John W. Kemp

Ion-transporting ATPases and matrix mineralization in cultured osteoblastlike cells

SummaryCultures of osteoblastlike cells obtained from the endosteal surfaces of rabbit long bones formed and mineralized an extracellular matrix when they were supplied daily with medium containing fresh ascorbate. No matrix formed without this supplementation. The matrix mineralized whether or not beta-glycerophosphate, a substrate of alkaline phosphatase, was added to the medium. The ion-transporting ATPase activities of untreated, ascorbate-treated, and ascorbate plus beta-glycerophosphate-treated cells were measured. Ascorbate-treated and ascorbate plus beta-glycerophosphate-treated cells had similar enzyme activities. The activities of the Ca2+-ATPase; Ca2+,Mg2+-ATPase; and alkaline phosphatase in treated cells were elevated over the activities in untreated cells. Na+,K+-ATPase activity was lower in treated than in untreated cells. HCO3−-ATPase activity was not changed by treatment. Alkaline phosphatase activity was 20 times higher in freshly isolated osteoblastlike cells than in cells grown to confluence in primary culture. In addition, subculturing further reduced the activity of this osteoblast-marker enzyme. The activities of the ion-transporting ATPases and alkaline phosphatase in second passage cells were similar to the activities of these enzymes in fresh, noncalcifying tissues. Nevertheless, second passage cells retain the ability to mineralize an extracellular matrix, and their ion-transporting ATPase and alkaline phosphatase activities are altered when the cells mineralize a matrix.

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.

Synthesis of urea-cycle intermediates from citrulline in brain

Abstract 1. 1. The biosynthesis in viro or urea-cycle intermediates in the cortex of rat brain from [ ureido - 14 C]citrulline has been studied. This amino acid was found to be readily converted to the succeeding intermediates of the urea cycle, namely, argininosuccinic acid, arginine, and urea. Several additional metabolites were also formed but were not identified. Results presented in this report indicate that the major part of the urea cycle is present in brain. 2. 2. The possibility that the urea cylce in brain may serve more to synthesize imperative metabolites or remove citrulline and argininosuccinic acid than to fix ammonia and permit its excretion as urea is inferred by the rapidity with which citrulline is metabolized to urea and other urea-cylce intermediates.

Effects of cortisol and fluoride on ion-transporting ATPase activities in cultured osteoblastlike cells

SummaryNa+,K+-ATPase, HCO3−-ATPase, Ca2+,Mg2+,-ATPase, Ca2+-ATPase, and alkaline phosphatase activities were measured in cultures of osteoblastlike cells treated with fluoride and cortisol separately and in combinations. Low concentrations of cortisol increased HCO3−-ATPase (10−11 to 10−18M cortisol) and alkaline phosphatase (10−11 to 10−9M cortisol) activities, but higher cortisol concentrations reduced these activities. Na+,K+-ATPase, Ca2+,Mg2+-ATPase, and Ca2+-ATPase activities tended only to be reduced by cortisol.Fluoride (10−6 and 5×10−6M) increased HCO3−-ATPase and alkaline phosphatase activities, but these activities were similar to controls in the presence of 10−5M fluoride. Ca2+,Mg2+-ATPase activity was decreased and Na+,K+-ATPase activity was increased as the concentration of fluoride increased (10−6 to 10−5M). Preliminary experiments with fluoride indicated that lower concentrations (10−7M) were without effect.Cortisol concentrations of 10−9 and 10−8M were chosen for studies with combinations of cortisol and fluoride because the effects of these concentrations on alkaline phosphatase activity were opposite, i.e. 10−9M increased whereas 10−8M decreased activity. Fluoride concentrations of 10−6, 5×10−6, and 10−5M were chosen because a peak of alkaline phosphatase activity occurred at 5×10−6M fluoride. Higher (10−4M) and lower (10−7M) fluoride concentrations were without effect.The effects of combinations of cortisol and fluoride depend on the enzyme activity measured. Fluoride (10−6M) combined with cortisol (10−9M) produced a peak of Na+,K+-ATPase activity. The increased activity obtained with all concentrations of fluoride alone was preserved when fluoride was combined with 10−8M cortisol, although the activity tended to be reduced at 5×10−6 and 10−5M fluoride. HCO3−-ATPase activity was increased by fluoride combined with 10−8M cortisol and decreased by fluoride combined with 10−9M cortisol compared to the activities obtained with fluoride alone. The decrease in Ca2+,Mg2+-ATPase activity caused by fluoride alone was prevented by 10−9 and enhanced by 10−8M cortisol, although all treatments produced the same activity at 10−5M fluoride. Ca2+-ATPase activity tended to be increased by combinations of fluoride and cortisol, but significantly so only at 10−5M fluoride in combinations with 10−8 and 10−9M cortisol. Alkaline phosphatase activity was increased by fluoride combined with 10−9M cortisol and decreased by fluoride combined with 10−8M cortisol compared to the activities obtained with fluoride alone.These results suggest that the abilities of bone cells to regulate ion transport (as reflected in their ion-transporting ATPase activities) are modulated by glucocorticoids and fluoride. Inasmuch as these cells may regulate the ionic composition and concentrations of the bone extracellular fluid (ECF) in vivo, the modulation of their activities by cortisol and fluoride may result in altered bone ECF composition.

