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(Stroke. 1998;29:2404-2411.)
© 1998 American Heart Association, Inc.

Original Contributions

Potent ₁-Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl) Piperidine Modulates Basal and N-Methyl-D-Aspartate–Evoked Nitric Oxide Production In Vivo

Anish Bhardwaj, MD; Masahiko Sawada, MD; Edythe D. London, PhD; Raymond C. Koehler, PhD; Richard J. Traystman, PhD; Jeffrey R. Kirsch, MD

From the Departments of Neurology (A.B.) and Anesthesiology and Critical Care Medicine (A.B., M.S., R.C.K., R.J.T., J.R.K.), Johns Hopkins University School of Medicine, and National Institute on Drug Abuse (E.D.L.), Baltimore, Md.

Correspondence to Anish Bhardwaj, MD, Neuroscience Critical Care Division, Meyer 8–140, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail abhardwa{at}welchlink.welch.jhu.edu

	Abstract

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Background and Purpose—-Receptor ligands ameliorate ischemic neuronal injury and modulate neuronal responses to N-methyl-D-aspartate (NMDA) receptor stimulation. Because NMDA-evoked synthesis of nitric oxide (NO) may play an important role in excitotoxic-mediated injury, we tested the hypothesis that -receptor ligands attenuate basal and NMDA-evoked NO production in the striatum in vivo.

Methods—Microdialysis probes were placed bilaterally into the striatum of halothane-anesthetized adult Wistar rats. Rats were divided into 7 treatment groups and perfused with artificial cerebrospinal fluid (aCSF) containing 3 µmol/L [¹⁴C]L-arginine for 2 to 3 hours followed by NMDA in various combinations with the following drugs: L-nitroarginine (L-NNA); the ₁-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP); the selective ₁-receptor antagonist 1-(cyclopropylmethyl)-4-(2'-oxoethyl) piperidine hydrobromide (DuP 734); and the noncompetitive NMDA receptor blocker MK-801 in aCSF. Right-left differences between [¹⁴C]L-citrulline in the effluent from rats treated with different drug combinations were assumed to reflect differences in NO production.

Results—After a 3-hour loading period with [¹⁴C]L-arginine, addition of 1 mmol/L NMDA increased [¹⁴C]L-citrulline recovery compared with aCSF alone. This NMDA-evoked increase was inhibited by 1 mmol/L of L-NNA and PPBP. Perfusion of 1 mmol/L of the ₁-receptor antagonist DuP 734 with 1 mmol/L PPBP augmented NMDA-evoked [¹⁴C]L-citrulline recovery compared with perfusion with PPBP and NMDA. MK-801 attenuated the basal as well as NMDA-evoked [¹⁴C]L-citrulline recovery. PPBP did not cause any further attenuation in the basal and NMDA-evoked [¹⁴C]L-citrulline recovery in the presence of MK-801.

Conclusions—These data indicate that a ₁-receptor ligand attenuates basal as well as NMDA-evoked NO production. Because the attenuated NO production was reversed by DuP 734, PPBP appears to act as an agonist at the ₁-receptor. Attenuated NO production by ₁-receptor agonists provides one possible mechanism for focal ischemic neuroprotection.

Key Words: excitotoxicity • ligands • microdialysis • nitric oxide • receptors, sigma • rats

	Introduction

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The -receptor ligands have been reported to modulate several neurotransmitter systems and affect behavior and cognition.^{1 2 3 4 5 6} These ligands have been shown to modulate neuronal responses to pharmacological N-methyl-D-aspartate (NMDA) receptor stimulation in vitro^{7 8} and to provide ischemic neuroprotection in vivo and in vitro.^{9 10 11 12 13 14} Recently, purification, molecular cloning, and high levels of expression of the ₁-binding sites in the sterol-producing tissues have been reported.¹⁵ We have previously demonstrated that the potent ₁-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) prevents early evidence of brain injury in rat¹² and cat¹³ models of transient focal ischemia. Similarly, (+)-pentazocine, another potent ₁-receptor ligand, afforded significant neuroprotection in ischemic injury in the rat model of transient focal ischemia.¹⁴ Thus, there appears to be a role for -receptors in modulating ischemic neuronal injury. Furthermore, it is postulated that this protective action is mediated by an alteration of NMDA receptor function.⁷

Excitotoxic mechanisms have been implicated in the propagation of ischemic neuronal injury.¹⁶ Nitric oxide (NO) plays a multifaceted role in the brain as a neurotransmitter^{17 18} and a regulator of cerebral blood flow.¹⁹ If present in abnormally high concentrations, NO may exert neurotoxic effects.^{16 20 21} Experiments in primary neuronal cultures implicate NO as a mediator of glutamatergic neurotoxicity acting through the NMDA receptors.¹⁶ Excessive stimulation of NMDA receptors allows influx of calcium ions into neurons and thereby stimulate NO synthase (NOS).²² NO is formed from arginine by NOS, which oxidizes a guanidino nitrogen of arginine, releasing NO and citrulline.¹⁷ Activity of NOS in vitro can be measured by the conversion of radiolabeled arginine to citrulline.¹⁷ 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. We have previously shown that NMDA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and metabotropic glutamate receptor stimulation enhances labeled citrulline recovery in the hippocampus.^{23 24 25} Because -ligands that provide neuroprotection from NMDA toxicity in vitro have little protective effect from kainate toxicity,²⁶ we focused on NMDA receptor activation in the present study.

