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S Grinstein

Publications and source records attributed to S Grinstein.

At least 145 records · Page 8Linked to original sources
PubMed1994

Cytokines in adenoviral disease in children: association of interleukin-6, interleukin-8, and tumor necrosis factor alpha levels with clinical outcome.

To explore the pathogenic mechanisms involved in adenovirus infection, we evaluated total levels of immunoglobulins, antiadenovirus antibodies, adenovirus-specific circulating immune complexes, and cytokines in serum samples obtained from 38 hospitalized children with adenovirus infection. According to their clinical findings and outcome, the infections were classified as follows: (1) moderate (group I, n = 10), (2) severe (group II, n = 12), and (3) fatal (group III, n = 16). About 60% of the children had elevated IgM levels. IgG-containing adenovirus-specific circulating immune complexes were initially detected in 7 of 16 group III patients, 4 of whom had low serum levels of the third component of complement. A decrease in initial antiadenovirus IgG antibodies was observed in 3 of 10 patients in group III. Serum interleukin-6 was not detected in group I (none of 10), but was present in group II (7 of 12, p = 0.016) and group III (13 of 16, p < 0.001). Interleukin-8 was detected in all groups; values in fatal cases were significantly higher than in surviving children. Tumor necrosis factor alpha was not observed in group I (none of 10) and was uncommon in group II (2 of 12) but was frequently detected in group III (9 of 15, p = 0.01). Interleukin-1 and interleukin-4 were rarely detected in serum samples. Increased concentrations of interleukin-6, interleukin-8, and tumor necrosis factor alpha were associated with hypoperfusion, febrile peaks, tonic-clonic seizures, and septic shock. In 5 of 10 patients in groups II and III, autoantibodies specific for smooth muscle were found. Our findings indicate that high serum values for interleukin-6, interleukin-8, and tumor necrosis factor alpha are associated with severity of adenovirus infection.

Adenovirus Infections, Human↗
PubMed1994

Release of nitric oxide during the experimental infection with Trypanosoma cruzi.

We analysed the production of nitric oxide (NO) intermediates by cells from BALB/c mice infected with either virulent (Tulahuén or RA) or avirulent (CA-1) strains of Trypanosoma cruzi. Peritoneal or spleen cells from mice infected with T. cruzi released NO when incubated without further stimuli. Cells from mice during the acute stage of infection accumulated higher levels of inducible NO synthase mRNA and produced both, before and after lypopolysaccharide stimulation, higher amounts of NO than cells from mice chronically infected with T. cruzi. NO synthesis showed similar kinetics in connection with all three strains of T. cruzi, but cells from mice inbred with the Tulahuén or RA strains released higher levels of IFN-gamma, an activator of the NO pathways, than cells from mice infected with the CA-1 strain. In vivo administration of L-Ng-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase, increased the susceptibility of mice to T. cruzi. We conclude that infection with T. cruzi induces NO production, and suggest that NO plays a role in the resistance against the parasite.

Amino Acid Oxidoreductases↗
PubMed1994

Development of a H(+)-selective conductance during granulocytic differentiation of HL-60 cells.

The NADPH oxidase is one of the main microbicidal systems of granulocytes. Stimulation of the oxidase during infection leads to a burst of metabolic acid generation. Potentially deleterious cytosolic acidification is prevented by the simultaneous activation of homeostatic H+ extrusion mechanisms, including a recently described H+ conductance. Studies in granulocytes from chronic granulomatous disease patients have suggested a relationship between the oxidase and the H+ conductive pathway. In this report we compared the expression of the H+ conductance and the NADPH oxidase during granulocytic differentiation of dimethyl sulfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated that the H(+)-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also estimated fluorimetrically, measuring changes in the cytosolic pH of suspended cells. Imposition of an inward protonmotive force failed to induce significant cytosolic acidification. In contrast, a sizable conductive H+ extrusion was detected in acid-loaded differentiated cells, consistent with the rectifying properties of the current measured electrophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulocytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH oxidase. These findings suggest that the H+ extrusion mechanisms required for the maintenance of the intracellular pH during granulocyte activation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conductance and the NADPH oxidase.

Amino Acid Sequence↗
PubMed1994

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations.

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

Animals↗
PubMed1994

Serine/threonine kinase activation in human neutrophils: relationship to tyrosine phosphorylation.

