Cloning of the genes for excitatory amino acid receptors.

Glutamate is the major excitatory neurotransmitter in the mammalian brain, with receptors on every neuron in the central nervous system; it has major roles in fast synaptic transmission and in the establishment of certain forms of memory. More than 20 years ago Olney and his colleagues described the 'Excitotoxic Hypothesis' which postulates that, in addition to its normal function in the healthy brain, glutamate can kill neurons by prolonged, receptor-mediated depolarization resulting in irreversible disturbances in ion homeostasis. Therefore, glutamate is a two-edged sword; in certain undefined, adverse conditions it undergoes a transition from neurotransmitter to neurotoxin. Its toxicity has been implicated in the death of neurons in ischemia, epilepsy, and the neurodegenerative disorders such as Alzheimer's, Huntington's, and Parkinson's disease. Recent advances in the molecular cloning of the genes for the glutamate family of receptors has revealed a plethora of receptor subtypes and an unexpected level of complexity in the mechanisms of receptor expression and function.

Expression of two glucose transporters, GLUT1 and GLUT3, in cultured cerebellar neurons: Evidence for neuron-specific expression of GLUT3.

The brain is dependent on glucose as an energy source and thus requires the expression of glucose transporter proteins to enable passage of glucose across both the endothelial cells of the blood-brain barrier and the plasma membranes of neurons and glia. The GLUT 1 isoform of the facilitative glucose transporter family is expressed in the blood-brain barrier; however, the major glucose transporter isoform(s) in neurons and glia have not been identified. We have investigated the expression of glucose transporters in cultured rat cerebellar granule neurons. Two isoforms, GLUT1 and GLUT3, were detected by Western and Northern blot analyses. Expression of both isoforms increased as neurons differentiated in culture, corresponding to an increase in glucose uptake. Localization of glucose transporters by immunofluorescence indicated the presence of both isoforms in neuronal processes and in the cell body. GLUT1 was detected in both plasma membrane and cytoplasm, whereas GLUT3 appeared only in plasma membrane. Significant GLUT3 expression was also detected in the neuronal cell lines PC12 and NG108 but not in primary cultured glia or C6 glioma cells. Our findings indicate that, in the rat brain, GLUT3 expression is predominantly in neurons, suggesting that this isoform may play a major role in neuronal glucose transport.

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons.

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: pharmacological characterization.

L-Glutamate neurotoxicity at the N-methyl-D-aspartate (NMDA) receptor was characterized in cultured cerebellar granule cells. When deprived of glucose for 40 min, these cells were killed by 20-60 microM L-glutamate. However, the neurons were resistant to glutamate at concentrations as high as 5 mM when glucose and Mg2+ were present throughout. Both competitive and non-competitive NMDA receptor antagonists completely blocked neurotoxicity due to glutamate and other NMDA receptor agonists. CPP [+/-)-3-(2-carboxypiperazin-4-yl)-prophyl-1-phosphonic acid) was the most effective competitive antagonist with full protection at 100 microM while MK-801 [+/-)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]-cyclohepten-5,10-imin e) was the most effective non-competitive antagonist with full protection at 20 nM. Other antagonists with higher selectivity for other subtypes of glutamate receptors were ineffective. We conclude that glutamate toxicity in energy-deprived cerebellar granule cells is mediated by NMDA receptors. Results are discussed in terms of an hypothesis offering an explanation for the transition of glutamate from neurotransmitter to neurotoxin which emphasizes the responsiveness of the receptor to agonists rather than focusing on the presence of high concentrations of agonist.

Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: role of the voltage-dependent magnesium block.

Results of the present report show that cerebellar neurons in primary culture are resistant to glutamate concentrations as high as 5 mM in the presence of glucose and Mg2+, but sensitive to glutamate concentrations lower than 35 microM when the neurons are deprived of glucose. Glutamate toxicity is also potentiated when Mg2+ is removed but glucose and EDTA are present; in this case, higher concentrations of glutamate (1 mM) are required for full toxicity. Glucose concentrations as low as 50 microM are fully protective against the toxicity of 100 microM glutamate; pyruvate and, to a lesser extent, lactate are also protective. Significantly, increasing concentrations of extracellular Mg2+ are fully protective against the toxicity of 100 microM glutamate in the absence of glucose and against the toxicity of 1 mM glutamate in the presence of glucose and EDTA. We interpret these results as support for our hypothesis that the pivotal event in glutamate's transition to neurotoxin is relief of the Mg2+ block of the N-methyl-D-aspartate (NMDA) receptor channel, which is known to be voltage-dependent. Partial depolarization in response to depletion of high-energy phosphates relieves the voltage-dependent block enabling glutamate to stimulate an excessive ion influx which results in the death of the neuron by a mechanism which is not yet understood. We propose that this mechanism may be operative in the neuronal damage associated with a variety of neurodegenerative disorders.

