(Stroke. 1997;28:850-857.)
© 1997 American Heart Association, Inc.
Articles |
Characterization of Ionotropic Glutamate Receptor–Mediated Nitric Oxide Production In Vivo in Rats
From the Departments of Neurology (A.B., R.N.I., D.F.H.), Anesthesiology/Critical Care Medicine (A.B., D.F.H., R.J.T., R.C.K.), and Pediatrics (F.J.N., R.N.I.), 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.
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Background and Purpose Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel–linked N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methylisoxazole-4-propionic
acid (AMPA) receptor subtypes on NO production in vivo in rat
hippocampus. We also studied whether dantrolene, a ryanodine calcium
channel inhibitor previously shown to attenuate
metabotropic glutamate receptor stimulation of NO production,
also attenuated ionotropic glutamate receptor–mediated stimulation of
NO production.
Methods Microdialysis probes were placed bilaterally into
the CA3 region of the hippocampus of pentobarbital-anesthetized
adult Sprague-Dawley rats and were perfused for 5 hours with artificial
cerebrospinal fluid (CSF) containing 3 µmol/L
[14C]L-arginine. Recovery of
[14C]L-citrulline in the effluent was used as
a marker of NO production. In 13 groups of rats, increases in
[14C]L-citrulline recovery were compared
between right- and left-sided probes perfused with no additional drugs
versus combinations of NMDA, AMPA, the NO synthase
inhibitor
N
-nitro-L-arginine methyl ester
(L-NAME), the noncompetitive glutamate receptor blocker MK-801, the
AMPA receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene.
Results Recovery of [14C]L-citrulline during perfusion with artificial CSF progressively increased to 272±73 fmol/min (±SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [14C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [14C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [14C]L-citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [14C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene.
Conclusions Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.
Key Words: hippocampus • N-methyl-D-aspartate • nitric oxide • rats
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Introduction |
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Nitric oxide plays a multifaceted role in the brain as a neurotransmitter1 2 and a regulator of cerebral blood flow.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 through 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 Furthermore, stimulation of NMDA receptors stimulates release of glutamate,8 which can then act on
other glutamate receptor subtypes. This amplifying effect was attenuated by NOS inhibition.9
It is well established that the hippocampus is one of the regions most vulnerable to ischemia.10 The synaptic release of glutamate is considered an important factor in the development of neuronal death. The hypothesis is supported by experiments in which surgical transection of glutamate-containing afferents to the hippocampus was performed, thereby inhibiting glutamatergic neurotransmission and leading to a decrease in ischemic cell damage ipsilateral to the damage.11 However, it is not fully understood which of the glutamate receptors activate the deleterious intracellular processes leading to neuronal death. Although studies in vitro emphasize involvement of the NMDA receptor complex,5 studies of forebrain ischemia in vivo generally find greater neuroprotection with antagonists of AMPA receptors.12 13 Therefore, it is useful to evaluate whether AMPA receptor activation stimulates NO production in vivo. Work by others indicates that kainate increases conversion of oxyhemoglobin to methemoglobin in hippocampal microdialysates14 and that AMPA increases cyclic GMP in cerebellar microdialysates.15
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.16 17 18 We have previously shown that NMDA enhances citrulline recovery in the hippocampus.16 Because basal and NMDA-induced increases in citrulline recovery were inhibited by the NOS inhibitor L-NAME but not by D-NAME, [14C]L-citrulline recovery appears to provide an indirect marker of NO production. Furthermore, we found that agonist stimulation of metabotropic glutamate receptor increased citrulline recovery and that dantrolene, an inhibitor of ryanodine-sensitive calcium channels, attenuated the metabotropic-induced response.17 It is also possible that release of intracellular calcium stores through ryanodine receptors amplifies NO production by NMDA and AMPA receptor activation.19 20
Using this technique, we tested the hypotheses that (1) stimulation of NMDA and AMPA receptors increases NO production in rat hippocampus in vivo; (2) antagonists of AMPA and NMDA receptors reduce basal NO production; (3) administration of an antagonist of AMPA receptors (CNQX) attenuates NMDA-evoked increases in NO production, whereas administration of an antagonist of NMDA receptors (MK-801) attenuates AMPA-evoked increase in NO production, thereby suggesting involvement of multisynaptic pathways; and (4) administration of the intracellular ryanodine calcium channel inhibitor dantrolene attenuates NMDA- and AMPA-evoked increases in NO production, as was found for metabotropic receptor activation.
