S. Y. 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.

Electrophysiological properties of osteoblastlike cells from the cortical endosteal surface of rabbit long bones

SummaryThe mean transmembrane potential of cultured osteoblastlike cells isolated from the cortical endosteal surface of rabbit long bones was −16.9±0.64 mV (n=335). Elevation of potassium concentration in medium caused a decrease in potential. As the external concentration of potassium reached 15 mmol/liter and above, there was a linear relationship between the potassium concentration in log scale and the membrane potential with a slope of −13 mV per 10-fold change in external potassium concentration. Dibutyryladenosine 3′,5′-cyclic monophosphate, parathyroid extract, hydrocortisone, and sodium fluoride all depolarized the membrane of osteoblast-like cells after both short (1–2 h) and long (24 h) exposures at suitable doses, whereas calcitonin and prostaglandin E2 hyperpolarized the membrane after long exposures. The Na+, K+ and Cl− concentrations of cultured osteoblastlike cells were 0.538, 0.984, and 0.358 mmol/g protein or 52.6, 96.3, and 35.0 mmol/liter cell water, respectively. The protein content of these cells was 8.18±0.6 g/100 g cells and the water content was 83.7 g/100 g cells. The above-mentioned chemical and hormonal preparations in doses that produced significant changes in the membrane potential of these cultured cells did not alter their electrolyte or protein contents 24 h after exposure. Intracellular pH of cultured osteoblastlike cells as determined by [14C]-dimethyloxazolidine-2,4-dione and3H2O averaged 7.03 ± 0.11 when the pH of culture medium was maintained at 7.4. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, and H+, and Cl− are actively transported out of the cells and K+ into the cells.

Studies on pH regulatory mechanisms in cultured astrocytes of DBA and C57 mice.

Summary: pH regulatory mechanisms in primary cultures of astrocytes from the cerebral cortex of neonatal audiogenic‐seizure‐susceptible DBA/2J (DBA) and genetically controlled C57BL/6J (C57) mice were studied with [14C]dimethyloxazolidine‐2‐4‐dione (DMO) and [3H]‐methyl‐D‐glucose (MDG). Effects of changing the concentration of Na+, K+, HCO3‐ or Cl‐ in medium, and/or of different transport blockers and metabolite inhibitor on intracellular pH (pHi) of cultured astrocytes were also studied. In nominal HCO3‐‐free HEPES‐buffered Hanks’balanced salt solution (HEPES HBSS), when the pH of medium (pHo) was maintained at 7.4, the steady‐ state pHi of cultured astrocytes from DBA mice was 6.98 ± 0.03, and that from C57 mice was 7.01 ± 0.03. When the cells were incubated in HBSS containing 25 mM HCO3‐ and equilibrated with 5% CO2 (HCO3‐ HBSS, pHo= 7.4), pHi of both DBA and C57 astrocytes was ∼0.1–0.15 pH units higher than that in HEPES HBSS. Reducing the pH or the Na+ concentration in media (pHo, [Na+]o) of either HEPES HBSS or HCO3‐ HBSS, pHi of both DBA and C57 astrocytes decreased markedly (0.25–0.45 pH units lower than the controls). The decrease in pH, was greater in HEPES HBSS than in HCO3‐ HBSS. Reducing the Cl‐ concentration ([Cl‐]o) in either HEPES or HCO3‐ HBSS, pHi of astrocytes increased by 0.05–0.1 pH units. Increasing the K+ concentration ([K+]o) of or adding Ba2+ to the media increased the pHi of both DBA and C57 astrocytes accordingly. SITS, an anion transport inhibitor, decreased the pHi of both DBA and C57 astrocytes in HCO3‐ HBSS but not in HEPES HBSS. It enhanced the response of pHi to reduction in pHi. Amiloride, a Na+‐H+ exchange inhibitor, decreased the pHi of both DBA and C57 astrocytes more in HEPES HBSS than in HCO3‐ HBSS. It enhanced the response of pHi to reduction in pHo and [Na+]o. Ouabain, an Na+, K+‐ATPase inhibitor, decreased the pHi of cultured astrocytes in HEPES HBSS, but not in HCO3‐ HBSS. It also enhanced the response of pHi to changing pHo and [Na+]o in HEPES HBSS. Acetazolamide, a carbonic anhydrase inhibitor, decreased the pHi of astrocytes in both HEPES and HCO3‐ HBSS. Both bumetanide, an Na+, K+/Cl‐ cotransport blocker, and KCN, a metabolic inhibitor, produced no significant effect on the steady‐state pH, or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.

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.

