Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive ß-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

Autocrine boost of NMDAR current in hippocampal CA1 pyramidal neurons by a PMCA-dependent, perisynaptic, extracellular pH shift.

The plasma membrane Ca(2+)-ATPase (PMCA) is found near postsynaptic NMDARs. This transporter is a Ca(2+)-H(+) exchanger that raises cell surface pH. We tested whether the PMCA acts in an autocrine fashion to boost pH-sensitive, postsynaptic NMDAR currents. In mouse hippocampal slices, NMDAR EPSCs in a singly activated CA1 pyramidal neuron were reduced when buffering was augmented by exogenous carbonic anhydrase (XCAR). This effect was blocked by the enzyme inhibitor benzolamide and mimicked by the addition of HEPES buffer. Similar EPSC reduction occurred when PMCA activation was prevented by dialysis of BAPTA or the PMCA inhibitor carboxyeosin. Using HEPES, BAPTA, or carboxyeosin, the effect of XCAR was completely occluded. XCAR similarly curtailed NMDAR EPSCs of minimal amplitude, but had no effect on small AMPAR responses. These results indicate that a significant fraction of the postsynaptic NMDAR current is reliant on a perisynaptic extracellular alkaline shift generated by the PMCA.

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.

Preemptive regulation of intracellular pH in hippocampal neurons by a dual mechanism of depolarization-induced alkalinization.

Numerous studies have documented the mechanisms that regulate intracellular pH (pH(i)) in hippocampal neurons in response to an acid load. Here, we studied the response of pH(i) to depolarization in cultured hippocampal neurons. Elevation of external K+ (6-30 mm) elicited an acid transient followed by a large net alkaline shift. Similar responses were observed in acutely dissociated hippocampal neurons. In Ca2+ -free media, the acid response was curtailed and the alkaline shift enhanced. DIDS blocked the alkaline response and revealed a prolonged underlying acidification that was highly dependent on Ca2+ entry. Similar alkaline responses could be elicited by AMPA, indicating that this rise in pH(i) was a depolarization-induced alkalinization (DIA). The DIA was found to consist of Cl- -dependent and Cl- -independent components, each accounting for approximately one-half of the peak amplitude. The Cl- -independent component was postulated to arise from operation of the electrogenic Na+ -HCO3- cotransporter NBCe1. Quantitative PCR and single-cell multiplex reverse transcription-PCR demonstrated message for NBCe1 in our hippocampal neurons. In neurons cultured from Slc4a4 knock-out (KO) mice, the DIA was reduced by approximately one-half compared with wild type, suggesting that NBCe1 was responsible for the Cl- -independent DIA. In Slc4a4 KO neurons, the remaining DIA was virtually abolished in Cl- -free media. These data demonstrate that DIA of hippocampal neurons occurs via NBCe1, and a parallel DIDS-sensitive, Cl- -dependent mechanism. Our results indicate that, by activating net acid extrusion in response to depolarization, hippocampal neurons can preempt a large, prolonged, Ca2+ -dependent acidosis.

Breaking Tradition to Bridge Bench and Bedside: Accelerating the MD-PhD-Residency Pathway.

PROBLEM: Physician-scientists are individuals trained in both clinical practice and scientific research. Often, the goal of physician-scientist training is to address pressing questions in biomedical research. The established pathways to formally train such individuals are mainly MD-PhD programs and physician-scientist track residencies. Although graduates of these pathways are well equipped to be physician-scientists, numerous factors, including funding and length of training, discourage application to such programs and impede success rates. APPROACH: To address some of the pressing challenges in training and retaining burgeoning physician-scientists, New York University Grossman School of Medicine formed the Accelerated MD-PhD-Residency Pathway in 2016. This pathway builds on the previously established accelerated 3-year MD pathway to residency at the same institution. The Accelerated MD-PhD-Residency Pathway conditionally accepts MD-PhD trainees to a residency position at the same institution through the National Resident Matching Program. OUTCOMES: Since its inception, 2 students have joined the Accelerated MD-PhD-Residency Pathway, which provides protected research time in their chosen residency. The pathway reduces the time to earn an MD and PhD by 1 year and reduces the MD training phase to 3 years, reducing the cost and lowering socioeconomic barriers. Remaining at the same institution for residency allows for the growth of strong research collaborations and mentoring opportunities, which foster success. NEXT STEPS: The authors and institutional leaders plan to increase the number of trainees who are accepted into the Accelerated MD-PhD-Residency Pathway and track the success of these students through residency and into practice to determine if the pathway is meeting its goal of increasing the number of practicing physician-scientists. The authors hope this model can serve as an example to leaders at other institutions who may wish to adopt this pathway for the training of their MD-PhD students.