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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 (weight, 300 to 450 g) were anesthetized with pentobarbital (60 mg/kg IP). Additional pentobarbital was given during the course of the experiment. Rats were tracheotomized and mechanically ventilated with a mixture of oxygen and nitrogen to maintain the following: arterial pH=7.35 to 7.40, PCO2
35 mm Hg, and
PO2150 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 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 8 mm from the dura).21 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 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 Justice22 and modified by Van
Wylen et al.23 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 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.23 Starting 1 hour after insertion, 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% N2/5% CO2 until O2 and
CO2 tensions were similar to CSF and brain
tissue.23
We estimated NO production 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 during 1-hour periods 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, 60 µL artificial CSF 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 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 13 treatment groups to receive
perfusates containing various combinations of NMDA, AMPA,
L-NAME, MK-801, CNQX, and dantrolene 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. Because recovery across the dialysis probe was approximately
10% 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 and AMPA to presumably
attain 10 to 100 µmol/L interstitial
concentrations.
In group 1 (n=7), perfusion with control CSF was compared with perfusion with 1 mmol/L L-NAME to demonstrate that the NOS inhibitor reduces basal citrulline recovery. To demonstrate that NMDA-enhanced citrulline recovery is attenuated by L-NAME, perfusion with control CSF was compared with perfusion with 1 mmol/L NMDA in group 2 (n=5), and perfusion with 1 mmol/L NMDA was compared with perfusion with 1 mmol/L NMDA+1 mmol/L L-NAME in group 3 (n=5). To test the hypothesis that MK-801 reduces basal as well as NMDA-enhanced NO production, perfusion with control CSF was compared with perfusion with 1 mmol/L MK-801 in group 4 (n=7), and perfusion with 1 mmol/L NMDA was compared with perfusion with 1 mmol/L NMDA+1 mmol/L MK-801 in group 5 (n=8). To test the hypothesis that NMDA-enhanced NO production is attenuated by CNQX and by dantrolene, perfusion with 1 mmol/L NMDA was compared with perfusion with 1 mmol/L NMDA+1 mmol/L CNQX in group 6 (n=7), and perfusion with 1 mmol/L NMDA was compared with perfusion with 1 mmol/L NMDA+1 mmol/L dantrolene in group 7 (n=7). To test the hypothesis that AMPA enhances NO production, perfusion with control CSF was compared with perfusion with 1 mmol/L AMPA in group 8 (n=7), and perfusion with 1 mmol/L AMPA was compared with perfusion with 1 mmol/L AMPA+1 mmol/L L-NAME in group 9 (n=7). To test the hypothesis that CNQX reduces basal as well as AMPA-enhanced NO production, perfusion with control CSF was compared with perfusion with 1 mmol/L CNQX in group 10 (n=7), and perfusion with 1 mmol/L AMPA was compared with perfusion with 1 mmol/L AMPA+1 mmol/L CNQX in group 11 (n=7). To test the hypothesis that AMPA-enhanced NO production is attenuated by MK-801 and by dantrolene, perfusion with 1 mmol/L AMPA was compared with perfusion with 1 mmol/L AMPA+1 mmol/L MK-801 in group 12 (n=7), and perfusion with 1 mmol/L AMPA was compared with perfusion with 1 mmol/L AMPA+1 mmol/L dantrolene in group 13 (n=7).
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. The brain was stored in 4% paraformaldehyde for 48 hours and then dissected to visualize the probe tracts.
