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S Grinstein

Publications and source records attributed to S Grinstein.

At least 307 records · Page 17Linked to original sources
PubMed1984

Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors.

The cytoplasmic pH (pHi) of human blood neutrophils was measured using trapped carboxyfluorescein derivatives. Cells were acid-loaded using propionate or by pretreatment with NH4+. Acid-loaded cells were found to regain near-normal pHi by means of a Na+-dependent process. A concomitant Na+ uptake was recorded as a change in cell volume. Both events were amiloride-sensitive, indicating involvement of a Na+/H+ antiport. Activation of Na+/H+ exchange was also observed with chemotactic factors. Studies of the pHi-dependence of the H+ extrusion rate indicate that chemotactic factors increase the [H+i] sensitivity of the antiport.

Amiloride↗
PubMed1984

Volume regulation of Chinese hamster ovary cells in anisoosmotic media.

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.

Animals↗
PubMed1984

Intracellular distribution of acridine derivatives in platelets and their suitability for cytoplasmic pH measurements.

The site and mechanism of accumulation of acridine derivatives into platelets and their isolated organelles were investigated. In addition, their suitability as indicators of cytoplasmic pH was analysed. Direct microscopic observation showed that quinacrine and 9-aminoacridine are concentrated inside organelles in platelets. Using fractionation studies, the acridine derivatives were found to accumulate particularly in dense and alpha-granules. Uptake into these organelles is driven by a pH differential across their membrane (acidic inside). Because of their cellular distribution, acridine derivatives were found to be poor indicators of cytoplasmic pH. In contrast, a poorly permeant dicarboxylated fluorescein derivative, generated in situ by cytosolic enzymes, is shown to be a more reliable probe of intracellular pH. The results are compared with previous reports of the use of 9-aminoacridine as a cytoplasmic pH probe in platelets and of quinacrine as a selective dense-granule marker.

Acridines↗
PubMed1984

Is cytoplasmic Ca2+ in lymphocytes elevated in cystic fibrosis?

An increased cytoplasmic Ca2+ concentration ( [Ca2+]i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca2+]i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca2+]i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca2+ stores were also comparable in both types of individual.

Adolescent↗
PubMed1984

The intracellular pH of quiescent and proliferating human and rat thymic lymphocytes.

We compared the cytoplasmic pH (pHi) of quiescent and actively cycling thymic lymphocytes. Human and rat thymocyte suspensions were fractionated by centrifugation on one-step albumin density gradients. The pellet was composed of small, quiescent cells and the interphase contained mostly larger, actively cycling cells with a high proliferation index. When measured using [14C]-dimethyloxazolidinedione (DMO), pHi in the large cells of both species was approximately 0.15 units more alkaline than in the small cells. However, these differences were not detectable when pHi was measured with carboxylated fluorescein derivatives generated in situ by cytoplasmic enzymes. This apparent discrepancy can be explained by compartmentation of DMO, which accumulates in the alkaline mitochondrial matrix. Comparison of the mitochondrial content of quiescent and cycling thymocytes by several methods showed that the latter contained over 2.5-fold more mitochondria per unit cell volume. Assuming a constant intramitochondrial pH, this difference can account for the observed accumulation of DMO (i.e., apparent cytoplasmic alkalinity) in the actively proliferating cells. Therefore, no evidence was found for the existence of differences in pHi between quiescent and proliferating lymphocytes. Moreover, caution must be exercised when comparing DMO partition data in cells with varying relative mitochondrial content.

Animals↗
PubMed1984

Role of membrane potential in the regulation of lectin-induced calcium uptake.

