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CARDIOVASCULAR

N-Methyl-D-aspartate Receptor Blockade Inhibits Cardiac Inflammation in the Mg²+-Deficient Rat

Division of Experimental Medicine, Departments of Biochemistry and Molecular Biology (M.I.T., J.J.C., I.T.M., W.B.W.), Neurosurgery (G.G.), and Medicine (W.B.W.), George Washington University Medical Center, Washington, DC

Received April 15, 2004; accepted May 27, 2004.

	Abstract

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Elevated plasma levels of the neuropeptide substance P (SP) precede the perivascular inflammatory infiltrate seen in hearts of Mg²⁺-deficient (MgD) animals. The N-methyl-D-aspartate (NMDA) receptor is found in neurons, and activation of this receptor participates in SP release; under normal circumstances, this release can be blocked by Mg²⁺. Therefore, we reasoned that blockade of the NMDA receptor with dizolcipine maleate (a noncompetitive NMDA receptor antagonist) would prevent SP release from C-fibers due to MgD. In this study, animals were implanted with slow-release pellets containing dizolcipine or placebo and were fed with diet sufficient in Mg²⁺ or deficient with only 9% of USDA-recommended Mg²⁺. SP immunostaining of dorsal root ganglia showed a time-dependent depletion of SP in the MgD animals, with a dramatic decrease of SP by week 2; this depletion was prevented by pretreatment with dizolcipine maleate. The significant increase in plasma prostaglandin E₂ levels during MgD was prevented by dizolcipine, and the loss of total red blood cell glutathione content was significantly attenuated by NMDA blockade after 3 weeks of MgD (p < 0.01 versus controls). Immunohistochemical and Western blot analyses of ventricular tissue demonstrated that NMDA receptor blockade abolished MgD-related increase of endothelium adhesion molecule CD54 (weeks 1 and 2; p < 0.05), and of monocyte/macrophage surface protein CD11b expression (week 3; p < 0.05). We conclude that NMDA receptor blockade with dizolcipine maleate prevented SP depletion and reduced perivascular inflammatory infiltrates, thus decreasing cardiac injury due to Mg²⁺ deficiency.

Evidence from our laboratory suggests that severe dietary Mg²⁺ restriction causes the release of substance P (SP) from neural stores and that specific antagonists of this neuropeptide receptor can ameliorate most of the cardiovascular pathology associated with this reduction (Weglicki et al., 1994a). Furthermore, Richardson et al. (2003) recently suggested that SP presence in the heart is limited to extrinsic nerve fibers only, most likely originating from neurons located in cervical and upper thoracic dorsal root ganglia (DRG). Activation of the N-methyl-D-aspartic acid (NMDA) receptor has been associated with SP release from the central processes of primary afferent neurons (whose bodies are located in the DRG) in the dorsal horn of the spinal cord (Liu et al., 1997), and with SP and calcitonin gene-related peptide release from peripheral afferent nerve endings (McRoberts et al., 2001). Under normal conditions, Mg²⁺ exerts a voltage-dependent block on the NMDA receptor channel (Crunelli and Mayer, 1984; Mayer et al., 1984), and reduced extracellular Mg²⁺ lowers the threshold for its physiological activation (Nowak et al., 1984). This enhanced activation of the NMDA receptor could be responsible for triggering the neurogenic cardiovascular inflammation associated with dietary Mg²⁺ restriction. Thus, the increased release of neurotransmitters, including SP, would possibly exaggerate cellular responses, resulting in increased production of oxidative stress mediators, such as cytokines, oxy-radicals and nitric oxide, which could subsequently cause significant tissue injury. Still, the specific role of NMDA receptor activation in the inflammatory response seen in severe Mg²⁺ deficiency remains unclear. Thus, we propose that blockade of the NMDA receptor with the noncompetitive antagonist dizolcipine maleate in dorsal root ganglia neurons would abolish cardiac inflammation due to severe dietary deficiency of Mg²⁺ in the rat.

