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2001 29: 208Toxicol PatholSantokh S. Gill and Olga M. PulidoImplications for Toxicology

Review Article: Glutamate Receptors in Peripheral Tissues: Current Knowledge, Future Research, and  

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TOXICOLOGIC PATHOLOGY, vol 29, no 2, pp 208–223, 2001Copyright C 2001 by the Society of Toxicologic Pathology

Laboratory Animal & Molecular Pathology

REVIEW ARTICLE

Glutamate Receptors in Peripheral Tissues: Current Knowledge,Future Research, and Implications for Toxicology

SANTOKH S. GILL AND OLGA M. PULIDO

Health Canada, Banting Building, Tunney’s Pasture, Ottawa, Ontario K1A 0L2, Canada

ABSTRACT

We illustrate the speci� c cellular distribution of different subtypes of glutamate receptors (GluRs) in peripheral neural and non-neural tissues. Someof the noteworthy locations are the heart, kidney, lungs, ovary, testis and endocrine cells. In these tissues the GluRs may be important in mediatingcardiorespiratory, endocrine and reproductive functions which include hormone regulation, heart rhythm, blood pressure, circulation and reproduction .Since excitotoxicity of excitatory amino acids (EAAs) in the CNS is intimately associated with the GluRs, the toxic effects may be more generalizedthan initially assumed. Currently there is not enough evidence to suggest the reassessment of the regulated safety levels for these products in foodsince little is known on how these receptors work in each of these organs. More research is required to assess the extent that these receptors participatein normal functions and/or in the development of diseases and how they mediate the toxic effects of EAAs. Non-neural GluRs may be involved innormal cellular functions such as excitability and cell to cell communication. This is supported by the wide distribution in plants and animals frominvertebrates to primates. The important tasks for the future will be to clarify the multiple biological roles of the GluRs in neural and non-neuraltissues and identify the conditions under in which these are up- or down-regulated. Then this could provide new therapeutic strategies to target GluRsoutside the CNS.

Keywords. Glutamate receptors; peripheral tissues; general injury mechanism; excitotoxicity

PERSPECTIVE

Food toxicology is the science of evaluating the safety ofchemicals that enter the human food chain either as natu-ral compounds, contaminants and/or during processing. Toassess chemical safety, tissues and organs are examined forstructural, chemical, or functional alterations. These inves-tigations help to establish the safety margins of such com-pounds for consumption by humans and animals as eitherfood or therapeutic products. Therefore, product safety re-quires continual reassessment as new information becomesavailable with advances in technology.

Glutamate (Glu) and aspartate (Asp) are naturally occur-ring amino acids found in the central nervous system (CNS)where they act as major excitatory neurotransmitters (20,21, 25, 36) by stimulating or exciting the postsynaptic neu-rons. These excitatory amino acids (EAAs) and their var-ious analogues can be neurotoxic, particularly when theyexcessively stimulate the same excitatory receptors—a phe-nomenon known as excitotoxicity (13, 20, 21, 25, 36, 63).This creates the potential to “over excite” neurons and causepossible neuronal damage. EAAs access thebrain tissueof the

Address correspondenc e to: Santokh S. Gill, Health Canada, Bant-ing Bldg., Tunney’s Pasture, Ottawa, Ontario K1A 0L2, Canada; email:santokh gill@hc-sc.gc.ca.

circumventricular organs that are located outside the bloodbrain barrier (BBB) (7, 67). Despite the BBB protectivemechanisms, the local or circulating concentrations of theseexcitatory compounds may induce damage leading to neuro-toxic exposure levels. The toxicity of each compound suchas domoic acid and Asp varies according to the potency, thechemical availability, the rate of absorption, the af� nity tospeci� c receptors and the particular anatomical target site. Inaddition, the susceptibility, genetic predisposition and healthstatus of the individual are also important factors.

Domoic acid is one of the most potent neurotoxins thatcan enter the food chain. It is manufactured by a sea phy-toplankton that is ingested and accumulated in the digestivesystem of seashells such as mussels. Domoic acid was foundto be the agent responsible for the outbreak of the lethal shell-� sh poisoning that occurred in Canada in 1987 (38, 42, 65,66, 80). Some of the survivors of this poisoning were leftwith severe residual memory de� cits. Previous work in ourlaboratory and from others has con� rmed that domoic acidpreferentially damages areas of the brain involved in mem-ory. The clinical manifestations of domoic acid intoxicationare related to its effect in the brain including severe seizures(38, 42, 65, 66, 79–81). However, the other clinical symp-toms such as: gastrointestinal disturbances, cardiovascularcollapse and cardiac arrhythmia (7, 38, 81, 93) have gainedless attention, until recently.

208 0192-6233/01$3.00 $0.00

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Vol. 29, No. 2, 2001 GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES 209

In the peripheral tissues, there is also a rich bed of nervecircuits and many cell tissues are also capable of conductingexcitatory impulses. This information together with the re-port of cardiovascular disturbances associated with domoicacid intoxications and other excitatory compounds in foodssuch as monosodium glutamate (MSG) prompted our initialinvestigation of the heart as a possible target organ (27–29,88, 93). Because it has been established that Glu or its ana-logues interact with the postsynaptic membrane of glutamatereceptors (GluRs) in the CNS, the existence of a similar rela-tionship was explored in other tissues and organs. This reviewsummarizes the current knowledge on the GluRs in periph-eral tissues, their distribution, potential role and the effectsthey can mediate. We hope this review will stimulate researchand provide additional information useful in formulating reg-ulatory decisions concerning the public health protection offoods and therapeutic products.

BACKGROUND

Excitatory Amino AcidsGlu and Asp are the most abundant dicarboxylic amino

acids in the brain and are believed to be the primary neuro-transmitters in the mammalian CNS (20, 21, 62). Althoughthese amino acids are primarily involved in intermediarymetabolism and other non-neuronal functions, their most im-portant role is as neurotransmitters. It is estimated Glu me-diates nearly 50% of all the synaptic transmissions in theCNS and its involvement is implicated in nearly all aspectsof normal brain function including learning, memory, move-ment, cognition and development (3, 20, 46–48, 51, 53, 62,73). At elevated concentrations, Glu acts as a neurotoxincapable of inducing severe neuronal damage. Hence, Glucan be considered to be a “2-edged sword” that undergoesa transition from a neurotransmitter to a neurotoxin. In ad-dition to the endogenous glutamate, there are naturally oc-curring substances, which have Glu-like excitatory proper-ties and potentially excitotoxic effects. Glu and its structuralanalogues (Figure 1) may enter the food supply during prepa-ration or processing as contaminants or additives (9, 42, 65,66, 69, 79, 81, 86, 88, 93). These analogues include MSG,L-aspartate, L-cysteine, related sulfur amino acids (ie ho-mocysteate), B-N -oxalyamino-L-alanine (BOAA or ODAP),B-N -methyl-amino-L-alanine (BMAA) and the potent seafood toxin domoic acid (20, 22, 38, 42, 59, 69, 86). A plethoraof � ndings in the past 2 decades has provided direct and cir-cumstantial evidence for abnormal glutamate (and its ana-logues) transmission in the etiology and pathophysiology ofmany neurological and psychiatric disorders such as epilepsy,schizophrenia, addiction, depression, anxiety, Alzheimer’s,Huntington’s, Parkinson’s and amyotrophic lateral sclerosis(3, 20, 21, 24, 25, 36, 46–48, 51).

