Glutamate Receptors in Peripheral Tissues - SAGE Journals

2 downloads 0 Views 3MB Size Report
ABSTRACT. We illustrate the specifi c cellular distribution of different subtypes of glutamate receptors (GluRs) in peripheral neural and non-neural tissues. Some.
Laboratory Animal & Molecular Pathology TOXICOLOGIC PATHOLOGY, vol 29, no 2, pp 208– 223, 2001 Copyright C 2001 by the Society of Toxicologi c 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 speciŽ c cellular distribution of different subtypes of glutamate receptors (GluRs) in peripheral neural and non-neura l tissues. Some of the noteworthy locations are the heart, kidney, lungs, ovary, testis and endocrine cells. In these tissues the GluRs may be important in mediating cardiorespiratory, 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 generalized than initially assumed. Currently there is not enough evidence to suggest the reassessment of the regulated safety levels for these products in food since little is known on how these receptors work in each of these organs. More research is required to assess the extent that these receptors participate in normal functions and/or in the development of diseases and how they mediate the toxic effects of EAAs. Non-neural GluRs may be involved in normal cellular functions such as excitability and cell to cell communication. This is supported by the wide distribution in plants and animals from invertebrates to primates. The important tasks for the future will be to clarify the multiple biological roles of the GluRs in neural and non-neura l tissues and identify the conditions under in which these are up- or down-regulated. Then this could provide new therapeutic strategies to target GluRs outside the CNS. Keywords. Glutamate receptors; peripheral tissues; general injury mechanism; excitotoxicity

PERSPECTIVE Food toxicology is the science of evaluating the safety of chemicals that enter the human food chain either as natural compounds, contaminants and/or during processing. To assess chemical safety, tissues and organs are examined for structural, chemical, or functional alterations. These investigations help to establish the safety margins of such compounds for consumption by humans and animals as either food or therapeutic products. Therefore, product safety requires continual reassessment as new information becomes available with advances in technology. Glutamate (Glu) and aspartate (Asp) are naturally occurring 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 neurons. These excitatory amino acids (EAAs) and their various analogues can be neurotoxic, particularly when they excessively stimulate the same excitatory receptors—a phenomenon known as excitotoxicity (13, 20, 21, 25, 36, 63). This creates the potential to “over excite” neurons and cause possible neuronal damage. EAAs access the brain tissue of the

circumventricular organs that are located outside the blood brain barrier (BBB) (7, 67). Despite the BBB protective mechanisms, the local or circulating concentrations of these excitatory compounds may induce damage leading to neurotoxic exposure levels. The toxicity of each compound such as domoic acid and Asp varies according to the potency, the chemical availability, the rate of absorption, the afŽ nity to speciŽ c receptors and the particular anatomical target site. In addition, the susceptibility, genetic predisposition and health status of the individual are also important factors. Domoic acid is one of the most potent neurotoxins that can enter the food chain. It is manufactured by a sea phytoplankton that is ingested and accumulated in the digestive system of seashells such as mussels. Domoic acid was found to be the agent responsible for the outbreak of the lethal shellŽ sh poisoning that occurred in Canada in 1987 (38, 42, 65, 66, 80). Some of the survivors of this poisoning were left with severe residual memory deŽ cits. Previous work in our laboratory and from others has conŽ rmed that domoic acid preferentially damages areas of the brain involved in memory. The clinical manifestations of domoic acid intoxication are related to its effect in the brain including severe seizures (38, 42, 65, 66, 79 – 81). However, the other clinical symptoms such as: gastrointestinal disturbances, cardiovascular collapse and cardiac arrhythmia (7, 38, 81, 93) have gained less attention, until recently.

Address correspondenc e to: Santokh S. Gill, Health Canada, Banting Bldg., Tunney’s Pasture, Ottawa, Ontario K1A 0L2, Canada; email: santokh [email protected].

208

0192-6233/01$3.00 $0.00

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

209

In the peripheral tissues, there is also a rich bed of nerve circuits and many cell tissues are also capable of conducting excitatory impulses. This information together with the report of cardiovascular disturbances associated with domoic acid intoxications and other excitatory compounds in foods such as monosodium glutamate (MSG) prompted our initial investigation of the heart as a possible target organ (27 – 29, 88, 93). Because it has been established that Glu or its analogues interact with the postsynaptic membrane of glutamate receptors (GluRs) in the CNS, the existence of a similar relationship was explored in other tissues and organs. This review summarizes the current knowledge on the GluRs in peripheral tissues, their distribution, potential role and the effects they can mediate. We hope this review will stimulate research and provide additional information useful in formulating regulatory decisions concerning the public health protection of foods and therapeutic products. BACKGROUND Excitatory Amino Acids Glu and Asp are the most abundant dicarboxylic amino acids in the brain and are believed to be the primary neurotransmitters in the mammalian CNS (20, 21, 62). Although these amino acids are primarily involved in intermediary metabolism and other non-neuronal functions, their most important role is as neurotransmitters. It is estimated Glu mediates nearly 50% of all the synaptic transmissions in the CNS and its involvement is implicated in nearly all aspects of normal brain function including learning, memory, movement, cognition and development (3, 20, 46 – 48, 51, 53, 62, 73). At elevated concentrations, Glu acts as a neurotoxin capable of inducing severe neuronal damage. Hence, Glu can be considered to be a “2-edged sword” that undergoes a transition from a neurotransmitter to a neurotoxin. In addition to the endogenous glutamate, there are naturally occurring substances, which have Glu-like excitatory properties and potentially excitotoxic effects. Glu and its structural analogues (Figure 1) may enter the food supply during preparation 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 homocysteate), B-N -oxalyamino- L -alanine (BOAA or ODAP), B-N -methyl-amino- L-alanine (BMAA) and the potent sea food toxin domoic acid (20, 22, 38, 42, 59, 69, 86). A plethora of Ž ndings in the past 2 decades has provided direct and circumstantial evidence for abnormal glutamate (and its analogues ) transmission in the etiology and pathophysiolog y of many 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 the species, developmental stage of the animal, type of agonist, duration of exposure to the agonist and the cellular expression of the GluR subtypes. An array of GluRs are known to be present on pre- and postsynaptic membranes that are used to transduce integrated signals using an increased ion  ux and second messenger pathways (Figure 2) (16, 17, 20, 25, 36, 62, 73). It is the excessive activation of these receptors that leads to neurotoxicity.

FIGURE 1.—Excitotoxic structural analogues of glutamate. Most of these compounds are known to mimic both the neuroexcitator y and neurotoxic properties of glutamate. ODAP is B-N -Oxalylamino-L-Alanine (it is also abbreviated BOAA60).

Glutamate Receptors: An Overview Numerous reviews are available on the GluRs in the CNS, their functional roles and implications on the pathobiology of neural injury and neuropsychiatric disorders (3, 20, 21, 24, 36, 48, 53, 62, 73). GluRs have been individually characterized by their sensitivity to speciŽ c glutamate analogues and by the features of the glutamate-elicited current. GluR agonists and antagonists are structurally similar to Glu, which allows them to bind onto the same receptors. Two classes of GluRs have been characterized based on the studies in the CNS: ionotropic (iGluRs) and metabotropic (mGluRs). Their cloning has revealed the molecular diversity of the gene families encoding various receptor types that are responsible for the pharmacological and functional heterogeneity in the brain. A brief resume is outlined in the present study because comprehensive treatment of these GluRs has been previously reviewed in detail (3, 16, 17, 20, 25, 36, 53, 62, 74). Ionotropic Glutamate Receptors The iGluRs contain integral cationic channels associated with ligand binding sites and they are known to mediate rapid synaptic transmission. The iGluR family is classiŽ ed into 3 major subtypes according to their sequence similarities, their electrophysiological properties and their afŽ nity to selective agonists: N -methyl-D -aspartate (NMDA), a -amino-3hydroxy-5-methyl-4-isoxazol e propionic acid (AMPA) and