Incubation of lymphocytes with mitogenic lectins triggers Ca2+ uptake. This increase in free cytoplasmic Ca2+ is postulated to be an important signal in the initiation of DNA synthesis. Transmembrane fluxes of monovalent ions and changes in membrane potential are also associated with lectin-induced activation of lymphocytes. We have examined the relationship between extra-cellular monovalent ion substitution, the associated electrical potential changes (measured with cyanine dyes), phytohemagglutinin-induced Ca2+ uptake (measured with Quin-2) and proliferation in human T cells. The results show that (1) the magnitude of the increase in free cytoplasmic Ca2+ concentration is correlated with the extent of the lymphoproliferative response, (2) lectin-induced Ca2+ fluxes are sensitive to membrane potential, decreasing with depolarization, and are likely conductive, and (3) the presence of extra-cellular Na+ during incubation with phytohemagglutinin is not essential to mitogenic triggering.

Calcium↗
PubMed1984

Comparative study of the major glycoproteins of the plasma membrane and secretory granule membranes of porcine platelets.

The major glycoproteins of porcine platelet plasma membranes and alpha-granule membranes were compared. Significant cross-contamination of the two preparations was ruled out by surface labeling and proteolysis experiments. At least four of the major glycoproteins of the alpha-granule membrane had counterparts in the plasma membranes with identical molecular weight and lectin-binding properties. Two of these (mol. wts 110,000 and 125,000) were further analysed by one-dimensional peptide mapping. The results confirmed that there are two distinct pools of identical glycoproteins: one on the surface membrane and the other on the alpha-granular membrane. The 110,000 and 125,000 mol. wt glycoproteins are probably equivalent to glycoproteins IIb and IIIa of the human platelet and may therefore be involved in fibrinogen binding.

Animals↗
PubMed1984

Monocytes are required to trigger Ca2+ uptake in the proliferative response of human t lymphocytes to staphylococcus aureus protein A.

We have used the T-cell mitogen Staphylococcus aureus protein A (SpA) to study the role of monocytes in the early events of T-lymphocyte activation. The mitogenic response of human peripheral blood mononuclear cells (PBM) was compared to the response of populations enriched for T cells by E-rosetting (PBM-E+). In response to SpA, the [3H]thymidine uptake of PBM-E+ was reduced by 80% compared to PBM. The reduced response of PBM-E+ was completely restored by the addition of irradiated PBM-E- or the monocyte-like human cell line U-937 but not by addition of irradiated PBM-E+. A direct interaction of SpA with monocytes is important since proliferative responses could be generated by preincubation of U-937 with SpA followed by washing and subsequent addition to PBM-E+; incubation of PBM-E+ with SpA followed by washing and subsequent addition of U-937 did not result in a proliferative response. To further delineate the role of the monocyte, we examined the ability of soluble SpA, U-937, or U-937 preincubated with SpA to trigger Ca2+ flux into T lymphocytes, an early step in initiation of the proliferative response. SpA-pretreated U-937, but neither SpA nor monocytes alone, triggered Ca2+ movement into the T lymphocytes. This defines a new role for the monocyte in the early events of T-lymphocyte activation.

Calcium↗
PubMed1984

Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive Na+/H+ antiport.

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i-regulatory mechanism.

Amiloride↗
PubMed1984

22Na+ fluxes in thymic lymphocytes. I. Na+/Na+ and Na+/H+ exchange through an amiloride-insensitive pathway.

The Na+ transport pathways of normal rat thymocytes were investigated. Na+ conductance was found to be lower than K+ conductance, which is consistent with reported values of membrane potential. In contrast, the isotopically measured Na+ permeability was greater than 10-fold higher than that of K+, which indicates that most of the flux is electroneutral. Cotransport with Cl- (or K+ and Cl-) and countertransport with Ca2+ were ruled out by ion substitution experiments and use of inhibitors. Countertransport for Na+ or H+ through the amiloride-sensitive antiport accounts for only 15-20% of the resting influx. In the presence of amiloride, 22Na+ uptake was increased in Na+-loaded cells, which suggests the existence of Na+/Na+ countertransport. Cytoplasmic pH determinations using fluorescent probes indicated that under certain conditions this amiloride-resistant system will also exchange Na+ for H+, as evidenced by an internal Na+-dependent acidification is proportional to internal [Na+] but inversely related to extracellular [Na+]. Moreover, 22Na+ uptake is inhibited by increasing external [H+]. The results support the existence of a substantial amiloride-insensitive, electroneutral cation exchange system capable of transporting Na+ and H+.