The neurotoxic effects of the EAAs are dependent on thespecies, developmental stage of the animal, type of agonist,duration of exposure to the agonist and the cellular expressionof the GluR subtypes. An array of GluRs are known to bepresent on pre- and postsynaptic membranes that are used totransduce integrated signals using an increased ion � ux andsecond messenger pathways (Figure 2) (16, 17, 20, 25, 36,62, 73). It is the excessive activation of these receptors thatleads to neurotoxicity.

FIGURE 1.—Excitotoxic structural analogues of glutamate. Most of these com-poundsareknown tomimic both the neuroexcitatory andneurotoxic properties ofglutamate. ODAP is B-N -Oxalylamino-L-Alanine (it is also abbreviated BOAA-60).

Glutamate Receptors: An OverviewNumerous reviews are available on the GluRs in the CNS,

their functional roles and implications on the pathobiology ofneural injury and neuropsychiatric disorders (3, 20, 21, 24, 36,48, 53, 62, 73). GluRs have been individually characterizedby their sensitivity to speci� c glutamate analogues and bythe features of the glutamate-elicited current. GluR agonistsand antagonists are structurally similar to Glu, which allowsthem to bind onto the same receptors.

Two classes of GluRs have been characterized based onthe studies in the CNS: ionotropic (iGluRs) and metabotropic(mGluRs). Their cloning has revealed the molecular diversityof the gene families encoding various receptor types that areresponsible for the pharmacological and functional hetero-geneity in the brain. A brief resume is outlined in the presentstudy because comprehensive treatment of these GluRs hasbeen previously reviewed in detail (3, 16, 17, 20, 25, 36, 53,62, 74).

Ionotropic Glutamate ReceptorsThe iGluRs contain integral cationic channels associated

with ligand binding sites and they are known to mediate rapidsynaptic transmission. The iGluR family is classi� ed into 3major subtypes according to their sequence similarities, theirelectrophysiological properties and their af� nity to selec-tive agonists: N -methyl-D-aspartate (NMDA), a -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and by guest on April 7, 2014tpx.sagepub.comDownloaded from

210 GILL AND PULIDO TOXICOLOGIC PATHOLOGY

FIGURE 2.—Schematic representation of the interaction betweeen the presynaptic and the postsynapti c terminal. The � gure shows vesicular release into the synapticspace, activation of the postsynapti c receptor systems, reuptake into the presynaptic terminal and surrounding glial cells. The excitatory amino acids (for exampleglutamate) activates the various glutamate receptors present in the postsynapti c membrane. This triggers the in� ux of Ca 2 from the extracellular environment to thesynaptic cleft. The accumulation of Ca 2 is crucial determinant of injury. This elevation of Ca 2 triggers the activation of several enzymes: calmodulin (CAM), proteinkinase C (PKC), nitric oxide synthase (NO synthase), phospholipase A2 (PLA2) and reactive oxygen species (ROS)—modi� ed from Harry (33) and Said (1999).

kainate (Ka) receptors (3, 20, 21, 36). The membrane chan-nels associated with these receptors exhibit varied pharmaco-logical and electrophysiological properties, including ionicchannel selectivity to sodium (Na ), potassium (K ) andcalcium (Ca 2) (3, 20, 21, 36, 62). The non-NMDA recep-tors control a nonselective cationic channel permeable to Naand K , whereas NMDA is more permeable to Ca 2 ions than

either AMPA or Ka (3, 20, 21, 36, 62). Recombinant tech-nology has identi� ed several gene families encoding iGluRs:AMPA family is composed of GluR 1-4 (GluR A-D); Ka fam-ily includes GluR 5-7 and Ka 1-2; NMDA includes NMDAR1 and NMDAR 2A-D. NMDAR 1 is the most tightly regu-lated neurotransmitter receptor by forming the channel whereother subunits (NMDA 2A-D) are involved in the receptor

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modulation. It is the most intensively studied and complexreceptor and is linked to the Na /Ca 2 ion channel that has5 distinct binding sites for endogenous ligands that in� uenceits opening (20, 21, 36, 62). These include 2 different agonistrecognition sites (1 for Glu and 1 for glycine), a polyamineregulatory site that promotes receptor activation. The re-maining sites separate recognition sites for Mg 2, Zn 2 andphencyclidine (PCP), which inhibit ion � ux through agonist-bound receptors (20, 21, 36, 62). The complexity of theseGluR families is further increased by alternate splicing, RNAediting and post-translational modi� cations such as phos-phorylation, glycosylation and palmitoylation. Each of thesemodi� cations is important in the regulation of channel func-tions. Within each family, GluRs subunits can also formhomo-oligomeric or heteroligomeric channels that exhibitdifferent functional properties depending on the subunit com-position. For example, the presence of the GluR 2 subunit de-creasesCa 2 permeability of AMPAchannels (20, 21, 36, 62).

Metabotropic Glutamate ReceptorsThe mGluRs exert their effects either on the second mes-

sengers or ion channels via the activation of the of GTP-binding proteins and regulate the synthesis of differentintracellular second messengers such as IP3, cAMP or cGMP(17, 21, 36, 62, 73). A single mGluR protein can cross-talkwith multiple second messengers in the same cell. As withiGluRs, the mGluRs are also classi� ed into 4 groups based onamino acid sequence similarities, agonist pharmacology andthe signal transduction pathways to which they are coupled.Group I (mGluR 1, 5, and 6) stimulates inositol phosphatemetabolism and mobilization of intracellular Ca 2. Group II(mGluR 2 and 3) and group III (mGLuR 4, 6–8) are cou-pled to adenyl cyclase (17, 21, 36, 62, 73). Group IV is cou-pled to to the activation of phospholipase D (PLD). The lat-ter class is more ef� ciently activated by L-cysteine-sul� nicacid (L-CSA) rather than Glu, which suggests that L-CSAmay serve as an endogenous agonist of this receptor (16).The mGluRs function is predominantly with the long-termaspects of cellular control by operating via G proteins andseveral second messenger systems. As with the iGluRs, themGluRs also have a unique distribution in the CNS and retina,

TABLE 1.—Distribution of glutamate receptors (GluRs) in peripheral tissues.

