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Cellular mechanisms underlying excitotoxicity
he cytotoxic action of excitaT tory amino acids is well docu- Cellularmechanismsunderlyingexcitotoxicity mented and of great clinical importance; establishing the underlying mechanism, an interesting experimental challenge, could be a significant step towards the development of treatments for seizure and anoxia-evoked pathological processes. In particular, differences between acute and delayed cytotoxic mechanisms could be especially important for the design of clinical treatments, since events such as status epilepticus and stroke almost inevitably involve a delay before treatment can be initiated. Probably more than 20% of neurons within the vertebrate CNS use L-glutamate, or a related amino acid, as a neurotransmitter to signal fast excitatory synaptic transmission; as a result, excitatory amino acids are not limited to information processing in any particular neuroanatomical substrate, and binding studies show the presence of excitatory amino acid receptors throughout the CNS. Furthermore, the cytoplasm of most vertebrate neurons contains millimolar concentrations of excitatory amino acids, which in addition to their role in intermediary metabolism, act as intracellular anions and contribute to the maintenance of internal osmotic pressure. In a sense, the brain is loaded with the seeds of its own destruction: excessive release of excitatory amino acids during abnormal synaptic transmission, or leakage of cellular contents following trauma, can in principle lead to the development of excitotoxicity. The similar pattern of cell loss following cerebral ischemia, episodes of status epilepticus, or experimental administration of excitotoxic amino acids suggests that direct investigation of the cellular mechanisms of action of excitatory amino acids will be an important step in elucidating the excitotoxic process. Recent advances in understanding the pharmacology and mechanism of action of excitatory amino acid receptor ion channels in the vertebrate CNS, together with experiments designed to examine the ionic mechanism of both excitatory amino acid-evoked depolarization and cytotoxic responses, all give clues as to the cellular mechanisms involved, and point towards the development of TINS- February1987110]
suitable treatment protocols. The important issues would seem to be: (1) elucidating the role of individual excitatory amino acidreceptor subtypes in specific pathophysiological models; (2) determining the ion fluxes associated with activation of each receptor subtype, and the cellular consequences these have for neuronal damage; (3) evaluating the degree to which processes secondary to activation of excitatory amino acid receptors contribute to the excitotoxic process: these include further synaptic release of L-glutamate subsequent to either the initial excitatory action of amino acids, or to depolarization of transmitter-releasing boutons following effiux of potassium into the extracellular fluid, such that the release of L-glutamate changes from a trickle to a torrent; the release of other non-amino acid transmitters via the above mechanisms could conceivably contribute to the excitotoxic process; and (4) knowing whether different firing patterns contribute to the potency of amino acids as excitotoxins. The excitatory action of individual acidic amino acids is quite characteristic - NMDA-receptor-preferring agonists evoke a burst discharge of action potentials, whereas kainic acid evokes a nondesensitizing, and therefore longlasting depolarization, associated with a precipitous fall in membrane resistance, and a loss of the action potential mechanism due to sodium channel inactivation. We now have many clues as to the importance of the above mechanisms. Physiological experiments on a variety of preparations show that excitatory amino acids activate non-selective cationic channels 1-4 permeable to both Na + and K +. Clearly the acute administration of excitatory amino acids, especially kainic acid, will generate large ionic fluxes, prinicipally influx of Na + ions through receptor-gated ion channels, and efflux of K + through these same channels and through voltage-activated potassium channels. Accompanying these ionic fluxes will be the passive movement of C1- and water molecules into the neuron. In two isolated preparations, cultures of rat fetal hippocampus a, and in-vitro chick retina 6, experimental evi~) 1987, Elsevier Science Publishers B,V., Amsterdam
