The rapid L- and D-aspartate uptake in cultured astrocytes.

Astrocytes in primary culture possess a rapid L-aspartate saturable transport system (K(m) = 93 microM; V(max) = 81 nmol/min/mg protein), which shows certain stereospecificity since V(max) was 36% lower for D-aspartate uptake. These are values obtained at short incubation time (15 seconds), to obtain approximate initial rate conditions. Metabolic energy inhibitors, rotenone and iodoacetate very potently inhibited the L- and D-aspartate uptake processes, indicating that the transport process is an active one. However, the accumulation of L-aspartate was "enhanced" by inhibitors of L-aspartate metabolism, such as the aspartate aminotransferase inhibitor, aminooxyacetate and L-methionine sulfoximine, an inhibitor of glutamine synthetase, whereas D-aspartate (a non-metabolizable analog of L-aspartate) uptake was not affected. The accumulated levels of L-aspartate in the presence of aminooxyacetate were similar to the levels of D-aspartate. These effects of L-aspartate metabolic inhibitors, suggest that due to metabolism of the the L-aspartate, short incubation time (eg., 15 seconds) is necessary to measure the initial rate of L-aspartate uptake, in order to obtain the "true" kinetic parameters.

Ionic dependence of adenosine uptake into cultured astrocytes.

Adenosine uptake in cultured astrocytes is dependent on various ions and energy metabolism. The Na(+)-gradient plays an important role, since nigericin, ouabain, amiloride and substitution of Na+ with choline inhibited adenosine uptake. The proton-gradient was of importance, since carbonylcyanide m-chlorophenylhydrozone (CCCP) and omeprazole also inhibited adenosine uptake. Furthermore, adenosine uptake was dependent on Cl- anion. Substitution of Cl- with isethionate, as well as DIDS or furosemide inhibited adenosine uptake. Adenosine uptake was also sensitive to Ca2+ gradient, removal of extracellular Ca2+ and calcimycin inhibited adenosine uptake. Adenosine uptake was not dependent on extracellular K+ and was not affected by valinomycin. Although, K(+)-channel openers (BRL 34195 and nicorandil) as well as the K(+)-channel antagonist, glyburide, inhibited adenosine uptake, the inhibitory effect of BRL 34915 was not antagonized by glyburide. Rotenone and 2,4-dinitrophenol also inhibited adenosine uptake. Ionic dependence and metabolic energy dependence of adenosine uptake suggest that uptake is primarily an active process.

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

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 approximately 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 pHi 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 pHo. 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 pHi or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.

Water and electrolyte contents, cell pH, and membrane potential of primary cultures of astrocytes from DBA, C57, and SW mice.

Some basic properties of primary cultures of astrocytes derived from the cerebral cortex of an audiogenic seizure-sensitive strain of mice, DBA/2J (DBA), were studied with different approaches. The results were compared with those of audiogenic seizure-resistant strains, C57BL/6J (C57) and Swiss Webster (SW). Contents of intracellular water, protein, and DNA of DBA astrocytes were 0.673 +/- 0.019 ml/g cells, 0.082 +/- 0.006 g/g cells, and 0.0072 +/- 0.0005 g/g cells, respectively. These results are not different from those of either C57 or SW astrocytes. Intracellular concentration of K+, Na+, and Cl- ([K+]1, [Na+]1, and [Cl-]1) derived from the flame photometric and from the radioisotope uptake data of DBA astrocytes were 120.4 +/- 8.5, 25.9 +/- 3.2, and 26.8 +/- 1.8 mM/L cell H2O, respectively. [Na+]1 and [Cl-]1 in DBA astrocytes were lower than those in C57 and SW astrocytes. In DBA astrocytes, SITS decreased the cell/medium ratio (C/M) of 36Cl- and increased the C/M of 125I-; ouabain increased the C/M of 22Na+ and decreased the C/M of 125I-; bumetanide decreased the C/M of both 36Cl- and 22Na+; and NaClO4 decreased the C/M of 125I-. Similar results were observed in both C57 and SW astrocytes. Intracellular pH (pHi) as determined with 14C-DMO of astrocytes in HEPES-buffered saline solution averaged 7.04 +/- 0.03 for DBA, 7.01 +/- 0.02 for C57, and 6.97 +/- 0.02 for SW mice when pH of medium was maintained at 7.4. Modification of ion (HCO3-, Cl-, Na+, and K+) concentration and pH of culture medium all changed the pHi of astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of elevated potassium on the ion content of mouse astrocytes and neurons.