210

GILL AND PULIDO

TOXICOLOGIC PATHOLOGY

FIGURE 2.—Schematic representation of the interaction betweeen the presynaptic and the postsynapti c terminal. The Ž gure shows vesicular release into the synaptic space, activation of the postsynapti c receptor systems, reuptake into the presynaptic terminal and surrounding glial cells. The excitatory amino acids (for example glutamate) activates the various glutamate receptors present in the postsynapti c membrane. This triggers the in ux of Ca 2 from the extracellular environmen t to the synaptic cleft. The accumulation of Ca 2 is crucial determinant of injury. This elevation of Ca 2 triggers the activation of several enzymes: calmodulin (CAM), protein kinase C (PKC), nitric oxide synthase (NO synthase), phospholipas e A2 (PLA2) and reactive oxygen species (ROS)—modiŽ ed from Harry (33) and Said (1999).

kainate (Ka) receptors (3, 20, 21, 36). The membrane channels associated with these receptors exhibit varied pharmacological and electrophysiological properties, including ionic channel selectivity to sodium (Na ), potassium (K ) and calcium (Ca 2 ) (3, 20, 21, 36, 62). The non-NMDA receptors control a nonselective cationic channel permeable to Na and K , whereas NMDA is more permeable to Ca 2 ions than

either AMPA or Ka (3, 20, 21, 36, 62). Recombinant technology has identiŽ ed several gene families encoding iGluRs: AMPA family is composed of GluR 1-4 (GluR A-D); Ka family includes GluR 5-7 and Ka 1-2; NMDA includes NMDAR 1 and NMDAR 2A-D. NMDAR 1 is the most tightly regulated neurotransmitter receptor by forming the channel where other subunits (NMDA 2A-D) are involved in the receptor

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

modulation. It is the most intensively studied and complex receptor and is linked to the Na /Ca 2 ion channel that has 5 distinct binding sites for endogenous ligands that in uence its opening (20, 21, 36, 62). These include 2 different agonist recognition sites (1 for Glu and 1 for glycine), a polyamine regulatory site that promotes receptor activation. The remaining sites separate recognition sites for Mg 2 , Zn 2 and phencyclidine (PCP), which inhibit ion  ux through agonistbound receptors (20, 21, 36, 62). The complexity of these GluR families is further increased by alternate splicing, RNA editing and post-translational modiŽ cations such as phosphorylation, glycosylation and palmitoylation. Each of these modiŽ cations is important in the regulation of channel functions. Within each family, GluRs subunits can also form homo-oligomeric or heteroligomeric channels that exhibit different functional properties depending on the subunit composition. For example, the presence of the GluR 2 subunit decreases Ca 2 permeability of AMPA channels (20, 21, 36, 62). Metabotropic Glutamate Receptors The mGluRs exert their effects either on the second messengers or ion channels via the activation of the of GTPbinding proteins and regulate the synthesis of different intracellular second messengers such as IP3 , cAMP or cGMP (17, 21, 36, 62, 73). A single mGluR protein can cross-talk with multiple second messengers in the same cell. As with iGluRs, the mGluRs are also classiŽ ed into 4 groups based on amino acid sequence similarities, agonist pharmacology and the signal transduction pathways to which they are coupled. Group I (mGluR 1, 5, and 6) stimulates inositol phosphate metabolism and mobilization of intracellular Ca 2 . Group II (mGluR 2 and 3) and group III (mGLuR 4, 6 – 8) are coupled to adenyl cyclase (17, 21, 36, 62, 73). Group IV is coupled to to the activation of phospholipase D (PLD). The latter class is more efŽ ciently activated by L-cysteine-sulŽ nic acid (L -CSA) rather than Glu, which suggests that L -CSA may serve as an endogenous agonist of this receptor (16). The mGluRs function is predominantly with the long-term aspects of cellular control by operating via G proteins and several second messenger systems. As with the iGluRs, the mGluRs also have a unique distribution in the CNS and retina,

211

which re ects a diversity of function in normal and pathological processes. These receptors have been shown to exert a wide variety of modulating effects on both excitatory and inhibitory synaptic transmission. This is expected if a receptor activation is coupled to multiple effector enzyme (17, 21, 36, 62, 73). The mGluRs have certain features that distinguish them from the iGluRs. First, the mGluRs modulate the activity of neurons rather than mediate fast synaptic neurotransmission. Second, the distribution of the mGluRs is highly diverse and heterogenous. Different subclasses are localized uniquely at both the anatomical and cellular levels. For example, mGluR 2 and 3 are found in high density in the cerebral cortex, whereas mGluR 4 is found in high density in the thalamus but not in the cortex and mGluR 6 is almost exclusively found in the retina. GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES Introduction A recent surge of publications supports the presence and functionality of GluRs outside the CNS in various tissues and species. Table 1 summarizes the distribution of the GluRs in peripheral tissues demonstrated by using various methodologies such as: RT-PCR, PCR, northern blots, western blots, immunohistochemistry and in situ hybridization. These tissues include adrenal medulla (85, 90), peripheral nerves— myelinated and unmyelinated (1, 15), bone (14, 26), bone marrow (26), bronchial smooth muscle, endocrine pancreas (4 – 6, 30, 32, 37, 45, 49, 54, 87), gut (8, 58, 77, 81, 86), esophagus (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 longitudinal muscle (56, 74, 75), autonomic and sensory ganglia (85), rat glaborous skin (10), kidney, spleens, ovaries, (26, 28, this study ), vagus and other cholinergic nerves (1), tachykinincontaining sensory nerves and vestibular tissues (18). In our studies, we have conducted a thorough analysis on the distribution of the GluRs in peripheral tissues of the rat where the antibodies and the methodology used have been previously described (27, 28, 29). Different subtypes of GluRs

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

Species

Organ

GluR 2/3, Ka 2, NMDAR 1, mGluR 5, mGluR 2/3, mGluR 1

Rat/monkey

Heart

GluR 2/3, Ka 2, NMDAR 1, mGluR 2/3

Rat/monkey

Ovary

GluR 2/3, Ka 2, NMDAR 1, GluR 2/3

Rat/monkey

Uterus

GluR 2/3, Ka 2, NMDAR 1, mGluR 2/3

Rat

Kidney

GluR 2/3, Ka 2, NMDAR 1, mGluR 2/3 GluR 2/3, Ka 2, NMDAR 1, mGluR 2/3

Rat

Testis

Rat

Gastrointestinal

GluR 2/3, Ka 2, NMDAR 1, mGluR 2/3

Rat

Others

Tissue/Cell type Atrium/septum conducting Ž bers, ganglia cells, nerve Ž bers, myocardiocytes , intercalated discs, blood vessels Corpus luteum, primordial follicles, theca, granulosa cells, oocyte, blood vessels, nerve Ž bers Exocervix, myometrium, endometrial glands, epithelium of fallopian tubes, nerve Ž bers Glomeruli, mesangium, podocytes , juxtaglomerula r apparatus, tubules Germinal epithelium, interstitial cells Enteroendocrine cells, parietal cells of the stomach, pancreatic islets, nerve Ž bers, ganglia cells, liver Lungs, spleen, bone marrow (megakaryocytes), mast cells, in ammatory cells

Methodology

References

IS, westerns, northerns, RT-PCR

27, 28, 56, 58, 68

IS

29, Gill et al (in preparation)

IS, westerns, RT-PCR, northerns

29, Gill et al (in preparation)

