Carbonic anhydrase activity of rabbit lungs

Pulmonary carbonic anhydrase (CA) activity was studied in rabbit lungs perfused with solutions containing no CA. Measurements were made of the amount of 14CO2 appearing in the expired gas following injections of H14CO3(-), 14CO2, or a 20:1 mixture of each into the pulmonary artery. The fraction of the injected label in the expired gas was only 17% greater for 14CO2 than for the mixture, suggesting that equilibration between H14CO3(-) and 14CO2 was nearly complete during the capillary transit time. Inhibition of pulmonary CA decreased excretion of H14CO3(-) and the mixture by 40 and 49% and increased the excretion of 14CO2 by 96%. Addition of CA to the perfusate had no effect. Thus, CO2 exchange is not significantly limited by pulmonary CA if inhibitors are absent. Tissue binding of [3H]acetazolamide injected into the pulmonary artery was diminished by 50% when acetazolamide concentrations reached 0.13 x 10(-6) M. Each liter of extravascular lung water contained 1.25 x 10(-6) mol of receptors for acetazolamide that were accessible to plasma during a single circulation. Binding of [3H]acetazolamide was also observed in lungs of anesthetized rabbits, suggesting that pulmonary CA is accessible to plasma in vivo as well as in situ.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.     

Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis

Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO(2) for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H(14)CO(-)(3) uptake in the presence of an outward NaCl gradient or inward H(+) gradient, showed no evidence for a Cl(-)- or H(+)- driven HCO(-)(3) transport. H(14)CO(-)(3) and (36)Cl(-) uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of HCO(-)(3) and Cl(-) conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC(50) = 20 nM) and ethoxyzolamide (IC(50) = 2.5 nM). A strong DIDS-sensitive H(+)-ATPase activity was observed (IC(50) = 14 microM). This activity was also highly sensitive to vanadate and allyl isothiocyanate, two inhibitors of P-type H(+)-ATPases. Present data suggest that HCO(-)(3) absorption by ectodermal cells is carried out by H(+) secretion by H(+)-ATPase, resulting in the formation of carbonic acid in the surrounding seawater, which is quickly dehydrated into CO(2) by a membrane-bound CA. CO(2) then diffuses passively into the cell where it is hydrated in HCO(-)(3) by a cytosolic CA.     

In situ characterization of carbonic anhydrase activity in isolated rat lungs

Lung carbonic anhydrase (CA) participates directly in plasma CO2-HCO3(-)-H+ reactions. To characterize pulmonary CA activity in situ, CO2 excretion and capillary pH equilibration were examined in isolated saline-perfused rat lungs. Isolated lungs were perfused at 25, 30, and 37 degrees C with solutions containing various concentrations of HCO3- and a CA inhibitor, acetazolamide (ACTZ). Total CO2 excretion was partitioned into those fractions attributable to dissolved CO2, uncatalyzed HCO3- dehydration, and catalyzed HCO3- dehydration. Approximately 60% of the total CO2 excretion at each temperature was attributable to CA-catalyzed HCO3- dehydration. Inhibition of pulmonary CA diminished CO2 excretion and produced significant postcapillary perfusate pH disequilibria, the magnitude and time course of which were dependent on temperature and the extent of CA inhibition. The half time for pH equilibration increased from approximately 5 s at 37 degrees C to 14 s at 25 degrees C. For the HCO3- dehydration reaction, pulmonary CA in situ displayed an apparent inhibition constant for ACTZ of 0.9-2.2 microM, a Michaelis-Menten constant of 90 mM, a maximal reaction velocity of 9 mM/s, and an apparent activation energy of 3.0 kcal/mol.     

Effect of tracer infusion site on measurement of bicarbonate-carbon dioxide metabolism in dogs

Ten dogs were given a primed infusion of H13CO3- for 220 min while under general anesthesia. Isotopic steady state was reached within 60 min in exhaled CO2, femoral arterial blood HCO3-, and femoral venous blood HCO3-. Halfway through each infusion study, the site of tracer infusion was changed either from the central aorta to a peripheral vein, or vice versa. The mean HCO3(-)-CO2 flux measured from blood HCO3- enrichments was 15.7 +/- 2.1 (SD) mmol X kg-1 X h-1. The mean fraction of tracer recovered in exhaled CO2 was 79 +/- 7% (SD) of the infused dose. No significant difference in either HCO3- flux or recovery of tracer was found between the venous and arterial infusions of tracer. These results indicate that when venous administration of HCO3- tracer is compared with central arterial infusion, the initial loss of tracer into expired CO2 is an unimportant consideration in experiments measuring HCO3- kinetics.     