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.

Barium plateau potentials of CA1 pyramidal neurons elicit all-or-none extracellular alkaline shifts via the plasma membrane calcium ATPase.

In many brain regions, synchronous neural activity causes a rapid rise in extracellular pH. In the CA1 region of hippocampus, this population alkaline transient (PAT) enhances responses from postsynaptic, pH-sensitive N-methyl-d-aspartate (NMDA) receptors. Recently, we showed that the plasma membrane Ca(2+)-ATPase (PMCA), a ubiquitous transporter that exchanges internal Ca(2+) for external H(+), is largely responsible for the PAT. It has also been shown that a PAT can be generated after replacing extracellular Ca(2+) with Ba(2+). The cause of this PAT is unknown, however, because the ability of the mammalian PMCA to transport Ba(2+) is unclear. If the PMCA did not carry Ba(2+), a different alkalinizing source would have to be postulated. Here, we address this issue in mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes. In Ba(2+)-containing, Ca(2+)-free artificial cerebrospinal fluid, a single antidromic shock to the alveus elicited a large (0.1-0.2 unit pH), "all-or-none" PAT in the CA1 cell body region. In whole cell current clamp of single CA1 pyramidal neurons, the same stimulus evoked a prolonged plateau potential that was similarly all-or-none. Using this plateau as the voltage command in other cells, we recorded Ba(2+)-dependent surface alkaline transients (SATs). The SATs were suppressed by adding 5 mM extracellular HEPES and abolished when carboxyeosin (a PMCA inhibitor) was in the patch pipette solution. These results suggest that the PAT evoked in the presence of Ba(2+) is caused by the PMCA and that this transporter is responsible for the PAT whether Ca(2+) or Ba(2+) is the charge carrying divalent cation.

Rapid rise of extracellular pH evoked by neural activity is generated by the plasma membrane calcium ATPase.

In hippocampus, synchronous activation of CA1 pyramidal neurons causes a rapid, extracellular, population alkaline transient (PAT). It has been suggested that the plasma membrane Ca(2+)-ATPase (PMCA) is the source of this alkalinization, because it exchanges cytosolic Ca(2+) for external H(+). Evidence supporting this hypothesis, however, has thus far been inconclusive. We addressed this long-standing problem by measuring surface alkaline transients (SATs) from voltage-clamped CA1 pyramidal neurons in juvenile mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes placed against the somata. In saline containing benzolamide (a poorly permeant carbonic anhydrase blocker), a 2-s step from -60 to 0 mV caused a mean SAT of 0.02 unit pH. Addition of 5 mM HEPES to the artificial cerebrospinal fluid diminished the SAT by 91%. Nifedipine reduced the SAT by 53%. Removal of Ca(2+) from the saline abolished the SAT, and addition of BAPTA to the patch pipette reduced it by 79%. The inclusion of carboxyeosin (a PMCA inhibitor) in the pipette abolished the SAT, whether it was induced by a depolarizing step, or by simulated, repetitive, antidromic firing. The peak amplitude of the "antidromic" SAT of a single cell averaged 11% of the PAT elicited by comparable real antidromic activation of the CA1 neuronal population. Caloxin 2A1, an extracellular PMCA peptide inhibitor, blocked both the SAT and PAT by 42%. These results provide the first direct evidence that the PMCA can explain the extracellular alkaline shift elicited by synchronous firing.