dence from ion substitution experiments suggests that osmotic disruption of the nerve cell membrane underlies the acute excitotoxic action of L-glutamate and kainic acid, via swelling due to transmitter-induced ion fluxes. Replacement of extracellular C1- with impermeable anions, or replacement of extracellular Na + with impermeable cations, spares neurons in these isolated preparations from excitatory amino acid-evoked cell death. That kainic acid should be especially excitotoxic is supported by physiological experiments also on isolated preparations - freshly dissociated horizontal cells from goldfish retina 7, and hippocampal neurons from young rats s. In each of these preparations, bath application of excitatory amino acids at known concentration via a rapid perfusion technique was used to study desensitization under voltage clamp. Although neither preparation responded to NMDA, kainic acid was unique in evoking nondesensitizing responseT'S; similar results have also been obtained in experiments on mouse spinal cord neurons in culture (Westbrook, G. L., unpublished observations). Thus, compared to L-glutamate, for example, kainate generates osmotically disruptive ion fluxes for a far longer period. In-vivo cellular uptake mechanisms limit the diffusion of certain amino acids within the neuropil, and this must be a factor in determining the potency of these agents as excitotoxins; the absence of a high-affinity uptake mechanism for kainic acid9 must certainly also contribute to its high potency as an excitotoxin. Furthermore, the especially high sensitivity of hippocampal CA3 pyramidal neurons to kainic acid appears to reflect the presence of a population of high-affinity receptors at which kainic acid is a specific ligand m, whereas in other areas of the CNS it is likely that both kainate and quisqualate act at another site of lower affinity. In summary, acute osmotic damage is easy to induce experimentally, especially with kainic acid, but requires massive and sustained receptor activation, and thus is likely to be a major factor only in those pathologies where 0378 - 5912/87/$02.00
Mark L. Mayer and Gary L. Westbrook
Laboratoryof Developmental Neurobiology, NICHO,NIH, Bethseda,MD 20892, USA.
59
prolonged release of excitatory amino acids can be expected. The activation of NMDA receptors has been shown to open ion channels functionally distinct from those linked to other excitatory amino acid receptors on vertebrate neurons. In particular, experiments on mouse spinal neurons in culture reveal NMDA-receptor activation to be associated with an increase in Ca 2+ permeability, as shown by Ca 2+ dependence of the NMDA reversal potential, and an increase in [Ca2+]i during application of NMDA but not kainate or quisqualate under voltage clamp n. The use of voltage clamp was critical to the interpretation of these experiments in order to prevent depolarization and the activation of voltage-dependent Ca 2+ channels 11. In addition, NMDAreceptor channels are permeable to Na + and K +, and thus with sustained activation might be expected to contribute to osmotically induced cell lysis. Aside from the biophysical interest of a receptorgated channel that shows appreciable Ca 2+ permeability (with 20 mM [Ca2+] o PCa~+/PNa+ ~ 4 versus 0.22 for nicotinic ACh receptor ion channels), these results suggest a second mechanism for excitotoxicity: direct entry of Ca 2+ through ion channels activated by L-glutamate. Ca 2+ dependence of a delayed excitotoxic response to L-glutamate in spinal cord cultures 12, without a fast osmotically induced cell lysis, provides direct experimental support for such a mechanism in CNS neurons. In addition, a Ca 2+dependent cytotoxic process has been unambiguously documented in locust muscle fibers la, in which L-glutamate is the transmitter at the neuromuscular junction. Ca 2+ entry into excitable cells is of course a normal physiological process responsible for neurosecretion, muscle contraction, and the modulation of excitability and second messenger systems, yet with sufficient influx, Ca z+ can become cytotoxic, probaby via activation of Cae+-dependent proteases, and an upset in the balance of activity in Ca2+-dependent enzyme systems responsible for protein phosphorylation, etc. For excitotoxicity to occur via this mechanism, Ca ~+ entry must be sufficient to overload normal homeostatic mechanisms. How60
ever, it is probable that with localized Ca z+ entry at synaptic sites, selective damage to individual dendrites could occur in less extreme cases, promoting degeneration of dendrites, while sparing nerve cell bodies. Another prominent feature of responses to NMDA - promotion of the burst discharge of action potentials - has also been explained by the results of electrophysiological experiments: physiological concentrations of Mge+ act to block NMDA-receptor-activated ion channels3'4, producing a voltage-dependent reduction of agonist-activated inward current with hyperpolarization. As a result, the membrane current-voltage relationship shows what is described as a region of negative slope conductance. This in turn leads to a regenerative (i.e. voltage-dependent) agonist-evoked current that sums with the intrinsic voltagedependent Na + and Ca 2+ conductance mechanisms in nerve cell membranes, leading to epileptiform-like discharge. This is known to be especially effective in promoting an