Isolation of various forms of sterol beta-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex.

The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.

Methionine sulfoximine shows excitotoxic actions in rat cortical slices.

Methionine sulfoximine (MSO) is a rare amino acid. It occurs in nature or as a by-product of some forms of food processing. A notable example of the latter was a former method for bleaching wheat flour, using nitrogen trichloride, the "agene process," in use for most of the first 50 years of this century. "Agenized" flour was found to be responsible for various neurological disorders in animals, and MSO was identified as the toxic factor. The agene process was subsequently discontinued in the United States and the United Kingdom circa 1950. MSO inhibits the synthesis of both glutathione and glutamine, and it is possible that its actions on the nervous system arise from alterations in the amount or distribution of these molecules. Structurally, MSO resembles glutamate, an observation that has also raised the possibility that it might have more direct glutamate-like actions on neurons. In the present investigation, we report excitatory and toxic actions of MSO in an in vitro preparation of adult rat cortex. Field potential recordings in this preparation show that MSO application evokes a sustained depolarization, which can be blocked by the N-methyl-D-aspartate (NMDA) antagonist L-(+)-2-amino-5-phosphonovalerate (AP5). However, competition assays using MSO on [3H]CGP-39653 (DL-(E)-2-amino-4-propyl-1-phosphono-3-pentenoate) binding in rat cortical homogenates show only 20% displacement of total binding, suggesting that MSO is acting indirectly, perhaps by releasing glutamate. To investigate this possibility, we measured glutamate release during MSO application. Time course and dose-response experiments with MSO showed significant [3H]glutamate release, which was partially attenuated by AP5. To assess cellular toxicity, we measured lactate dehydrogenase (LDH) release from cortical sections exposed to MSO. MSO treatment led to a rapid increase in LDH activity, which could be blocked by AP5. These data suggest that MSO acts by increasing glutamate release, which then activates NMDA receptors, leading to excitotoxic cell death. These data suggest the possibility that MSO in processed flour had excitotoxic actions that may have been contributing factors to some human neuronal disorders.

A novel fluorescent GSH-adduct binds to the NMDA receptor.

In an attempt to develop various fluorescent probes to label glutathione (GSH) receptors, we have serendipitously synthesized a probe that binds to and antagonizes the NMDA receptor. Probe 1, a GSH adduct, displaces the competitive NMDA antagonist [3H]-CGP 39653 with a higher affinity than NMDA or cysteine in rat synaptic membranes. In recording experiments from a rat cortical 'wedge' preparation, Probe 1 reversibly blocks both NMDA- and cysteine-induced depolarization. In mixed astrocyte-neuron tissue culture preparations, Probe 1 labels parts of both cell bodies as well as processes. The present data suggest that Probe 1 binds to the NMDA receptor and antagonizes channel function.

Glutathione and signal transduction in the mammalian CNS.

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Regulation of ionotropic receptors by protein phosphorylation.

The regulation of synaptic signal transduction is of central importance to our understanding of normal and abnormal nervous system function. One mechanism by which signal transduction can be affected is the modification of cellular sensitivity by alterations of transmembrane receptor properties. For G-protein coupled receptors, protein phosphorylation is intimately involved in many stages of receptor regulation. This appears to be true for ionotropic receptors as well. Evidence of a role for protein kinase and protein phosphatase activity in the multi-staged ionotropic receptor regulation cascade is presented and a comparison to G-protein coupled receptor regulation is considered.

Glutathione-induced sodium currents in neocortex.