Endogenous alkaline transients boost postsynaptic NMDA receptor responses in hippocampal CA1 pyramidal neurons.

In hippocampus, activation of the Schaffer collaterals generates an extracellular alkaline transient both in vitro and in vivo. This pH change may provide relief of the H+ block of NMDA receptors (NMDARs) and thereby increase excitability. To test this hypothesis, we augmented extracellular buffering in mouse hippocampal slices by adding 2 microM bovine type II carbonic anhydrase to the superfusate. With addition of enzyme, the alkaline transient elicited by a 10 pulse, 100 Hz stimulus train was reduced by 33%. At a holding potential (V(H)) of -30 mV, the enzyme decreased the half-time of decay and charge transfer of EPSCs by 32 and 39%, respectively, but had no effect at a V(H) of -80 mV. In current clamp, a 10 pulse, 100 Hz stimulus train gave rise to an NMDAR-dependent afterdepolarization (ADP). Exogenous enzyme curtailed the ADP half-width and voltage integral by 20 and 25%, respectively. Similar reduction of the ADP was noted with a brief 12 Hz stimulus train. The effect persisted in the presence of GABAergic antagonists or the L-type Ca2+ channel blocker methoxyverapamil hydrochloride but was absent in the presence of the carbonic anhydrase inhibitor benzolamide or when the exogenous enzyme was heat inactivated. The effects of the enzyme in voltage and current clamp were noted in 0 Mg2+ media but were abolished when (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate was included in the patch pipette. These results provide strong evidence that endogenous alkaline transients are sufficiently large in the vicinity of the synapse to augment NMDAR responses.

Regulation of postsynaptic Ca2+ influx in hippocampal CA1 pyramidal neurons via extracellular carbonic anhydrase.

Synchronous neural activity causes rapid changes of extracellular pH (pH(e)) in the nervous system. In the CA1 region of the hippocampus, stimulation of the Schaffer collaterals elicits an alkaline pH(e) transient in stratum radiatum that is limited by extracellular carbonic anhydrase (ECA). When interstitial buffering is diminished by inhibition of ECA, the alkalosis is enhanced and NMDA receptor (NMDAR)-mediated postsynaptic currents can be augmented. Accordingly, the dendritic influx of Ca2+ elicited by synaptic excitation may be expected to increase if ECA activity were blocked. We tested this hypothesis in the CA1 stratum radiatum of hippocampal slices from juvenile rats, using extracellular, concentric pH- and Ca2+-selective microelectrodes with response times of a few milliseconds, as well as Fluo-5F imaging of intracellular Ca2+ transients. Brief stimulation of the Schaffer collaterals elicited an alkaline pH(e) transient, a transient decrease in free extracellular Ca2+ concentration ([Ca2+]e), and a corresponding transient rise in free intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ECA with benzolamide caused a marked amplification and prolonged recovery of the pH(e) and [Ca2+]e responses, as well as the dendritic [Ca2+]i transients. The increase in amplitude caused by benzolamide did not occur in the presence of the NMDAR antagonist APV, but the decay of the responses was still prolonged. These results indicate that ECA can shape dendritic Ca2+ dynamics governed by NMDARs by virtue of its regulation of concomitant activity-dependent pH(e) shifts. The data also suggest that Ca2+ transients are influenced by additional mechanisms sensitive to shifts in pH(e).

Intracellular pH Measurements in Glioblastoma Cells Using the pH-Sensitive Dye BCECF.

The regulation of pH in glioblastoma (GBM) has received significant attention, because it has been linked to tumor metabolism and the stem cell phenotype. The variability in blood perfusion and oxygen tension within tumors suggests that ambient pH values fluctuate across different tumor territories. This chapter describes a detailed protocol for measuring intracellular pH in patient-derived GBM cells in vitro, using the fluorescent pH sensitive dye BCECF.