Receptor subtypes Species Organ Tissue/Cell type Methodology References

GluR 2/3, Ka 2, NMDAR 1, Rat/monkey Heart Atrium/septum conducting � bers, IS, westerns, northerns, 27, 28, 56, 58, 68mGluR 5, mGluR 2/3, ganglia cells, nerve � bers, RT-PCRmGluR 1 myocardiocytes , intercalated

discs, blood vesselsGluR 2/3, Ka 2, NMDAR 1, Rat/monkey Ovary Corpus luteum, primordial follicles, IS 29, Gill et al (in

mGluR 2/3 theca, granulosa cells, oocyte, preparation)blood vessels, nerve � bers

GluR 2/3, Ka 2, NMDAR 1, Rat/monkey Uterus Exocervix, myometrium, IS, westerns, RT-PCR, 29, Gill et al (inGluR 2/3 endometrial glands, epithelium northerns preparation)

of fallopian tubes, nerve � bersGluR 2/3, Ka 2, NMDAR 1, Rat Kidney Glomeruli, mesangium, IS, westerns, RT-PCR, 27, 29

mGluR 2/3 podocytes , juxtaglomerular northernsapparatus, tubules

GluR 2/3, Ka 2, NMDAR 1, Rat Testis Germinal epithelium, IS, westerns, RT-PCR, 27, 29, 45mGluR 2/3 interstitial cells northerns

GluR 2/3, Ka 2, NMDAR 1, Rat Gastrointestinal Enteroendocrine cells, parietal cells IS, westerns, RT-PCR, 5, 6, 8, 29, 37, 47, 57,mGluR 2/3 of the stomach, pancreatic islets, northern blot 73, 74, 82, 85

nerve � bers, ganglia cells, liverGluR 2/3, Ka 2, NMDAR 1, Rat Others Lungs, spleen, bone marrow IS, westerns, RT-PCR, 14, 26, 27, 29, 31,

mGluR 2/3 (megakaryocytes), mast cells, northern blots 67, 69, 70in� ammatory cells

which re� ects a diversity of function in normal and patho-logical processes. These receptors have been shown to exerta wide variety of modulating effects on both excitatory andinhibitory synaptic transmission. This is expected if a recep-tor activation is coupled to multiple effector enzyme (17, 21,36, 62, 73).

The mGluRs have certain features that distinguish themfrom the iGluRs. First, the mGluRs modulate the activity ofneurons rather than mediate fast synaptic neurotransmission.Second, the distribution of the mGluRs is highly diverse andheterogenous. Different subclasses are localized uniquely atboth the anatomical and cellular levels. For example, mGluR2 and 3 are found in high density in the cerebral cortex,whereas mGluR 4 is found in high density in the thalamus butnot in the cortex and mGluR 6 is almost exclusively found inthe retina.

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

IntroductionA recent surge of publications supports the presence and

functionality of GluRs outside the CNS in various tissues andspecies. Table 1 summarizes the distribution of the GluRs inperipheral tissues demonstrated by using various methodolo-gies such as: RT-PCR, PCR, northern blots, western blots,immunohistochemistry and in situ hybridization. These tis-sues include adrenal medulla (85, 90), peripheral nerves—myelinated and unmyelinated (1, 15), bone (14, 26), bonemarrow (26), bronchial smooth muscle, endocrine pancreas(4–6, 30, 32, 37, 45, 49, 54, 87), gut (8, 58, 77, 81, 86), esoph-agus (this study), hepatocytes (27, 78), heart (27, 28, 55, 69,87), taste buds (12, 35), keratinocytes (55), lungs (29, 34, 70,77), pituitary (41, 86), pineal gland (52), ileal longitudinalmuscle (56, 74, 75), autonomic and sensory ganglia (85), ratglaborous skin (10), kidney, spleens, ovaries, (26, 28, thisstudy), vagus and other cholinergic nerves (1), tachykinin-containing sensory nerves and vestibular tissues (18). In ourstudies, we have conducted a thorough analysis on the dis-tribution of the GluRs in peripheral tissues of the rat wherethe antibodies and the methodology used have been previ-ously described (27, 28, 29). Different subtypes of GluRs

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212 GILL AND PULIDO TOXICOLOGIC PATHOLOGY

were observed in the heart, spleen, testis and kidney (27, 28,29). These � ndings and that of others clearly supports theview that GluRs are widely present in peripheral tissues andhave a speci� c cellular distribution.

Some of the GluRs isolated from peripheral tissues havebeen cloned and sequenced (10, 12, 14, 24, 27, 32, 36,87). These sequences correspond with GluRs that have beencloned in the CNS. Further physiological and pharmacolog-ical experiments support the hypothesis that GluR receptorsin the periphery have similar properties to those in the CNS orexpressed in host cells transfected with cloned subunits (28,30, 36, 56, 71, 74, 84, 87). For example, the AMPA recep-tors in the rat pancreas respond to L-glutamate, AMPA andkainate. These receptors were blocked by competitive antag-onists, 6-cyano-7-nitroquinoxaline (CNQX) and potentiatedby cyclothiazide (87). These properties are also shared byneuronal AMPA receptors. In addition, the stimulation ofcultured rat myocardial cells by L-glutamate leads to an in-crease in the intracellular Ca 2 oscillation frequency (88).Similar physiological studies with agonists and antagonistsof the NMDAR 1 receptor in the pig ileum have shown thatthese receptors are similar to those characterized in the CNS(73). NMDAR 1 receptors in the pig ileum were also blockedby Mg 2 ions and competitively antagonized by DL-2-amino-5-phosphonovaleric acid.

Glutamate Receptors in the Heart and Cardiac ArrhythmiaWe have extensively investigated the presence of the GluRs

in the rat (27, 28). The preferential localization of GluRswas seen within the conducting system, nerve terminals andintramural ganglia cells (Figure 3). Similar distribution, butwith enhanced de� nition was observed in the conducting sys-tems in the monkey heart (in preparation, 57). These � ndingslikely re� ect the higher level of anatomical differentiationof the conducting system in nonhuman primates versus therodents as illustrated by using the neural markers PGP 9.5and NF (57). The presence of the GluRs in areas speci� callyinvolved in the conduction of impulses suggests their involve-ment in the control of heart rhythm. Therefore, the presenceof the GluRs in the heart implies that this organ may be animportant target site for compounds such as domoic acid,which is a known ligand for these receptors.