The present report demonstrates that glutathione (GSH), a tripeptide composed of glutamate, glycine and cysteine (gamma-L-glutamyl-L-cysteinyl-glycine) and best known as a free radical scavenger, elicits a large fast depolarizing potential when applied to cortical slices. This potential is maximally larger than that produced by either NMDA or AMPA. Like AMPA, the GSH current appears to be carried by sodium ions, but cannot be blocked by the glutamate receptor antagonists AP5 or DNQX. In addition, removal of external calcium or blockade of potassium currents by TEA does not diminish the GSH-induced potential. Together, these results suggest that GSH acts through its own receptor-mediated channels, independently of the known EAA receptors, and that its receptors may be a key, and previously unknown, component of cortical excitatory neurotransmission.

gamma-Aminobutyric acidA receptor regulation by a chloride-dependent kinase and a sodium-dependent phosphatase.

gamma-Aminobutyric acidA (GABAA) receptors are linked to ion channels which mediate many aspects of neural inhibition. Although the effects of phosphorylation on GABAA receptor function have been widely studied, the actual role of phosphorylation in the regulation of these receptors still remains controversial. In recent reports, we have described the effects of phosphorylating/dephosphorylating enzymes on the regulation of GABAA receptors in a rat cortical slice preparation (Shaw et al., Mol. Neuropharmacol., 2 (1992) 297-302; Shaw and Lanius, Dev. Brain Res., 70 (1992) 153-161; Pasqualotto et al., Neuroreport, 4 (1993) 447-450) and predicted that ionic co-factors are involved in mediating the regulation of GABAA receptors by kinases and phosphatases. In the present report, the effects of chloride, sodium, potassium, and calcium were examined alone and in the presence of cAMP-dependent protein kinase (protein kinase A) or alkaline phosphatase. The results showed a decrease in [3H]SR 95531 (GABAA receptor antagonist) binding after incubation with chloride alone; this decrease was further enhanced in the presence of protein kinase A. Both effects could be blocked by a protein kinase A inhibitor. Conversely, an increase in [3H]SR 95531 binding was observed after incubation with sodium alone; this increase was further enhanced in the presence of alkaline phosphatase. In both cases these increases in binding could be blocked by sodium orthovanadate, a phosphatase inhibitor. Potassium was ineffective under all conditions; calcium showed enzyme-independent effects at low concentrations only. These results suggest the existence of a novel chloride-dependent protein kinase which may have significant sequence homology to protein kinase A, and a novel sodium-dependent phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent expression, phosphorylation and function of neurotransmitter receptors: pharmacological implications.

In recent years, a number of experimental controversies have arisen in the pharmacological sciences literature. Two possibly related examples concern the role of phosphorylation in receptor regulation and the occurrence of paradoxical effects of neurally active drugs in patients. In this article, Ruth Lanius and colleagues review these two issues and suggest that some of the recently reported age-dependent aspects of neurotransmitter receptor regulation and function may explain drug action. An appreciation of the dynamic interactions involved in receptor regulation, especially during development, may offer novel perspectives on neural function.

A novel mechanism of AMPA receptor regulation: ionically triggered kinases and phosphatases.

We have postulated elsewhere (Shaw CA and Lanius RA. Dev Brain Res 70, 153-161 (1992)) that the kinase/phosphatase regulation of AMPA receptors is mediated by specific ions. Using an in vitro cortical slice preparation we have now examined the roles of calcium (Ca2+), chloride (Cl-), potassium (K+), and sodium (Na+) in the regulation of AMPA receptors. Ca2+ led to a concentration-dependent decrease in [3H]-CNQX binding which could be blocked by a general protein kinase inhibitor (H-7) and a protein kinase A inhibiting peptide. Tamoxifen, a relatively specific protein kinase C inhibitor, had no effect. In contrast, Cl- led to concentration-dependent increases in [3H]-CNQX binding which could be blocked by both sodium-ortho-vanadate, a tyrosine residue selective phosphatase inhibitor, and sodium-beta-D-glycerol phosphate, a serine residue selective phosphatase blocker. K+ and Na+ had no effect on [3H]-CNQX binding. These results suggest that Ca2+ and Cl- may be acting as signals which trigger kinase(s) and phosphatase(s) involved in the regulation of AMPA receptors.

Regulation of GABAA and AMPA receptors by agonist and depolarizing stimulation requires phosphatase or kinase activity.

