Extracellular Carbonic Anhydrase Research Articles

Branchial membrane-associated carbonic anhydrase activity maintains CO2 excretion in severely anemic dogfish.

Plasma CO(2) reactions in Pacific spiny dogfish (Squalus acanthias) have access to plasma and gill membrane-associated carbonic anhydrase (CA). Acute severe experimental anemia and selective CA inhibitors were used to investigate the role of extracellular CA in CO(2) excretion. Anemia was induced by blood withdrawal coupled to volume replacement with saline. Lowering hematocrit from 14.2 +/- 0.4% (mean +/- SE; N = 31) to 5.2 +/- 0.1% (N = 31) had no significant impact on arterial or venous CO(2) tensions (Pa(CO(2)) and Pv(CO(2)), respectively) over the subsequent 2 h. PCO(2) was maintained despite the reduction in red cell number and a significant 32% increase in cardiac output (V(b)), both of which have been found to cause Pa(CO(2)) increases in teleost fish. By contrast, treatment of anemic dogfish with the CA inhibitors benzolamide (1.3 mg/kg) or F3500 (50 mg/kg), to selectively inhibit extracellular CA, elicited rapid and significant increases in Pa(CO(2)) of 0.68 +/- 0.17 Torr (N = 6) and 0.53 +/- 0.11 Torr (N = 7), respectively, by 30 min after treatment. These findings provide a functional context in which extracellular CA in dogfish contributes substantially to CO(2) excretion. Additionally, the apparent lack of effect of V(b) changes on PCO(2) suggests that, in contrast to teleost fish, CO(2) excretion in dogfish does not behave as a diffusion-limited system.

Bacteria biomass and carbonic anhydrase activity in some karst areas of Southwest China

To determine the role of microbes in karst processes, it is necessary to investigate the ecological distribution and characteristics of soil microorganisms in karst areas. In this paper, a preliminary study was carried out in two different karst areas of Southwest China: Nongla and the Guilin Yaji Karst Experimental Site (Guangxi). Soil samples from 10–20 cm in depth were analyzed for the number of bacteria, and the predominant bacteria were identified. Analysis showed that the amount of soil bacteria correlated highly with characteristics of the karst ecosystems, including their different geochemical environments and vegetation. The predominance of Azotobacteraceae colonies showed that the soil fertility of both types of karst areas may be improving. Also, the origin of carbonic anhydrase (CA), which could accelerate karst processes, was explored. The CA-producing bacteria were screened, and activities of extracellular and intracellular CA were measured. Obvious differences existed in intracellular and extracellular CA activities of soil bacteria between the two karst ecosystems with different vegetation conditions. This suggests that the activity of CA from soil bacteria in the two different karst areas was also correlated with karst ecosystem characteristics, including their different geochemical environments and vegetation features.

Inorganic Carbon Utilization of the Freshwater Red Alga Compsopogon coeruleus (Balbis) Montagne (Compsopogonaceae, Rhodophyta) Evaluated by in situ Measurement of Chlorophyll Fluorescence

To explore the inorganic carbon utilization of the freshwater red alga Compsopogon coeruleus, photosynthetic rates in response to increasing of bicarbonate concentration, the addition of alkaline HEPES buffer (pH 8.8), acid HEPES buffer (pH 4.0) and the extracellular carbonic anhydrase inhibitor (acetazolamide, AZ), respectively, were examined in situ by using a submersible pulse amplitude modulated (PAM) fluorometer. Among the treatments, adding acid HEPES buffer significantly reduced photosynthetic rates of the alga, while others showed no effect. Accordingly, we concluded that C. coeruleus had less or no inorganic carbon (Ci) limitation in its natural habitat. The alga might have higher affinity for bicarbonate and directly uptake bicarbonate as main Ci source without the aid of extracellular carbonic anhydrase.

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA, HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (Ci) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO2 production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO3− as a source of Ci for its photosynthesis via the mediation of the external CA. CO2 concentrations in the culture media affected its photosynthetic properties. A high level of CO2 (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO2 level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO2 was a reduction in the photosynthetic CO2 affinity. Decreased activity of the external CA in the high CO2 grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO2‐limited photosynthesis with the half‐saturating concentration of Ci exceeding that of seawater, did not operate active HCO3− uptake but used it via the extracellular CA for its photosynthetic carbon fixation.