Using modifications of this technique in vivo, we tested the hypotheses that (1) NMDA-evoked NO production is attenuated by the ₁-receptor ligand PPBP, (2) attenuation of NMDA-evoked NO production by PPBP can be reversed by the selective ₁-receptor antagonist 1-(cyclopropylmethyl)-4-(2'-oxoethyl) piperidine hydrobromide (DuP 734), and (3) PPBP attenuates basal and NMDA-evoked NO production by its interaction with the NMDA receptor.

	Materials and Methods

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General Preparation and Animal Surgery
The experimental protocol was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University and conforms to the National Institutes of Health guidelines for the care and use of animals in research. Adult male Wistar rats weighing 300 to 450 g were anesthetized with halothane in oxygen. Anesthesia was maintained with halothane (1.0% to 2.0%) in oxygen-enriched air (35% to 40%) to maintain arterial pH (7.35 to 7.40) and arterial partial pressures of CO₂ (35 mm Hg) and O₂ (150 mm Hg). Cannulas were inserted into the tail artery to monitor arterial blood pressure, heart rate, and arterial blood gases. Temperature was monitored with a rectal probe and was maintained at 37°C to 38°C with a heating lamp. 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 striatum (0.5 mm anterior and 2.5 mm lateral to the bregma; depth 6 mm from the dura).²⁷ A 2x2-mm² 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 animals were then removed from the stereotaxic apparatus and allowed a 60-minute postsurgical equilibration period before the experiment began.

Microdialysis
Dialysis probes used in these studies were made according to the description of Johnson and Justice²⁸ and modified by Van Wylen et al²⁹ and as described previously.^{23 24 25} The probes consisted of a single hollow dialysis fiber, one end of which was sealed with epoxy. The dialysis membrane diameter was 300 µm and had a molecular mass cutoff of 5 kDa. Two hollow silica perfusion tubes were inserted into the dialysis fiber so that their ends were 3 mm apart. The distance between the tips constituted the effective dialyzing area of the cannula.²⁹ Starting 1 hour after insertion, the cannulas were perfused at a rate of 1 µL/min. The concentration of artificial cerebrospinal fluid (aCSF) was as follows (mmol/L): NaCl 131.8, NaHCO₃ 24.6, CaCl₂ 2.0, KCl 3.0, MgCl₂ 0.65, urea 6.7, and dextrose 3.7. The aCSF was filtered, warmed to 37°C, and bubbled with 95% N₂/5% CO₂ until O₂ and CO₂ tensions were similar to those of aCSF and brain tissue.²⁹

Estimation of NO Production
We estimated NO production using modifications of the assay described by Bredt et al.¹⁷ Arginine is converted to equimolar concentrations of citrulline and NO by the action of NOS.¹⁷ During continuous infusion of aCSF containing 3 µmol/L [¹⁴C]L-arginine, 20-µL effluent dialysate samples were collected during 20-minute epochs and assayed for [¹⁴C]L-citrulline content. Samples were diluted with 200 µL water and 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), 3 mmol/L EDTA, and 1 mL water. Radioactivity of the flow through the column was quantified by liquid scintillation spectroscopy. To determine resin efficiency of arginine trapping, 20 µL aCSF containing 3 µmol/L [¹⁴C]L-arginine (not used for dialysis) was diluted in 200 µL water, poured over a column, and washed as above. Specific activity was corrected for counting efficiency and background activity and expressed as femtomoles per minute of perfusion. As an internal control, 100 µL aCSF not used for dialysis was directly assayed for activity to ensure that consistent concentrations of [¹⁴C]L-arginine were added to the aCSF.

Experimental Groups
Rats were divided into 7 treatment groups to receive perfusates containing various combinations of NMDA, L-nitroarginine (L-NNA), the ₁-receptor ligand PPBP, the selective ₁-receptor antagonist DuP 734, and the noncompetitive NMDA receptor antagonist MK-801 in aCSF containing [¹⁴C]L-arginine. In all experimental groups, the perfusion with labeled arginine lasted 6 hours. The agonists NMDA and PPBP were added at 3 hours of perfusion to permit time for loading of the cells with labeled arginine. The inhibitors and antagonists (L-NNA, DuP 734, MK-801) were added at 2 hours of perfusion to permit tissue delivery before challenging with an agonist. Within each group, the particular drug combination was randomly assigned to either right or left striatum, and the effluent concentrations of [¹⁴C]L-citrulline were compared on a paired basis. Because recovery across the dialysis probe is 15% to 20% in vitro and because radial diffusion is expected to rapidly reduce the concentration of administered drugs in surrounding tissue, we infused 1 mmol/L concentration of NMDA to presumably attain 20 to 200 µmol/L interstitial concentrations.

In group 1 (n=8), perfusion with control aCSF was compared with perfusion with 1 mmol/L NMDA to demonstrate that NMDA enhances citrulline recovery in the striatum. To demonstrate that NMDA-evoked citrulline recovery is attenuated by the NOS inhibitor L-NNA, perfusion with 1 mmol/L NMDA was compared with perfusion with 1 mmol/L NMDA+1 mmol/L L-NNA in group 2 (n=7). In group 3 (n=8), perfusion with 1 mmol/L PPBP was compared with aCSF perfusion to test the hypothesis that PPBP attenuates basal labeled citrulline recovery. To test the hypothesis that NMDA-evoked NO production is attenuated by PPBP, perfusion with 1 mmol/L NMDA was compared with 1 mmol/L NMDA+1 mmol/L PPBP in group 4 (n=8). To test the hypothesis that PPBP does not attenuate basal citrulline recovery in the presence of NMDA receptor blockade, perfusion with 1 mmol/L MK-801 was compared with perfusion of 1 mmol/L MK-801+1 mmol/L PPBP in group 5 (n=8). In group 6 (n=8), 1 mmol/L NMDA+1 mmol/L MK-801 was compared with a perfusion of 1 mmol/L NMDA+1 mmol/L MK-801+1 mmol/L PPBP to test the hypothesis that PPBP does not attenuate NMDA-enhanced citrulline recovery in the presence of NMDA receptor blockade. To test the hypothesis that the attenuation of NMDA-evoked citrulline recovery by PPBP can be reversed by the selective ₁-receptor blocker DuP 734, perfusion with 1 mmol/L NMDA+1 mmol/L PPBP was compared with perfusion with 1 mmol/L NMDA+1 mmol/L PPBP+1 mmol/L DuP 734 in group 7 (n=7).

