pH Transients Research Articles

NMDA Receptor-Dependent Afterdepolarizations Are Curtailed by Carbonic Anhydrase 14: Regulation of a Short-Term Postsynaptic Potentiation

In the hippocampus, extracellular carbonic anhydrase (Car) speeds the buffering of an activity-generated rise in extracellular pH that impacts H(+)-sensitive NMDA receptors (NMDARs). We studied the role of Car14 in this brain structure, in which it is expressed solely on neurons. Current-clamp responses were recorded from CA1 pyramidal neurons in wild-type (WT) versus Car14 knock-out (KO) mice 2 s before (control) and after (test) a 10 pulse, 100 Hz afferent train. In both WT and KO, the half-width (HW) of the test response, and its number of spikes, were augmented relative to the control. An increase in presynaptic release was not involved, because AMPAR-mediated EPSCs were depressed after a train. The increases in HW and spike number were both greater in the Car14 KO. In 0 Mg(2+) saline with picrotoxin (using a 20 Hz train), the HW measures were still greater in the KO. The Car inhibitor benzolamide (BZ) enhanced the test response HW in the WT but had no effect on the already-prolonged HW in the KO. With intracellular MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]-cyclohepten-5,10-imine maleate], the curtailed WT and KO responses were indistinguishable, and BZ caused no change. In contrast, the extracellular alkaline changes evoked by the train were not different between WT and KO, and BZ amplified these alkalinizations similarly. These data suggest that Car14 regulates pH transients in the perisynaptic microenvironment and govern their impact on NMDARs but plays little role in buffering pH shifts in the broader, macroscopic, extracellular space.

Mathematical modeling of the role of carbonic anhydrase II and IV on the influx of CO2 in a Xenopus oocyte

Exposing oocytes to a solution containing CO2/HCO3 causes the familiar fall in intracellular pH (pHi) and a rise in surface pH (pHS) followed by a decay. Musa‐Aziz et al (ASN, 2005) examined the effects of carbonic anhydrases (CAs) on pH transients caused by CO2 influx. They found that injecting CAII into oocytes or expressing CAIV on the oocyte surface accelerates the initial rate of pHi acidification (dpHi/dt) and amplifies the height of the pHS spike (ΔpHS). They proposed that both enzymes enhance CO2 influx by maximizing CO2 gradients across the plasma membrane. We use a mathematical model of a Xenopus oocyte—which accounts for CO2, carbonic acid (H2CO3), HCO3, protons, and a multitude of non‐HCO3 buffer pairs—to investigate the above findings. We simulate the experimental protocol in which the CO2/HCO3 solution is delivered from the bulk to the oocyte surface by raising exponentially the concentrations of CO2, H2CO3 and HCO3 in the bulk and assuming that initially no CO2, H2CO3 and HCO3 are present in the extracellular unconvected fluid. We use the experimental data for 1.5% CO2/10mM HCO3 (pHo=7.5) to find parameter values that simultaneously match ΔpHS, the time that it takes for pHS to reach its peak (tP), dpHi/dt, and the time delay in the pHi decay. We validate the model against data collected when exposing oocytes to 5% CO2/33mM HCO3 and to 10% CO2/66mM HCO3. The model confirms that CAII and CAIV enhance CO2 influx.