increase in [Ca2+]i and could potentially contribute to the excitotoxic action of NMDA. A consistent finding in several experimental models of excitotoxicity is the requirement of an intact afferent input, or excitotoxic action at a distance remote from the site of application 14. This strongly suggests a role for synaptic release of endogenous excitatory transmitters. Of particular interest is the observation that septal (cholinergic) afferents are required for excitotoxic destruction of granule and CA1 pyramidal neurons following intraventricular injection of kainic acid 14. It is unclear whether this reflects a direct effect of ACh on excitability (e.g. initiation of burst discharge via suppression of potassium currents), or a direct cytotoxic action of ACh via an unknown mechanism. Together, the above results suggest at least two direct excitotoxic mechanisms for excitatory amino acids (i.e. linked to ion flux through receptor-operated ion channels): osmotically induced dammage, and Cae+-mediated damage. In addition, these experiments raise the issues of interactions between the excitatory actions of Lglutamate and its analogues with intrinsic regenerative excitatory
mechanisms, the role of glial uptake in controlling the extracellular accumulation of excitatory transmitters, and the possible release of transmitters from nerve terminals subject to pathologically induced transmembrane ion gradients. Disruption of amino acid uptake during anoxia may in fact be an important factor in mediating excitotoxic damage during cerebral anoxia: uptake mechanisms for amino acids are energy dependent, and require a physiological transmembrane ion concentration gradient. Disruption of these gradients during anoxia probably contributes to the accumulation of u-glutamate in the extraneuronal environment. In addition, a rise in intracellular Na + may be expected to interfere with Na+-Ca 2+ exchange, leading to an increase in [Ca2+]i and release of L-glutamate from synaptic boutons. In dissociated cultures prepared from fetal rat hippocampus, the non-specific excitatory amino acid receptor antagonist y-D-glutamylglycine protects against neuronal death evoked by a period of nitrogen-induced anoxia 16, suggesting a critical role for the release of L-glutamate under these conditions. A major problem of clinical relevance is to elucidate which of the above mechanisms contributes to neuronal cell death during naturally occurring pathological events. Some surprising results have come from the development of experimental models utilizing a period of cerebral anoxia 17, evoked by ligating the carotid arteries, or the induction of hypoglycemic coma, evoked by insulin overdose tS, both of which produce extensive neuronal atrophy, which is greatly reduced by the selective NMDA-receptor antagonist AP7 (2-amino-7-phosphonoheptanoic acid). These results suggest that anoxia or a temporary halt of oxidative metabolism might not cause neuronal damage via osmotic overload due to activation of kainate or quisqualate receptors, but rather that activation of NMDA receptors, which are selectively linked to Ca2+-permeable ion channels, switch on the cytotoxic process. However, it is conceivable that potentiation of responses linked to activation of kainate or quisqualate receptors secondary to NMDAreceptor-evoked Ca e+ influx, as suggested by a general model of TINS- February 1987 [10]
long-term potentiation 19, might indirectly lead to AP7-sensitive osmotic overload via kainate or quisqualate receptors. Future experiments with more specific antagonists might be used to address this, although in view of the results obtained in experiments with locust muscle ~a, it is unnecessary to postulate this indirect mechanism, since Ca2+ entry through NMDAreceptor channels is a sufficient cytotoxic stimulus. Several drug companies are currently in the process of developing NMDA-receptor-specific antagonists, targeted not at the receptor itself, but at the ion channel or an allosteric site, based on observations that phencyclidine and ketamine, which cross the blood-brain barrier more efficiently than AP7, also block responses to NMDA 2° in a use-dependent and non-competitive manner 2~'22. Of relevance to the role of NMDA receptors in cytoxic processes is the difference in potency of Lglutamate at NMDA and nonNMDA receptors: L-glutamate is a very potent ligand at NMDA receptors and generates physiological responses at sub-micromolar concentrations (Mayer,
M. L. and Westbrook, G. L., unpublished observation), whereas considerably higher concentrations are required for an action at quisqualate-sensitive receptors 8. Although there are many details yet to be elucidated, a cellular explanation for excitotoxocity is emerging. It is probably naive to expect that any one process will be exclusively involved in a particular syndrome, yet the results with AP7 suggest some hope for the production of clinically useful drugs in the forseeable future.