	Materials and Methods

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Experimental Protocol
All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by the National Institutes of Health (publication 85-23, revised 1996). Male Sprague-Dawley outbred rats (Harlan, Indianapolis, IN) about 7 weeks old were housed under 12-h light/dark cycle, with free access to bidistilled water and food. After 1-week adaptation, animals were randomly assigned to the different experimental groups. Dizolcipine maleate (also known as MK-801; Sigma-Aldrich, St. Louis, MO) was obtained as powder and then formulated as slow-release pellets (Innovative Research of America, Sarasota, FL), equivalent to a release dose of 0.5 mg/kg/day. Pellet matrix alone was used as placebo. Pellets were subcutaneously implanted on day 1 as described previously (Weglicki et al., 1994b). After the surgery, animals were fed with rat chow containing 100 or 9% of RDA of Mg²⁺ content. Four treatment groups were examined in this study: animals implanted with placebo pellets were fed control diet (MgS; n = 15) or rat chow with 9% of RDA Mg²⁺ (MgD; n = 15); and rats implanted with dizolcipine maleate pellets were fed control diet (S-Diz; n = 9) or rat chow with 9% of RDA Mg²⁺ (D-Diz; n = 16). Treatment lasted 1, 2, or 3 weeks, when the animals were sacrificed with an overdose of ether. Of 55 animals used in the study, only one died during ether anesthesia. Once removed, hearts were dissected immediately: one-half were snap-frozen in liquid N₂ and the other half was embedded in OCT compound and frozen in methyl-butane cooled in dry ice. Upper thoracic and lower cervical DRG were removed and snap-frozen in liquid N₂. All tissue samples were stored at –70°C until further use.

Plasma PGE₂ and Red Blood Cell (RBC) Glutathione
When under ether anesthesia, heparinized blood samples were withdrawn from vena cava. The RBCs were centrifuged within 20 min to be separated from the plasma. The plasma samples were quickly frozen and stored at –70°C for later analysis of PGE₂. Total RBC glutathione was determined by the enzymatic recycling method as described previously (Mak et al., 1994, 1996). Red cell hemoglobin was determined by the cyanmethemyoglobin method using Sigma Diagnostics hemoglobin reagent. Plasma PGE₂ levels were determined quantitatively by a PGE₂ enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN).

Immunohistochemistry
Immunofluorescence Assays. OCT-embedded cardiac and DRG tissues were sliced into 8- to 10-µm sections. In situ localization of SP and ICAM was accomplished with indirect fluorescent immunohistochemistry. Primary antibodies against SP (1:500; rabbit) and ICAM (1:50; mouse) were acquired from Chemicon International (Temecula, CA). Air-dried tissue sections were rinsed in Tris-buffered saline (pH 7.5) and incubated 45 min in blocking buffer (10% goat serum, 1% bovine serum albumin, 0.05% Triton-100 in Tris-buffered saline). All antibodies were diluted in blocking buffer before addition to tissue sections. After overnight incubation with primary antibody at 4°C, tissue sections were washed and incubated at room temperature in a dark humidity chamber with secondary antibody conjugated with fluorescein isothiocyanate, Alexa 488, or Texas Red-Alexa 590 (1:500; Molecular Probes, Eugene, OR) for 1 h. Once washed and dried, the slides were mounted using Vectashield H-1000 (Vectra, Cheshire, UK) as mounting medium. Samples were then examined under a fluorescence microscope (Olympus BX60; Olympus America Inc., Melville, NY), and multiple microphotographs taken with an Evolution Color MP camera (Media Cybernetics, Silver Spring, MD). Negative controls were included in all assays.

