Depolarization blunts the oxidative burst of human neutrophils. Parallel effects of monoclonal antibodies, depolarizing buffers, and glycolytic inhibitors

The anti-neutrophil mAb PMN 7C3 and IIC4 inhibited the respiratory burst of neutrophils as measured by the generation of superoxide anion or hydrogen peroxide in response to PMA, serum-treated zymosan, and FMLP. To examine the effect of these mAb on neutrophil transmembrane potential, a fluorescent probe was used in a continuous assay. Compared with control cells, antibody-treated neutrophils were partially depolarized at rest and had a blunted response when stimulated. The F(ab)2 fragment of PMN 7C3 had similar effects on both the respiratory burst and transmembrane potential, whereas the Fab fragment did not. The unrelated antineutrophil mAb 31D8 had no effect on either the respiratory burst or on transmembrane potential. Neutrophils suspended in high potassium buffers also exhibited partial depolarization of the resting cell membrane and a blunted depolarization response to stimuli and produced less superoxide anion and hydrogen peroxide in response to stimuli than did control cells in physiologic buffer. Exposure of neutrophils to 2-deoxy-D-glucose resulted in dose- and time-dependent depression of the respiratory burst. 2-Deoxy-D-glucose also caused depolarization of the resting membrane and impaired subsequent stimulus-induced depolarization. Similar effects were seen with addition of iodoacetamide or depletion of glucose. The parallel effects of anti-neutrophil mAb, depolarizing buffers, and glycolytic inhibitors on both neutrophil membrane depolarization and activation of the respiratory burst indicate a close association between these two events. The evidence suggests that the inhibitory effects of these antibodies are mediated through partial membrane depolarization which interferes with signal transduction on subsequent stimulation of the cells. The impairment in oxidative responses to phorbol esters as well as to receptor-dependent activating agents points to interruption at a distal step, e.g., subsequent to Ca2+ mobilization.     

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.     

Nonselective inhibition of neutrophil functions by sphinganine

Sphinganine has been proposed to be a specific inhibitor of protein kinase C. In the present study we have evaluated whether sphinganine is a convenient tool to probe for the role of protein kinase C in neutrophil function. Human neutrophils were loaded with the fluorescent probe quin2 and then tested in parallel for cytosolic free Ca2+, [Ca2+]i, membrane potential changes, O2- production, and exocytosis of primary granules (containing beta-glucuronidase) in response to various stimuli. In addition to inhibiting O2- production and exocytosis in a dose-dependent manner, sphinganine also blocked formyl-methionyl-leucyl-phenylalanine-induced [Ca2+]i, transients. Furthermore, sphinganine inhibited exocytosis elicited by the calcium ionophore ionomycin. Although sphinganine blocked O2- production due to phorbol 12-myristate 13-acetate, the most striking finding was that the drug rendered the cells leaky. Thus, at similar concentrations as those inhibiting cellular functions, sphinganine was shown to lead to cell permeabilization, as assessed by release of quin2 and cytoplasmic markers into the extracellular medium, and changes in plasma membrane potential. We conclude, therefore, that sphinganine does not appear to be a suitable compound for the evaluation of the involvement of protein kinase C in neutrophil activation.     

Surfactant releases internal calcium stores in neutrophils by G protein-activated pathway

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.     

Actin polymerization in neutrophils is triggered without a requirement for a rise in cytoplasmic Ca2+.