Selected references 1 Habtitz, J. J. and Langmoen, I. A. (1982) J. PhysioL (London) 325, 317331 2 Mayer, M. L. and Westbrook, G. L. (1984) J. Physiol. (London) 354, 2953 3 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A. (1984) Nature 307, 462-465 4 Mayer, M. L., Westbrook, G. L. and Guthrie, P.B. (1984) Nature 309, 261-263 5 Rothman, S. M. (1985) J. Neurosci. 5, 1483-1489 6 0 l n e y , J. W., Price, M. T., Samson, L. and Labruyere, J. (1986) Neurosci. Lett. 65, 65-7! 7 Ishida, A, T. and Neyton, J. (1985) Proc. Natl Acad. 5ci. USA 82, 18371841 8 Kiskin, N. I., Khrishtal, O. A. and
9 10 11
12 13
14 15 16 17 18 19 20 21 22
Tsyndrenko, A.Y. (1986) Neurosci. Lett. 63,225-230 Johnston, G. A. R., Kennedy, S. M. E. and Twitchin, B. (1979)J. Neurochem. 32, 121-127 Foster, A. C., Mena, E. E., Monaghan, D.T. and Cotman, C.W. (1981) Nature 289, 73-75 MacDermott, A~ B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J. L. (1986) Nature 321, 519522 Choi, D. W. (1985) Neurosci. Left. 58, 293-297 Duce, I. R., Donaldson, P. L. and Usherwood, P. N. R. (1983) Brain. Res. 263, 77-87 Coyle, J. T. (1983) J. Neurochem. 41, 1-11 Nadler, J. V., Evenson, D. A. and Smith, E.M. (1981) Brain Res. 205, 405-410 Rothman, S. (1984) J. Neurosci. 4, 1884-1891 Simon, R. P., Swan, J. H., Griffiths, T. and Meldrum, B.S. (1984) Science 226, 850-852 Wieloch, T. (1985) Science 230, 681-683 Baudry, M. and Lynch, G. (1980) Exp. Neurol. 68, 202-204 Anis, N. A., Berry, S. C., Burton, N. R. and Lodge, D. (1983)Br. J. Pharmacol. 79, 565-575 Honey, C. R., Miljkovic, Z. and Macdonald, J. F. (1985) Neurosci. Left. 61, 135-139 Wong, E. H. F., Kemp, J. A., Priestly, T., Knight, A. R., Woodruff, G. N. and Iversen, L. L. (1986) Proc. Natl Acad. Sci. USA 83, 7104-7108
,i
a number of years evidence accumulated that the nico- Whatdoesphosphorylationdo for thenicotinic Ftinic~orhasacetylcholine receptor (AChR) is phosphorylated in vivo ~ and that acetylcholinereceptor? it can be phosphorylated by enzymes present in the electric organ of Torpedo californica 2-4, yet no functional effect of phosphorylation had been found. On the other hand, there is circumstantial evidence that phosphorylation affects the function of other membrane channels 5. Unfortunately, no one had been able to obtain evidence that phosphorylation of a channel itself had any direct influence on its function, rather than acting indirectly via a regulatory protein. Now, Huganir et al. 6 have phosphorylated receptor-rich membrane fragments from Torpedo californica, purified the AChR, reconstituted them into lipid vesicles, and shown that a measured increase in phosphorylation of the AChR correlates well with an increased rate of desensitization. Desensitization of the AChR is a phenomenon in which the receptor becomes unresponsive to activation when it is exposed to an T I N S - February 1987 [10J
activating ligand for a relatively long period of time (tens of milliseconds or longer) 7. It occurs with purified AChR6 and so apparently requires neither metabolic energy nor the presence of any cell components in addition to the AChR. Desensitization results in AChR whose channels remain closed even when ACh is bound to the receptor. Unfortunately, desensitization is a relatively poorly understood process. Only recently has it become apparent that there are probably three or more 'desensitized' states 8-1°, based on the rates at which desensitization develops and recovers. Physiological data have suggested that desensitized AChR bind agonist with higher affinity than resting AChR, and direct binding measurements n have shown that this is true for at least some of the desensitized states. Spectroscopic studies le have given evidence consistent with the idea that the
desensitized AChR has a different conformation than normal. At present, there is some uncertainty as to how much resting desensitization exists, i.e. whether an appreciable amount of the physically present AChR is unresponsive before exposure to transmitter. The methods used by Huganir et al. take advantage of the fact that the electric organ of the marine ray Torpedo californica is a very rich source of AChR. They prepared receptor-rich membranes, and phosphorylated them in vitro. They then" purified the AChR and reconstituted the purified receptors (control or phosphorylated) into liposomes. The functional properties of the reconstituted purified AChR were studied using rapid mixing techniques developed in George Hess' laboratory (see references in Ref. 6). They measured the initial rate of ACh-elicited ion flux into vesicles, and determined how the
© 1987,ElsevierSciencePublishersB.V.,Amsterdam 0378-5912/87/$02.00
Joe Henry Steinbach Departmentsof Anesthesiologyand ofAnatomyand Neurobiology, Washington UnivemtySchoolof Medicine,5t Louis, MO, USA. John Zempel MedicalScienb'st TrainingProgram, Washington UniversitySchoolof Medicine,StLouis, MO, USA.
