Response of F344 rats to inhalation of subclinical levels of sarin: exploring potential causes of Gulf War illness.

Subclinical, repeated exposures of F344 rats to sarin resulted in brain alterations in densities of chlonergic receptor subtypes that may be associated with memory loss and cognitive dysfunction. The exposures also depressed the immune system. The rat appears to be a good model for studying the effects of subclinical exposure to a nerve gas.

Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils.

An initial overload of intracellular Ca2+ plays a critical role in the delayed death of hippocampal CA1 neurons that die a few days after transient ischemia. Without direct evidence, the prevailing hypothesis has been that Ca2+ overload may recur until cell death. Here, we report the first measurements of intracellular Ca2+ in living CA1 neurons within brain slices prepared 1, 2, and 3 days after transient (5 min) ischemia. With no sign of ongoing Ca2+ overload, voltage-dependent Ca2+ transients were actually reduced after 2-3 days of reperfusion. Resting Ca2+ levels and recovery rate after loading were similar to neurons receiving no ischemic insult. The tetrodotoxin-insensitive Ca spike, normally generated by these neurons, was absent at 2 days postischemia, as was a large fraction of Ca2+-dependent spike train adaptation. These surprising findings may lead to a new perspective on delayed neuronal death and intervention.

Lead stimulates lymphocyte proliferation through enhanced T cell-B cell interaction.

We have studied the in vitro effects of lead (Pb) as Pb-acetate (0. 1-1000 ppm) on the activation of rat spleen (SP) cells. At a concentration of 0.5 to 200 ppm, Pb augmented the uptake of [3H]thymidine, progression of SP cells through the cell cycle, and allogeneic and syngeneic mixed lymphocyte reactions. However, at concentrations above 200 ppm, Pb inhibited the proliferation of these cells. To understand the cellular and molecular basis of these responses, we examined the effects of Pb on the proliferation of isolated T and/or B cell populations. Pb failed to stimulate the proliferation of isolated T and B cells; however, the addition of gamma-irradiated B cells to T cell cultures or irradiated T cells to B cell cultures resulted in Pb-induced incorporation of [3H]thymidine. On the other hand, macrophages were unable to reconstitute this response. Pb also induced a significant rise in the intracellular concentration of inositol 1,4,5-trisphosphate in SP cells; however, unlike the activation of lymphocytes through the antigen receptors, Pb did not significantly stimulate protein tyrosine kinase activity. These observations suggest that Pb facilitates the T cell-B cell interaction-dependent proliferation of lymphocytes through a signaling pathway(s) independent of the antigen receptor.

Effects of nicotine on the immune response. II. Chronic nicotine treatment induces T cell anergy.

Previously, we have shown that both T and B lymphocytes from chronically nicotine-treated (NT) animals exhibit tolerance to activation by Ags (ligation of Ag receptors), as indicated by their decreased ability to mobilize intracellular calcium and, at least in T cells, arrest of cells in the G0/G1 phase of the cell cycle. Herein, we demonstrate that NT T cells significantly lose their ability to up-regulate inositol trisphosphate synthesis in response to TCR ligation or nonspecific activation of G proteins by AIF-4. However, increases in cAMP concentrations of T cells following activation of G protein-sensitive adenylate cyclase by cholera or pertussis toxin were not significantly affected by the nicotine treatment. Interestingly, compared with control T cells, the background levels of inositol trisphosphate were significantly elevated in NT T cells, indicating some degree of activation in these cells. This inference was further supported by observations that naive T cells from NT animals exhibit tyrosine phosphorylation of several substrates, including phospholipase C-gamma1, which were either absent or underphosphorylated in unstimulated control T cells. Moreover, when, after 4-wk nicotine treatment, nicotine pumps were removed and serum cotinine levels fell to background, inhibition of the Ab-forming cells and Ca2+ responses continued for at least 2 more wk. These results suggest that chronic in vivo nicotine exposure leads to T cell anergy and may contribute to nicotine/cigarette smoke-induced immunosuppression.

Intracellular calcium signaling induced by thapsigargin in excitable and inexcitable cells.