After 6 hours of perfusion, rats were killed with an intravenous injection of potassium chloride while still anesthetized, and dialysis probes were perfused with methylene blue. The brain was stored in 4% paraformaldehyde for 48 hours and then dissected to visualize the probe tracts.

Immunohistochemical Studies
Naive rats were given intravenous heparin and were perfused through the ascending aorta with 4% paraformaldehyde and 1% acrolein. Brains were removed, frozen en bloc, and stored at -80°C until processing. Immunohistochemical experiments were performed on 40-µm floating sections of tissue. The tissue was processed with the use of a peroxidase/antiperoxidase detection method with 3,3'-diaminobenzidine as the chromogen. Incubation with or without the primary antibody was performed at 4°C for 48 hours on a rocker plate. Nonspecific binding was inhibited by preincubating tissues with 4% goat serum. The neuronal and endothelial NOS antibodies were polyclonal rabbit antibodies that recognized a 155- and 135-kDa protein, respectively.³⁰

Materials
[¹⁴C]L-Arginine (317 mCi/mmol) was obtained from Amersham; L-NNA, NMDA, and MK-801 were obtained from Sigma Chemical Co; PPBP was prepared by Dr Kenji Hashimoto at the National Institute of Drug Abuse, and DuP 734 was obtained from Dupont Co. The endothelial NOS antibody was obtained from Transduction Laboratories.

Statistical Analysis
Within each group, the effluent citrulline data were analyzed by 2-way ANOVA; the 2 treatments delivered to the 2 striata were 1 within-subject factor, and the 6 hourly collections were a second within-subject factor. If the overall effect of treatment or the treatmentxtime interaction was significant, comparisons of mean values between the 2 treatments at individual time points were made by orthogonal contrasts. P<0.05 was considered significant. Data are presented as mean±SEM.

	Results

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At baseline the mean arterial blood pressure (105±5), arterial PCO₂ (38±1.5) and PO₂ (136±8), pH (7.37±.01), and rectal temperature (37.5±0.1°C) were within normal physiological ranges in all groups and remained unchanged during the entire experimental period (data not shown).

Immunohistochemistry of the neuronal and endothelial isoforms of NOS demonstrated the presence of the neuronal isoform in neurons and the endothelial isoform in the endothelium of blood vessels in the striatum of Wistar rats (Figure 1).

View larger version (150K): [in this window] [in a new window]   Figure 1. Photomicrograph demonstrating positive immunocytochemical staining for neuronal (A) and endothelial (B) NOS at high magnification (x100) in the naive rat striatum.

In the microdialysis studies, all groups of animals were perfused with aCSF containing labeled arginine bilaterally in the striatum for 3 hours before addition of NMDA or PPBP to "load" the cells with the labeled substrate. In group 1, perfusion with [¹⁴C]L-arginine in aCSF resulted in time-dependent increases in [¹⁴C]L-citrulline in the effluent. These time-dependent increases were presumed to represent the time required for transport of labeled arginine into the tissue and the time required for transport of labeled citrulline back to the probe. Upon switching the perfusion to NMDA, labeled citrulline recovery was markedly increased during the first 20-minute collection period, and this increase was sustained for the entire 3-hour experimental period compared with aCSF perfusion alone (Figure 2A). In group 2, perfusion with L-NNA after the second hour decreased labeled citrulline recovery during the first collection period compared with contralateral perfusion with aCSF alone. Upon switching to the combined perfusion of NMDA+L-NNA after the third hour, labeled citrulline recovery remained completely suppressed compared with contralateral perfusion with NMDA alone (Figure 2B).

View larger version (24K): [in this window] [in a new window]   Figure 2. [14C]L-Citrulline recovery in dialysis effluent from probes perfused with control aCSF in 1 striatum compared with 1 mmol/L NMDA on contralateral side starting at 3 hours (arrow) until the end of the experiment (n=8 rats) (A) and 1 mmol/L NMDA on 1 side compared with 1 mmol NMDA+1 mmol L-NNA (n=7 rats); treatment with L-NNA commenced 1 hour before (dashed arrow) treatment with NMDA (B). Values are mean±SEM; *P<0.05 between treatments.

In group 3, perfusion with PPBP decreased labeled citrulline recovery by the first collection period compared with perfusion with aCSF alone (Figure 3A). Citrulline recovery remained below basal levels for the entire 3-hour PPBP perfusion period. In group 4, combined perfusion with NMDA+PPBP produced a rapid decrease in labeled citrulline recovery in contrast to the increase seen with NMDA alone (Figure 3B). Citrulline recovery remained significantly suppressed throughout the subsequent 3-hour perfusion period.