Immunoperoxidase Staining Protocol. Vectastain Elite ABC kit immunoperoxidase system (Vector Laboratories, Burlingame, CA) was used for CD11b localization on ventricular sections. Sections were always washed between steps. Frozen cardiac tissue sections (8 µm in thickness) were treated with 3% hydrogen peroxide for 3 min to quench endogenous peroxidase and incubated 10 min with normal serum followed by overnight incubation with mouse anti-rat -M Integrin (CD11b) antibody (1:200; Chemicon International). Next day, sections were incubated 10 min with biotinylated secondary antibody, followed by 5-min incubation with Vectastain Elite ABC reagent, and finally submerged in peroxidase substrate solution to optimal stain development and counterstained with hematoxylin before mounting and examination under the microscope.

Western Blot Analyses of Ventricular Tissue
Snap-frozen heart tissue (n = 4–6/treatment group) was homogenized at 4°C with a tissue homogenizer in 5 volumes of radioimmunoprecipitation assay buffer, centrifuged at 33,000g for 15 min, and the pellets were discarded (Tejero-Taldo et al., 2002). Supernatant samples containing 50 or 75 µg of proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane as described previously (Tejero-Taldo et al., 2002). Membranes were probed overnight with specific antibodies against ICAM (1:100; mouse monoclonal; Serotec, Oxford, UK) and CD11b (1:200, goat; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with horseradish peroxidase-conjugated donkey anti-mouse (1:2000; Amersham Biosciences Inc., Piscataway, NJ) or goat (1:17,500; Santa Cruz Biotechnology, Inc.) antibodies. Membranes were developed using enhanced chemoluminescence (ECL Plus; Amersham Biosciences Inc.), and exposed to X-ray film. Optical densitometric evaluation was performed using a Personal Densitometer SI (Amersham Biosciences Inc.) and computerized analysis system (ImageQuant version 5.2; Amersham Biosciences Inc.). Arbitrary units calculated by the software for each band were normalized against background.

Statistical Analysis
Data are expressed as means ± S.E.M. Differences among treatment groups were analyzed with parametric one-way analysis of variance. When statistical significance was reached (p < 0.05), the analysis was completed with Bonferroni's multiple comparison test. All statistical analyses were performed using GraphPad Prism (version 3.03; GraphPad Software Inc., San Diego, CA).

	Results

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Substance P Immunofluorescence Changes in Dorsal Root Ganglia with NMDA Receptor Blockade in the Mg²⁺-Deficient Rat. Frozen sections (5–8 µm; n = 2–4/rat) of lower cervical and upper thoracic DRG were immunostained with SP antibodies at the end of weeks 1, 2, and 3 of the experimental protocol. A constant and diffuse pattern of positive green fluorescence is appreciable in MgS animals at all times (Fig. 1). Severe MgD resulted in apparent decrease of fluorescence over time, with almost nonexisting green staining by the end of week 2 (MgD; Fig. 1). Concurrent treatment in vivo with dizolcipine maleate (NMDA receptor antagonist) did not cause any apparent changes on SP immunostaining of DRG (S-Diz; Fig. 1), importantly, it seemed to prevent the loss of fluorescent staining in DRG of MgD rats at weeks 2 and 3 of the experimental protocol (D-Diz; Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1. SP immunostaining of the dorsal root ganglia. Cervical and upper thoracic DRG collected from placebo animals on control diet (MgS; 4–6) or on diet low in Mg2+ (MgD; 4–6) and from animals treated with dizolcipine both on control diet (S-Diz; 3) or on MgD diet (D-Diz; 4–6) were cut into 8-µm sections. DRG sections were indirectly immunostained for SP with antibody conjugated with Alexa 488 (green fluorescence). Microphotographs are arranged by treatment group (columns) and length of treatment (rows). Original magnification, 10x.

