Biomedical subjects

S Grinstein

Publications and source records attributed to S Grinstein.

At least 163 records · Page 9Linked to original sources

PubMed1993

Regulation of tyrosine phosphorylation in neutrophils by the NADPH oxidase. Role of reactive oxygen intermediates.

Neutrophils possess a multicomponent NADPH-oxidase that produces large quantities of superoxide, which can in turn generate other reactive oxygen intermediates. Superoxide and its dismutation product, hydrogen peroxide, are powerful oxidants. Because the activity of certain tyrosine kinases and phosphatases can be affected by their redox state, we considered the possibility that endogenously generated reactive oxygen intermediates (ROI) may alter phosphotyrosine formation and thereby function as intra- or intercellular messengers in neutrophils. Exposure of human neutrophils to exogenous oxidants such as diamide induced marked tyrosine phosphorylation of several cellular proteins. More importantly, activation of the NADPH oxidase in permeabilized neutrophils, by direct stimulation of GTP-binding proteins, also resulted in enhanced tyrosine phosphorylation. The latter was NADPH-dependent, paralleled by production of superoxide, and was inhibited by diphenylene iodonium, an inhibitor of the flavoprotein component of the oxidase. Neutrophils, from a patient with chronic granulomatous disease, which are deficient in the production of ROI, demonstrated no such phosphotyrosine accumulation. We conclude that ROI produced by the NADPH oxidase can regulate tyrosine phosphorylation in granulocytes, possibly by effects of oxidation-sensitive tyrosine kinases and/or phosphatases.

Animals↗

PubMed1993

Cytosolic [Ca2+] homeostasis and tyrosine phosphorylation of phospholipase C gamma 2 in HL60 granulocytes.

Activated phagocytes produce large amounts of reactive oxygen intermediates, including peroxides. In addition to their microbicidal effect, it has recently been suggested that reactive oxygen species play a role as intracellular messengers. The mechanism of action remains unknown, but peroxides have been reported to increase tyrosine phosphorylation, an effect potentiated by vanadate. In this report we studied the effects of a combination of H2O2 and vanadate on Ca2+ homeostasis in granulocytic HL60 cells. The peroxides induced a transient elevation of cytosolic [Ca2+] associated with release from internal stores. Ca2+ mobilization was accompanied by increased generation of inositol 1,4,5-trisphosphate, implicating phospholipase C (PLC). A sizable increase in phosphotyrosine accumulation by several polypeptides in the M(r) 20,000 to 250,000 range preceded the [Ca2+] changes. We therefore considered the possibility that tyrosine phosphorylation of a phospholipase mediates the observed effects. Differentiated (granulocytic) HL60 cells did not have detectable levels of PLC gamma 1 but had substantial PLC gamma 2. Immunoprecipitation and immunoblotting experiments demonstrated that PLC gamma 2 becomes tyrosine-phosphorylated upon treatment of the cells with peroxides of vanadate. If associated with activation, such phosphorylation of PLC gamma 2 can account for the rise in [Ca2+]. Although capable of mobilizing internal Ca2+ stores, the peroxides failed to produce the sustained [Ca2+] increase predicted by the "capacitative" model. Mn2+ influx determinations indicated that this is due to impairment of divalent cation entry by the peroxides, uncoupling the plasma membrane from the internal stores. Changes in [Ca2+] homeostasis could mediate some of the messenger actions of reactive oxygen species.

Amino Acid Sequence↗

PubMed1993

Abnormal activation of H+ conductance in NADPH oxidase-defective neutrophils.

To combat bacterial infection, phagocytes generate superoxide (O2-) and other microbicidal oxygen radicals. NADPH oxidase, the enzyme responsible for O2- synthesis, is deficient in chronic granulomatous disease (CGD) patients. Although O2- generation is accompanied by a large burst of metabolic acid production, intracellular pH (pHi) remains near neutrality due to the concomitant stimulation of H+ extrusion. Three major pathways contribute to pHi regulation in activated phagocytes: Na+/H+ exchange, vacuolar-type H+ pumps, and a H+ conductance. The present study analyzed the relationship between activation of the NADPH oxidase and stimulation of the H+ extrusion mechanisms in human blood neutrophils. Phorbol ester-induced activation of Na+/H+ exchange and H+ pumping occurred normally in cells from CGD patients. Unlike normal individuals, however, CGD patients were unable to activate the H+ conductive pathway. Thus, activation of the H+ conductance appears to be contingent on the assembly of a functional NADPH oxidase. These findings imply a dual role of the NADPH oxidase in O2- synthesis and in the regulation of pHi. The oxidase (or some components thereof) may itself undertake H+ translocation or, alternatively, may signal the activation of a separate H+ conducting entity.