61
suitable treatment protocols. The important issues would seem to be: (1) elucidating the role of individual excitatory amino acidreceptor subtypes in specific pathophysiological models; (2) determining the ion fluxes associated with activation of each receptor subtype, and the cellular consequences these have for neuronal damage; (3) evaluating the degree to which processes secondary to activation of excitatory amino acid receptors contribute to the excitotoxic process: these include further synaptic release of L-glutamate subsequent to either the initial excitatory action of amino acids, or to depolarization of transmitter-releasing boutons following effiux of potassium into the extracellular fluid, such that the release of L-glutamate changes from a trickle to a torrent; the release of other non-amino acid transmitters via the above mechanisms could conceivably contribute to the excitotoxic process; and (4) knowing whether different firing patterns contribute to the potency of amino acids as excitotoxins. The excitatory action of individual acidic amino acids is quite characteristic - NMDA-receptor-preferring agonists evoke a burst discharge of action potentials, whereas kainic acid evokes a nondesensitizing, and therefore longlasting depolarization, associated with a precipitous fall in membrane resistance, and a loss of the action potential mechanism due to sodium channel inactivation. We now have many clues as to the importance of the above mechanisms. Physiological experiments on a variety of preparations show that excitatory amino acids activate non-selective cationic channels 1-4 permeable to both Na + and K +. Clearly the acute administration of excitatory amino acids, especially kainic acid, will generate large ionic fluxes, prinicipally influx of Na + ions through receptor-gated ion channels, and efflux of K + through these same channels and through voltage-activated potassium channels. Accompanying these ionic fluxes will be the passive movement of C1- and water molecules into the neuron. In two isolated preparations, cultures of rat fetal hippocampus a, and in-vitro chick retina 6, experimental evi~) 1987, Elsevier Science Publishers B,V., Amsterdam
dence from ion substitution experiments suggests that osmotic disruption of the nerve cell membrane underlies the acute excitotoxic action of L-glutamate and kainic acid, via swelling due to transmitter-induced ion fluxes. Replacement of extracellular C1- with impermeable anions, or replacement of extracellular Na + with impermeable cations, spares neurons in these isolated preparations from excitatory amino acid-evoked cell death. That kainic acid should be especially excitotoxic is supported by physiological experiments also on isolated preparations - freshly dissociated horizontal cells from goldfish retina 7, and hippocampal neurons from young rats s. In each of these preparations, bath application of excitatory amino acids at known concentration via a rapid perfusion technique was used to study desensitization under voltage clamp. Although neither preparation responded to NMDA, kainic acid was unique in evoking nondesensitizing responseT'S; similar results have also been obtained in experiments on mouse spinal cord neurons in culture (Westbrook, G. L., unpublished observations). Thus, compared to L-glutamate, for example, kainate generates osmotically disruptive ion fluxes for a far longer period. In-vivo cellular uptake mechanisms limit the diffusion of certain amino acids within the neuropil, and this must be a factor in determining the potency of these agents as excitotoxins; the absence of a high-affinity uptake mechanism for kainic acid9 must certainly also contribute to its high potency as an excitotoxin. Furthermore, the especially high sensitivity of hippocampal CA3 pyramidal neurons to kainic acid appears to reflect the presence of a population of high-affinity receptors at which kainic acid is a specific ligand m, whereas in other areas of the CNS it is likely that both kainate and quisqualate act at another site of lower affinity. In summary, acute osmotic damage is easy to induce experimentally, especially with kainic acid, but requires massive and sustained receptor activation, and thus is likely to be a major factor only in those pathologies where 0378 - 5912/87/$02.00
Mark L. Mayer and Gary L. Westbrook
Laboratoryof Developmental Neurobiology, NICHO,NIH, Bethseda,MD 20892, USA.