NMDA Blockade-Related Changes in Mg²⁺ Deficiency-Induced Systemic Stress. Total RBC glutathione and plasma PGE₂ concentration were used as indexes of systemic stress and were measured at the end of week 3 of treatment. Severe MgD resulted in a significant depletion of RBC glutathione from 6.11 ± 0.30 µmol/g Hb in MgS controls to 3.09 ± 0.22 (p < 0.001). Concurrent treatment with dizolcipine maleate of animals on control diet (S-Diz) had no effect on total RBC glutathione levels (5.86 ± 0.46 µmol/g Hb), but it provided significant attenuation of RBC glutathione loss in the deficient animals (D-Diz: 4.19 ± 0.35 µmol/g Hb, p < 0.05; Fig. 2A).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Indexes of systemic stress. A, total RBC glutathione, measured at 3 weeks of treatment (n = 3–6), as explained under Materials and Methods. B, plasma concentration of PGE2, measured at the end of week 3 (n = 3–6), as described under Materials and Methods. MgS, placebo animals on control diet (4–6); MgD, placebo-treated animals on MgD diet (4–6); S-Diz, rats on control diet treated with dizolcipine (3); D-Diz, dizolcipine-treated rats on MgD diet (4–6). ***, p < 0.001 versus MgS; , p < 0.05 and , p < 0.01 versus MgD.

Plasma concentration of PGE₂ was also measured in all animals at the end of week 3. Compared with MgS (336 ± 44 pg/ml sample), PGE₂ plasma concentration in the MgD animal reached almost 300% of control levels (930 ± 122 pg/ml sample, p < 0.001; Fig. 2B). NMDA receptor blockade had no effect on plasma PGE₂ levels (343 ± 53 pg/ml sample) in control animals but completely prevented its elevation in the MgD animal (343 ± 62 pg/ml sample, p < 0.01; Fig. 2B).

Changes in the Endothelial Adhesion Molecule ICAM Expression Levels in the Mg²⁺-Deficient Rat. Adhesion molecules expressed on the surface of endothelium bind specifically to certain white blood cells, thus starting the extravasation process. ICAM (CD54) is an endothelial adhesion molecule that specifically binds cells of the monocyte/macrophage line, thus promoting their migration to extravascular spaces. Immunohistochemical analysis of ICAM presence in the ventricles of MgD animals showed a very apparent increase in ICAM immunostaining by the end of week 1, reaching peak staining intensity by the end of week 2 (Fig. 3). Western blot analyses of ventricular tissue showed a significant 1.5-fold increase in ICAM protein expression in the MgD animal by the end of week 1 (p < 0.05 versus MgS), increasing to 2-fold by the end of week 2 (p < 0.05 versus MgS; data not shown), and then returning to control levels by the end of week 3.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Texas-Red indirect immunostaining of the adhesion molecule CD54 (ICAM) in ventricular myocardium. Cardiac ventricle sections were immunoprobed for CD54 (ICAM-Red) presence with antibody bound to Texas-Red. MgS, placebo animals on control diet (4–6); MgD, placebo-treated animals on MgD diet (4–6); S-Diz, rats on control diet treated with dizolcipine (3); D-Diz, dizolcipine-treated rats on MgD diet (4–6). Microphotographs are arranged by treatment group (columns) and length of treatment (rows). Original magnification, 20x.

Concurrent treatment with dizolcipine maleate of control animals resulted in no appreciable changes in ICAM immunostaining of ventricular sections compared with matched controls, and throughout the length of the experimental protocol (Fig. 3). Also, no difference in ICAM protein expression was found on Western blot analyses between placebo- and dizolcipine-treated control animals (data not shown). Dizolcipine treatment of MgD animals, however, resulted in a marked decrease in ICAM immunostaining of the heart, compared with MgD placebo animals, at the end of weeks 1 and 2 (Fig. 3). Western blot analyses also revealed that dizolcipine prevented ICAM protein expression increase in MgD animals by the end of weeks 1 (p < 0.05 versus MgD) and 2 (p < 0.05 versus MgD; data not shown).