View larger version (23K): [in this window] [in a new window]   Figure 3. [14C]L-Citrulline recovery in dialysis effluent from probes perfused with control aCSF in 1 striatum compared with 1 mmol/L PPBP on contralateral side starting at 3 hours until the end of the experiment (A) or 1 mmol/L NMDA and 1 mmol/L NMDA+1 mmol/L PPBP in the contralateral striatum (B). Arrow indicates the time of switching treatments. Values are mean±SEM (n=8 rats); *P<0.05 between treatments.

In group 5, perfusion of MK-801 bilaterally after the second hour decreased basal labeled citrulline recovery. There was no further decrease upon switching to perfusion with MK-801+PPBP after the third hour compared with MK-801 alone (Figure 4A). In group 6, perfusion of MK-801 bilaterally after the second hour decreased basal labeled citrulline recovery bilaterally (Figure 4B). Upon switching the perfusion to NMDA+MK-801, there was a small time-dependent increase in labeled citrulline recovery. Coadministration of PPBP with NMDA failed to attenuate this increase in the presence of MK-801.

View larger version (22K): [in this window] [in a new window]   Figure 4. [14C]L-Citrulline recovery in dialysis effluent from striatal probes perfused bilaterally with 1 mmol/L MK-801 starting after 2 hours of [14C]L-arginine perfusion (dashed arrow). A, 1 mmol/L PPBP was added to the infusate of 1 side after 3 hours of perfusion (solid arrow). B, 1 mmol/L NMDA was added to the infusate on 1 side and 1 mmol/L NMDA+1 mmol/L PPBP was added to the contralateral side after 3 hours of perfusion (solid arrow). There were no differences between treatment sides. Values are mean±SEM (n=8 rats).

In group 7, perfusion with DuP 734 after the second hour did not alter basal citrulline recovery (Figure 5). Coadministration of NMDA+PPBP after the third hour in the absence of DuP 734 rapidly decreased citrulline recovery. This decrease was not observed in the presence of DuP 734 (Figure 5).

View larger version (24K): [in this window] [in a new window]   Figure 5. [14C]L-Citrulline recovery in dialysis effluent from probes perfused with 1 mmol/L NMDA+1 mmol/L PPBP compared with 1 mmol/L NMDA+1 mmol/L PPBP+1 mmol/L DuP 734. Treatment with DuP 734 was started ipsilaterally after the second hour (dashed arrow) and continued through the sixth hour. NMDA+PPBP was added bilaterally after the third hour (solid arrow) of perfusion. Values are mean±SEM (n=7 rats); *P<0.05 between treatments.

Neuronal injury was assessed by cresyl violet staining 24 hours after removal of bilateral microdialysis probes in 3 rats. A margin of increased cellularity surrounding the probe tract was seen both on the side perfused for 6 hours with aCSF and on the side perfused for 6 hours with 1 mmol/L NMDA (Figure 6). There was no apparent difference in the thickness of this cell boundary layer between sides, and there was no observable necrosis beyond this boundary layer.

View larger version (112K): [in this window] [in a new window]   Figure 6. Photomicrographs demonstrating cresyl violet staining of the striatum after infusion of aCSF (A) and 1 mmol/L NMDA (B) for 6 hours. Animals were maintained for 24 hours after removal of probes, before perfusion with phosphate-buffered saline and 4% paraformaldehyde. A margin of increased cellularity surrounding the probe tract is seen both on the side perfused for 6 hours with aCSF and on the side perfused for 6 hours with 1 mmol/L NMDA, with no apparent difference in the thickness of this glial boundary layer and no observable necrosis beyond this boundary layer.

	Discussion

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The notable findings of this study are as follows: (1) NMDA enhanced citrulline recovery in the striatal microdialysates, and this enhancement is completely suppressed by the NOS inhibitor L-NNA; (2) NMDA-evoked citrulline recovery was markedly attenuated by the ₁-receptor ligand PPBP; (3) PPBP did not attenuate basal or NMDA-evoked citrulline recovery in the presence of NMDA receptor blocker MK-801; and (4) the ₁-receptor antagonist DuP 734 reversed the attenuating effect of PPBP on the NMDA-evoked citrulline recovery.

Technical Considerations
In this study, the tips of the microdialysis probes were localized in the caudate-putamen complex of the striatum, as confirmed by postmortem infusion of methylene blue dye. It is well established that the striatum is highly vulnerable to ischemia and excitotoxic glutamatergic injury.^{31 32 33} Using well-characterized antibodies, we confirmed the presence of neuronal and endothelial isoforms of NOS in the striatum, demonstrating that abundant NO synthetic mechanisms are present in the striatum. In the present study, NMDA enhanced striatal citrulline recovery, which was attenuated by the NOS inhibitor L-NNA. This observation confirms our previous findings^{23 25 34} demonstrating NMDA-evoked NO production in the rat hippocampus and lamb neocortex. The increase in radiolabeled citrulline recovery with NMDA receptor stimulation is assumed to reflect increased NO production because the responses were inhibited by nitroarginine in all 3 brain regions assayed.