Amiloride↗
PubMed1984

22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site.

22Na+ flux and cytoplasmic pH (pHi) determinations were used to study the reversibility, symmetry, and mechanism of activation of the Na+/H+ exchange system in rat thymic lymphocytes. In acid-loaded cells, the antiport can be detected as an Na+-induced, amiloride-sensitive alkalinization. At pHi greater than or equal to 7.0, amiloride-sensitive net H+ fluxes are not detectable. To investigate whether at this pHi the transporter is operative in a different mode, e.g., Na+/Na+ exchange, 22Na+ uptake was measured as a function of pHi. The results indicate that the antiport is relatively inactive at pHi greater than or equal to 7.0. Comparison of the rates of H+ efflux (or equivalent OH- uptake) and Na+ uptake indicate that Na+/Na+ countertransport through this system is negligible at all values of pHi and that the Na+:H+ stoichiometry is 1:1. Measurements of pHi in Na+-loaded cells suspended in Na+-free medium revealed an amiloride-sensitive cytoplasmic acidification, which is indicative of exchange of internal Na+ for external H+. The symmetry of the system was analyzed by measuring the effect of extracellular pH (pHo) on Na+ efflux. Unlike cytoplasmic acidification, lowering pHo failed to activate the antiport. The results indicate that the amiloride-sensitive Na+/H+ exchanger is reversible but asymmetric. The system is virtually inactive at pHi greater than or equal to 7.0 but can be activated by protonation of a modifier site on the cytoplasmic surface. Activation can also occur by depletion of cellular Na+. It is proposed that Na+ may also interact with the modifier site, stabilizing the unprotonated (inactive) form.

Amiloride↗
PubMed1984

Responses of lymphocytes to anisotonic media: volume-regulating behavior.

The regulatory responses elicited in lymphoid cells suspended in anisotonic media are reviewed. The immediate response approximates osmometric behavior. In addition, in hypotonic media, the initial osmometric swelling is followed by a regulatory volume decrease (RVD), which is associated with KCl loss. The volume-induced effluxes of K+ and Cl- are mediated by two independent conductive pathways. Ca2+-depletion experiments and studies of inhibitor susceptibility suggest that Ca2+ may mediate the activation of the K+ pathway. The responses of the two main lymphocyte subpopulations to hypotonic challenge are different. RVD is much more rapid in T- than in B-cells, regardless of their tissue of origin. Under certain conditions, shrunken lymphocytes will regain their initial volume. This regulatory volume increase (RVI) is due to NaCl uptake, followed by a secondary exchange of Na+ for K+ via the Na+-K+ pump. Na+ is primarily taken up in exchange for H+ through an amiloride-sensitive pathway, whereas Cl- enters in exchange for HCO-3 (or OH-). Anion and cation fluxes responsible for RVI are electroneutral. Some of the volume-sensitive pathways can also be activated in isotonic cells. The conductive K+ pathway is activated by Ca2+ plus ionophore A23187, and the Na+-H+ exchanger can be activated by cytoplasmic acidification. The responses of lymphocytes to anisotonic challenge are compared with those of other cells, and the possible significance of the volume-induced fluxes is discussed.

Animals↗
PubMed1984

Amiloride-sensitive Na+-H+ exchange in platelets and leukocytes: detection by electronic cell sizing.