Changes in the Presence of Inflammatory Cells in the Heart of Mg²⁺-Deficient Rats Treated with Dizolcipine Maleate. Western blot analyses and immunohistochemical localization on ventricular sections of the monocyte/macrophage cell surface marker CD11b (M-Integrin) were used to characterize white blood cell infiltration in ventricular myocardium. Bright field horseradish peroxidase staining (brown) counterstained with hematoxylin was used to establish CD11b presence and tissue localization. A light perivascular staining is appreciable on control animals at all experimental times (data not shown). By the end of week 1, CD11b staining seemed already to have increased in MgD animals in a pattern compatible with small focal perivascular infiltrates, further increasing through week 2 to a much more generalized positive staining by the end of week 3 (Fig. 4B). Ventricular tissue homogenates were analyzed by Western blotting for the presence of CD11b, showing an almost 2-fold increase in CD11b expression by the end of week 1 (data not shown); at the end of week 3, the CD11b levels were a100-fold higher than in controls (p < 0.001 versus MgS; Fig. 4E).

View larger version (72K): [in this window] [in a new window]   Fig. 4. Immunohistochemical localization and Western blot analyses of M integrin (CD11b): expression in ventricular myocardium after 3 weeks of treatment. Tissue sections (8 µm) were indirectly probed for CD11b and visualized with an immunoperoxidase staining protocol (brown in microphotograph; see Materials and Methods) counterstained with hematoxylin (blue). Representative micrographs for MgS (A), MgD (B), S-Diz (C), and D-Diz (D) experimental groups are presented. E, Western blot analysis of CD11b protein expression in ventricular myocardium at the end of 3 weeks of treatment with representative bands. MgS, placebo animals on control diet (4–6); MgD, placebo-treated animals on MgD diet (4–6); S-Diz, rats on control diet treated with dizolcipine (3); D-Diz, dizolcipine-treated rats on MgD diet (4–6). ***, p < 0.001 versus MgS. , p < 0.05 and , p < 0.01 versus MgD.

Treatment with dizolcipine of control animals for 3 weeks produced no appreciable changes on ventricular immunostaining with CD11b antibodies compared with diet controls (Fig. 4, A and C). However, Western blot analysis of CD11b expression showed a 5.8-fold increased expression in S-Diz animals compared with diet controls (MgS; Fig. 4E). Concurrent treatment with dizolcipine of MgD rats (D-Diz) decreased CD11b immunostaining of cardiac ventricles (Fig. 4). Western blot analysis of ventricle homogenates of D-Diz animals showed a significant decrease in CD11b expression from 100-fold in MgD animals to a mere 6-fold by the end of week 3 (p < 0.001 versus MgD; Fig. 4E).

	Discussion

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We examined the role of NMDA receptor-mediated SP release on systemic stress and cardiac inflammatory disease secondary to severe MgD, by using the noncompetitive NMDA receptor antagonist dizolcipine maleate. MgD resulted in loss of SP immunofluorescence in DRG, decreased RBC glutathione content, and a significant elevation of plasma PGE₂. Concurrently, in the heart, MgD led to increased ICAM expression and CD11b+ cell infiltration. Dizolcipine blockade of NMDA receptors prevented SP immunofluorescence loss in DRG, ameliorated the decrease in RBC glutathione, and prevented plasma PGE₂ elevation. In the MgD heart, dizolcipine prevented ICAM expression increase and greatly reduced CD11b+ cell infiltration.

Prior studies from our laboratory identified SP as a key mediator in the pathological changes seen during severe MgD (Weglicki et al., 1994a,b). Early increase in SP plasma levels during severe MgD was accompanied by increased plasma calcitonin gene-related peptide, thus suggesting a neural origin for SP (Weglicki et al., 1994a). In the present study, we examined the presence of SP immunofluorescence in the DRG of MgD rats. We found a decrease in SP immunofluorescence already apparent by the end of week 1 that was further apparent at the end of week 2, with SP immunofluorescence being almost nonexistent by the end of week 3. However, taken alone, these data do not directly implicate neural SP in MgD pathology. Thus, based upon the fact that presynaptic NMDA receptors mediate, at least in part, SP release from neural terminals (Juranek and Lembeck, 1996; Liu et al., 1997; Marvizon et al., 1997), we treated MgD animals with the noncompetitive NMDA-receptor blocker dizolcipine maleate. Blockade of the NMDA receptor in MgD animals resulted in conservation of SP immunofluorescence in DRG of these animals throughout the length of the experiment. Furthermore, NMDA receptor blockade clearly ameliorated the inflammatory changes seen in MgD hearts, preventing ICAM expression increase and reducing CD11b positive cellular migration into the cardiac tissue. NMDA blockade also ameliorated the RBC glutathione loss and PGE₂ plasma concentration elevation.