Citrulline recovery is only an indirect marker of NO production in vivo because of the complex compartmental kinetics involving (1) diffusion of labeled arginine across the dialysis membrane, (2) diffusion through the tortuous interstitial space, (3) cellular uptake of labeled arginine, (4) cellular efflux of labeled citrulline, and (5) diffusion of labeled citrulline back to and across the dialysis membrane. The time-dependent increase in recovery of labeled citrulline during control perfusion with aCSF is presumed to reflect this complex kinetic process rather than an actual increase in NO synthesis over time. In our previous studies with microdialysis in hippocampus,^{23 24 25} we started perfusion with various combinations of agonists and antagonists simultaneously with labeled arginine. In the present study on striatum, we altered the experimental paradigm to include a 3-hour period of labeled arginine perfusion before adding an agonist. This 3-hour loading period allowed basal citrulline recovery to increase to levels that permitted decreases as well as increases to be detected with various pharmacological challenges. Moreover, we were able to reduce the sampling time from 60 to 20 minutes and thereby to improve the time resolution. We found that agents that inhibit citrulline recovery, such as L-NNA, PPBP, and MK-801, did so during the first 20-minute collection with no further decreases on subsequent samples. If the sum of the response time for the delivery of drugs through the dialysis probes plus the response time for labeled citrulline to be transported back across the dialysis membrane was in the order of tens of minutes, the first 20-minute collection value would be intermediate between the baseline and steady state values. Our observations that the value obtained from first 20-minute sample was close to the steady state response of these inhibitors indicate that the overall response time of the microdialysis system is less than a few minutes. This rapid response is consistent with the rapid decrease in cyclic GMP in microdialysates reported to result from nitroarginine infusion^{35 36} and with the rapid spread of infused radiolabeled sucrose from the dialysis probe (1-mm diameter spread by 14 minutes).³⁵

We used bilateral microdialysis perfusion in a paired experimental design to reduce interanimal variability arising from physiological factors, such as arterial blood pressure, blood gases, and depth of anesthesia. This approach assumes that the response to NMDA would normally be equivalent on the 2 sides in the absence of administration of an antagonist to only 1 side. Because treatments were randomly assigned to the left or right striatum, we did not believe it was necessary to perform an additional control series in which both sides received NMDA alone. It is conceivable that there are transcallosal effects between the striatum that would influence the magnitude of the evoked responses, but it seems unlikely that this effect would have a major qualitative impact on left-right comparisons with unilateral antagonist administration. When N-nitro-D-arginine methyl ester was given to 1 hippocampus as a control for the NOS inhibitor N-nitro-L-arginine methyl ester, the NMDA-evoked increases in citrulline recovery were equivalent on the 2 sides.²³

The 56 rats used in this study were not randomly assigned to the 7 experimental groups. Rather, the experiments for each group were performed over a 1- to 2-week period so that the same lot of labeled arginine and Dowex resin was used for each group to reduce within-group variability. Some differences in the absolute levels of citrulline recovery before addition of NMDA or PPBP are evident between different groups that received similar treatments. Some of this variability may be due to differences in the efficiency of arginine trapping by the Dowex column or to biological variability.

Although PPBP blocked the increase in citrulline recovery during the first hour of NMDA perfusion, citrulline recovery eventually began to increase from the low basal level (Figure 3B). Because NMDA is not rapidly metabolized, the tissue concentration of NMDA may have eventually increased to a level sufficient to partially counteract the inhibitory effect of PPBP. Even in the presence of MK-801, there is a small increase in citrulline recovery over time (Figure 4B). However, the increase in citrulline with PPBP+NMDA during the last 2 hours shown in Figure 3B is greater than that in another similarly treated group shown in Figure 5. As discussed above, interassay variability and anesthetic and physiological status of the animal may contribute to some of these differences between animal groups. Therefore, emphasis is placed on within-group comparisons.

Variability may also arise from tissue injury resulting from placement of a 300-µm diameter probe. We evaluated histology 24 hours after probe removal to permit time for any neuronal injury to mature. A rim of dense cellularity was observed around the probe tract that had been perfused with aCSF. However, the area of drug delivery and sampling by the microdialysis probe was much greater than this dense cellular rim. Autoradiography revealed that labeled sucrose spreads over a 1-mm diameter cylinder by 14 minutes of dialysis perfusion,³⁵ and we have found that labeled arginine spreads 3 mm by 1 hour of infusion.²⁵ Although probe insertion can cause transient disruption of the blood-brain barrier and an increase in the extracellular space in the immediate vicinity around the probe,^{36 37} the volume of tissue sampled by the probe probably extends well beyond the injured volume.

Injection of NMDA into the lateral ventricle of adult rats can cause injury to neurons and the blood-brain barrier in neighboring tissue.^{33 38} However, with infusion of 1 mmol/L into the dialysis probe, we did not observe substantial increases in the area of gliosis surrounding the probe tract or neuronal injury beyond the rim of gliosis. Thus, the amount of NMDA delivered into the tissue appeared to be below the toxic range.

-Receptor Ligand Effects
Others^{7 9 10 11} as well as our group have demonstrated that -receptor ligands ameliorate injury in animal models of transient focal ischemia. The mechanism of this protection is not completely understood. Several mechanisms of protection for -receptor ligands are supported by studies in vitro. For example, -receptor ligands inhibit ischemia-induced glutamate release in vitro, but they do not block glutamate release caused by high potassium or calcium.³⁹ In addition, -receptor ligands, particularly ₁-receptor ligands, reduce NMDA-induced increase in intracellular calcium in isolated neurons.⁴⁰ In hippocampal slices, there was more than an additive protective effect of combined administration of an NMDA receptor antagonist and a -receptor ligand,³⁹ presumably by decreasing the ratio of endogenously released glutamate to antagonist at the receptor. -Receptor ligands that protect against glutamate toxicity in neuronal cell culture decrease evoked increases in cyclic GMP without directly inhibiting NOS.⁸ Other neurotransmitter systems may also be involved in the neuroprotection seen with -receptor ligands. For example, (+)-pentazocine, as well as other -receptor ligands, inhibits stimulated striatal dopamine release.⁵ In vivo, -receptor ligands can prevent cortical spreading depression.⁴¹ In our previous studies, PPBP ameliorated ischemic injury in the cat model of transient focal ischemia, and this neuroprotection was not afforded by a more favorable redistribution of cerebral blood flow or effect on temperature.¹³ In the rat model of transient focal ischemia, (+)-pentazocine decreased infarction volume, whereas (-)-pentazocine did not demonstrate any neuroprotection.¹⁴ In the rat brain, (+)-pentazocine is a potent ligand for the ₁-receptor,⁴² while (-)-pentazocine appears to be a less selective drug that has activity primarily at µ-,-, and -opioid receptors.⁴³ Many -receptor ligands have alternative receptor effects that are concentration dependent. In this regard, both PPBP and (+)-pentazocine are considered selective ₁-receptor ligands,⁴⁴ but at high concentrations (+)-pentazocine is also an inhibitor of the NMDA receptor binding at the phencyclidine site.⁴⁵ Therefore, in the present study we used PPBP as the prototype of the ₁-receptor ligands and studied its effects on basal and NMDA-evoked NO production in vivo.

