Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages.

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.

Simultaneous detection of free radical release and membrane current during phagocytosis.

Stimulation of macrophages induces the "respiratory burst" response which is associated with the generation of superoxide (O2-), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O2- was temporally related to changes in membrane potential and transmembrane current. Release of O2- at the single cell level was photometrically monitored during phagocytosis of immune complexes while simultaneously measuring whole-cell current. Membrane depolarization and the generation of a non-selective current followed an increase in O2- production with a variable lag time which was correlated with the state of cellular maturation in culture. In the absence of phagocytosis, the exposure of macrophages to O2- generated by a xanthine-xanthine oxidase reaction activated a non-selective current similar to that seen after phagocytosis. These results provide the first demonstration of the relationship between free radical release and the ensuing electrophysiological signaling events which are linked to particle engulfment in phagocytic cells.

Elevation in intracellular calcium activates both chloride and proton currents in human macrophages.

The transition of a resting macrophage into the activated state is accompanied by changes in membrane potential, cytoplasmic pH, and intracellular calcium (Ca(i)). Activation of Cl- as well as H(+)-selective currents may give rise to stimulus-induced changes in membrane potential and counteract changes in intracellular pH (pHi) which have been observed to be closely associated with respiratory burst activation and superoxide production in macrophages. We carried out whole-cell voltage clamp experiments on human monocyte-derived macrophages (HMDMs) and characterized currents activated following an elevation in Ca(i) using isosmotic pipette and bath solutions in which Cl- was the major permeant species. Ca(i) was elevated by exposing cells to the Ca2+ ionophore A23187 (1-10 microM) in the presence of extracellular Ca2+ or by internally exchanging the patch-electrode solution with ones buffered to free Ca2+ concentrations between 40 and 2,000 nM. We have identified two Ca(2+)-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl- conductance that showed variable inactivation at depolarized potentials and a H+ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 microM). Current activation for both Ca(2+)-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 microM). Inclusion of the autophosphorylated, Ca2+/calmodulin-dependent protein kinase in the pipette in the presence of ATP induced a rapidly activating current similar to that observed following an elevation in Ca(i). Activation of both conductances would contribute to the changes in membrane potential which accompany stimulation-induced activation of macrophages as well as counteract the decrease in pHi during sustained superoxide production.

ATP-sensitive K+ channel opener acts as a potent Cl- channel inhibitor in vascular smooth muscle cells.

We describe the activation of a K+ current and inhibition of a Cl- current by a cyanoguanidine activator of ATP-sensitive K+ channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 microM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 microM). The U83757-induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl-ammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 microM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where EK = -82 mV and ECl = 0 mV, the reversal potential of the U83757-sensitive current was approximately -70 mV in those experiments where an increase in membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl- channel blocker as well as a K+ channel opener. In experiments in which Cl- current activation was specifically brought about by cellular swelling and performed in solutions where Cl- was the major permeant ion, U83757 (300 nM to 300 microM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K(+)-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl- currents as well as the activation of a K(+)-selective current.