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S Grinstein

Publications and source records attributed to S Grinstein.

At least 325 records · Page 18Linked to original sources
PubMed1983

The electrochemical H+ gradient of platelet secretory alpha-granules. Contribution of a H+ pump and a Donnan potential.

Experiments were performed to determine the internal pH and membrane potential of platelet alpha-granules. Fluorescence microscopy showed accumulation of weak bases, indicative of an acidic interior, inside secretory vesicles in intact platelets and in isolated alpha-granules. Weak base uptake was pH-dependent and NH+4-sensitive. In isolated alpha-granules suspended in medium buffered at pH 7.2, a delta pH, the difference between internal and external pH, of 1.2 (inside acidic) was measured by [14C]methylamine distribution. Uptake of isotopic or fluorescent amines was reduced by H+/cation exchange via ionophores and by addition of NH+4, but also by increasing the ionic strength suggesting that delta pH is partly due to a Donnan potential. Transmembrane potential measurements by fluorescent or radioactive ion distribution indicated that in the absence of ATP, granules are internally negative. When measured with 86Rb+, this potential could be entirely collapsed by increasing the ionic strength. Addition of ATP X Mg in the absence of permeating anions made the intragranular space more positive, as expected from inward electrogenic H+ pumping. The results are compatible with the coexistence of sealed and leaky subpopulations of alpha-granules. Internal acidity was generated in sealed granules in vivo by a H+-pumping ATPase, whereas in leaky granules acidity is a consequence of an internally negative Donnan potential.

Ammonia↗
PubMed1983

Measurement of sidedness of isolated plasma-membrane vesicles: quantitation of actin exposure by DNase I inactivation.

A rapid and simple method is described to determine the orientation of isolated plasma membrane vesicle populations. The method is based on quantitation of the exposure of actin, an extrinsic membrane component bound exclusively to the cytoplasmic surface. Actin accessibility was determined using DNase I as a nonpermeating probe. The activity of this enzyme is lost upon complexation with actin, and inhibition can be conveniently monitored as the change in the rate of DNase-mediated hyperchromic reaction. Exposed actin is determined in intact vesicles, and total actin in detergent-lysed membranes; the ratio of these values equals the sum of inside-out and leaky vesicles. Sealed right-side-out vesicles are calculated by difference. Using thymocyte plasma membrane vesicles as a model system, we determined that 54% of the membrane is sealed in a right-side-out configuration. This value is consistent with independent determinations of inside-out membranes in the same preparation, obtained by 5'-nucleotidase latency measurements, and with reported values in other lymphocyte membrane preparations.

Actins↗
PubMed1983

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes.

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.

Animals↗
PubMed1983

Isolation and partial characterization of platelet alpha-granule membranes.

Porcine alpha-granules, prepared by a modification of pre-existing methods, were found to be essentially homogeneous by transmission electron microscopy. Freeze-fractured samples of isolated granules revealed numerous intramembranous particles on the EF (exoplasmic fracture) surface and to a lesser extent on the PF (protoplasmic fracture) surface whereas the PS (protoplasmic) surface was relatively smooth. The granules appear to be sealed, as evidenced by: a) the retention of their electron dense core material; b) the inability of impermeant labels to react with the granule contents, and c) the finding that the intragranular proteins are refractory to mild hydrolysis by externally added proteases. Membranes were isolated by alkali extraction of the granules and used for biochemical characterization. Approximately 87% of the protein, but only insignificant amounts of phospholipid were removed by this procedure, which yielded membrane vesicles devoid of the dense core. The membranes contain one major and several minor polypeptides of molecular weights ranging from 28,000 to 230,000, as determined by polyacrylamide gel electrophoresis. The major polypeptide contains carbohydrate residues. The exposure of specific proteins on the cytoplasmic surface of the granule membrane was determined by a combination of surface-specific labeling and proteolysis of intact granules, followed by membrane isolation and analysis. In sealed granules, only a limited number of bands are modified by the reagents whereas most of them are affected following granule lysis, indicating asymmetry in their transmembrane disposition. The fraction eluted by alkali extraction was also analyzed and found to contain nine major polypeptides of molecular weights ranging from 230,000 to 43,000. These are compared to the weights of the macromolecules believed to be secreted from alpha-granules, as determined by radioimmunological techniques.