IS, westerns, RT-PCR, northerns

27, 29

IS, westerns, RT-PCR, northerns IS, westerns, RT-PCR, northern blot

27, 29, 45

IS, westerns, RT-PCR, northern blots

14, 26, 27, 29, 31, 67, 69, 70

5, 6, 8, 29, 37, 47, 57, 73, 74, 82, 85

212

GILL AND PULIDO

were observed in the heart, spleen, testis and kidney (27, 28, 29). These Ž ndings and that of others clearly supports the view that GluRs are widely present in peripheral tissues and have a speciŽ c cellular distribution. Some of the GluRs isolated from peripheral tissues have been cloned and sequenced (10, 12, 14, 24, 27, 32, 36, 87). These sequences correspond with GluRs that have been cloned in the CNS. Further physiological and pharmacological experiments support the hypothesis that GluR receptors in the periphery have similar properties to those in the CNS or expressed in host cells transfected with cloned subunits (28, 30, 36, 56, 71, 74, 84, 87). For example, the AMPA receptors in the rat pancreas respond to L-glutamate, AMPA and kainate. These receptors were blocked by competitive antagonists, 6-cyano-7-nitroquinoxalin e (CNQX) and potentiated by cyclothiazide (87). These properties are also shared by neuronal AMPA receptors. In addition, the stimulation of cultured rat myocardial cells by L-glutamate leads to an increase in the intracellular Ca 2 oscillation frequency (88). Similar physiological studies with agonists and antagonists of the NMDAR 1 receptor in the pig ileum have shown that these receptors are similar to those characterized in the CNS (73). NMDAR 1 receptors in the pig ileum were also blocked by Mg 2 ions and competitively antagonized by DL -2-amino5-phosphonovaleri c acid. Glutamate Receptors in the Heart and Cardiac Arrhythmia We have extensively investigated the presence of the GluRs in the rat (27, 28). The preferential localization of GluRs was seen within the conducting system, nerve terminals and intramural ganglia cells (Figure 3). Similar distribution, but with enhanced deŽ nition was observed in the conducting systems in the monkey heart (in preparation, 57). These Ž ndings likely re ect the higher level of anatomical differentiation of the conducting system in nonhuman primates versus the rodents as illustrated by using the neural markers PGP 9.5 and NF (57). The presence of the GluRs in areas speciŽ cally involved in the conduction of impulses suggests their involvement in the control of heart rhythm. Therefore, the presence of the GluRs in the heart implies that this organ may be an important 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 and cardiac nerve Ž bers, which are known to be components of the peripheral autonomic nervous system, prompted us to investigate the presence of GluRs in other tissues. Glutamate Receptors in Kidney and Electrolyte – Water Homeostasis In the kidney, the wide distribution of NMDAR 1 and the presence of mGluR 2/3 and GluR 2/3 in the juxtaglomerular apparatus (JGA) and proximal tubules (Figures 4a, b, c, and d), suggests that these receptors may be involved in electrolytes and water homeostasis. The strong visualization of immunoreactivity with anti-mGluR 2/3 and anti-GluR 2/3 within the granular cells of the afferent arteriole suggests a potential involvement in the control of renin release (18, Figures 4b and c). The renin-angiotensin system is a major hormonal system involved in the regulation of electrolyte,  uid balance and blood pressure (39). Pharmacological and biochem-

TOXICOLOGIC PATHOLOGY

ical evidence also supports the presence of the dopamine receptors—D1A , D1B , D2 , and D3 within the kidney (58, 72, 94). Like the GluRs, the dopamine receptors were also specifically distributed. D1A and D1B are both reported to be present in the renal vasculature, renal proximal and distal convoluted tubules, cortical and collecting ducts. In contrast, D1A is not present in JGA apparatus and the ascending loop of Henle, whereas D1B is present in these regions. Experimental data suggests that dopamine receptors are involved in renal hemodynamics, ion transport and renin secretion (58, 72, 94). In addition to the GluRs and the dopamine receptors, the GABA receptors are also found in the kidney. The GABAA and GABA B receptors have been localized to the renal cortex (11). Therefore, GABA might also in uence the renal functions in the kidney cortex rather than the medulla. Whether or not there is co-localization or co-functionality of these receptors needs to be determined. Glutamate Receptors in Sex Organs and Reproduction We have shown the differential distribution of GluRs in reproductive organs of the male (Figures 4e – i) and female rat (Figures 5 and 6). In the testis, these receptors have a speciŽ c afŽ nity for different structures. There is intense antimGluR 2/3 immunolabelling of the head of the mature spermatids/spermatozoa, interstitial cells and myoid cells. AntiNMDAR 1 has a strong afŽ nity for the germinal epithelium, particularly the spermatogonia adjacent to the basal lamina and the more mature spermatids near the lumen (Figure 4e), whereas the anti-GluR 2/3 immunostain is limited to the cells in the interstitial spaces. This differential distribution suggests that GluRs may be involved in spermatogenesis, spermatozoa motility and testicular development—each linked to a speciŽ c receptor. To support our hypothesis, we are investigating the presence and distribution of GluRs in rat testes during various developmental stages. Previously, it has been shown that speciŽ c binding sites for [ 3 H]-TCP, a ligand that labels a binding site within the NMDA receptor ion channel, has been demonstrated on membranes of mammalian spermatozoa. Morever Cl -independent [3 H]glutamate binding, which could be partially displaced by NMDA and AP5, has also been detected in seminal vesicles (20). Further, the results of Lara and Bastos-Ramos (44) suggest the noradrenergic neurons innervating the rat vas deferens are controlled by a glutamate-dependent excitatory process in the ganglia. They showed that a single dose of kainate to the ganglia induced a decrease in the norepinephrine content of the vas deferens. Binding studies using [3 H]-glutamate to the membrane fraction showed that the binding was saturable. This binding was inhibited by different analogues according to different potencies L -glutamate > kainate > quisqualate N -methyl-D -aspartate. Thus, it is likely that the vas deferens has a glutamatergic excitatory mechanism for the control of their activity and these are responsible for the depolarizing potential on the noradrenergic neurons. This mechanism might be important for the contractile activity of the vas deferens and hence in the control of seminal  uid (44). In the rat female reproductive system, GluRs also have a unique distribution within each organ (Figures 5 and 6). Each antibody has a differential afŽ nity to speciŽ c structures in the ovaries, the fallopian tubes, the cervix, the myometrium and the endometrium (Figures 5 and 6). In the ovary (Figure 6)

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

213

FPO

FIGURE 3.—Photomicrograph s of the rat heart perfused with 4% PFA and processed for immunohistochemistry. Microwaved parafŽ n sections were immunostained with speciŽ 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 Ž 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 approximately 100 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 Ž 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 the intercalated discs (ID) (27, 28).

214

GILL AND PULIDO

TOXICOLOGIC PATHOLOGY

FPO

FIGURE 4.—Photomicrographs of the rat kidney and testis perfused with 4% PFA and processed for immunohistochemistr y. Microwaved parafŽ n sections were immunostained with speciŽ c antibodies to GluRs, using LAB/avidin biotin method and DAB as chromogen. A) Immunostainin g with anti-NMDAR 1 is seen in the distal tubule, proximal tubule, and glomeruli, particularly in the mesangium and podocytes (shown by an arrow). B) Anti-mGluR 2/3 strong immunostain in the convoluted proximal tubules and the JGA (arrow). C) Higher magniŽ cation of the JGA, showing anti-mGluR 2/3 dark cytoplasmic staining of the granular cells in the 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 afŽ nity for the germinal epithelium, particularly the spermatogoni a adjacent to the basal lamina and the more mature spermatids near the lumen. F) & G) Anti-GluR 2/3 immunostai n is limited to the interstitial 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); interstitial spaces (IS); seminiferus tubules (ST); spermatogonium (SP); spermatids (SP) (29).

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

215

FPO

FIGURE 5.—Immunohistochemica l analysis of various subtypes of GluRs. ParafŽ n sections of rat uterus Ž xed with 4% PFA, immunostained using the LAB/avidin biotin method and DAB as chromogen. Each subtype of GluR Abs tested has a differential speciŽ c distribution. Anti-mGluR shows preferential binding to the most superŽ cial layer of the stratiŽ 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 afŽ nity for the endometrial glands, myometrium (D) and the ciliated epithelium of the fallopian tube (G, H). Abbreviations: exocervica l epithelium (CE); myometrium (M); endometrial glands (EG); stroma (S); fallopian tube or oviduct (FT); lumen (L); ciliated epithelium (this study).

216

GILL AND PULIDO

TOXICOLOGIC PATHOLOGY

FPO

FIGURE 6.—Rat ovaries Ž xed in 4% PFA and stained with H&E (A). Microwaved parafŽ n sections were immunostained with speciŽ c antibodies to various subtypes of GluRs, using the LAB/avidin biotin method and DAB as chromogen . Immunostain with anti-GluR 2/3 shows a wide distribution throughout the ovary, including stroma; 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 mature follicles, oocytes, and corpus luteum. Anti-mGluR 2/3 also shows afŽ nity for the corpus luteum and oocytes (E, F), but is very faint for other structures. Anti-NMDAR 1 has strong selective afŽ nity for the oocyte (G). Nerve Ž 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 Ž bers (NF) (this work).