Tyrosine phosphorylation is among the earliest responses of neutrophils to chemotactic peptides. Tyrosine phosphorylated proteins comigrate with serine/threonine kinases of 65 and 72 kDa (PK65 and PK72), which are activated concomitantly by the chemoattractants. Studies were designed to test whether tyrosine phosphorylation is required for activation of PK65 and PK72. Pretreatment of cells with the tyrosine kinase inhibitors erbstatin or genistein prevented both phosphotyrosine accumulation and activation of PK65 and PK72. In nondenaturing lysates, PK65 and PK72 became spontaneously inactivated in parallel with rapid endogenous tyrosine dephosphorylation. Spontaneous dephosphorylation and inactivation of PK65 and PK72 were prevented in denatured lysates. Under these conditions, dephosphorylation could be induced by exogenous phosphotyrosine phosphatase 1B. PK65 and PK72 activation persisted despite virtually complete tyrosine dephosphorylation. Moreover, immunoprecipitation experiments indicated that PK65 and PK72 are not themselves tyrosine phosphorylated. We concluded that tyrosine phosphorylation is a necessary upstream event in the activation of the serine/threonine kinases. However, once the posttranslational modification that renders PK65 and PK72 active has occurred, tyrosine phosphorylation is no longer required for maintenance of their kinase activity.

Enzyme Activation↗
PubMed1994

Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function.

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp91-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H+ translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H+ extrusion. CGD cells expressing diminished amounts of cytochrome displayed H+ conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+. Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity.

Female↗
PubMed1994

Na+/H+ antiport: modulation by ATP and role in cell volume regulation.

Na+/H+ antiport is a major determinant of intracellular pH (pHi) and also plays an important role in the maintenance of cellular volume. Na+/H+ exchange through NHE-1, the ubiquitous isoform of the antiporter, is accelerated by cytosolic acidification and also by osmotically induced cell shrinking, thereby promoting recovery of the physiological pHi and volume, respectively. Although hydrolysis of ATP is not required for transport of ions through the antiporter, metabolic depletion exerts a marked inhibitory effect. Depletion of ATP also prevents osmotic activation and volume regulation. Contrary to earlier suggestions, however, changes in the phosphorylation state of the antiporter itself are not involved in the effects of either metabolic depletion or osmotic stimulation. Nevertheless, the cytosolic carboxy-terminal segment of the antiporter, which contains the major phosphorylation sites, is essential for the ATP dependence as well as for osmotic activation. It is conceivable that this domain interacts with ancillary phosphorylated or nucleotide-binding proteins, with the cytoskeleton and/or with specific phospholipids, which modulate the rate of transport. Nucleotide dependence and osmotic sensitivity have been compared in three different isoforms of the antiporter, heterologously expressed in fibroblastic cells. Like NHE-1, NHE-2 and NHE-3 were severely inhibited by depletion of ATP. In contrast, whereas NHE-2 was stimulated by osmotic shrinkage, NHE-3 was inhibited. The possible physiological significance of the ATP-dependence and osmotic responsiveness of the antiporter isoforms is discussed.

Adenosine Triphosphate↗
PubMed1994

Oxidant stress inhibits pH regulatory mechanisms in murine peritoneal macrophages.

BACKGROUND: Maintenance of cytoplasmic pH (pHi) close to the physiologic range is vital to normal cellular homeostasis. We have previously reported that a vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism. Since the inflammatory microenvironment is frequently characterized by the influx of cells known to release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pHi regulation in peritoneal macrophages. Specifically, the effect of hydrogen peroxide on V-ATPase-mediated proton extrusion from acid-loaded macrophages was investigated. METHODS: Thioglycollate-elicited murine peritoneal macrophages were exposed to varying concentrations of hydrogen peroxide and examined for their ability to recover from an acid-load. pHi was studied by preloading cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carboxyfluorescein, and monitoring changes in fluorescence under various conditions using a fluorescence spectrometer. RESULTS: Hydrogen peroxide caused a time- and dose-dependent decrease in proton pump-mediated pHi recovery in peritoneal macrophages. This effect occurred without cytotoxicity and was a specific effect as evidenced by the ability of catalase to reverse the inhibition. Since hydrogen peroxide is known to deplete intracellular ATP, a substrate for V-ATPase activity, we hypothesized that ATP depletion may underlie the effect. These studies showed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular glutathione in vivo by using diethyl maleate increased the sensitivity of V-ATPase activity to oxidant stress. CONCLUSIONS: Oxidant stress within the inflammatory milieu impairs macrophage pHi regulation. This effect is magnified by depletion of intracellular antioxidants, as occurs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory states.

Adenosine Triphosphate↗
PubMed1993

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.

Actins↗
PubMed1993

Chloroquine inhibits glucose-transporter recruitment induced by insulin in rat adipocytes independently of its action on endomembrane pH.