Animals↗
PubMed1983

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification.

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.

Acid-Base Equilibrium↗
PubMed1983

Volume-induced anion conductance in human B lymphocytes is cation independent.

Peripheral blood T lymphocytes swollen in hypotonic media regain normal size by releasing internal KCl through activation of conductive K+ and Cl- pathways. In contrast, no regulatory volume decrease (RVD) is observed in tonsillar B lymphocytes. The volume-induced K+ permeability is minimal in these cells, but little was known about the effect of swelling on anion transport and its coupling to cation translocation. The induction of an anion-conductive pathway on swelling of B cells was demonstrated in the following way: 1) when an exogenous cation ionophore was added, hypotonic swelling was followed by secondary volume changes, the direction of which was dictated by the electrochemical gradients of the diffusible ions; 2) the rate of 36Cl efflux was markedly increased by swelling; and 3) upon swelling, the membrane potential approached the Cl- equilibrium potential. Quinine, which blocks K+ transport and RVD in swollen T cells, has no effect on volume-induced anion fluxes. Consequently, secondary volume changes could be elicited by gramicidin in cells pretreated with quinine. It can be concluded that in B cells, swelling leads to activation of a conductive Cl- permeability but not of K+ permeability. These and other findings support the view that the Cl- pathway functions independently of the K+ pathway.

Anions↗
PubMed1983

Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists.

The importance of Ca++ in the initiation of lymphocyte activation and mitogenesis has been supported by several studies. Because calmodulin functions as the intracellular mediator of the effects of Ca++, it likely plays a major role in the regulation of lymphocyte function. We have examined the effects of known calmodulin antagonists, the phenothiazines, on lectin-induced T cell mitogenesis and have shown a central role for Ca++ uptake in the expression of a phenothiazine-sensitive stage after lectin activation. The drug effects were observed only if the cells were previously activated by PHA or the ionophore A23187, and only in the presence of Ca++. These effects were restricted to a defined time period (5 hr) after lectin activation. The data support the concept that calmodulin is the target for the phenothiazine effects and demonstrate the permissive role of Ca++ in the mediation of these events.

Adult↗
PubMed1982

Increased anion permeability during volume regulation in human lymphocytes.

Peripheral blood lymphocytes (p.b.ls) readjust their volumes after swelling in hypotonic media. An essential component of the regulatory response is an increase in K+ and Cl- permeability. No evidence was found for a tightly coupled co-transport of K+ and Cl-. The flux of either ion proceeds normally in the virtual absence of the transported counterion. Furthermore, alterations in membrane potential recorded during the phase of volume readjustment can be qualitatively accounted for by an increase in Cl- conductance. In tonsillar lymphocytes, a failure of the K+-permeability is nevertheless increased upon swelling. This further suggests that K+ and Cl- are transported during volume regulation through independent pathways. Cytoplasmic free Ca2+ appears to be involved in regulatory volume decrease. K+ and Cl-. Moreover, swelling and shrinking can be induced in isotonic K+-rich and K+-free media, respectively, by the Ca2+ ionophore. The ion flux and volume changes produced by either swelling or internal Ca2+ can be inhibited by similar concentrations of quinine and phenothiazines. The inhibitory activity of the latter drugs, which are powerful antagonists of calmodulin, suggests the participation of this Ca2+-regulator protein in volume regulation.

Anions↗
PubMed1982

Possible role of H+--alkali cation countertransport in secretory granule swelling during exocytosis.

