Search PubMed⌕ Search

Biomedical subjects

N Brookes

Publications and source records attributed to N Brookes.

At least 19 recordsLinked to original sources
PubMed2006

Guiding practice development using the Tidal Commitments.

The Tidal Model of Mental Health Recovery has contributed to the transformation of nursing practice at the Royal Ottawa Hospital (ROH), a psychiatric and mental health facility in Ontario, Canada. Ten commitments affirm the core values of the Tidal Model. These commitments guide person-centred, collaborative, strength-based practice and they facilitate Tidal teaching. In this paper we illustrate fidelity to the values, principles and processes of the model and the commitments while implementing the model. We share how some of the commitments are realized in our Tidal teaching and provide examples of successes and challenges.

Attitude of Health Personnel↗
PubMed2001

Transfer of glutamine between astrocytes and neurons.

The export of glutamine from astrocytes, and the uptake of glutamine by neurons, are integral steps in the glutamate-glutamine cycle, a major pathway for the replenishment of neuronal glutamate. We review here the functional and molecular identification of the transporters that mediate this transfer. The emerging picture of glutamine transfer in adult brain is of a dominant pathway mediated by system N transport (SN1) in astrocytes and system A transport (SAT/ATA) in neurons. The participating glutamine transporters are functionally and structurally related, sharing the following properties: (a) unlike many neutral amino acid transporters which have proven to be obligate exchangers, these glutamine transporters mediate net substrate transfer energized by coupling to ionic gradients; (b) they are sensitive to small pH changes in the physiological range; (c) they are susceptible to adaptive and humoral regulation; (d) they are related structurally to the AAAP (amino acid and auxin permeases) family of transporters. A key difference between SN1 and the SAT/ATA transporters is the ready reversibility of glutamine fluxes via SN1 under physiological conditions, which allows SN1 both to sustain a glutamine concentration gradient in astrocytes and to mediate the net outward flux of glutamine. It is likely that the ASCT2 transporter, an obligate exchanger of neutral amino acids, displaces the SN1 transporter as the main carrier of glutamine export in proliferating astrocytes.

Amino Acid Transport Systems↗
PubMed1999

The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux.

Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14C]glutamine (Km = 70 microM) that was Na(+)-dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.

Amino Acid Sequence↗
PubMed1998

Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes.

We describe an unconventional response of intracellular pH to NH4Cl in mouse cerebral astrocytes. Rapid alkalinization reversed abruptly to be replaced by an intense sustained acidification in the continued presence of NH4Cl. We hypothesize that high-velocity NH4+ influx persisted after the distribution of ammonia attained steady state. From the initial rate of acidification elicited by 1 mM NH4Cl in bicarbonate-buffered solution, we estimate that NH4+ entered at a velocity of at least 31.5 nmol.min-1.mg protein-1. This rate increased with NH4Cl concentration, not saturating at up to 20 mM NH4Cl. Acidification was attenuated by raising or lowering extracellular K+ concentration. Ba2+ (50 microM) inhibited the acidification rate by 80.6%, suggesting inwardly rectifying K+ channels as the primary NH4+ entry pathway. Acidification was 10-fold slower in rat hippocampal astrocytes, consistent with the difference reported for K+ flux in vitro. The combination of Ba2+ and bumetanide prevented net acidification by 1 mM NH4Cl, identifying the Na(+)-K(+)-2Cl- cotransporter as a second NH4+ entry route. NH4+ entry via K+ transport pathways could impact "buffering" of ammonia by astrocytes and could initiate the elevation of extracellular K+ concentration and astrocyte swelling observed in acute hyperammonemia.

Acids↗
PubMed1997

Intracellular pH as a regulatory signal in astrocyte metabolism.

An intracellular alkalinization is observed in mammalian astrocytes in response to repetitive neuronal activity in vivo. The alkalinizing effect of potassium released from electrically active neurons predominates over many other influences on the intracellular pH of astrocytes, including acid loads induced by glutamate and ammonium. There is evidence that this pH signal, elicited by neuronal activity, may facilitate glucose utilization and glutamine formation in astrocytes. This short review surveys the mechanisms of the intracellular pH changes induced in astrocytes by extracellular potassium, glutamate, and ammonium. It then focuses upon the regulatory effects of these pH changes on glucose utilization, as estimated primarily by rates of deoxyglucose phosphorylation, and on the uptake and metabolism of glutamate. Many studies in this field exploit the advantages of astrocyte cell culture. However, the conditions of cell culture can diminish expression of the intracellular alkalinization induced by potassium and thus lead to underestimation of the metabolic significance of this pH signal.