In adipocytes, stimulation of glucose transport by insulin is mediated largely by translocation of the GLUT4 isoform of glucose transporters from an intracellular store to the plasma membrane. Most endomembrane compartments are endowed with H(+)-pumping ATPases, and the resulting luminal acidification is thought to play a role in vesicular traffic. Chloroquine (Clq), a permeant weak base, was used to test whether endomembrane pH is an important factor in GLUT4 translocation. Under conditions chosen to optimize Clq uptake, the weak base precluded insulin-induced GLUT4 translocation and the associated stimulation of glucose transport. Clq also effectively dissipated the delta pH of acidic endomembrane compartments, assessed fluorimetrically. To define whether the intracellular GLUT4 storage compartment is acidic, immunoadsorption and immunoblotting experiments were performed to determine whether glucose transporters and vacuolar-type H+ pumps coexist in the same membranes. Unexpectedly, H+ pumps were not detectable in vesicles bearing GLUT4. Moreover, dissipation of endomembrane delta pH by monensin failed to inhibit insulin-stimulated GLUT4 translocation and hexose transport. Finally, the inhibitory effect of Clq persisted in the presence of monensin. We conclude that GLUT4 resides in an intracellular compartment devoid of H+ pumps. The insertion of this compartment into the plasmalemma is not regulated by transmembrane pH gradients. Clq impairs the stimulation of glucose transport by blocking translocation of GLUT4 by a pH-independent mechanism. Clq may provide a useful tool to elucidate the signalling or fusion steps involved in insulin-induced GLUT4 translocation.

Adipocytes↗
PubMed1993

Plasma membrane recycling in CFTR-expressing CHO cells.

The hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) participates in plasma membrane recycling was tested experimentally. Using CHO cells, we determined the effects of CFTR expression and of elevated intracellular cAMP on exocytosis, measured as the incorporation into the plasma membrane of endosomes pre-labelled with biotinylated wheat-germ agglutinin (WGA). CFTR expression was without effect on the rate of exocytosis. Furthermore, cAMP did not affect endosomal recycling to the plasma membrane in either CFTR-expressing or control cells. These findings suggest that CFTR is not involved in regulating plasma membrane recycling in all cells.

Animals↗
PubMed1993

Ionomycin activates electrogenic Ca2+ influx in rat thymic lymphocytes.

Increases in intracellular Ca2+ concentration ([Ca2+]i) after addition of the carboxylic Ca(2+)-transporting ionophore ionomycin have been reported to be inhibited by depolarization in mouse T- and B-lymphocytes, a result attributed to effects on the plasma-membrane Ca2+ pump. The present experiments were undertaken to test the hypothesis that activation of electrogenic Ca2+ uptake, due to release of Ca2+i from intracellular pools, underlies the membrane-potential (Em)-sensitivity of ionomycin-mediated Ca2+ uptake in T-lymphocytes. To address this proposal we have compared the characteristics of Ca2+ influx induced by ionomycin with those of Ca2+ influx activated by release of Ca2+ from intracellular stores (store-regulated Ca2+ uptake, SRCU). The endosomal Ca(2+)-ATPase inhibitor thapsigargin was used to bring about loss of Ca2+ from intracellular pools. In support of this hypothesis, we report that: (1) ionomycin releases Ca2+ from a thapsigargin-sensitive endosomal pool, a pool previously demonstrated to regulate electrogenic SRCU in rat thymic lymphocytes, (2) Ca2+ and Mn2+ uptake mediated by low doses of ionomycin are inhibited by compounds previously reported to inhibit SRCU, and (3) in the absence of SRCU, ionomycin-mediated Ca2+ uptake displays no sensitivity to Em. We conclude that activation of electrogenic SRCU can adequately account for the Em-sensitivity of Ca2+ flux previously attributed to alterations in Ca(2+)-pump activity. Such a mechanism of action may underlie previous reports of electrogenic Ca2+ transport mediated by ionomycin in other tissues.

Animals↗
PubMed1993

Role of isoprenylation in intracellular pH regulation of granulocytes.

Lovastatin was used to study the role of isoprenylated GTP-binding proteins in the regulation of intracellular pH in granulocytic HL-60 cells. The cytosolic acidification that accompanies stimulation by chemoattractants was completely eliminated by lovastatin. The subsequent activation of Na+/H+ exchange was partially inhibited by the isoprenylation antagonist. In contrast, the osmotic activation of the Na+/H+ antiport was unaffected. The data indicate that the osmotically- and receptor-induced activation of the antiport utilize divergent signalling pathways. The results also provide evidence supporting the notion that cytosolic acidification by chemoattractants results from accumulation of metabolic acid generated during the respiratory burst.

GTP-Binding Proteins↗
PubMed1993

Hepoxilin A3 inhibits the rise in free intracellular calcium evoked by formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4.

