Form Approved UMENTATION j OMB No. 0704-0188 72 n is @sI Imated to average I tiour per resoons. inctuding the time for (qvjewins ntruclltiun. searching existing data siources etang and rviewing trie collection o4 information send coiuet reg(Aiding this~ burden estimate or any otther asoect of this AD~x-A 241 608-4~cin 04&s udt ahngtoen-Hviand ugt hpeRmnP.""IeaaO.~.WSig~ C253 Scinttii buren, o Watingon feedqaner Ser i tercorete for information Operations and Recinx 1215 ,offersoñ ~~ ~1991-Reprint _______________________ Y4. TITLE AND-SUBTITLE 5. FUNDING NUMBERS
5. 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]0 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 D600.6. 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
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 mV 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~ and inactivation by goat antisera to human C5 but not C3. 17% 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 [fl-aminoethyl ether]N, N'-tetraacetic acid (Mg [2.5 mM]-EGTA [5.0 raM]) blocked the CSaevoked potential changes, whereas colchine (10 -6 M) and cytochalasin B (3.0 p,g/ 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 electrophysiological properties of guinea pig peritoneal macrophages cultured in vitro were studied using standard intracellular recording techniques. The mean transmembrane potential, input resistance and time constant recorded from these cells were -13.1 mV, 143 Mohms, and 18 msec respectively. The majority of macrophages exhibited spontaneous hyperpolarizations (HA) of 4-8 seconds in duration and 10-50 mV in amplitude. Mouse peritoneal macrophages and human monocyte-derived macrophages manifested similar HA. HA could be induced by either mechanical stimulation or application of hyperpolarizing currents of 2-8 namps. HA had a mean reversal potential of -53 mV. Increasing the extracellular [K+] 10-fold resulted in a 50 mV shift in reversal potential. Addition of EGTA (1.5 mM) inhibited both spontaneous and evoked macrophage HA in the presence of excess Mg++. The divalent cation ionophore, A23187 induced prolongation of HA at low concentration (0.6 X 10(-6) M) and resulted in sustained hyperpolarization at higher concentration (2.0 X 10(-6) M). Addition to EGTA to cells treated with A23187 abolished HA. These data indicate that: (1) cultured macrophages from a variety of species exhibit spontaneous and induced HA, (2) development of HA is related to an increase in membrane permeability to K+, and (3) Ca++ may regulate the spontaneous and evoked electrical activity of the macrophage membrane presumably by affecting K+ permeability.
Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.
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