Potassium is tightly regulated within the extracellular compartment of the brain. Nonetheless, it can increase 3- to 4-fold during periods of intense seizure activity and 10- to 20-fold under certain pathological conditions such as spreading depression. Within the central nervous system, neurons and astrocytes are both affected by shifts in the extracellular concentration of potassium. Elevated potassium can lead to a redistribution of other ions (e.g., calcium, sodium, chloride, hydrogen, etc.) within the cellular compartment of the brain. Small shifts in the extracellular potassium concentration can markedly affect acid-based homeostasis, energy metabolism, and volume regulation of these two brain cells. Since normal neuronal function is tightly coupled to the ability of the surrounding glial cells to regulate ionic shifts within the brain and since both cell types can be affected by shifts in the extracellular potassium, it is important to characterize their individual response to an elevation of this ion. This review describes the results of side-by-side studies conducted on cortical neurons and astrocytes, which assessed the effect of elevated potassium on their resting membrane potential, intracellular volume, and their intracellular concentration of potassium, sodium, and chloride. The results obtained from these studies suggest that there exists a marked cellular heterogeneity between neurons and astrocytes in their response to an elevation in the extracellular potassium concentration.

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

In astrocytes, 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. In neurons, 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. In astrocytes, pHi increased when [K+]o was increased. In neurons, 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 both astrocytes and neurons, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

pH regulation after acid load in primary cultures of mouse astrocytes.

Intracellular pH (pHi) recovery in primary cultures of mouse astrocytes after acid-loading was studied with the ion transport inhibitors (amiloride, SITS, acetazolamide, ouabain and bumetanide), and by reducing the concentration of Na+ or Cl- in HCO3- -free HEPES-buffered (HEPES) and in HCO3-/CO2 Hanks' balanced salt solution (HBSS). The pHi of astrocytes exposed to 15 mM NH4Cl decreased abruptly and began to recover slowly after 5 min. Exposure of the cells to NH4Cl for 2 min and reincubation in HEPES HBSS decreased pHi further within 1-2 min after removal of NH4Cl; pHi then recovered toward the control value. Cultures exposed to HCO3-/CO2 HBSS (10 mM/2%) showed changes in pHi in the opposite direction. These responses are unique to astrocytes and differ from those occurring in most other cells. Recovery of pHi after NH4Cl prepulse was markedly inhibited in low-Na+ and in amiloride-containing HEPES HBSS. Ouabain also reduced pHi recovery rate; however, SITS, acetazolamide and bumetanide did not. Therefore, Na(+)-H+ exchange is the major process for pHi recovery from acidification in HCO3- -free solution. In HCO3-/CO2 HBSS pHi recovery was markedly inhibited by SITS and acetazolamide, but not by amiloride, ouabain, or bumetanide. The inhibitory effect of SITS on pHi recovery was enhanced in low-Na+ HBSS. These results indicate that both Na+ and HCO3- are directly related to pHi recovery in HCO3-/CO2 solution after acid-load. Low-Cl HEPES HBSS and low-Cl HCO3-/CO2 HBSS media did not alter pH recovery rate. Thus, pHi recovery after acid-load is not Cl- -dependent, and therefore, does not involve a Na(+)-dependent Cl- -HCO3- exchange process. It appears that mouse astrocytes possess 3 acid-regulating systems: Na(+)-H+ exchange, Na(+)-HCO3- co-transport and Na(+)-independent Cl- -HCO3- exchange.

Vagal stimulation reduces the severity of maximal electroshock seizures in intact rats: use of a cuff electrode for stimulating and recording.

