PubMed Health⌕ Search

Biomedical subjects

E K Gallin

Publications and source records attributed to E K Gallin.

At least 19 recordsLinked to original sources
PubMed1992

G-protein activators induce a potassium conductance in murine macrophages.

The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.

4-Aminopyridine↗
PubMed1992

Comparative effects of particulate and soluble glucan on macrophages of C3H/HeN and C3H/HeJ mice.

In order to compare both the actions of soluble glucan (glucan-F) and particulate glucan (glucan-P) on macrophages and the responsiveness of macrophages from C3H/HeJ and C3H/HeN mice to these immunomodulators, interleukin-1 (IL-1) levels, phagocytosis and superoxide production were monitored after an in vitro exposure to glucan-F or glucan-P. A 2 or 20 h exposure to either glucan preparation decreased the ability of both C3H/HeJ and C3H/HeN macrophages to ingest zymosan. In contrast, glucan-P, but not glucan-F, decreased (after a 20 h exposure) the uptake of both IgG opsonized erythrocytes and latex beads. Furthermore, glucan-P, but not glucan-F was as effective as zymosan (after a 1 h exposure) in inducing superoxide release by macrophages isolated from both C3H/HeN and C3H/HeJ mice. While the effects of glucan-P on PMA-induced superoxide release and IL-1 levels were similar in macrophages from C3H/HeJ and C3H/HeN mice, glucan-F was ineffective at enhancing PMA-induced superoxide release or increasing IL-1 levels in C3H/HeJ mice. Thus (1) the effects of glucan-P on phagocytosis of opsonized erythrocytes and latex beads are not mimicked by glucan-F and (2) while macrophages from C3H/HeJ mice respond normally (as compared with C3H/HeN macrophages) to glucan-P, they are hyporesponders to glucan-F. These findings indicate that the activation of macrophages by glucan-P involves different (or additional) pathways from those activated by glucan-F.

Animals↗
PubMed1992

Comparison of apical and basal surfaces of confluent endothelial cells: patch-clamp and viral studies.

The distribution of inwardly rectifying (Ki) and calcium-activated (KCa) potassium channels on the apical and basal surfaces of bovine aortic endothelial cells (BAECs) was examined by inverting BAEC monolayers onto polylysine-coated cover slips. To monitor cellular polarity, we examined human red blood cell adherence (hemadsorption) to the influenza virus protein, hemagglutinin (HA), and virus budding on the surface of infected BAECs. Hemadsorption and virus budding occurred on the apical surface but were not apparent on the basal surface of monolayers 1 and 5 h after inversion, although cellular HA antigen localization confirmed that all monolayers were infected. In contrast, by 9.5 and 24 h after inversion, hemadsorption was evident on the "new" apical surface. Single-channel patch-clamp analysis revealed the presence of both Ki and KCa channels on the apical surface and basal surface of BAEC monolayers 2-5 h after inversion. K channel conductance and kinetics were similar regardless of the surface monitored. This nonenzymatic mechanical technique of exposing the basal surface of endothelium provides a useful tool to study the distribution of ion channels in endothelium and in other polarized cell types grown in tissue culture.

Animals↗
PubMed1990

Characterization of human phagocytic cell receptors for C5A and platelet activating factor expressed in Xenopus oocytes.

Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.

Animals↗
PubMed1990

The formyl peptide chemoattractant receptor is encoded by a 2 kilobase messenger RNA. Expression in Xenopus oocytes.

Activation of the formyl peptide chemoattractant receptor (FPCR) of phagocytic cells mobilizes intracellular calcium stores and affects the plasma membrane potential. Affinity crosslinking of FPCR has demonstrated a 60-80 kDa glycoprotein, with core peptide of 32 kDa. It is not known whether functional FPCR is this single peptide or requires multiple subunits. We used Xenopus oocyte expression system to determine the size of mRNA required for synthesis of functional FPCR. Injection of oocytes with poly(A)+ RNA from HL60 cells differentiated to the granulocyte phenotype resulted in acquisition of formyl peptide-specific responses (inward transmembrane current with a reversal potential consistent with a chloride conductance, and calcium efflux). FPCR activity expressed in oocytes had a ligand concentration dependence, ligand structure dependence and pertussis toxin sensitivity similar to those reported in phagocytic cells. When RNA was size fractionated, a single peak of FPCR activity at 2 kilobases was observed after injection of mRNA into oocytes. Our data strongly suggest that FPCR is composed of a single-sized polypeptide.

