Recovery from NMDA-induced intracellular acidification is delayed and dependent on extracellular bicarbonate

1996 ◽  
Vol 270 (2) ◽  
pp. C593-C599 ◽  
Author(s):  
L. M. Canzoniero ◽  
S. L. Sensi ◽  
D. W. Choi
Keyword(s):  
Intracellular Ph ◽  
Cortical Neurons ◽  
Nmda Antagonist ◽  
Disulfonic Acid ◽  
Intracellular Acidification ◽  
Subsequent Recovery ◽  
Cultured Cortical Neurons ◽  
Acidification Recovery ◽  
Dose Dependent ◽  
Stimulation Of

A 30-s exposure to N-methyl-D-aspartate (NMDA) produced a dose-dependent and long-lasting (10-20 min) reduction in intracellular pH in cultured cortical neurons, detected by the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This intracellular acidification could be blocked by addition of the NMDA antagonist, D-(-)-2-amino-5-phosphonovalerate, or by removal of extracellular Ca2+. Removal of extracellular HCO3- markedly impaired recovery from NMDA-induced intracellular acidification. Recovery was also impaired when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, inhibitors of HCO3- transport, were added to the cultures immediately after NMDA exposure. In contrast, the Na+/H+ exchange blocker, 5-(N-ethyl-N-isopropyl)amiloride, did not affect pH recovery. Removal of extracellular Cl- partially prevented pH recovery after NMDA stimulation. In addition, extracellular HCO3- increased intracellular Na+ after NMDA exposure, consistent with HCO3- activation of a Na(+)-dependent exchanger. These results demonstrate that stimulation of cortical neuronal NMDA receptors is followed by long-lasting intracellular acidification and that the presence of extracellular HCO3- is important in the subsequent recovery of normal intracellular pH, likely acting at least in part via the Na(+)-dependent Cl-/HCO3- exchanger.

scholarly journals Glucose Deprivation Neuronal Injury in vitro is Modified by Withdrawal of Extracellular Glutamine

1990 ◽  
Vol 10 (3) ◽  
pp. 337-342 ◽  
Author(s):  
Hannelore Monyer ◽  
Dennis W. Choi
Keyword(s):  
Cortical Neurons ◽  
Excitatory Amino Acids ◽  
Neuronal Loss ◽  
Nmda Antagonist ◽  
Neuronal Injury ◽  
Glucose Deprivation ◽  
Glutamine Concentration ◽  
Energy Substrate ◽  
Cultured Cortical Neurons

Cultured cortical neurons deprived of glucose in a defined solution containing 2 m M glutamine became acutely swollen and went on to degenerate over the next day; this neuronal loss could be substantially attenuated by an N-methyl-D-aspartate (NMDA) antagonist. Removal of extracellular glutamine produced two effects: an increase in overall neuronal injury and a decrease in the protective effect of an NMDA antagonist. Both effects of glutamine removal were glutamine concentration dependent (EC50 for both ∼300 μ M) and not reversed by substitution of equimolar concentrations of alanine or arginine. These observations suggest that glucose deprivation neuronal injury may be tonically regulated by the presence of extracellular glutamine. We speculate that glutamine may reduce overall injury by serving as an energy substrate in the absence of glucose, but may increase NMDA receptor-mediated injury by serving as a precursor for transmitter excitatory amino acids.

Excitatory amino acid receptor-mediated transmission in geniculocortical and intracortical pathways within visual cortex

1991 ◽  
Vol 66 (1) ◽  
pp. 293-306 ◽  
Author(s):  
L. J. Larson-Prior ◽  
P. S. Ulinski ◽  
N. T. Slater
Keyword(s):  
Visual Cortex ◽  
Amino Acid ◽  
Cortical Neurons ◽  
Excitatory Amino Acid ◽  
Nmda Antagonist ◽  
Receptor Subtypes ◽  
Excitatory Amino Acid Receptor ◽  
Visual Cortical ◽  
Stimulation Of