Animals↗
PubMed1996

Characterization of L-alpha-aminoadipic acid transport in cultured rat astrocytes.

The mechanism of the selective gliotoxicity of L-alpha-aminoadipate (L-alpha AA) is thought to involve its entry into glia as a substrate for glutamate transporters or, alternatively, its ability to inhibit glial glutamate transport. To clarify the properties of L-alpha AA as a transport substrate, we explored the ionic dependence, kinetics and pharmacology of L-[3H] alpha AA uptake in rat cortical astrocytes. We observed two components of saturable L-alpha AA uptake, one Na(+)-dependent and the other Na(+)-independent. These components exhibited the characteristics of system X-AG, the widespread family of Na(+)-cotransporters of aspartate and glutamate, and system x-c, a Cl(-)-dependent glutamate/cystine exchanger, respectively. The K(m) value of Na(+)-dependent L-alpha AA uptake was 629 +/- 42 microM, and Vmax was 62 +/- 4 nmol.min-1.mg-1 protein, which was more than twice the capacity of Na(+)-dependent glutamate uptake. The kinetic parameters of Na(+)-dependent L-alpha AA uptake (K(m) of 20 +/- 2 microM, Vmax of 1.7 +/- 0.4 nmol.min-1.mg-1 protein did not differ from the values for Na(+)-independent glutamate uptake, indicating that L-alpha AA and glutamate are equally good substrates for system x-c.

2-Aminoadipic Acid↗
PubMed1996

Potassium-induced stimulation of glutamate uptake in mouse cerebral astrocytes: the role of intracellular pH.

The Na(+)-glutamate cotransporters are believed to countertransport OH- and K+. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO3- dependent. Also, it was reported recently that raising extracellular [K+] alkalinizes astrocyte cytoplasm by an HCO3- dependent mechanism. Here, we have compared glutamate uptake in HCO3(-)-buffered and HCO3(-)-depleted solutions at varying [K+]. We observed a pronounced stimulation of glutamate uptake by extracellular K+ (3-24 mM) that was substantially HCO3- dependent and affected preferentially the uptake of high concentrations (> 25 microM) of glutamate. Stimulation of uptake by low extracellular [K+] (1.5-3 mM) was less dependent on HCO3-. Potassium-induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba2+ and amiloride on glutamate uptake, as well as the HCO3(-)-dependent stimulatory effects of K+ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH-. A "bimodal" scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.

Animals↗
PubMed1996

Glutamine transport in mouse cerebral astrocytes.

We measured initial influx and exchange of [14C]glutamine in primary astrocyte cultures in the presence and absence of Na+. Kinetic analysis of transport in Na+ -free solution indicated two saturable Na+ -independent components, one of which was identifiable functionally as system L1 transport. In the presence of Na+, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na+ -dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by alpha-(methylamino) isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na+ -dependent uptake, preferential inhibition of an Li+ -intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and alpha-(methylamino) isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na+, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 microM glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li+ in place of Na+.

Amino Acids↗
PubMed1996

Mercuric chloride uncouples glutamate uptake from the countertransport of hydroxyl equivalents.