Signaling between intracellular Ca2+ stores and cell membrane channels or transporters is important to Ca(2+)-based second messenger systems. Two hypotheses, the capacitative and the Ca(2+)-induced Ca(2+)-influx models have been proposed to explain aspects of this signaling. In this study, we examined the applicability of these models in neuroendocrine (PC12), neuronal (dorsal root ganglion), immune (spleen), and fibroblast (3T3) cells. We used thapsigargin (TPG) to deplete specific intracellular Ca2+ stores and to increase the cytoplasmic Ca2+ concentration ([Ca2+]), and Ca2+ free medium to prevent Ca2+ influx and lower cytoplasmic [Ca2+]. We demonstrate that, although TPG causes an increase of [Ca2+]i in all cells examined, the subsequent stimulation of Ca2+ influx varies from high in spleen, to moderate in 3T3 and PC12, to undetectable in DRG cells. All cell types exhibited Ca2+ influx when Ca2+ was added to the medium following an exposure to Ca(2+)-free medium. Without added provisions, the two aforementioned hypotheses are inadequate in explaining the TPG-induced Ca(2+)-influx in all cell types. These results support the hypothesis of the existence of unique Ca2+ channels or transporters in spleen cells that operate subsequent to TPG treatment and are distinct from the voltage-gated Ca2+ channels and Ca(2+)-activated non-selective cation channels present in excitable cells.

Alcohol-induced changes in the immune response: immunological effects of chronic ethanol intake are genetically regulated.

Experimental evidence on the immunomodulating effects of ethanol is contradictory and, in animals, the immunological effects of long-term alcohol intake may depend on the age of animal, amount of alcohol consumed, and nutritional composition of the administered diet. In this study, immunological effects of pair-feeding a 35% ethanol-containing Bio-Serv liquid diet for 6 weeks were evaluated using two major histocompatibility complex (MHC)-compatible inbred strains of rats (F344 and LEW). Food intake, rate of gain in body weight, and percentages of B cells, T cells, and T cell subtypes were not affected by ethanol intake. Also, proliferative responses of lymphocytes to T and B cell mitogens were similar in control and ethanol-fed groups of the two strains. Ethanol consumption had no significant influence on spleen weights and the antibody plaque-forming cell (PFC) response in F344 rats; however, in LEW rats, ethanol ingestion leads to a significant decrease (about 16%; p < 0.012) in spleen weight and a > 75% reduction in the PFC response. These results suggest that a non-MHC-encoded gene(s) regulates the ethanol-mediated immunosuppression of the PFC response. Thus, LEW-F344 combination may provide an excellent model to characterize genetic factors which determine sensitivity/resistance to immunological effects of ethanol ingestion.

Activation and modulation of calcium-activated non-selective cation channels from embryonic chick sensory neurons.

We have shown that calcium-activated non-selective (CAN) channels from embryonic chick sensory neurons are permeable to both Na+ and K+ and are not blocked by TTX, TEA, or 4-AP. These neuronal CAN channels are activated by sub-micromolar cytoplasmic Ca2+ with negative cooperativity. The effect of Ca2+ is to decrease the closed times of the channel with little effect on the time the channel remains open. Isolated neuronal CAN channels can be phosphorylated by cAMP-dependent protein kinase (PKA). The effect of phosphorylation is to shorten channel open time and to minimize the effect of Ca2+ on channel closed time.

Increased natural killer cell activity in a model of immunoglobulin A nephropathy secondary to chronic alcohol consumption.

We investigated natural killer (NK) cell activity in an animal model of ethanol-induced immunoglobulin A (IgA) nephropathy. Two groups, of 10 rats each, received a continuous intragastric infusion of liquid diet through a permanent cannula for 6 weeks. The alcoholic group was infused additionally with intragastric ethanol, representing from 32% to 40% of the caloric requirement. The group of control rats received an isocaloric diet supplemented with glucose instead of alcohol. IgA nephropathy was observed in all the alcoholic rats but in none of the controls. NK cell activity was investigated in the two groups by measuring the cytotoxicity of spleen cells using the chromium release method. NK cell activity was found to be significantly increased in the alcoholic rats. In view of the known modulation of IgA synthesis by NK cells, we suggest that increased NK cell activity may be a contributing factor to the high levels of circulating IgA seen in IgA nephropathy secondary to chronic alcohol consumption.

Decrease in locus coeruleus [3H]idazoxan binding site density in genetically epilepsy-prone (GEPR) rats.

