mmol/L NMDA on one side and 1 mmol/L NMDA plus 1 mmol/L D-NAME on the contralateral side (n=6 rats) (B). Values are mean±SEM; *P<.05 between treatments.

In eight rats, there was no difference between right- and left-side citrulline recovery when both sides were perfused with artificial CSF containing 3 µmol/L [¹⁴C]L-arginine without stimulation by exogenous drugs.

In the 14 probes in which [¹⁴C]L-citrulline recovery was measured in vitro both before and after the experiment, recovery was 9.5±1.7% before implantation and 5.4±1.3% after removal.

Levels of mean arterial pressure, arterial blood gases, pH, and rectal temperatures were stable throughout the experiment. Values obtained during the fifth hour of perfusion are summarized in the Table.

View this table: [in this window] [in a new window]   Table 1. Physiological Data at Hour 5 Perfusion

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The significant finding of this study is that stimulation of adenosine receptors attenuates basal as well as NMDA-enhanced NO production in the hippocampus in vivo as measured indirectly by conversion of arginine to citrulline. These data are also consistent with the hypothesis that this action of adenosine is mediated via stimulation of the neuroinhibitory A₁ receptor.

Cerebral microdialysis measures neurochemical changes occurring in the brain extracellular fluid, a microenvironment that serves as a communication between neurons, glia, and the vasculature. In this study, the tips of the dialysis probes were localized in the CA3 region of the hippocampus, as confirmed by postmortem infusion of methylene blue dye. Neurons containing NOS have been conspicuously identified by NADPH diaphorase staining in the pyramidal area of the CA3 hippocampal region.³⁶ We confirmed this observation immunohistochemically with an antibody to the neuronal isoform of NOS (Fig 1). In addition, the endothelial isoform of NOS appears to be present in hippocampal pyramidal neurons.³⁷ It is also possible that NO is produced by astrocytes.³⁸ Thus, the necessary NO synthetic mechanisms are present. Brain tissue in vitro responds to stimulation of NMDA and non-NMDA glutamate receptor with increased conversion of arginine to citrulline and increased cGMP concentrations that are inhibitable by NO scavengers and NOS inhibitors.^{1 27} The present data demonstrate that NO production, as measured by [¹⁴C]L-citrulline recovery, was enhanced by NMDA infusion in the hippocampus. The postulated mechanism is that stimulation of the NMDA receptors allows the influx of ionized calcium into neurons, which then stimulates production of NO by NOS.¹ In addition, NMDA receptor stimulation can cause downstream glutamate release¹⁰ and potentially stimulate distant NOS-containing neurons via either NMDA or non-NMDA glutamate receptors. Infusion of the NOS inhibitor L-NAME markedly attenuated NMDA-enhanced [¹⁴C]L-citrulline recovery. This result confirms previous results in lamb cortex, where L-NAME reduced citrulline recovery under both basal and NMDA-stimulated conditions,³⁰ and is consistent with the premise that labeled citrulline is derived from labeled arginine by the catalytic activity of NOS.

Activity of [¹⁴C]L-citrulline in the dialysis effluent increased over time in controls. This is attributable to (1) the diffusion of [¹⁴C]L-arginine away from the dialysis probe over time, uptake into cells, and time-dependent recruitment of distant cells capable of producing NO and (2) the time-dependent extrusion of radiolabeled citrulline from the cell and diffusion back to and across the dialysis membrane. In addition, an unknown fraction of labeled citrulline may have been recycled back to arginine. Therefore, this microdialysis technique does not provide a precise instantaneous measure of NOS activity. Furthermore, there was considerable intergroup variability in the absolute amount of citrulline recovery during control perfusion or during perfusion with NMDA. Interanimal variability could arise from factors including depth of anesthesia and efficiency of radiolabeled arginine removal by the Dowex column. Potential factors contributing to intra-animal variability or right/left differences could arise from local tissue trauma and dialysis membrane recovery. Although we attempted to control these factors, they may have contributed to variability in the magnitude and rate of increase of labeled citrulline recovery. Despite this variability, we were able to detect statistical differences between treatment groups by using a paired experimental design. We assumed that the modulatory effect of infusate treatments on citrulline recovery was a local phenomenon. However, we cannot exclude transcallosal interactions such that NMDA excitation could lead to NOS inhibition contralaterally or A₁ receptor stimulation could augment NOS activity contralaterally.

