Biomedical subjects

S Grinstein

Publications and source records attributed to S Grinstein.

At least 127 records · Page 7Linked to original sources

PubMed1995

Volume regulation in leukocytes: requirement for an intact cytoskeleton.

Animal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL-60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL-60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F-actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F-actin content of the different cells was determined by NBD-phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 +/- 0.01 for CB and 0.48 +/- 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL-60 cells or human neutrophils. To study the effects of an increase in F-actin on volume regulation, neutrophils were treated with the chemoattractant f-Met-Leu-Phe or with an antibody (Ab) to beta 2 integrins followed by a cross-linking secondary Ab. Despite an increase in F-actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation.

Actins↗

PubMed1995

Chemoattractant-induced activation of vacuolar H+ pumps and of an H(+)-selective conductance in neutrophils.

Upon binding to their receptors on the surface of neutrophils, chemotactic peptides elicit a burst of metabolic activity. The excess acid generated by this burst must be rapidly extruded in order to maintain intracellular pH and preserve normal microbicidal responses. Recently, H(+)-pumping vacuolar-type ATPases (V-pumps) and a H(+)-selective conductance were described in the membrane of neutrophils. However, these systems are virtually quiescent in resting cells. In this report, we analyzed whether the V-pumps and the conductance become active and contribute to pH regulation following cell activation by chemoattractants. Formyl-Met-Leu-Phe (fMLP) was found to stimulate V-pumps, as assessed by the appearance of bafilomycin-sensitive H+ extrusion. Concomitantly, the chemoattractant also activated the H+ conductance, detected as a voltage-dependent and Zn(2+)-sensitive net H+ efflux. In both cases, activation was prevented by treatment with competing antagonistic peptides or with pertussis toxin, implying mediation by a receptor coupled to a heterotrimeric G protein. The signalling pathways downstream of the G proteins were also investigated. Stimulation of neither the V-pump nor the conductance required activation of protein kinase C. An elevation of cytosolic calcium ([Ca2+]i) comparable to that induced by fMLP did not suffice to trigger either transporter. Moreover activation of the conductance remained unaffected when the chemoattractant-induced increase in [Ca2+]i was precluded. In contrast, stimulation of the V-pump was substantially (approximately 50%) depressed when [Ca2+]i was prevented from rising. Tyrosine phosphorylation of several polypeptides accompanies stimulation by fMLP. Prevention of phosphotyrosine accumulation resulted in a pronounced inhibition of H(+)-pumping and of the H+ conductance. Together, these data indicate that engagement of surface receptors by chemotactic peptides can lead to stimulation of two voltage-sensitive pH regulatory pathways, a pump and a conductance, by a pathway that requires tyrosine phosphorylation. Both pathways are capable of sizable H+ extrusion, thereby contributing to pH regulation during the metabolic burst.

Calcium↗

PubMed1995

Epstein-Barr virus (EBV) latent membrane protein (LMP) in tumor cells of Hodgkin's disease in pediatric patients.

Twenty-nine out of 31 consecutive pediatric patients with Hodgkin's disease treated at our hospital from 1988 to 1992 were studied. The selection criterion was the availability of sufficient formalin-fixed, paraffin-embedded tissue for analysis. Patient age ranged from 3 to 15 years with a median age of 7 years. Lymph node biopsies were examined for the presence of Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) in malignant cells by peroxidase immunolabeling. LMP positivity was present in 10/15 (67%) of mixed cellularity, 1/6 (17%) of lymphocyte predominance, 0/7 (0%) of nodular sclerosis, and 1/1 (100%) of lymphocyte depletion. Positive cases by age range were: 10/12 (83%) for 3-6 years and 2/17 (11%) for 7-15 years. The association between EBV and Hodgkin's disease in children appeared to be more frequent in patients with mixed cellularity and those in the 3-6 age range, through examples of EBV-positive tumors were found in other histologic subtypes, stages and ages. Findings indicate that Hodgkin's disease in children is at least as strongly linked to EBV as in adults. Furthermore, we suggest that the EBV is associated with a subgroup of patients which can be defined on the basis of the age at diagnosis.