Biological Transport, Active↗

PubMed1993

Okadaic acid-induced actin assembly in neutrophils: role of protein phosphatases.

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of 32P into cellular proteins using SDS-PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid.

Actins↗

PubMed1993

Relative roles of Na+/H+ exchange and vacuolar-type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages.

Two distinct mechanisms have been shown to mediate cytoplasmic pH (pHi) recovery in acid-loaded peritoneal macrophages (M phi s): Na+/H+ exchange and H+ extrusion by vacuolar-type (V-type) H+ ATPases. The present studies examined the relative roles of these two systems in maintaining pHi and cell function. Measurements of M phi pHi and superoxide (O2-) production in response to stimulation with 12-O-tetradecanoyl phorbol 13-acetate (TPA) were made at physiological or acidic extracellular pH (pHo) levels. The V-type H+ ATPase inhibitor, bafilomycin A1, and the potent Na+/H+ exchange inhibitor, N-ethyl-N-propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pHi regulation and cell function. At pHo 7.35, the complementary activities of the Na+/H+ antiport and the V-type H+ ATPase mediate pHi homeostasis. At pHo 6.7, maintenance of pHi depends primarily on H+ ATPase activity: bafilomycin A1 reduced pHi from 6.8 +/- 0.02 in control cells to 6.59 +/- 0.01 (P < 0.01) while EPA was without effect. The functional importance of V-type H+ ATPase-activity in preserving pHi homeostasis at acidic extracellular pH levels was reflected by the impairment of O2- production at pHo 6.70 when H+ ATPase activity was inhibited: bafilomycin A1 reduced O2- production from 13.9 +/- 1.0 to 9.3 +/- 0.6 nmoles/10(6) cells/40 min, in control and bafilomycin A1-treated cells, respectively (P < 0.05), while EPA had no effect. In subsequent studies, pHi was independently manipulated using the ionophore nigericin. Lowering pHi from 6.80 to 6.60 reduced O2- production from 15.3 +/- 1.8 to 9.8 +/- 1.6 nmoles/10(6) cells/40 min (P < 0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ ATPases at low pHo could account for the associated impairment of O2- production. In a more profoundly acidic environment (pHo 6.35), H+ ATPases remained active in regulating pHi, but could not preserve a sufficiently physiological pHi to support respiratory burst activity. V-type H+ ATPases constitute the dominant mechanism by which the pHi of peritoneal M phi s is maintained in an acidic extracellular environment.

Adenosine Triphosphatases↗

PubMed1993

Effect of anti-gamma-interferon and anti-interleukin-4 administration on the resistance of mice against infection with reticulotropic and myotropic strains of Trypanosoma cruzi.

We studied the effect of in vivo administration of anti-gamma-IFN and anti-IL-4 monoclonal antibodies on the resistance of mice against myotropic and reticulotropic strains of Trypanosoma cruzi. Anti-gamma-IFN treatment augmented the susceptibility of mice when infected with the reticulotropic RA and Tulahuén strains of T. cruzi but did not alter the course of infection with the myotropic CA-I strain of the parasite. In vivo administration of anti-IL-4 enhanced the resistance of mice when infected with either Tulahuén or RA strains but did not affect the course of parasitemia when infected with CA-I. The possible biological relevance of these observations is discussed.

Animals↗

PubMed1993

A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages.

Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H+ extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H+ conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na+ and HCO3(-)-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H+ (equivalent) pathway. This notion was confirmed by patch-clamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H+ (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intra-and/or extracellular media, (b) the reversal potential of the tail currents approached the H+ equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn2+ sensitive and had similar time course and potential dependence. The peak whole-cell current displayed marked outward rectification and was exquisitely H+ selective. At constant voltage, the H+ permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H+ extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these cells undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H+ extrusion required for maintenance of pHi.

Animals↗

PubMed1993

Proton conductance of the plasma membrane: properties, regulation, and functional role.

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.

Animals↗

PubMed1993

Proton currents in human granulocytes: regulation by membrane potential and intracellular pH.

