Glutamate and Acute Lung Injury 47
From: Cell Signaling in Vascular Inflammation Edited by: J. Bhattacharya © Humana Press Inc., Totowa, NJ
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Glutamate Receptor Activation in the Pathogenesis of Acute Lung Injury
Sami I. Said
SUMMARY
The excitatory amino acids glutamate and aspartate, acting on glutamate receptors, exert important physiological functions in the central nervous system. But overactivation of these receptors can produce neuronal cell injury and death. Recent studies show that the glutamate agonist N-methyl-NN D-aspartate (NMDA) can trigger acute lung injury (ALI), manifested by high-permeability pulmonary edema, and that NMDA receptor subtypes are expressed in normal rat lung. These findings suggest that glutamate signal- ing may be involved in the pathogenesis of ALI and, as such, may be a novel target for the prevention or attenuation of this condition.
Key Words:Glutamate receptors; lung injury; NMDA; excitotoxicity; excitatory amino acids.
1. GLUTAMATE IS A PHYSIOLOGICAL NEUROTRANSMITTER
The amino acids glutamate and aspartate, abundantly present in the mammalian central nervous system (CNS), are the major excitatory neurotransmitters. These excitatory amino acids (EAAs), acting on glutamate receptors, play an important role in many physiological functions, including learning, memory, development, and other forms of synaptic plasticity (1). More recently, glutamate has received some recognition as a neurotransmitter in the peripheral nervous system, and glutamate receptors have been detected in several sites outside the CNS (2,3).
2. GLUTAMATE IS POTENTIALLY TOXIC TO NEURONS: EXCITOTOXICITY Despite its physiological role as a neurotransmitter, glutamate can be lethal to neurons upon in- tense exposure (4). Overactivation of glutamate receptors has been implicated in neuronal degenera- tion and loss in such acute conditions as hypoxia-ischemia, hypoglycemia, head injury, stroke, and prolonged epileptic seizures, as well as in chronic neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and acquired im- munodeficiency syndrome (AIDS) dementia (4). In most instances, neuronal cell loss is attributable to excitotoxicity, a term derived from its mediation by excitatory amino acid receptors.
3. GLUTAMATE RECEPTORS
Several classes of glutamate receptors, widely distributed throughout the CNS, have been identi- fied (5). Already cloned and characterized are three subtypes of ionotropic receptors, classified ac- cording to their activation by specific agonists: N-methyl-D-aspartate (NMDA), AMPA (F-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) and kainate receptors, and a family of het-
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erogeneous, G protein-coupled metabotropic receptors. These receptors regulate the activity of mem- brane enzymes and ion channels, and act through different second-messenger systems (Fig. 1). The NMDA receptor has been the focus of much attention because of its implication in excitotoxic (4), as well as neuroexcitatory, events.
4. THE NMDA RECEPTOR
The NMDA receptor forms an ion channel whose permeability to Ca2+is increased upon ligand binding (6). Calcium permeability can be blocked by Mg2+. Functional NMDA receptors in the brain are formed by combinations of two subunits: NR1 and NR2. NR1 exists as a family of eight isoforms formed by alternative splicing at three exons (N1, C1, C2), and contains a binding site for the cofac- Fig. 1. Schematic representation of signaling pathways and enzymatic reactions triggered by glutamate re- ceptor activation that lead to cell death. CaM, calmodulin; DAG, diacylglycerol; Ins(1,4,5)P3, inositol 1,4,5 trisphosphate; NMDA, N-methyl-NN D-aspartate; NO, nitric oxide; PARP, poly(ADP-ribose) polymerase; PKC, protein kinase C; PLA2and PLC, phospholipase A2and C, respectively; ROS, reactive oxygen species; VSCC, voltage-sensitive Ca2+ channels. (Reproduced with permission from ref. 17.)
Glutamate and Acute Lung Injury 49
tor glycine. The NR2 subfamily consists of four individual isoforms: NR2A, NR2B, NR2C, NR2D, and contains the glutamate (NMDA) binding site. NMDA receptor isoforms are differentially ex- pressed in different regions of the brain, where they presumably mediate site-specific functions (5).
5. GLUTAMATE TOXICITY IN THE LUNG: EXCITOTOXICITY AS A MECHANISM OF LUNG INJURY
As mentioned above, other investigators have reported the presence of NMDA-type glutamate receptors in nonneuronal tissues, such as pancreatic-islet G-cells, megakaryocytes (2,3), and cerebral microvasculature. Our interest in the potential occurrence of glutamate toxicity in the lung, and its pathophysiological significance, began with the demonstration that the excitotoxin NMDA caused acute lung injury in the rat (7). The injury was in the form of high-permeability edema, marked by increased lung weight, increased protein content in bronchoalveolar lavage (BAL) fluid, and elevated airway and pulmonary artery pressures. The injury was prevented by NMDA receptor blockade and was nitric oxide (NO) dependent (7).