Exchange of labeled bicarbonate and carbon dioxide with erythrocytes suspended in an elutriator

An elutriator was used to study exchange of labeled CO2 and bicarbonate with erythrocytes. Rabbit erythrocytes were suspended by centrifugation in a stream of fluid and exposed to transient injections of an extracellular indicator (125I-albumin or 22Na+), a water indicator (3H2O), and H14CO3- and/or 14CO2. Diffusion of indicators into erythrocytes was judged by comparison of initial concentrations of diffusible and extracellular indicators in the elutriator outflow. It was possible to conduct these experiments at normal hematocrits because any carbonic anhydrase released from erythrocytes by hemolysis was washed away in the elutriator flow, and ambient pH, PO2, and PCO2 were kept constant by the inflow of fresh fluid. Equilibration of HCO3- with erythrocytes was complete during the 7- to 10-s transit time through the chamber. After this exchange was irreversibly inhibited by the anion exchange inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), addition of carbonic anhydrase (100 mg/dl) accelerated exchange, but acetazolamide (20 mg/dl) was without effect. These observations were consistent with the absence of carbonic anhydrase on the surface of the erythrocytes.     

Hepatocyte heterogeneity in response to icosanoids. The perivenous scavenger cell hypothesis

1. The metabolic and hemodynamic effects of prostaglandin F2 alpha, leukotriene C4 and the thromboxane A2 analogue U-46619 were studied during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion and in a system of two rat livers perfused in sequence. 2. The stimulatory effects of prostaglandin F2 alpha (3 microM) on hepatic glucose release, perfusion pressure and net Ca2+ release were diminished by 77%, 95% and 64%, respectively, during retrograde perfusion when compared to the antegrade direction, whereas the stimulation of 14CO2 production from [1-14C]glutamate by prostaglandin F2 alpha (which largely reflects the metabolism of perivenous hepatocytes) was lowered by only 20%. Ca2+ mobilization and glucose release from the liver comparable to that seen during antegrade perfusion could also be observed in retrograde perfusions; however, higher concentrations of the prostaglandin were required. 3. The glucose, Ca2+ and pressure response to leukotriene C4 (20 nM) or the thromboxane A2 analogue U-46619 (200 nM) of livers perfused in the antegrade direction were diminished by about 90% during retrograde perfusion. Sodium nitroprusside (20 microM) decreased the pressure response to leukotriene C4 (20 nM) and U-46619 (200 nM) by about 40% and 20% in antegrade perfusions, respectively, but did not affect the maximal increase of glucose output. 4. When two livers were perfused antegradely in series, such that the perfusate leaving the first liver (liver I) entered a second liver (liver II), infusion of U-46619 at concentrations below 200 nM to the influent perfusate of liver I increased the portal pressure of liver I, but not of liver II. At higher concentrations of U-46619 there was also an increase of the portal pressure of liver II and with concentrations above 800 nM the pressure responses of both livers were near-maximal [19.6 +/- 0.8 (n = 7) cm H2O and 16.5 +/- 1.1 (n = 8) cm H2O for livers I and II, respectively]. There was a similar behaviour of glucose release from livers I and II in response to U-46619 infusion. When liver I was perfused in the retrograde direction, a significant pressure or glucose response of liver II (antegrade perfusion) could not be observed even with U-46619 concentrations up to 1000 nM. 5. Similarly, the perfusion pressure increase and glucose release induced by leukotriene C4 (10 nM) observed with liver II was only about 20% of that seen with liver I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