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

the distribution of GluRs within the follicles varies at different stages of their maturation. In the rat anti-NMDAR 1 and to some extent anti-GluR 2/3 and anti-mGluR 2/3, have a remarkable selective afŽ nity for the oocyte. GluR 2/3 and mGluR 2/3, but not NMDAR 1 are visualized in the corpus luteum. In the uterus (Figure 5), anti-GluR 2/3 showed strong afŽ nity for the ciliated epithelium of the fallopian tubes, smooth muscle of the myometrium and endometrial glands, whereas anti-NMDAR 1 is more selective to the endometrial glands and the exocervical epithelium. This suggests that these receptors may be involved in ovulation, fertilization, implantation of the ovum and excitability of the uterus. To examine if similar preferential distributions exist in higher mammals, we have tested the location of GluRs in the sex organs of nonhuman primates, using ovary, uterus and fallopian tubes of Macaca fascicularis. In this species, the corpus luteum and oocytes display intense, selective immunolabelling with anti-NMDAR 1 and anti-GluR 2/3 (manuscript in preparation). The presence of the GluRs within the reproductive organs and the known functional inhibitory/excitatory effects of GABA/GABA receptors (22) suggest that similar excitatory – inhibitory neurotransmission interplay may also be present in the reproductive organs using GluRs as mediators. Therefore reproductive functions—such as gonadal maturation; steroidal sex hormone regulation; maturation, motility, and excitability of the spermatozoa; ovulation; fertilization; excitability 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 toxicological implications. Glutamate Receptors in Neuroendocrine Tissues and 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 presence of GluRs in other endocrine tissues. These include the pancreas, pituitary, pineal gland, adrenal gland and kidney. The differential distribution of the GluR subunits in the pancreas has already been described (37, 54, 87). Liu et al (49) showed that GluR 1 and GluR 4 were mainly localized to insulin-secreting cells in the central mass of the pancreatic islet, whereas GluR 2/3 was preferentially localized in the peripheral rim composed of non – insulin-secreting islet cells. It appears that insulin- and non – insulin-secreting cells express different AMPA receptor subunits, which may be used to mediate their hormone secretion, as was suggested earlier by Bertrand (5, 6). Weaver et al (87) showed that the AMPA receptors were located in the a , b , and PP cells but were generally absent from the c cells, whereas kainate receptors were expressed in the a and c cells although they were not found in b or PP cells. These observations add to the evidence that these receptors may be involved in the regulation of hormone secretion (4 – 6, 37, 86). Studies of Weaver et al (87) show that Glu depolarizes islet cells when Glu serum levels are elevated. Intracellular Ca 2 measurements and electrophysiologica l recordings showed that kainate, AMPA and NMDA elicited increases in Ca 2 in single b -pancreatic cells and depolarized them. In addition, kainate and AMPA stimulated the release of insulin whereas NMDA did not (36). This stimulatory effect was dependent on the glucose con-

217

centration: Glu stimulated insulin release in the presence of a glucose concentration of 8.3 mM but not in the presence of a low concentration (2.8 mM). Hence, Glu is a potentiator of glucose-induced insulin release. In addition to the GluRs, the presence of GABA receptors has also been reported in the pancreas (11, 76, 86). Therefore, the Ž nal activity of these cells is probably determined by the balance in the activities of both GluR and GABA receptors. It is therefore conceivable that these receptors are involved in the pathophysiolog y of the pancreas. Glutamate Receptors in the Gastro-Intestinal (GI) Tract Experimental evidence from several labs and from this laboratory (Figure 7) have shown the GluRs to be present in the stomach, duodenum and descending colon (8, 56, 74, 75, 83). Immunohistochemical analysis showed that GluRs antibodies had poor afŽ nity for the esophagus with the exception of anti-NMDAR 1, which preferentially stained the less mature cells within the basal layer of the stratiŽ ed squamous epithelium (Figure 7). In the stomach mucosa, parietal cells were stained with anti-GluR 2/3, anti-NMDAR 1 and anti-mGluR 2/3. Mast cells throughout the GI tract show strong staining with anti-NMDAR 1. All the antibodies showed some afŽ nity for enteroendocrine cells, ganglia cells and nerve Ž bers throughout the GI tract. Our Ž ndings with the NMDAR 1 are similar to those described by Burns et al (8). We show the presence of NMDAR 1 expression in the stomach, duodenum, ileum and descending colon. Previous studies have shown that Glu, through the action of NMDAR 1, induces contraction of the ileal longitudinal smooth muscle/myenteric plexus (74). The myenteric plexus is a layer of neurons innervating the gastrointestinal tract, which is largely responsible for gastrointestinal motility. The role of NMDA receptors in intestinal motility was conŽ rmed by the capability of Glu, Asp, L -homocysteate and NMDA (but not kainate or quisqualate) to cause muscle contraction. The contraction was blocked by NMDA antagonists (noncompetitively by Mg 2 and by phencyclidine-like drugs such as etoxadrol, dextromethorphan, and MK801) but not by kainate or quisqualate antagonists. The order of potencies for the contractile effects is: L -glutamate > aspartate > L-homocysteate > NMDA > D glutamate (73). In the CNS, electrophysiological L-glutamate and L -aspartate are less potent than NMDA due to their avid uptake. The results of Shannon and Sawyer (74) suggest an absence of the uptake processes of these compounds in the myenteric plexus. These results are in agreement with Moroni et al (56). The studies of Tsai et al (82, 83) showed that Glu and Asp are both involved in regulating acid secretion in the stomach. However, their mode of action is different. They found that Asp was more speciŽ c than Glu in regulating acid secretion. This was attributed to the fact that Glu is a general agonist for all types of GluRs, yet Asp is a potent agonist for NMDAR receptors. Glutamate Receptors in Other Tissues Patton 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 demonstrated that bone cells have the potential to express many of the molecules associated with the glutamate-mediated

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 parafŽ n sections immunostained using the LAB/avidin biotin method and DAB as chromogen . Each subtype of GluRs Ab tested has a speciŽ c distribution. A & B) Anti-GluR 2/3 immunostaining of the afŽ 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 afŽ nity for GC and NF throughou t the tract. D) Pancreatic islets stained with anti-GluR 2/3. The intensity of the 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); goblet cells (G).

signalling. Chenu et al (14) documented that all mature cell types (osteoblasts, osteocytes, and osteoclasts) express one or more of the GluRs subunits. The blockade of NMDA receptors with antagonists resulted in inhibition of osteoblast formation, 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 identiŽ ed in bone (14), further supporting the view that neuroexcitatory amino acids may play a role in paracrine signalling in bone cells. The GLAST performs an essential function during glutamate-mediated synaptic neurotransmission by acting as a high afŽ nity uptake system to remove released Glu from the synaptic cleft, thus preventing overstimulation of the postsynaptic glutamate receptors. Thus, the presence of GLAST in the bone and other tissues supports the theory that Glu signalling may have a role outside the CNS. More recently, the presence of the glutamate receptors NMDAR 1 and NMDAR 2 were demonstrated in bone mar-

row of the rat, human megakaryocyte and the MEG-01 clonal megakaryoblastic cell line (see Table 1). An interesting observation is that the level of glycosylation in these cells is reduced or absent as compared to the CNS-NMDA type. It is speculated that glycosylation provides stability to the NMDA receptor in the synaptic membrane for the precise orientation and localization in the CNS. However, in the megakaryocyte, the receptors are distributed evenly across the cell surface, which allows for multidirectional agonist stimulation. These receptors are probably involved in signalling, which is reinforced by the Ž ndings that megakaryocyte exist in intimate contact with cells bearing the glutamate-transported proteins GLT-1 and GLAST, which are found on mononuclear bone marrow cells and osteoblasts, respectively. These transporters are essential requirements for functional glutamate-mediated communications. Genever et al (26) showed that NMDAR 1 activity was necessary for phorbol myristate acetate (PMA) induced differentiation of megakaryoblastic cells; NMDAR 1