We found that administration of either PPBP or the noncompetitive NMDA antagonist MK-801 alone rapidly suppressed citrulline recovery. These observations suggest that tonic activation of NMDA receptors is a major stimulus to NOS activity under basal conditions in striatum and that ₁-receptor ligands are potent inhibitors of this basal activity. Moreover, coadministration of PPBP with NMDA in the microdialysis infusate not only inhibited the increase in citrulline recovery but rapidly decreased citrulline recovery to below basal levels. Thus, ₁-receptor ligands are potent inhibitors of NMDA-evoked activation of NO production.

-Receptor ligands may act through -receptors or directly on the NMDA receptor ion channel. For example, -receptor ligands have been reported to noncompetitively inhibit currents generated by NMDA receptors expressed in Xenopus oocytes,⁴⁶ thereby suggesting that these ligands can act directly on the NMDA receptor channel complex independent of -receptors. Our observation that PPBP produced no additional decrement of citrulline recovery in the presence of MK-801 supports this possibility. However, MK-801 by itself reduced basal citrulline recovery to extremely low levels. Any effect of PPBP, independent of NMDA receptor modulation, is difficult to detect in this situation. Moreover, a specific ₁-receptor antagonist reversed the suppressive effect of PPBP on NMDA-evoked citrulline recovery. We therefore believe that the primary site of action of PPBP is on ₁-receptors rather than directly on the NMDA receptor complex. -Receptors may act to modulate the signal transduction pathway between NMDA receptors and NOS activation.

In conclusion, this study demonstrates that the ₁-receptor ligand PPBP attenuates basal and NMDA-evoked NO production in the striatum, as measured indirectly by conversion of arginine to citrulline. This attenuation is possibly mediated by an interaction of the ₁-receptor with the signal transduction pathway between the NMDA receptor complex and the NOS enzyme. If it is assumed that excessive NO production during and after ischemia is neurotoxic, this inhibition may represent one mechanism by which ₁-receptor ligands exert their neuroprotective effect.

	Acknowledgments

 
This work was supported in part by US Public Health Service National Institutes of Health grant NS20020. Dr Bhardwaj is supported in part by the American Heart Association Clinician Scientist Award and the Richard S. Ross Clinician Scientist Award from the Johns Hopkins University School of Medicine.

Received April 14, 1998; revision received June 15, 1998; accepted August 11, 1998.