The role of neuronal energy in the neurotoxicity of excitatory amino acids.

Excitatory amino acids, acting at receptors such as the N-methyl-D-aspartate (NMDA) subtype, are good candidates for a major role in the neuronal death characteristic of Alzheimer's disease. Recent evidence from studies with cultured neurons suggests that perturbations in the energy metabolism of the neuron may be involved in the transition of NMDA agonists from neurotransmitters to neurotoxins via a mechanism that involves relief of the voltage-dependent Mg++ block of the NMDA channel.

Energy-related neurotoxicity at the NMDA receptor: a possible role in Alzheimer's disease and related disorders.

Using rat cerebellar granule cells in primary culture as our model system, we have shown that excitatory amino acids (EAAs) become neurotoxic via the NMDA (N-methyl-D-aspartate) receptor when neuronal energy levels are compromised. Omission of glucose, exclusion of oxygen, or inclusion of inhibitors of oxidative phosphorylation or of Na+/K+-ATPases enables NMDA receptor agonists to express their neurotoxic potential. Both competitive and noncompetitive NMDA receptor antagonists are potent blockers of EAA neurotoxicity, with MK-801 fully effective at 20 nM. We interpret these results as indicating that glucose metabolism, ATP production, and functioning ion pumps are necessary to generate a resting potential sufficient to maintain the voltage-dependent Mg++ block of the NMDA receptor channel; relief of the block enables EAAs to act persistently at the NMDA receptor causing an excessive ion influx which leads to neuronal death by a mechanism not yet understood. These findings are discussed in the context of the potential role for NMDA receptor-mediated neurotoxicity in Alzheimer's disease and related disorders.

Neurotoxicity at the N-methyl-D-aspartate receptor in energy-compromised neurons. An hypothesis for cell death in aging and disease.

Our results demonstrated that the neurotoxicity of glutamate and closely related agonists was mediated by the NMDA receptor in rat cerebellar granule cells. Evidence was presented to support our hypothesis that the pivotal event in the transition of these EAAs from neurotransmitters to neurotoxins is relief of the voltage-dependent Mg++ block of the NMDA channel due to changes in membrane potential which can be caused by depletion of highly phosphorylated nucleotides or by other depolarizing stimuli. Persistent stimulation of NMDA receptors whose channels are unblocked by Mg++ can permit excessive influx of Na+ and Ca++ and neuronal death can follow by a mechanism not yet understood. Glutamate is not toxic at kainate receptors although they are present on these cells. These findings underline the potential importance of perturbations in energy metabolism in a variety of neurodegenerative disorders and in the normal process of aging which share the common feature of the loss of neurons.

Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor appears to play a pivotal role in enabling glutamate to express its neurotoxic potential in a variety of neurological disorders. Our results show that the transition of glutamate from neurotransmitter to neurotoxin is facilitated when cellular energy is limited in cultured cerebellar neurons. Omission of glucose, exclusion of oxygen, or inclusion of inhibitors of oxidative phosphorylation or of the sodium/potassium pump, enables the excitatory amino acids glutamate or NMDA to express their neurotoxic potential. We interpret these results as demonstrating that glucose metabolism, ATP production, and functioning Na+,K+-ATPases are necessary to generate a resting potential sufficient to maintain the voltage-dependent Mg2+ block of the NMDA receptor channel; relief of the Mg2+ block enables the excitatory amino acids to act persistently at the NMDA receptor, resulting in the opening of ion channels and subsequent neuronal damage. These findings are discussed in the context of perturbations or abnormalities which lead to decreased availability or utilization of glucose and oxygen in the brain which may trigger endogenous excitatory amino acids to become neurotoxic by this mechanism.

cGmp synthesis in cultured cerebellar neurons is stimulated by glutamate via a Ca2+-mediated, differentiation-dependent mechanism.

Sensitivity of cyclic GMP synthesis to stimulation by excitatory amino acids, depolarizing agents, and divalent cation ionophores develops during the differentiation of cerebellar neurons in culture; in each case calcium influx appears responsible for activating guanylate cyclase. The developmental event being followed is not the appearance of either the soluble or the particulate form of the enzyme since both are present throughout. The possible role of a differentiation-dependent calcium-modulating protein is discussed.

Uptake of imipramine in neurons cultured from rat cerebellum.