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

receptor cell blockade by speciŽ c antagonists, MK-801 or DAP5; inhibited PMA-mediated increases in cell size; CD41 expression; and adhesion of MEG-01 cells. Several mGluRs—mGluR 1, 2, 3, and 5—were also reported in the thymic stromal cell line (TC1S) and in the thymocytes. Using RT-PCR and western blotting, it was demonstrated that the thymic stromal cell line TC1S expressed mGluR 2, 3, and 5. Thymocytes expressed mGluR 1, 3, and 5. Fluorescence Activated Cell Sorter (FACS) analysis illustrated that majority of the unfractionated thymocytes (70%) showed the presence of mGluR 5, whereas 50% of the cells expressed mGluR 3 and only 15% expressed mGluR 1. In contrast, isolated CD4 /CD8 , double negative thymocyte precursor, expressed mGluR 3 (45%) and mGluR 1 (40%), whereas mGLuR 5 was barely detectable. Therefore, it was hypothesized that changes in mGluRs subtype expressions may be related to T-cell maturation stages (31). GluRs are also expressed in other lymphoid tissues and in ammatory inŽ ltrates (Table 1). The iGluRs (27) and mGluRs (77) were also present in the liver. Sureda et al (77) showed mGluR 5 in the primary hepatocytes stimulated the hydrolysis of inositol phospholipid. The effects of mGluRs agonists, 1S,3R-ACPD and quisqualate, was examined on anoxia-induced cell damage. A time-dependent decline was observed on the viability and was maximal after 90 minutes. Both 1S,3R-ACPD and quisqualate shortened this time course of anoxia-induced cell damage, reducing signiŽ cantly the viability of primary hepatocytes. In contrast, the agonist 4C3HPG for the type 11 mGluR had no effect. The hypothesis is that this receptor is activated by the Glu present in the portal blood and may contribute to the liver damage under adverse conditions. Glutamate Receptors as Possible Mediators of Cell and Tissue Injury and Pathology The view that Glu or related excitatory amino acids can cause neuronal injury as a result of overexcitation is called excitotoxicity. The GluRs are known to act as mediators of in ammation and cellular injury through a common injury pathway (46, 47, 48, 51; see Figure 2). In the CNS, the stimulation of GluRs triggers an excessive in ux of calcium into neurons through the ion channels, which mediates neural injury (13, 20, 21, 22, 46, 47, 48, 52). Because the iGluRs are ion-gated channels selective to Na , K , and Ca 2 , any sustained stimulation of the GluRs results in osmotic damage due to the entry of excessive ions and water. This increases the intracellular Ca 2 concentration which is crucial to the determinant of injury. It is this high concentration of Ca 2 that triggers the activation of several enzyme pathways and signalling cascades including as phospholipases , protein kinase C, proteases, protein phosphatases, nitric acid synthases and the generation of free radicals (3 – 13, 19, 20, 46, 48, 51). The destabilization of Ca 2 homeostasis also causes the translocation of protein kinase C (PKC) from the cytoplasm to the membrane. This leads to the phosphorylation of the membrane proteins via PKC promoting the destabilization of the regulatory mechanisms for Ca 2 homeostasis, which mediates toxicity (13, 20, 21). On activation of phospholipase A2 , arachidonic acid (with its metabolites and platelet-activating factors) is generated. Platelet-activating factors increase the

219

neuronal calcium levels by stimulating the release of Glu. Arachidonic acid potentiates NMDA evoked currents and inhibits the reabsorption of Glu into astrocytes and neurons. This further exacerbates the situation by a positive feedback mechanism where free radicals are formed during arachidonic acid metabolism, leading to further phospholipase A2 activation. This results in an increased concentration of extracellular glutamates which contributes to the sustained activation of the GluRs (13, 16, 20, 21). As a consequence, cysteine transport is inhibited causing a decrease of intracellular reducing sulphydryls and the generation of oxygen radicals, which results in cell death. In addition to enzymes of the cell cytosol, the nuclear enzymes are also activated by increase of Ca 2 . For example, Ca 2 may activate endonucleases that result in condensation of nuclear chromatin and eventually DNA fragmentation and nuclear breakdown, a pathologic process known as apoptosis. Free radicals also contribute to DNA fragmentation. The increased concentration of Ca 2 raises the nitric oxide via the calmodulin activation of nitric oxide synthetases, which generates oxygen radicals. Nitric oxide has been observed in peripheral tissues, ganglion cells, nerve Ž bers, cardiocytes, and myocytes in the heart of pig and rat. Our work has demonstrated the presence of the glutamate receptors in the same structures. Liu et al (49) showed the colocalization of nitric oxide and AMPA receptors in ganglion cells of the pancreas. Because the nitric oxide is calcium- and calmodulindependent and the AMPA receptors are Ca 2 permeable, it is possible that nitric oxide is activated through the AMPA receptors. We propose that the mechanism involved in injury in the CNS may be a basic mechanism for injury in all tissues. This is supported by the Ž nding that excessive activation of the NMDAR 1 in the lungs induces acute edema and lung injury as seen in “adult respiratory distress syndrome” (70, 71). This injury can further be modulated by blockage of one of three critical steps: NMDA 1 binding, inhibition of NO synthesis, or activation of poly (ADP-ribose) polymerase (70, 71). Our results showed immunolabelling for various GluRs in bronchial epithelium, blood vessels of the lungs, mast cells, and in ammatory cells. This supports the view that they play a role in airway responses to injury and in ammation. All the antibodies tested for the subtypes of GluRs showed afŽ nity to mast cells in all tissues analyzed, particularly in the lungs and gastrointestinal tract. Their presence in the airway structures such as the larynx, esophagus and mast cells also implicate the GluRs in the mediation of asthmatic episodes (2, 70, 71). The excitation of GluRs in the air passages therefore may be important in airway in ammation and hyperreactivity observed in bronchial asthma (60). Their presence also could explain the enhancement of acute asthmatic attacks by glutamate-containing foods (2). These researchers did blind placebo-controlled experiments where subjects with asthma received MSG in tablet form. Their studies showed that MSG did indeed provoke asthma, which in some cases was severe and life-threatening. Mast cells are known to be found in the connective tissues throughout the body, most abundantly in the submucosa tissues and the dermis. Purcell et al (68) showed that spermidine-induced release from the mast cells are dependent on the presence of Ca 2 in the external mileau. The in ux of Ca 2 is known to be accompanied by NMDAR 1 activation.

220

GILL AND PULIDO

This increased intracellular Ca 2 concentration initiates the exocytotic degranulation process in mast cells. Spermine is a natural polyamine, and the opening of the ion channel associated with NMDAR receptors is facilitated by binding sites for polyamines. In neuronal tissue, polyamine triggers histamine secretion through interaction with a polyamine site associated with an NMDAR 1 macrocomplex. Therefore, spermine can modulate activation of the macrocomplex, either through action at polyamine-binding sites in the lung or at other sites. The antagonists of NMDAR 1, MK801, blocked this release of histamine secretion that was induced by the natural polyamine-spermine. If the NMDAR 1 is present, as supported by (68, 70, 71), then it opens up the possibility that EAAs can also in uence allergic reactions. Glu and related EAA agonists induce neurotoxic damage in the CNS, which occurs under conditions of hypoxia or ischemia and is believed to be due to the increased intracellular Ca 2 concentration (13, 20, 21, 25, 36, 48). Intestinal mucosa damage has also been reported after hypoxia and ischemia (61). CONSIDERATIONS ON THE PHYLOGENY OF GLUTAMATE RECEPTORS Recent data suggests that putative iGluRs exist in plants, in both monocotyledons and dicotyledons. These observations are based on northern and heterologous Southern blot analysis (43). Preliminary data indicates that the GluRs are involved in light signal transduction. Therefore, it is possible that signalling between cells by EAAs may have evolved from primitive mechanisms before the divergence of plants and animals. GluRs and other similar signalling systems are actually ancestral methods of communication, common to plants and animals alike. This is supported by the fact that these receptors are present in mollusc (77), leech (19), and Oreochromis sp (freshwater Ž sh, 88) and C elegans (bluegreen algae, 50). In plants, the GluR-like receptors respond to the same antagonist as the GluRs in the CNS. The DNA sequence data also shows variable (60% – 16%) homology to the glutamate receptors characterized in the mammalian system. CONCLUSIONS AND FUTURE RESEARCH CONSIDERATIONS In summary, GluRs have a wide and a unique distribution in peripheral tissues. These receptors are pharmacologically similar to their counterparts in the CNS, although the possibility that there are subtle distinctions such as glycosylation cannot be ignored. The presence of the GluRs in peripheral tissues may provide explanations for the autonomic disturbances (GI, salivation, cardiovascular, vomiting) that have been reported in animals dosed with potent excitotoxins such as domoic acid (38, 66, 67, 79, 80, 88, 93). Because the EAA excitotoxicity is intimately associated with the GluRs, the toxic effects may be more generalized than initially assumed, particularly in the light that GluRs are widely present in peripheral tissues that are not protected by the blood brain barrier (7, 67). Excitotoxicity depends on the intracellular Na 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 toxic effects (13, 20, 21). Based on the pattern of anatomical distribution, we suggest that these receptors may be important for the mediation of functions such as hormone regulation, heart

