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.     

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.     

Extracellular carbonic anhydrase of skeletal muscle associated with the sarcolemma

We report here 1) the synthesis and properties of a new macromolecular carbonic anhydrase inhibitor, Prontosil-dextran, 2) its application to determine the localization of a previously described extracellular carbonic anhydrase in skeletal muscle, and 3) the application of a recently published histochemical technique using dansylsulfonamide to the same problem. Stable macromolecular inhibitors of molecular weights of 5,000, 100,000 and 1,000,000 were produced by covalently coupling the sulfonamide Prontosil to dextrans. Their inhibition constants towards bovine carbonic anhydrase II are 1-2 X 10(-7) M. The Prontosil-dextrans, PD 5,000, PD 100,000, and PD 1,000,000, were used in studies of the washout of H14CO3-) from the perfused rabbit hindlimb. This washout is slow due to the presence of an extracellular carbonic anhydrase and can be markedly accelerated by PD 5,000 but not by PD 100,000 and PD 1,000,000. Since PD 5,000 is accessible to the entire extracellular space and PD 100,000 and PD 1,000,000 are confined to the intravascular space, we conclude that the extracellular carbonic anhydrase of skeletal muscle is located in the interstitium. The histochemical studies show a strong staining of the sarcolemma of the muscle fibers with high oxidative capacity. It appears likely, therefore, that the extracellular carbonic anhydrase of skeletal muscle is associated with muscle plasma membranes with its active site directed toward the interstitial space.     

CO2 and bicarbonate exchange in the rat liver

Several forms of carbonic anhydrase (CA) have been detected in hepatocytes. The distribution of these enzymes appears to be heterogeneous in the hepatic lobule, and the specific isoenzyme that predominates is influenced by sex steroid levels in the animal. In the present study, experiments were conducted in isolated male rat livers perfused with erythrocyte-free solutions, which were devoid of CA to see if there were sufficient tissue CA activity accessible to the plasma to ensure equilibration between labeled HCO3- and CO2 during a single passage from the portal vein to the hepatic vein. After injection of H14CO3- into the portal vein, emergence of the 14C label from the hepatic vein was slightly more rapid than after injections of 14CO2. After infusion of 5-250 microM of acetazolamide, an inhibitor of CA, H14CO3- was virtually confined to the extracellular space during a single transit through the organ, whereas the outflow of 14CO2 was very prolonged, suggesting that some of the 14C had been "trapped" within the hepatic cells as H14CO3-. Inhibition of CA activity in the intact organ with low doses of acetazolamide suggests the presence of a readily inhibitable isoenzyme of CA on the surface of the hepatocytes, which is directly accessible to both HCO3- and acetazolamide. The outflow patterns of 14CO2 and H14CO3- became the same after infusion of erythrocyte CA into the portal vein. On the basis of the pH of the perfusate and the cellular distribution of 14CO2 and H14CO3- in the presence of CA, an intracellular pH value of 7.26 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary carbonic anhydrase in the garter snake, Thamnophis sirtalis

This study examined whether the snake lung possesses intravascular carbonic anhydrase (CA). Lungs were perfused with control salines and with salines containing CA inhibitors. Perfusion with control salines resulted in a stable CO(2) excretion, whereas CA inhibitors significantly reduced pulmonary CO(2) excretion. Membrane-permeable and membrane-impermeable CA inhibitors produced comparable decreases in CO(2) excretion, suggesting that extracellular, intravascular CA participated in the pulmonary CO(2)-HCO3(-)-H(+) reactions. Treatment of lungs with phosphatidylinositol specific-phospholipase C (PI-PLC) significantly decreased CO(2) excretion, indicating that CA was connected to the luminal endothelial cell membrane by a phosphatidylinositol glycan linkage. Taken together, these results are the first to demonstrate the presence of membrane-bound, intravascular CA (CA IV) in the snake lung.     