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.

Effect of CO2 concentrations on the activity of photosynthetic CO2 fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum

The growth and activity of photosynthetic CO2 uptake and extracellular carbonic anhydrase (CAext) of the marine diatom Skeletonema costatum were investigated while cultured at different levels of CO2 in order to see its physiological response to different CO2 concentrations under either a low (30 μmol · m−2 · s−1) or high (210 μmol · m−2 · s−1) irradiance. The changes in CO2 concentrations (4–31 μmol/L) affected the growth and net photosynthesis to a greater extent under the low than under the high light regime. CAext was detected in the cells grown at 4 μmol/L CO2 but not at 31 and 12 μmol/L CO2, with its activity being about 2.5-fold higher at the high than at the low irradiance. Photosynthetic CO2 affinity (1/ K1/2(CO2)) of the cells decreased with increased CO2 concentrations in culture. The cells cultured under the high-light show significantly higher photosynthetic CO2 affinity than those grown at the low-light level. It is concluded that the regulations of CAext activity and photosynthetic CO2 affinity are dependent not only on CO2 concentration but also on light availability, and that the development of higher CAext activity and CO2 affinity under higher light level could sufficiently support the photosynthetic demand for CO2 even at low level of CO2.

Carbonic Anhydrase Activity of Alkalophilic Cyanobacteria from Soda Lakes

The activity and intracellular partition of carbonic anhydrase (CA) were studied in alkalophilic cyanobacteria, an inhabitant of soda lakes at pH 9–10. In the homogenates of Rhabdoderma lineare, Rhabdoderma sp., and Microcoleus chthonoplastes, high activity of CA was found, similar to that in eukaryotic microalgae. The activity of CA calculated on the basis of chlorophyll and protein was higher for the soluble (sCA) than for membrane (mCA) protein fraction. Intact cells of all cyanobacteria under investigation also showed CA activity that implies the presence of extracellular form(s). The extracellular CA in benthic M. chthonoplastes was localized, at least partly, in a vast glycocalix (gCA) as shown by Western blotting and the measurement of enzyme activity in the isolated glycocalix preparations. Probing gCA from M. chthonoplastes with the antibodies against thylakoid CA from Chlamydomonas reinhardtii (Cah3) demonstrated that gCA belongs to the α-type of enzyme and has the structure identical to that of Cah3. The extracellular CA of M. chthonoplastes manifested the maximum activity at pH 7 and 10, but not at pH 6 and 9. An increase in medium pH from 7.2 to 9.6 resulted only in slight alkalization of the cytoplasm in R. lineare, from 7.1 to 7.5. It follows that true alkalophils can maintain the pH inside the cell at the near-neutral level in spite of high pH (10.2) level in the cultural medium.

External HCO(3) (-) dehydration maintained by acid zones in the plasma membrane is an important component of the photosynthetic carbon uptake in Ruppia cirrhosa.

Ruppia cirrhosa, a temperate seagrass growing in brackish water, featured a high capacity for HCO(3) (-) utilisation, which could operate over a wide pH range (from 7.5 up to 9.5) with maintained efficiency. Tris buffer inhibited this means of HCO(3) (-) utilisation in a competitive manner, while addition of acetazolamide, an inhibitor of extracellular carbonic anhydrase activity, caused a 40-50% inhibition. A mechanism involving periplasmic carbonic anhydrase-catalysed HCO(3) (-) dehydration in acid zones, followed by a (probably diffusive) transport of the formed CO(2) across the plasma membrane was thus, at least partly, responsible for the HCO(3) (-) utilisation. This mechanism, which comprises a CO(2)-concentrating mechanism (CCM) associated with the plasma membrane, is thus shown for the first time in an aquatic angiosperm. Additional mechanisms involved in the Tris-sensitive HCO(3) (-) utilisation could be direct HCO(3) (-) uptake (e.g., in an H(+)/HCO(3) (-)symport) or (more likely) non-catalysed HCO(3) (-) dehydration in the acid zones. Based on these results, and on earlier investigations on Zostera marina, a general model for analysis of HCO(3) (-) utilisation mechanisms of seagrasses is suggested. In this model, three 'systems' for HCO(3) (-) utilisation are defined which are characterised (and can to some extent be quantified) by their capability to operate at high pH in combination with their response to acetazolamide and Tris. Some consequences of the fact that HCO(3) (-) utilisation and osmoregulation probably depend on the same energy source (ATP via H(+)-ATPase in the plasma membrane) are discussed.