A new method was developed to detect the activity of the Na+-H+ exchange system as changes in cell volume. The cytoplasmic pH of isolated cells in suspension was lowered by incubation in Na-propionate medium, due to permeation of the protonated acid. This resulted in activation of Na+-H+ countertransport, measurable either as a Na+-dependent alkalinization or as an increase in 22Na+ uptake, both of which are amiloride sensitive. The continued operation of the antiport on prolonged exposure to Na-propionate results in a considerable increase in Na+ (and presumably propionate-) content. This is accompanied by an osmotic water shift and cell swelling, detectable by electronic sizing. This method was used to investigate the presence of the Na+-H+ exchanger in human platelets, neutrophils, lymphocytes, and monocytes as well as in cultured cell lines of B and T lymphoblasts and of macrophages. All these cell types displayed an amiloride-sensitive swelling when suspended in Na-propionate media. The results suggest the ubiquity of the Na+-H+ exchange system in the plasma membrane of nucleated mammalian blood cells.

Amiloride↗
PubMed1983

The total and free concentrations of Ca2+ and Mg2+ inside platelet secretory granules. Measurements employing a novel double null point technique.

The inorganic ion contents of platelet alpha-granules were determined by a combination of neutron activation analysis and flame photometry. Total concentrations were estimated using intragranular water spaces measured by isotope dilution. To measure the free concentrations of Ca2+ and Mg2+ we developed a novel double null point titration technique. The method requires independent determinations of transmembrane delta pH and the availability of two divalent cation-H+ exchange ionophores with different Ca2+/Mg2+ selectivity ratios. A23187 and the halogenated analog 4-bromo-A23187 were used for this purpose. Absolute delta pH was measured by methylamine distribution, while relative pH changes induced by the ionophores were monitored with 9-aminoacridine. The free concentrations of Ca2+ and Mg2+ were found to be 12 and 326 microM, respectively. These values are markedly lower than the calculated total concentrations of these cations, i.e. 32 mM for Ca2+ and 172 mM for Mg2+.

Animals↗
PubMed1983

Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration.

An increase in cytoplasmic free [Ca2+], [Ca2+]i, has been suggested as the trigger for the permeability changes that bring about cell volume restoration following exposure to anisotonic media. This idea was directly tested in human peripheral lymphocytes undergoing regulatory volume decrease following a hypotonic dilution of the suspension. [Ca2+]i was measured with the intracellularly trapped fluorescent indicator, quin2, and showed no measureable change on hypotonic swelling or during the subsequent volume decrease. Moreover, even though the incorporated quin2 adds significant Ca-buffering to the cytoplasm, regulatory volume decrease occurred normally in the quin2-loaded cells. It appears that alterations in [Ca2+]i are not involved in these processes of volume regulation. An intracellularly trapped derivative of fluorescein, bis(carboxyethyl)carboxyfluorescein, was used to monitor cytoplasmic pH, which also showed no change during regulatory volume decrease.

Body Fluids↗
PubMed1983

Studies on the Ca2+-induced lysis of platelet alpha-granules.

Platelet alpha-granules have been reported to lyse upon addition of submillimolar Ca2+ (J. Van der Meulen and S. Grinstein, J. Biol. Chem. 257, 5190). Similar observations in parotid granules have been attributed to extensive lipid hydrolysis. Experiments were performed to assess the role of lipases and proteases in Ca2+-induced lysis of alpha-granules. No differences were detected between lipids of Ca2+-treated and control granules by two-dimensional thin-layer chromatography. Moreover, several phospholipase inhibitors were without effect on Ca2+-induced lysis. Similarly, the polypeptide patterns of control and treated granules were identical and protease inhibitors failed to prevent lysis. In contrast, lysis could be suppressed by increasing the osmolarity of the medium or by substitution with nonpermeating ions. Lysis was unaffected by quinine, amiloride, furosemide, or tetraethylammonium but was inhibited by 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a powerful inhibitor of anion transport. The data suggest that Ca2+-induced lysis of alpha-granules does not result from wholesale hydrolysis of either lipids or proteins. Instead, the results are consistent with a Ca2+-mediated change in membrane permeability. In the presence of permeating ions, this leads to entry of salt and osmotically obliged water with consequent swelling and eventual lysis.

Biological Transport↗