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called "concentric" ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

Kinetics of activity-evoked pH transients and extracellular pH buffering in rat hippocampal slices.

The kinetics of activity-dependent, extracellular alkaline transients, and the buffering of extracellular pH (pH(e)), were studied in rat hippocampal slices using a fluorescein-dextran probe. Orthodromic stimuli generated alkaline transients < or = 0.05 pH units that peaked in 273 +/- 26 ms and decayed with a half-time of 508 +/- 43 ms. Inhibition of extracellular carbonic anhydrase (ECA) with benzolamide increased the rate of rise by 25%, doubled peak amplitude, and prolonged the decay three- to fourfold. The slow decay in benzolamide allowed marked temporal summation, resulting in a severalfold increase in amplitude during long stimulus trains. Addition of exogenous carbonic anhydrase reduced the rate of rise, halved the peak amplitude, but had no effect on the normalized decay. A simulation of extracellular buffering kinetics generated recoveries from a base load consistent with the observed decay of the alkaline transient in the presence of benzolamide. Under control conditions, the model approximated the observed decays with an acceleration of the CO2 hydration-dehydration reactions by a factor of 2.5. These data suggest low endogenous ECA activity, insufficient to maintain equilibrium during the alkaline transients. Disequilibrium implies a time-dependent buffering capacity, with a CO2/HCO3- contribution that is small shortly after a base load. It is suggested that within 100 ms, extracellular buffering capacity is about 1% of the value at equilibrium and is provided mainly by phosphate. Accordingly, in the time frame of synaptic transmission, small base loads would generate relatively large changes in interstitial pH.

Fabrication and use of high-speed, concentric h+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording.

Ion-selective microelectrodes (ISMs) have been used extensively in neurophysiological studies. ISMs selective for H(+) and Ca(2+) are notable for their sensitivity and selectivity, but suffer from a slow response time, and susceptibility to noise because of the high electrical resistance of the respective ion exchange cocktails. These drawbacks can be overcome by using a "coaxial" or "concentric" inner micropipette to shunt the bulk of the ion exchanger resistance. This approach was used decades ago to record extracellular [Ca(2+)] transients in cat cortex, but has not been subsequently used. Here, we describe a method for the rapid fabrication of concentric pH- and Ca(2+)-selective microelectrodes useful for extracellular studies in brain slices or other work in vitro. Construction was simplified compared with previous implementations, by using commercially available, thin-walled borosilicate glass, drawing an outer barrel with a rapid taper (similar to a patch pipette), and by use of a quick and reliable silanization procedure. Using a piezoelectric stepper to effect a rapid solution change, the response time constants of the concentric pH and Ca(2+)-electrodes were 14.9 +/- 1.3 and 5.3 +/- 0.90 ms, respectively. Use of these concentric ISMs is demonstrated in rat hippocampal slices. Activity-dependent, extracellular pH, and [Ca(2+)] transients are shown to arise two- to threefold faster, and attain amplitudes two- to fourfold greater, when recorded by concentric versus conventional ISMs. The advantage of concentric ISMs for studies of ion transport and ion diffusion is discussed.

Functional demonstration of surface carbonic anhydrase IV activity on rat astrocytes.

