Y. C. Yen-Chow

Effects of Phenytoin on Primary Glial Cell Cultures

Summary: The activity of enzymes involved in anion and cation transport, the concentration of intracellular potassium (K+i), and the transmembrane potential (Em) were determined following acute and chronic exposure of primary astroglial cultures to micromolar concentrations of phenytoin (PHT). Na+, K+ ‐ATPase activity of homogenates of cultured glial cells was determined in the presence of an increasing K+ concentration (1–20 mM). Acutely, PHT had little effect on the K+ activation pattern of Na+, K+ ‐ATPase. In contrast, the percentage of Na+, K+ ‐ATPase activated by elevating the K+ concentration was dose dependently increased by chronic PHT treatment. This effect was accompanied by a marked increase in K+i and a significant membrane hyperpolarization. The acute effect of PHT on the Em was biphasic, characterized by membrane hyperpolarization at concentrations of 10‐6‐10‐5M; at concentrations between 10‐5 and 10‐4M, the Em progressively returned to control values. These results suggest that glial cells acutely and chronically treated with therapeutic concentrations of PHT have an enhanced capacity to control elevated extracellular potassium levels. Return of the Em to control values at PHT concentrations > 10‐5M suggests that these cells are less able to regulate extracellular potassium. These data can partially explain the excitatory effects of PHT at high therapeutic concentrations.

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K+]o was increased from 1.2 to 10 mM, [K+]i and [Cl−]i were increased, whereas [Na+]i was decreased. As [K+]o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K+]o was increased from 60 to 122 mM, an increase in [K+]i and [Cl−]i and a decrease in [Na+]i were observed.Inneurons, as [K+]o was increased from 1.2 to 2.8 mM, [Na+]i and [Cl−]i were decreased, whereas [K+]i was increased. As [K+]o was increased from 2.8 to 30 mM, [K+]i, [Na+]i and [Cl−]i showed no significant change. When [K+]o was increased from 30 to 122 mM, [K+]i and [Cl−]i were increased, whereas [Na+]i was decreased. Inastrocytes, pHi increased when [K+]o was increased. Inneurons, there was a biphasic change in pHi. In lower [K+]o (1.2–2.8 mM) pHi decreased as [K+]o increased, whereas in higher [K+]o (2.8–122 mM) pHi was directly related to [K+]o. In bothastrocytes andneurons, changes in [K+]o did not affect the extracellular water content, whereas the intracellular water content increased as the [K+]o increased. Transmembrane potential (Em) as measured with Tl-204 was inversely related to [K+]o between 1.2 and 90 mM, a ten-fold increase in [K+]o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The Em values measured with Tl-204 were close to the potassium equilibrium potential (Ek) except those in neurons at lower [K+]o. However, they were not equal to the chloride equilibrium potential (ECl) at [K+]o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K+]o produced different changes in [K+]i, [Na+]i, [Cl−]i, and pHi in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K+]o, in such a way to maintain a more suitable environment for neurons.

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more125I− than36Cl− from the medium. The average cell/medium ratio of125I− of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of36Cl− of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both125I− and36Cl− calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting125I− uptake whereas furosemide is more effective in inhibiting36Cl− uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na+]o but was not significantly affected by changes of [Cl−]o or [HCO3−]o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl−]o and [HCO3−]o and was not affected [Na+]o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na+]o between 60 and 140 mM and inversely related to [HCO3−]o between 10 and 40 mM, but it was not affected by [Cl−]o. Radiochloride uptake by neurons was directly related to [Cl−]o and to [Na+]o between 60 and 140 mM and was not affected by [HCO3−]o. However, in sodium-free medium both125I− and36Cl− uptakes into neurons were higher than those in [Na+]o between 5 and 60 mM. These results indicate that uptake of125I− and36Cl− into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.

MEMBRANE POTENTIALS, ELECTROLYTE CONTENTS, CELL pH, AND SOME ENZYME ACTIVITIES OF FIBROBLASTS

SummaryThe resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10−5 to 10−4M), hydrocortisone (10−7 to 10−6M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl− concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na+, K+-, HCO3−-, and Ca++, Mg++-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl−, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl−, and H+ are actively transported out of the cells and K+ into the cells.