1. To determine whether conductive pathways contribute to the H+ efflux from granulocytes, we used the whole-cell patch-clamp technique combined with microfluorimetric determinations of cytosolic pH (pHi) in single, dimethylsulphoxide-differentiated HL-60 cells. 2. In voltage-clamp mode, depolarization of the cell from the resting potential (around -60 mV) to +60 mV caused an increase in pHi that was accompanied by a sizeable outward current. 3. Ion substitution experiments and analysis of the reversal potential of tail currents indicated that the outward current is carried largely by H+ ions. 4. Full activation of the H+ current occurred within 1-2s after depolarization and deactivation within 100-200 ms upon repolarization. 5. This H+ conductance was strongly dependent on pHi, being larger at acidic pH. In addition, at low pHi the threshold for voltage activation of the H+ conductance was shifted to more negative values. 6. Addition of millimolar concentrations of Cd2+ and Zn2+ to the bath solution reduced the maximum H+ conductance and shifted the voltage dependence of the H+ conductance to more positive potentials. The effects were reversible. 7. In conclusion, our results demonstrate that granulocytic HL-60 cells possess a voltage-gated and pHi-sensitive H+ conductance. Because both a depolarization and a cytosolic acidification occur during the activation of granulocytes, this conductance may play a role in pHi homeostasis of granulocytes during microbial killing.

Cations, Divalent↗

PubMed1993

[DNA identification of human papillomavirus in anogenital condyloma of a male population].

Human Papillomaviruses (HPV) are the cause of benign human anogenital lesions where HPV 6 and HPV 11 are most commonly found. Conversely, HPV 16, 18, 31 and 33 are frequently detected in genital carcinomas and are thus considered as oncogenic types. In order to analyze the prevalence of specific HPV types in an Argentine male population, 43 anogenital lesions from different patients with diagnosis of condyloma acuminata were analyzed. These lesions were localized in different regions of the male genitalia comprising the corona glandis, urethral meatus, skin of the penis, scrotum and anus. The biopsies were screened for the presence of HPV 6, 11, 16, 18, 30, 31 and 33 by Southern blot at different stringent conditions of hybridization (Tm -48 degrees C and Tm -20 degrees C). HPV DNA was found in 41 examined cases (95.3%) with a clear prevalence of HPV 6 and HPV 11 types (51.2% and 23.3% respectively). Six samples (14.0%) were positive only under nonstringent conditions of hybridization. Mixed infections between HPV 16, 18, 30, 31, 33 or a HPV 30 related type with HPV 6 or HPV 11 were detected in 8 specimens (18.6%). Only one case was between HPV 16 and HPV 30. Two additional samples were only positive for HPV 30. Experiments in progress about the prevalence of HPV types in female lesions as well as in normal subjects will contribute to complete the description of the epidemiology of these infections in Argentina.

Adult↗

PubMed1993

Involvement of multiple kinases in neutrophil activation.

Production of reactive oxygen metabolites by the NADPH oxidase is an essential mechanism underlying the microbicidal role of phagocytes. Receptor-mediated activation of the oxidase was originally thought to be mediated by calcium and/or by protein kinase C (PKC). However, recent evidence suggests that additional signalling pathways exist. In this article the possible role of tyrosine phosphorylation is discussed. In addition, results obtained using an in vitro kinase renaturation assay are described. The latter assay revealed the existence of at least four serine/threonine kinases that are activated in cells stimulated with chemoattractants. One of these, of molecular weight 41,000 was identified as a member of the ERK or MAP-kinase family. The existence of multiple, possibly redundant or synergistic signaling pathways is considered.

Animals↗

PubMed1992

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.

Animals↗

PubMed1992

Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation.

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.

Adult↗

PubMed1992

Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.

Activation of human neutrophils by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) induces tyrosine phosphorylation of several polypeptides, including a prominent band of approximately 41 kDa. A polypeptide of identical electrophoretic mobility was recognized by a monoclonal antibody raised against a sequence corresponding to amino acids 325-345 of ERK-1, one of a family of mitogen-activated protein (MAP) kinases. To establish the possible identity of these polypeptides, extracts from control and fMLP-treated cells were immunoprecipitated with immobilized antiphosphotyrosine antibodies. Reactivity with anti-ERK-1 antibodies was observed only in the precipitate of chemoattractant-stimulated cells. These data imply that a MAP kinase constitutes at least part of the tyrosine-phosphorylated 41-kDa polypeptide. By using an in vitro renaturation assay, treatment of intact cells with fMLP was found to stimulate several protein kinases, including one of approximately 41 kDa. Renaturation of samples immunoprecipitated with antiphosphotyrosine antibodies revealed the presence of an active protein kinase in chemoattractant-stimulated, but not in control cells. The immunoprecipitated kinase comigrated with the 41-kDa tyrosine phosphorylated polypeptide and the anti-ERK-1 reactive band. We conclude that a MAP kinase closely related or identical to ERK-1 is tyrosine phosphorylated and activated when human neutrophils are stimulated by chemotactic peptides. The rapid phosphorylation of this kinase, which is apparent within seconds, is compatible with a role in the activation of the respiratory burst and/or other neutrophil responses.

