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R Spanier

Publications and source records attributed to R Spanier.

17 recordsLinked to original sources
PubMed1991

The modulation of rat brain Na(+)-Ca2+ exchange by K+.

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.

Animals↗
PubMed1991

Molecular and mechanistic heterogeneity of the Na(+)-Ca2+ exchanger.

1. Studying the effect of K+ on Na(+)-Ca2+ exchange in rat brain SPMs revealed that a consistent stimulation was obtained. This stimulation persisted also when FCCP was included in the K(+)-containing reaction mixture to minimize the effect of membrane potential on the electrogenic process. 2. Using Rb+ as a K+ analogue revealed that it was cotransported with Ca2+ in a Na+ gradient-dependent manner. The ratio between the amount of Ca2+/Rb+ transported in rat brain SPMs in a Na+ gradient-dependent manner suggests that not all the Na(+)-Ca2+ exchangers in that preparation cotransport Rb+ (K+) with Ca2+. This is supported also by the finding that Na+ gradient-dependent Ca2+ influx can proceed in rat brain SPMs in the complete absence of K+ although to a lesser extent. 3. Protein purification studies and immunological characterization indicate that a 70-kDa protein is consistently detected in rat brain SPMs. Immunological characterization of the proteins expressed in the 14-18 S mRNA-injected Xenopus oocyte in conjunction with Na+ gradient dependent Ca2+ uptake activity or in the same mRNA-fortified reticulocyte lysate suggest that proteins of about 70 kDa are specifically synthesized. 4. Torpedo electric organ Na(+)-Ca2+ exchanger differs at least in two respects from the rat brain Na(+)-Ca2+ exchanger: It has a low affinity to Na+ (K0.5 = 170 mM), and it reaches maximal activity between 15-20 degrees C. Reconstitution studies suggest that the temperature difference might reflect a difference in the proteins themselves rather then a difference in membrane fluidity due to a difference in the membrane lipid composition.

Animals↗
PubMed1987

Immunological identification of the synaptic plasma membrane Na+-Ca2+ exchanger.

The protein moiety responsible for Na+-Ca2+ exchange activity was identified in synaptic plasma membranes (SPM). This was done by raising polyclonal antibodies in rabbits against each one of the detectable proteins present in the purified preparation containing the enriched specific transport activity. Two of the antibody preparations bound specifically to native SPM: antibodies which were raised against the 70,000-Da protein (the most prominent species consistently present in the purified preparation) and antibodies raised against a 33,000-Da protein (inconsistently present in variable amounts in the purified preparation). Both antibodies bound exclusively to a protein of 70,000 Da in native SPM. When, however, the purified 33,000- and 70,000-Da proteins were used as antigens, each one of the antibody preparations bound to both proteins. In addition, both antibody preparations immunoprecipitated Na+ gradient-dependent Ca2+ transport activity from detergent-solubilized SPM. This was obtained by incubation of solubilized SPM with a complex containing antibodies bound to Protein A-Sepharose beads, reconstitution of the material excluded from the beads, and determination of the residual transport activity. The decrease in Na+ gradient-dependent Ca2+ transport activity paralleled the amount of antibody bound to Protein A-Sepharose beads and could reach 82% as compared to the activity remaining in control experiments using preimmune sera. In comparison, ATP-dependent Ca2+ transport activity was unimpaired. These results indicate that the 70,000-Da protein in SPM contains the catalytic Na+-Ca2+ antiport activity. The presence of the 33,000-Da protein in some preparations and its properties may be explained by its being either a degradation product or a subunit of the 70,000-Da protein.

Adenosine Triphosphate↗
PubMed1984

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles.

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.

Animals↗
PubMed1984

Restoration of doxorubicin responsiveness in doxorubicin-resistant P388 murine leukaemia cells.

