Studies on the prevention of nigericin action in neuroblastoma X glioma hybrid (NG108-15) cells.

Electrophysiological analysis of neuroblastoma X glioma hybrid (NG108-15) cells was used as an in vitro neuronal model system to evaluate antagonists of the K+-selective carboxylic ionophore, nigericin. Changes in membrane electrical characteristics induced by nigericin with and without the simultaneous administration of antagonists were measured using intracellular microelectrode techniques. Bath application of nigericin (3 microM) produced a severe hyperpolarization and blocked the generation of action potentials in response to electrical stimulation. Simultaneous administration of nigericin plus the Na+-K+ pump inhibitor ouabain or drugs known to influence Ca++ signaling in cells, i.e., quinidine, compound R24571, verapamil or haloperidol, was able to significantly attenuate the hyperpolarization. All antagonists acted in a concentration-dependent manner. However, nigericin plus maximally effective concentrations of ouabain (1 microM), verapamil (3 microM) and haloperidol (3 and 10 microM) resulted in moderate-to-severe depolarization by the end of 24 min. superfusions, suggesting that the concentrations of antagonists were excessive and that NG108-15 cell damage had occurred. In addition, none of the compounds studied was able to effectively prevent nigericin-induced blockade of action potentials. Thus, none of these antagonists appears suitable for transition to in vivo antidotal protection studies.

Prevention of monensin-induced hyperpolarization in NG108-15 cells.

The NG 108-15 (neuroblastoma X glioma hybrid) cell line was used as an in vitro neuronal model to evaluate potential antagonists of the Na+-selective carboxylic ionophore monensin. Changes in membrane electrical characteristics induced by monensin with and without the simultaneous administration of antagonists were measured using intracellular microelectrode techniques. Bath application of monensin (3 microM) produced a hyperpolarization of approximately = 35 mV. Monensin also altered the generation of action potentials in response to electrical stimulation in 14 of 24 (58%) exposed cells, as evident in a partial or complete loss of action potentials or in an alteration of action potential waveform. The antagonists used were Na+-K+ pump inhibitor ouabain (1-3 microM), the Ca2+dependent K+ channel blocker quinine (3-30 microM) or drugs known to influence Ca2+ signaling in cells, i.e., trifluoperazine (3-10 microM), verapamil (1-10 microM) or chlorpromazine (3-30 microM). On a molar basis, ouabain was the most and trifluoperazine the least effective of the antagonists. Quinine, verapamil and chlorpromazine all prevented the development of the hyperpolarization in an approximate concentration-dependent manner. However, none of these drugs was able to block the effects of monensin on action potentials. Indeed, high concentrations of the antagonists that were most effective in preventing the hyperpolarization accentuated impairments in action potential generation and also reduced input resistance in many cells. Thus, none of these antagonists appears suitable for transition to in vivo antidotal protection studies.

Comparative effects of carboxylic ionophores on membrane potential and resistance of NG108-15 cells.

Comparative analyses were conducted to determine the effects of Na(+) (monensin, MON), K(+) (nigericin, NIG) and Ca(2+) (A23187) selective carboxylic ionophores on differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Alterations in membrane potential (V(m)), input resistance (Rin) and electrically induced action potential generation were measured using intracellular microelectrode techniques in cells treated with 0.1-30 microM MON and NIG and 0.1-10 microM A23187. Responses to the ionophores were similar in that membrane hyperpolarization and unchanged R(in) predominated with all three compounds. However, significant differences between the ionophores were also detected. MON- and A23187-induced hyperpolarization was generally maintained throughout the 24-min superfusion whereas that produced by NIG diminished with time or was replaced by depolarization. In addition, action potential generation was blocked by NIG, whereas MON had no effect and action potential alterations were evident only with the highest A23187 concentration (10 microM). This study represents the initial comprehensive analysis of the effects of carboxylic ionophores on membrane electrical characteristics of an intact cell system and forms the basis for subsequent work using NG108-15 cells as a model system to evaluate potential therapeutic treatments against the carboxylic ionophores.

Effects of protonophores on membrane electrical characteristics in NG108-15 cells.