Autoradiography
Autoradiography was used to assess the radial
spread of label away from the dialysis probe as a function of perfusion
duration. Rats were prepared and cannulas placed in the same manner as
for control experiments described above. Infusions of CSF containing
3 µmol/L [14C]L-arginine at 1 µL/min
were performed to 1, 2, 3, 4, and 5 hours. Two or three animals were
studied for each infusion duration, with two cannulas placed
bilaterally in the hippocampus in each animal. At the end of the
designated infusion period, the animals were killed with potassium
chloride under anesthesia. The brain was removed, rinsed
with saline, divided into three coronal blocks, frozen in dry ice, and
stored at -70°C until sectioning. The middle coronal block
containing the cannula track was sectioned at a thickness of 20
µm in a cryostat at -20°C. Three contiguous sections taken at
200-µm intervals through the full thickness of the block were
thaw-mounted on triple-subbed slides. After it was air-dried and
equilibrated to room temperature, the middle section of each level was
placed on tritium-sensitive film (Leica Ultrofilm) along with standards
of 14C. Films were developed after incubation at -4°C
for 1 week and analyzed through a high-resolution CCD video
camera (Sierra Scientific) on an illuminator with variable
intensity (Northern Light). Displayed images were digitized and
quantitated with a microcomputer imaging device system (Imaging
Research Inc.). After calibration for each film for optical density
units and linear distance, each section was analyzed for
diameter. Diameter was determined as the short axis of the image,
viewed within an overlay of tissue border for consistency
of orientation. Sections were excluded if the image appeared to have
lost reasonable preservation of tissue architecture. Neighboring
sections were stained with cresyl violet to verify tissue architecture
and orientation of cannulas. The maximum value of the short axis
diameter was determined for each cannula site studied.
Materials
[14C]L-Arginine (317 mCi/mmol) was
obtained from New England Nuclear Products; L-NAME, NMDA, and
AMPA were obtained from Sigma Chemical Co; and MK-801, CNQX, and
dantrolene were obtained from Research Biochemical
International.
Statistical Analysis
Within each group, the effluent citrulline data were
analyzed by two-way ANOVA; the two treatments delivered to the
two hippocampi were one within-subject factor, and the five 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 two treatments at individual
time points were made by orthogonal contrasts.
P<.05 was considered significant. Data are presented as mean±SEM.
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Arterial blood pressure, PCO2, pH, and rectal temperature were within the normal physiological range in all groups, and all rats were well oxygenated.
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 are 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. In the
side perfused with 1 mmol/L L-NAME, recovery of labeled citrulline
was significantly attenuated from the second through the fifth hour of
perfusion compared with the control side (Fig 1
). In
group 2, perfusion with 1 mmol/L NMDA increased citrulline
recovery from the second to the fifth hour compared with perfusion
with artificial CSF alone (Fig 2). In group 3, combined
perfusion with 1 mmol/L NMDA+1 mmol/L L-NAME reduced the
citrulline recovery compared with 1 mmol/L NMDA alone (Fig 2).
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In group 4, perfusion with 1 mmol/L MK-801 attenuated the
citrulline recovery compared with CSF alone (Fig 3). In
group 5, combined perfusion with 1 mmol/L NMDA+1 mmol/L
MK-801 attenuated the citrulline recovery compared with NMDA alone (Fig 3).
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In group 6, perfusion with 1 mmol/L NMDA+1 mmol/L CNQX
attenuated the citrulline recovery compared with perfusion with 1
mmol/L NMDA (Fig 4). In group 7, combined perfusion with
1 mmol/L NMDA+1 mmol/L dantrolene did not significantly
change the citrulline recovery compared with perfusion with 1
mmol/L NMDA alone (Fig 4).
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In group 8, perfusion with 1 mmol/L AMPA increased the citrulline
recovery compared with perfusion with CSF alone (Fig 5).
In group 9, combined perfusion with 1 mmol/L AMPA+1 mmol/L
L-NAME attenuated the citrulline recovery compared with AMPA alone (Fig 5).
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In group 10, perfusion with 1 mmol/L CNQX attenuated the
citrulline recovery from the second to the fifth hour compared with
perfusion with artificial CSF alone (Fig 6). In group
11, combined perfusion of 1 mmol/L AMPA+1 mmol/L CNQX reduced
the citrulline recovery compared with 1 mmol/L AMPA alone (Fig 6).