J. Zabara showed that repetitive vagal stimulation (VS) prevents or ameliorates convulsive seizures in dogs. We have studied the effects of VS on maximal electroshock seizures (MES) in intact rats: (1) A 5 wire cuff electrode was developed for stimulating and recording from the vagus. Compound action potentials (AP) were recorded and strength-duration curves obtained for A and C fibers. There is a monotonic relationship with a negative slope between heart rate (HR) and AP amplitude. C fibers remain excitable for 25 days after cuff implant. (2) The anticonvulsant efficacy of VS is directly related to the fraction of vagal C fibers stimulated and the frequency of stimulation. (3) The anticonvulsant efficacy of VS has been established using two rat models of human epilepsy. VS abolishes the extensor component of the tonic phase of a MES and shortens or prevents tonic seizures induced by pentylenetetrazol (PTZ). (4) VS appears to act via release of large quantities of the inhibitory mediators GABA and glycine throughout large volumes of the brain. (5) It is rational to test VS in man as a treatment for intractable seizures.

Relation between pH regulation and iodide transport in turtle thyroid glands.

Mechanisms of pH recovery after alkalinization and acidification by exposing or prepulsing turtle thyroid slices with a Hanks' balanced salt solution (HBSS) containing NH4Cl or CO2 were studied by examining the effects of amiloride, 4-acetamido-4'-isocyanostilbene-2,2'-disulphonic acid (SITS), frusemide and acetazolamide, and of reducing the concentration of Na+ or Cl- in the incubation medium. When alkalinization was produced either during exposure to NH4Cl or after a CO2 pulse, the pH in thyroid slices rose rapidly and then recovered gradually. Addition of SITS (0.1 mmol/l) or reduction of the Cl- concentration markedly inhibited pH recovery. However, amiloride (0.1 mmol/l) and low Na+ in the medium had no significant effect on recovery from alkalinization induced by NH4Cl exposure or by a CO2 pulse. These data suggest that pH recovery from alkalinization in turtle thyroid gland is achieved by an exchange of internal HCO3- for external Cl-. When acidification was accomplished by either exposure to CO2 or removal of NH4Cl, the pH of thyroid slices fell rapidly and then recovered gradually. If amiloride was added or the Na+ concentration in the medium was reduced, the pH recovery was greatly attenuated. However, SITS and low Cl- in the medium did not affect the recovery from an acid load in turtle thyroid slices. These results suggest that pH recovery from acidification in turtle thyroid gland is achieved by an exchange of internal H+ for external Na+. Both frusemide and acetazolamide prevented the pH recovery in turtle thyroid slices during exposure to and withdrawal from NH4Cl. These results suggest that besides the Na(+)-H+ and Cl(-)-HCO3- exchange processes, other mechanisms may also be involved in pH regulation in turtle thyroid glands. Simultaneous uptakes into turtle thyroid slices of 125I- and 22Na+ and of 125I- and 36Cl- were studied during and following exposure to NH4Cl in the absence and presence of different transport inhibitors, such as frusemide, amiloride, SITS and acetazolamide. When the thyroid slices were exposed to HBSS containing 30 mmol/l NH4Cl (alkalinization phase), the tissue/medium (T/M) ratios of 125I- increased gradually, reached the highest point in 10 min, and were maintained at this level for the next 20 min. The T/M ratios of 22Na+ and 36Cl- of thyroid slices also slowly increased after exposure to NH4Cl.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation of cancellous bone to aging and immobilization in the rat: a single photon absorptiometry and histomorphometry study.