Amino Acid Sequence↗
PubMed1990

Effect of adherence, cell morphology, and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells.

The effects of adherence, cell morphology, and lipopolysaccharide on electrical membrane properties and on the expression of the inwardly rectifying K conductance in J774.1 cells were investigated. Whole-cell inwardly rectifying K currents (Ki), membrane capacitance (Cm), and membrane potential (Vm) were measured using the patch-clamp technique. Specific Ki conductance (GKi, whole-cell Ki conductance corrected for leak and normalized to membrane capacitance) was measured as a function of time after adherence, and was found to increase almost twofold one day after plating. Membrane potential (Vm) also increased from -42 +/- 4 mV (n = 32) to -58 +/- 2 mV (n = 47) over the same time period. GKi and Vm were correlated with each other; GL (leak conductance normalized to membrane capacitance) and Vm were not. The magnitudes of GKi and Vm 15 min to 2 hr after adherence were unaffected by the presence of 100 microM cycloheximide, but the increase in GKi and Vm that normally occurred between 2 and 8 hr after adherence was abolished by cycloheximide treatment. Membrane properties were analyzed as a function of cell morphology, by dividing cells into three categories ranging from small round cells to large, extremely spread cells. The capacitance of spread cells increased more than twofold within one day after adherence, which indicates that spread cells inserted new membrane. Spread cells had more negative resting membrane potentials than round cells, but GKi and GL were not significantly different. Lipopolysaccharide-(LPS; 1 or 10 micrograms/ml) treated cells showed increased Cm compared to control cells plated for comparable times. In contrast to the effect of adherence, LPS-treated cells exhibited a significantly lower GKi than control cells, indicating that the additional membrane did not have as high a density of functional GKi channels. We conclude that both adherence and LPS treatment increase the total surface membrane area of J774 cells and change the density of Ki channels. In addition, this study demonstrates that membrane area and density of Ki channels can vary independently of one another.

Animals↗
PubMed1990

Heat induces intracellular acidification in human A-431 cells: role of Na(+)-H+ exchange and metabolism.

The resting intracellular pH (pHi) of A-431 cells at 37 degrees C in Na+ Hanks' solution is 7.23 +/- 0.02. In the presence of amiloride (100 microM) pHi decreases to 7.08 +/- 0.03. Hyperthermia induces a temperature- and time-dependent intracellular acidification of 0.2 pH units in either bicarbonate-free or bicarbonate-buffered solutions. After heat treatment (45 degrees C, 10 min) pHi returns to normal 1 h after incubation at 37 degrees C. The activity of the Na(+)-H+ exchanger was examined in heated and unheated cells in the absence of bicarbonate. Unheated cells recover from an acid load in a [Na+]o-dependent and amiloride-sensitive manner. The apparent Michaelis constant for extracellular Na+ is 38 +/- 9 mM, and the apparent mean affinity constant for amiloride is 11 +/- 3 microM. In heated cells the apparent affinity of the Na(+)-H+ exchanger for extracellular Na+ is not changed, but the maximal recovery rate is approximately 40% slower than that of unheated cells. The rate of recovery from acid loading returns to normal 2 h after heat treatment. [Na+]i and intrinsic buffering power in heated cells are the same as those in unheated cells. Decreases in both intracellular ATP and lactic acid are observed in heated cells. 2-Deoxy-D-glucose and sodium azide induce an intracellular acidification but prevent most of the acidification induced by heat. Heat treatment causes no further acidification in cells that are acidified by both amiloride and 2-deoxy-D-glucose together. These data are the first to suggest that thermally induced intracellular acidification is due to both an inhibition of Na(+)-H+ exchange and an inhibition of metabolic pathways.

Amiloride↗
PubMed1989

Evidence for a Ca-activated inwardly rectifying K channel in human macrophages.