The effects of certain compounds on the in vitro growth rate and the sensitivity to doxorubicin of P388 murine leukaemia cell line and of a doxorubicin-resistant subline (P388/ADR) were studied. The calcium channel blocking activity of these compounds was evaluated by measuring their effects on the sodium-dependent and membrane potential-dependent calcium uptake in synaptic plasma membrane vesicles. At non-inhibitory concentrations, verapamil, dipyridamole, meclizine and nicardipine were highly active in restoring the sensitivity to doxorubicin of P388/ADR cells. Moderately active were propranolol, N-(beta-diethylaminoethyl)-N-(beta-hydroxy-beta-phenylethyl)-2,5-dich loranaline (MDL-6792), thioridazine and chlorocyclizine, while nifedipine, guanethidine, phentolamine, chloroquine and papaverine had zero or only minimal synergistic activity to doxorubicin in this cell line. Doxorubicin synergistic activity could not be demonstrated in the parent drug-sensitive cell line. No sodium-dependent or membrane potential-dependent calcium uptake could be demonstrated in vesicles prepared from plasma membranes of either cell line. There is no correlation between the ability of these compounds to inhibit calcium uptake in synaptic vesicles and their potency in restoring the sensitivity of P388/ADR cells to doxorubicin.

Animals↗
PubMed1984

Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes.

A [Na+]-gradient-dependent Ca2+ transporter from brain synaptic plasma membranes has been isolated, purified, and reconstituted into brain phospholipid vesicles. The purification was achieved by sucrose-gradient centrifugation after solubilization of the synaptic membranes in cholate in the presence of a 30-fold excess (by weight) of added brain phospholipids and [Na+]-gradient-dependent Ca2+ loading of the reconstituted vesicles. A 128-fold increase in specific activity of [Na+]-gradient-dependent Ca2+ uptake per mg of protein has been obtained. The purified and reconstituted vesicles took up Ca2+ only in response to an outward-oriented [Na+] gradient. The Ca2+ uptake could be inhibited by dissipation of the [Na+] gradient with nigericin. Successful purification was based on the initial [Na+]-gradient dependency of the Ca2+-transport process, the magnitude of the [Na+]-gradient-dependent uptake, and the presence of purified brain phospholipids. Analysis of the sucrose-gradient-purified reconstituted vesicles on NaDodSO4/polyacrylamide gels showed that the activity coincided with enriched appearance of a 70,000-Da protein.

Animals↗
PubMed1983

The inhibition of Na-dependent Ca uptake by verapamil in synaptic plasma membrane vesicles.

This study on the effect of verapamil on the Na+-dependent Ca2+ uptake system (Na+-Ca2+ exchanger) of synaptic plasma membrane (SPM) vesicles showed that verapamil inhibited the Na+-dependent Ca2+ uptake in a dose dependent manner. A 50% inhibition was obtained with 175 microM verapamil. Calcium uptake in SPM vesicles is dependent on the Na+ gradient across the membrane and on the membrane potential. Both components of the Ca2+ uptake system were inhibited by verapamil. Na+-coupled GABA uptake in SPM vesicles was also inhibited by verapamil in a similar dose-dependent fashion. It seems therefore that verapamil inhibits Na+ gradient driven processes in synaptic plasma membrane vesicles.

Animals↗
PubMed1980

Calcium transport systems in nerve terminals. Studies on membrane vesicles.

A) Synaptosomal vesicles are closed membranous structures isolated by differential gradient centrifugation from lysed nerve terminals. 1. They are capable of transporting calcium in an ATP dependent process. 2. The calcium transport system is not inhibited by H+ ionophores, oligomycin, ruthenium red and atractyloside. 3. Calcium can be taken up into synaptosomal vesicles at very low calcium ion concentrations (below 0.5 microM Ca++) and the kinetics of calcium transport exhibits a sigmoidal dependency on calcium ion concentrations, with a cooperativity of about 2. 4. The divalent cation-activated ATPase associated with synaptosomal vesicle calcium transport exhibits Michaelis-Menten kinetics, with a Km for ATP of 19.13 +/- 5.8 microM ATP. 5. The synaptosomal vesicle ATP-dependent Ca carrier has been purified and reconstituted into phospholipid membranes. Two proteins associated with the ATP-dependent Ca transport were identified on SDS containing Laemmli gels with molecular weights of 94 000 and 140 000. B) Another type of membrane vesicle derived probably from synaptosomal plasma membrane has also been isolated. 1. This membrane vesicle takes up calcium when a sodium gradient (high inside) is set up across its membrane. 2. Magnesium is a competitive inhibitor of the Na dependent Ca uptake. 3. The Na dependent Ca uptake is not inhibited by tetrodotoxin or ouabain.

Adenosine Triphosphate↗