Studies were conducted to determine the effects of bath application of the protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Membrane resting potential (Vm), input resistance (R(in)) and electrically induced action potential generation were measured using intracellular micro-electrode techniques. Both compounds produced concentration-dependent depolarization rather than the hyperpolarization commonly found with other central mammalian neurons. CCCP and FCCP also reduced R(in) and disrupted the generation of action potentials in a concentration-dependent manner. The contribution of the observed alterations to the in vivo toxicity of these compounds remains to be established.

Effects of neutral ionophores on membrane electrical characteristics of NG108-15 cells.

The effects of several K(+)-selective neutral ionophores on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells were examined. Specifically, alterations in membrane resting potential (V(m)), input resistance (R(in)) and electrically-induced action potential generation were determined upon bath application of enniatin (0.1-10 microg/ml), nonactin (0. 1-10 microM) and valinomycin (0.1-10 microM). Although some cells exhibited a slight hyperpolarization and/or reduced R(in), i.e. membrane electrical correlates of enhanced K(+) loss, neither V(m) nor R(in) were significantly altered by any of the ionophores. However, valinomycin and especially nonactin affected action potentials induced by electrical stimulation. This was apparent in the ablation of action potentials in some cells and in the occurrence of degenerative changes in action potential shape in others. The simultaneous administration of the neutral ionophores and the protonophore CCCP or the superfusion of enniatin, nonactin or valinomycin in high (50 mM) glucose-containing physiological solution did not yield more extensive alterations in V(m) or R(in). These data suggest that the neutral ionophores are unable to materially enhance K(+) flux above the relatively high resting level in NG108-15 cells. Thus, alterations in action potentials appear to be unrelated to K(+) transport activity.

Gramicidin toxicity in NG108-15 cells: protective effects of acetamidine and guanidine.

Studies were conducted using a novel in vitro approach to investigate the efficacy of acetamidine hydrochloride (ACE) and guanidine hydrochloride (GUAN), previously shown to block gramicidin D (GRAM) channels in artificial membranes, in preventing the toxic effects of GRAM in NG108-15 (neuroblastoma x glioma hybrid) cells. Specifically, intracellular microelectrode techniques were employed to examine changes in membrane resting potential (Vm) and input resistance (Rin). At 1 micromol/L, ACE significantly reduced loss of Vm induced by 1 or 10 microg/ml GRAM, although higher concentrations of ACE did not afford enhanced antagonism. GUAN, in contrast, produced a concentration-dependent antagonism of GRAM-induced Vm and Rin loss, with high concentrations (10 or 100 micromol/L) completely preventing diminutions in both Vm and Rin. In control cells superfused without GRAM, ACE produced a direct, concentration-dependent reduction in Vm and Rin, whereas GUAN hyperpolarized NG108-15 cells but did not alter Rin. These data represent the initial demonstration of the reversal of GRAM toxicity in an intact cell system.

The distribution of [125I]ricin in mice following aerosol inhalation exposure.

Studies were conducted to examine the uptake and redistribution of [125I]ricin from the lungs of mice following nose-only aerosol inhalation exposure. Radiolabelled contents were measured in lung and various extra-pulmonary tissues 15 min through 30 h following 10 min aerosol exposures. Pharmacokinetic analyses were performed on whole-organ data obtained for lungs, stomach, liver and spleen. Radioactivity within the lungs, maximal at 15 min post-exposure, was eliminated in a biexponential fashion with a long beta half-life (approximately 40 h). Large amounts of radiolabel were also found within the gastrointestinal tract. Radiolabel within the stomach exhibited an absorption phase and two-compartment elimination. Radiolabel content of many other tissues, including known accumulation sites for intravenously administered toxin, was significantly (p < 0.05) increased (relative to 15 min post-exposure) in association with the early elimination of radiolabel from the lungs, but levels in these tissues were very low and did not increase after 4 h post-exposure. The only exception was our sample of trachea, which showed delayed elevations in radiolabel (peak at 24 h); this pattern was attributable to the contained thyroid (not removed at necropsy) and its trapping of free [125I] released upon tissue [125I]ricin degradation. The overall data indicate that ricin administered by aerosol inhalation is delivered to both respiratory and gastrointestinal tracts; however, it is not extensively transported from either tract to other potential target sites. Ricin delivered to the lungs is primarily sequestered within the lungs until degradation. Only small amounts of ricin delivered to the gastrointestinal tract are absorbed into the circulation.