Alkalinization Transients in Individual Mitochondria

The pH within the mitochondrial matrix (pHmito) is an important bioenergetic parameter well studied in isolated mitochondria but poorly characterized in intact cells, and all in situ studies available report spatially averaged measurements of pHmito. We used a new pH-sensitive GFP-based fluorescent probe targeted to the mitochondrial matrix, mito-SypHer, to study pHmito homeostasis at the level of single mitochondria in intact living cells. We observed that individual mitochondria undergo spontaneous alkalinization transients. The pH transients occurred randomly in time and space and had a characteristic profile, with a rapid onset (time to peak 1.6±0.1 sec), a slower decay (τ=8.5±0.6 sec), and an average amplitude of 0.38±0.05 pH units. The pH transients were absent in Rho-0 cells that lack a functional respiratory chain, were abrogated by protonophores, occurred concomitantly with transient mitochondrial depolarization events measured with TMRE, and their frequency was strongly decreased by respiratory chain inhibitors. These kinetics and functional properties resemble the “superoxide flashes” previously reported in single mitochondria with a pericam-derived probe. However, the fluorescence of purified mito-SypHer was not altered by all the ROS tested including superoxide, and in live cells increasing the pH buffering power of mitochondria with NH4Cl decreased the amplitude and slowed the kinetics of the transients, confirming that these were caused by protons. The pH spikes were not spatially restricted to single mitochondria but were also observed in clusters of interconnected mitochondria and their spatial extent was altered by enforced fusion or fission of mitochondria. Our data indicate that in live cells individual mitochondria undergo spontaneous basification transients that require functional OXPHOS machinery. The synchronicity between the spontaneous pH spikes and depolarization events suggests that H+ extrusion by the respiratory chain complexes or by the reverse mode of the ATP-synthase might be transiently enhanced during bouts of depolarization.

Concentration and fractionation by isoelectric trapping in a micropreparative‐scale multicompartmental electrolyzer having orthogonal pH gradients. Part 2

A micropreparative-scale multicompartmental electrolyzer called ConFrac has been developed and tested for isoelectric trapping separations. ConFrac can be operated in pass-by-pass mode or recirculating mode, using either asymmetrical feeding (feed enters only the anodic or the cathodic flow-through compartment) or symmetrical feeding (feed enters both the anodic and the cathodic flow-through compartment). Symmetrical feeding results in higher processing rates and is the preferred operation mode. Residence time in the flow-through compartments is set as a compromise between processing rate and temperature rise in the effluent. Ampholytic components have been isolated from 5 to 50 mL volumes of micromolar feed solutions and hundredfold concentrated into 100-μL collection compartments. Samples containing ampholytic analytes in highly conducting salt solutions were readily desalted and fractionated in ConFrac in one operation. pH transients formerly observed in other isoelectric trapping devices were observed in ConFrac as well. The pH transients were caused by the unequal ion transmission rates of the anodic- and cathodic-side buffering membranes.

Characterization of Local pH Changes in Brain Using Fast-Scan Cyclic Voltammetry with Carbon Microelectrodes

Transient local pH changes in the brain are important markers of neural activity that can be used to follow metabolic processes that underlie the biological basis of behavior, learning and memory. There are few methods that can measure pH fluctuations with sufficient time resolution in freely moving animals. Previously, fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the measurement of such pH transients. However, the origin of the potential dependent current in the cyclic voltammograms for pH changes recorded in vivo was unclear. The current work explored the nature of these peaks and established the origin for some of them. A peak relating to the capacitive nature of the pH CV was identified. Adsorption of electrochemically inert species, such as aromatic amines and calcium could suppress this peak, and is the origin for inconsistencies regarding in vivo and in vitro data. Also, we identified an extra peak in the in vivo pH CV relating to the presence of 3,4-dihydroxyacetic acid (DOPAC) in the brain extracellular fluid. To evaluate the in vivo performance of the carbon-fiber sensor, carbon dioxide inhalation by an anesthetized rat was used to induce brain acidosis induced by hypercapnia. Hypercapnia is demonstrated to be a useful tool to induce robust in vivo pH changes, allowing confirmation of the pH signal observed with FSCV.