Adolescent↗

PubMed1995

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

Animals↗

PubMed1995

Ion transport and the function of phagocytic cells.

Inorganic ions constitute the predominant osmolytes of blood cells and are therefore the main determinant of the cellular volume. In leukocytes, maintenance of constant cell size is accomplished by modulating the ionic permeability of the membrane. In addition, the plasmalemmal ionic permeability dictates the transmembrane potential, which influences a variety of cellular responses. Inorganic ions are also essential for the regulation of the intracellular pH and play an active role in signal transduction during phagocyte activation. This chapter reviews recent progress in the area of ion transport in phagocytic leukocytes, with special reference to the physiologic implications to the activation process.

Animals↗

PubMed1995

Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress.

Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be activated by shrinking after an acid load. Jointly, these results suggest that the H+ affinity and high cooperativity of the antiporter, earlier attributed to a single "modifier site," can be varied independently and are probably controlled by different regions of the molecule. The data indicate that volume or osmolarity-sensitive site(s) exist between the NH2-terminus and residue 566. This putative volume-sensitive site is therefore different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors.

Animals↗

PubMed1995

Signaling events in monocytes and macrophages.

The cellular signaling events leading to the systemic inflammatory response syndrome and sepsis in monocytes/macrophages activated by lipopolysaccharide (LPS) are well understood. LPS is a glycolipid component of Gram-negative bacterial cell wall. It exerts its effect through the lipid A moiety. LPS binds to monocytes/macrophages via a membrane-bound receptor, CD14, an interaction which is optimized in the presence of plasma factors, LPS-binding protein, and septin. Although LPS is known to bind to other receptors, the roles of these receptors in transmembrane signaling and activation of monocytes/macrophages are not as well understood as is that of the CD14 receptor. Intracellular events in response to LPS stimulation are mediated by phospholipase (PL) C, protein kinases, PLA2, and PLD. Activation of PLC by LPS results in the release of diacylglycerol and inositol 1,4,5-trisphosphate. The former mediates the stimulation of protein kinase C, and the latter induces an increase in intracellular calcium concentration. LPS stimulation of monocytes/macrophages also results in the phosphorylation and activation of several protein kinases, including protein tyrosine kinases which mediate cytokine production, and mitogen-activated protein kinase which activates cytosolic PLA2 to release arachidonate. LPS also plays a role in cellular proliferation and differentiation. Upregulation of the secretory form of PLA2 has also been documented in response to LPS. PLD is stimulated by LPS to release phosphatidic acid (PA). PA can activate the respiratory burst by increasing diacylglycerol production and by modulating the effects of guanine nucleotide-binding proteins. Therapeutic strategies to decrease the clinical effects of sepsis would logically include agents which block at initial receptor-ligand interaction, as well as those which attenuate the intracellular events that follow LPS stimulation. Early in vivo studies are promising, but clearly much work remains to be done.

Animals↗

PubMed1995

An iron-binding Trypanosoma cruzi urinary antigen.

An 80-kDa Trypanosoma cruzi urinary antigen (UAg) was affinity-purified from the urine of infected dogs. We demonstrated that UAg is structurally and functionally related to proteins belonging to the transferrin family, as shown by amino acid sequence and iron binding experiments. Nevertheless, monoclonal antibodies raised against UAg specifically and selectively recognized this parasite's circulating antigen. The existence of an 80-kDa T. cruzi antigen co-migrating with UAg could be confirmed when epimastigotes were metabolically labelled with [35S] methionine and then immunoprecipitated with the above mentioned antibodies. We conclude that UAg is an iron-binding T. cruzi component eliminated in the urine of the infected host.

Amino Acid Sequence↗

PubMed1994

Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP.

Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild-type (wt) and mutant (delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all delta F508 CFTR and 45-80% of wt CFTR were rapidly degraded with a similar half-life (t1/2 approximately 0.5 h). The remaining wt CFTR attained a protease-resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease-resistant wt CFTR. Intracellular degradation of delta F508 CFTR and of incompletely folded wt CFTR occurs in a non-lysosomal, pre-Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease-sensitive form which is partially converted by an ATP-dependent process to a more mature form that is protease-resistant and capable of leaving the ER. The inability delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre-Golgi compartment.