Buffering of the brain extracellular fluid is catalyzed by carbonic anhydrase (CA) activity. Whereas the extracellular isoform CA XIV has been localized exclusively to neurons in the brain, and to glial cells in the retina, there has been uncertainty regarding the form or forms of CA on the surface of brain astrocytes. We addressed this issue using physiological methods on cultured and acutely dissociated rat astrocytes. Prior work showed that the intracellular lactate-induced acidification (LIA) of astrocytes is diminished by benzolamide, a poorly permeant, nonspecific CA inhibitor. We demonstrate that pretreatment of astrocytes with phosphatidylinositol-specific phospholipase C (PI-PLC) results in a similar inhibition of the mean LIA (by 66 +/- 3%), suggesting that the glycosylphosphatidylinositol-anchored CA IV was responsible. Pretreatment of astrocytes with CA IV inhibitory antisera also markedly reduced the mean LIA in both cultured cortical (by 46 +/- 4%) and acutely dissociated hippocampal astrocytes (by 54 +/- 8%). Pre-immune sera had no effect. The inhibition produced by PIPLC or CA IV antisera was not significantly less than that by benzolamide, suggesting that the majority of detectable surface CA activity was attributable to CA IV. Thus, our data collectively document the presence of CAIV on the surface of brain astrocytes, and suggest that this is the predominant CA isoform on these cells.

Failure and function of intracellular pH regulation in acute hypoxic-ischemic injury of astrocytes.

Astrocytes can die rapidly following ischemic and traumatic injury to the CNS. Brain acid-base status has featured prominently in theories of acute astrocyte injury. Failure of astrocyte pH regulation can lead to cell loss under conditions of severe acidosis. By contrast, the function of astrocyte pH regulatory mechanisms appears to be necessary for acute cell death following the simulation of transient ischemia and reperfusion. Severe lactic acidosis, and the failure of astrocytes to regulate intracellular pH (pH(i)) have been emphasized in brain ischemia under hyperglycemic conditions. Direct measurements of astrocyte pH(i) after cardiac arrest demonstrated a mean pH(i) of 5.3 in hyperglycemic rats. In addition, both in vivo and in vitro studies of astrocytes have shown similar pH levels to be cytotoxic. Whereas astrocytes exposed to hypoxia alone may require 12-24 h to die, acidosis has been found to exacerbate and speed hypoxic loss of these cells. Recently, astrocyte cultures were exposed to hypoxic, acidic media in which the large ionic perturbations characteristic of brain ischemia were simulated. Upon return to normal saline ("reperfusion"), the majority of cells died. This injury was dependent on external Ca2+ and was prevented by inhibition of reversed Na(+)-Ca2+ exchange, blockade of Na(+)-H+ exchange, or by low pH of the reperfusion saline. These data suggested that cytotoxic elevation of [Ca2+]i occurred during reperfusion due to a sequence of activated Na(+)-H+ exchange, cytosolic Na+ loading, and resultant reversal of Na(+)-Ca2+ exchange. The significance of this reperfusion model to ischemic astrocyte injury in vivo is discussed.

Role of Na+-H+ and Na+-Ca2+ exchange in hypoxia-related acute astrocyte death.

Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20-40-min exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., "reperfusion"). Astrocyte death required external Ca2+ and was blocked by KB-R7943, an inhibitor of reversed Na+-Ca2+ exchange, suggesting that injury was triggered by a rise in [Ca2+]i. In the present study, we confirmed the elevation of [Ca2+]i during reperfusion and studied the role of Na+-Ca2+ and Na+-H+ exchange in this process. Upon reperfusion, elevation of [Ca2+]i was detectable by Fura-2 and was blocked by KB-R7943. The low-affinity Ca2+ indicator Fura-FF indicated a mean [Ca2+]i rise to 4.8+/-0.4 microM. Loading astrocytes with Fura-2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca2+. Injury was prevented by the Na+-H+ exchange inhibitors ethyl isopropyl amiloride or HOE-694, and the rise of [Ca2+]i at the onset of reperfusion was blocked by HOE-694. Acidic reperfusion media was also protective. These data are consistent with Na+ loading via Na+-H+ exchange, fostering reversal of Na+-Ca2+ exchange and cytotoxic elevation of [Ca2+]i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.

Carbonic anhydrase IV and XIV knockout mice: roles of the respective carbonic anhydrases in buffering the extracellular space in brain.