Photosynthetic bicarbonate utilization inPorphyra haitanensis (Bangiales, Rhodophyta)

The activities of carbonic anhydrase (CA) and photosynthesis ofPorphyra haitanensis were investigated in order to see its photosynthetic utilization of inorganic carbon source. Both intra- and extra-cellular CA activities existed in the thallus. CA inhibitors, acetazolamide (AZ) and ethoxy-zolamide (EZ), remarkably depressed the photosynthetic oxygen evolution in seawater of pH 8.2 and 10.0, and EZ showed stronger inhibition than AZ. The observed net photosynthetic rate in seawater of pH 8.2 was much higher than that of CO2 supply theoretically derived from spontaneous dehydration of HCO3 −.P. haitanensis also showed a rather high pH compensation point (9.9). The results demonstrated thatP. haitanensis could utilize bicarbonate as the external inorganic carbon source for photosynthesis. The bicarbonate utilization was closely associated with HCO3 − dehydration catalyzed by extracellular CA activity. The inorganic carbon composition in seawater could well saturate the photosynthesis ofP. haitanensis. The low Km value and compensation points for inorganic carbon reflected the existence of CO2-concentrating mechanism in this alga.

Sources of inorganic carbon for phytoplankton in the eastern Subtropical and Equatorial Pacific Ocean

We present the results of a field study examining inorganic carbon utilization by phytoplankton assemblages in the eastern Subtropical and Equatorial Pacific Ocean. Data from isotope disequilibrium experiments demonstrate that HCO3- was the principal form of inorganic carbon taken up by all of the in situ phytoplankton populations we sampled. In a cyanobacteria‐dominated assemblage, HCO3- uptake occurred chiefly through a direct transmembrane transport mechanism. Diatom‐dominated assemblages expressed extracellular carbonic anhydrase and transported CO2 derived from the catalyzed dehydration of HCO3-. Direct HCO3- transport by the diatoms may have also occurred. In a 3‐d incubation experiment, we observed the CO2‐dependent regulation of inorganic C uptake in diatom‐dominated phytoplankton assemblages. Phytoplankton assemblages grown at 150 ppm CO2 possessed external carbonic anhydrase activity and took up HCO3- following its dehydration to CO2. In contrast, the assemblages cultured with 750 ppm CO2 appeared to lack external carbonic anhydrase activity and rely solely on CO2 as an exogenous source of carbon for photosynthesis. The CO2 effect on inorganic C utilization occurred in the absence of a detectable difference in phytoplankton growth rates between the 150 and 750 ppm CO2 treatments. Our field data provide compelling evidence that HCO3- utilization is prevalent in natural marine phytoplankton communities and is regulated by ambient CO2 concentrations. We discuss the ecological and biogeochemical implications of these results.

Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii.

We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.

The energy source for CO 2 transport in the marine microalga Nannochloris atomus

CO2 fluxes in the marine microalga Nannochloris atomus were studied by mass spectrometry using inhibitors and artificial acceptors of photosynthetic electron transport to investigate the energy source for CO2 uptake. This algal species is capable of taking up CO2 from the external medium by active transport but lacks active HCO(3)(-) transport and extracellular carbonic anhydrase. The capacity of cells to take up CO2 was a function of photosynthetic photon flux density. Dark respiration rates were also dependent upon the light intensity during the preceding illumination period, indicating the presence of light-enhanced dark respiration. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea to illuminated cell suspensions that had been allowed to concentrate inorganic carbon internally during photosynthesis caused a rapid burst of CO2, demonstrating that active CO2 transport had been abolished. A similar response was obtained when cell suspensions were treated with 2,5-dibromo-6-isopropyl-3methyl-1,4-benzoqinone or hydroxylamine. When methyl viologen was used to drain electrons from ferredoxin, cells were still able to take up CO2 from the external medium, although C-fixation decreased with time. These results demonstrate that active CO2 transport in N. atomus is supported by photosynthetic linear electron transport.