Adenosine Triphosphate↗

PubMed1994

Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants.

In addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.

Cells, Cultured↗

PubMed1994

Assessment of the contribution of the cytochrome b moiety of the NADPH oxidase to the transmembrane H+ conductance of leukocytes.

Phagocytic cells can kill microorganisms by synthesizing superoxide. Activation of the NADPH oxidase that generates superoxide is accompanied by a large intracellular burst of metabolic acid production. Despite the excess acid generation, cytosolic pH (pHi) remains near neutrality due to the concomitant stimulation of several homeostatic H+ extrusion mechanisms including a recently described H(+)-conductive pathway. Activation of the conductance by phorbol esters is defective in neutrophils of chronic granulomatous disease (CGD) patients lacking the transmembrane cytochrome b subunits of the NADPH oxidase. This finding suggests that the oxidase itself undertakes H+ translocation or that, alternatively, assembly of the oxidase is required to activate a separate H+ conducting entity. To distinguish between these possibilities, the presence of the conductive pathway was assessed in unstimulated normal and CGD cells by manipulating pHi and the transmembrane potential. Using fluorimetric determinations of pHi, a conductive, Zn(2+)-sensitive alkalinization was observed in neutrophils from both normal and cytochrome b-deficient CGD donors. The electrophysiological properties of the conductance were defined in purified blood monocytes using the whole cell configuration of the patch clamp. Depolarizing pulses induced slowly activating outward currents in cells from both normal and cytochrome b-deficient individuals. The elicited currents were potentiated by cytosolic acidification and did not inactivate within the times tested. As in control leukocytes, the reversal potential of tail currents in the CGD cells closely approximated the H+ equilibrium potential and was unaffected by substitution of the major ionic components of the external bathing medium. At all voltages tested, the magnitude of the evoked currents was comparable in normal and CGD cells. The results indicate that, like macrophages and granulocytes, human monocytes display a voltage-gated highly H(+)-selective conductance. More importantly, our findings imply that the conductive pathway is present in cells devoid of cytochrome b. Therefore, the defective activation of the conductive pathway by protein kinase C agonists in CGD cells is not due to the physical absence of the transporter. Instead we propose that the oxidase functions in a regulatory capacity, facilitating the opening of a distinct H+ conductance during cellular stimulation.

Biological Transport↗

PubMed1994

Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation.

Four distinct isoforms of the mammalian Na+/H+ exchanger (NHE) have been identified by molecular cloning. Three of these (NHE-1, NHE-2, and NHE-3) have been shown to be functionally active by heterologous expression. Their kinetic and pharmacological properties are well documented, yet comparatively little is known about their regulation. In this report, rat NHE-1, NHE-2, and NHE-3 were stably transfected into antiporter-deficient Chinese hamster ovary cells to study their role in cellular proliferation and their regulation by nucleotides and cell volume. Their ability to influence cell proliferation was assessed by measuring the growth of antiporter-deficient cells and of the different transfectants in media of varying pH. While antiporter-deficient cells were unable to grow at acidic pH levels, all three isoforms supported proliferation under these conditions. Therefore, while the epithelia-specific isoforms (NHE-2 and NHE-3) are thought to play primarily a role in transcellular ion transport, they can also contribute to intracellular pH (pHi) homeostasis and have a permissive role in cell growth. The activity of the three isoforms was markedly inhibited by depletion of cellular ATP. In the pHi 6.0-7.2 range, decreases in the affinity for internal H+ and/or the maximal rate of transport accounted for the inhibitory effect, depending on the isoform. The osmotic responsiveness of the three isoforms was also compared. As reported earlier, NHE-1 was stimulated by hypertonicity. Under similar conditions, NHE-2 was also stimulated to a comparable extent. Conversely, both isoforms were inhibited in hypotonic media. In contrast, NHE-3 was markedly inhibited by hypertonic cell shrinking but was unaffected by hypotonicity. Osmotic inhibition of NHE-3 was rapid, reversible, and apparent throughout the pH range studied. Osmotic inhibition of NHE-3 may play a role in the physiology and pathophysiology of epithelia.