Equilibrium of CO2 reactions in the pulmonary capillary

Steady-state CO2 excretion was measured in isolated blood-free rabbit lungs perfused with bicarbonate solutions. CO2 in the expired ventilation was either present initially in the perfusate as dissolved CO2 or produced from bicarbonate during pulmonary capillary transit. The two components were separated by measurement of simultaneous acetylene excretion. Bovine carbonic anhydrase and acetazolamide were sequentially added to the perfusate to determine the effects of maximal enzyme catalysis and inhibition of native lung carbonic anhydrase on CO2 production. Control CO2 production was significantly greater than that observed during inhibition of native lung carbonic anhydrase, confirming previous observations that bicarbonate has access to the tissue enzyme. Addition of excess carbonic anhydrase increased CO2 production by a statistically, but not physiologically, significant amount. These data demonstrate that CO2 reactions outside the erythrocyte attain 97% completion during pulmonary capillary transit. Under control and catalyzed conditions, alveolar and venous CO2 tens ions and pH were essentially identical to equilibrium values determined by in vitro tonometry.     

Influence of proton availability on intracapillary CO2-HCO3(-)-H+ reactions in isolated rat lungs.

Transcapillary CO2 exchange entails a transient perfusate CO2-HCO3(-)-H+ disequilibrium, leading to net loading or unloading of blood HCO3-. Perfusate reequilibration may or may not reach completion during the time of capillary transit, depending on the rate of intracapillary CO2-HCO3(-)-H+ reactions. Failure to reestablish equilibrium within the "open" capillary system leads to continued reequilibration in the "closed" postcapillary vasculature with resultant shifts in postcapillary perfusate PCO2, pH, and [HCO3-]. In the present study, we determined the effects of perfusate nonbicarbonate buffer capacity (beta) on intracapillary CO2-HCO3(-)-H+ reactions in isolated saline-perfused rat lungs. Effects of beta on the rate of transcapillary CO2 excretion (VCO2) and the magnitude of the postcapillary perfusate pH disequilibrium were measured as a function of luminal vascular carbonic anhydrase (CA) activity. The data indicate that beta markedly influenced the kinetics and dynamics of intravascular CO2-HCO3(-)-H+ reactions. beta affected VCO2 and the relative enhancement of VCO2 by luminal vascular CA. The data emphasize the inadequacies of using traditional "equilibrium" models of the CO2-HCO3(-)-H+ system to investigate capillary CO2 transport and exchange, even in organs (e.g., lungs) that contain significant luminal vascular CA activity.     

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.     

Total CA activity in isolated perfused guinea pig lung by 18O-exchange method

The rate of exchange of 18O between alveolar CO2 and lung water was measured in isolated perfused guinea pig lungs to quantify carbonic anhydrase (CA) activity. The average lung CA activity, with a reaction velocity constant of 5.32 +/- 2.2 s-1, is sufficient to accelerate CO2 reactions in lung water by two orders of magnitude over the uncatalyzed rate at 22 degrees C and a PCO2 of 40 Torr. Three sulfonamide inhibitors of CA with different human erythrocyte membrane permeabilities were used to determine the availability of the enzyme to the perfusate. Ethoxzolamide, the most permeable at 0.1 microM (100 times its inhibition constant, of Ki) inhibited 85% of enzyme activity after exposure of the lung for 3 min and 94% of enzyme activity after 30 min, whereas 1.25 microM (320 times its Ki) acetazolamide (1/165 as permeable) only inhibited CA 28% at 3 min and 75% at 30 min. Benzolamide (less than 1/1,000 as permeable) at 4 microM (1,000 times its Ki) inhibited only approximately 17% of control CA activity by 5 min and 48% by 30 min after the start of perfusion. These data indicate the CA available to pulmonary capillary plasma is approximately 10% of the total lung CA activity, in agreement with published measurements on the homogenized lung.     

Thromboxane release from irradiated perfused rat lungs: role of oncotic agents

Isolated lungs from 20 Gray (Gy) whole body irradiated rats were perfused with Krebs-Ringer bicarbonate plus 3% bovine serum albumin (KRB-BSA). The pulmonary effluent showed a 99% (p less than .05) increase in immunoassayable thromboxane B2 (iTXB2) release compared with non-irradiated lungs. Since both arachidonic acid and cyclooxygenase products bind to albumin, studies were performed to determine if omission or substitution of this protein oncotic agent would alter the radiation-induced increase in pulmonary iTXB2 release. Irradiated, isolated lungs perfused with media from which the BSA was omitted (KRB) did not demonstrate the radiation-induced increase in pulmonary iTXB2 release. Similarly, irradiated lungs perfused with media in which Dextran 70 (KRB plus 3% Dextran 70, KRB-Dextran 70) was substituted for BSA also did not show the radiation-induced increase in pulmonary effluent iTXB2 levels. These studies demonstrate the importance of including albumin as the oncotic agent in perfused organ systems when studying cyclooxygenase product release.     

