Intracellular potentials were measured, using a piezoelectric electromechanical transducer to impale Ehrlich ascites tumor cells with capillary microelectrodes. In sodium Ringer's, the potential immediately after the penetration was -24±7 mV, and decayed to a stable value of about -8 mV within a few msec. The peak potentials disappeared in potassium Ringer's and reappeared immediately after resuspension in sodium. Ringer's, whereas the stable potentials were only slightly influenced by the change of medium. The peak potential is in good agreement with the Nernst potential for chloride. This is also the case when cell sodium and potassium have been changed by addition of ouabain. It is concluded that the peak potentials represent the membrane potential of the unperturbed cell, and that chloride is in electrochemical equilibrium across the cell membrane.The membrane potential of about -11 mV previously reported corresponds to the stable potential in this study, and is considered as a junction potential between damaged cells and their environment. Similar potential differences were recorded between a homogenate of cells and Ringer's.The apparent membrane resistance of Ehrlich cells was about 70 Ωcm(2). This is two orders of magnitude less than the value calculated from(36)Cl fluxes, and may, in part, represent a leak in the cell membrane.For comparison, the influence of an eventual leak on measurements in red cells and mitochondria is discussed.
Like most other red cells, the giant erythrocytes of Amphiuma means possess a system for rapid exchange of chloride across the membrane. Also, there are indications that the net transport of chloride in these cells is slow. The size of Amphiuma erythrocytes allows direct measurements of membrane potential with microelectrodes. The present work exploits the possibility that such measurements can be used to give a quantitative estimate of the chloride conductance (GCl) of the Amphiuma red cell membrane. The membrane potential was measured as a function of extracellular chloride concentration (5-120mM), using an impermeant anion (Para-amino-hippurate) as a substitute. Furthermore, the effect of different pH values (6.0-7.2) was studied. For each extracellular chloride concentration the membrane potential was determined at a pH at which hydroxyl, hydrogen, and bicarbonate ions were in electrochemical equilibrium. From these membrane potentials and the corresponding chloride concentrations in the medium (at constant intracellular ion concentrations), the GCl of the membrane was calculated to be 3.9 x 10-7 omega-1 cm-2. This value is some six orders of magnitude smaller than that calculated from the rate of tracer exchange under equilibrium conditions. The experimental strategy used gives the values for a "partial transference number" which takes into account only ions which are not in electrochemical equilibrium. Whereas this approach gives a value for GCl, it does not permit calculation of the overall membrane conductance. From the calculated value of GCl it is possible to estimate that the maximal value of the combined conductances of hydroxyl (or proton) and bicarbonate ions is 0.6 x 10-7 omega-1 cm-2. The large discrepancy between the rate of exchange of chloride and its conductance is in agreement with measurements on human and sheep red cells employing the ionophore valinomycin to increase the potassium conductance of the membrane. The results in the present study were, however, obtained without valinomycin and an accompaning assumption of a constant field in the membrane. Therefore, the present measurements give independent support to the above mentioned conclusions.
Summary. The erythrocyte of Amphiuma means was chosen as a model for elucidation of membrane properties of red cells because the large size of this cell permitted direct measurements of plasma membrane potential. In the 30-sec period following micropuncture and withdrawal of the electrode the plasma membrane reseals and hyperpolarizes to a value of about -50 inV. The hyperpolarization is followed by a gradual return to the unperturbed potential of -15 mV. The magnitude of the hyperpolarization is strongly reduced by an increase in extracellular K concentration and is therefore related to an increase in relative K permeability. The transference number for K is calculated to have a maximal value of about 0.6. However, it is not yet clear whether the hyperpolarization can be solely attributed to a rise in K permeability, or whether there is a concomitant decline in C1 permeability as well. The magnitude of the hyperpolarization is unaffected by the presence of either ouabain or oligomycin.Extracellular Ca is prerequisite to the observed hyperpolarization and presumably acts by permitting the membrane to seal, and having entered the cell during the leak period, causes an increase in the relative K permeability. This permeability change resembles the Ca-induced rise in K permeability seen in metabolically depleted human red cells and red cell ghosts. An important difference, however, is that the Amphiuma red cells used in the present study are neither poisoned nor metabolically depleted so that the Ca effect is not prevented by the presence of cellular ATP as seems to be the case in human erythrocytes. The transient nature of the hyperpolarization may be related to the active transport of Ca out of the cell.
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