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G Rudnick

Publications and source records attributed to G Rudnick.

At least 73 records · Page 4Linked to original sources
PubMed1985

Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells.

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.

Animals↗
PubMed1984

Osmotic properties and internal pH of isolated rat parotid secretory granules.

Secretory granules of the rat parotid have been purified rapidly by differential centrifugation in iso-osmotic sucrose. Despite low-level contamination by other organelles, this fraction is predominantly secretory granules, based on biochemical and morphological characterization. Measurements of intragranular water space show that isolated granules swell in hypo-osmotic media, and begin to release alpha-amylase and other content proteins below 250 mosM. Exposure of granules to selected anions also causes lysis, increasing in the order sulfate less than phosphate less than or equal to gluconate less than or equal to isethionate less than or equal to acetate less than or equal to chloride much less than thiocyanate. The internal pH of isolated parotid granules is approximately 6.8 as measured by the distributions of either [3H]acetate or [14C]methylamine. Varying the external medium pH from 5.0 to 8.0 results in little change in the measured internal pH. Parotid granules have a substantial internal buffering capacity, i.e. a decrease of 85-140 mM intragranular H+ is needed to raise the internal pH by 1.0 unit. Finally, results obtained using acridine orange staining suggest that the secretory granule internal pH in situ is similar to that measured in isolated granules.

Animals↗
PubMed1984

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases.

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.

Adenosine Triphosphatases↗
PubMed1984

Spiperone: evidence for uptake into secretory granules.

Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution.

Animals↗
PubMed1983

Hydrogen ion cotransport by the renal brush border glutamate transporter.

Sodium ion dependent glutamate transport into rabbit renal brush border membrane vesicles is stimulated by low external pH and inhibited by low internal pH. Imposition of a pH difference (delta pH) (interior alkaline) across the vesicle membrane drives glutamate accumulation in the absence of other driving forces. This process requires Na+ but is not due to generation of an Na+ gradient in response to delta pH. Internal K+ stimulates both the rate and extent of glutamate accumulation, although K+ is not absolutely required for transport. Internal H+ inhibits the rate of glutamate accumulation by decreasing this K+ stimulation. Conversely, external K+ inhibits glutamate influx, and this inhibition can be overcome by lowering the external pH. These results indicate that H+ is cotransported with glutamate and suggest the possibility that, in the absence of internal K+, H+ can also fulfill the requirement for a countertransported ion.

Animals↗
PubMed1983

The serotonin transporter-imipramine "receptor".

The platelet plasma membrane serotonin transporter requires Na+ for two reactions, serotonin transport and imipramine binding. Although imipramine binding has been thought to reflect the same process required for serotonin binding prior to transport (Talvenheimo, J., Nelson, P.J., and Rudnick, G. (1979) J. Biol. Chem. 254, 4631-4635), binding and transport display markedly different responses to Na+. Imipramine binding (and competitive inhibition of transport) apparently requires two sodium ions which bind with a KD of 300 +/- 70 meq/liter. The total number of sites (Bmax) is the same at all Na+ concentrations, but the affinity for imipramine increases from 7.3 x 10(6) M-1 at 20 meq/liter to 110 x 10(6) M-1 at 200 meq/liter. Na+ acts, at least in part, by decreasing the rate of imipramine dissociation from its binding site. Serotonin binding displaces imipramine from its site on the membrane. In contrast to imipramine binding, this displacement is a simple, hyperbolic function of Na+ concentration with a KD for Na+ of 400 +/- 100 meq/liter, which suggests that only one Na+ is required. Serotonin transport is also much less responsive to Na+ concentration. Over the same concentration range in which the affinity for imipramine increases 15-fold, the affinity for serotonin increases only 2-fold. Despite the lack of Na+ effect on the Bmax for imipramine binding, the Vmax for serotonin transport increases as a simple saturable function of Na+ with a KM (Na+) of 52 meq/liter. Thus, substrate translocation as well as binding requires Na+. Since serotonin is cotransported with Na+, the serotonin gradient accumulated depends on the coupling stoichiometry and the magnitude of the Na+ gradient imposed. From the response of the serotonin gradient to imposed Na+ gradients, we calculated a serotonin:Na+ cotransport stoichiometry of 0.9. Taken together, the results suggest that serotonin and imipramine bind either to the same site or to mutually exclusive sites, but maximal imipramine binding requires two sodium ions, while maximal serotonin binding and translocation requires only one.

Animals↗
PubMed1983

Acidification of macrophage and fibroblast endocytic vesicles in vitro.

We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1- to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20 degrees C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium chloride. Acidification was not affected by inhibitors of the mitochondrial F1, F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or K+. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and secretory granules.

Adenosine Triphosphatases↗
PubMed1982

The role of chloride ion in platelet serotonin transport.

Chloride ion is required for platelet serotonin transport. This anion requirement is relatively specific for Cl-. Only Br- fully substitutes, while SCN-, NO2-, and NO3- are much less effective. Serotonin influx requires external Cl-, and efflux requires internal Cl-. Although internal Cl- is not required for transport and has little effect on the initial rate of influx, it decreases the steady state level of serotonin accumulation. Chloride ion does not merely fulfill a need for a permanent ion since it is required for exchange of internal with external serotonin. Moreover, serotonin accumulation still requires external Cl- when a K+ diffusion potential (interior negative) is imposed across the vesicle membrane with valinomycin. Furthermore, Cl- lowers the Km and raises the Vmax for transport, suggesting direct effects on the transporter. These results strongly suggest serotonin-Cl- co-transport across the platelet plasma membrane.

