Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium

1. (1) Evidence is presented which indicates that the carbocyanine dye (3,3′ dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across the plasma membrane of the ciliated protozoan Paramecium.

2. (2) The dye at low concentrations ( 1 μM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response.

3. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration.

4. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential.

5. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods.

6. (6) Both cations which induce a negative chemotactic response in Paramecium (K⁺, Na⁺, Ba²⁺) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.

Intrinsic cone adaptation modulates feedback efficiency from horizontal cells to cones.

Processing of visual stimuli by the retina changes strongly during light/dark adaptation. These changes are due to both local photoreceptor-based processes and to changes in the retinal network. The feedback pathway from horizontal cells to cones is known to be one of the pathways that is modulated strongly during adaptation. Although this phenomenon is well described, the mechanism for this change is poorly characterized. The aim of this paper is to describe the mechanism for the increase in efficiency of the feedback synapse from horizontal cells to cones. We show that a train of flashes can increase the feedback response from the horizontal cells, as measured in the cones, up to threefold. This process has a time constant of approximately 3 s and can be attributed to processes intrinsic to the cones. It does not require dopamine, is not the result of changes in the kinetics of the cone light response and is not due to changes in horizontal cells themselves. During a flash train, cones adapt to the mean light intensity, resulting in a slight (4 mV) depolarization of the cones. The time constant of this depolarization is approximately 3 s. We will show that at this depolarized membrane potential, a light-induced change of the cone membrane potential induces a larger change in the calcium current than in the unadapted condition. Furthermore, we will show that negative feedback from horizontal cells to cones can modulate the calcium current more efficiently at this depolarized cone membrane potential. The change in horizontal cell response properties during the train of flashes can be fully attributed to these changes in the synaptic efficiency. Since feedback has major consequences for the dynamic, spatial, and spectral processing, the described mechanism might be very important to optimize the retina for ambient light conditions.     

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[β-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 μg/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     

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (ΔV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.     

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.     

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Effect of arachidonic acid and the chemotactic factor F-Met-Leu-Phe on cation transport in rabbit neutrophils

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of ⁴⁵Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of ⁴⁵Ca. Arachidonate also increases the uptake of ²²Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to ⁴⁵Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.     

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 μM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100mM KCl. An increase in ⁴⁵CaCl₂ accumulation from solutions of low concentrations (1 μM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242–248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [³H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K⁺-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.     

Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by adp-ribose, the major product generated by the CD38 enzyme reaction

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD(+). Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD(+) analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.     

Calcium and flagellar response during the chemotaxis of bracken spermatozoids

The attraction of fern spermatozoids by secretions from the female reproductive structures, and by salts of malic acid, has long been known as a classic example of precise chemotactic orientation by motile cells. Spermatozoids of the bracken fern are attracted by the partially ionized form of malic acid, bimalate ion, and also by calcium ions. Both calcium and bimalate ions must be present for chemotactic response and for response to voltage gradients, Spermatozoids swimming up a bimalate concentration gradient swim in helical paths of abnormally small radius; if they accidentally swim down the concentration gradient the radii of their path helices become abnormally large. These observations suggest that changes in the direction of flagellar beating in response to the rate of change of bimalate ion concentration with time may be the basis for chemotactic orientation. A “coupled diffusion” hypothesis for chemo-reception is presented, which postulates a membrane carrier which can only circulate freely in the membrane if it binds both bimalate and calcium ions. This hypothesis could explain time-differentiation of the stimulus, the coupling of a specific stimulus — bimalate ions — to a general mediator of intracellular response — calcium ions — and the quantitative relationship between response of the spermatozoids and the chemical potential of “calcium bimalate.”     

Involvement of envelope-bound calcium in the transient depolarization of the Escherichia coli cytoplasmic membrane induced by bacteriophage T4 and T5 adsorption

We previously showed that adsorption of bacteriophages T4 and T5 to their respective outer membrane receptors induced a partial depolarization of the cytoplasmic membrane. As these membrane potential changes were independent of phage properties, we proposed that phage adsorption triggered the emission of a signal which must be transmitted between the two membranes. We show here that these two phages use different mechanisms of transmission of this stimulation signal. In the case of T4, but not of T5, a specific requirement for envelope-bound calcium was found. Indeed, addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the membrane potential changes induced by T4. This envelope-bound calcium became accessible to the chelator only as a consequence of phage adsorption and remained in this state during the depolarization and repolarization. Membrane potential changes again occurred if calcium was added after the addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phage. The same concentration (300 microM) of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the T4-induced depolarization between multiplicities of infection of 6 and 30. This suggests that phage adsorption triggers both a conformational change of membrane components, the number of which reflects the number of stimuli (phages), and the liberation of a definite amount of calcium. This liberated calcium would, in turn, activate these modified membrane components to induce the depolarization. The fact that depolarization may be induced several times after a unique adsorption implies that these membrane components remain irreversibly modified.     

