The NMDA receptor ion channel: a site for binding of Huperzine A.

Huperzine A (HUP-A), first isolated from the Chinese club moss Huperzia serrata, is a potent, reversible and selective inhibitor of acetylcholinesterase (AChE) over butyrylcholinesterase (BChE) (Life Sci. 54: 991-997). Because HUP-A has been shown to penetrate the blood-brain barrier, is more stable than the carbamates used as pretreatments for organophosphate poisoning (OP) and the HUP-A:AChE complex has a longer half-life than other prophylactic sequestering agents, HUP-A has been proposed as a pretreatment drug for nerve agent toxicity by protecting AChE from irreversible OP-induced phosphonylation. More recently (NeuroReport 8: 963-968), pretreatment of embryonic neuronal cultures with HUP-A reduced glutamate-induced cell death and also decreased glutamate-induced calcium mobilization. These results suggest that HUP-A might interfere with and be beneficial for excitatory amino acid overstimulation, such as seen in ischemia, where persistent elevation of internal calcium levels by activation of the N-methyl-D-aspartate (NMDA) glutamate subtype receptor is found. We have now investigated the interaction of HUP-A with glutamate receptors. Freshly frozen cortex or synaptic plasma membranes were used, providing 60-90% specific radioligand binding. Huperzine A (< or =100 microM) had no effect on the binding of [3H]glutamate (low- and high-affinity glutamate sites), [3H]MDL 105,519 (NMDA glycine regulatory site), [3H]ifenprodil (NMDA polyamine site) or [3H]CGS 19755 (NMDA antagonist). In contrast with these results, HUP-A non-competitively (Hill slope < 1) inhibited [3H]MK-801 and [3H]TCP binding (co-located NMDA ion channel PCP site) with pseudo K(i) approximately 6 microM. Furthermore, when neuronal cultures were pretreated with HUP-A for 45 min prior to NMDA exposure, HUP-A dose-dependently inhibited the NMDA-induced toxicity. Although HUP-A has been implicated to interact with cholinergic receptors, it was without effect at 100 microM on muscarinic (measured by inhibition of [3H]QNB or [3H]NMS binding) or nicotinic [3H]epibatidine binding) receptors; also, HUP-A did not perturb adenosine receptor binding [3H]PIA or [3H]NECA). Therefore, HUP-A most likely attenuates excitatory amino acid toxicity by blocking the NMDA ion channel and subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites. Thus, on the one hand, HUP-A could be used as a pretreatment against OPs and it might also be a valuable therapeutic intervention in a variety of acute and chronic disorders by protecting against overstimulation of the excitatory amino acid pathway. By blocking NMDA ion channels without psychotomimetic side-effects, HUP-A may protect against diverse neurodegenerative states observed during ischemia or Alzheimer's disease.

RS-100642-198, a novel sodium channel blocker, provides differential neuroprotection against hypoxia/hypoglycemia, veratridine or glutamate-mediated neurotoxicity in primary cultures of rat cerebellar neurons.

The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), in in vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 micro M. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS-100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium [Ca(2+)]i) mobilization revealed that glutamate caused immediate and sustained increases in [Ca(2+)]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca(2+)]i mobilization produced by veratridine or KCl depolarization. QX-314 produced similar effects on glutamate-, veratridine- or KCl-induced [Ca(2+)]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca(2+)]i, and attenuating the sustained increase in [Ca(2+)]i mobilization. By using different in vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na(+) channels.

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A.

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.

Neuroprotection produced by the NAALADase inhibitor 2-PMPA in rat cerebellar neurons.