59
prolonged release of excitatory amino acids can be expected. The activation of NMDA receptors has been shown to open ion channels functionally distinct from those linked to other excitatory amino acid receptors on vertebrate neurons. In particular, experiments on mouse spinal neurons in culture reveal NMDA-receptor activation to be associated with an increase in Ca 2+ permeability, as shown by Ca 2+ dependence of the NMDA reversal potential, and an increase in [Ca2+]i during application of NMDA but not kainate or quisqualate under voltage clamp n. The use of voltage clamp was critical to the interpretation of these experiments in order to prevent depolarization and the activation of voltage-dependent Ca 2+ channels 11. In addition, NMDAreceptor channels are permeable to Na + and K +, and thus with sustained activation might be expected to contribute to osmotically induced cell lysis. Aside from the biophysical interest of a receptorgated channel that shows appreciable Ca 2+ permeability (with 20 mM [Ca2+] o PCa~+/PNa+ ~ 4 versus 0.22 for nicotinic ACh receptor ion channels), these results suggest a second mechanism for excitotoxicity: direct entry of Ca 2+ through ion channels activated by L-glutamate. Ca 2+ dependence of a delayed excitotoxic response to L-glutamate in spinal cord cultures 12, without a fast osmotically induced cell lysis, provides direct experimental support for such a mechanism in CNS neurons. In addition, a Ca 2+dependent cytotoxic process has been unambiguously documented in locust muscle fibers la, in which L-glutamate is the transmitter at the neuromuscular junction. Ca 2+ entry into excitable cells is of course a normal physiological process responsible for neurosecretion, muscle contraction, and the modulation of excitability and second messenger systems, yet with sufficient influx, Ca z+ can become cytotoxic, probaby via activation of Cae+-dependent proteases, and an upset in the balance of activity in Ca2+-dependent enzyme systems responsible for protein phosphorylation, etc. For excitotoxicity to occur via this mechanism, Ca ~+ entry must be sufficient to overload normal homeostatic mechanisms. How60
ever, it is probable that with localized Ca z+ entry at synaptic sites, selective damage to individual dendrites could occur in less extreme cases, promoting degeneration of dendrites, while sparing nerve cell bodies. Another prominent feature of responses to NMDA - promotion of the burst discharge of action potentials - has also been explained by the results of electrophysiological experiments: physiological concentrations of Mge+ act to block NMDA-receptor-activated ion channels3'4, producing a voltage-dependent reduction of agonist-activated inward current with hyperpolarization. As a result, the membrane current-voltage relationship shows what is described as a region of negative slope conductance. This in turn leads to a regenerative (i.e. voltage-dependent) agonist-evoked current that sums with the intrinsic voltagedependent Na + and Ca 2+ conductance mechanisms in nerve cell membranes, leading to epileptiform-like discharge. This is known to be especially effective in promoting an increase in [Ca2+]i and could potentially contribute to the excitotoxic action of NMDA. A consistent finding in several experimental models of excitotoxicity is the requirement of an intact afferent input, or excitotoxic action at a distance remote from the site of application 14. This strongly suggests a role for synaptic release of endogenous excitatory transmitters. Of particular interest is the observation that septal (cholinergic) afferents are required for excitotoxic destruction of granule and CA1 pyramidal neurons following intraventricular injection of kainic acid 14. It is unclear whether this reflects a direct effect of ACh on excitability (e.g. initiation of burst discharge via suppression of potassium currents), or a direct cytotoxic action of ACh via an unknown mechanism. Together, the above results suggest at least two direct excitotoxic mechanisms for excitatory amino acids (i.e. linked to ion flux through receptor-operated ion channels): osmotically induced dammage, and Cae+-mediated damage. In addition, these experiments raise the issues of interactions between the excitatory actions of Lglutamate and its analogues with intrinsic regenerative excitatory