1. A preparation of turtle (Chrysemys picta and Pseudemys scripta) brain in which the integrity of the intracortical and geniculocortical pathways in visual cortex are maintained in vitro has been used to differentiate the excitatory amino acid (EAA) receptor subtypes involved in geniculocortical and intracortical synapses. 2. Stimulation of the geniculocortical fibers at subcortical loci produces monosynaptic excitatory postsynaptic potentials (EPSPs) in visual cortical neurons. These EPSPs are blocked by the broad-spectrum EAA receptor antagonist kynurenate (1-2 mM) and the non-N-methyl-D-aspartate (NMDA) antagonist 6, 7-dinitroquinoxaline-2,3-dione (DNQX, 10 microM), but not by the NMDA antagonist D,L-2-amino-5-phosphonovalerate (D,L-AP-5, 100 microM). These results indicate that the geniculocortical EPSP is mediated by EAAs that access principally, if not exclusively, EAA receptors of the non-NMDA subtypes. 3. Stimulation of intracortical fibers evokes compound EPSPs that could be resolved into three components differing in latency to peak. The component with the shortest latency was not affected by any of the EAA-receptor antagonists tested. The second component, of intermediate latency, was blocked by kyurenate and DNQX but not by D,L-AP-5. The component of longest latency was blocked by kynurenate and D,L-AP-5, but not by DNQX. These results indicate that the compound intracortical EPSP is comprised of three pharmacologically distinct components that are mediated by an unknown receptor, by quisqualate/kainate, and by NMDA receptors, respectively. 4. Repetitive stimulation of intracortical pathways at 0.33 Hz produces a dramatic potentiation of the late, D,L-AP-5-sensitive component of the intracortical EPSP. 5. These experiments lead to a hypothesis about the subtypes of EAA receptors that are accessed by the geniculocortical and intracortical pathways within visual cortex.

Control of intracellular pH during regulatory volume decrease in HL-60 cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1057-C1066 ◽  
Author(s):  
K. R. Hallows ◽  
D. Restrepo ◽  
P. A. Knauf
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Regulatory Volume Decrease ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Volume Dependence ◽  
Supportive Role ◽  
22Na Uptake ◽  
Volume Decrease ◽  
Stimulation Of

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.

Intracellular pH dependence of buffer capacity and anion exchange in the parietal cell

1989 ◽  
Vol 257 (5) ◽  
pp. G741-G747 ◽  
Author(s):  
E. Wenzl ◽  
T. E. Machen
Keyword(s):  
Anion Exchange ◽  
Intracellular Ph ◽  
Anion Exchanger ◽  
Buffer Capacity ◽  
Ph Dependence ◽  
Disulfonic Acid ◽  
Rates Of Change ◽  
Ethanesulfonic Acid ◽  
Increased Activity ◽  
Stimulation Of

When parietal cells (PC) are stimulated with histamine, the anion exchanger rate increases three to five times to compensate for alkaline loading induced by H+-K+ adenosinetriphosphatase (ATPase) and to provide Cl for acid secretion. It has been hypothesized that this increased activity is caused by the increase in intracellular pH (pHi) that often occurs in stimulated PC (from 7.1 to a maximum of 7.3). The dependence of the anion exchanger on pHi was studied using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions were used because the anion exchanger can transport OH- (or HCO3) in exchange for Cl- even with [HCO3]o = 200 microM. It was found that when solutions were changed either from NaCl to Cl- free or Cl- free to NaCl, rates of change of pHi (delta pH/delta t) were strongly dependent on pHi: nearly 0 at pHi 6.6 and 1.25 pH/min at pHi 8.0. To convert these pHi changes into anion flux rates, the intrinsic buffer capacity (beta i) was determined over the same pHi range by making small changes of [NH4]o to determine the resulting changes of [NH4]i and pHi (i.e., beta i = delta[NH4]i/delta pHi) in PC that had been pretreated with 1 mM amiloride and 200 microM [H2]4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [to block Na+-H+ and Cl- -OH-(HCO3-) exchange]. beta i was also strongly dependent on pHi: at pHi 6.5 beta i = 48 mM/pH, and this decreased as pHi increased; at pHi 7.75 beta i = 8 mM/pH. The derived anion fluxes (i.e., JOH = beta i x delta pH/delta t) were roughly linearly related to pHi between 6.6 (JOH near 0) and 8.1 (JOH = 13 mM/min). Between pHi 7.1 and 7.3, the range normally observed during stimulation of PC, rates of anion exchange increased by 75%. This pHi sensitivity cannot explain the 300-500% increase in anion exchanger activity observed during secretagogue stimulation of PC.

Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance

1985 ◽  
Vol 248 (5) ◽  
pp. C542-C549 ◽  
Author(s):  
M. J. Kushmerick ◽  
R. A. Meyer
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Intracellular Ph ◽  
Contractile Activity ◽  
Surface Coil ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Nuclear Magnetic Resonance Spectra ◽  
Stimulation Of ◽  
Nuclear Magnetic

Phosphorus nuclear magnetic resonance spectra at 145 MHz were obtained with a surface coil from the gastrocnemius-plantaris muscles of anesthetized rats. Phosphocreatine (PCr) and inorganic phosphate (Pi) contents and intracellular pH were measured before, during, and after periods of contractile activity induced by twitch or tetanic stimulation of the sciatic nerve. Reduced levels of PCr, increased levels of Pi, and intracellular acidification were achieved in a graded fashion with increased rates of twitch stimulation from 2 to 10 Hz without detectable changes in the ATP content. In all cases, the decrease in PCr was matched by a stoichiometric increase in Pi content. The time constant of resynthesis of PCr averaged 30 s, was five times faster than the restoration of intracellular pH to control levels, and was independent of the degree of intracellular acidosis at the beginning of recovery.

scholarly journals Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

1993 ◽  
Vol 13 (5) ◽  
pp. 827-840 ◽  
Author(s):  
Yibing Ou-Yang ◽  
Pekka Mellergård ◽  
Bo K. Siesjö
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Cortical Neurons ◽  
Ph Sensitive ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Rat Cortical Neurons ◽  
Acid Extrusion

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi was 7.00 ± 0.17 (mean ± SD) and 7.09 ± 0.14 in nominally HCO3− -free and HCO3−-containing solutions, respectively, and was dependent on extracellular Na+ and Cl−. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pHi decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3− -free and HCO3− -containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5- N-methyl- N-isobutylamiloride. In most cells pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or removal of Cl−. The rapid regulation of pHi seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl−. The results are compatible with the presence of at least two different pHi-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl−/HCO3− exchanger, mediating loss of HCO3− from the cell.

Differential Stimulation of Somatostatin but Not Neuropeptide Y Gene Expression by Quinolinic Acid in Cultured Cortical Neurons

2002 ◽  
Vol 65 (3) ◽  
pp. 998-1006 ◽  
Author(s):  
Y. C. Patel ◽  
J.-L. Liu ◽  
A. Warszynska ◽  
G. Kent ◽  
D. N. Papachristou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Neuropeptide Y ◽  
Quinolinic Acid ◽  
Cortical Neurons ◽  
Cultured Cortical Neurons ◽  
Stimulation Of

NH4Cl activates AE2 anion exchanger in Xenopus oocytes at acidic pHi

1997 ◽  
Vol 272 (4) ◽  
pp. C1232-C1240 ◽  
Author(s):  
B. D. Humphreys ◽  
M. N. Chernova ◽  
L. Jiang ◽  
Y. Zhang ◽  
S. L. Alper
Keyword(s):  
Intracellular Ph ◽  
Anion Exchanger ◽  
Sodium Acetate ◽  
Xenopus Oocytes ◽  
Renal Medulla ◽  
Disulfonic Acid ◽  
Acidic Ph ◽  
Intracellular Acidification ◽  
Extracellular Acidification ◽  
Exchange Activity

In the course of experiments to define regulation by intracellular pH (pHi) of the AE2 anion exchanger expressed in Xenopus oocytes, we discovered an unexpected regulation of AE2 by NH4+. Intracellular acidification produced by extracellular acidification or produced by equimolar substitution of NaCl with sodium acetate each inhibited AE2 activity. In contrast, intracellular acidification by equimolar substitution of NaCl with NH4Cl activated AE2-associated, trans-anion-dependent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- influx and efflux. Regulation by NH4+ was isoform specific, since neither erythroid nor kidney AE1 was activated. AE2 activation was maximal at <5 mM NH4Cl; was not mimicked by extracellular KCl, chloroquine, or polyamines; and was insensitive to amiloride, bumetanide, barium, and gadolinium. Whether NH4Cl acts directly on AE2 or on another target remains to be determined. Activation of AE2 by NH4+ may serve to sustain Cl-/HCO3- exchange activity in the presence of acidic pH in renal medulla, colon, abscesses, and other AE2-expressing acidic locales exposed to elevated NH4+ concentration.