The present study examined the neuroprotective actions of the N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) in four in vitro models of neurotoxicity. Using neuron-enriched primary cultures derived from rat embryo (E15) cerebellum, 2-PMPA afforded 100% neuroprotection from injuries induced by hypoxia (EC(50)=8.4 microM). In contrast, against glutamate or N-methyl-D-aspartate (NMDA) injury, 2-PMPA was less potent and its efficacy limited to a maximum of 46% and 16%, respectively. 2-PMPA was not effective against veratridine-induced injury. Also, the less potent analog of 2-PMPA, 2-[phosphonomethyl]succinic acid (2-PMSA), was ineffective. Unlike 2-PMPA, the endogenous NAALADase substrate and mGlu(3) receptor agonist N-acetyl-aspartyl-glutamate (NAAG) was neuroprotective against all four injury mechanisms and compared to 2-PMPA, exhibited a different "phosphate effect" on neuroprotection. These results confirm the superior efficacy of 2-PMPA to protect against injury caused by cellular anoxia, and are discussed relative to upstream modulation of hyperglutamatergic activity vs. downstream modulation of metabotropic receptors as possible targets for ischemia/stroke therapy.

Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies.

We investigated the dose-response and time-course of c-fos antisense oligodeoxynucleotide (ASO) treatment against excitatory amino acid (EAA)-induced neurotoxicity in rat hippocampal neurons. Glutamate (in vitro) or NMDA (in vivo) produced significant neuronal degeneration. Neuroprotection produced by 30 min or 4 h pretreatment with c-fos ASO in cultured hippocampal neurons was dose-dependent. In vivo, bilateral intrahippocampal injections of c-fos ASO (0.025 nmol/site) was neuroprotective when administered 30 min before or after NMDA treatment. However, 4 h pretreatment was ineffective. A higher dose (0.125 nmol) of c-fos ASO was neurotoxic and failed to afford neuroprotection regardless of the treatment schedule. Collectively, these results demonstrate a neuroprotective effect of c-fos ASO against EAA-induced neuronal injury supporting a causative role of c-fos expression in EAA neurotoxicity.

Huperzine A, a potential therapeutic agent for dementia, reduces neuronal cell death caused by glutamate.

Huperzine a, a potential therapeutic agent for Alzheimer's disease, inhibits acetylcholinesterase in primary cultures derived from forebrain, hippocampus, cortex and cerebellum of embryonic rat brain. Glutamate induces cell death in cultures from all these brain regions. Maximum cell toxicity was observed in cerebellar cultures. Pretreatment of cell cultures with Huperzine A reduced cell toxicity, as evidenced by cytotoxicity assay and general morphology. Huperzine A pretreatment also reduced glutamate-induced calcium mobilization, but did not affect elevations in intraneuronal free Ca2+ ([Ca]i) caused by KCl or (-)Bay K 8644. The data suggest that Huperzine A could be a potent neuroprotective agent not only where cholinergic neurons are impaired, but also under conditions in which glutamatergic functions are compromised.

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain.

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 microM), or glutamate (75 microM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca2+ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca2+ were maximal in neurons from cerebellum and minimal in neurons from cortex. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15-35% was observed following 4 h of DDG pretreatment, increasing to 60-85% protection after 24 h of DDG pretreatment. Although the mechanism of DDG's neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.

Regulation of neuronal thyroid hormone receptor alpha 1 mRNA by hydrocortisone, thyroid hormone and retinoic acid.

The regulation of thyroid hormone receptor alpha 1 (TR alpha 1) mRNA by hydrocortisone (HC), thyroid hormone (T3) and retinoic acid (RA) has been studied in mixed and neuronal primary cultures of cells dissociated from fetal rat cerebra. Steady-state levels of TR alpha 1 mRNA were increased as much as 5-fold at 13 days of development by 0.3 microM HC in both mixed and neuronal-enriched cultures. The regulation of TR alpha 1 mRNA by HC appears to be mainly limited to neurons. This conclusion is based on two observations. First, stimulation by HC occurs in cultures highly enriched in neurons at approximately the same time and extent as that seen in mixed-cell cultures (containing neurons, oligodendroglia and astroglia). Second, the stimulation reaches a peak at 13 days in mixed-cell cultures, an age at which neurons but not glia differentiate. In most cases, neither T3 (20 nM) nor RA (10(-7) M) stimulated an increase in TR alpha 1 mRNA steady-state levels. The exception was that RA increased TR alpha 1 mRNA levels at 13 days in culture, but at no other stage of development. In both types of cultures, RA and T3 separately and together produced as much as a complete inhibition of the stimulation by HC. Regulation by T3, when it occurred, was always negative. The fact that T3 can strongly repress the induction of TR alpha 1 mRNA by HC demonstrates that T3 can function through negative cooperativity as a potent regulator of its own receptor in brain.