Studies in model systems, as well as observations in intact cells, suggest that osmotic swelling of secretory granules is an essential step in exocytosis. A model is proposed whereby the low pH recorded in most secretory organelles could provide the driving force for granule swelling. The model assumes that during stimulation an exchange of H+ for alkali cations is triggered across the granule membrane. The outgoing H+ are rapidly replaced by the internal buffering capacity with a concomitant osmotic gain. The exchange is independent of anions and could be triggered by cytoplasmic Ca2+. The H+ -pump is responsible for the delta pH but independent of the exchange mechanism.

Cytoplasmic Granules↗
PubMed1982

Ca2+-induced lysis of platelet secretory granules.

Platelet secretory granules were used for in vitro studies bearing on the mechanism of exocytosis. A morphologically homogeneous preparation of alpha-granules was obtained from porcine platelets. The granules were shown to be osmotically fragile, as determined by electron microscopy, turbidity measurements, and the release of protein upon hypotonic lysis. Lysis could also be induced in isotonic media by addition of gramicidin to granules suspended in NaCl or KCl. Increasing the osmotic strength of the medium prevented this effect. The antibiotic had no effect when added to granules in sucrose solution. Submillimolar concentrations of Ca2+ drastically reduced the turbidity of granule suspensions while releasing protein to the supernatant. Electron micrographs showed that Ca2+-treated granules lost most of their electron-dense core, indicating that lysis had occurred. Mg2+ and other divalent cations failed to induce this response. The effect occurred in NaCl or KCl but not in sucrose or sorbitol solutions. Increasing the osmotic content of the medium with sucrose suppressed the Ca2+-induced lysis. It is suggested that Ca2+ increases the ionic permeability of the granule membrane leading to solute influx followed by osmotically coupled water uptake and, therefore, swelling and lysis. During platelet activation, Ca2+-induced granule swelling could provide the driving force for membrane fusion leading to exocytosis.

Animals↗
PubMed1982

Calmodulin binding to platelet plasma membranes.

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind 125I-labeled calmodulin in vitro in the presence of Ca2+. Binding is largely reduced by replacing Ca2+ by Mg2+ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent Kd of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 microM trifluoperazine or 50 microM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149000 was detected. Binding to this band was Ca2+-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca2+ + Mg2+)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.

Blood Platelets↗
PubMed1982

Volume regulation by human lymphocytes: characterization of the ionic basis for regulatory volume decrease.

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5-0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K+ decreased during RVD, while Na+ content remained unchanged. Similarly, the efflux of 86Rb (used as a K+ analog) increased upon dilution, whereas 22Na efflux was not altered. 86Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na+- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K+-free media, and also in Na+-free media when Li+, choline+, or Tris+ were the substitutes. In high K+ or RB+ hypotonic media shrinking was absent and a second swelling phase was observed. Cs+ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K+ concentration was varied and, with 50 mM K+, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K+, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na+ entry into the cell, but this activation is not essential for RVD.

Cations, Monovalent↗
PubMed1982

Volume regulation by human lymphocytes. Role of calcium.

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++-containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).

Calcimycin↗
PubMed1982

Volume-induced increase of anion permeability in human lymphocytes.

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.

Anions↗
PubMed1982

Volume regulation by human lymphocytes. Identification of differences between the two major lymphocyte subpopulations.

Following exposure to hypotonic media, human peripheral blood lymphocytes swell initially but restore their isotonic volume within minutes. In contrast, tonsillar lymphocytes demonstrate a similar initial phase of swelling but fail to restore their isotonic volume. We have studied the ionic basis for this second or regulatory volume decrease (RVD) phase using lymphocytes from peripheral blood, tonsil, and thymus. RVD was characterized by 86Rb efflux and a decrease in K+ content. The increase in K+ permeability in response to hypotonic challenge was characteristic for T lymphocytes obtained from peripheral blood, tonsil, or thymus. B lymphocytes showed only a modest increase in K+ permeability and consequently little RVD. The data confirm that the response of peripheral blood and tonsillar lymphocytes to a hypotonic environment can be accounted for by differences in the proportions of T and B cells, and the differential behaviour of B and T lymphocytes is based on differences in membrane permeability to K+ upon swelling.

B-Lymphocytes↗