Cell-attached patch studies of cultured human macrophages demonstrate that exposure to ionomycin induces inward-rectifying single-channel currents that differ from the voltage-dependent 28 pS inward-rectifying K currents previously described in these cells (J. Membr. Biol. 103: 55-66, 1988). With 150 mM KCl in the electrode and NaCl Hanks' solution in the bath, the ionomycin-induced single-channel conductance for inward currents was 37 pS, and the reversal potential was 57 mV. Channel activity was often associated with a shift in the base-line current level indicating that the cell membrane potential hyperpolarized. The ability of ionomycin to induce channel activity depended on extracellular [Ca] supporting the view that the channels were gated by calcium. Ionomycin-induced channels were permeable to K, relatively impermeable to Cl or Na, exhibited bursting kinetics, and had no apparent voltage dependence. Barium (3 mM in the patch electrode) did not significantly block the ionomycin-induced channel at rest but blocked channel activity when the patch was hyperpolarized beyond the resting membrane potential. Exposure of macrophages to platelet-activating factor, which is known to increase intracellular [Ca] [( Ca]i) (J. Cell Biol. 103: 439-450, 1986), also transiently induced channel activity. In excised patches with 3 microM [Ca]i bursting inward-rectifying channels with a 41 pS conductance were noted that probably correspond to the ionomycin-induced channels present in cell-attached patches. Increasing [Ca]i from 10(-8) to 3 x 10(-6) M induced inward-rectifying channel activity in previously quiescent excised patches.(ABSTRACT TRUNCATED AT 250 WORDS)

Barium↗
PubMed1988

Inositol 1,4,5-trisphosphate concentrations increase after adherence in the macrophage-like cell line J774.1.

Several properties of macrophages change when suspended cells become adherent. To determine the intracellular signals involved in these changes, concentrations of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were monitored during adherence of J774.1 cells, a macrophage-like cell line. When cells grown in suspension were allowed to adhere to a glass surface, there was a transient increase in InsP3 that reached a peak between 100 and 120 s after plating. Inositol mono- and bis-phosphate concentrations were also elevated 100 and 120 s after plating. Analysis of isomer distribution showed significant 3-fold increases in Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] at 100 s after plating. These values were maintained at 120 s, with the additional appearance of a 4-fold increase in inositol 1,3,4-trisphosphate. The adherence-induced generation of Ins(1,4,5)P3 was decreased, and Ins(1,3,4,5)P4 formation was blocked, in Ca2+-free medium. However, doubling intracellular [Ca2+] by addition of the Ca2+ ionophore ionomycin (1 microM) did not increase Ins(1,4,5)P3 in suspended cells. Adherence of J774.1 cells to fibronectin-coated glass also induced an increase in InsP3.

Animals↗
PubMed1988

Inwardly rectifying whole-cell and single-channel K currents in the murine macrophage cell line J774.1.

Inward currents in the murine macrophage-like cell line J774.1 were studied using the whole-cell and cell-attached variations of the patch-clamp technique. When cells were bathed in Na Hanks' (KCl = 4.5 mM, NaCl = 145 mM), and the electrode contained Na-free K Hanks' (KCl = 145 mM) single-channel currents were observed at potentials below -40 mV which showed inward rectification, were K-selective, and were blocked by 2.5 mM Ba in the pipette. Single-channel conductance was 29 pS, and was proportional to the square root of [K]o. Channels manifested complex kinetics, with multiple open and closed states. The steady-state open probability of the channel was voltage dependent, and declined from 0.9 to 0.45 between -40 and -140 mV. When hyperpolarizing voltage pulses were repetitively applied in the cell-attached patch mode, averaged single-channel currents showed inactivation. Inactivation of inwardly rectifying whole-cell current was measured in Na Hanks' and in two types of Na-free Hanks': one with a normal K concentration (4.5 mM) and the other containing 145 mM K. Inactivation was shown to have Na-dependent and Na-independent components. Properties of single-channel current were found to be sufficient to account for the behavior of the macroscopic current, except that single-channel current showed a greater degree of Na-independent inactivation than whole-cell current.

