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N Brookes

Publications and source records attributed to N Brookes.

At least 37 records · Page 2Linked to original sources
PubMed1989

High-affinity uptake of L-kynurenine by a Na+-independent transporter of neutral amino acids in astrocytes.

L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.

Amino Acids↗
PubMed1988

Neutral amino acid transport in astrocytes: characterization of Na+-dependent and Na+-independent components of alpha-aminoisobutyric acid uptake.

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.

Amino Acids↗
PubMed1988

Specificity and reversibility of the inhibition by HgCl2 of glutamate transport in astrocyte cultures.

Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2-deoxyglucose uptake. Uptake of 2-deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+-ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated X-AG. Acute exposure to 0.5 microM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2-deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 microM as chlorides). The inhibitory action of 0.5 microM HgCl2 on glutamate transport was rapidly reversible. The action of 1-2 microM HgCl2 was progressive when exposures were extended to 1-3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.

Animals↗
PubMed1986

Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry.

This study was undertaken to measure the effect of maximal stimulation of sodium pump activity on the rate of energy metabolism in mouse cerebral astrocytes. The rate of uptake of 3H-2-deoxyglucose (3H-2-DG) was measured in astrocyte cultures sodium-loaded either by incubation in a K+-deficient solution or by use of the carboxylic sodium ionophore monensin. Sodium-loading by the first method caused 3H-2-DG uptake to increase by 80%, but the effect was brief (about 5 min) compared with the period of uptake measurement (20 min). In contrast, the presence of monensin (20 microM) caused a sustained 3.4-fold increase in the rate of 3H-2-DG uptake. The concentration-response relationship for monensin indicated a Kd of 1.5 microM and a maximum uptake enhancement of approximately fourfold. The monensin-stimulated uptake of 3H-2-DG was totally inhibited by incubation of the cultures in either K+-free or Na+-free solutions, or in the presence of ouabain (0.4 mM), indicating that the enhancement of uptake was the result of Na+ influx and sodium pump activation. These results raise the possibility that astroglia contribute significantly to regional variations in glucose consumption associated with functional activity in the brain. Ultrastructural analysis showed that sodium-loading in K+-free solution caused swelling confined to the trans face of Golgi stacks. However, monensin (5 microM) caused swelling of the entire Golgi stack, with progressively more severe swelling from cis to trans cisternae and formation of cytoplasmic vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Determinants of deoxyglucose uptake in cultured astrocytes: the role of the sodium pump.

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.

Animals↗
PubMed1984

Interactions of bupivacaine with ionic channels of the nicotinic receptor. Analysis of single-channel currents.

Bupivacaine and its quaternary derivative, bupivacaine methiodide, were studied on acetylcholine (ACh)-activated single-channel currents recorded in myoballs from neonatal rat muscles using the patch-clamp technique. Under control conditions, the ACh-induced channels had three conductance states, 10, 20, and 33 pS, at a temperature of 10 degrees. The intermediate conductance state (20 pS) was the most prevalent. Moreover, an excessive number of very short events was observed which contributed to a deviation of the channel open-time distribution from a single-exponential function. At 20 degrees, the amplitude of these currents was increased (Q10 = 1.4), and the mean channel open time was decreased (Q10 = 3). Bupivacaine and its quaternary derivative (5-50 microM), when inside the patch micropipette with ACh, caused shortening of the channel open time, but the single-channel conductance remained unchanged at all concentrations studied. In the presence of bupivacaine, there was a loss of voltage dependence of the mean channel open time seen under control conditions; i.e., the shortening of the channel open time was more pronounced at more negative potentials. The plot of the reciprocal of mean channel open time versus bupivacaine concentration was linear. Similar effects were observed when bupivacaine was added to the bathing medium in both cell-attached and inside-out patch conditions, but in this case the onset of the drug action occurred at a later time and its potency was lower. Application of bupivacaine methiodide via the bathing medium after the establishment of the gigaohm seals, however, had no effect on the kinetics of ACh-activated single channels under both patch conditions (cell-attached and inside-out). The patch-clamp results indicated that the charged form of bupivacaine blocks the open state of ACh-activated ionic channels interacting with sites at the extracellular segment of the ACh receptor-ionic channel complex and creating a species with little or no conductance. A sequential model can be used to explain the interactions of these noncompetitive antagonists of the ACh receptor-ionic channel complex with the open channel. This interpretation of the action of bupivacaine and its quaternary analogue as open channel blockers also was reached based on an analysis of macroscopic events in nicotinic synapses of frog muscle.