Nine-month-old female rats were double-labeled with bone markers and subjected to right hindlimb immobilization or served as control for 0, 2, 10, 18, or 26 weeks. The right limb was immobilized against the abdomen, thus unloading it, while the left limb was overloaded during ambulation. Single photon absorptiometry and cancellous bone histomorphometry were performed on dissected intact femur and 20-microns-thick undecalcified specimens of the proximal tibial metaphysis. In the unloaded limb, immobilization-induced muscle and cancellous bone loss occurred rapidly before 10 weeks and stabilized at 50% less bone mass after 18 weeks. Unloading caused a negative bone balance from a combination of elevated bone resorption and depressed bone formation. At 2, 10, and 18 weeks of immobilization, the ratios of bone resorption to bone formation surfaces were 1.6, 1.5, and 1.3, respectively; at 26 weeks, the ratio was 1. The bone loss was accompanied by poorer trabecular architecture (trabecular number decreased and trabecular separation increased), reaching the maximum at 18 weeks and stabilizing thereafter. These observations are in general agreement with Frost's postulate for mechanical effects on lamellar bone remodeling, and the findings on disuse osteoporosis in man. Therefore, the one-legged immobilization model can be useful in studies of the mechanisms of structural adaptation to mechanical usage.

Effects of vagal stimulation on experimentally induced seizures in rats.

Repetitive stimulation of the vagus nerve inhibits chemically induced seizures in dogs. We report here the results and conclusions from studies designed to answer some of the immediate questions raised by this finding. (1) Maximal stimulation of vagal C fibers at frequencies greater than 4 Hz prevents or reduces chemically and electrically induced seizures in young male rats. (2) Antiepileptic potency is directly related to the fraction of vagal C fibers stimulated. (3) Vagal stimulation shortens but does not shut down a chemical seizure once it has begun. (4) In rats, optimal stimulus frequency is approximately 10-20 Hz; duration of stimulus, 0.5-1 ms; and stimulus strength, 0.2-0.5 mA/mm2 of nerve cross-section. These results, when taken together with similar results obtained from dogs, monkeys, and humans, strongly suggest that periodic stimulation of the vagus nerve using appropriate stimulation parameters is a powerful method for preventing seizures. The data from the literature suggest that the antiepileptic actions of vagal stimulation are largely mediated by widespread release of GABA and glycine in the brainstem and cerebral cortex. The probable pathway is via projections from the nucleus of the solitary tract to the reticular formation and thence by diffuse projections to the cortex and other areas. Intermittent vagal stimulation has the potentiality of reducing the number and/or the intensity of seizures in patients with intractable epilepsy. These results indicate that feasibility studies in humans should be continued and expanded.

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

Primary cultures of both mouse astrocytes and neurons accumulate more 125I- than 36Cl- from the medium. The average cell/medium ratio of 125I- of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of 36Cl- of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both 125I- and 36Cl- 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 inhibiting 125I- uptake whereas furosemide is more effective in inhibiting 36Cl- 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 both 125I- and 36Cl- uptakes into neurons were higher than those in [Na+]o between 5 and 60 mM. These results indicate that uptake of 125I- and 36Cl- into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.

Beta-DL-methylene-aspartate, an inhibitor of aspartate aminotransferase, potently inhibits L-glutamate uptake into astrocytes.

[3H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analogues L- and D-aspartic acid, DL-threo-beta-hydroxy-aspartic acid-beta-hydroxymate (IC50's: 136, 259, 168, and 560 microM, respectively) and by beta-DL-methylene-aspartate, a suicide inhibitor of aspartate aminotransferase (IC50: 524 microM), and by the endogenous sulphur-containing amino acid L-cysteinesulfinic acid (IC50: 114 microM), [3H]Glutamate uptake was not significantly affected by either N-methyl-D-aspartate or DL-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate and L-cysteinesulfinic acid (but excluding L-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.

Mechanisms of tolerance to the anticonvulsant effects of acetazolamide in mice: relation to the activity and amount of carbonic anhydrase in brain.