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In group 12, combined perfusion with 1 mmol/L AMPA+1 mmol/L
MK-801 attenuated the citrulline recovery compared with AMPA alone
(Fig 7). In group 13, combined perfusion with 1
mmol/L AMPA+1 mmol/L dantrolene did not significantly change the
citrulline recovery compared with AMPA alone (Fig 7).
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Analysis of autoradiograms revealed that the
maximum short axis of the 14C label surrounding cannula
tracks approached 3 mm by 1 hour of
[14C]L-arginine perfusion and changed very
little with longer perfusion times (Table).
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Discussion |
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The significant findings of this study are as follows: (1) MK-801 and CNQX reduce basal citrulline recovery, suggesting that tonic activity of NMDA and AMPA receptors contributes to basal NO production in rat hippocampus; (2) enhanced citrulline recovery with NMDA is partially inhibited by the AMPA antagonist CNQX, whereas enhanced citrulline recovery with AMPA is partially inhibited by the NMDA antagonist MK-801, thereby suggesting a multisynaptic amplification of NO production with ionotropic glutamate receptor activation; and (3) NMDA- or AMPA-enhanced citrulline recovery is not attenuated by dantrolene, suggesting that intracellular ryanodine calcium channels do not act to amplify NO production with ionotropic glutamate receptor activation.
The tip of the microdialysis probe was verified to be in the CA3 region of the hippocampus, where we previously confirmed the presence of neuronal NOS.16 The endothelial isoform may also be present in pyramidal neurons.24 Although the source of NO with glutamate receptor stimulation is likely to be from neurons containing either isoform of NOS, we cannot exclude some contribution from astrocytes. Astrocytes also express glutamate receptors, and immunologic NOS can be induced in cultured astrocytes.25 However, changes in citrulline recovery were evident within 2 to 3 hours of NMDA or AMPA perfusion. It is unclear whether adequate immunologic NOS would be expressed by that time. The increase in radiolabeled citrulline recovery with glutamate receptor stimulation is assumed to reflect increased NO production because the responses are inhibited by L-NAME. In addition, we previously showed that the NMDA response was not inhibited by the stereoisomer D-NAME, thereby supporting the specificity for NOS inhibition.16
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. In addition, some portion of labeled citrulline that is released may be taken up by other cells and recycled back to arginine. The time-dependent increase in recovery of labeled citrulline with control perfusion of artificial CSF is presumed to reflect this complex kinetic process rather than an actual increase in NO synthesis over time. Another underlying assumption of the technique is that alterations in citrulline recovery with various glutamate agonists and antagonists reflect changes in NO synthesis and not changes in arginine and citrulline transport kinetics. Other measures of NO production, such as increased cyclic GMP in microdialysis effluent with NMDA perfusion15 and increased methemoglobin formation during microdialysis perfusion with hemoglobin plus kainate,14 support the assumption that increased citrulline recovery with NMDA and AMPA perfusion of hippocampus reflects increased NO production.
Our autoradiography results indicate that the label spread radially from the dialysis probe with a maximum diameter of approximately 3 mm by 1 hour of perfusion. The lack of further spread of label with perfusion as long as 5 hours suggests that the cellular uptake limits further diffusion away from the probe site. It was difficult to quantify changes in the spatial concentration profile over time because the plane of section was not perfectly parallel to the probe tract and the probe tract intersected the plane of section at an unknown angle.
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. In addition, the 88 rats used in this study were not randomly assigned to the 13 experimental groups. Rather, the experiments for each individual 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 are evident between different groups receiving similar treatments, eg, control CSF, NMDA, or AMPA. Some of this variability may be due to differences in the efficiency of arginine trapping by the Dowex column or to biological variability.