The cotransport of sodium and glutamate by system X(AG)- is believed to be coupled to the countertransport of potassium and hydroxyl ion equivalents. Accordingly, the uptake of glutamate or D-aspartate in astrocytes is accompanied by an intracellular acidification. Here, we report that HgCl2 blocks the glutamate-induced acidification with an approximate 50% inhibitor concentration (IC50) of 55 nM, an order of magnitude below its IC50 for inhibition of glutamate uptake. At 100 nM HgCl2, glutamate-induced acidification was abolished, whereas glutamate uptake was unaffected. D-Aspartate-induced acidification was equally sensitive to HgCl2, indicating that HgCl2 blocked a transporter-mediated, rather than a receptor-mediated, acidification. Unaltered responses to acute acid and alkaline loads showed that HgCl2 was not acting indirectly via a change in pH regulation. We conclude that HgCl2 acted directly on the glutamate transporter to uncouple the uptake of glutamate from the export of hydroxyl equivalents. In contrast, two other sulfhydryl reagents, p-chloromercuribenzensulfonate and N-ethylmaleimide, failed to discriminate between glutamate-induced acidification and glutamate uptake. An additional effect of > or = 100 nM HgCl2, in this case shared by p-chlormercuribenzenesulfonate, was transient intracellular acidification. There is evidence that glutamate transport is regulated by intracellular pH. Mercuric mercury may disrupt the regulation of glutamate transport at lower concentrations than those that block transport.

4-Chloromercuribenzenesulfonate↗
PubMed1994

K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO3- cotransport.

Raising extracellular K+ concentration ([K+]o) induces an alkaline shift of intracellular pH (pHi) in astrocytes. The mechanism of this effect was examined using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+]o from 3 to 12 mM increased pHi by 0.28 pH units in 26 mM HCO(3-)-buffered solution. In nominally HCO(3-)-free solution (containing approximately 95 microM HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na(+)-HCO3- cotransport. The relationship between pHi and log[K+]o was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pHi elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na(+)-HCO3- cotransporter was outward at rest and was reversed by depolarization.

Animals↗
PubMed1993

Extracellular potassium regulates the glutamine content of astrocytes: mediation by intracellular pH.

Based upon previous evidence that glutamine formation in astrocytes is pH-sensitive and that raised extracellular K+ alkalinizes astrocytic cytoplasm, it was hypothesized that extracellular K+ might regulate glutamine formation. In this study, the free glutamine content of mouse cerebral astrocytes incubated with 0.1 mM glutamate and 0.1 mM ammonium increased by 80-90% when the extracellular K+ concentration was raised from 3 to 12 mM. The corresponding K(+)-induced intracellular alkalinization of +0.13 pH units only partially reversed a glutamate-induced intracellular acidification of -0.24 pH units. By comparison, adjustment of extracellular pH from 7.4 to 7.8 shifted intracellular pH by +0.25 pH units, fully reversing the glutamate-induced acidification and increasing glutamine content by 120-180%. The effect of K+ on intracellular pH increased to +0.25 pH units in bicarbonate-buffered solution, suggesting that the regulation of glutamine formation by extracellular K+ is enhanced in the presence of bicarbonate.

Ammonia↗
PubMed1993

Interaction between the glutamine cycle and the uptake of large neutral amino acids in astrocytes.

When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two- to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.

Amino Acids↗
PubMed1992

Regulation of the glutamine content of astrocytes by cAMP and hydrocortisone: effect of pH.

It was reported recently that the glutamine content of astrocytes incubated with glutamate and ammonium is steeply dependent on the pH of the solution. The present study shows that pretreatment of astrocytes with dibutyryl cAMP or with hydrocortisone, conditions that induce glutamine synthetase activity, increased glutamine content 2.4-fold and 5.3-fold, respectively. Nevertheless, a shift of pH from 7.4 to 7.8 increased glutamine content further by 2.7-fold and 3.0-fold, respectively. The net rates of uptake of glutamate and export of glutamine varied narrowly compared to these very large changes in glutamine content.

Animals↗
PubMed1992

In vitro evidence for the role of glutamate in the CNS toxicity of mercury.

Intoxication with elemental mercury vapor or with methylmercury results in the accumulation of mercuric mercury (Hg2+) in the brain. Submicromolar concentrations of Hg2+ were shown previously to inhibit glutamate uptake in astrocyte cultures selectively and reversibly. This finding suggests that blockade of the inactivation of synaptically released glutamate is a potential mechanism of the CNS toxicity of Hg2+. The present study shows further that Hg2+ (< or = 1 microM): (i) markedly inhibits the clearance of extracellular glutamate both by astrocyte cultures and by spinal cord cultures; (ii) reduces glutamine content and export in astrocyte cultures; (iii) has little effect on neuronal viability in spinal cord cultures in the absence of excitotoxic accumulations of glutamate; (iv) does not impair the sensitivity of neurons to the excitotoxic action of glutamate. Also, it is noted that Hg2+ (< or = 1 microM) has not been shown to impair transmitter release acutely in existing studies of presynaptic actions. Thus, the available evidence from in vitro studies is consistent with the hypothesis that low concentrations of mercuric mercury in the brain can cause neurotoxicity by selectively inhibiting the uptake of synaptically released glutamate, with consequent elevation of glutamate levels in the extracellular space.