PH transients in hydroxyapatite chromatography columns—Effects of operating conditions and media properties

pH transitions occur in hydroxyapatite (HAP) columns that are subject to step changes in salt concentration, which have been shown to be controlled by proton exchange on the HAP surface. The pH temporarily decreases before gradually returning to the feed value when the salt concentration increases, potentially compromising the stability of the HAP when either the magnitude or duration of the pH drop is excessive. The opposite happens when the salt concentration decreases. In this work we address the effects of several key variables: the flow rate, the particle size, the use of salt gradients instead of steps, the use of different co-buffers, the surface area of the HAP, and the use of a slightly alkaline wash prior to increasing the salt concentration. Flow rate and particle size were found to have virtually no effect, demonstrating that the pH transitions are equilibrium rather than kinetically driven. Salt gradients resulted in smaller pH drops compared to steps since the exchanged protons are diluted over the gradient volume. MES and histidine used as co-buffers were effective at reducing the duration of the pH transitions but did not affect their magnitude. The same result was found when comparing HAP samples with different surface areas, with the lower surface area HAP yielding much shorter duration but similar pH drops and rises. Finally, washing the HAP column with a pH 7.5 buffer prior to the salt step was found to dramatically reduce the subsequent pH drop. In general, there was good agreement between these results and predictions based on our previously developed model.

PH Transients in hydroxyapatite chromatography columns—Experimental evidence and phenomenological modeling

Hydroxyapatite (HAP) columns, widely used for chromatographic separation of proteins and other biomolecules because of their unique selectivity and ability to resolve complex mixtures, exhibit limited stability at acidic conditions requiring careful control of pH. Even with buffered solutions, however, unintended pH transients can occur when the salt concentration varies. For example, the pH temporarily decreases below the feed value when the salt concentration increases and increases above the feed value when the salt concentration is decreased. The intensity and duration of these transients depend on the particular buffer used and the magnitude of the salt concentration step, but in extreme cases the pH can drop by as much as 1.5 pH units creating conditions where the HAP stability is potentially compromised. This work examines the mechanisms leading to pH transients in HAP columns generated by salt steps. The pH excursions are similar to those observed for weak cation exchange columns, but are accompanied by a transient evolution of phosphate which temporarily decreases below the feed value when the salt concentration is increased and increases sharply when the salt concentration is reduced before returning to the feed value. A phenomenological model is developed to describe this behavior by considering the reversible uptake of sodium ions by the P-sites and binding of phosphate ions by the C-sites. The interplay of these two adsorption mechanisms results in complex pH patterns that are consistent with those observed experimentally. In addition to helping understand the underlying mechanisms, the model also provides a useful tool to predict the effects of different buffers and salt concentration and develop corrective measures that can reduce the intensity and duration of the pH transients such as the addition of unretained co-buffers.

Highlights From The Literature

Highlights From The Literature

Letter to the editor: The use of extracellular, ion-selective microelectrodes to study the function of heterologously expressed transporters in Xenopus oocytes

to the editor: In a recent issue of American Journal of Physiology-Cell Physiology, Blanchard et al. (1) published an elegant article in which they report that they have “developed the ion-trap technique” that “uses a large ISE [ion-selective electrode] … gently pushed against the surface of an oocyte … [to isolate] a small volume of extracellular solution (the trap) in which transport-dependent changes in ionic concentrations can be detected.” The authors cite others' use of extracellular ISEs, including work from our own laboratory published by Endeward et al. (4), in which we used a large-tipped H+-selective electrode to monitor the depletion of H+ on the surface of a Xenopus oocyte during the influx of CO2 through aquaporin 1, due to the reaction HCO3− + H+ → CO2 + H2O. Blanchard et al. (1) might also have noted the work of Parker et al. (5), in which we used a large-tipped Cl−-selective electrode to measure transporter-mediated efflux of Cl− from oocytes. In both sets of experiments from our laboratory, Dr. Musa-Aziz positioned a 5–15 μM ISE tip at the oocyte surface and then advanced it ∼40–60 μm further to create a dimple. She subsequently monitored the appearance of ions in (or disappearance from) the unstirred layer trapped between the oocyte surface and the abutting electrode tip. This approach is essentially the same as that first described by de Hemptinne and Huguenin (3) in 1984 to measure pH transients (on the surface of muscle) caused by CO2/HCO3− fluxes and then implemented somewhat differently by Chesler (2) in 1986 to monitor pH transients (in the restricted extracellular space near neurons) caused by NH3/NH4+ and CO2/HCO3− fluxes. Blanchard et al. (1) have coined the phrase “ion-trap” and are unique in applying this technology to study transporters under voltage clamp. They attempted to distinguish their work from ours by stating that Endeward et al. (4) used a “simple static [i.e., immobile] extracellular ISE,” which is, in fact, the approach used by all of the aforementioned groups. To our knowledge, the “ion-trap” technique itself was first developed by de Hemptinne and Huguenin (3) and first exploited to study the molecular function of heterologously expressed transporters by our laboratory (4, 5).