Acute and chronic acetazolamide administration in DBA and C57 mice: effects of age

Summary: The clinical utility of the carbonic anhydrase (CA) inhibitor acetazolamide (ACTZ) is limited because of rapid development of tolerance to its effects. Tolerance is thought to develop as a result of glial cell proliferation and/or increased CA synthesis. DBA mice, susceptible to audiogenic seizures (AGSs) in an age‐dependent manner, have increased CA activity as compared with C57 (non‐audiogenic seizure susceptible) mice at 21 and 110 days of age. The present work utilized ACTZ to help determine the relationship between increased CA activity in brain and AGSs in DBA mice. Also, minimal electroshock seizure threshold (EST) was measured at various ages in DBA and C57 mice to determine age‐related changes in CNS excitability. EST was significantly lower in DBA as compared with C57 mice at 18 days and between 40 and 115 days of age, suggesting that DBA mice remain hyperexcitable to electrical stimulation after they develop resistance to AGSs. ACTZ ED50s against maximal electroshock seizures (MES) were significantly higher in DBA as compared with C57 mice at 26, 36, and 115 days of age. This finding correlates with higher CA activity in this strain at 110 days of age, noted previously. However, at 21 days of age, when CA activity is also higher in DBA versus C57 mice, there were no significant differences in ACTZ ED50s against MES between the strains. ACTZ ED50s against AGSs in DBA mice were considerably lower than ACTZ ED50s against MES in either strain, suggesting that a particular fraction of CA is intimately involved in the production of AGSs. Chronic administration of ACTZ to both strains of mice at 26 and 114 days of age demonstrated that C57 mice were able to develop tolerance to this drug as demonstrated by increases in CA activity and ED50s. In contrast, DBA mice were not able to develop tolerance, probably because they are already producing CA at a maximal rate and cannot further induce synthesis of this enzyme.

Subcellular Distribution of Carbonic Anhydrase and Na+,K+- and HCO3--ATPases in Brains of DBA and C57 Mice

Summary: DBA/2J mice are susceptible to audiogenic seizures (ASs) in an age‐dependent manner, susceptibility being maximal at 21 days of age and declining thereafter. DBA, as compared with AS‐resistant C57BL/6J (C57) mice, had higher carbonic anhydrase (CA) activity in cerebral cortex, brainstem, and cerebellum homogenates at 21 days of age. CA activity was also increased in cyto‐solic (82%), microsomal (167%), and myelin (68%) subcellular fractions from cerebral cortex, and in cytosolic (51%) and mitochondrial (102%) fractions from brainstem of DBA mice at 21 days of age. In addition, DBA mice had a higher Na+, K+‐ATPase activity in myelin from cerebral cortex, and a lower HCO3‐‐ATPase activity in mitochondria from brainstem. The differences in CA activity in the cerebral cortex and in HCO3‐‐ATPase were not present at 110 days of age, when DBA mice are no longer susceptible to ASs. Because CA and HCO3‐‐ATPase are involved in maintaining a proper ionic environment for neuronal function, these data suggest that alterations in activity of these enzymes are related to the age‐dependent changes in AS susceptibility in DBA mice.

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.

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.

Identification of Acetylcholine in Sympathetic Ganglia by Chemical and Physical Methods

Extracts of sympathetic ganglionic chains contain a substance which behaves like acetylcholine biologically, chromatographically, and electrophoretically. The melting point of the tetrachloroaurate salt of this substance is identical to that of acetylcholine tetrachloroaurate. No other choline esters have been detected in these extracts. Perfusion of sympathetic ganglia with C14-choline indicates that C14-acetylcholine is released.

A comparison of thyroid function in DBA/2J and C57BL/6J mice.

Summary: DBA/2J mice exhibit audiogenic seizure susceptibility (AGSS) and lower electroshock seizure thresholds compared with C57BL/6J mice. Thyroid function, including thyroxine (T4), 3,5,3′‐triiodothyronine (T3), and thyrotropin (TSH) concentrations, T4/T3 ratio, and iodide uptake, of DBA and C57 mice were compared. Thyroid function was also assessed in relation to AGSS and severity in DBA mice. DBA mice have a larger thyroidal pool of iodide due to increased iodide uptake and possibly decreased release, but not to an increased organification rate. This increased iodide uptake exists until about 40 days of age. DBA mice also have a decreased radiochlo‐ride space and increased thyroid Weight, indicative of enhanced TSH activity. The DBA mice show high T4 and TSH concentrations and a high T4/T3 ratio between the ages of 20 and 40 days. Beginning at 40 days of age the DBA mice have high T4, TSH, and T3 concentrations leading to a T4/T3 ratio approximating the C57 ratio. At any age, DBA mice demonstrating clonic/tonic seizures in response to auditory stimulation have hormone concentrations similar to their 21‐day‐old counter‐ parts with seizures. Mice that show decreased response to auditory stimulation have hormone concentrations similar to the older age group. The increased thyroid activity of DBA mice is the result of enhanced TSH secretion. The increased TSH production is due to adaptations corresponding to the different age and AGSS. A decreased conversion of T4 to T3 by 3,3,5′‐monodeiodinase, is responsible for the increase in TSH due to loss of T3 negative feedback on the anterior pituitary gland. By 40 days of age, the Type 15′‐deiodinase matures whereas the brain deiodinase activity remains subnormal. Therefore, even though the body has sufficient T3, the brain does not, accounting for the high T4, T3, and TSH concentrations post‐40 days. The evidence suggests that the increased seizure susceptibility and varying patterns are influenced by the relative amounts of T4 and T3 in the brain. These amounts are dependent on the activity of the 5′‐monodeiodinase.