Animals↗
PubMed1992

Effect of pH on glutamine content derived from exogenous glutamate in astrocytes.

A shift in pH from 7.4 to 7.8 in the incubation solution caused a 3.4-fold increase in the free glutamine content of mouse cerebral astrocytes that were incubated with glutamate (100 microM) and ammonium (100 microM). This large and reversible steady-state increase in glutamine content was accompanied by smaller transient increases in the following: (a) net formation of glutamine; (b) clearance of glutamate from the incubation solution; and (c) glutamate content. The content of glutamine was reduced markedly by omission of either glutamate or ammonium from the incubation solution, or by inhibition of glutamine synthetase activity with methionine sulfoximine. The rate at which glutamine was exported from the astrocytes was unaffected by the pH change. The effects of pH on the concentration of free ammonia or on glutamate uptake do not appear to mediate the increase in glutamine content. Uptake of exogenous glutamine was little affected by the pH change. Therefore, possible mediation of the effect by an increase in intracellular pH must be considered. The response to altered pH described here may provide a cellular basis for the increased level of brain glutamine observed in hyperammonemia.

Amino Acids↗
PubMed1992

Effect of intracellular glutamine on the uptake of large neutral amino acids in astrocytes: concentrative Na(+)-independent transport exhibits metastability.

To examine whether the concentration gradient of glutamine (Gln) drives concentrative Na(+)-independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in "Gln-depleted" and "Gln-replete" cultures. Uptake (30 min in Na(+)-free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70-150% greater in Gln-replete cultures. Phenylalanine uptake was not affected. All of these NAA trans-stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gln-replete cultures declined. Also, uptake of Gln itself was enhanced in Gln-replete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gln-depleted cultures either with Gln or with histidine. It is suggested that substrate-induced asymmetry and molecular hysteresis in the Na(+)-independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gln (15- to 29-fold at 1 mM in Na(+)-free buffer). The other NAA were four to six times less concentrated. At least two Na(+)-dependent transport systems also supported the concentration gradient of Gln in regular buffer.

Amino Acids↗
PubMed1989

Inhibition of amino acid transport and protein synthesis by HgCl2 and methylmercury in astrocytes: selectivity and reversibility.

The previously reported observation that submicromolar concentrations of HgCl2 inhibit glutamate uptake reversibly in astrocytes, without effect on 2-deoxyglucose uptake, suggested that elemental mercury vapor, which is oxidized to mercuric mercury in the brain, might cause neurodegenerative change through the mediation of glutamate excitotoxicity. Here, selectivity is explored further by measuring the inhibition of other amino acid transporters and protein synthesis as a function of HgCl2 concentration. The properties of MeHgCl were compared under identical conditions, and some morphological correlates of function were examined. Inhibition of amino acid transport by HgCl2 was selective, whereas MeHgCl was nonselective. The 50% inhibitory concentrations of HgCl2 for uptake of alpha-aminoisobutyric acid by system A, uptake of alpha-aminoisobutyric acid or kynurenine by a system L variant, and uptake of gamma-aminobutyric acid were all two- to fourfold greater than that for uptake of glutamate. The submicromolar concentrations of HgCl2 that inhibited glutamate transport also inhibited protein synthesis, but in a rapidly reversible fashion, and elicited only discrete ultrastructural changes (heterochromatin, increased numbers of lysosomal bodies, and increased complexity of cell surface). In contrast, inhibition of protein synthesis by MeHgCl was acutely (1-h) irreversible and became marked only at concentrations higher than those that elicited gross morphologic change in the form of "bleb"-like swellings. The results lend support to the proposed excitotoxic mediation of mercury vapor neurotoxicity and reveal a sharp contrast between the effects of HgCl2 and MeHgCl on astrocytes.

Amino Acids↗