Chemoreception inParamecium tetraurelia: acetate and folate-induced membrane hyperpolarization

Summary Acetic and folic acids hyperpolarize the membrane potential ofParamecium tetraurelia in a concentration-dependent manner. The membrane responses are accompanied by small changes in cell resistance, and are significantly reduced by increasing extracellular cation concentrations, suggesting that the attractants bring about the membrane potential change by increasing cell permeability to cations. The inability to show a reversal potential for the hyperpolarization to attractants suggests that the effects of cations on the response are non-specific, however. The possible roles of Ca⁺⁺, K⁺, and Na⁺ in the attractant-induced responses were further investigated by applying acetate and folate to cells with genetic defects in specific ion conductances, by collapsing the driving forces for these ions, and by testing the effects of ion channel blockers on the responses. These studies suggest that the membrane responses to attractants are not due to the direct effects of increased or decreased membrane permeability to cations.Attempts to block the acetate and folate-induced hyperpolarization by collapsing surface potential or using a mutant with reduced surface charge were inconclusive, as were studies on the possible role of attractant transport in the membrane responses.We hypothesize that the membrane hyperpolarization may be due to either the indirect effects of increased calcium permeability, to extrusion of calcium through activation of a calcium pump, or to a proton efflux.     

Functional ion channel formation by mouse macrophage IgG Fc receptor triggered by specific ligands

The mouse macrophage Fc gamma 2b/gamma 1R has previously been purified with the aid of the monoclonal antibody 2.4G2. That this Fc gamma R functions as a ligand-dependent ion channel is supported by the following evidence. Employing [3H]tetraphenylphosphonium ([3H]Ph4P+) as a probe for membrane potential changes in intact cells, we found a biphasic change in membrane potential following treatment with immune complexes, monoclonal antibody 2.4G2 IgG and 2.4G2 Fab-Sephadex particles. We observed an immediate depolarization followed by prolonged hyperpolarization. [3H]Ph4P+ uptake experiments with plasma membrane vesicles prepared from J774 macrophages showed that binding of ligands to the FcR led to transmembrane monovalent cation flow. Similar [3H]Ph4+ uptake experiments were done with phospholipid vesicles containing purified and reconstituted Fc gamma 2b/gamma. Following challenge with specific ligands, transmembrane monovalent cation flow was observed. Purified FcR was reconstituted into planar lipid bilayers; exposure to ligands led to transient bilayer conductance increase. THe conductance change was resolved into single channel events. Quin-2 measurements showed an increase of free cytosolic calcium levels in macrophages following exposure of cells to different ligands of the FcR. An optimal range of calcium was found to be required for phagocytosis, below and above which inhibition of ingestion occurred.     

Phorbol myristate acetate promotes entry of calcium in hypophysial cells GH3 B6 via potential dependent calcium channels

10 GH3/B6 cells were patched-clamped using a pipette containing NMG as internal cation, 2 mM ATP and 100 microM leupeptin. Whole-cell calcium or barium currents were recorded prior and after PMA (10(-8) or 10(-7) M). PMA increased the inward calcium current at potential levels close to threshold in 8 cells; 7 cells only exhibited an increase in transitory calcium current at potential levels close to threshold; in one cell, both transitory and conventional calcium currents were increased. 2 cells did not respond to PMA.     

Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.     

Calcium movement and membrane potential changes in the early phase of neutrophil activation by phorbol myristate acetate: a study with ion- selective electrodes

To quantitate calcium movements and membrane potential changes in stimulated neutrophils, we have measured net fluxes of Ca2+ and of the lipophilic cation tetraphenyl phosphonium by a very sensitive ion- selective electrode system. Activation of neutrophils by 3 X 10(-8) M phorbol 12-myristate, 13-acetate induces a release of approximately 20% of total cell calcium, with an initial lag period of less than 10 s. The Ca2+ outflux is markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurs by activation of the ATP-driven Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induces a transiently increased exchange of medium 45Ca with cell calcium, which is measurable a few seconds after cell exposure to the stimulant and peaks at approximately 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) results in a marked depolarization, with a lag period of approximately 60 s. The rate and extent of depolarization are reduced by 40 and 65%, respectively, in a low Na+ medium but are not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarizes neutrophils without either modifying their resting oxidative metabolism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibits no effect on the oxidative metabolism of neutrophils, does not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.     

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.