These observations raised the possibility that glutamate (NMDA) toxicity could be a novel mecha- nism in the pathogenesis of acute lung injury, as seen in the acute respiratory distress syndrome (ARDS) (8). To validate this hypothesis, at least two criteria need to be fulfilled. First, NMDA toxic- ity must be shown to be involved in lung injury induced by other than the application of NMDA itself. Secondly, NMDA receptors should be expressed in the lung. To test the first requirement, we demonstrated that the NMDA receptor antagonist MK-801 reduced acute pulmonary injury due not only to NMDA itself, but also to the oxidants paraquat and xanthine + xanthine oxidase (9). These findings suggested that endogenous activation of NMDA receptors, probably by glutamate released from damaged cells, could play a role in oxidant lung injury. Data from neuroscience have shown that intracellular glutamate levels are a thousand-fold greater than extracellular levels, and that glutamate released from injured neurons acts as a source of excitotoxic injury of other cells (10). The significance of this mechanism, by which glutamate toxicity can be perpetuated and amplified, has yet to be explored in the lung.
6. NMDA RECEPTORS ARE EXPRESSED IN THE LUNG
The observation that high concentrations of the glutamate agonist NMDA induced high- perme- ability pulmonary edema implied the presence of NMDA receptors in the lung, particularly in the alveolar-capillary area. For direct evidence, we used a variety of experimental approaches to identify the subtypes of NMDA receptors expressed in the lung, and to localize them to specific regions of the lungs and airways. First, with the help of immunhistochemical techniques, we demonstrated the pres- ence of NMDAR 2B in airway neurons (11). We then showed by autoradiography the localization of specific binding of NMDA receptor antagonist MK-801 to alveolar walls and bronchial smooth- muscle and epithelial cells (12). More recently, the use of RT-PCR revealed the constitutive expres- sion of the NR1 and four NMDAR2 receptor subunits in rat lung. mRNA for the NR1 and NMDAR 2D subunits were constitutively expressed in the peripheral lung, mid-lung, and central-lung regions, as well as in alveolar macrophages. NMDAR 2A and 2B expression was not detectable in any of the lung regions examined, while NMDAR 2C was present in peripheral and mid-lung regions. Western blot analysis confirmed the presence of NR1 isoforms with C2 sites in peripheral rat lung. It is likely that the localization of these receptor subunits in the lung is largely nonneuronal, particularly in peripheral lung, which is devoid of neurons (13).
7. HOW DOES GLUTAMATE KILL CELLS?
As stated before, activation of the NMDA receptor opens up a receptor-gated Ca2+channel, caus- ing the rapid influx of Ca2+into the cell. In neurons, the sharp elevation of Ca2+concentration induces the activation of several enzyme pathways and signaling cascades, including various protein kinases, phospholipases, lipoxygenase, cyclooxygenase, proteases and NOS, and the generation of reactive
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oxygen species (ROS, oxygen free radicals) and peroxynitrite, a toxic reaction product of NO and superoxide. These reaction cascades culminate in lipid peroxidation, DNA strand breaks, and activa- tion of caspases and poly (ADP-ribose) polymerase (PARP). As a result of these events, the affected cells die, through both apoptosis and necrosis (Fig. 1) (14,15). In the lung, we have shown that acute NMDA exposure increases NO production and activates caspase-3 (15). Furthermore, both NO syn- thase (NOS) inhibitors and PARP inhibitors block the development of high-permeability pulmonary edema in response to NMDA, and injury is attenuated or delayed in both neuronal NOS- and PARP- knockout mice compared to wild-type (16).
8. CONCLUSIONS
Glutamate, an excitatory neurotransmitter that can be lethal to neurons upon hyperactivation of NMDA receptors, can also induce acute lung injury when added directly to perfused rat lungs. In addition, we have provided evidence that NMDA-type glutamate receptors are present in the lungs, and that activation of these receptors plays a major role in the pathogenesis of oxidant lung injury.
We postulate that signaling via NMDA receptors may be a pathogenetic factor in the acute lung injury of ARDS (and may also be causally related to bronchial asthma [17]). Recognition of the potential significance of glutamate toxicity could lead to the introduction of novel therapeutic ap- proaches. The physiological role(s) of glutamate signaling in the lung remains to be determined.
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