碳酸酐酶Ⅵ的生理功能与酶缺陷性疾病

碳酸酐酶(carbonic anhydrase,CA)催化可逆的水合反应CO2+H2O?ΗCO3?+H+,参与维持pH值平衡、CO2与离子的转运、细胞凋亡等生理过程。碳酸酐酶VI(CA-VI)作为该类含锌酶中惟一的细胞分泌型碳酸酐酶,在哺乳动物及人的唾液腺、乳腺、泪腺、支气管等腺体中表达,对维持口腔、上消化道和呼吸道的生理功能起重要作用。     

An external delta-carbonic anhydrase in a free-living marine dinoflagellate may circumvent diffusion-limited carbon acquisition

The oceans globally constitute an important sink for carbon dioxide (CO(2)) due to phytoplankton photosynthesis. However, the marine environment imposes serious restraints to carbon fixation. First, the equilibrium between CO(2) and bicarbonate (HCO(3)(-)) is pH dependent, and, in normal, slightly alkaline seawater, [CO(2)] is typically low (approximately 10 mum). Second, the rate of CO(2) diffusion in seawater is slow, so, for any cells unable to take up bicarbonate efficiently, photosynthesis could become carbon limited due to depletion of CO(2) from their immediate vicinity. This may be especially problematic for those dinoflagellates using a form II Rubisco because this form is less oxygen tolerant than the usually found form I enzyme. We have identified a carbonic anhydrase (CA) from the free-living marine dinoflagellate Lingulodinium polyedrum that appears to play a role in carbon acquisition. This CA shares 60% sequence identity with delta-class CAs, isoforms so far found only in marine algae. Immunoelectron microscopy indicates that this enzyme is associated exclusively with the plasma membrane. Furthermore, this enzyme appears to be exposed to the external medium as determined by whole-cell CA assays and vectorial labeling of cell surface proteins with (125)I. The fixation of (14)CO(2) is strongly pH dependent, suggesting preferential uptake of CO(2) rather than HCO(3)(-), and photosynthetic rates decrease in the presence of 1 mm acetazolamide, a non-membrane-permeable CA inhibitor. This constitutes the first CA identified in the dinoflagellates, and, taken together, our results suggest that this enzyme may help to increase CO(2) availability at the cell surface.     

Purification and partial characterization of high and low activity carbonic anhydrase isoenzymes from Malaclemys terrapin centrata.

1. High activity (CA C) and low activity (CA B) carbonic anhydrase isoenzymes have been purified from turtle erythrocytes. 2. The two isoenzymes differed in CO2 hydration specific activity by 36-fold. 3. The low activity isoenzyme contained one half-cystine residue, whereas the high activity isoenzyme contained four half-cystines and required a reducing environment to maintain activity. Both isoenzymes contained zinc. 4. Molecular weights of 28,500 and 30,400 daltons were established for the low and high activity isoenzymes respectively. 5. Both isoenzymes were inhibited by acetazolamide, but only the high activity isoenzyme was inhibited by parachloromercuribenzoate. 6. The low activity isoenzyme was present in the erythrocytes at about 8-10 times the concentration of the high activity isoenzyme. 7. The high activity isoenzyme cross-reacted with antibodies prepared against pure chicken carbonic anhydrase C.     

Uptake of lactate by the liver: effect of red blood cell carriage

Multiple-indicator dilution experiments with labeled lactate were performed in the livers of anesthetized dogs. A mixture of (51)Cr-labeled erythrocytes, [(3)H]sucrose, and L-[1-(14)C]lactate or a mixture of (51)Cr-labeled erythrocytes, [(14)C]sucrose, and L-[2-(3)H]lactate was injected into the portal vein, and samples were obtained from the hepatic vein. Data were evaluated using a model comprising flow along sinusoids, exchange of lactate between plasma and erythrocytes and between plasma and hepatocytes, and, in the case of L-[1-(14)C]lactate, metabolism to H[(14)C]O(-)(3) within hepatocytes. The coefficient for lactate efflux from erythrocytes was 0.62 +/- 0.24 s(-1), and those for influx into and efflux from hepatocytes were 0.44 +/- 0.13 and 0.14 +/- 0.07 s(-1), respectively. The influx permeability-surface area product of the hepatocyte membrane for lactate (P(in)S, in ml x s(-1) x g(-1)) varied with total flow rate (F, in ml s(-1) x g(-1)) according to P(in)S = (3.1 +/- 0.5)F + (0.021 +/- 0.014). Lactate in plasma, erythrocytes, and hepatocytes was close to equilibrium, whereas lactate metabolism was rate limiting.     

Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic anhydrase, bicarbonate, and unstirred layers

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

Effects of dextran-bound inhibitors on carbonic anhydrase activity in isolated rat lungs

Effects of macromolecular Prontosil-dextran inhibitors (PD) on carbonic anhydrase (CA) activity in isolated rat lungs were studied. Isolated lungs were perfused with Krebs-Ringer bicarbonate (KRB) solutions containing no inhibitor, PD 100,000 (mol wt 100,000), PD 5,000 (mol wt 5,000), or low-molecular-weight inhibitors (Prontosil or acetazolamide). The time course of effluent perfusate pH equilibration was measured in a stop-flow pH electrode apparatus. Pulmonary CO2 excretion (Vco2) was monitored by continuously recording expired CO2 concentration. The lungs were ventilated with room air and perfused at 37 degrees C with KRB prebubbled with 5% CO2- 20% O2- 75% N2. The results obtained show that both the low-molecular-weight inhibitors and PD's caused postcapillary pH disequilibria (delta pH) in effluent perfusate. However, only acetazolamide and Prontosil caused a reduction in Vco2. These results suggest that there is an intravascular CA, presumably associated with endothelial cell membranes, that is accessible to all inhibitors used and is responsible in part for equilibration of the CO2- HCO3- -H+ reactions in the perfusate but, under the conditions used, does not affect CO2 excretion; and there is an extravascular (possibly intracellular) CA that can be inhibited by low-molecular-weight inhibitors, is primarily responsible for enhanced CO2 transfer across the alveolar-capillary barrier (perhaps via facilitation of CO2 diffusion), and is in part responsible for pH equilibration.     

Light-induced stimulation of carbonic anhydrase activity in pea thylakoids

Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier.     

Hepatocyte heterogeneity in response to extracellular ATP

1. The metabolic and hemodynamic effects of extracellular ATP in perfused rat liver were compared during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion. ATP in concentrations up to 100 microM was completely hydrolyzed during a single liver passage regardless of the perfusion direction. 2. The ATP(20 microM)-induced increases of glucose output, perfusion pressure and ammonium ion release seen during antegrade perfusions were diminished by 85-95% when the perfusion was in the retrograde direction, whereas the amount of Ca2+ mobilized from the liver was decreased by only 60%. The maximal rate of initial K+ uptake following ATP was dependent on the amount of Ca2+ mobilized regardless of the direction of perfusion. In the presence of UMP (1 mM), an inhibitor of ATP hydrolysis by membrane-bound nucleotide pyrophosphatase, the effect of the direction of perfusion on the glycogenolytic response to ATP (20 microM) was largely diminished. 3. For a maximal response of glucose output, Ca2+ release and perfusion pressure to extracellular ATP, concentrations of about 20 microM, 50 microM and 100 microM were required during antegrade perfusion, respectively. These maximal responses could also be obtained during retrograde perfusion, but higher ATP concentrations were required (120 microM, 80 microM, above 200 microM, respectively). 4. 14CO2 production from [1-14C]glutamate which occurs predominantly in the perivenous hepatocytes capable of glutamine synthesis was stimulated by extracellular ATP (20 microM); it was only slightly affected by the direction of perfusion. In antegrade perfusions, ATP (20 microM) increased 14CO2 production from 88 to 162 nmol g-1 min-1, compared to an increase from 91 to 148 nmol g-1 min-1 in retrograde perfusion. 5. The data are interpreted to suggest that (a) extracellular ATP is predominantly hydrolyzed by a small hepatocyte population located at the perivenous outflow of the acinus; (b) glycogenolysis to glucose is predominantly localized in the periportal area; (c) contractile elements (sphincters) exist near the inflow of the sinusoidal bed; (d) a considerable portion of the Ca2+ mobilized by ATP is derived from liver cells that do not contribute to hepatic glucose output.