Analysis of Cl- -HCO3(-) exchange during recovery from intracellular acidosis in cardiac Purkinje strands

1984 ◽  
Vol 246 (5) ◽  
pp. C391-C400 ◽  
Author(s):  
B. Vanheel ◽  
A. de Hemptinne ◽  
I. Leusen
Keyword(s):  
Steady State ◽  
Membrane Permeability ◽  
Intracellular Ph ◽  
Significant Role ◽  
Exchange Mechanism ◽  
Disulfonic Acid ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Acid Extrusion

The possible role of a Cl- -HCO3(-) exchange mechanism in the recovery from intracellular acidosis of isolated cardiac Purkinje strands was investigated. Intracellular pH (pHi) was measured using double-barreled pH-sensitive microelectrodes. Acidifications were produced by withdrawing 20 meq NH+4 from the superfusate. Experiments were performed in normal CO2-HCO3(-)-buffered, in HCO3(-)free, and in Cl-free solutions and also in the presence of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a blocker of Cl--HCO3(-) exchange. In the absence of external HCO3(-), the apparent rate of acid extrusion following induced acidification was only slightly decreased, but the observed effect does not necessarily imply the intervention of a Cl--HCO3(-) exchange mechanism. SITS had little effect on the response to acidification. In zero-Cl- solutions, recovery of pHi from acidosis was not impaired. These observations suggest that in Purkinje fibers, [Cl-]i-[HCO3(-)]o exchange plays no significant role in recovery from intracellular acidification. Moreover, additional evidence is presented in favor of a passive HCO3(-) efflux at steady-state pHi in the normal superfusate. The apparent membrane permeability to HCO-3 was estimated to be 3.2 X 10(-8) cm X s-1.

Inhibition of Both Na/H and Bicarbonate-Dependent Exchange is Required to Prevent Recovery of Intracellular pH in Single Cardiomyocytes Exposed to Metabolic Stress

1999 ◽  
Vol 19 (2) ◽  
pp. 99-107 ◽  
Author(s):  
Oscar K. Serrano ◽  
Aleksandar Jovanovic ◽  
Andre Terzic
Keyword(s):  
Intracellular Ph ◽  
Cellular Response ◽  
Metabolic Stress ◽  
Disulfonic Acid ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Hypoxic Injury ◽  
Combined Use ◽  
Novel Strategy ◽  
Necessary And Sufficient

Although tight regulation of intracellular pH (pHi) is critical for the survival under stress, paradoxically a slowed recovery of pHi under hypoxic injury may be cardioprotective. In this study, we investigated the recovery of pHi after hypoxia-induced intracellular acidosis in cardiomyocytes loaded with the H+-sensitive dye SNARF-1. Exposure of single cardiomyocytes to 2,4-dinitrophenol (DNP), an inhibitor of mitochondrial oxidative phosphorylation, induced significant intracellular acidification. However, within 10–12 min upon removal of DNP, cardiomyocytes restituted their intracellular H+ concentration. The presence either of 5-N-ethyl-N-isopropylamiloride (EIPA) an inhibitor of Na/H antiporter, or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of bicarbonate-dependent exchange, did not modify the cellular response to DNP. But, combined use of EIPA and DIDS prevented the restitution of intracellular pH following removal of DNP. This study, thus, demonstrated, for the first time, that blockade of both Na/H and bicarbonate-dependent exchange is necessary and sufficient to maintain the hypoxia-induced intracellular acidification. Therefore, concomitant blockade of both pH-regulating mechanisms deserves to be further considered as a novel strategy against hypoxia-reoxygenation injury in the heart.

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