The presence of GluR in the intramural ganglia cells andcardiac nerve � bers, which are known to be components ofthe peripheral autonomic nervous system, prompted us toinvestigate the presence of GluRs in other tissues.

Glutamate Receptors in Kidney and Electrolyte–WaterHomeostasis

In the kidney, the wide distribution of NMDAR 1 and thepresence of mGluR 2/3 and GluR 2/3 in the juxtaglomeru-lar apparatus (JGA) and proximal tubules (Figures 4a, b, c,and d), suggests that these receptors may be involved in elec-trolytes and water homeostasis. The strong visualization ofimmunoreactivity with anti-mGluR 2/3 and anti-GluR 2/3within the granular cells of the afferent arteriole suggests a po-tential involvement in the control of renin release (18, Figures4b and c). The renin-angiotensin system is a major hormonalsystem involved in the regulation of electrolyte, � uid bal-ance and blood pressure (39). Pharmacological and biochem-

ical evidence also supports the presence of the dopaminereceptors—D1A, D1B, D2, and D3 within the kidney (58, 72,94). Like the GluRs, the dopamine receptors were also specif-ically distributed. D1A and D1B are both reported to be presentin the renal vasculature, renal proximal and distal convo-luted tubules, cortical and collecting ducts. In contrast, D1Ais not present in JGA apparatus and the ascending loop ofHenle, whereas D1B is present in these regions. Experimentaldata suggests that dopamine receptors are involved in re-nal hemodynamics, ion transport and renin secretion (58, 72,94). In addition to the GluRs and the dopamine receptors, theGABA receptors are also found in the kidney. The GABAAand GABAB receptors have been localized to the renal cortex(11). Therefore, GABA might also in� uence the renal func-tions in the kidney cortex rather than the medulla. Whetheror not there is co-localization or co-functionality of thesereceptors needs to be determined.

Glutamate Receptors in Sex Organs and ReproductionWe have shown the differential distribution of GluRs in

reproductive organs of the male (Figures 4e– i) and femalerat (Figures 5 and 6). In the testis, these receptors have aspeci� c af� nity for different structures. There is intense anti-mGluR 2/3 immunolabelling of the head of the mature sper-matids/spermatozoa, interstitial cells and myoid cells. Anti-NMDAR 1 has a strong af� nity for the germinal epithelium,particularly the spermatogonia adjacent to the basal laminaand the more mature spermatids near the lumen (Figure 4e),whereas the anti-GluR 2/3 immunostain is limited to the cellsin the interstitial spaces. This differential distribution sug-gests that GluRs may be involved in spermatogenesis, sper-matozoa motility and testicular development—each linkedto a speci� c receptor. To support our hypothesis, we areinvestigating the presence and distribution of GluRs in rattestes during various developmental stages. Previously, ithas been shown that speci� c binding sites for [3H]-TCP, aligand that labels a binding site within the NMDA recep-tor ion channel, has been demonstrated on membranes ofmammalian spermatozoa. Morever Cl -independent [3H]-glutamate binding, which could be partially displaced byNMDA and AP5, has also been detected in seminal vesi-cles (20). Further, the results of Lara and Bastos-Ramos (44)suggest the noradrenergic neurons innervating the rat vas def-erens are controlled by a glutamate-dependent excitatory pro-cess in the ganglia. They showed that a single dose of kainateto the ganglia induced a decrease in the norepinephrine con-tent of the vas deferens. Binding studies using [3H]-glutamateto the membrane fraction showed that the binding was sat-urable. This binding was inhibited by different analoguesaccording to different potencies L-glutamate > kainate >quisqualate N -methyl-D-aspartate. Thus, it is likely thatthe vas deferens has a glutamatergic excitatory mechanismfor the control of their activity and these are responsible forthe depolarizing potential on the noradrenergic neurons. Thismechanism might be important for the contractile activity ofthe vas deferens and hence in the control of seminal � uid (44).

In the rat female reproductive system, GluRs also have aunique distribution within each organ (Figures 5 and 6). Eachantibody has a differential af� nity to speci� c structures in theovaries, the fallopian tubes, the cervix, the myometrium andthe endometrium (Figures 5 and 6). In the ovary (Figure 6)

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FIGURE 3.—Photomicrographs of the rat heart perfused with 4% PFA and processed for immunohistochemistry. Microwaved paraf� n sections were immunostainedwith speci� c antibodies to GluRs, using LAB/avidin biotin method and DAB as chromogen . A) Anti-GluR 2/3 staining in the ganglia (GC) and conducting � bers (CF)of the atrium. Inset illustrates western blot with the same antibody using crude membrane extracts from brain (lane 1), heart (lane 2). Both bands were approximately100 Kd. B) Anti-Ka 2 immunostaining in GC and cardiocytes (MYOF). C) Anti-GluR 2/3 staining in the CF of the septum. D) Anti-NMDAR 1 staining in nerve� bers (NF), MYOF, and GC. E) GC showing cytoplasmic distribution of the stain at the periphery of the cells. F) Anti-mGluR 5 showing preferential stain in theintercalated discs (ID) (27, 28).

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FIGURE 4.—Photomicrographs of the rat kidney and testis perfused with 4% PFA and processed for immunohistochemistr y. Microwaved paraf� n sections wereimmunostained with speci� c antibodies to GluRs, using LAB/avidin biotin method and DAB as chromogen. A) Immunostaining with anti-NMDAR 1 is seen inthe distal tubule, proximal tubule, and glomeruli, particularly in the mesangium and podocytes (shown by an arrow). B) Anti-mGluR 2/3 strong immunostain in theconvoluted proximal tubules and the JGA (arrow). C) Higher magni� cation of the JGA, showing anti-mGluR 2/3 dark cytoplasmic staining of the granular cells inthe wall of the afferent arteriole. D) Anti-GluR 2/3 stain distribution is similar to the anti-mGluR 2/3. E) Anti-NMDAR 1 strong af� nity for the germinal epithelium,particularly the spermatogonia adjacent to the basal lamina and the more mature spermatids near the lumen. F) & G) Anti-GluR 2/3 immunostain is limited to theinterstitial spaces. H) Intense anti-mGluR2/3 immunolabelling of the head of the mature spermatids/spermatozoa, and I) Interstitial and myoid cells. Abbreviations:afferent arteriole (AA); distal convoluted tubule (D); proximal convoluted tubule (PT); mesangium (M); glomeruli (G); juxtaglomerular apparatus (JGA); interstitialspaces (IS); seminiferus tubules (ST); spermatogonium (SP); spermatids (SP) (29). by guest on April 7, 2014tpx.sagepub.comDownloaded from