TOXICOLOGIC PATHOLOGY

rhythm, blood pressure, circulation and reproduction. Some of the noteworthy locations are the heart, kidney, lungs, ovary, testis and endocrine cells—suggesting they play a role on cardiorespiratory, endocrine and reproductive functions. Furthermore, they could potentially turn the tissues where they are present into target sites for the toxic effect of glutamate – like-products. Many of these are known toxins that contaminate foods and others are used as food additives or enhancers during processing. Currently, there is not enough evidence to suggest the reassessment of the regulated safety levels for these products in food since little is known about how these receptors work in each of these organs. However, it has been shown that MSG can trigger severe asthma (2). The relatively high concentrations of the endogenous EAAs in the blood and other tissue  uids (100 – 200 uM ) suggests that peripheral GluRs may be constantly saturated and therefore would argue against a physiologica l role. The true nature of the GluRs – ligand interaction in each tissue is not known. It is possible that some ligands have more potentiating ability than others. From the toxicological point of view, it is known that various EAAs contaminants have different potencies. Some of these would have the ability to replace the weaker ligand. In addition to the potentiating ability of the compound of interest, the true local concentrations of endogenous EAAs at peripheral tissues are not known. Currently, more research will be needed to assess the extent that these receptors participate in normal functions and in the development of diseases and 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 dopamine receptors have been localized to heart, liver, and kidney (58, 63, 90, 94). The GABA receptors are reported in the following tissues—heart, spleen, liver, lung, small and large intestine, stomach, adrenal, testis, ovary, and urinary bladder (11). Therefore, it appears that the circuitry that is present in the brain may also be present in the peripheral tissues. In addition, these receptors may be colocalized and interacting to produce the Ž nal outcome. In addition to food safety, a growing area of interest is the development of therapeutic products speciŽ cally designed to interact with synaptic transmission of the GluRs in the CNS (16, 42, 71, 95). It is established that a myriad of pre- and postsynaptic mechanisms exist by which iGluRs and mGluRs could modulate cell functions in the CNS. Therefore, selective agonists and antagonists could be used to modulate glutamatergic neuronal transmissions in very select areas of the CNS (70, 94). For example, it has been suggested that NMDAR 1 antagonists may be useful in preventing tolerance to opiate analgesia and helping control withdrawal symptoms from addictive drugs. Also, the overactivation of NMDAR 1 has been suggestive as the causal factor in chronic disease such as Huntington’s, Alzheimer’s, Parkinson’s , HIV-related neuronal injury and amyotrophic lateral sclerosis. Therefore, antagonists of NMDAR 1 receptor function are expected to be useful in the treatment of some of these diseases. Ample research demonstrating the presence of the GluR, dopamine and GABA receptors in peripheral tissues suggests that these antagonists might have modulating functions in the peripheral organs and tissues. GluRs in peripheral tissues could also be targets for pharmacological manipulations. For example,

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

GluRs in pancreatic islets offers a potential target for therapeutic intervention to Ž ne-tune insulin or glucagon secretion (37, 86). The presence of these receptors in osteoblasts and the demonstration that NMDAR activation is effective in inhibiting bone reabsorption in vitro may contribute to the development of new therapeutics for osteoporosis (14). Of all NMDA subunits, NMDAR 1 has been shown to be the most widely distributed in the CNS (3, 20, 21, 24, 25, 36, 46 – 48, 51). It is the excessive activation of this receptor that is known to cause neuronal damage. Our data and that of others also supports that the NMDAR 1 also has a wide distribution in peripheral tissues (13, 27 – 29, 30). This supports a possible role in cell injury outside the CNS for this receptor. In conclusion, it is evident that GluRs have a cell speciŽ c distribution in neural and nonneural tissues. In these locations, they may play a pathophysiologica l role or as targeteffector sites for excitatory compounds in foods or therapeutic products. The wide distribution in plants and animals from invertebrate to primates suggests that GluRs may also represent a primitive signalling system. Further research is required to assess the signiŽ cance and the role of the GluRs and their impact in various Ž elds. ACKNOWLEDGMENTS We would like to thank Peter McGuire for his technical support with the immunohistochemistry and Peter Smyth for preparations of the histological sections. In addition, we thank Meghan Murphy, Paul Rowsell, and Dr Tim Schrader for their diligent reading of the initial drafts of this manuscript. We are also indebted to Dr R. Mueller for providing the Macaca fascicularis specimens. R EFERENCES 1. Aas P, Tanso R, Fonnum F (1989). Stimulation of peripheral cholinergic nerves by glutamate indicates a new peripheral glutamate receptor. Eur J Pharmacol 164: 93 – 102. 2. Allen DH, Delohery H, Baker G (1987). Monosodium L -glutamate-induced asthma. J Allergy Clin Immunol 80: 530 – 537. 3. Asztely F, Gustafasson B (1996). Ionotropic glutamate receptors: Their possible role in the expression of hippocampal synaptic plasticity. Mol Neurobiol 12: 1– 11. 4. Barb CR, Campbell RM, Armstrong JD, Cox NM. (1996). Aspartate and glutamate modulation of growth hormone secretion in the pig: Possible site of action. Domestic Animal Endocrinol 13: 81 – 90. 5. Bertrand G, Gross R, Puech R, Loubatieres-Mariana MM, Bockaert J (1992). Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas. Br J Pharmacol 106: 354 – 359. 6. Bertrand G, Gross R, Puech R, Loubatieres-Mariana MM, Bockaert J (1993). Glutamate stimulates glucagon secretion via an excitatory amino acid receptor of the AMPA subtype in rat pancreas. Br J Pharmacol 237: 45 – 50. 7. Bruni JE, Bose R, Pinsky C, Gavin G (1991). Circumventricular organ origin of domoic acid-induced neuropatholog y and toxicology. Brain Res Bull 26: 419 – 424. 8. Burns GA, Stephens KE, Benson JA (1994). Expression of mRNA for N methyl- D-aspartate (NMDAR 1) receptor by the enteric neurons of the rat. Neurosci Lett 170: 87 – 90. 9. Butcher SP, Sandberg M, Hagberg H, Hamberger A (1987). Cellular origins of endogenou s amino acids released into the extracellular  uid of the rat striatum during severe insulin-induced hypoglycemia . J Neurochem 48: 722 – 723.

