Immunologic localization and kinetic characterization of a Na+/Ca2+ exchanger in neuronal and non-neuronal cells.

The plasma membrane Na+/Ca2+ exchanger is believed to play a role in the regulation of Ca2+ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na+/Ca2+ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The Vmax was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted approximately 90% of the exchanger activity in the primary neurons and approximately 70 and 50% of the activity in NCB-20 and CV-1 cells respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate receptor changes in brain synaptic membranes during chronic alcohol intake.

Ethanol inhibits the receptor-ion channels activated by the glutamate analog N-methyl-D-aspartate (NMDA) in mammalian brain neurons. The high degree of sensitivity of these receptors to the acute effects of ethanol has led to the development of hypotheses that brain neurons may adapt to chronic exposure to ethanol either by increasing the numbers of NMDA receptors, or by expressing receptors with decreased sensitivity to ethanol, or by expressing receptors with increased affinity for the endogenous activator of these entities, L-glutamic acid. Studies performed with experimental animal models of chronic ethanol intake are indicative of an adaptive response that entails the expression in neuronal membranes of greater numbers of L-glutamate and NMDA receptors. When similar studies were performed with synaptic membranes isolated from the brains of human alcoholics, there was an apparent increase in the total number of glutamate receptors, but a decrease in the density of the subpopulation of these receptors that are activated by NMDA. These results are interpreted to suggest that there are more subtle and complex adaptations made in neurons in the brains of human alcoholics as compared with those occurring in animal models of chronic alcoholism.

Glutamate receptor changes in brain synaptic membranes from human alcoholics.

Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membrane-enriched fraction and the study of L-[3H]glutamic acid and 3-((+-)-2-carboxypiperazin-4-yl)-[1,2(3H)propyl-1-phosphonic acid ([3H]CPP) binding sites. The pharmacological characteristics of L-[3H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-D-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population of L-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates or L-[3H]glutamate binding to synaptic membranes. In order to determine whether some of the changes in L-[3H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [3H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [3H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics.(ABSTRACT TRUNCATED AT 250 WORDS)

Transferrin, iron, and dermatophytes. I. Serum dematophyte inhibitory component definitively identified as unsaturated transferrin.

The factor present in normal human serum which inhibits growth of dematophytic fungi is characterized and identified. Serum inhibitory factor (SIF) is nondialyzable, heat stable at 56 degrees C. for 4 hours, and fungistatic. SIF was found to be an inhibitor of the dermatophyte genera Trichophyton, Microsporium, and Epidermophyton as well as the dimorphic yeast Candida albicans. SIF activity directly correlated with a serum's unbound iron-binding capacity (UIBC) in that lower UIBC'S were less inhibitory. Addition of iron to serum neutralized the inhibitory activity and this neutrilization was shown to be specific for iron since zinc, magnesium, managnese, and copper failed to alter serum inhibitory activity. Furthermore, addition of purified iron-free transferrin to a neutralized serum restored the SIF activity in parallel with the UIBC. Removal of transferrin from serum by affinity chromatography was confirmed by polyacrylamide gel electrophoresis and iron-binding assays. Transferrin-free serum produced by this procedure was shown to lack inhibitory activity. These data indicate that SIF is unsaturated transferrin and strongly suggests that it inhibits dermatophytes by binding iron which many organisms need for growth.