mechanisms, the role of glial uptake in controlling the extracellular accumulation of excitatory transmitters, and the possible release of transmitters from nerve terminals subject to pathologically induced transmembrane ion gradients. Disruption of amino acid uptake during anoxia may in fact be an important factor in mediating excitotoxic damage during cerebral anoxia: uptake mechanisms for amino acids are energy dependent, and require a physiological transmembrane ion concentration gradient. Disruption of these gradients during anoxia probably contributes to the accumulation of u-glutamate in the extraneuronal environment. In addition, a rise in intracellular Na + may be expected to interfere with Na+-Ca 2+ exchange, leading to an increase in [Ca2+]i and release of L-glutamate from synaptic boutons. In dissociated cultures prepared from fetal rat hippocampus, the non-specific excitatory amino acid receptor antagonist y-D-glutamylglycine protects against neuronal death evoked by a period of nitrogen-induced anoxia 16, suggesting a critical role for the release of L-glutamate under these conditions. A major problem of clinical relevance is to elucidate which of the above mechanisms contributes to neuronal cell death during naturally occurring pathological events. Some surprising results have come from the development of experimental models utilizing a period of cerebral anoxia 17, evoked by ligating the carotid arteries, or the induction of hypoglycemic coma, evoked by insulin overdose tS, both of which produce extensive neuronal atrophy, which is greatly reduced by the selective NMDA-receptor antagonist AP7 (2-amino-7-phosphonoheptanoic acid). These results suggest that anoxia or a temporary halt of oxidative metabolism might not cause neuronal damage via osmotic overload due to activation of kainate or quisqualate receptors, but rather that activation of NMDA receptors, which are selectively linked to Ca2+-permeable ion channels, switch on the cytotoxic process. However, it is conceivable that potentiation of responses linked to activation of kainate or quisqualate receptors secondary to NMDAreceptor-evoked Ca e+ influx, as suggested by a general model of TINS- February 1987 [10]
long-term potentiation 19, might indirectly lead to AP7-sensitive osmotic overload via kainate or quisqualate receptors. Future experiments with more specific antagonists might be used to address this, although in view of the results obtained in experiments with locust muscle ~a, it is unnecessary to postulate this indirect mechanism, since Ca2+ entry through NMDAreceptor channels is a sufficient cytotoxic stimulus. Several drug companies are currently in the process of developing NMDA-receptor-specific antagonists, targeted not at the receptor itself, but at the ion channel or an allosteric site, based on observations that phencyclidine and ketamine, which cross the blood-brain barrier more efficiently than AP7, also block responses to NMDA 2° in a use-dependent and non-competitive manner 2~'22. Of relevance to the role of NMDA receptors in cytoxic processes is the difference in potency of Lglutamate at NMDA and nonNMDA receptors: L-glutamate is a very potent ligand at NMDA receptors and generates physiological responses at sub-micromolar concentrations (Mayer,
M. L. and Westbrook, G. L., unpublished observation), whereas considerably higher concentrations are required for an action at quisqualate-sensitive receptors 8. Although there are many details yet to be elucidated, a cellular explanation for excitotoxocity is emerging. It is probably naive to expect that any one process will be exclusively involved in a particular syndrome, yet the results with AP7 suggest some hope for the production of clinically useful drugs in the forseeable future.
