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An antisense (AS) oligodeoxynucleotide based on a conserved sequence in the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) was used to decrease expression of this Ca(2+) transporter in primary neuronal cultures. Two AS oligo applications decreased NCX activity by approximately 40% within 12-24 h, and neither sense (S) or missense (MS) oligos altered NCX activity. The reduced NCX expression was confirmed by immunoblots and enzyme-linked immunosorbent assays (ELISAs). Resting [Ca(2+)](i) levels were 20% higher in AS-treated neurons and showed a slower return to baseline levels following activation of Ca(2+) influx by N-methyl-D-aspartate (NMDA). These results suggest that NCX plays a significant role in maintaining neuronal Ca(2+) homeostasis and in restoring baseline Ca(2+) levels following depolarization.

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.

Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca2+. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36-72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca2+, Mg2+-ATPases. The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.

We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.

The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N-methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5-50 mM). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg2+, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg2+ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg2+ site has been shown to increase the binding of N-[1-(2-thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(+/-)-carboxypiperazine-4-yl)-[1,2]-propyl-1-phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg2+ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg2+ can be clarified and any relationship between Mg2+ regulation and ethanol inhibition established.

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.

The possibility that regulation of Ca2+ levels in brain nerve terminals is altered as the brain ages was examined in synaptosomes from adult and aged Fischer 344 rats. Free intrasynaptosomal [Ca2+]i was monitored with fura-2 as synaptosomes were depolarized with KCl, veratridine and ibotenic acid. With all three depolarizing agents, synaptosomes from aged animals reached higher free Ca2+ levels, and the maximal Ca2+ increases (delta Ca2+) estimated from computer assisted-fitting of the curves, ranged from 35% to 80% greater in synaptosomes from aged animals. The total Ca2+ content of the brain and of synaptosomes was also found to be considerably higher in aged than in adult animals. These results suggest that the aging process in brain is accompanied by alterations in both dynamic aspects of Ca2+ handling in nerve endings and the overall content of Ca2+ in the brain and synaptic terminals.

Procedures were developed for measurement of Na+/Ca2+ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca2+ in the presence of a Na+ gradient. The kinetic characteristics of the Na+/Ca2+ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The Kact for Ca2+ was estimated to be 32 microM for hippocampal and 17 microM for cortical tissue. The maximal rate of Ca2+ uptake (Vmax) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na+ gradient, and was optimal in the high pH range. Therefore, membranes in which Na(+)-dependent Ca2+ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system.

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.

We have recently reported that ethanol and other n-alkanols added to in vitro assays inhibit the activity of the Na+-Ca2+ exchange system in brain synaptic plasma membrane vesicles. The present studies were undertaken to determine whether in vivo chronic ethanol administration leads to alterations in this Na+-Ca2+ antiporter that might be indicative of an adaptive response to alcohol. The Na+-dependent Ca2+ transport activity in the plasma-membrane fractions obtained was measured at various Ca2+ concentrations. Results of these experiments revealed that a 3-week ethanol regimen brought about a significant increase in the Na+-dependent Ca2+ transport activity only in the membrane fraction enriched in synaptic junctional complexes. These membranes showed a near doubling in the maximal transport activity of the antiporter in alcohol-treated compared with the control animals. Changes in the kinetic parameters were reversible as the Na+-Ca2+ exchange activity in these membranes from animals maintained on alcohol for 3 weeks and then withdrawn for 1 week was indistinguishable from that of membranes from control animals. Thus it appears that ethanol-treated animals make a reversible adaptation in their neuronal cell membranes to compensate for the acute effects of ethanol on the Na+-Ca2+ antiporter.

The studies presented above add further support to the notion that one of ethanol's most important sites of activity may be the plasma membrane of cells. In addition, they provide an example of how ethanol's effects on these membranes may affect the function of protein molecules associated with nerve cell membranes such as the receptive sites for the putative excitatory neurotransmitter L-glutamic acid. The result of chronic ethanol administration is an apparently enhanced sensitivity to glutamate action in the CNS as measured by either the increases in L-glutamate binding activity or the increased Ca2+ mobilization as a result of glutamate interaction with the synaptosomal membranes. Both of these processes exhibit an enhancement of the maximum response with little or no change in the KD for binding of L-glutamic acid to its receptor site. Finally, it was demonstrated by means of in vivo experiments that there is an apparent sensitization to glutamate during the induction of ethanol dependence, and that this enhanced sensitivity may be causally linked to some of the signs of the post withdrawal CNS hyperexcitability.

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.

