Cellular energy metabolism, trans-plasma and trans-mitochondrial membrane potentials, and pH gradients in mouse neuroblastoma

Abstract

(mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143 mV.Assessment of the electrical potential and ion gradients across subcellular compartments in the intact cell is an important goal that has not been pursued directly; isolation of subcellular compartments and subsequent in vitro studies have been the predominant approach, although possible redistribution of ionic species during isolation raises doubts with respect to the validity of this procedure. In this communication, we present a quantitative approach to the evaluation of the transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell. The requisite information for such evaluations includes: (i) relative volumes of the subcellular compartment and the cell; (ii) a known (measured) value of trans-plasma membrane electrical potential and pH gradient; and (iii) measured total concentrations of ions and weak acids/bases that distribute passively in the cell and in the compartment. These distributions give estimates of the cytosol-to-extracellular space gradient plus the compartment-tocytosol gradients.We have used this approach to evaluate the total trans-mitochondrial protonmotive force (the electrical plus pH gradients across the mitochondrial membrane) in mouse neuroblastoma Hanks' medium (3) containing 10 mM Hepes (pH 7.4) and 0.05% bovine serum albumin and finally resuspended in the same medium at 25-30 mg wet weight/ml. These suspensions were passed through a wire mesh (20-to 40-,m pore size diameter) in order to produce suspension homogeneity. Suspensions treated in this manner showed no clumps (no more than three cells grouped together) under a microscope. The dry weights of the cell suspension and the medium were determined for each preparation.For microelectrode measurements, cells were grown in Nunclon plastic petri dishes for 2-3 days as described above. Measurements of membrane potential were made while most of the cells were still in the exponential growth phase.Microelectrode Measurements. The medium in the petri dish was poured off and replaced by Hanks' medium containing 10 mM Hepes (pH 7.4) and 0.05% bovine serum albumin. The dish was placed on an inverted high-power microscope with a warmed stage (20-25°C). The electrode assembly was advanced by a remote stepping-motor hydraulic system. The microscope and the electrode carrier were contained within an electrically shielded cage and a humidified chamber. Lighting was via a fiber optic, heat-filtered high intensity source (Leitz, West Germany). After the lid of the petri dish was removed, the electrode was inserted under direct vision into randomly selected cells adhering to the floor of the dish. Only those Abbreviation: TPMP, triphenylmethyl phosphonium ion. Thomas (5). Some electrodes were ground to a chisel point on an optically flat grinding machine to allow easier penetration of the cell membrane.Isotope Distribution Measurement...

Transmembrane electrical potential measurements in rat brain synaptosomes

“…The permeability of the synaptosome membrane to TPMP is similar to that observed in other nervous tissue [27], equilibration of the probe requiring - [25][26][27][28][29][30] min. The rate of TPMP accumulation is substantially increased upon the addition of TPB.…”

“…The rate of TPMP accumulation is substantially increased upon the addition of TPB. TPB (at PM levels) has been shown to increase the rate of uptake of lipophilic cations into erythrocytes [28], lymphocytes [28] and neuroblastoma [27]. Furthermore, a decrease of the internal dipole potential of lipid bilayers has been shown with lo-' M TPB, enhancing cation permeation by one order of magnitude [29].…”

“…The consequences of subcellular compartments with significant membrane potentials have been discussed in [27] and are under further evaluation for synaptosomes.…”

“…Binding of TPMP results in an increased apparent intracellular TPMP concentration and therefore a larger calculated negative potential. Although there are as yet no suitable methods for directly measuring binding, an indirect assessment was made in cultured neuroblastoma [27] by assuming that voltage-independent binding of TPMP was responsible for its presence in cells suspended in high K+ medium (depolarized). Introducing this binding correction, gave a calculated value of membrane potential which was in excellent agreement with that obtained from microelectrode measurements.…”

alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

“…The release of both cantly greater in the presence than in lower efficacy of a-latrotoxin on transn conditions could be correlated with (Fig. 5, values in parentheses) (15,19,21,(26)(27)(28)(29) or neurobl lizing radioisotopes of the lipophilic c Ph3MeP+ (15,19,21,(27)(28)(29)31). Thei to the extent to which the cations in( significant potential across the inner X mitochondria.…”