Adenosine Triphosphate↗

PubMed1994

Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages.

The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.

Animals↗

PubMed1994

Chemotactic peptides induce phosphorylation and activation of MEK-1 in human neutrophils.

Extracellular signal-regulated kinase (Erk) (mitogen-activated protein (MAP) kinase) is rapidly activated when neutrophils are stimulated. Several isoforms of MAP/Erk kinase (MEK), a kinase capable of phosphorylating and activating Erk, have been identified, but their distribution and differential roles in leukocytes are unknown. We studied the effect of chemotactic stimulation on MEK-1, using isoform-specific antibodies. MEK-1 was found to be phosphorylated on serine and threonine residues in unstimulated human neutrophils. Stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced serine/threonine phosphorylation of MEK-1, while reducing its electrophoretic mobility. MEK-1 activity, measured as autophosphorylation or as phosphorylation of a glutathione S-transferase-Erk fusion protein, was undetectable in unstimulated cells but became evident after treatment with chemoattractant. Phosphorylation and activation of MEK-1 were rapid and transient, peaking after 1-2 min and returning to base line by 10 min. Experiments using electropermeabilized cells indicated that elevation of cytosolic Ca2+ is not required for activation of MEK-1 by fMLP. Moreover, MEK-1 was not stimulated by either platelet-activating factor or thapsigargin, which increase Ca2+ to levels comparable with those attained in chemoattractant-activated cells. In contrast, activation of MEK-1 was induced by phorbol esters, and the stimulatory effect of fMLP was blocked by an antagonist of protein kinase C. Stimulation of MEK-1 was also blocked by concentrations of erbstatin that prevent the fMLP-induced accumulation of tyrosine-phosphorylated proteins. The data suggest that MEK-1 is largely responsible for the activation of Erk in chemoattractant-stimulated neutrophils and that protein kinase C and/or tyrosine kinases mediate this effect, whereas elevated cytosolic Ca2+ is not essential.

Amino Acid Sequence↗

PubMed1994

Activation of multiple pH-regulatory pathways in granulocytes by a phosphotyrosine phosphatase antagonist.

Activated phagocytes undergo a massive burst of metabolic acid generation, yet must be able to maintain their cytosolic pH (pHi) within physiological limits. Peroxides of vanadate (V(4+)-OOH), potent inhibitors of phosphotyrosine phosphatases, have recently been shown to produce activation of the respiratory burst in HL60 granulocytes. We therefore investigated the effects of V(4+)-OOH on pHi homoeostasis in HL60 granulocytes, using a pH-sensitive fluorescent dye. V(4+)-OOH stimulation induced a biphasic pH change: a transient cytosolic acidification followed by a significant alkalinization. The initial acidification was prevented by inhibition of the NADPH oxidase and was absent in undifferentiated cells lacking oxidase activity. Analysis of the alkalinization phase demonstrated the involvement of the Na+/H+ antiporter, and also provided evidence for activation of two alternative H(+)-extrusion pathways: a bafilomycin-sensitive component, likely reflecting vacuolar-type H(+)-ATPase activity, and a Zn(2+)-sensitive H(+)-conductive pathway. Our results indicate that V(4+)-OOH stimulation not only activated the NADPH oxidase but concomitantly stimulated H(+)-extrusion pathways, enabling the cells to compensate for the massive production of intracellular H+ associated with the respiratory burst.

Culture Media↗

PubMed1994

ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization.