Regulation of neuronal differentiation by retinoic acid alone and in cooperation with thyroid hormone or hydrocortisone.

Cultures highly enriched in neurons obtained from embryonic mouse cerebra were used to demonstrate that: (1) at the optimum concentration of 10(-8) M retinoic acid stimulated the neurons to produce axon- and dendrite-like structures as determined by phase contrast and fluorescent microscopy; (2) the same concentration of retinoic acid stimulated acetyl cholinesterase and choline acetyltransferase activities; (3) treatment of neurons of either prenatal or neonatal equivalent age with retinoic acid produced a sustained stimulation of neuronal differentiation, and (4) retinoic acid cooperatively stimulated neuronal differentiation with either thyroid hormone or hydrocortisone.

Regulation of neuronal differentiation in neuron-enriched primary cultures from embryonic rat cerebra by platelet activating factor and the structurally related glycerol ether lipid, dodecylglycerol.

Primary cultures enriched in neurons dissociated from embryonic rat cerebra were used to demonstrate that platelet activating factor and the structurally related ether glycerolipid, dodecylglycerol, are readily taken up in small amounts by neurons and that they stimulate the differentiation of neurons. The stimulation of neuronal differentiation was observed as a precocious development of axon-like extensions which correlated with a concentration-dependent increase in neuronal-specific enzyme activities. This stimulation of morphological and neurochemical factors by either platelet activating factor or dodecylglycerol was almost completely abolished by triazolam, a known inhibitor of platelet activating factor function. Neither platelet activating factor nor dodecylglycerol at the concentrations used to achieve stimulation of neuronal differentiation compromised the plasma membrane, as indicated by the lack of leakage of cytoplasmic lactic acid dehydrogenase.

Degrees of cooperativity between triiodothyronine and hydrocortisone in their regulation of the expression of myelin basic protein and proteolipid protein during brain development.

Cultures of cells dissociated from embryonic mouse cerebra were used to demonstrate: (1) that the developmental expression of the mRNA of proteolipid protein is dependent on thyroid hormone; (2) that the expression of the mRNA of proteolipid protein is stimulated not only by triiodothyronine but also by hydrocortisone, which achieve their respective stimulations by an additive and uncompetitive mechanism; (3) the stimulation of the net accumulation of the mRNA of myelin basic protein by hydrocortisone and triiodothyronine is also cooperative, additive, and uncompetitive, and (4) the stimulation of the net accumulation of myelin basic protein, during development by hydrocortisone, is completely dependent on the presence of thyroid hormone. These results suggest that the regulation of the synthesis of myelin basic protein by hydrocortisone requires the presence of triiodothyronine at a posttranscriptional event, but not for transcription itself.

Synergism between penicillin G and the antimicrobial ether lipid, rac-1-dodecylglycerol, acting below its critical micelle concentration.

rac-1-Dodecylglycerol (DDG) and penicillin G (Pen G) act synergistically to dramatically lower the minimum inhibitory concentration (MIC) of each other in four Gram-positive bacteria studied. At one-half its MIC, DDG ether lowered the MIC of Pen G 10- to 80-fold. Under the same conditions, Pen G lowered the MIC of DDG 4- to 7.5-fold. The critical micelle concentration of DDG was determined to be 7.93 mg/ml (0.0305 mM), which is approximately two-fold greater than the minimum inhibitory concentration of DDG determined in the presence of a protein-free chemically defined medium. This finding suggests that DDG is not killing bacteria through its detergent action. Pen G also did not alter the critical micelle concentration of DDG, which indicates that the synergism between these two agents is not related to micelle formation.