Blotting, Western↗

PubMed1992

The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to defective Cl- conductance in epithelial cells. While the CFTR gene product has been detected in the plasma membrane, its presence and functional role in the membranes of intracellular compartments remain to be established. The purpose of the present experiments was to functionally localize CFTR in the endosomal membrane and to test the role of the associated Cl- conductance in the regulation of endosomal pH (pH(en)). When using conductive protonophores, the net H+ flux across the endosomal membrane of Chinese hamster ovary (CHO) cells is limited by the movement of counterions. Thus, ionic permeability could be estimated indirectly, from the changes in pH(en) determined fluorimetrically. Measurements in situ and in a cell-free microsomal preparation indicate the presence of a protein kinase A (PKA)-activated anion conductance in endosomes from CHO cells transfected with CFTR, but not in endosomes from wild-type or mock-transfected cells. In endosomes isolated from CFTR-expressing cells, the stimulatory effect of PKA was diminished by a specific peptide inhibitor of PKA, by alkaline phosphatase treatment or by a monoclonal antibody against the second nucleotide binding fold of CFTR. Increasing counterion permeability by phosphorylation of CFTR or by addition of valinomycin failed to alter the rate or extent of endosomal acidification in situ. Our observations indicate that functional CFTR, susceptible to activation by PKA, is present in endosomes of transfected CHO cells. More importantly, the data suggest that factors other than counterion permeability are the major determinants of pH(en).

Alkaline Phosphatase↗

PubMed1992

Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation.

To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.

Adenosine Triphosphate↗

PubMed1992

Lipopolysaccharide impairs macrophage cytoplasmic pH regulation under conditions simulating the inflammatory microenvironment.

Within the acidic inflammatory milieu, macrophages (m phi s) must maintain their cytoplasmic pH (pHi) within a range conducive to optimal function. It was previously shown that metabolism of L-arginine at concentrations present in vitro in RPMI medium (1.14 mM) impairs the ability of m phi s to regulate pHi. However, concentrations of L-arginine in vivo reportedly range from approximately 100 microM in serum to less than or equal to 50 microM in wounds. To investigate the potential in vivo relevance of this inhibition, m phi pHi regulation was examined following incubation with low concentrations of L-arginine that mimic the inflammatory microenvironment, in the presence or absence of lipopolysaccharide (LPS). pHi regulation was evaluated as the ability of thioglycolate-elicited murine peritoneal m phi s to recover from an imposed cytoplasmic acid load. The m phi pHi was measured using a pH-sensitive fluorescent probe. Following incubation for 2 h in the absence of LPS, the pHi recovery rate was equivalent in cells incubated with and without L-arginine. Coincubation with LPS, however, resulted in marked inhibition of pHi recovery at L-arginine concentrations as low as 12.5 microM. The inhibition was not due to LPS alone, since LPS without L-arginine was not inhibitory. Inhibition of pHi recovery was observed at LPS concentrations ranging from 10 ng/ml to 10 micrograms/ml. The L-arginine-dependent inhibition was apparent within 60 min of exposure to LPS, in both freshly harvested cells and cells preincubated for 2 h in the absence of L-arginine and then exposed to both L-arginine and LPS. Under conditions mimicking the in vivo setting, LPS-stimulated L-arginine metabolism impairs m phi pHi regulation. Modulation of pHi by this mechanism may compromise m phi function within the acidic microenvironment of inflammation.

Animals↗

PubMed1992

Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families.

To examine the role of enteric adenoviruses (EAV) in an urban area of Buenos Aires (Argentina), we prospectively studied faecal samples from 49 families of newborns. These were monitored weekly for diarrhoea for 2 years. A total of 180 samples from cases of diarrhoea and 766 samples obtained during diarrhoea-free periods were studied by dot-blot hybridization with an EAV-specific DNA probe. EAV were found in 6/180 (3.3%) cases of diarrhoea and 6/766 (0.8%) asymptomatic samples (P < 0.015). Incidence of EAV was 3.9 cases per 100 person-years in children < 60 months old. EAV-related diarrhoeas were slight and of short duration. In addition, 129 faeces from hospital out-patients, 1-30 months old, were also studied. EAV was identified in 7/129 cases (5.4%). These cases were 9.5 +/- 3.5 months old and the diarrhoea was mild or severe, of 3 +/- 1.5 days of duration. We suggest that EAV are low-risk causes of diarrhoea under natural conditions, although a few children may develop more severe diarrhoea. The diagnosis of EAV needs to be considered in these patients.

Adenovirus Infections, Human↗