ATP is not hydrolyzed during the transport cycle of the Na+/H+ exchanger (NHE), yet depletion of the nucleotide drastically reduces the rate of cation exchange. The mechanism underlying this inhibition was investigated in fibroblasts transfected with NHE-1, the growth factor-sensitive isoform of the antiport. NHE-1 was found to be phosphorylated in serum-starved, unstimulated cells. Acute ATP depletion induced a profound inhibition of transport without detectable changes in NHE-1 phosphorylation. Analysis of cells transfected with truncated mutants of NHE-1 indicated that the carboxyl-terminal cytosolic domain of the antiport is required for expression of its ATP dependence. To define whether inhibition of Na+/H+ exchange resulted from internalization of NHE-1, extracellularly exposed proteins were labeled with impermeant biotin derivatives. The proportion of NHE-1 exposed to the surface was comparable before and after ATP depletion. Immunofluorescence determinations revealed focal accumulations of NHE-1 on the membrane of untreated cells. NHE-1 redistributed following ATP depletion, showing a more homogeneous localization. F-actin, which co-localizes with the antiport in untreated cells, also redistributed when cells were ATP depleted. These findings suggest an interaction of NHE-1 with the cytoskeleton. Accordingly, disassembly of actin filaments with cytochalasin D induced redistribution of the antiport. However, Na+/H+ exchange activity was unaltered by cytochalasin D. We propose that ancillary proteins confer ATP sensitivity to the antiporter and may also mediate its association with the cytoskeleton. Depletion of the nucleotide would alter the interaction between NHE-1 and the putative regulator, inhibiting Na+/H+ exchange and inducing subcellular redistribution. However, disruption of the cytoskeleton at distal sites, such as induced by cytochalasins, is insufficient to inactivate the antiport.

Adenosine Triphosphate↗

PubMed1994

Integration of human papillomavirus type 6a DNA in a tonsillar carcinoma: chromosomal localization and nucleotide sequence of the genomic target region.

Human papillomavirus type 6a (HPV 6a) DNA was detected in a tonsillar carcinoma both as integrated and episomal molecules, and one viral-cellular junction was molecularly cloned (Bercovich et al., J. Gen Virol., 72: 2569-2572, 1991). The cellular sequence was used as a probe for the isolation of a cosmid from a normal human genomic DNA library. A 2.7-kilobase subclone including the integration site was sequenced. It was shown to contain sequences with similarities to the E2 and L2 regions of human papillomaviruses, a 5' truncated long interspersed repeated DNA element type 1 retrotransposon, and a fragment of an O-repeat element. The chromosomal localization of the integration site was determined to be at region 24 of the long arm of chromosome 10 (10q24). This is the region where the fragile site is located in which HPV 18 DNA is integrated in the cell line FEP18-5. In addition it contains the site of breakpoints affecting protooncogenes Hox11 and Lyt10. Other genes related to cell division and DNA repair have also been mapped to this chromosomal band. Analysis of genomic DNA of cell lines and patients using 10q24-derived probes is presented. The integration of human papillomavirus type 6 DNA into chromosome 10q24 may have disrupted a cellular gene critical for normal cell growth, which further analysis should help to identify.

Base Sequence↗

PubMed1994

Arachidonic acid stimulates the plasma membrane H+ conductance of macrophages.

When activated, phagocytes undergo a large burst of metabolic acid production. Deleterious cytosolic acidification is prevented by extrusion of H+ (equivalents) through specific transport systems, including a recently described H+ conductive pathway. The conductance can be activated by cytosolic acidification and by depolarization, events known to occur during phagocyte activation. It is possible, however, that the conductance is also directly stimulated by agonists or second messengers. In this report, spectroscopic and electrophysiological determinations were used to assess the effects of arachidonic acid (AA), a potent phagocyte stimulant, on the plasmalemmal H+ conductance of murine macrophages. AA greatly enhanced the slowly activating H+ currents and the cytosolic alkalinization triggered by depolarizing pulses. The H+ current in AA-treated cells appeared at more negative potentials and its activation and deactivation became faster. The ionic selectivity, outward rectification, and pharmacological properties of the stimulated current were identical to those of the basal current, suggesting that AA acts by facilitating the activation of the endogenous H+ conductive pathway, rather than by exerting a protonophoric effect. Experiments using specific inhibitors suggested that the effects of AA are not mediated by lipo- or cyclooxygenase. Comparison of the effects of a variety of fatty acids supported this conclusion. The order of potency to stimulate the conductance was: AA > palmitoleate approximately palmitelaidate > linoleate > oleate > elaidate. Saturated fatty acids were inactive. This sequence shows striking similarity with the ability of these lipids to stimulate the NADPH oxidase. The results indicate that, simultaneously with the activation of metabolic acid generation, phagocyte agonists also directly activate regulatory H+ extrusion, thereby favoring maintenance of intracellular pH in the physiological range.

Animals↗