RBC glutathione, a key index of systemic oxidative stress, was depleted significantly in the MgD rats probably due to excessive oxy-radical production by the activated inflammatory cells (Mak et al., 2003). In the present study, blockade of the NMDA receptor partially, but significantly, attenuated the glutathione loss, suggesting a direct link between NMDA activation and the inflammatory cascade leading to elevated cellular production of free radicals. In a previous study (Mak et al., 2003), we observed that the loss of the glutathione was effectively prevented by SP-receptor blockade. The combined data suggest that SP released from the neurons subsequent to NMDA receptor activation plays a critical role in promoting systemic oxidative stress. We have reported that the elevated PGE₂ during MgD is derived, in part, from the activated endothelium and that its induction is subjected to the similar cascade of events governed by SP (Mak et al., 2003). The present study seems to suggest that such SP-governed cascade leading to PGE₂ elevation is completely linked to the upstream event of NMDA receptor activation.

During MgD, inflammatory cell infiltration of the myocardium is already apparent after only 1 week on MgD diet (Kurantsin-Mills et al., 1997). Because a change in vascular endothelium, with increased expression of adhesion molecules, usually precedes white blood cell migration into the heart (Steinhoff et al., 1991), we studied ICAM presence in ventricular tissue. We observed a significant increase in ICAM at 1 and 2 weeks, with a return to baseline levels by the end of week 3, in the MgD animal. Furthermore, we examined inflammatory cell infiltration of the ventricular tissue by monitoring CD11b+ cells and found a steady increase in CD11b+ cells in the MgD animal. Blocking SP release from neural stores with dizolcipine prevented the increase of ICAM expression in the heart and also greatly decreased the presence of CD11b+ cells in the myocardium, suggesting a prominent role of neural SP in the pathogenesis of cardiac inflammation secondary to severe MgD.

In summary, blockade of the NMDA receptor with dizolcipine prevented the loss of SP immunofluorescence in the DRG of MgD animals and prevented the appearance of systemic oxidative stress and cardiac inflammation. We conclude that local cardiac inflammation and systemic stress in the Mg²⁺-deficient rat are the result of SP release from nerve stores due to the decreased inhibition by Mg ions on the NMDA receptor.

	Acknowledgements

 
We recognize the outstanding technical assistance provided by Rili Ahmed and Armida Torres-Duarte during the completion of the present work.

	Footnotes

 
This study was supported by the National Institutes of Health National Heart, Lung, and Blood Institute Grant HL-62282. Results from this study were partially presented at the International Society for Heart Research 2003 North American Section meeting in Mystic, CT: Tejero-Taldo MI, Chmielinska J, Gonzalez G, Ghouse RR, Mak IT, and Weglicki WB (2003) NMDA receptor blockade abolishes cardiac inflammation in the Mg-deficient rat (Abstract). J Mol Cell Cardiol 35:A21.

doi:10.1124/jpet.104.070003.

ABBREVIATIONS: MgD, Mg²⁺ deficiency; RDA, recommended daily allowance; DRG, dorsal root ganglion; NMDA, N-methyl-D-aspartate; PGE₂, prostaglandin E₂; RBC, red blood cell; ICAM, intercellular adhesion molecule.

Address correspondence to: Dr. M. Isabel Tejero, Division of Experimental Medicine, Department of Biochemistry and Molecular Biology, George Washington University Medical Center, 2300 I St., NW, Washington, DC 20037. E-mail: phymit{at}gwumc.edu

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