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FIGURE 5.—Immunohistochemica l analysis of various subtypes of GluRs. Paraf� n sections of rat uterus � xed with 4% PFA, immunostained using the LAB/avidinbiotin method and DAB as chromogen. Each subtype of GluR Abs tested has a differential speci� c distribution. Anti-mGluR shows preferential binding to themost super� cial layer of the strati� ed squamous epithelium of the exocervix (A), whereas the full thickness of the epithelium is stained with anti-NMDAR 1 (C)and remains unstained with anti-GluR 2/3 (E). Cross-section of the body of the uterus stained with H&E, depicting the myometrium and endometrial glands (B).Endometrial glands show moderate staining with anti-NMDAR 1 (F). Anti-Glu R 2/3 has strong af� nity for the endometrial glands, myometrium (D) and the ciliatedepithelium of the fallopian tube (G, H). Abbreviations: exocervical epithelium (CE); myometrium (M); endometrial glands (EG); stroma (S); fallopian tube or oviduct(FT); lumen (L); ciliated epithelium (this study).

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FIGURE 6.—Rat ovaries � xed in 4% PFA and stained with H&E (A). Microwaved paraf� n sections were immunostained with speci� c antibodies to various subtypesof GluRs, using the LAB/avidin biotin method and DAB as chromogen . Immunostain with anti-GluR 2/3 shows a wide distribution throughout the ovary, includingstroma; corpus luteum; and within the follicles, the granular cells, theca, and oocytes (B, C, D). The intensity of the stain varies and is higher within the more maturefollicles, oocytes, and corpus luteum. Anti-mGluR 2/3 also shows af� nity for the corpus luteum and oocytes (E, F), but is very faint for other structures. Anti-NMDAR1 has strong selective af� nity for the oocyte (G). Nerve � ber within the suspensory ligament stained with all the antibodies, anti-GluR 2/3 (H). Abbreviations: ovary(OV); follicles (FL); oocyte (Oc); granulosa cells (Gc); theca (T); corpus luteum (CL); nerve � bers (NF) (this work).

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the distribution of GluRs within the follicles varies at dif-ferent stages of their maturation. In the rat anti-NMDAR 1and to some extent anti-GluR 2/3 and anti-mGluR 2/3, havea remarkable selective af� nity for the oocyte. GluR 2/3 andmGluR 2/3, but not NMDAR 1 are visualized in the cor-pus luteum. In the uterus (Figure 5), anti-GluR 2/3 showedstrong af� nity for the ciliated epithelium of the fallopiantubes, smooth muscle of the myometrium and endometrialglands, whereas anti-NMDAR 1 is more selective to the en-dometrial glands and the exocervical epithelium. This sug-gests that these receptors may be involved in ovulation, fer-tilization, implantation of the ovum and excitability of theuterus. To examine if similar preferential distributions existin higher mammals, we have tested the location of GluRsin the sex organs of nonhuman primates, using ovary, uterusand fallopian tubes of Macaca fascicularis. In this species, thecorpus luteum and oocytes display intense, selective immuno-labelling with anti-NMDAR 1 and anti-GluR 2/3 (manuscriptin preparation).

The presence of the GluRs within the reproductive or-gans and the known functional inhibitory/excitatory effects ofGABA/GABA receptors (22) suggest that similar excitatory–

inhibitory neurotransmission interplay may also be present inthe reproductive organs using GluRs as mediators. There-fore reproductive functions—such as gonadal maturation;steroidal sex hormone regulation; maturation, motility, andexcitability of the spermatozoa; ovulation; fertilization; ex-citability of the fallopian tubes; implantation of the ovum;and excitability of the myometrium—may be all affected.This warrants testing as it has important therapeutic and tox-icological implications.

Glutamate Receptors in Neuroendocrine Tissuesand Hormone Secretion

In addition to the gonads, we and other researchers (5,6, 37, 41, 49, 53, 54, 84, 85, 87, 91) have shown the pres-ence of GluRs in other endocrine tissues. These include thepancreas, pituitary, pineal gland, adrenal gland and kidney.The differential distribution of the GluR subunits in the pan-creas has already been described (37, 54, 87). Liu et al (49)showed that GluR 1 and GluR 4 were mainly localized toinsulin-secreting cells in the central mass of the pancreaticislet, whereas GluR 2/3 was preferentially localized in theperipheral rim composed of non– insulin-secreting islet cells.It appears that insulin- and non–insulin-secreting cells ex-press different AMPA receptor subunits, which may be usedto mediate their hormone secretion, as was suggested earlierby Bertrand (5, 6). Weaver et al (87) showed that the AMPAreceptors were located in the a , b , and PP cells but weregenerally absent from the c cells, whereas kainate receptorswere expressed in the a and c cells although they were notfound in b or PP cells. These observations add to the evidencethat these receptors may be involved in the regulation of hor-mone secretion (4–6, 37, 86). Studies of Weaver et al (87)show that Glu depolarizes islet cells when Glu serum lev-els are elevated. Intracellular Ca 2 measurements and elec-trophysiological recordings showed that kainate, AMPA andNMDA elicited increases in Ca 2 in single b -pancreatic cellsand depolarized them. In addition, kainate and AMPA stim-ulated the release of insulin whereas NMDA did not (36).This stimulatory effect was dependent on the glucose con-

centration: Glu stimulated insulin release in the presence of aglucose concentration of 8.3 mM but not in the presence ofa low concentration (2.8 mM). Hence, Glu is a potentiator ofglucose-induced insulin release. In addition to the GluRs, thepresence of GABA receptors has also been reported in thepancreas (11, 76, 86). Therefore, the � nal activity of thesecells is probably determined by the balance in the activitiesof both GluR and GABA receptors. It is therefore conceiv-able that these receptors are involved in the pathophysiologyof the pancreas.