Carbonic Anhydrases CA4 and CA14 Both Enhance AE3-Mediated Cl−–HCO3−Exchange in Hippocampal Neurons

Carbonic anhydrase (CA) activity in the brain extracellular space is attributable mainly to isoforms CA4 and CA14. In brain, these enzymes have been studied mostly in the context of buffering activity-dependent extracellular pH transients. Yet evidence from others has suggested that CA4 acts in a complex with anion exchangers (AEs) to facilitate Cl(-)-HCO(3)(-) exchange in cotransfected cells. To investigate whether CA4 or CA14 plays such a role in hippocampal neurons, we studied NH(4)(+)-induced alkalinization of the cytosol, which is mitigated by Cl(-) entry and HCO(3)(-) exit. The NH(4)(+)-induced alkalinization was enhanced when the extracellular CAs were inhibited by the poorly permeant CA blocker, benzolamide, or by inhibitory antibodies specific for either CA4 or CA14. The NH(4)(+)-induced alkalinization was also increased with inhibition of anion exchange by 4,4*-diisothiocyanostilbene-2,2*-disulfonic acid, or by eliminating Cl(-) from the medium. No effect of benzolamide was seen under these conditions, in which no Cl(-)-HCO(3)(-) exchange was possible. Quantitative PCR on RNA from the neuronal cultures indicated that AE3 was the predominant AE isoform. Single-cell PCR also showed that Slc4a3 (AE3) transcripts were abundant in isolated neurons. In hippocampal neurons dissociated from AE3-null mice, the NH(4)(+)-induced alkalinization was much larger than that seen in neurons from wild-type mice, suggesting little or no Cl(-)-HCO(3)(-) exchange in the absence of AE3. Benzolamide had no effect on the NH(4)(+)-induced alkalinization in the AE3 knock-out neurons. Our results indicate that CA4 and CA14 both play important roles in the regulation of intracellular pH in hippocampal neurons, by facilitating AE3-mediated Cl(-)-HCO(3)(-) exchange.

Simulation of desalting that occurs during isoelectric trapping separations

Simul 5, the simulation program based on the 1-D model of electrophoresis has been extended to simulate isoelectric trapping (IET) separations that take place in recirculating multicompartmental electrolyzers (MCEs). In the extended Simul 5, the simulated separation space between the anode and cathode can be divided into multiple segments to represent the anode compartment, separation compartment(s) and the cathode compartment. The compartments may have identical or different cross sections. A new algorithm simulates convective mixing that occurs in the recirculating MCEs where the distances between the buffering membranes are short and the velocities of tangential flows through the compartments, orthogonal to the electric field, are high. The intensity of simulated mixing can be independently controlled in each compartment. pH transients that were reported to occur during the desalting step in IET separations were simulated with the extended Simul 5 program: the main features of the experimental results were reproduced by the simulations. The simulations shed light on the possible causes of uneven anion and cation removal rates, pH transients and the transient invasion of the separation compartment by components of the electrode solutions that might occur during the desalting step.