Animals↗
PubMed1988

Patch-clamp studies in human macrophages: single-channel and whole-cell characterization of two K+ conductances.

Human peripheral blood monocytes cultured for varying periods of time were studied using whole-cell and single-channel patch-clamp recording techniques. Whole-cell recordings revealed both an outward K current activating at potentials greater than 20 mV and an inwardly rectifying K current present at potentials negative to -60 mV. Tail currents elicited by voltage steps that activated outward current reversed near EK, indicating that the outward current was due to a K conductance. The I-V curve for the macroscopic outward current was similar to the mean single-channel I-V curve for the large conductance (240 pS in symmetrical K) calcium-activated K channel present in these cells. TEA and charybdotoxin blocked the whole-cell outward current and the single-channel current. Excised and cell-attached single-channel data showed that calcium-activated K channels were absent in freshly isolated monocytes but were present in greater than 85% of patches from macrophages cultured for greater than 7 days. Only 35% of the human macrophages cultured for greater than 7 days exhibited whole-cell inward currents. The inward current was blocked by external barium and increased when [K]o increased. Inward-rectifying single-channel currents with a conductance of 28 pS were present in cells exhibiting inward whole-cell currents. These single-channel currents are similar to those described in detail in J774.1 cells (L.C. McKinney & E.K. Gallin, J. Membrane Biol. 103:41-53, 1988).

Barium↗
PubMed1987

Exposure to gamma-irradiation increases phorbol myristate acetate-induced H2O2 production in human macrophages.

Cell number, protein, and phorbol myristate acetate (PMA)-induced H2O2 production were measured in cultured human peripheral blood monocytes for six days after exposure to varying doses of gamma-radiation. Both the number of adherent cells and the protein per dish decreased with increasing radiation doses. The dose of radiation decreasing the number of adherent cells by 37% on days 4 and 6 postirradiation was 29 Gy. Four hours postirradiation there was a small decrease in PMA-induced H2O2 production for doses of 7.5 Gy or greater; levels returned to normal by eight hours and increased at 24 hours postirradiation. By day 4 postirradiation significant increases in PMA-induced H2O2 production were noted at all radiation doses (2.5 to 50 Gy). This increase was not due to a shift in the PMA dose-response curve, a change in the time course of the PMA response, or an effect of decreased cell density on the assay system. Superoxide levels were not significantly changed in cells exposed to 20 Gy. Catalase, glutathione peroxidase, and superoxide dismutase levels also were unchanged. Culturing irradiated cells with gamma-interferon increased PMA-induced H2O2 release, which indicated that irradiated cells retained their capacity to respond to gamma-interferon. These data demonstrate that irradiation affects the PMA-induced H2O2 production of human monocytes in a time- and dose-dependent manner. An increase in the release of reactive oxygen intermediates by the macrophage may play a role in enhancing the deleterious effects of radiation in vivo.

Azides↗
PubMed1986

Ionic channels in leukocytes.

In the past several years, with the advent of the patch clamp technique, the field of leukocyte electrophysiology has grown considerably. With the exceptions of the neutrophil and the eosinophil, electrophysiological and biochemical techniques have been used to characterize a number of types of voltage- or ligand-gated ionic channels in the different classes of leukocytes. This article reviews each of the ionic channels described in leukocytes and their functional relevance. It should be emphasized that this is by no means a final listing of the ionic channels in leukocytes, but merely a summary of the studies to date. It is highly likely that there are many other ionic channels in leukocytes with important functional implications that have not yet been discovered.

Animals↗
PubMed1985

Enhanced activity of the macrophage-like cell line J774.1 following exposure to gamma radiation.

Exposure of the macrophage-like cell line J774.1 to 20 gray of cobalt-60 gamma radiation resulted in a block of tritiated thymidine incorporation, along with an increase in cell "activation," as assessed by increases in lysosomal enzyme and ectoenzyme content, PMA-induced H2O2 production, and NBT staining, ingestion of E(IgG), spreading, and membrane ruffling. These changes are evident within 1 day postradiation and peak at 4 days postradiation.

Animals↗
PubMed1985

Differential expression of inward and outward potassium currents in the macrophage-like cell line J774.1.