Mesenteric mast cell degranulation in acute T-2 toxin poisoning.

T-2 toxin-induced alterations in rat mesenteric mast cell granulation were measured by cytophotometric analyses of the metachromatic reaction of mast cell granules with azure B. Hypogranulation (diminution of metachromatic material) was observed 8 h following injections of T-2 toxin (0.5-1.5 LD50, i.p.). These data suggest that mast cell activation occurs during acute T-2 intoxication and raise the possibility that mast cell mediators may contribute to toxin-induced cardiovascular collapse.

Neuronal RNA in Pick's and Alzheimer's diseases. Comparison of disease-susceptible and disease-resistant cortical areas.

Comparative neuronal RNA analyses were conducted in disease-prone (frontal association, Brodmann's area 9) vs relatively disease-resistant (primary visual, occipital area 17) cortex of patients with autopsy-proved Pick's disease (PD) and Alzheimer's disease (AD). Azure B-RNA staining and scanning-integrating microdensitometry were used to determine total RNA contents of pyramidal neurons in layers 3 and 5. In both PD and AD (1) significant (15% to 47%) RNA loss was detected in neurons of both cortical areas and layers relative to those of aged, nondemented controls, and (2) RNA loss was not markedly enhanced in the damaged frontal cortex relative to that in the preserved occipital cortex. Neuronal RNA depletion was generally more marked in PD than AD. However, this impairment does not appear to be related to the formation of classic neuropathological abnormalities in either disease.

Neuronal RNA in relation to Alz-50 immunoreactivity in Alzheimer's disease.

Both defective nucleic acid metabolism/protein synthesis and the expression of an abnormal antigen recognized by the Alz-50 antibody may be involved in the neuronal degeneration of Alzheimer's disease (AD). A multiparametric analysis involving Alz-50 immunocytochemistry and azure B--RNA microdensitometry was developed to compare nucleic acid alterations in Alz-50-positive neurons to those in Alz-50-negative neurons of the brain of AD patients. Alz-50-immunoreactive neurons of the hippocampal endplate (Rose's H3-H5 fields) and the subiculum exhibited significantly lower (by approximately 30%) total RNA contents than negative neurons of the corresponding region. The mean RNA content of Alz-50-positive neurons of the AD brain was also reduced in comparison to that of Alz-50-negative neurons of age-matched, nondemented controls, whereas there were no significant differences between negative neurons of AD patients and controls. The hippocampus of nondemented controls was also found to contain Alz-50-immunoreactive neurons, although 20- to 30-fold fewer than the hippocampus of AD patients. In the controls, there was also a tendency toward reduced RNA levels in Alz-50-positive versus -negative neurons. These data suggest a relationship between Alz-50 immunoreactivity and defective nucleic acid metabolism in the AD brain.

Astrocyte RNA in relation to neuronal RNA depletion in Alzheimer's disease.

A new double-staining procedure, in which the techniques of immunocytochemistry of glial fibrillary acidic protein (GFAP) and quantitative microdensitometry of azure B-RNA were combined, was used to study nucleic acid alterations in fibrous astrocytes in Alzheimer's disease (AD). RNA contents of GFAP-positive cells of the hippocampal endplate (Rose's H3-H5 fields) and the dentate gyrus molecular layer were determined in ten autopsy-proven AD patients (ages 51-88) and ten age-matched, non-demented controls. In addition, RNA contents of pyramidal neurons of the endplate were examined. While there were no differences in RNA contents of astrocytes of either region between AD patients and controls, neuronal RNA was markedly depleted. These data suggest that astrocytes maintain protein synthetic capabilities in AD and that RNA loss is limited to the neuronal compartment.

In situ analysis of neostriatal RNA and chromatin in Alzheimer's disease.