Behavior of the Inadvertent pH Transient Formed by a Salt Gradient in the Ion-Exchange Chromatography of Proteins

The inadvertent pH transient produced when a stepwise change in salt concentration is used as the eluent in ion-exchange chromatography was studied theoretically using a local-equilibrium theory and experimentally using both strong-base and weak-base anion-exchange column packings. The accuracy of the local-equilibrium theory was verified by comparing it to a full numerical solution of the governing partial differential equations obtained using the method of characteristics. The predictions from the local-equilibrium theory were observed to largely agree with experimental results. Detailed comparisons of experimental results and the local-equilibrium theory permitted the observed trends for the pH transients to be interpreted in terms of the physical properties of the column packing and mobile phase. The results of this study are useful for the design of ion-exchange processes using salt gradient elution where it is desired to limit the exposure of eluted proteins to the inadvertent pH transient caused by the salt gradient.

Glucose Regulation of Pre-steady State Kinetics of ATP Hydrolysis by Na,K-ATPase

The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na+ (120 mM) and K+ (30 mM) concentrations, magnitudes of both H+ release and H+ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H+ absorption at high Na+ concentrations, whereas it was ineffective at low Na+. H+ release was decreased significantly in the presence of 1 mM glucose at Na+ concentrations ranging from 30 mM to 120 mM. Similar to the control, K+ did not show any effect on either H+ release or H+ absorption at all tested combinations of Na+ and K+ concentrations. Pre-steady state H+ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K+ (by 30 ms) to the mixture (enzyme+120 mM Na++ATP+glucose) showed that only small fractions of population absorb H+ in the absence of K+. No H+ absorption was observed in the absence of Na+. Delayed mixing of Na+ or K+ did not have any effect on H+ release. Effect of 2-deoxy-D-glucose on H+ absorption and release was almost the same as that of glucose at all combinations of Na+ and K+ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reached the E-P(3Na+) stage and that K+ plays an important role in the conversion of E1.ADP.P(3Na+) to H+.E1~(3Na+) complex.

PH transients during salt removal in isoelectric trapping separations: A curse revisited

The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH 7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (mu(anion)/mu(cation)) and the pH difference between pH 7 and the pH of the buffering membranes (|pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|). When |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1, mu(anion)/mu(cation)<1 leads to an acidic pH transient, mu(anion)/mu(cation) = 1 eliminates the pH transient and mu(anion)/mu(cation)>1 leads to a basic pH transient. When mu(anion)/mu(cation) = 1, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|<1 leads to a basic pH transient, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1 eliminates the pH transient and |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| value, pH transients caused by dissimilar anion and cation mobilities can be avoided.

PH transitions in cation exchange chromatographic columns containing weak acid groups

Complex pH transitions occur in cation exchange columns used for protein chromatography during equilibration and salt elution steps when the stationary phase contains weak acid groups even if the mobile phase is buffered and the buffering species do not interact with the stationary phase. In this work, we present a local equilibrium model to predict the magnitude and duration of these pH transients. The model equations are solved by the method of characteristics and by numerical simulations using an equilibrium-dispersive model. By incorporating an explicit description of the dissociation of the weakly ionogenic groups in the resin, we show that counterion binding in the column can be predicted for different buffer systems based on a single experimental resin titration curve without having to resort to empirically defined adsorption equilibrium constants. Model predictions based on these assumptions are found to be in excellent agreement with experimental results obtained for three different resins containing varying concentrations of weak acid groups. Four common buffer systems, acetate, citrate, MES, and phosphate are considered with both step and gradient changes in salt concentration at pH 5.5. Each buffer yields a different pH excursion behavior. We demonstrate that when the counterion concentration is kept constant in each of these buffers, which is needed to attain identical protein adsorption behavior, the magnitude of the pH transitions occurring during salt steps is nearly independent of the buffer system. On the other hand, the duration of the pH transitions is smallest for MES suggesting that this buffer system is preferable where pH variations are to be prevented.