Radioiodide Uptake in Brain, CSF, Thyroid, and Salivary Glands of Audiogenic Seizure Mice

Summary: DBA/2J (DBA) mice are susceptible to audiogenic seizures (ASs) in an age‐dependent manner. Anion transport as measured by radioiodide uptake was determined in thyroid gland, salivary gland, skeletal muscle, cerebral cortex, cerebellum, brainstem, and CSF from these mice at various ages. Anion transport was also determined in C57BL/6J(C57) mice, an AS‐resistant strain. In thyroid, DBA mice had an enhanced ability to concentrate iodide at 21 days of age when they have maximal AS susceptibility, as compared with the same‐aged C57 mice. This difference in thyroid function was less marked at 40 days of age, when DBA mice are less AS susceptible, and was absent at 110 days of age, when DBA mice are AS resistant. In brain, differences in iodide uptake were also noted between these two strains of mice at 21 days of age. DBA mice had an increased concentration of iodide in CSF, an indication that they have a defect in the transport of iodide out of the CSF across the choroid plexus. In addition, DBA mice had a lower ratio of cerebral cortex to CSF iodide, which suggests that DBA mice have a defect in the transport of this anion into cerebral cortical cells from brain interstitial fluid. These differences in iodide transport in brain decreased with age as the AS susceptibility of DBA mice decreased. These results suggest a relation between anion transport in thyroid gland, cerebral cortex, and choroid plexus and AS susceptibility in DBA mice at 21 days of age.

Effects of dibutyryl cyclic AMP on Na+,K(+)-ATPase activity and intracellular Na+ and K+ in primary cultures of astrocytes from DBA and C57 mice.

Summary: Effects of chronic treatment of dibutyryl cyclic AMP (db‐cyclic AMP) on Na+, K+‐ATPase activity in cell homogenates and intracellular N a f and K+ contents [(Na+)i and (K+)i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure‐susceptible DBA and audiogenic seizure‐resistant C57 mice. Na+, K+‐ATPase activity in cell homogenates was greater and (Na+)i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K+)i between astrocytes from DBA and C57 mice. Addition of db‐cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na+, K+‐ATPase activity in cell homogenates and decreased (Na+)i, but had no significant effect on (K+)i in astrocytes from either DBA or C57 mice. Chronic treatment with db‐cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K+ and Na+ concentration in medium altered Na+, K+‐ATPase activity in cell homogenates as well as (Na+)i and (K+)i in cultured astrocytes of both DBA and C57 mice. Changes in (Na+)i and (K+)i at different K+ concentrations in medium paralleled those in Na+, K+‐ATPase activity in cell homogenates. Results indicate that the ability to transport Na+ across the cell membrane and the response of Na+, K+‐ATPase to db‐cyclic AMP and to the changes in K + in medium of cultured astrocytes from audiogenic seizure‐susceptible DBA mice are sufficient.

Extracellular (36C1) Space, Electrolyte, Protein, and DNA Content in Brain of DBA and C57 Mice: Effects of Age

Summary: DBA/2J (DBA) mice are susceptible to audiogenic seizures (AGSs) in an age‐dependent manner, susceptibility being maximal at 21 days and absent at 110 days of age. Previous studies have demonstrated that there is a decrease in anion transport and an increase in carbonic anhydrase (CA) activity in brain from DBA mice as compared with C57BL/6J (C57, non‐AGS) mice at 21 days. Since these results suggest that there are alterations in cellular and electrolyte composition of brain from DBA mice, the present work was directed toward determining electrolyte content, extracellular space, and DNA content of brain from DBA and C57 mice at 21 and 110 days of age.

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.

Effects of Glutamate, N‐Methyl‐d‐Aspartate, High Potassium, and Hypoxia on Unit Discharges in CA1 Area of Hippocampal Slices of DBA and C57 Mice

Summary We studied effects of l‐glutamate, N‐methyl‐d‐aspartate (NMDA), high K+, and hypoxia on spontaneous unit discharges in stratum pyramidale of CA1 region of hippocampal slices in DBA and C57 mice aged 3–4 and 5–6 weeks. Application of l‐glutamate (0.5–2.0 mM), NMDA (5–20 μM), high K+ (8.5 mM), and a brief period of hypoxia (1 min) to the perfused artificial cerebrospinal fluid (ACSF) all produced different degrees of spontaneous high‐frequency discharges from CA1 area of hippocampal slices of both DBA and C57 mice. Two types of responses recorded extracellularly occurred after these manipulations: high‐frequency repetitive single spikes and bursts of multiple population spikes. The rate and type of responses from CA1 region of hippocampal slices after these manipulations were different and depended on the strain and age of mice and the nature of manipulations. In general, hippocampal slices from audiogenic seizure‐susceptible DBA mice were more sensitive than those from audiogenic seizure‐resistant C57 mice, and hippocampal slices from younger animals were more susceptible than those from older ones. Thus, DBA mice aged 3–4 weeks of age were most susceptible and C57 mice aged 5–6 weeks were least susceptible to all these pharmacological, ionic, and hypoxic manipulations. Bursts of multiple population spikes were the most common responses in DBA mice and in younger animals, and repetitive single spikes were the predominant responses in C57 mice and in older animals. In all groups of animals, the average spontaneous discharge rate was highest after l‐glutamate perfusion, next highest after NMDA, and lowest after high K+ and hypoxia. The latency of the appearance of spontaneous epileptiform activity from CA1 region of hippocampal slices was long (>2 min) after NMDA perfusion and short (<1 min) after l‐glutamate, high K+ and hypoxia. The duration of the increased spontaneous discharges was short (−1 min) after l‐glutamate perfusion, long (>3 min) after high K+ and hypoxia, and between short and long after NMDA perfusion. These results suggest that age and strain of animal and nature of stimulus precipitate different patterns of epileptiform activity in CNS.