Scanning-integrating microdensitometry of azure B-RNA- and Feulgen-Schiff-stained tissue sections was used to measure neostriatal neuronal RNA levels and susceptibility of neuronal and oligodendrocyte chromatin to acid hydrolysis in Alzheimer's disease (AD) patients and controls. AD was associated with neuronal RNA depletion (17 to 23%) in both caudate nucleus and putamen. While neuronal chromatin was found to be more acid-labile than that of the oligodendrocytes, there were no differences in either cell type between AD and controls. These data support the existence of a macromolecular disturbance (RNA loss) occurring within neostriatal neurons, perhaps related to the extrapyramidal dysfunction of AD, but fail to demonstrate that an alteration in chromatin is responsible for this effect.

Neuronal RNA in relation to neuronal loss and neurofibrillary pathology in the hippocampus in Alzheimer's disease.

Topographic analyses were performed on the distribution of neuronal RNA loss in relation to local neuronal loss and neurofibrillary degeneration in the hippocampal region of brains of patients with Alzheimer's disease (AD). Compared to age-matched controls, pyramidal neuronal RNA was depressed (p less than 0.0001) in all areas of the hippocampus examined in AD, viz., the endplate (33%), Rose's H2 field (30%), Rose's H1 field (37%) and the subiculum (46%). Significant neuronal loss was observed in Rose's H1 field and the subiculum but not in the endplate or Rose's H2 field. The frequency of neurofibrillary tangle-bearing neurons was enhanced in all four regions of AD brains, the number of involved neurons being markedly greater in Rose's H1 field and the subiculum than in the endplate and Rose's H2 field. Overall, the data indicate the existence of a generalized disturbance in RNA metabolism within pyramidal neurons in the hippocampus in AD, occurring in regions of minimal (endplate, Rose's H2 field) as well as those of extensive (Rose's H1 field, subiculum) pathological alterations. Although there is focal accentuation of RNA depletion in the latter, the marked RNA depletion in regions of minimal pathologic change suggests that this effect is largely unrelated to local neuronal loss or neurofibrillary degeneration.

Scanning cytophotometric analysis of brain neuronal nuclear chromatin changes in acute T-2 toxin-treated rats.

Male Sprague-Dawley rats (200 g) were injected intraperitoneally with T-2 toxin, a trichothecene mycotoxin protein synthesis inhibitor, at dosages of 0.75, 1.0, 1.5, and 6.0 mg/kg (1 LD50 = 0.9 mg/kg) before decapitation at 8-hr postexposure. Correlative data were obtained on changes in physicochemical properties of nuclear chromatin, chromatin dispersion, and nuclear volume of cerebrocortical (layer III) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage, and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity, i.e., neuronal nuclear functioning. Conversely, increased F-DNA yield, chromophore area, and nuclear volume signify enhanced neuronal activation. At 8 hr following T-2 toxin exposure, cerebrocortical and striatal neurons exhibited a dose-dependent decrease in F-DNA hydrolyzability, i.e., impaired chromatin activity, and increases in both chromatin dispersion and nuclear volume. Microscopic observation revealed no gross evidence of T-2 induced neurotoxicity. These data indicate that T-2 toxin elicits both neurochemical injury and adaptive or compensatory processes simultaneously. The toxicological importance of observed nuclear alterations and the role of impairments in central nervous system metabolism in acute T-2 toxicity remain to be ascertained.

Brain neuronal chromatin responses in acute soman intoxicated rats.

Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50 = 135 micrograms/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.

Metabolism-permeability coupling in the normal rabbit aorta.

Microfluorimetric and quantitative cytoenzymatic techniques were employed to examine regional variations in endothelial macromolecular uptake as related to inner mural intermediary metabolism in aortas from normocholesterolemic rabbits. Concomitant reductions in luminal fluorescein isothiocyanate-conjugated bovine serum albumin (FITCBSA) accumulation and succinate (SDH), lactate (LDH) and glucose-6-phosphate (G-6-PDH) dehydrogenase activities were evidenced from ascending to upper abdominal segments. Further diminution of FITCBSA accumulation was observed in the lower abdominal aorta, whereas there was a corresponding elevation of enzyme activities. Highly significant but not exceedingly close correlations were obtained between luminal FITCBSA uptake and inner mural SDH and LDH activities. However, much of the associated variability was attributable to the lower abdominal segment, where there was a microscopically-discernible augmentation in adventitial surface FITCBSA accumulation. The overall data provide direct in vivo support for the concept that endothelial macromolecular transport is coupled to mural oxidative demands, but also indicate that luminal metabolism-permeability relationships are influenced by factors such as extensiveness of the vasoral network and wall thickness. Details of the metabolism-permeability coupling hypothesis and observations implicating metabolic events as basic causative factors underlying vascular pathogenesis are discussed.