Cannabinoid modulation of electrically evoked pH and oxygen transients in the nucleus accumbens of awake rats

Cannabinoid receptors have been implicated in the regulation of blood flow in the cerebral vasculature. Because the nucleus accumbens (NAc) shows high levels of central cannabinoid receptor 1 (CB1) expression we examined the effects of cannabinoids on the local transient alkaline shifts and increases in extracellular oxygen induced by electrical stimulation of the medial forebrain bundle (MFB) in conscious animals. These changes result from increases in cerebral blood flow (CBF) and metabolism in the NAc that are evoked by the stimulation. Oxygen and pH changes were monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the NAc of freely moving rats. Administration of the cannabinoid receptor agonist WIN55,212-2 potently inhibited extracellular oxygen and pH changes, an effect that was reversed and prevented by pre-treatment with the CB1 receptor antagonists SR141716A and AM251. The effects on pH following WIN55,212-2 were similar to those following nimodipine, a recognized vasodilator. When AM251 was injected alone, the amplitude of electrically evoked pH shifts was unaffected. Administration of AM404 and VDM11, endocannabinoid transport inhibitors, partially inhibited pH transients in a CB1 receptor-dependent manner. The present findings suggest that CB1 receptor activation modulates changes in two well-established indices of local blood flow and metabolism resulting from electrically evoked activation of ascending fibers. Although endogenous cannabinoid tone alone is not sufficient to modify these responses, uptake blockade demonstrates that the system has the potential to exert CB1-specific effects similar to those of full agonists.

Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells.

Our aim was to test the hypothesis that depolarization-induced intracellular pH (pH(i)) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH-sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole-cell patch-clamped rat cerebellar Purkinje cells. In the absence of added CO(2)-HCO(3)(-), depolarization to +20 mV for 1 s caused large (approximately 0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (approximately 0.06 pH units) and slower. The pH(i) shifts were smaller in the presence of 5 % CO(2)-25 mM HCO(3)(-)-buffered saline (approximately 0.08 pH units in the dendrites and approximately 0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 microM) doubled the size of the dendritic acid shifts in the presence of CO(2)-HCO(3)(-), indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mM) inhibited the depolarization-evoked acid shifts. We investigated more physiological pH(i) changes by evoking modest bursts of action potentials (approximately 10 s duration) in CO(2)-HCO(3)(-)-buffered saline. Such neuronal firing induced an acidification of approximately 0.11 pH units in the fine dendritic regions, but only approximately 0.03 pH units in the soma. There was considerable variation in the size of the pH(i) shifts between cells, with dendritic acid shifts as large as 0.2-0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pH(i) changes (pH microdomains) might act as important signals in synaptic function.

Chronic metabolic acidosis upregulates rat kidney Na-HCO cotransporters NBCn1 and NBC3 but not NBC1.

Several members of the Na-HCO cotransporter (NBC) family have recently been identified functionally and partly characterized, including rkNBC1, NBCn1, and NBC3. Regulation of these NBCs may play a role in the maintenance of intracellular pH and in the regulation of renal acid-base balance. However, it is unknown whether the expressions of these NBCs are regulated in response to changes in acid-base status. We therefore tested whether chronic metabolic acidosis (CMA) affects the abundance of these NBCs in kidneys using two conventional protocols. In protocol 1, rats were treated with NH(4)Cl in their drinking water (12 +/- 1 mmol. rat(-1). day(-1)) for 2 wk with free access to water (n = 8). Semiquantitative immunoblotting demonstrated that whole kidney abundance of NBCn1 and NBC3 in rats with CMA was dramatically increased to 995 +/- 87 and 224 +/- 35%, respectively, of control levels (P < 0.05), whereas whole kidney rkNBC1 was unchanged (88 +/- 14%). In protocol 2, rats were given NH(4)Cl in their food (10 +/- 1 mmol. rat(-1). day(-1)) for 7 days, with a fixed daily water intake (n = 6). Consistent with protocol 1, whole kidney abundances of NBCn1 (262 +/- 42%) and NBC3 (160 +/- 31%) were significantly increased compared with controls (n = 6), whereas whole kidney rkNBC1 was unchanged (84 +/- 17%). In both protocols, immunocytochemistry confirmed upregulation of NBCn1 and NBC3 with no change in the segmental distribution along the nephron. Consistent with the increase in NBCn1, measurements of pH transients in medullary thick ascending limb (mTAL) cells in kidney slices revealed two- to threefold increases in DIDS- sensitive, Na(+)-dependent HCO uptake in rats with CMA. In conclusion, CMA is associated with a marked increase in the abundance of NBCn1 in the mTAL and NBC3 in intercalated cells, whereas the abundance of NBC1 in the proximal tubule was not altered. The increased abundance of NBCn1 may play a role in the reabsorption of NH in the mTAL and increased NBC3 in reabsorbing HCO.