Glutamate Receptors in the Gastro-Intestinal (GI) TractExperimental evidence from several labs and from this lab-

oratory (Figure 7) have shown the GluRs to be present in thestomach, duodenum and descending colon (8, 56, 74, 75, 83).Immunohistochemical analysis showed that GluRs antibod-ies had poor af� nity for the esophagus with the exception ofanti-NMDAR 1, which preferentially stained the less maturecells within the basal layer of the strati� ed squamous epithe-lium (Figure 7). In the stomach mucosa, parietal cells werestained with anti-GluR 2/3, anti-NMDAR 1 and anti-mGluR2/3. Mast cells throughout the GI tract show strong stainingwith anti-NMDAR 1. All the antibodies showed some af� n-ity for enteroendocrine cells, ganglia cells and nerve � bersthroughout the GI tract. Our � ndings with the NMDAR 1 aresimilar to those described by Burns et al (8). We show thepresenceof NMDAR 1 expression in the stomach, duodenum,ileum and descending colon. Previous studies have shown thatGlu, through the action of NMDAR 1, induces contraction ofthe ileal longitudinal smooth muscle/myenteric plexus (74).The myenteric plexus is a layer of neurons innervating thegastrointestinal tract, which is largely responsible for gas-trointestinal motility. The role of NMDA receptors in intesti-nal motility was con� rmed by the capability of Glu, Asp,L-homocysteate and NMDA (but not kainate or quisqualate)to cause muscle contraction. The contraction was blockedby NMDA antagonists (noncompetitively by Mg 2 and byphencyclidine-like drugs such as etoxadrol, dextromethor-phan, and MK801) but not by kainate or quisqualate an-tagonists. The order of potencies for the contractile effectsis: L-glutamate > aspartate > L-homocysteate > NMDA > D-glutamate (73). In the CNS, electrophysiological L-glutamateand L-aspartate are less potent than NMDA due to their aviduptake. The results of Shannon and Sawyer (74) suggest anabsence of the uptake processes of these compounds in themyenteric plexus. These results are in agreement with Moroniet al (56).

The studies of Tsai et al (82, 83) showed that Glu and Aspare both involved in regulating acid secretion in the stomach.However, their mode of action is different. They found thatAsp was more speci� c than Glu in regulating acid secretion.This was attributed to the fact that Glu is a general agonist forall types of GluRs, yet Asp is a potent agonist for NMDARreceptors.

Glutamate Receptors in Other TissuesPatton et al (64) showed the presence of different GluRs

subunits-GluR 2/3, NMDAR 1, mGluR 2, 4, 5, and 7 in bone.Further, using the agonists MK801 and AP5, they demon-strated that bone cells have the potential to express manyof the molecules associated with the glutamate-mediated by guest on April 7, 2014tpx.sagepub.comDownloaded from

218 GILL AND PULIDO TOXICOLOGIC PATHOLOGY

FPO

FIGURE 7.—Photographs show the immunohistochemica l localization of GluRs within the gastro-entero-pancreati c system. Microwaved paraf� n sections im-munostained using the LAB/avidin biotin method and DAB as chromogen . Each subtype of GluRs Ab tested has a speci� c distribution. A & B) Anti-GluR 2/3immunostaining of the af� nity for the parietal cells (PC) and the endocrine cells (EC) of the stomach mucosa. C) Anti-NMDAR 1 staining of the ganglion cells (GC)and the EC cells of the bowel. All the Abs showed some af� nity for GC and NF throughout the tract. D) Pancreatic islets stained with anti-GluR 2/3. The intensity ofthe stain varied with the subtype GluR 2/3 > mGluR 2/3 > NMDAR 1. None of the Abs stained with the exocrine pancreas. Anti-GluR 2/3, but not the other Abs,stained the wall of the blood vessels in the pancreas (D). Abbreviations: parietal cells (PC) of the stomach; enteroendocrin e cells (EC); ganglion cells (GC); gobletcells (G).

signalling. Chenu et al (14) documented that all mature celltypes (osteoblasts, osteocytes, and osteoclasts) express oneor more of the GluRs subunits. The blockade of NMDA re-ceptors with antagonists resulted in inhibition of osteoblastformation, suggesting that NMDAR 1 are functional in bone.In particular, NMDAR 1 was most abundant on bone cells.Glutamate/aspartate transporter (GLAST) has also been iden-ti� ed in bone (14), further supporting the view that neuroex-citatory amino acids may play a role in paracrine signalling inbone cells. The GLAST performs an essential function duringglutamate-mediated synaptic neurotransmission by acting asa high af� nity uptake system to remove released Glu from thesynaptic cleft, thus preventing overstimulation of the postsy-naptic glutamate receptors. Thus, the presence of GLASTin the bone and other tissues supports the theory that Glusignalling may have a role outside the CNS.

More recently, the presence of the glutamate receptorsNMDAR 1 and NMDAR 2 were demonstrated in bone mar-

row of the rat, human megakaryocyte and the MEG-01 clonalmegakaryoblastic cell line (see Table 1). An interesting ob-servation is that the level of glycosylation in these cells isreduced or absent as compared to the CNS-NMDA type. It isspeculated that glycosylation provides stability to the NMDAreceptor in the synaptic membrane for the precise orientationand localization in the CNS. However, in the megakaryocyte,the receptors are distributed evenly across the cell surface,which allows for multidirectional agonist stimulation. Thesereceptors are probably involved in signalling, which is rein-forced by the � ndings that megakaryocyte exist in intimatecontact with cells bearing the glutamate-transported proteinsGLT-1 and GLAST, which are found on mononuclear bonemarrow cells and osteoblasts, respectively. These transportersare essential requirements for functional glutamate-mediatedcommunications. Genever et al (26) showed that NMDAR 1activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDAR 1

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receptor cell blockade by speci� c antagonists, MK-801 or D-AP5; inhibited PMA-mediated increases in cell size; CD41expression; and adhesion of MEG-01 cells.

Several mGluRs—mGluR 1, 2, 3, and 5—were also re-ported in the thymic stromal cell line (TC1S) and in the thy-mocytes. Using RT-PCR and western blotting, it was demon-strated that the thymic stromal cell line TC1S expressedmGluR 2, 3, and 5. Thymocytes expressed mGluR 1, 3, and5. Fluorescence Activated Cell Sorter (FACS) analysis illus-trated that majority of the unfractionated thymocytes (70%)showed the presence of mGluR 5, whereas 50% of the cellsexpressed mGluR 3 and only 15% expressed mGluR 1. Incontrast, isolated CD4 /CD8 , double negative thymocyteprecursor, expressed mGluR 3 (45%) and mGluR 1 (40%),whereas mGLuR 5 was barely detectable. Therefore, it washypothesized that changes in mGluRs subtype expressionsmay be related to T-cell maturation stages (31). GluRs arealso expressed in other lymphoid tissues and in� ammatoryin� ltrates (Table 1).