Deficits in norepinephrine synthesis, transmitter level, turnover and reuptake have been reported in the brain of genetically epilepsy-prone (GEPR) rats. We investigated the hypothesis that these alterations may trigger a compensatory downregulation of locus coeruleus alpha 2-adrenergic receptors and an upregulation of postsynaptic alpha 2-adrenergic receptor density in forebrain regions of GEPR rat brain. alpha 2-adrenergic receptor density was measured in the locus coeruleus and 7 forebrain regions of control and GEPR rats by in vitro [3H]idazoxan autoradiography. Specific [3H]idazoxan binding site density was decreased significantly in the locus coeruleus of both GEPR-3 and GEPR-9 rats compared to controls. No significant differences in specific [3H]idazoxan binding were observed in the 7 forebrain regions of GEPR-9 rats compared to control. Reduced locus coeruleus alpha 2-adrenergic receptor density in GEPR rats may produce a net increase in locus coeruleus noradrenergic cell firing, an effect which could, in part, offset the impact of reduced noradrenergic influence in GEPR rat forebrain. Additionally, decreased norepinephrine levels in GEPR rat brain may be a long-term consequence of reduced alpha 2-adrenergic receptor-mediated inhibition of locus coeruleus firing activity.

Evidence of altered T-lymphocyte number and proliferative responses in genetically epilepsy-prone rats.

Genetically epilepsy-prone (GEPR-9) rats exhibit decreased antibody plaque-forming cell responses following immunization. We examined the hypothesis that this immunosuppression was due to deficits in the number or proliferative responses of T-lymphocytes. Splenocyte responses to concanavalin A and pokeweed mitogen were significantly greater in GEPR-9 rats than controls. Flow cytometric analysis indicated that GEPR-9 rats possess an increase in T-cells associated with the T-helper phenotype. The increased proportion of T-helper cells in GEPR-9 rats may underlie their enhanced proliferative responses to T-cell mitogens. These results clearly indicate that the failure of the GEPR-9 rat to respond to a T-dependent antigen in vivo is not due to a lack of T-helper activity.

Perinatal hypothyroidism decreases hippocampal mossy fiber zinc density in rats.

The effect of perinatal hypothyroidism on hippocampal mossy fiber zinc density was examined in rats. Timed pregnant Sprague-Dawley rat dams were given water containing either 0.02% propylthiouracil (PTU) or vehicle from gestational day 18 until their litters were weaned on postnatal day 31. Hippocampal mossy fiber zinc density was reduced by 75% in both the dorsal and ventral hippocampal formation CA3 stratum lucidum region of 31-day-old PTU-treated rats compared to untreated controls. Perinatal hypothyroidism did not alter hippocampal tissue zinc concentration, indicating that the PTU-induced reduction in mossy fiber zinc was not a consequence of reduced hippocampal zinc concentration. At 120 days of age, 3 months after discontinuation of PTU treatment, hippocampal mossy fiber zinc density remained significantly reduced by 33-45% in PTU-treated rats compared to control. These data indicate that perinatal hypothyroidism causes a long-lasting reduction in hippocampal mossy fiber zinc density.

Decrease in hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats.

Specific [3H]vinylidene kainic acid binding to the kainate-sensitive subtype of glutamate receptor was studied in brain of 31-day-old non-epileptic Sprague-Dawley control and two colonies of genetically epilepsy-prone rats using in vitro autoradiographic techniques. At 37.5 nM [3H]vinylidene kainic acid, specific [3H]vinylidene kainic acid binding was reduced significantly by 18 and 22% in dorsal and ventral hippocampal formation stratum lucidum of 31-day-old genetically epilepsy-prone-9 rats compared with non-epileptic controls. Hippocampal [3H]vinylidene kainic acid binding was reduced in genetically epilepsy-prone-3 rats by 15 and 18%, but these reductions were not statistically significant. Saturation of [3H]vinylidene kainic acid binding studies indicated that the total number of ventral hippocampal [3H]vinylidene kainic acid binding sites was decreased by 21% in genetically epilepsy-prone-3 rats and 28% in genetically epilepsy-prone-9 rats. The reduction in ventral hippocampal [3H]vinylidene kainic acid binding in genetically epilepsy-prone rats resembles the reduction in ventral hippocampal [3H]vinylidene kainic acid binding sites observed in perinatal hypothyroid rats. As genetically epilepsy-prone rats are hypothyroid during the neonatal period, the reduction in hippocampal [3H]vinylidene kainic acid binding in the genetically epilepsy-prone rats may be a consequence of a hypothyroid-induced defect in the development or maturation of the hippocampal mossy fiber projection in genetically epilepsy-prone rats. An alternative hypothesis is that the putative occurrence of spontaneous limbic seizures in genetically epilepsy-prone rats may lead secondarily to a reduction in hippocampal [3H]vinylidene kainic acid binding sites.