Selected references 1 Habtitz, J. J. and Langmoen, I. A. (1982) J. PhysioL (London) 325, 317331 2 Mayer, M. L. and Westbrook, G. L. (1984) J. Physiol. (London) 354, 2953 3 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A. (1984) Nature 307, 462-465 4 Mayer, M. L., Westbrook, G. L. and Guthrie, P.B. (1984) Nature 309, 261-263 5 Rothman, S. M. (1985) J. Neurosci. 5, 1483-1489 6 0 l n e y , J. W., Price, M. T., Samson, L. and Labruyere, J. (1986) Neurosci. Lett. 65, 65-7! 7 Ishida, A, T. and Neyton, J. (1985) Proc. Natl Acad. 5ci. USA 82, 18371841 8 Kiskin, N. I., Khrishtal, O. A. and
9 10 11
12 13
14 15 16 17 18 19 20 21 22
Tsyndrenko, A.Y. (1986) Neurosci. Lett. 63,225-230 Johnston, G. A. R., Kennedy, S. M. E. and Twitchin, B. (1979)J. Neurochem. 32, 121-127 Foster, A. C., Mena, E. E., Monaghan, D.T. and Cotman, C.W. (1981) Nature 289, 73-75 MacDermott, A~ B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J. L. (1986) Nature 321, 519522 Choi, D. W. (1985) Neurosci. Left. 58, 293-297 Duce, I. R., Donaldson, P. L. and Usherwood, P. N. R. (1983) Brain. Res. 263, 77-87 Coyle, J. T. (1983) J. Neurochem. 41, 1-11 Nadler, J. V., Evenson, D. A. and Smith, E.M. (1981) Brain Res. 205, 405-410 Rothman, S. (1984) J. Neurosci. 4, 1884-1891 Simon, R. P., Swan, J. H., Griffiths, T. and Meldrum, B.S. (1984) Science 226, 850-852 Wieloch, T. (1985) Science 230, 681-683 Baudry, M. and Lynch, G. (1980) Exp. Neurol. 68, 202-204 Anis, N. A., Berry, S. C., Burton, N. R. and Lodge, D. (1983)Br. J. Pharmacol. 79, 565-575 Honey, C. R., Miljkovic, Z. and Macdonald, J. F. (1985) Neurosci. Left. 61, 135-139 Wong, E. H. F., Kemp, J. A., Priestly, T., Knight, A. R., Woodruff, G. N. and Iversen, L. L. (1986) Proc. Natl Acad. Sci. USA 83, 7104-7108
,i
a number of years evidence accumulated that the nico- Whatdoesphosphorylationdo for thenicotinic Ftinic~orhasacetylcholine receptor (AChR) is phosphorylated in vivo ~ and that acetylcholinereceptor? it can be phosphorylated by enzymes present in the electric organ of Torpedo californica 2-4, yet no functional effect of phosphorylation had been found. On the other hand, there is circumstantial evidence that phosphorylation affects the function of other membrane channels 5. Unfortunately, no one had been able to obtain evidence that phosphorylation of a channel itself had any direct influence on its function, rather than acting indirectly via a regulatory protein. Now, Huganir et al. 6 have phosphorylated receptor-rich membrane fragments from Torpedo californica, purified the AChR, reconstituted them into lipid vesicles, and shown that a measured increase in phosphorylation of the AChR correlates well with an increased rate of desensitization. Desensitization of the AChR is a phenomenon in which the receptor becomes unresponsive to activation when it is exposed to an T I N S - February 1987 [10J
activating ligand for a relatively long period of time (tens of milliseconds or longer) 7. It occurs with purified AChR6 and so apparently requires neither metabolic energy nor the presence of any cell components in addition to the AChR. Desensitization results in AChR whose channels remain closed even when ACh is bound to the receptor. Unfortunately, desensitization is a relatively poorly understood process. Only recently has it become apparent that there are probably three or more 'desensitized' states 8-1°, based on the rates at which desensitization develops and recovers. Physiological data have suggested that desensitized AChR bind agonist with higher affinity than resting AChR, and direct binding measurements n have shown that this is true for at least some of the desensitized states. Spectroscopic studies le have given evidence consistent with the idea that the
desensitized AChR has a different conformation than normal. At present, there is some uncertainty as to how much resting desensitization exists, i.e. whether an appreciable amount of the physically present AChR is unresponsive before exposure to transmitter. The methods used by Huganir et al. take advantage of the fact that the electric organ of the marine ray Torpedo californica is a very rich source of AChR. They prepared receptor-rich membranes, and phosphorylated them in vitro. They then" purified the AChR and reconstituted the purified receptors (control or phosphorylated) into liposomes. The functional properties of the reconstituted purified AChR were studied using rapid mixing techniques developed in George Hess' laboratory (see references in Ref. 6). They measured the initial rate of ACh-elicited ion flux into vesicles, and determined how the
© 1987,ElsevierSciencePublishersB.V.,Amsterdam 0378-5912/87/$02.00
Joe Henry Steinbach Departmentsof Anesthesiologyand ofAnatomyand Neurobiology, Washington UnivemtySchoolof Medicine,5t Louis, MO, USA. John Zempel MedicalScienb'st TrainingProgram, Washington UniversitySchoolof Medicine,StLouis, MO, USA.
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