J774.1 cells, a mouse-derived macrophage-like tumour cell line, were voltage clamped using whole-cell patch-clamp techniques. Cells were maintained in suspension cultures and plated at varying times before recording. The average zero-current potential of long-term adherent (greater than 24 h) cells was -77.6 mV. A tenfold increase in [K]o produced a 49 mV shift in zero-current potential. Freshly plated cells (less than 24 h) expressed two voltage-dependent currents: an outward current expressed transiently from 1 to 12 h post-plating and an inward current expressed 2-4 h post-plating which persisted in 100% of long-term adherent cells. Inward current was dependent upon voltage, time and [K]o 1/2, similar to the anomalous rectifier of other tissues. The conductance activated at potentials negative to -50 mV and plateaued at potentials negative to -110 mV. Inactivation was evident at potentials negative to -100 mV. Both the rate and extent of inactivation increased with hyperpolarization. Inward rectification was blocked by external BaCl2 or CsCl. The outward current was time- and voltage-dependent. The instantaneous I/V curves derived from tail experiments reversed at the potassium equilibrium potential (EK). A tenfold change of [K]o shifted the reversal potential 52 mV, indicating that the current was carried by potassium. This conductance activated at potentials positive to -50 mV, plateaued at potentials positive to -10 mV and inactivated completely with an exponential time course at all potentials. At voltages positive to -25 mV the rate of inactivation was independent of voltage. The outward current was blocked by 4-aminopyridine or D600. During the first 10 min after attaining a whole-cell recording, the conductance/voltage relation of the outward current shifted to more negative voltages and peak conductance showed a slight increase; recordings then stabilized. The voltage dependence of the inward current did not shift with time but wash-out of inward current was observed in some cells. The J774.1 cell line can serve as a model for the study of the role of voltage-dependent ionic conductances in macrophages.

Action Potentials↗
PubMed1977

Interaction of chemotactic factors with human macrophages. Induction of transmembrane potential changes.

The electrophysiology of chemotactic factor interaction with cultured human macrophages was investigated with standard intracellular recording techniques. In initial studies, E. coli endotoxin-activated serum, added to cell cultures during intracellular recordings, caused membrane hyperpolarizations which were greater than 30 s in duration, 10-50 mV in amplitude, and associated with decreased membrane resistance. Control serum produced smaller hyperpolarizations lasting 10-20 s and 5-30 m V in amplitude. Endotoxin-activated human serum deficient in the third complement component (C3) did not produce hyperpolarizations unless the serum was reconstituted with C3 before activation. Fractionation of normal activated serum by molecular seive chromatography (G-75 Sephadex) indicated that only fractions that eluted with an estimated molecular weight of 12,500 produced membrane potential changes. The active material that was chemotactic for the macrophages was identified as the small molecular weight cleavage product of C5, C5a, by heat stability (30 min at 56 degrees C) and inactivation by goat antisera to human C5 but not C3. 17 percent of macrophages stimulated with C5a exhibited a biphasic response characterized by a small (2-6 mV), brief (1-10 s) depolarization associated with a decreased membrane resistance preceding the larger and prolonged hyperpolarizations. Magnesium-ethylene glycol bis[beta-aminoethyl ether]N,N'-tetraacetic acid (Mg [2.5 mM]-EGTA [5.0 mM]) blocked the C5a-evoked potential changes, whereas colchine (10(- 6)M) and cytochalasin B (3.0 mug/ml did not. Hydrocortisone sodium succinate (0.5 mg/ml) decreased the percentage of cells responding to C5a. In related studies, synthetic N-formyl methionyl peptide (f-met-leu-phe), which had chemotactic activity for cultured macrophages, produced similar membrane potential changes. Repeated exposure of macrophages to C5a or f- met-leu-phe resulted in desensitization to the same stimulus. Simultaneous photomicroscope and intracellular recording studies during macrophage stimulation with chemotactic factor demonstrated that the membrane potential changes preceded membrane spreading, ruffling, and pseudopod formation. These observations demonstrate that ion fluxes associated with membrane potential changes are early events in macrophage activation by chemotactic factors

Chemotaxis↗