221

10. Carlton SM, Hargett GL, Coggeshall RE (1995). Localization and activation of glutamate receptors in unmyelinated axons of rat glaborous skin. Neurosci Lett 197: 25 – 28. 11. Castelli MP, Ingianna A, Stefanini E, Gess GL (1999). Distribution of GABAB receptors mRNAs in the rat brain and peripheral tissues. Life Sci 64: 1321 – 1328. 12. Chaudhari N, Yang H, Lamp C, Delay E, Cartford C, Than T, Roper S (1996). The taste of monosodium glutamate: Membrane receptors in taste buds. J Neurosci 16: 3817 – 3826. 13. Choi, DW (1992). Excitotoxic cell death. J Neurobiol 23: 1261 – 1276. 14. Chenu C, Serre CM, Raynal C, Burt-Pichat B, Delmas PD (1997). Glutamate receptors are expressed by bone cells and are involved in bone reabsorption. Bone 22(4): 295 – 299. 15. Coggeshall RE, Carlton SM (1998). Ultrastructural analysis of NMDA, AMPA, and kainate receptors on myelinated and unmyelinated axons in the periphery. J Comp Neuro 391: 78 – 86. 16. Conn PJ, Pin JP (1997). Pharmacology and functions of metabotrophic receptors. Ann Rev Pharmacol Toxicol 37: 205 – 237. 17. Cunningham MD, Ferkany JW, Enna SJ (1994). Excitatory amino acid receptors: A gallery of new targets for pharmacologica l intervention. Life Sci 54: 135 – 148. 18. Demenes D, Lleixa A, Dechesne CJ (1995). Cellular and subcellular localization of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system. Brain Res 671: 83 – 94. 19. Dierkes PW, Hochstrate P, Schlue WR (1996). Distribution and functional properties of glutamate receptors in the leech central nervous system. J Neurophys 75: 2312 – 2321. 20. Dingledine R, Borges K, Bowie D, Traynelis SF (1999). The glutamate receptor ion channels. Pharmacol Rev 51: 7– 61. 21. Dingledine R, McBain CJ (1994). Excitatory amino acids transmitters. In: Basic Neurochemistry. Siegal GJ, Agronoff RW, Albers BW, Molinof PB (eds). Raven Press, New York, pp 367 – 387. 22. Erdo SL (1990). In GABA: Outside the CNS. Erdo SL (ed). Springer-Verlag, New York, pp 183– 197. 23. Erdo SL (1991). Excitatory amino acid receptors in the mammalian periphery. TIBS 121: 426 – 429. 24. Farooqui AA, Horrocks LA (1994). Involvemen t of glutamate receptors, lipases, and phospholipase s in long-term potentiation and neurodegeneration . J Neurosci Res 38: 6 – 11. 25. Gasic GP, Hollmann M (1992). Molecular neurobiology of glutamate receptors. Ann Rev Physiol 54: 507 – 536. 26. Genever PG, Wilkinson DJP, Patton AJ, Peet NM, Hong Y, Mathur A, Erusalimsky JD Skerry TM (1999). Expression of a functional N -methyl- D aspartate-type glutamate receptor by bone marrow megakaryocytes . Blood 93: 2876 – 2883. 27. Gill SS, Pulido OM, Mueller RW, McGuire PF (1998). Molecular and immunologica l characterization of the ionotropic glutamate receptors in the rat heart. Brain Res Bull 46: 429 – 435. 28. Gill SS, Pulido OM, Mueller RW, McGuire PF (1999). Immunological characterization of the metabotrophic glutamate receptors in the rat heart. Brain Res Bull 48: 143 – 146. 29. Gill SS, Pulido OM, Mueller RW, McGuire PF (2000). Potential target Sites in peripheral tissues for excitatory neurotransmissio n and excitotoxicity. Toxicol Pathol 28: 277 – 284. 30. Gonoi T, Mizuno N, Inagaki N, Kuromi H, Seino Y, Miyazaki J, Seino S (1994). Functional neuronal ionotropic glutamate receptors are expressed in the non-neurona l cell line MIN6. J Biol Chem 269: 16989 – 16992. 31. Grazia U, Storto M, Battaglia G, Felli MP, Maroder M,Gulino A, Nicoletti F, Ragona G, Screptani I, Calogero A (1999). Evidence for the expression of metabotrophic receptors in the thymic cells. 29th Annual Meeting Miami Beach, Fl. Oct. 23 – 28. Society of Neuroscience. P. 449. Abstract 177.16. 32. Hardy M, Younkin D, Tang CM, Pleasure J, Shi QY, Williams M, Pleasure D (1994). Expression of non-NMDA glutamate receptor channel genes by clonal human neurons. J Neurochem 63: 482 – 489. 33. Harry GJ (1999). Basic principles of disturbed CNS and PNS functions. In: Introduction to Neurobehaviora l Toxicology: Food and Environment.

222

34.

35.

36. 37.

38.

39.

40. 41.

42.

43. 44.

45. 46.

47. 48. 49.

50.

51. 52.

53. 54.

55.

56.

57.

GILL AND PULIDO

Niesink RJM, Jaspers RMA, Kornet LMW, van Ree JM, Tislosn HA (ed). CRC Press, Boca Raton, pp 115 – 162. Haxhiu MA, Erokwu B, Dreshaj IA (1997). The role of excitatory amino acids in airway re ex responses in anaesthetized dogs. J Auton Nerv Sys 67: 192 – 199. Hayashi Y, Zviman MM, Brand JG, Teeter JH, Restrepo, D (1996). Measurement of membrane potential and [Ca2 ] in cell ensembles: Application to the study of glutamate taste in mice. Biophys J 71: 1057 – 1070. Hollmann M, Heinemann S (1994). Cloned glutamate receptors. Annu Rev Neurosci 17: 31 – 108. Inagaki N, Kuromi H, Gonoi T, Okamoto Y, Ishida H, Seino Y, Kaneko T, Iwanaga T, Seino S (1995). Expression and role of ionotropic glutamate receptors in pancreatic islet cells. FASEB J 9: 686 – 691. Iverson F, Truelove J, Tryphonas L, Nera EA (1990). The toxicology of domoic acid administered systemically to rodents and primates. Can Dis Wkly Rep 16(Suppl. 1E): 15 – 19. Jackson EK, Branch RA, Margoius HS, Oates JA (1985). Physiological functions of the renal prostaglandin , renin and kallikrein systems. In: The Kidney-Physiology and Pathology. Seldin DW, Giebisch G (ed). Raven Press, New York, pp 613– 644. Janeway CA, Travers P (1994). Immunobiology : The immune system in health and disease. Robertson M (ed). Garland Publishing Inc, New York. Kiyama H, Sato K, Tohyama M (1993). Characteristic localization of nonNMDA type glutamate receptor subunits in the rat pituitary gland. Mol Br Res 19: 262 – 268. Krogsgaard-Larsen P, Hansen JJ (1992). Naturally occurring excitatory amino acids as neurotoxins and leads in drug design. Toxicol Lett 64/65: 409 – 416. Lam HM, Chiu J, Hsieh MH, Meisel L, Oliveira IC, Shin M, Coruzzi G (1998). Glutamate receptor genes in plants. Nature 396: 125 – 126. Lara H, Bastos-Ramos W (1988). Glutamate and kainate effects on the noradrenergi c neurons innervating rat vas deferens. J Neurosci Res 19: 239 – 244. Lindstrom P, Ohlsson L (1992). Effects of N -Methyl-DL-aspartate on isolated rat somatotrophs . Endocrinology 131: 1903 – 1907. Lipton SA (1993). Prospects for clinically tolerated NMDA antagonists: Open-channe l blockers and alternative redox states of nitric oxide. Trends Neuorsci 16: 527 – 532. Lipton SA, Gendelman HE (1995). Dementia associated with the acquired immunodeŽ ciency syndrome. N Engl J Med 332: 934 – 940. Lipton SA, Rosenberg PA (1994). Excitatory amino acids as a Ž nal common pathway for neurologi c disorders. N Engl J Med 330: 613 – 622. Liu PH, Tay SSW, Leong SK (1997). Localization of glutamate receptors subunits of the a -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type in the pancreas of newborn guinea pig. Pancreas 14: 360 – 368. Maricq AV, Peckol E, Driscoll M, Bargmann CI (1995). Mechanosensor y signalling in C. elegans mediated by the GLR1 glutamate receptor. Nature 378: 78 – 81. Meldrum BS (1994). The role of glutamate in epilepsy and other central nervous disorders. Neurology 44: 14 – 23. Mick G (1995). Non-N -methyl- D-aspartate glutamate receptors in glial cells and neurons of the pineal gland in a higher primate. Neuroendocrinology 61: 256 – 264. Miller S, Kesslak JP, Romano C, Cotman CW (1996). Roles of metabotropic receptors in brain plasticity and pathology. Ann NY Acad Sci 757: 460 – 474. Molnar E, Varadi A, McIlhinney RAJ, Ashcroft SJH (1995). IdentiŽ cation of functional ionotrophic glutamate receptor proteins in the pancreatic Bcells and in the islets of Langerhans. FEBS Lett 371: 253 – 257. Morhenn VB, Waleh NS, Mansbridge JN, Unson D, Zolotorev A, Cline P, Toll L (1994). Evidence for an NMDA receptor subunit in human keratinocytes and rat cardiocytes. Eur J Pharmacol 268: 409 – 414. Moroni F, Luzzi S, Micheli SF, Zilleti L (1986). The presence of N -methylD-aspartate type receptors for glutamic acid in the guinea pig myenteric plexus. Neurosci Lett 68: 57 – 62. Mueller R, Gill S, Pulido O, Kapal K, Smyth P (1996). Demonstration and differential localization of glutamate receptors in the rat and monkey (Macaca fascicularis) FASEB J 9: LB146.