Modulation by extracellular pH of low- and high-voltage-activated calcium currents of rat thalamic relay neurons.

The effects of changes in the extracellular pH (pH(o)) on low-voltage- (LVA) and high-voltage- (HVA) activated calcium currents of acutely isolated relay neurons of the ventrobasal thalamic complex (VB) were examined using the whole cell patch-clamp technique. Modest extracellular alkalinization (pH 7.3 to 7.7) reversibly enlarged LVA calcium currents by 18.6 +/- 3.2% (mean +/- SE, n = 6), whereas extracellular acidification (pH 7.3 to 6.9) decreased the current by 24.8 +/- 3.1% (n = 9). Normalized current amplitudes (I/I(7.3)) fitted as a function of pH(o) revealed an apparent pK(a) of 6.9. Both, half-maximal activation voltage and steady-state inactivation were significantly shifted to more negative voltages by 2-4 mV on extracellular alkalinization and to more positive voltages by 2-3 mV on extracellular acidification, respectively. Recovery from inactivation of LVA calcium currents was not significantly affected by changes in pH(o). In contrast, HVA calcium currents were less sensitive to changes in pH(o). Although extracellular alkalinization increased maximal HVA current by 6.0 +/- 2.0% (n = 7) and extracellular acidification decreased it by 11.9 +/- 0.02% (n = 11), both activation and steady-state inactivation were only marginally affected by the moderate changes in pH(o) used in the present study. The results show that calcium currents of thalamic relay neurons exhibit different pH(o) sensitivity. Therefore activity-related extracellular pH transients might selectively modulate certain aspects of the electrogenic behavior of thalamic relay neurons.

Interstitial Carbonic Anhydrase (CA) Activity in Brain Is Attributable to Membrane-Bound CA Type IV

We tested the hypothesis that extracellular membrane-bound carbonic anhydrase (CA) type IV is responsible for the regulation of interstitial pH (pH(o)) transients in brain. Rat hippocampal slices were incubated in phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the link of CA IV to the external face of plasma membranes. Then evoked alkaline pH(o) shifts were studied in a recording chamber, using pH microelectrodes. Incubation fluid was saved for later analysis. The ability to buffer a rapid alkaline load was reduced markedly in PI-PLC-treated tissue as compared with adjacent, paired control slices. The effect of benzolamide (a poorly permeant CA inhibitor) on evoked pH(o) shifts was diminished greatly in the PI-PLC-treated tissue, consistent with the washout of interstitial CA. Treatment of the incubation fluid with SDS abolished nearly all of the CA activity in fluid from controls, whereas an SDS-insensitive component remained in the fluid from PI-PLC-treated slices. These data suggested that CA type II (which is blocked by SDS) leaked from injured glial cells in both slice preparations, whereas CA type IV (which is insensitive to SDS) was liberated selectively into the fluid from PI-PLC-treated tissue. Western blot analysis was consistent with this interpretation, demonstrating a predominance of CA IV in the incubation fluid from PI-PLC-treated tissue and variable amounts of CA II in fluid from PI-PLC-treated and control slices. These results demonstrate that interstitial CA activity brain is attributable principally to membrane-bound CA IV.