Cytophotometric analysis of neuronal chromatin and RNA changes in oxotremorine-treated rats.

Neuronal nucleic acid responses were examined within the rat striatum and sensorimotor cortex (layer V) following single intraperitoneal injections of the central cholinergic-muscarinic agonist oxotremorine (0.1, 0.7, or 1.0 mg/kg). After stoichiometric Feulgen and azure B staining of brain sections, scanning-integrating microdensitometry was used to quantify Feulgen-deoxyribonucleic acid levels, changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA yield) and azure B-ribonucleic acid (RNA) content of neurons on an individual basis. Changes in neuronal nuclear and nucleolar volumes were also determined histometrically. Within the striatum and sensorimotor cortex, oxotremorine produced marked dose-dependent elevations in both F-DNA yield and RNA content. These metabolic increases were typically paralleled by elevations in nuclear and nucleolar volumes. The data demonstrate that the oxotremorine-induced central muscarinic activation is associated with dose-related enhancements in neuronal chromatin template activity, RNA content, and protein synthetic capacity.

Soman intoxication in hypothyroid rats: alterations in brain neuronal RNA, acetylcholinesterase and survival.

Studies were conducted to investigate relationships among soman (pinacolyl methylphosphonofluoridate) induced seizure activity, central metabolic impairments and lethality in normal vs thyroid-deficient rats. Quantitative cytophotometric measurements of individual cerebrocortical (layer V) and striatal neuron RNA contents were made following dosages of 0.5, 0.9 and 1.5 LD(50) soman (LD(50) = 135 ?g/kg, sc). Hypothyroidism was associated with a marked diminution of overt convulsive activity and reduced susceptibility to lethal actions of soman as indicated by enhanced 24- and 48-h survival rates at 0.9, 1.2 and 1.5 LD(50). Hypothyroidism per se produced RNA depletion in both cortical and striatal neurons. Soman treatment diminished cortical RNA to essentially the same extent in thyroid-deficient rats as in euthyroids, whereas there was no further reduction of striatal neuron RNA. It was found that amelioration of convulsive activity and lethal- ity in hypothyroid rats was accompanied by reduced cerebral acetylcholinesterase (AChE, EC 3.1.1.7) inactivation, and that plasma cholinesterase (EC 3.1.1.8) and aliesterase (EC 3.1.1.1) levels were significantly higher in hypothyroid than in euthyroid saline-control rats. The overall data indicate that soman- induced central metabolic impairments can occur independent of paroxysmal neural activity and lethal actions of the agent. Resistance to soman observed with thyroid deficiency may be due in large part to increased binding to plasma enzymes and diminished delivery of soman to AChE in vital cholinergic sites.

Perikaryal RNA changes within neurons of the caudate-putamen and substantia nigra in soman intoxicated rats.

Quantitative azure B cytophotometry was employed to monitor neuronal ribonucleic acid (RNA) metabolism within cholinergic and noncholinergic brain compartments following single sc injections of either 0.5, 0.9 or 1.5 LD(50) soman (pinacolyl methylphosphonofluoridate). Dose-dependent losses in neuronal RNA were observed within the cholinergic caudate-putamen (CP) and dopaminergic substantia nigra pars compacta (SNPC), whereas levels of RNA were generally maintained or elevated within the gabaergic substantia nigra pars reticulata (SNPR). CP acetylcholinesterase was inhibited in a dose-dependent fashion. These neuronal RNA changes are perhaps related to seizure activity. The overall data lend support to the hypothesis that alterations in noncholinergic activity contribute to certain manifestations of soman neurotoxicity, such as seizures, which probably stem from an impairment in the functional integrity of both excitatory and inhibitory neuronal elements.