Previous studies have implicated extracellular carbonic anhydrases (CAs) in buffering the alkaline pH shifts that accompany neuronal activity in the rat and mouse hippocampus. CAs IV and XIV both have been proposed to mediate this extracellular buffering. To examine the relative importance of these two isozymes in this and other physiological functions attributed to extracellular CAs, we produced CA IV and CA XIV knockout (KO) mice by targeted mutagenesis and the doubly deficient CA IV/XIV KO mice by intercrossing the individual null mice. Although CA IV and CA XIV null mice both are viable, the CA IV nulls are produced in smaller numbers than predicted, indicating either fetal or postnatal losses, which preferentially affect females. CA IV/XIV double KO mice are also produced in fewer numbers than predicted and are smaller than WT mice, and many females die prematurely before and after weaning. Electrophysiological studies on hippocampal slices on these KO mice showed that either CA can mediate buffering after synaptic transmission in hippocampal slices in the absence of the other, but that eliminating both is nearly as effective as the CA inhibitor, benzolamide, in blocking the buffering seen in the WT mice. Thus, both CA IV and CA XIV contribute to extracellular buffering in the central nervous system, although CA IV appears to be more important in the hippocampus. These individual and double KO mice should be valuable tools in clarifying the relative contributions of each CA to other physiological functions where extracellular CAs have been implicated.

Regulation and modulation of pH in the brain.

The regulation of pH is a vital homeostatic function shared by all tissues. Mechanisms that govern H+ in the intracellular and extracellular fluid are especially important in the brain, because electrical activity can elicit rapid pH changes in both compartments. These acid-base transients may in turn influence neural activity by affecting a variety of ion channels. The mechanisms responsible for the regulation of intracellular pH in brain are similar to those of other tissues and are comprised principally of forms of Na+/H+ exchange, Na+-driven Cl-/HCO3- exchange, Na+-HCO3- cotransport, and passive Cl-/HCO3- exchange. Differences in the expression or efficacy of these mechanisms have been noted among the functionally and morphologically diverse neurons and glial cells that have been studied. Molecular identification of transporter isoforms has revealed heterogeneity among brain regions and cell types. Neural activity gives rise to an assortment of extracellular and intracellular pH shifts that originate from a variety of mechanisms. Intracellular pH shifts in neurons and glia have been linked to Ca2+ transport, activation of acid extrusion systems, and the accumulation of metabolic products. Extracellular pH shifts can occur within milliseconds of neural activity, arise from an assortment of mechanisms, and are governed by the activity of extracellular carbonic anhydrase. The functional significance of these compartmental, activity-dependent pH shifts is discussed.

Surface carbonic anhydrase activity on astrocytes and neurons facilitates lactate transport.

A number of studies have provided physiological evidence for extracellular carbonic anhydrase (CA) in brain. Association of extracellular CA with glia has been limited to functional studies of gliotic slices and retinal Muller cells. While astrocytes contain intracellular CA, there has been no direct evidence for surface CA on these cells. In fact, some morphological studies suggest that the extracellular CA in brain parenchyma resides on neurons, not glia. There has been no functional demonstration of extracellular CA activity on CNS neurons, however. Here we capitalized on the H(+) dependence of inward lactate transport to reveal functional extracellular CA activity on cultured astrocytes and acutely isolated hippocampal pyramidal neurons. Exposure to 20 mM L-lactate produced a rapid acidification of astrocytes that was reversibly blocked by 10 microM benzolamide. The lactate-induced acidification (LIA) was also blocked by a dextran-conjugated CA inhibitor. In CO(2)/HCO(3) (-)-free, HEPES-buffered media, the LIA was largely unaffected. Acutely dissociated hippocampal pyramidal neurons underwent a similar LIA that was reversibly blocked by benzolamide. Surface CA is likely to facilitate lactate transport by enabling rapid replenishment (i.e., buffering) of surface H(+) required for inward lactate-H(+) cotransport. These results demonstrate functional surface CA for the first time on individual mammalian astrocytes and neurons and suggest that this enzyme may play a role in the utilization of monocarboxylate substrates such as lactate and pyruvate by the brain.