The iGluRs (27) and mGluRs (77) were also present inthe liver. Sureda et al (77) showed mGluR 5 in the primaryhepatocytes stimulated the hydrolysis of inositol phospho-lipid. The effects of mGluRs agonists, 1S,3R-ACPD andquisqualate, was examined on anoxia-induced cell damage.A time-dependent decline was observed on the viabilityand was maximal after 90 minutes. Both 1S,3R-ACPD andquisqualate shortened this time course of anoxia-induced celldamage, reducing signi� cantly the viability of primary hep-atocytes. In contrast, the agonist 4C3HPG for the type 11mGluR had no effect. The hypothesis is that this receptoris activated by the Glu present in the portal blood and maycontribute to the liver damage under adverse conditions.

Glutamate Receptors as Possible Mediators of Celland Tissue Injury and Pathology

The view that Glu or related excitatory amino acids cancause neuronal injury as a result of overexcitation is calledexcitotoxicity. The GluRs are known to act as mediators ofin� ammation and cellular injury through a common injurypathway (46, 47, 48, 51; see Figure 2). In the CNS, thestimulation of GluRs triggers an excessive in� ux of calciuminto neurons through the ion channels, which mediates neuralinjury (13, 20, 21, 22, 46, 47, 48, 52). Because the iGluRsare ion-gated channels selective to Na , K , and Ca 2, anysustained stimulation of the GluRs results in osmotic damagedue to the entry of excessive ions and water. This increasesthe intracellular Ca 2 concentration which is crucial to thedeterminant of injury. It is this high concentration of Ca 2 thattriggers the activation of several enzyme pathways and sig-nalling cascades including as phospholipases , protein kinaseC, proteases, protein phosphatases, nitric acid synthases andthe generation of free radicals (3–13, 19, 20, 46, 48, 51). Thedestabilization of Ca 2 homeostasis also causes the translo-cation of protein kinase C (PKC) from the cytoplasm to themembrane. This leads to the phosphorylation of the mem-brane proteins via PKC promoting the destabilization of theregulatory mechanisms for Ca 2 homeostasis, which medi-ates toxicity (13, 20, 21). On activation of phospholipase A2,arachidonic acid (with its metabolites and platelet-activatingfactors) is generated. Platelet-activating factors increase the

neuronal calcium levels by stimulating the release of Glu.Arachidonic acid potentiates NMDA evoked currents and in-hibits the reabsorption of Glu into astrocytes and neurons.This further exacerbates the situation by a positive feedbackmechanism where free radicals are formed during arachi-donic acid metabolism, leading to further phospholipase A2activation. This results in an increased concentration of ex-tracellular glutamates which contributes to the sustained ac-tivation of the GluRs (13, 16, 20, 21). As a consequence,cysteine transport is inhibited causing a decrease of intra-cellular reducing sulphydryls and the generation of oxygenradicals, which results in cell death. In addition to enzymesof the cell cytosol, the nuclear enzymes are also activatedby increase of Ca 2. For example, Ca 2 may activate en-donucleases that result in condensation of nuclear chromatinand eventually DNA fragmentation and nuclear breakdown,a pathologic process known as apoptosis. Free radicals alsocontribute to DNA fragmentation.

The increased concentration of Ca 2 raises the nitric ox-ide via the calmodulin activation of nitric oxide synthetases,which generates oxygen radicals. Nitric oxide has been ob-served in peripheral tissues, ganglion cells, nerve � bers, car-diocytes, and myocytes in the heart of pig and rat. Our workhas demonstrated the presence of the glutamate receptors inthe same structures. Liu et al (49) showed the colocalization ofnitric oxide and AMPA receptors in ganglion cells of the pan-creas. Because the nitric oxide is calcium- and calmodulin-dependent and the AMPA receptors are Ca 2 permeable, itis possible that nitric oxide is activated through the AMPAreceptors. We propose that the mechanism involved in injuryin the CNS may be a basic mechanism for injury in all tissues.This is supported by the � nding that excessive activation ofthe NMDAR 1 in the lungs induces acute edema and lung in-jury as seen in “adult respiratory distress syndrome” (70, 71).This injury can further be modulated by blockage of one ofthree critical steps: NMDA 1 binding, inhibition of NO syn-thesis, or activation of poly (ADP-ribose) polymerase (70,71). Our results showed immunolabelling for various GluRsin bronchial epithelium, blood vessels of the lungs, mast cells,and in� ammatory cells. This supports the view that they playa role in airway responses to injury and in� ammation. Allthe antibodies tested for the subtypes of GluRs showed af� n-ity to mast cells in all tissues analyzed, particularly in thelungs and gastrointestinal tract. Their presence in the airwaystructures such as the larynx, esophagus and mast cells alsoimplicate the GluRs in themediation of asthmatic episodes (2,70, 71). The excitation of GluRs in the air passages thereforemay be important in airway in� ammation and hyperreactiv-ity observed in bronchial asthma (60). Their presence alsocould explain the enhancement of acute asthmatic attacks byglutamate-containing foods (2). These researchers did blindplacebo-controlled experiments where subjects with asthmareceived MSG in tablet form. Their studies showed that MSGdid indeed provoke asthma, which in some cases was severeand life-threatening.

Mast cells are known to be found in the connective tis-sues throughout the body, most abundantly in the submu-cosa tissues and the dermis. Purcell et al (68) showed thatspermidine-induced release from the mast cells are dependenton the presence of Ca 2 in the external mileau. The in� ux ofCa 2 is known to be accompanied by NMDAR 1 activation. by guest on April 7, 2014tpx.sagepub.comDownloaded from

220 GILL AND PULIDO TOXICOLOGIC PATHOLOGY

This increased intracellular Ca 2 concentration initiates theexocytotic degranulation process in mast cells. Spermine is anatural polyamine, and the opening of the ion channel asso-ciated with NMDAR receptors is facilitated by binding sitesfor polyamines. In neuronal tissue, polyamine triggers his-tamine secretion through interaction with a polyamine site as-sociated with an NMDAR 1 macrocomplex. Therefore, sper-mine can modulate activation of the macrocomplex, eitherthrough action at polyamine-binding sites in the lung or atother sites. The antagonists of NMDAR 1, MK801, blockedthis release of histamine secretion that was induced by thenatural polyamine-spermine. If the NMDAR 1 is present, assupported by (68, 70, 71), then it opens up the possibility thatEAAs can also in� uence allergic reactions.

Glu and related EAA agonists induce neurotoxic damagein the CNS, which occurs under conditions of hypoxia orischemia and is believed to be due to the increased intracel-lular Ca 2 concentration (13, 20, 21, 25, 36, 48). Intestinalmucosa damage has also been reported after hypoxia andischemia (61).