Photosynthetic bicarbonate utilization in Porphyra haitanen-sis (Bangiales, Rhodophyta)

The activities of carbonic anhydrase (CA) and photosynthesis of Porphyra haitanensis were investigated in order to see its photosynthetic utilization of inorganic carbon source. Both intra- and extra-cellular CA activities existed in the thallus. CA inhibitors, acetazolamide (AZ) and ethoxy- zolamide (EZ), remarkably depressed the photosynthetic oxygen evolution in seawater of pH 8.2 and 10.0, and EZ showed stronger inhibition than AZ. The observed net pho- tosynthetic rate in seawater of pH 8.2 was much higher than that of CO 2 supply theoretically derived from spontaneous dehydration of HCO 3 - . P. haitanensis also showed a rather high pH compensation point (9.9). The results demonstrated that P. haitanensis could utilize bicarbonate as the external inorganic carbon source for photosynthesis. The bicarbonate utilization was closely associated with 3 HCO - dehydration catalyzed by extracellular CA activity. The inorganic carbon composition in seawater could well saturate the photosynthe- sis of P. haitanensis. The low K m value and compensation points for inorganic carbon reflected the existence of CO 2 - concentrating mechanism in this alga.

AN ECOLOGICAL BASIS FOR EXTRACELLULAR CARBONIC ANHYDRASE IN MARINE UNICELLULAR ALGAE

Extracellular carbonic anhydrase (CAe) is expressed by many, but not all, autotrophic species of aquatic unicellular protists. We measured CAe activities in unicellular marine algae characteristic of either high nutrient spring, fall, and winter blooms or low nutrient summer populations to provide ecological/evolutionary information about the enzyme. Highest activities occurred in spring bloom and opportunistic diatoms exposed to long photoperiods (16 h) when pH was highest and CO2 was lowest. Lower activities were recorded for a fall‐bloom diatom exposed to the long photoperiod, and lowest values were found under all culture conditions for one diatom and a number of flagellated species typical of summer low nutrient environments. Other potential sources of variance in measurements of CAe activity were examined. Maximum activities of CAe were recorded for the diatom, Skeletonema costatum (Greville) Cleve, during late exponential phase of growth and within 8 h of the beginning of the photoperiod. We concluded that ecological factors are important in determining CAe activities in marine unicellular protists. Potential functions of CAe in the metabolism of marine unicellular algae are discussed.

The effects of exogenous extracellular carbonic anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering capacity.

The buffering capacity (beta) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3- within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma beta (-3.9+/-0.3 mmol 1(-1) pH unit(-1)), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) elevated plasma beta (-12.1+/-1.1 mmol 1(-1) pH unit(-1)). An injection of 5 mg kg(-1) carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (-0.23+/-0.05 Torr) or high (-0.20+/-0.05 Torr) plasma beta; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3- dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3- to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl-/HCO3- exchange governs HCO3- accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl-/HCO3- exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3- dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma beta was elevated in the present study was insufficient to drive a substantially increased component of HCO3- dehydration through the plasma.

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres.

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3- buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+-lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (DeltapHs, DeltapHi) of pH changes were quantified. From dpHi/dt, DeltapHi and the total buffer factor (BFtot) the lactate fluxes (mM min-1) and intracellular lactate concentrations ([lactate]i) were estimated. 3. BFtot was obtained as the sum of the non-HCO3- buffer factor (BFnon-HCO3) and the HCO3- buffer factor (BFHCO3). BFnon-HCO3 was 35 +/- 4 mM pH-1 for the EDL (n = 14) and 86 /- 16 mM pH-1 for the soleus (n = 14). 4. In soleus, 10 mM cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mM DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5. BZ (0.01 mM) and AZ (0.1 mM), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and pHs by about 40 %-150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %-50 % and reduced [lactate]i by about 40 %. The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7. BZ (0.01 mM) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF(non-HCO3). The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Role of basolateral carbonic anhydrase in proximal tubular fluid and bicarbonate absorption.