The mechanism by which tolerance develops to the anticonvulsant effects of acetazolamide (AZM) was investigated in Swiss-Webster mice. The effects of single and six daily doses of 40 mg or 200 mg/kg AZM on electroshock seizure threshold (EST), maximal electroshock (MES) seizure pattern, and on the activity and total amount of carbonic anhydrase II (CAII) in various subcellular fractions (cytosol, microsomes, and myelin) of cerebral cortex, cerebellum, and brainstem were assessed. The activity of CAII was measured by microassay, and the total amount was measured by immunoassay methods developed in this laboratory. From the activity (units per microgram of protein) and total amount (nanograms per microgram protein) data, the specific activity (units per nanogram CAII) of the enzyme was calculated. With multiple doses, tolerance developed to both elevation of the EST and modification of the MES pattern noted with single doses of AZM. Accompanying the development of tolerance to the anticonvulsant effects of AZM was an increase in both the activity and specific activity of CAII in the various subcellular fractions and areas of the brain. The effects were dose dependent. Tolerance to the EST-elevating effects of AZM correlated with increases in the activity, total amount, and specific activity of CAII in the myelin fraction of the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium-stimulated calcium uptake in astrocytes and its potent inhibition by nimodipine.

Elevation of the extracellular potassium concentration above its "resting" level of 5.4 mM stimulated uptake of 45Ca2+ in primary cultures of astrocytes. This effect was only observed when cells were exposed to excess potassium shortly after their exposure to 45Ca2+ and was potently inhibited (IC50 congruent to 3 nM) by the calcium channel blocker nimodipine. In contrast, nimodipine exerted little effect on unstimulated basal uptake of 45Ca2+. These findings suggest that the therapeutic benefit of calcium channel blockers in epilepsy may result in part from the ability of these drugs to prevent calcium entry into astrocytes during seizures when the extracellular potassium is elevated four- to fivefold above normal.

Hypothesis: the classification of epileptic seizures according to systems of the CNS.

Several comprehensive classifications of the epilepsies and epileptic syndromes have been proposed and the purpose of the last one (1985) was to supplement the International Classification of Epileptic Seizures as accepted by the General Assembly of the International League Against Epilepsy in 1981. These efforts represent a major contribution to a difficult problem, which must be continued as further scientific advances permit. Categorization according to clinical seizure type is a logical approach, inasmuch as response to antiepileptic medication depends more on seizure type than on etiological or other factors. A limitation of this approach, however, has arisen as a result of the Commission's failure to relate clinical seizure types to the underlying neurophysiology that mediates them. This has resulted in the categorization of absence as a generalized form of seizure along with generalized tonic-clonic seizures (GTCS). On the other hand, temporal lobe seizures (including automatisms) have been categorized as a complex form of partial seizures, which emphasizes their focal origin. It is the belief of the authors that a further clarification might be achieved, if a differentiation of seizure types is made with respect to the underlying CNS circuitry which mediates the sustained discharge that determines the clinical manifestations of each type. Focal origins, while significant, assume a secondary importance, since they do not explain the sustained discharge that mediates clinical seizure types, except in the case of elementary partial seizures (EPS) that remain discretely localized. In contradistinction to GTCS that essentially involve the entire CNS, absence and complex partial seizures (CPS) should be categorized as limited forms of seizures, mediated by bilateral, regional systems.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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 radiochloride 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 counterparts 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 I 5'-deiodinase matures whereas the brain deiodinase activity remains subnormal.(ABSTRACT TRUNCATED AT 250 WORDS)

Seizure susceptibility in DBA and C57 mice: the effects of various convulsants.

Convulsive dose 50s (CD50s) for various convulsive drugs and minimal and maximal electroshock seizure thresholds were determined in DBA and C57 mice. DBA mice had lower maximal electroshock seizure thresholds (MESTs, 15%) and CD50s for homocysteine thiolactone (HTL, 23%) and bicuculline (69%), and a higher CD50 for pentylenetetrazol (PTZ) at 3 weeks of age, the age of maximal audiogenic seizure (AGS) susceptibility. At 8 weeks, when DBA mice are not susceptible to AGSs, significant differences were a lower minimal electroshock seizure threshold (mEST, 37%) and maximal EST (MEST) (19%), lower CD50s for N-methyl-D-aspartate (NMDA) (39%), kainic acid (KA, 50%), HTL (32%), strychnine (37%), and a higher CD50 for nicotine (55%) in DBA mice. Based on these data it is suggested that pathways involving NMDA and KA receptors are responsible for increased susceptibility to seizure initiation (mEST), and are opposed by glycine pathways, and that opposing GABA and cholinergic systems at higher CNS levels are involved in seizure spread (AGSs and MEST) in these mice. Latency patterns indicate that nicotine, strychnine, PTZ and bicuculline have high blood-brain barrier (BBB) penetrability. Picrotoxin and the excitatory amino acid receptor agonists had longer latencies, suggesting low BBB penetrability. Age-related changes in latency, however, give evidence that difficulty in drug penetration of the BBB is not responsible for differences observed in CD50s between strains.