TOXICOLOGIC PATHOLOGY

58. O’Connell DP, Aherne AM, Lane E, Felder RA, Carey RM (1998). Detection of dopamine receptors D1A subtype-speci Ž c mRNA in rat kidney by in situ hybridization. Am J Physiol 275: F232 – F241. 59. Olney JW (1989). Excitotoxicity: An overview. Can Dis Wkly Rep 16(Suppl 1E): 49 – 58. 60. Olney JW (1994). Excitotoxins in foods. NeuroToxicology 15: 535 – 544. 61. Otamiri T (1988). Quinacrine prevention of intestinal ischaemic mucosal damage is partly mediated through inhibition of intraluminal phospholipas e A2 . Agents and Actions 25: 378 – 384. 62. Ozawa S, Kamiya H, Tsuzuki K (1998). Glutamate receptors in the mammalian central nervous system. Prog Neurobiol 54: 581 – 618. 63. Ozono R, O’Connell DP, Vaughan C, Botkin SJ, Walk SF, Felder RA, Carey RM (1996). Expression of the subtype 1A dopamine receptor in the rat heart. Hypertension 27: 693 – 703. 64. Patton AJ, Genever PG, Birch MA, Peet NM, Grabowski P, Rands RS, Wilkinson DJP, Howarth S, Suva LJ, and Skerr TM (1997). Glutamate signalling in human and rat bone cells. Bone 2: 76S. 65. Peng YG, Taylor TB, Finch RE, Switzer RC, Ramsdell JS (1994). Neuroexcitatory and neurotoxic actions of the amnesic shellŽ sh poison, domoic acid. NeuroReport 5: 981 – 985. 66. Perl TM, Bedard L, Kosatsky T, Hockin JC, Todd ECD, Remis RS (1990). An outbreak of toxic encephalopath y caused by eating mussels contaminated with domoic acid. N Engl J Med 322: 1775 – 1780. 67. Price MD, Olney JW, Lowry OH, Buchsbaum S (1981). Uptake of exogenous glutamate and aspartate by circumventricula r organs but not other regions of brain. J Neurochem 36: 1734 – 1780. 68. Purcell WM, Doyle KM, Westgate C, Atterwill CK (1996). Characterization of a functional polyamine site on rat mast cells: Association with a NMDA receptor macrocomplex. J Neuroimmuno l 65: 49 – 53. 69. Rockhold RW, Acuff CG, Clower BR (1989). Excitotoxin-induced myocardial necrosis. Eur J Pharmacol 166: 571 – 576. 70. Said SI (1999). Glutamate receptors and asthmatic airway disease. Trends Biochem Sci 20: 132 – 135. 71. Said SI, Berisha HI, Pakbaz H (1996). Excitotoxicity in the lung: N -methylD-aspartate induced, nitric oxide-dependent , pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase. Proc Natl Acad Sci USA 93: 4688 – 4692. 72. Sanada H, Yao L, Jose PA, Carey RM, Felder RA (1997). Dopamine D3 receptors in rat juxtaglomerula r cells. Clin Exper Hypertension 19: 93 – 105. 73. Schoepp DD (1994). Novel function for subtypes of metabotropic glutamate receptors. Neurochem Int 24: 439 – 449. 74. Shannon HE, Sawyer BD (1989). Glutamate receptors of the N -methyl- Daspartate subtype in the myentric plexus of the guinea pig ileum. J Pharmacol Exp Ther 251: 518 – 523. 75. Sninsky CA, Brooderson RJ, Broome TA, Bergeron RJ (1994). Evidence for an N -methyl- D-aspartate (NMDA) receptor in the GI tract of guinea pigs: Studies with diethylhomospermin e (DEHSPM). Gastroenterology 106: A569. 76. Sorenson RL, Garry DG, Brelje TC (1991). Structural and functional considerations of GABA in islets of Langerhans. Diabetes 40: 1365 – 1374. 77. Stumer T, Amar M, Harvey RJ, Bermudez I, Minnen JV, Darlison MG (1996). Structure and pharmacologica l properties of a molluscan glutamategated cation channel and its likely role in the feeding behavior J Neurosci 16: 2869 – 2880. 78. Sureda F, Copani A, Bruno V, Knopel T, Meltzger G, Nicoletti F (1997). Metabotropic glutamate receptor agonists stimulate polyphosphoinositid e hydrolysis in primary cultures of rat hepatocytes . Eur J Pharmacol 338: R1– R2. 79. Teitelbaum J, Zatorre RS, Carpenter S, Gendron D, Evans AC, Gjedde A, Cashman NR (1990). Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med 322: 1781 – 1787. 80. Truelove J, Mueller R, Pulido O, Iverson F (1996). Subchroni c toxicity study of domoic acid in the rat. Food Chem Toxicol 34: 525 – 529. 81. Tryphonas L, Truelove J, Iverson F, Todd ECD, Nera EA (1990). Neuropathology of experimental domoic acid poisoning in non-human primates and rats. Can Dis Wkly Rep 16(Suppl 1E): 75 – 81.

Vol. 29, No. 2, 2001

GLUTAMATE RECEPTORS IN PERIPHERAL TISSUES

82. Tsai LH, Lee YJ, Wu JY (1994). Effect of L -glutamate acid on acid secretion and immunohistochemica l localization of glutamatergic neurons in the rat stomach. J Neurosci Res 38: 188 – 195. 83. Tsai LH, Lee YJ, Wu JY (1999). Effect of excitatory amino acid neurotransmitters on acid secretion in the rat stomach. J Biomed Sci 6: 36 – 44. 84. Villalobos C, Nunez L, Garcia-Sancho J (1996). Functional glutamate receptors in a subpopulatio n of anterior pituitary cell. FASEB J 10: 654 – 660. 85. Watanabe M, Mishina M, Inoue Y (1994). Distinct gene expression of the N -methyl- D -aspartate receptor channel subunit in peripheral neurons of the mouse sensory ganglia and adrenal gland. Neurosci Lett 165: 183 – 186. 86. Watters MR (1995). Organic neurotoxins in seafoods. Clin Neurol Neurosurg 97: 119 – 124. 87. Weaver CD, Yao TL, Powers AC, Verdoorn TA (1996). Differential expression of glutamate receptor subtypes in rat pancreatic islets. J Biol Chem 271: 12977 – 12984. 88. Winter CR, Baker RC (1996). L -Glutamate induced changes in intracellular calcium oscillation frequency through non-classical glutamate receptor binding in cultured rat myocardial cells. Life Sci 57: 1925 – 1934.

223

89. Wu YM, Kung SS, Chow WC (1996). Determination of relative abundanc e of splicing variants of Oreochromis glutamate receptors by quantitative reverse-transcriptas e PCR. FEBS Lett 390: 157 – 160. 90. Yamaguchi I, Jose PA, Mouradian M, Canessa LM, Monsma FJ, Sibley DR,Takeyasu K, Felder RA (1993). Expression of dopamine D1A receptor in proximal tubule of rat kidneys. Am J Physiol 264: F280 – F285. 91. Yoneda Y, Ogita K (1986). Localization of [ 3 H-] glutamate binding sites in rat adrenal medulla. Brain Res 383: 387 – 391. 92. Zagon IS, Hurst WF, McLaughlin PJ (1997). IdentiŽ cation of [ Met5 ]enkephalin in developing , adult and renewing tissues by reversed-phas e high performance liquid chromatograph y and radioimmunoassa y. Life Sci 61: 363 – 370. 93. Zautcke JL, Schwartz JA, Mueller EJ (1986). Chinese restaurant syndrome: A review. Ann Emerg Med 15: 1210 – 1213. 94. Zhang H, Qiao Z, Zhao Y, Zhao R (1996). Transcription of dopamine DA1 receptor mRNAs in rat heart. Meth Find Clin Pharmacol 18: 183 – 187. 95. Ziyu L, Becker J, Noe CR (1998). Functional expression of recombinant N -methyl- D-aspartate receptors in the yeast Saccharomyce s cerevisiae. Eur J Biochem 252: 391 – 399.