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human neutrophils through a pertussis toxin-sensitive, extracellular calcium-independent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-dependent influx of calcium is observed. In an effort to investigate further the role of these compounds in the human neutrophil, we investigated their potential effects on the action of known agonists such as formyl-methionine-leucine-phenylalanine (fMLP), platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepoxilis dose-dependently inhibited the increases in [Ca2+]i induced by fMLP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin did not cause any measurable change in [Ca2+]i. The extent of inhibition of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond its threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a constant rate of calcium leakage from stores), hepoxilin A3 increased further this rate of calcium leakage, indicating that hepoxilin acts by rapidly draining calcium from stores. Its potential (additional) thapsigargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins may serve an important negative regulatory function in the agonist-induced mobilization of calcium in these cells by depleting calcium stores.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells.

Deletion of the phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most prevalent mutation in cystic fibrosis (CF). This mutation (delta F508CFTR) leads to a reduced cAMP-sensitive Cl- conductance in epithelial cells. While the mutant protein can function as a Cl- channel, it seems to be misprocessed and unable to accumulate at normal levels in the plasma membrane. Under conditions where the biosynthetic block of delta F508CFTR is not complete, the residence time of delta F508CFTR in the plasma membrane is a critical determinant of the cAMP-sensitive Cl- conductance. To assess the stability of the mutant and wild-type CFTR, we compared their functional half-lives at the plasma membrane of transfected Chinese hamster ovary cells. The plasma membrane Cl- conductance was assessed by patch-clamp recordings and/or by fluorimetric determinations of the membrane potential. Accumulation of delta F508CFTR in the plasma membrane was promoted by growing the transfected cells at reduced temperature (24-28 degrees C), and was verified by immunoblotting and by detecting the appearance of a plasmalemmal cAMP-activated Cl- conductance. Subsequently increasing the temperature to 37 degrees C inhibited further delivery of newly synthesized delta F508CFTR to the surface membrane. By studying the time dependence of the disappearance of the Cl- conductance, the functional half-life of the mutant protein at the plasma membrane was determined to be < 4 h, which is considerably shorter than the half-life of wild-type CFTR (> 24 h). The latter was estimated by terminating protein synthesis or secretion with cycloheximide or brefeldin A, respectively. Inhibition of protein synthesis did not alter the rate of disappearance of delta F508CFTR at 37 degrees C, validating the difference in turnover between mutant and wild-type CFTR. These results indicate that the structural abnormality of delta F508CFTR affects not only the delivery of the protein to the plasma membrane, but also its stability therein. Moreover, they suggest that overcoming the processing block at the endoplasmic reticulum may not suffice to restore normal Cl- conductance in CF.

Animals↗
PubMed1993

Receptor-mediated activation of multiple serine/threonine kinases in human leukocytes.

Activation of the microbicidal response of phagocytes requires cytosolic ATP and is associated with extensive protein phosphorylation, suggesting the involvement of protein kinases in the signal transduction cascade. An in vitro renaturation assay was used to identify the protein kinase(s) activated by chemoattractants in human blood neutrophils. Four distinct kinases were activated by the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine with molecular masses of 72, 65, 49, and 41 kDa (designated PK72, PK65, PK49, and PK41, respectively). PK72 and PK65 were activated very rapidly (5-15 s), yet transiently. By comparison, PK49 and PK41 responded in a slower, more sustained manner. Treatment of extracts of activated cells with alkaline phosphatase reverted the stimulation of the kinases, suggesting that phosphorylation is the post-translational modification that underlies activation of the kinases. Stimulation of PK72 and PK65 by chemoattractant was independent of calcium and protein kinase C. In contrast, elevation of cytosolic free calcium levels was sufficient and appeared to be necessary for full activation of PK49 and PK41. While phorbol esters can mimic the effects of formyl-methionyl-leucyl-phenylalanine on PK49 and PK41, inhibition of protein kinase C by staurosporine did not prevent the receptor-mediated activation of these kinases. PK41 most likely corresponds to the Erk-1 isoform of mitogen-activated protein (MAP) kinase. Accordingly, PK41 effectively phosphorylated myelin basic protein, known to be a good substrate for Erk-1. The electrophoretic mobility of PK49 is similar to that of MAP kinase-kinase (MAP/Erk kinase). However, immunoprecipitation experiments indicated that PK49 is not MAP/Erk kinase. The identity of this and other kinases remains to be defined, but possible candidates are discussed. In addition to autophosphorylating, PK72, PK65, and PK41 were shown to effectively phosphorylate exogenous substrates. These kinases may therefore play a role in signal transduction during stimulation by chemoattractants.

Base Sequence↗