Imipramine was taken up by rat cerebellar neurons in primary culture. The process was dependent on time, temperature and pH and was reduced in the presence of the adenosine triphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide or in the absence of glucose. Uptake approached saturation at 100 microM imipramine where approximately 42 nmol of the drug accumulated intracellularly per mg cell protein. Propranolol, but not serotonin, competed for imipramine uptake and uptake was inhibited by the 'lysosomotropic' amine chloroquine and by the Na+/H+ ionophore monensin, both of which dissipate proton gradients. Neurons were fractionated on Percoll gradients and the fractions exposed to [3H]imipramine. MgATP-dependent accumulation of [3H]imipramine was found mainly in fractions enriched for dense lysosomes. We conclude that imipramine was taken up by cerebellar neurons in primary culture and accumulated at high concentrations in intracellular compartments.

Transport of beta-adrenergic antagonists in the absence of beta-adrenergic receptors in rat pituitary tumor cells.

We have demonstrated that the rat pituitary tumor cell line GH3 has a carrier-mediated active transport system for the beta-adrenergic antagonist dihydroalprenolol (DHA). Transport of DHA in GH3 was saturable, with an apparent Km of 1.4 microM, was temperature and pH dependent, and was inhibited by the ionophore monensin and the amine transport inhibitor reserpine. Propranolol competed for DHA transport, but not in a stereoselective fashion. The tricyclic antidepressant imipramine also competed for DHA transport, but catecholamines or serotonin did not. This amine transport system in GH3 cells appeared to be identical to the one we recently described in several other cell types; however, analysis in those cells was complicated by the fact that they contain beta-adrenergic receptors which bind beta-adrenergic ligands. In this report we show that GH3 cells do not possess detectable beta-adrenergic receptors, based on their inability to bind the partial agonist CGP-12177, their inability to bind nanomolar concentrations of DHA in a saturable, stereospecific manner, and their failure to produce cAMP in response to stimulation by beta-adrenergic agonists. Characterization of the amine transport system in GH3 cells clearly distinguishes it from receptor-mediated phenomena and should facilitate our efforts to fully understand its mechanism and significance.

Differentiation between amine transport and beta-adrenergic receptor-mediated binding in cultured mammalian cells.

We have found that several types of cultured mammalian cells, including both normal and transformed human, rat, and mouse cell lines, have an active transport system for a diverse group of structurally related compounds possessing an amine group and various types of aromatic ring structures. Ligands such as the beta-adrenergic antagonists (-)-[3H] dihydroalprenolol (DHA), (-)-[3H]propranolol, and (-)-[125I] iodocyanopindolol, and the tricyclic antidepressant [3H]imipramine, which are used to assess cell surface receptors for catecholamines and serotonin, appear to be actively transported into cells rather than simply bound to cell surface sites or accumulated by passive diffusion. DHA transport was competed by many structurally related amines including imipramine and certain alpha-and beta-adrenergic ligands, but not by catecholamines or serotonin. Ligand transport in HeLa cells was saturable at micromolar levels, selective, nonstereospecific, temperature- and pH-dependent, and sensitive to the ionophore monensin and the amine transport inhibitor reserpine, thus indicating dependence on a carrier system driven by a transmembrane proton gradient. In C6 glioma cells, amine transport was clearly distinguishable from beta-adrenergic receptor binding which could be measured with the recently developed hydrophilic beta-blocker (+/-)-[3H] 4-(3-tertiarybutylamino-2-hydroxy-propoxy)-benzimidazole-2-on hydrochloride (CGP-12177); binding of this ligand met rigorous pharmacological criteria, was not influenced by monensin or reserpine, and, therefore, did not appear to be transported. Membrane vesicles from HeLa and C6 cells transported DHA but not CGP-12177 via a MgATP-dependent mechanism which was inhibited by N,N'-dicyclohexylcarbodiimide, monensin, and reserpine, indicating a carrier system driven by a proton gradient maintained by a proton-pumping ATPase.

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component.

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture.

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.

Phospholipid methylation: a possible mechanism of signal transduction across biomembranes.

The conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is catalyzed by two methyltransferases with S-adenosylmethionine as the methyl donor. PC formed by transmethylation is further metabolized by phospholipase A2. The synthesis and degradation of methylated phospholipids are involved in regulating the number of the beta-adrenergic receptors and their coupling to adenylate cyclase in rat reticulocytes, HeLa cells, and rat astrocytoma cells. Methylation of the phospholipids in these cells is stimulated by binding of agonists to the beta-adrenergic receptors. Accumulation of phosphatidyl-N-monomethylethanolamine causes an increase in membrane fluidity and enhances the coupling of the receptors to adenylate cyclase. Agents that inhibit phospholipid methylation decrease the number of receptors in intact HeLa cells, while increased phospholipid methylation unmasks cryptic receptors. Conversely, the degradation of methylated phospholipids appears to be closely associated with the desensitization of the beta-adrenergic receptors following prolonged stimulation with isoproterenol. Inhibition and stimulation of phospholipase A2 causes inhibition and stimulation of this desensitization process.