The actions of adenosine are mediated via the presynaptic A₁ receptor, the A₂ receptor, and possibly an A₃ receptor.³⁹ Presynaptically, A₁ receptor stimulation inhibits neurotransmitter release by directly suppressing neuronal firing.^{16 17} Postsynaptically, adenosine inhibits evoked synaptic potentials and after discharges.²⁴ The concentration of extracellular adenosine is increased during hypoxia, synaptic stimulation, and application of glutamate in vitro.²⁶ The NMDA subtype of glutamate receptor is necessary for glutamate-evoked elevation of adenosine, and this effect is Ca²⁺ dependent.²⁶ 2-CADO is partially selective for the A₁ receptor, although it retains A₂ activity.⁴⁰ 2-CADO has been shown to protect against long-term development of ischemic cell loss in the rat hippocampus, possibly because of activation of A₁ receptors.⁴¹ In our study, infusion of 0.1 mmol/L 2-CADO attenuated basal as well as 1 mmol/L NMDA induced [¹⁴C]L-citrulline recovery, suggesting that NMDA-mediated NO release can be modulated by adenosine agonists. To determine whether the action of 2-CADO was mediated via stimulation of the A₁ receptor, we used perfusion with the highly selective A₁ receptor antagonist DPCPX.⁴² Attenuation of NMDA-enhanced [¹⁴C]L-citrulline recovery caused by 2-CADO was reversed by 0.1 mmol/L DPCPX. This additionally supports the hypothesis that 2-CADO attenuates NMDA-mediated NO production via the A₁ receptor. Infusion of NMDA plus 0.01 mmol/L 2-CADO infusion did not decrease [¹⁴C]L-citrulline recovery compared with 1 mmol/L NMDA alone, demonstrating that the threshold for this effect was 0.1 mmol/L in the present system. Furthermore, infusion of the highly selective adenosine A₁ agonist S(-)-ENBA⁴³ also attenuated the NMDA-induced [¹⁴C]L-citrulline recovery. The effect was attained at the same concentration as for 2-CADO. Therefore, we conclude that A₁ receptors can exert a considerable modulatory role on NO production in vivo.

Activation of NMDA receptors can lead to release of various neurotransmitters, including glutamate.^{10 11} On the basis of electrophysiological measurements in hippocampal slices, Manzoni et al²⁶ suggested that stimulation of NMDA receptors also causes adenosine release from interneurons and that adenosine acts at distant excitatory synapses to reduce the release of glutamate. Thus, NMDA may be acting both (1) directly on NOS-containing neurons or astrocytes via NMDA receptors and (2) indirectly to stimulate release of glutamate, which then acts on NOS-containing neurons or astrocytes via NMDA or non-NMDA glutamate receptors. Assuming that the adenosine analogues used in our study are acting presynaptically, then our data are consistent with NMDA receptor activation amplifying NO production through the release of other neurotransmitters and with adenosine A₁ receptor modulation of this amplification process.

In conclusion, this study demonstrates that NMDA-enhanced NO production in the hippocampus can be measured in vivo by measuring labeled citrulline recovery after labeled arginine infusion. Additionally, adenosine analogues with action specifically at the A₁ receptor attenuate NO production induced by NMDA. Assuming that excessive NO production during and after ischemia is neurotoxic, this may represent one mechanism by which adenosine exerts its neuroprotective effect. Selective A₁ agonists may have potential use as neuroprotective agents.

	Selected Abbreviations and Acronyms

 

2-CADO = 2-chloroadenosine CSF = cerebrospinal fluid D-NAME = N-nitro-D-arginine methyl ester DPCPX = 8-cyclopentyl-1,3-dipropylxanthine L-NAME = N-nitro-L-arginine methyl ester NMDA = N-methyl-D-aspartate NO = nitric oxide NOS = nitric oxide synthase S(-)-ENBA = (2S)-N6-[2-endo-norbornyl]adenosine

	Acknowledgments

 
This work was supported in part by a grant from the National Institutes of Health (NS-20020) and a beginning Grant-in-Aid from the Maryland Affiliate of the American Heart Association, part by a National Stroke Association fellowship career development award (Dr Bhardwaj), a Merck clinician scientist award from Johns Hopkins University School of Medicine and a clinician investigator development award from the National Institutes of Health (NS-1742) (Dr Northington). The authors wish to thank the laboratory of Dr T.M. Dawson for generously providing the antibody for immunohistochemical studies.

Received March 13, 1995; revision received May 30, 1995; accepted June 15, 1995.

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