Using a live in vitro slice preparation of adult rat neocortex we have examined the role of phosphorylation and dephosphorylation in the regulation of GABAA and AMPA receptors. Alkaline phosphatase increased binding levels for both receptors while protein kinase A had the opposite effect. For both receptor populations, phosphatase effects were blocked by sodium beta-D-glycerol phosphate and sodium vanadate (phosphatase inhibitors) while kinase actions were blocked by a protein kinase inhibitor. Increases in cell depolarizations by veratridine led to an increase in labelled GABAA receptors, but to decreases in labelled AMPA receptors. Increases in binding were differentially blocked by the two phosphatase inhibitors, sodium beta-D-glycerol phosphate and sodium vanadate while decreases in binding were blocked by protein kinase inhibitor. Agonist stimulation of GABAA and AMPA receptors led to a decrease in receptor binding which could be blocked in both cases by protein kinase inhibitor. These data show that certain cortical excitatory and inhibitory amino acid receptors can be regulated by the action of phosphorylating/dephosphorylating enzymes and further suggest that the regulation induced by cell depolarization and agonist stimulation is based on similar mechanisms.

Citrulline in the rat brain: immunohistochemistry and coexistence with NADPH-diaphorase.

The presence in the brain of the urea cycle intermediate citrulline in the absence of a complete urea cycle has never been adequately explained. In an attempt to clarify this problem, we developed antibodies to citrulline and determined the distribution of citrulline-immunoreactivity in fixed sections of rat brain using immunoperoxidase and indirect immunofluorescence techniques. Citrulline-positive neurons were found to have a restricted distribution within the brain. A few cells were present in the cortex and corpus callosum. A large population of strongly stained cells was diffusely scattered throughout the striatum, nucleus accumbens and olfactory tubercle. Less strongly stained cells were detected in the supraoptic and paraventricular nuclei of the hypothalamus, the dorsal raphe, and the laterodorsal and pedunculopontine tegmental nuclei of the pons. The citrulline-immunoreactive cells were similar to those previously shown to contain NADPH-diaphorase activity, and double staining experiments indicated that citrulline-immunoreactivity was present in a subpopulation of NADPH-diaphorase-positive neurons. We have recently identified NADPH-diaphorase as a nitric oxide synthase. Thus the presence of citrulline in these cells suggests that it is formed within the brain as a coproduct during nitric oxide formation from arginine.

Galanin and NADPH-diaphorase coexistence in cholinergic neurons of the rat basal forebrain.

The distribution of the enzyme NADPH-diaphorase in the rat basal forebrain was examined in relation to the neuropeptide galanin and the neurotransmitter synthetic enzyme choline acetyltransferase. Immunoperoxidase staining permitted camera lucida mapping of galanin and choline acetyltransferase distributions in serial sections through the basal forebrain for comparison with adjacent sections prepared for NADPH-diaphorase histochemistry. Photographs of sections subjected to indirect immunofluorescence for galanin and choline acetyltransferase were compared to photographs of the same sections taken after NADPH-diaphorase histochemistry. This permitted the direct investigation of co-localization within the cholinergic basal forebrain. The distributions of choline acetyltransferase- and galanin-immunoreactive neurons in the basal forebrain agreed with previous descriptions. NADPH-diaphorase histochemistry selectively stained a population of magnocellular basal forebrain neurons with a distribution similar to that observed with galanin immunohistochemistry. Double and triple staining experiments indicated that NADPH-diaphorase labels a majority of the magnocellular cholinergic neurons in the medial septum and diagonal band nuclei. Most of these neurons also contain galanin immunoreactivity. However, small populations of galanin-positive/diaphorase-negative or diaphorase-positive/galanin-negative cholinergic neurons were also observed. In the more caudal portions of the cholinergic basal forebrain, very few galanin or NADPH-diaphorase-positive neurons were observed. Thus, galanin and NADPH-diaphorase coexist in the majority of cholinergic basal forebrain neurons in the regions innervating limbic structures. The neocortically projecting cholinergic cells in the caudal basal forebrain appear to lack these other neurochemical markers.