Labeled citrulline recovery was attenuated by MK-801 and to a lesser degree by CNQX compared with control CSF alone. This indicates that there is a basal NO production through tonic activation of both NMDA and AMPA receptors. These results are consistent with decreased basal citrulline recovery during infusion of 2-chloroadenosine,16 18 which is thought to decrease glutamate release by stimulating presynaptic adenosine receptors. Moreover, the present experiments were performed with the use of pentobarbital anesthesia, which is expected to decrease spontaneous neural activity. Thus, in the unanesthetized state, the contribution of these glutamate receptors to tonic NO production may be greater.
The NMDA-enhanced citrulline recovery was markedly attenuated by MK-801 and partially attenuated by CNQX. The AMPA-enhanced citrulline recovery was partially attenuated by the addition of MK-801 and partially attenuated by CNQX. These data suggest that ionotropic glutamate receptor–mediated NO production may involve NMDA and AMPA acting through multisynaptic pathways rather than solely on NOS neurons. NMDA receptor stimulation may facilitate further downstream glutamate release,8 which in turn could act on AMPA receptors on NOS-containing neurons. Likewise, AMPA receptor stimulation could enhance downstream glutamate release, which could then act on NMDA receptors on NOS-containing neurons.
Stimulation of NMDA receptors on NOS neurons is expected to increase calcium influx and increase NOS activity. Some AMPA receptor subtypes also permit calcium entry.26 In addition, AMPA-induced depolarization is likely to open voltage-sensitive calcium channels and permit calcium entry.20 The increase in calcium arising from either NMDA or AMPA receptor stimulation may be amplified by calcium-induced calcium release from intracellular stores. To test this possibility, we used dantrolene, which inhibits intracellular ryanodine-sensitive calcium channels and calcium-induced calcium release. In cultured neurons, dantrolene has been reported to attenuate the intracellular calcium response to glutamate and NMDA but not to AMPA.19 20 27 Ryanodine-sensitive calcium channels have been demonstrated in the hippocampus.28 In cortical and hippocampal slices, use of caffeine to stimulate release of calcium from intracellular stores failed to significantly increase NOS activity.29 In an experimental paradigm similar to that used in the present study, we previously found that 1 mmol/L dantrolene infused in the microdialysis did not decrease citrulline recovery below control levels, thereby indicating that ryanodine-sensitive calcium channels do not make a major contribution to basal NO production under this experimental condition.17 However, this dose of dantrolene did attenuate citrulline recovery when metabotropic glutamate agonist was infused.17 Thus, the dose and route of delivery of dantrolene used in the present study were sufficient to exert a positive pharmacological effect on citrulline recovery. However, dantrolene did not attenuate NMDA- or AMPA-enhanced citrulline recovery in the present study. These results suggest that stimulation of ryanodine-sensitive calcium channels is not a prominent mechanism for amplifying NO production with either NMDA or AMPA receptor activation, in contrast to the amplified NO production seen with metabotropic activation.
In conclusion, this study demonstrates that AMPA as well as NMDA enhances NO production in the rat hippocampus. The increase in NO production with either agonist is possibly amplified by stimulation of other ionotropic glutamate receptors through multisynaptic activation. Amplification of NO production by intracellular ryanodine-sensitive receptors appears to be of minor importance when ionotropic glutamate receptors are stimulated.
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Acknowledgments |
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This study was supported in part by US Public Health Service (PHS) National Institute of Neurological Disorders and Stroke (NINDS) grant P01-NS20020. Dr Bhardwaj was supported in part by the Clinician Scientist Award from the American Heart Association and the Richard S. Ross Clinician Scientist Award from the Johns Hopkins University School of Medicine. Dr Northington was supported by US PHS NINDS grant K08-1742 and by a Grant-in-Aid from the American Heart Association with funds supplied by the Maryland Affiliate. Dr Ichord was supported in part by the Merck Clinician Scientist Award from the Johns Hopkins University School of Medicine and by US PHS NINDS grant K08-NS01805. Autoradiographic facilities were provided by the neuroscience laboratory of Dr Michael Johnston at the Kennedy Krieger Institute, Baltimore, Md.
Received September 11, 1996; revision received November 12, 1996; accepted December 12, 1996.
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