Acetylcholine↗
PubMed1984

The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. II. Patch clamp studies.

Patch clamping of myoballs to record single channels was performed to examine the interaction of the anticholinesterase agent pyridostigmine (Pyr) with the acetylcholine (ACh) receptor-ion channel complex. Single ACh channel currents were recorded from tissue-cultured muscle cells of neonatal rats (myoballs). Pyr (50-100 microM) decreased the frequency of channel-opening events activated by ACh, and induced a modified form of the ACh channel currents. Channel conductance was lower in the presence of Pyr, and channel lifetime remained unaltered or only slightly prolonged. In addition, channel openings were frequently interrupted by fast flickers in the presence of Pyr. Higher concentrations (200 microM-1 mM) of the drug induced irregular waves of bursting activity during the initial phase of the application, and, subsequently, significantly reduced the frequency of channel openings. Infrequent channel openings with low conductance were observed in the patch when the micropipette was filled with Pyr alone. These results suggest that, in addition to its anticholinesterase activity, Pyr reacts with the ACh receptor, and both alone or in combination with ACh induces an altered, desensitized species of the nicotinic receptor-ion channel complex.

Animals↗
PubMed1980

The use of spinal cord cell cultures in the study of neurotoxicological agents.

Primary cell cultures of mouse embryo spinal cords were used to study the biological effects of exposure to organophosphate esters and neurotoxic amino acids. The effects of exposure were correlated with markers of cholinergic function. The purpose of this study was to examine the mechanisms whereby xenobiotics produce neurotoxicologic damage.

Animals↗
PubMed1980

Development of muscarinic receptor binding in spinal cord cell cultures and its reduction by glutamic and kainic acids.

The density of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites in dissociated cell cultures of mouse spinal cord increased almost 4-fold between the 5th and 21st days of maturation in vitro. The mean specific binding of [3H]QNB in 21-day cultures was 340 pmol/g protein. Up to 79% of [3H]QNB binding sites were lost within a few days after exposure of the cultures to 1 mM glutamate. This neurotoxic action of glutamate showed a steep concentration dependence in the range 0.1-1 mM and did not vary with the duration of exposure of the cultures from 10 min to 24 h. Kainic acid was only moderately more potent than glutamate in reducing [3H]QNB binding. The susceptibility of 8-day cultures to glutamate-induced loss of [3H]QNB binding was less than that of 12- and 19-day cultures. These findings suggest that 80% or more of muscarinic binding sites in the spinal cord cell cultures are on neurons and that the sensitivity of these neurons to the toxic action of glutamate increases with their maturation in vitro.

Aging↗
PubMed1971

Diffusion of labelled substances through isolated rat diaphragm.

1. The rate of diffusion of [(14)C] sucrose through rat diaphragm muscle is dependent on the period of preliminary soaking of the muscle in Krebs solution at 36 degrees C. The ratio of the length of the diffusion path through the diaphragm to its geometrical thickness appears to fall from 4.39+/-0.38 (S.E. of the mean) to 2.23+/-0.18 after about 6 h of soaking.2. The area of the diaphragms available for diffusion is apparently constant at 0.3-0.4 of their total area.3. The measured thickness of the diaphragms increases by about 20% on soaking.4. Entry of [(131)I] iodide into the muscle fibres is detected as a fall in both steady state and non-steady state rates of diffusion through the diaphragm.5. Entry of [(14)C-methyl] decamethonium into the muscle cells is not evident from such diffusion studies in normal Krebs solution.

Animals↗