	References

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1. Maurice T, Privat A. SA4503, a novel cognitive enhancer with sigma₁ receptor agonist properties, facilitates NMDA receptor-dependent learning in mice. Eur J Pharmacol. 1997;328:9–18.[Medline] [Order article via Infotrieve]
2. Maurice T, Lockhart BP. Neuroprotective and anti-amnesic potentials of sigma receptor ligands. Prog Neuropyschopharmacol Biol Psychiatry. 1997;21:69–102.[Medline] [Order article via Infotrieve]
3. Bouchard P, Maurice T, St-Pierre S, Privat A, Quirion R. Neuropeptide Y and the calcitonin gene-related peptide attenuate learning impairments induced by MK-801 via a sigma receptor-related mechanism. Eur J Neurosci. 1997;9:2142–2151.[Medline] [Order article via Infotrieve]
4. Gonzalez-Alvear GM, Werling LL. Sigma receptor regulation of norepinephrine release from rat hippocampal slices. Brain Res. 1995;673:61–69.[Medline] [Order article via Infotrieve]
5. Gonzalez-Alvear GM, Werling LL. Regulation of [³H]dopamine release from rat striatal slices by sigma receptor ligands. J Pharmacol Exp Ther. 1994;271:212–219.[Abstract/Free Full Text]
6. Matsuno K, Matsunaga K, Senda T, Mita S. Increase in extracellular acetylcholine level by sigma ligands in rat frontal cortex. J Pharmacol Exp Ther. 1993;265:851–859.[Abstract/Free Full Text]
7. Carter C, Benavides J, Legendre P, Vincent JD, Noel F, Thuret F, Lloyd KG, Arbilla S, Zivkovic B, MacKenzie ET. Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents, II: evidence for N-methyl-D-aspartate receptor antagonist properties. J Pharmacol Exp Ther. 1988;247:1222–1232.[Abstract/Free Full Text]
8. Lesage As, De Loore KL, Peeters L, Leysen JE. Neuroprotective sigma ligands interfere with the glutamate-activated NOS pathway in hippocampal cell culture. Synapse. 1995;20:156–164.[Medline] [Order article via Infotrieve]
9. Lysko PG, Feuerstein G. Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: protection by SKF 10,047. Neurosci Lett. 1990;120:217–220.[Medline] [Order article via Infotrieve]
10. Contreras PC, Gray NM, Ragan DM, Lanthorn TH. BMY-14802 protects against ischemia-induced neuronal damage in the gerbil. Life Sci. 1992;51:1145–1149.[Medline] [Order article via Infotrieve]
11. O'Neill M, Caldwell M, Earley B, Canney M, O'Halloran A, Kelly J, Leonard BE, Junien JL. The sigma receptor ligand JO 1784 (igmesine hydrochloride) is neuroprotective in the gerbil model of global cerebral ischemia. Eur J Pharmacol. 1995;283:217–225.[Medline] [Order article via Infotrieve]
12. Takahashi H, Kirsch JR, Hashimoto K, London ED, Koehler RC, Traystman RJ. PPBP [4-phenyl-1-(4-phenylbutyl) peperidine] decreases brain injury after transient focal ischemia in rats. Stroke. 1996;27:2120–2123.[Abstract/Free Full Text]
13. Takahashi H, Kirsch JR, Hashimoto K, London ED, Koehler RC, Traystman RJ. PPBP [4-phenyl-1-(4-phenylbutyl) peperidine], a potent -receptor ligand, decreases brain injury after transient focal ischemia in cats. Stroke. 1995;26:1676–1682.[Abstract/Free Full Text]
14. Takahashi H, Traystman RJ, Hashimoto K, London ED, Kirsch JR. Postischemic brain injury is affected stereospecifically by pentazocine in rats. Anesth Analg. 1997;85:353–357.[Abstract]
15. Hanner M, Moebius FF, Flanorfer A, Knaus HG, Striessnig J, Kempner E, Glossman H. Purification, molecular cloning, and expression of the mammalian sigma₁-binding site. Proc Natl Acad Sci U S A. 1996;93:8072–8077.[Abstract/Free Full Text]
16. Dawson VL, Dawson TM, Bartley DA, Uhl GR, Snyder SH. Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J Neurosci. 1993;13:2651–2661.[Abstract]
17. Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role of nitric oxide. Nature. 1990;347:768–770.[Medline] [Order article via Infotrieve]
18. Izumi Y, Clifford DB, Zorumski CF. Inhibition of long-term potentiation by NMDA-mediated nitric oxide release. Science. 1992;257:1273–1276.[Abstract/Free Full Text]
19. Iadecola C, Pelligrino DA, Moskowitz MA, Lassen N. Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab. 1994;14:175–192.[Medline] [Order article via Infotrieve]
20. Huang Z, Huang PL, Panahian N, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science. 1994;265:1883–1885.[Abstract/Free Full Text]
21. Zhang J, Dawson VL, Dawson TM, Snyder SH. Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science. 1994;263:687–690.[Abstract/Free Full Text]
22. Garthwaite J, Garthwaite G, Palmer RMJ. NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur J Pharmacol. 1989;172:413–416.[Medline] [Order article via Infotrieve]
23. Bhardwaj A, Northington FJ, Koehler RC, Steifel T, Hanley DF, Traystman RJ. Adenosine modulates N-methyl-D-aspartate–stimulated hippocampal nitric oxide production in vivo. Stroke. 1995;26:1627–1633.[Abstract/Free Full Text]
24. Bhardwaj A, Northington FJ, Ichord RN, Hanley DF, Traystman RJ, Koehler RC. Characterization of ionotropic receptor-mediated nitric oxide production in vivo in rats. Stroke. 1997;28:850–857.[Abstract/Free Full Text]
25. Bhardwaj A, Northington FJ, Martin LJ, Hanley DF, Traystman RJ, Koehler RC. Characterization of metabotropic glutamate receptor-mediated nitric oxide production in vivo. J Cereb Blood Flow Metab. 1997;17:153–159.
26. Lockhart BP, Soulard P, Benicourt C, Privat A, Junien J-L. Distinct neuroprotective profiles for ligands against N-methyl-D-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies. Brain Res. 1995;675:110–120[Medline] [Order article via Infotrieve]
27. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed. New York, NY: Academic Press Inc; 1986.
28. Johnson RD, Justice JB. Model studies for brain dialysis. Brain Res Bull. 1983;10:567–571.[Medline] [Order article via Infotrieve]
29. Van Wylen DGL, Park TS, Rubio R, Berne RM. The effect of local infusion of adenosine and adenosine analogues on local cerebral blood flow. J Cereb Blood Flow Metab. 1989;9:556–562.[Medline] [Order article via Infotrieve]
30. Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM, Snyder SH. Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of mammalian CNS together with NADPH diaphorase. Neuron. 1991;7:615–624.[Medline] [Order article via Infotrieve]
31. Benveniste H, Drejer J, Schousboe A, Diemer NH. Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient focal ischemia monitored by intracerebral microdialysis. J Neurochem. 1984;43:1369–1374.[Medline] [Order article via Infotrieve]
32. Fonnum F. Glutamate: a neurotransmitter in mammalian brain. J Neurochem. 1984;42:1–11.[Medline] [Order article via Infotrieve]
33. Dietrich WD, Alonso O, Halley M, Busto R, Globus MYT. Intraventricular infusion of N-methyl-D-aspartate, II: acute neuronal consequences. Acta Neuropathol (Berl). 1992;84:630–637.[Medline] [Order article via Infotrieve]
34. Northington FJ, Tobin JR, Koehler RC, Traystman RJ. In vivo production of nitric oxide with NMDA-induced cerebral hyperemia in newborn sheep. Am J Physiol. 1995;38:H215–H222.
35. Vallebuona F, Raiteri M. Extracellular cGMP in the hippocampus of freely moving rats as an index of nitric oxide (NO) synthase activity. J Neurosci. 1994;14:134–139.[Abstract]
36. Benveniste H. Brain microdialysis. J Neurochem. 1989;52:1667–1679[Medline] [Order article via Infotrieve]
37. Dykstra KH, Hsiao JK, Morrison PF, Bungay PM, Mefford IN, Scully MM, Dedrick RL. Quantitative examination of tissue concentration profiles associated with microdialysis. J Neurochem. 1992;58:931–940.[Medline] [Order article via Infotrieve]
38. Dietrich WD, Alonso O, Halley M, Busto R, and Globus MYT. Intraventricular infusion of N-methyl-D-aspartate, I: acute blood-brain barrier consequences. Acta Neuropathol (Berl). 1992;84:621–629.[Medline] [Order article via Infotrieve]
39. Lobner D, Lipton P. -Ligands and non-competitive NMDA antagonists inhibit glutamate release during cerebral ischemia. Neurosci Lett. 1990;117:169–174.[Medline] [Order article via Infotrieve]
40. Hayashi T, Kagaya A, Takebayashi M, Shimizu M, Uchitomi Y, Motohashi N, Yamawaki S. Modulation of -ligands of intracellular free Ca⁺⁺ mobilization by N-methyl-D-aspartate in primary culture of rat frontal cortical neurons. J Pharmacol Exp Ther. 1995;275:207–214.[Abstract/Free Full Text]
41. Willette RN, Lysko PG, Sauermelch CF. A comparison of (+)SF&F 10047 and MK-801 on cortical spreading depression. Brain Res. 1994;648:347–351.[Medline] [Order article via Infotrieve]
42. Cagnotto A, Bastone A, Mennini T. [³H](+)-Pentazocine binding to the rat brain ₁-receptors. Eur J Pharmacol. 1994;266:131–138.[Medline] [Order article via Infotrieve]
43. Picker MJ, Craft RM, Negus SS, Powell KR, Mattox SR, Jones SR, Hargrove BK, Dykstra LA. Intermediate efficacy µ opioids: examination of their morphine-like stimulus effects and response rate-deceasing effects in morphine-tolerant rats. J Pharmacol Exp Ther. 1992;263:668–681.[Abstract/Free Full Text]
44. Hashimoto K, London ED. Further characterization of [³H]ifenprodil binding to receptors in rat brain. Eur J Pharmacol. 1993;236:159–163.[Medline] [Order article via Infotrieve]
45. Lyengar S, Dilworth VM, Mick SJ, Contrras PC, Monahan JB, Rao TS, Wood PL. Sigma receptors modulate both A9 and A10 dopaminergic neurons in the rat brain: functional interaction with NMDA receptors. Brain Res. 1990;524:322–326.[Medline] [Order article via Infotrieve]
46. Whittemore ER, Ilyin VI, Woodward RM. Antagonism of N-methyl-D-aspartate receptors by sigma site ligands: potency, subtype-selectivity and mechanisms of inhibition. J Pharmacol Exp Ther. 1997;282:326–338.[Abstract/Free Full Text]

