H. Manev

Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity.

Glutamate is an important excitatory amino acid at many central nervous system synapses. After its release from presynaptic nerve terminals, glutamate transiently binds to specific neuronal membrane receptors, which transduce its signal by the generation of intracellular second messengers before being rapidly cleared from the synapse. However, during ischemia, the glutamate concentration at synapses surrounding the focal lesion can be increased for sustained periods of time, resulting in abusive stimulation of glutamate receptors that can eventually be neurotoxic. To develop drugs capable of selectively blocking the pathological effects of glutamate in neurons surrounding ischemic lesions while leaving the physiological actions of glutamate in nonlesioned areas of the brain unaffected, it is essential to delineate glutamate‐induced intracellular events that are specific to receptor abuse. This article describes the intracellular sequelae of physiological and pathological glutamate receptor activation and suggests potential targets for such receptor abuse‐dependent antagonists (RADAs).— Manev, H.; Costa, E.; Wroblewski, J. T.; Guidotti, A. Abusive stimulation of excitatory amino acid receptors: a strategy to limit neurotoxicity: FASEB J. 4: 2789‐2797; 1990.

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate‐induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate‐induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose‐dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK‐801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N‐acetyl‐Leu‐Leu‐ norleucinal) prevented glutamate‐induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate‐induced neurotoxicity is independent of calpain I activation.

Inhibition of glutamate-induced cell death by sodium nitroprusside is not mediated by nitric oxide

Pretreatment of primary cultures of cerebellar granule cells with sodium nitroprusside (SNP) protected these neurons from delayed death induced by glutamate and N-methyl-D-aspartate (NMDA). This neuroprotective effect was not mimicked by S-nitroso-N-acetylpenicillamine (SNAP) which like SNP stimulates guanylate cyclase via a nitric oxide (NO) related mechanism. In contrast, neuroprotection was achieved with potassium ferrocyanide, a compound structurally related to SNP, but devoid of NO. On the other hand, kainate-induced neurotoxicity was not protected but potentiated by SNP. This effect of SNP was not mimicked by SNAP, potassium ferrocyanide and potassium ferricyanide. We conclude that neuroprotective properties of SNP on glutamate- and NMDA-induced neurotoxicity are not due to the release of NO and activation of guanylate cyclase, but are determined by the ferrocyanide portion of the SNP molecule.

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced. Ca2(+)-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 microM glutamate for 15 min, at 22 degrees C, in the absence of Mg2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC50 300 microM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 microM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA-(20 microM) stimulated current was inhibited by 700 microM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at -50 mV, 100 microM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg2(+)-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N‐methyl‐d‐aspartate (NMDA)‐sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate‐induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate‐triggered neuronal death, but the glutamate‐induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.

Protection by Natural and Semisynthetic Gangliosides from Ca2+-Dependent Neurotoxicity Caused by Excitatory Amino Acid (EAA) Neurotransmitters

Neuronal death is a frequent occurrence during nervous system ontogenesis and continues, though at a slower rate, throughout life. EAA neurotransmitters, when added (in appropriate concentrations) to primary neuronal cultures, are the only neurotransmitters known to cause neuronal death by destabilizing homeostasis of free Ca2+ (Olney, 1969; Coyle, 1987; Vaccarino et al., 1987; Favaron et al., 1988; Connor et al., 1988). In neurons, EAA increase Ca2+ influx through specific cationic channel activation, thereby stimulating Ca2+-dependent enzymes, including protein kinase C (PKC) (Wroblewski and Danysz, 1989). Presumably, these actions of EAAs can be mediated at NMDA-, kainate- or quisqualate-sensitive synaptic receptors (Wroblewski and Danysz, 1989). Normally, intraneuronal Ca2+ homeostasis is maintained by the equilibration of free Ca2+, with Ca2+ compartmentalized in endoplasmic reticulum and mitochondria and the operation of channels and pumps allowing Ca2+ influx (cationic channels) and efflux (Nat-Ca2+ exchanger and ATPase-operated Ca2+ pumps) (Carafoli, 1987). It is, therefore, not surprising that many have attempted to evaluate whether EAA receptors are also operative in physiologically programmed neuronal death. In central nervous system, an alteration of EAA transmission may occur in a variety of acute (stroke, ischemia, and CNS traumas) or chronic (autoimmune responses, Huntington’s chorea, amyotrophic lateral sclerosis (ALS), olivopontocerebellar atrophy, lathyrism, and the Guamanian complex) neuropathological processes (Rothman, 1984; Kostic et al., 1989; Spencer et al., 1987; Plaitakis et al., 1988).