Stimulation of rat neutrophils with the peptide fMetLeuPhe caused (i) the appearance of a 40 kDa protein in the Triton-X-100-insoluble cytoskeleton, (ii) the disappearance of DNAase inhibition from the cytosol and (iii) the appearance of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin) binding sites. All three observations were consistent with a rapid and transient assembly of polymerized actin, peaking at approximately 5 s and returning to near resting levels within 40 s. By experimentally depleting the cells of Ca2+ and increasing the cytoplasmic Ca2+ buffering capacity, the peptide-induced Ca2+ transient was reduced from a peak of 900 nM to 250 nM, without inhibiting actin polymerization, and this peak was sustained for at least 2 min. A further dissociation between the triggering of actin polymerization and peptide-induced Ca2+ elevation and oxidase activation was demonstrated at low concentrations of peptide (1-100 pM), actin polymerization being triggered without an elevation in Ca2+ or activation of the oxidase. Two other agents which induced actin polymerization, phorbol 12-myristate 13-acetate and latex beads, failed to elevate cytoplasmic Ca2+. It was therefore concluded that neither Ca2+ nor those intracellular messengers which act with Ca2+ to trigger the neutrophil oxidase are responsible for triggering actin polymerization in neutrophils.     

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.     

Effect of cetiedil on the superoxide-generating system of porcine neutrophils

Cetiedil, alpha-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-1H-azepin-1-yl)-ethyl ester, was found to inhibit the generation of superoxide (O2-) by porcine neutrophils exposed to various stimulators. The concentration of cetiedil required for 50% inhibition was about 45 microM when neutrophils were stimulated by phorbol myristate acetate. Cetiedil not only decreased the rate of generation of O2-, but prolonged the lag time prior to the production of O2-. The inhibitory effect of cetiedil on the O2(-)-generating activity of the NADPH oxidase in the membrane vesicles was less than that on whole cells; the concentration of cetiedil necessary for 50% inhibition was about 250 microM. To study the mechanism of cetiedil's effect on the membrane, the transmembrane potential of neutrophils and the intracellular free Ca2+ concentration were monitored by using fluorescence probes, diS-C3-(5), and quin-2, respectively. Cetiedil caused depolarization of the membrane potential and increased the intracellular free Ca2+. These results indicate that integrity of ionic distribution is necessary to activate the O2(-)-generating system of neutrophils.     

Pulsed electric current enhances the phorbol ester induced oxidative burst in human neutrophils.

Oxidative burst (OB) response in human neutrophils, measured with chemiluminescence (CL), has been used to determine whether pulsed electric current (PEC) might induce a functional response in these electrically nonexcitable cells, and also whether it might modify cellular response to tumor-promoting phorbol ester (PMA). Five minutes of PEC treatment caused no significant changes in neutrophil CL levels in HBSS (1.2 mM Ca2+ concentration) as well as in HBSS-EGTA, where the extracellular Ca2+ concentration was reduced to less than 30 nM. The CL level of PMA-activated neutrophils in HBSS was 52% higher than in HBSS-EGTA. In HBSS the CL level, after the combined PMA and PEC treatment, was 53% higher than in PMA-alone-treated neutrophils. Activation of the OB in HBSS-EGTA with PMA and PEC was 13% higher than in solely PMA treated neutrophils. The results suggest that in neutrophil OB response, the PEC effect is closely related with cellular calcium mobilization, since depletion of extracellular Ca2+ decreased the PEC effect.     

Auranofin modulated cytoplasmic free calcium in neutrophils by mobilizing intracellular calcium and inhibiting protein kinase

The effect of the lipophilic gold compound, auranofin (AUR) on the calcium homeostasis of human neutrophils treated with or without n-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. In agreement with previous reports, FMLP induced a rapid release of intracellular Ca2+ stores followed by a smaller influx of extracellular Ca2+. AUR and staurosporine enhanced while phorbol 12-myristate 13-acetate suppressed the secondary influx of Ca2+. Mn2(+)-quenching-of-fluorescence studies indicate that phorbol 12-myristate 13-acetate incubation blocked cation entry. AUR or staurosporine potentiation of FMLP effects on cytoplasmic free Ca2+ [( Ca2+]i) was attributed to suppression of negative feedback effects of protein kinase C. AUR (5-45 microM) per se induced a slow release of internal Ca2+ stores followed by a delayed influx of extracellular Ca2+. Control studies showed that AUR did not induce the formation of inositol 1,4,5-trisphosphate, lyse cells, or promote dye leakage. Dithiothreitol suppressed the AUR effect. AUR triggered biphasic but smaller increases in [Ca2+]i of neutrophil cytoplasts. Studies with permeabilized neutrophils showed that AUR directly released Ca2+ from internal stores. By comparison, gold sodium thiomalate, which had no effect on intact cells, also released Ca2+ from permeabilized cells. Present results indicate that AUR modulated [Ca2+]i directly by mobilized Ca2+ from multiple storage sites and indirectly by inhibiting protein kinase C.     

Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.     

The relationship between mitogen-induced membrane potential changes and intracellular free calcium in human T-lymphocytes

We have investigated the effects of mitogenic lectins on human T-lymphocytes, isolated from peripheral blood, and cells from the T-cell clone, HPB-ALL, using the fluorescent dyes, bis-thiobarbiturate tri-methineoxonol (bisoxonol) and quin2 to sense changes in membrane potential and intracellular free [Ca2+], respectively. The resting potential of both cell types is close to the K+ equilibrium potential. Changes from the resting level occur when mitogenic concentrations of either concanavalin A or phytohaemagglutinin are added. T-lymphocytes undergo a decrease in emission, maximal at 1 to 2 min, corresponding to a small membrane hyperpolarization. This is followed by a depolarization to approximately the resting level. HPB-ALL cells, on the other hand, respond to the mitogens by a sustained increase in fluorescence, denoting a depolarization, that is maximal at 4 to 5 min and 7 to 9 min, respectively. The Ca2+-dependence of these phenomena indicates that the membrane potential response, in both cell types, is the resultant of two opposing effects: a Ca2+-sensitive ion movement tending to hyperpolarize the cells and a Ca2+-insensitive effect that generates a depolarization. Our results suggest that Ca2+-activated K+ channels are responsible for the first effect and that an inward Na+ movement accounts for the depolarization signal in T-lymphocytes. In HPB-ALL cells only part of the depolarization is Na+-dependent. Although the effects elicited by phytohaemagglutinin occur more slowly than those produced by concanavalin A, similar membrane potential and [Ca2+]i changes occur.     

Regulation of ANF receptor internalization: involvement of extracellular calcium.

Receptor mediated internalization of 125I-ANF (99-126) and the underlying mechanism was studied in PC12 cells. Phosphorylation of PC12 cell plasma membrane proteins at 0 degrees C or 37 degrees C was not altered in presence of ANF (99-126) or c-ANF (4-23). Exposure of cells to phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) did not alter the endocytic rate or extent of 125I-ANF (99-126) internalization. When cells were treated with a combination of PMA and the calcium ionophore A23187, internalization was not stimulated. Incubation with A23187 (10 microM) alone decreased 125I-ANF (99-126) internalization by 22% in Ca2+ containing medium. Cell surface binding increased 10% in the presence of Ca2+ compared to Ca2+ free medium, irrespective of the presence of A23187. Ca2+ appears to play an important role in the binding of ANF to the receptor and initiation of ligand-receptor complex internalization. Activation of protein kinase C or receptor phosphorylation is not an essential step in initiating ANF receptor internalization.     

Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils

The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA-induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 X 10(-8) to 1.0 X 10(-5) M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 X 10(-6) M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 X 10(-6) M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex.     

Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium

The addition of low concentrations of phorbol 12-myristate 13-acetate to rabbit neutrophils induces cell aggregation, degranulation, increased oxygen consumption and an increase in the amount of actin associated with the cytoskeleton without a rise in the level of intracellular free calcium as measured using the fluorescent probe quin-2. The ability of phorbol 12-myristate 13-acetate to initiate neutrophil responses similar to those produced by the chemotactic factor without causing a rise in the level of intracellular free calcium suggests two possibilities; that there is a second messenger in addition to calcium or that it activates the cells at a point distal to calcium mobilization. The possible role of diacylglycerol in neutrophil activation is discussed.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Membrane potential modulates divalent cation entry in rat parotid acini

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.     

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.