Editorial Comment

Frank M. Faraci, PhD, Guest Editor

Department of Internal Medicine Cardiovascular Division University of Iowa College of Medicine Iowa City, Iowa

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Under normal conditions, NO is produced in brain by the neuronal isoform of NOS (nNOS).¹ A major stimulus for production of NO by nNOS in neurons is activation of glutamate receptors. For example, recent molecular analysis revealed physical coupling (via postsynaptic density proteins) of nNOS to one subtype of glutamate receptor, the NMDA receptor.² Activation of NMDA receptors increases activity of nNOS, resulting in extracellular release of NO.³ Inhibitors of NMDA receptors or NOS attenuate basal and stimulated NO production in a variety of models (see References 4 and 5 for examples), and local dilatation of cerebral arterioles in response to activation of NMDA receptors is mediated by NO.⁶ Excessive activation of NMDA receptors can produce neurotoxicity and may contribute to brain damage after ischemia.

The present study makes a new contribution in this area by providing evidence that ₁-receptor ligands attenuate production of NO by nNOS under basal conditions and in response to activation of NMDA receptors. Previous studies suggested that -receptor ligands have neuroprotective effects in models of focal ischemia. Thus, inhibition of production of NO by nNOS is a possible mechanism by which 1-receptor agonist protects brain after ischemia.

Received April 14, 1998; revision received June 15, 1998; accepted August 11, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell.. 1993;75:1273–1286.[Medline] [Order article via Infotrieve]
2. Brenman JE, Christopherson KS, Craven SE, McGee AW, Bredt DS. Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein. J Neurosci.. 1996;16:7407–7415.[Abstract/Free Full Text]
3. Garthwaite J, Charles SL, Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature.. 1988;336:385–388.[Medline] [Order article via Infotrieve]
4. Luo D, Knezevich S, Vincent SR. N-methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study. Neuroscience.. 1993;57:897–900.[Medline] [Order article via Infotrieve]
5. Vallebuona F, Raiteri M. Extracellular cGMP in the hippocampus of freely moving rats as an index of nitric oxide (NO) synthase activity. J Neurosci. 1994;14:134–139.
6. Faraci FM, Breese KR. Nitric oxide mediates vasodilatation in response to activation of N-methyl-D-aspartate receptors in brain. Circ Res.. 1993;72:476–480.[Abstract/Free Full Text]

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