Effects of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells.

Iodide uptake by primary cultures of turtle thyroid follicular cells is directly proportional to the Na+ concentration and is inversely proportional to the HCO3- concentration in culture medium, but is not affected by the Cl- concentration. Addition of 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS; 10 mumol/l and higher doses) to medium containing different concentrations of Na+ (5-140 mmol/l), HCO3- (0-40 mmol/l) and Cl- (120 mmol/l) generally enhanced iodide uptake by the cultured cells; however, there was no significant effect in Na+-free and in low Cl- (90 mmol/l and less) medium. The inhibitory effects on iodide uptake of ouabain, frusemide and perchlorate were attenuated by DIDS which also antagonized the stimulatory effects on iodide uptake of TSH, although both DIDS and TSH increased the 125I- cell/medium ratio when they were given alone. At doses of 100 mumol/l and higher, DIDS lowered the intracellular pH of cultured cells when the pH of the medium was maintained at a constant level. It also increased the intracellular Cl- concentration, but had no effect on intracellular Na+ or K+. The input and specific resistances of cell membranes in cultured thyroid cells and in isolated thyroid slices increased (decreased conductance) after adding DIDS to the perfusion fluids. Both Na+/K+- and HCO3(-)-ATPase activities in homogenates of turtle thyroid tissue were inhibited by DIDS. Results from this investigation demonstrate (1) that in addition to preventing the leak of iodide from thyroid cells, DIDS may act to increase the sensitivity of the Na+-anion carrier to I- and thereby increases iodide uptake, and (2) that a HCO3(-)-Cl- exchange system is present in the thyroid cell membrane and appears to be linked to the transport of iodide into thyroid cells.

The role of bone cells in increasing metaphyseal hard tissue in rapidly growing rats treated with prostaglandin E2.

The skeletal effects of graded doses of prostaglandin E2 (PGE2) given to weanling Sprague-Dawley rats for 3 weeks were investigated to elucidate the role of bone cells in increasing hard tissue mass. Decalcified (3 micron) sections were quantified in the light microscope by point hit and intersect counting using a Merz grid. Hard tissue mass (bone and calcified cartilage) and osteoblast, osteoclast and osteoprogenitor cell numbers were counted in metaphyseal tissue bands 0.24, 0.48, 0.72, 1.20, 1.68, 2.16, 2.64, 3.12, 3.60 and 4.08 mm from the growth plate metaphyseal junction. Changes were different and more marked in the secondary spongiosa than the primary spongiosa of the proximal tibial metaphysis of treated rats. In the primary spongiosa of rats treated with 3 or 6 mg PGE2/kg/d (1) an increase in bone and hard tissue masses and (2) a decrease in osteoclasts, osteoprogenitor cell numbers and surface to volume ratio was observed. In the secondary spongiosa (lower metaphysis) of rats treated with 2 same dose levels (1) an increase in bone mass, calcified cartilage cores, and hard tissue mass and perimeter, an elevation of osteoprogenitor cell and osteoblast numbers, a depression of osteoclast, osteoclast nuclei numbers and surface to volume ratio and new sites of intramembranous ossification (woven bone formation) originating from the cortico-endosteal envelope was observed. In this growing rat skeletal model, we showed that PGE2 increases metaphyseal calcified tissue mass by depressing hard tissue resorption and stimulating the replication and differentiation of osteoblast precursors to form new foci of woven bone.(ABSTRACT TRUNCATED AT 250 WORDS)