Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry

Abstract

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388DI and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (Es) frequently reported. Upon microelectrode entry into the cell (P388DI), the recorded potential drops to a peak value (Ep) (mean -37 rnV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (En) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (Es) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (Eh) is --30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U. V., A. M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288) for other cells. It indicates that the microelectrode introduces a leakage into the membrane, causing a change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of Ep, E,, Eh, Es, and membrane resistance (Rm) measured for the other macrophages were similar to those of P388DI. From these results we conclude that Eo is a better estimate of the true membrane potential of macrophages than Es, and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.

Oscillatory hyperpolarizations and resting membrane potentials of mouse fibroblast and macrophage cell lines.

“…Giant L cells and giant macrophages were obtained by exposing the two cell lines on day 3 of culture to 20-30 Gy X-irradiation (Whitmore, Till, Gwatkin, Siminovitch & Graham, 1958;Nelson & Peacock, 1973 (Ince, Ypey, Diesselhoff-Den Dulk, Visser, De Vos & Van Furth, 1983a). The experimental set-up and bathing solution were as described elsewhere (Ince et al 1983b). Glass micro-electrodes filled with 4 M-K acetate and with tip resistances ranging (Re) from 100 to 400 MO were used.…”

“…Cells were cultured on 24 mm glass cover-slips in culture medium at 37 0C in a 5 % C02 humidified atmosphere, as described elsewhere (Ince et al 1983b). L cells (a mouse fibroblast cell line), were used for experiments after 3-5 days of culture.…”

“…None the less, properties of L cells, such as phagocytosis (Okada, Tsuchiya, Yada, Yano & Yawo, 1981), cell motility (Tsuchiya, Okada, Yano, Inouye, Saski & Doida, 1981 a), and pinocytosis (Tsuchiya, Okada, Yano, Murai, Miyahara & Tanaka, 1981 b) have been associated with such hyperpolarizations, and in macrophages these hyperpolarizations have been associated with chemotaxis (Gallin & Gallin, 1977) and phagocytosis (Kouri, Noa, Diaz & Niubo, 1980). Recently, we analysed the fast potential transient seen after impalement (Lassen, Nielson, Pape & Simonsen, 1971) into macrophages and found (Ince, Ypey, Van Furth & Verveen, 1983b) that penetratation by a microelectrode induces a leakage conductance across the membrane and that the membrane potentials of macrophages (mean -40 mV) are two to three times more negative than has generally been thought on the basis of sustained potential measurements (-15 to -25 mV). In the present study we measured fast potential transients upon impalement in L cells and macrophages in order to determine the origin of spontaneous hyperpolarizations and to assess the effect of the leakage conductance induced by the micro-electrode on membrane potential measurements.…”

Development of a delayed outward-rectifying K+ conductance in cultured mouse peritoneal macrophages.

“…Cultured macrophages derived from human monocytes have an excitable membrane (1) and changes in membrane potential are among the earliest detectable events upon stimulation of phagocytosis (2). So far, direct membrane potential or current measurements have been made with the use of intracellular glass microelectrodes (1,3,4). This type of electrode, however, seriously hampers the study of ionic currents in small cells such as macrophages (3).…”

“…So far, direct membrane potential or current measurements have been made with the use of intracellular glass microelectrodes (1,3,4). This type of electrode, however, seriously hampers the study of ionic currents in small cells such as macrophages (3). Therefore, we have applied the patch clamp technique (5), which allows high resolution of ionic currents in small cells (diameter, <20 jtm) up to high frequencies (<10 kHz).…”