Isolated synaptic plasma membrane vesicles developed an internal negative membrane potential (delta psi) following loading with potassium succinate and incubation in NaCl, sodium succinate, or Tris succinate media. Membrane delta psi was monitored by measuring triphenyl[3H]methylphosphonium ion ([3H]TPMP+) accumulation by these vesicles. Estimates of delta psi ranged from --6.9 mV for vesicles incubated in sodium succinate to --28 mV for membranes incubated in NaCl. Intravesicular TPMP+ accumulation was strongly dependent on the K+ diffusion potential and was enhanced by the K+ ionophore valinomycin and by the adenosine analog 2-chloroadenosine (2-Cl-Ado). The stimulation of TPMP+ influx by 2-Cl-Ado was dependent on the concentration of this agent, independent of Cl- fluxes, and sensitive to inhibition by the methylxanthine theophylline. The increase of delta psi of the synaptic membrane vesicles caused by 2-Cl-Ado paralleled the hyperpolarization of neurons produced by adenosine and 2-Cl-Ado in physiological systems.

The studies described above have provided evidence for the presence of a high affinity, glutamate binding glycoprotein which has some of the pharmacologic characteristics of the recognition site of neuronal glutamate receptors. In addition, evidence was presented for the distinct molecular nature of this glutamate binding protein as compared to that of the kainic acid binding macromolecule in synaptic membranes and also, for the differential characteristics of the glutamate binding sites as compared to the glutamate transport carriers. Finally, information was presented which is suggestive of the presence and functional integrity of glutamate receptor.ion channel complexes in synaptic membrane preparations.

Synaptic plasma membranes isolated from rat brain exhibited a Ca2+ transport process that was strictly dependent on the presence of Mg2+ and activated by ATP hydrolysis. The characteristics of this ATP-activated transport process included a high affinity for Ca2+ and ATP with the Kact for these two substrates being 0.7 and 5 microM, respectively, and a lower affinity for Mg2+, Kact = 54 microM. The estimated constants for ATP-activated Ca2+ transport into synaptic membrane vesicles and the dependence of such transport on Mg2+ were indicative that such transport was related to the previously described high affinity (Ca2+ + Mg2+)-ATPase in synaptic membranes. An ATP- and Mg2+-dependent Ca2+ transport process with very similar kinetic characteristics was present also in a general microsomal membrane fraction obtained from brain tissue. The synaptic and microsomal membrane ATP-activated transport processes exhibited differences in their sensitivity to vanadate inhibition. Interaction with vanadate was fairly complex and best analyzed by a two-component model. Thus, the estimated Ki values for vanadate were 0.2 and 6.6 microM for the synaptic membranes and 0.7 and 13.8 microM for the microsomes. Since the microsomal membranes contain a substantial population of intraneuronal endoplasmic reticulum vesicles, the effects of vanadate on Ca2+ transport into intraneuronal membrane organelles, other than mitochondria, was determined in saponin-permeabilized synaptosomes. The estimated Ki values for vanadate inhibition of Ca2+ transport activity were 0.7 and 13 microM. The accumulation of Ca2+ into synaptic plasma membrane vesicles was readily reversed by activation of the Na+-Ca2+ exchange carrier, whereas the Ca2+ associated with intrasynaptosomal organelles was not affected by changes in [Na+]. Thus, there are at least two ATP-dependent Ca2+ transporting processes localized on two distinct neuronal membranes, one on the plasma membrane and the second on intraneuronal membranes.

The effects of ethanol on Na+-dependent CA2+ fluxes have been examined in resealed synaptic membrane vesicles assayed at 3 different temperatures. Sodium chloride-loaded vesicles were preincubated with various concentrations of ethanol for 120 sec prior to being diluted into a 45CaCl2-containing medium in the presence or absence of an outward-directed Na+ gradient. The effect of ethanol on Na+-dependent Ca2+ transport measured at 23 degrees C was biphasic. However, when the assay was conducted either at 16 degrees C or at 35 degrees C, all ethanol concentrations tested (10-300 mM) produced only inhibition of Ca2+ influx. The role of membrane fluidization in the ethanol-induced inhibition was explored by determining the effects of incorporating various fatty acids into the membranes. Membrane fluidizing agents such as cis-vaccenic acid stimulated Ca2+ influx whereas trans-vaccenic and saturated fatty acids had little effect. The fluidizing effect of incorporating cis-vaccenic acid into the membranes was confirmed with electron paramagnetic resonance (EPR) spectroscopy. The data obtained from these studies suggest that the inhibition of Ca2+ fluxes produced by alcohol and local anesthetics is not the result of a general increase in bulk phase synaptic membrane fluidity.