Membrane-bound carbonic anhydrase (CA) is critical to renal acidification. The role of CA activity on the basolateral membrane of the proximal tubule has not been defined clearly. To investigate this issue in microperfused rabbit proximal straight tubules in vitro, we measured fluid and HCO(3)(-) absorption and cell pH before and after the extracellular CA inhibitor p-fluorobenzyl-aminobenzolamide was applied in the bath to inhibit only basolateral CA. This inhibitor was 1% as permeant as acetazolamide. Neutral dextran (2 g/dl, molecular mass 70,000) was used as a colloid to support fluid absorption because albumin could affect CO(2) diffusion and rheogenic HCO(3)(-) efflux. Indeed, dextran in the bath stimulated fluid absorption by 55% over albumin. Basolateral CA inhibition reduced fluid absorption ( approximately 30%) and markedly decreased HCO(3)(-) absorption ( approximately 60%), both reversible when CA was added to the bathing solution. In the presence of luminal CA inhibition, which reduced fluid ( approximately 16%) and HCO(3)(-) ( approximately 66%) absorption, inhibition of basolateral CA further decreased the absorption of fluid (to 74% of baseline) and HCO(3)(-) (to 22% of baseline). CA inhibition also alkalinized cell pH by approximately 0.2 units, suggesting the presence of an alkaline disequilibrium pH in the interspace, which would secondarily block HCO(3)(-) exit from the cell and thereby decrease luminal proton secretion (HCO(3)(-) absorption). These data clearly indicate that basolateral CA has an important role in mediating fluid and especially HCO(3)(-) absorption in the proximal straight tubule.

Photosynthetic utilisation of carbon and light by two tropical seagrass species as measured in situ

In situ measurements of seagrass photosynthesis in relation to inorganic carbon (Ci) availability, increased pH and an inhibitor of extracellular carbonic anhydrase were made using an underwater pulse amplitude modulated (PAM) fluorometer. By combining the instrument with a specially designed Perspex chamber, we were able to alter the water surrounding a leaf without removing it from the growing plant. Responses to Ci within the chamber showed that subtidal plants of the seagrasses Cymodocea serrulata and Halophila ovalis had photosynthetic rates that were limited by the ambient Ci concentration depending on the irradiance that was available during short-term photosynthesis–irradiance trials. Relative electron transport rates (RETRs) at light saturation (up to 500 μ mol photons m−2 s−1) increased by 66–100% when the Ci concentration was increased from ca. 2.2 to 6.2 mM. On the other hand, intertidal plants of the same species exhibited a much lesser limitation of photosynthesis by Ci at any irradiance (up to 1500 μ mol photons m−2 s−1). Both species were able to use HCO−3 efficiently, and there was stronger evidence for direct uptake of HCO−3 rather than extracellular dehydration of HCO−3 to CO2 prior to Ci uptake. Subtidally, H. ovalis and C. serrulata grew to 10 and 12 m, respectively, where ambient irradiances were approximately 16 and 11% of those at the surface. Maximum RETRs (at light saturation) were lower for these deep-growing plants than for the intertidally growing ones. For both species, the onset of light saturation of photosynthesis (Ek) occurred at approximately 100 μ mol photons m−2 s−1 for the deep water populations, which was four and two times lower than for the shallow populations of C. serrulata and H. ovalis, respectively. This, and the differences in maximal photosynthetic rates (RETRmax), reflects an acclimation of the deep-growing populations to the lower light environment. The results presented here show that photosynthesis, as measured in situ, was limited by the availability of Ci for the deeper growing plants in Zanzibar, while the intertidally growing plants photosynthesised at close to Ci saturation. The latter result is contrary to previous conclusions regarding Ci limitations for these intertidal plants, and, in general, our findings highlight the need for performing similar experiments in situ rather than under laboratory conditions.