Animals↗
PubMed1982

Bioenergetics of serotonin transport by membrane vesicles derived from platelet dense granules.

A population of membrane vesicles derived from platelet-dense granules was isolated from platelet osmotic lysates by density gradient centrifugation. The preparation is relatively free from contamination by mitochondrial and soluble enzymatic activities and contains only traces of serotonin and adenine nucleotides, but retains significant amounts of Na+-dependent serotonin transport (plasma membrane) activity. In the presence of ATP and in the absence of Na+, these vesicles accumulate serotonin to intravesicular concentrations over 100 times that of the medium. Transport is a saturable process (Km = 1.15 microM) inhibited by reserpine, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, ammonia, and nigericin (in the presence of external potassium), but not by imipramine. When diluted into assay medium at pH 8.5, the interior of these vesicles remains relatively acidic. Addition of ATP further induced generation of a membrane potential (interior positive). The vesicles accumulate maximal concentrations of serotonin only when the vesicle interior is both acidic and positive with respect to the medium. These results are consistent with a transporter which catalyzes countertransport of at least two protons for each serotonin cation accumulated.

Adenosine Triphosphate↗
PubMed1982

Coupling of transmembrane proton gradients to platelet serotonin transport.

A pH difference (acid inside) across the platelet plasma membrane increases both the rate and extent of serotonin accumulation inside plasma membrane vesicles. Even in the absence of other transmembrane ion gradients, this pH difference (delta pH) serves as the sole driving force for serotonin accumulation, leading to a serotonin concentration 18-fold higher inside the vesicle. This process requires Na+ and is blocked by imipramine, indicating that it is mediated by the serotonin transporter. At physiological pH, internal K+ is counter-transported with serotonin, and high internal K+ stimulates transport maximally. Internal K+ also blocks the delta pH stimulation of serotonin transport. Conversely, low internal pH (5.6) inhibits the ability of internal K+ to stimulate transport. This apparent competition between K+ and protons suggests that delta pH drives serotonin accumulation through counter-transport with protons, and that serotonin is transported in its cationic form.

Animals↗
PubMed1981

Anion-dependent sodium ion conductance of platelet plasma membranes.

External stimulate Na+ efflux from platelet plasma membrane vesicles. Efflux is apparently electrogenic since K+ diffusion potentials induced with valinomycin (interior positive) accelerate and potentials of the opposite polarity (interior negative) inhibit. In the presence of stimulatory anions, voltage-dependent Na+ efflux is much faster than Na+-Na+ exchange in the absence of an induced membrane potential. Anions stimulate voltage-dependent efflux in the following order: SCN- greater than I- greater than NO3- greater than Br- approximately acetate approximately Cl- greater than F- approximately SO42- greater than HPO42-, gluconate, and isethionate. Thiocyanate, the most stimulatory anion, increases Na+ efflux 20-fold in the presence of a membrane potential (interior positive). Stimulation of efflux by Cl- is a saturable phenomenon with a K0.5 of 41 mM and a maximal 2-3-fold stimulation over the basal level of efflux. Neither basal nor valinomycin-stimulated efflux was influenced by the presence of the platelet-aggregating agents thrombin, epinephrine, or ADP in the presence of fibrinogen.

Animals↗
PubMed1980

Solubilization of the platelet plasma membrane serotonin transporter in an active form.

The activity of the serotonin transporter of platelet plasma membrane may be assessed by its ability to bind [N-methyl-3H]imipramine, even in the presence of detergents which disrupt the membrane and inactivate serotonin transport. Of the 21 detergents which were tested for their ability to solubilize the transporter in an active form, some, such as Triton X-100, inactivate imipramine-binding activity in a manner which is difficult or impossible to reverse, while inactivation by others, such as cholate, is readily reversed by gel filtration. Of the detergents which do not inactivate imipramine binding, most, such as the Tween series, also fail to solubilize significant amounts of the transporter. Of all the detergents tested, only one, digitonin, solubilized specific imipramine-binding activity. Imipramine binds to the digitonin-solubilized transporter with slightly reduced affinity, but in all other respects binding to intact and digitonin-solubilized membranes is identical. Thus, binding is, in both cases, saturable and Na+-dependent, and is reversed by serotonin. Furthermore, inhibitors of serotonin transport but not of serotonin-mediated platelet aggregation block imipramine binding to the soluble preparation. Sodium-dependent binding to the soluble extract is insensitive to K+, suggesting that separate sites on the serotonin transporter mediate the effects of effects of Na+ and K+.

Animals↗
PubMed1980

Evidence for two distinct serotonin transport systems in platelets.

Two transport systems for serotonin were measured independently in osmotic lysates of porcine platelets. After pre-equilibration with potassium phosphate and dilution into NaCl medium, only the plasma membrane transport system was observed. Under these conditions, serotonin accumulation is inhibited by imipramine, but not by reserpine or norepinephrine, and requires high (100 meq/liter) concentrations of Na+. In the presence of ATP at low (10 meq/liter) concentrations of Na+, only the transport system of the intracellular serotonin storage organelles was observed. Under these conditions, transport is inhibited by reserpine, norepinephrine, and epinephrine, but not by imipramine, and requires either ATP or an artificially imposed pH gradient (interior acidic) across the organelle membrane. These and other results suggest that accumulation of serotonin in platelet storage organelles is coupled predominantly to the pH gradient across the membrane.

Animals↗
PubMed1979

Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport.

Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.

Animals↗