Effect of hydrocortisone on myelin basic protein in developing primary brain cultures.

The hormones hydrocortisone (HC) and triiodothyronine (T3) are known to regulate myelinogenic parameters in cultures of brain cells. However, the effect of glucocorticoids on the myelin-specific metabolite, myelin basic protein, has not been previously studied. In the present studies we show that the concentrations of myelin basic protein (MBP) in developing primary cultures from mouse cerebra are significantly higher in HC (0.3 microM)-treated as compared to untreated cultures after 15 days in vitro. Further, this effect of HC on MBP appears to be T3-dependent. Since HC stimulates oligodendroglia to produce MBP, the effect of HC on the activities of the enzymes, glutamine synthetase which is primarily associated with astrocytes, and acetylcholinesterase, which is primarily associated with neurons was was determined. HC stimulated both enzymes, suggesting that all 3 cell types may be regulated by HC.

Correlation between inhibition of myelin basic protein (arginine) methyltransferase by sinefungin and lack of compact myelin formation in cultures of cerebral cells from embryonic mice.

Sinefungin, a known inhibitor of protein methylation, inhibited the myelin basic protein (arginine) methyltransferase activity in homogenates of cultured cerebral cells from embryonic mice. Fifty percent inhibition was achieved with 25 microM sinefungin. Electron microscopic examination of the myelin fraction, isolated by gradient density centrifugation and obtained from untreated cells, revealed numerous ringlike multilamellar membranous substructures that had a major dense line periodicity, compactness, and the general appearance expected of myelin obtained by the same technique from whole brain. Cells treated with 30 microM sinefungin, which inhibits myelin basic protein methyltransferase in broken cell preparations about 60%, produced ringlike structures that were devoid of multilamellar periodicity and compactness reminiscent of the vacuolated myelin observed in subacute combined degeneration and in nitrous-oxide- or cycloleucine-treated animals in which methyltransferase activity is also inhibited. The sinefungin-induced change in multilamellar periodicity cannot be attributed to a lack of myelin basic protein, since the ratio of myelin basic protein to total protein did not decrease in sinefungin-treated cells. This primary culture system should be useful for further evaluating the hypothesis that the methylation of myelin basic protein is related to the formation of compact myelin.

Inhibition of peptidoglycan synthesis of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT by the antibacterial agent dodecyl glycerol.

Dodecyl glycerol inhibits the synthesis of the peptidoglycans of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT. This metabolic regulation represents the second known mode by which dodecyl glycerol expresses antibacterial activity. The first mode of action of dodecyl glycerol was shown to stimulate autolysin activity which degrades cell-wall peptidoglycan (Ved HS, Gustow E, Mahadevan V and Pieringer RA, 1984, J. Biol. Chem. 259, 8115-8121).

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790.

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).

The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol.

Treatment of Streptococcus faecium ATCC 9790 with 3.5 micrograms/ml of dodecylglycerol produces a nonwall entity found in the 25,000 X g supernatant cell fraction which activates the autolysin activity of S. faecium. The stimulation of the autolysin activity by dodecylglycerol mimics the activation of the autolysin from a latent to an active form by trypsin and other proteolytic enzymes. This stimulation of autolytic activity by dodecylglycerol can be reversed by specific proteinase inhibitors. Dodecylglycerol also markedly stimulates the proteinase activity endogenous to S. faecium, and this stimulation can be reversed by several proteinase inhibitors. It is concluded that one primary antibacterial mode of action of dodecylglycerol is to stimulate the proteinase of S. faecium which activates the cell's autolysin and thereby prevents bacterial growth.