CONSIDERATIONS ON THE PHYLOGENYOF GLUTAMATE RECEPTORS

Recent data suggests that putative iGluRs exist in plants,in both monocotyledons and dicotyledons. These observa-tions are based on northern and heterologous Southern blotanalysis (43). Preliminary data indicates that the GluRs areinvolved in light signal transduction. Therefore, it is possi-ble that signalling between cells by EAAs may have evolvedfrom primitive mechanisms before the divergence of plantsand animals. GluRs and other similar signalling systems areactually ancestral methods of communication, common toplants and animals alike. This is supported by the fact thatthese receptors are present in mollusc (77), leech (19), andOreochromis sp (freshwater � sh, 88) and C elegans (blue-green algae, 50). In plants, the GluR-like receptors respondto the same antagonist as the GluRs in the CNS. The DNA se-quence data also shows variable (60%–16%) homology to theglutamate receptors characterized in the mammalian system.

CONCLUSIONS AND FUTURE RESEARCH CONSIDERATIONS

In summary, GluRs have a wide and a unique distributionin peripheral tissues. These receptors are pharmacologicallysimilar to their counterparts in the CNS, although the possi-bility that there are subtle distinctions such as glycosylationcannot be ignored. The presence of the GluRs in peripheraltissues may provide explanations for the autonomic distur-bances (GI, salivation, cardiovascular, vomiting) that havebeen reported in animals dosed with potent excitotoxins suchas domoic acid (38, 66, 67, 79, 80, 88, 93). Because theEAA excitotoxicity is intimately associated with the GluRs,the toxic effects may be more generalized than initially as-sumed, particularly in the light that GluRs are widely presentin peripheral tissues that are not protected by the blood brainbarrier (7, 67). Excitotoxicity depends on the intracellularNa and Cl ions and in the in� ux of Ca 2. It is the Ca 2

in� ux that is thought to be the ultimate trigger of the toxiceffects (13, 20, 21). Based on the pattern of anatomical distri-bution, we suggest that these receptors may be important forthe mediation of functions such as hormone regulation, heart

rhythm, blood pressure, circulation and reproduction. Someof the noteworthy locations are the heart, kidney, lungs, ovary,testis and endocrine cells—suggesting they play a role oncardiorespiratory, endocrine and reproductive functions. Fur-thermore, they could potentially turn the tissues where theyare present into target sites for the toxic effect of glutamate–

like-products. Many of these are known toxins that contami-nate foods and others are used as food additives or enhancersduring processing. Currently, there is not enough evidenceto suggest the reassessment of the regulated safety levels forthese products in food since little is known about how thesereceptors work in each of these organs. However, it has beenshown that MSG can trigger severe asthma (2). The rela-tively high concentrations of the endogenous EAAs in theblood and other tissue � uids (100–200 uM) suggests thatperipheral GluRs may be constantly saturated and thereforewould argue against a physiological role. The true nature ofthe GluRs– ligand interaction in each tissue is not known. It ispossible that some ligands have more potentiating ability thanothers. From the toxicological point of view, it is known thatvarious EAAs contaminants have different potencies. Someof these would have the ability to replace the weaker ligand.In addition to the potentiating ability of the compound of in-terest, the true local concentrations of endogenous EAAs atperipheral tissues are not known. Currently, more researchwill be needed to assess the extent that these receptors partic-ipate in normal functions and in the development of diseasesand how they mediate the toxic effects of EAAs.

In addition to the GluRs, enkephalin (91), the dopamine(58, 63, 90, 94), and the GABA receptors (11, 37, 76, 87)have also been described in peripheral tissues. The dopaminereceptors have been localized to heart, liver, and kidney (58,63, 90, 94). The GABA receptors are reported in the follow-ing tissues—heart, spleen, liver, lung, small and large intes-tine, stomach, adrenal, testis, ovary, and urinary bladder (11).Therefore, it appears that the circuitry that is present in thebrain may also be present in the peripheral tissues. In addi-tion, these receptors may be colocalized and interacting toproduce the � nal outcome.

In addition to food safety, a growing area of interest is thedevelopment of therapeutic products speci� cally designed tointeract with synaptic transmission of the GluRs in the CNS(16, 42, 71, 95). It is established that a myriad of pre- andpostsynaptic mechanisms exist by which iGluRs and mGluRscould modulate cell functions in the CNS. Therefore, selec-tive agonists and antagonists could be used to modulate glu-tamatergic neuronal transmissions in very select areas of theCNS (70, 94). For example, it has been suggested that NM-DAR 1 antagonists may be useful in preventing tolerance toopiate analgesia and helping control withdrawal symptomsfrom addictive drugs. Also, the overactivation of NMDAR1 has been suggestive as the causal factor in chronic diseasesuch as Huntington’s, Alzheimer’s, Parkinson’s , HIV-relatedneuronal injury and amyotrophic lateral sclerosis. Therefore,antagonists of NMDAR 1 receptor function are expected tobe useful in the treatment of some of these diseases. Ampleresearch demonstrating the presence of the GluR, dopamineand GABA receptors in peripheral tissues suggests that theseantagonists might have modulating functions in the periph-eral organs and tissues. GluRs in peripheral tissues could alsobe targets for pharmacological manipulations. For example, by guest on April 7, 2014tpx.sagepub.comDownloaded from

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GluRs in pancreatic islets offers a potential target for thera-peutic intervention to � ne-tune insulin or glucagon secretion(37, 86). The presence of these receptors in osteoblasts andthe demonstration that NMDAR activation is effective in in-hibiting bone reabsorption in vitro may contribute to the de-velopment of new therapeutics for osteoporosis (14). Of allNMDA subunits, NMDAR 1 has been shown to be the mostwidely distributed in the CNS (3, 20, 21, 24, 25, 36, 46–48,51). It is the excessive activation of this receptor that is knownto cause neuronal damage. Our data and that of others alsosupports that the NMDAR 1 also has a wide distribution inperipheral tissues (13, 27–29, 30). This supports a possiblerole in cell injury outside the CNS for this receptor.

In conclusion, it is evident that GluRs have a cell speci� cdistribution in neural and nonneural tissues. In these loca-tions, they may play a pathophysiologica l role or as target-effector sites for excitatory compounds in foods or thera-peutic products. The wide distribution in plants and animalsfrom invertebrate to primates suggests that GluRs may alsorepresent a primitive signalling system. Further research isrequired to assess the signi� cance and the role of the GluRsand their impact in various � elds.

ACKNOWLEDGMENTS

We would like to thank Peter McGuire for his technicalsupport with the immunohistochemistry and Peter Smyth forpreparations of thehistological sections. In addition, we thankMeghan Murphy, Paul Rowsell, and Dr Tim Schrader for theirdiligent reading of the initial drafts of this manuscript. Weare also indebted to Dr R. Mueller for providing the Macacafascicularis specimens.

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