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 perfusate buffer capacity on capillary CO2-HCO3(-)-H+ reactions: theory.

The importance of perfusate nonbicarbonate buffer capacity (beta nonHCO3) to intracapillary CO2-HCO3(-)-H+ reactions was assessed by theoretical analysis of CO2 exchange in saline-perfused pulmonary capillaries. Time courses for perfusate PCO2, [HCO3-], and [H+] were computed for capillaries containing different activities of luminal vascular carbonic anhydrase and different amounts of perfusate nonbicarbonate buffers. Mobilization of perfusate HCO3- toward CO2 during capillary transit is determined by the availability of HCO3- and H+. A supply of protons from the nonbicarbonate buffer pool is necessary to maintain a high rate of HCO3- dehydration. The analyses indicate that beta nonHCO3 has marked nonlinear effects on transcapillary CO2 exchange and intravascular pH equilibration. These nonlinear effects differ from those previously computed for CO2 reactions in an open system because the present model system consists of a sequential combination of open (within capillary proper) and closed (within postcapillary vasculature) systems. The role of luminal vascular carbonic anhydrase in capillary CO2 reactions is strongly dependent on beta nonHCO3. Perfusate nonbicarbonate buffer capacity must be considered when the results of experimental studies of transcapillary CO2 exchange and/or intravascular pH equilibration are interpreted.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

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.     

Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver.

1. Addition of 1-chloro-2,4-dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione-S-conjugate [S-(2,4-dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Anisotonic perfusion did not affect total S-conjugate formation, but release of the S-conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S-conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume-regulatory net K+ uptake. 2. Basal steady-state release of oxidized glutathione (GSSG) into bile was 1.30 +/- 0.12 nmol.g-1.min-1 (n = 18) during normotonic (305 mOsmol/l) perfusion and was 3.8 +/- 0.3 nmol.g-1.min-1 in the presence of t-butylhydroperoxide (50 mumol/l). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t-butylhydroperoxide (50 mumol/l)-stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/l) increased. Anisotonic exposure was without effect on t-butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver-cell swelling due to addition of glutamine plus glycine (2 mmol/l, each). 3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1-14C]glucose by about 80%, whereas 14CO2 production from [6-14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1-14C]glucose by about 40%, whereas 14CO2 production from [6-14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1-14C]glucose was also observed in presence of t-butylhydroperoxide (50 mumol/l), which increased 14CO2 production from [1-14C]glucose by about 40%. 4. t-Butylhydroperoxide (50 mumol/l) was without significant effect on volume-regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/l) or hypertonic (385 mOsmol/l) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t-butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose-phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide-induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione-S-conjugates into the sinusoidal circulation at the expense of biliary secretion.     

Acid-base and respiratory properties of a buffered bovine erythrocyte perfusion medium

Current research in organ physiology often utilizes in situ or isolated perfused tissues. We have characterized a perfusion medium associated with excellent performance characteristics in perfused mammalian skeletal muscle. The perfusion medium consisting of Krebs-Henseleit buffer, bovine serum albumin, and fresh bovine erythrocytes was studied with respect to its gas-carrying relationships and its response to manipulation of acid-base state. Equilibration of the perfusion medium at base excess of -10, -5, 0, 5, and 10 mmol X L-1 to humidified gas mixtures varying in their CO2 and O2 content was followed by measurements of perfusate hematocrit, hemoglobin concentration, pH, Pco2, Cco2, Po2, and percent oxygen saturation. The oxygen dissociation curve was similar to that of mammalian bloods, having a P50 of 32 Torr (1 Torr = 133.3 Pa), Hill's constant n of 2.87 +/- 0.15, and a Bohr factor of -0.47, showing the typical Bohr shifts with respect to CO2 and pH. The oxygen capacity was calculated to be 190 mL X L-1 blood. The carbon dioxide dissociation curve was also similar to that of mammalian blood. The in vitro nonbicarbonate buffer capacity (delta [HCO3-] X delta pH-1) at zero base excess was -24.6 and -29.9 mmol X L-1 X pH-1 for the perfusate and buffer, respectively. The effects of reduced oxygen saturation on base excess and pH of the medium were quantified. The data were used to construct an acid-base alignment diagram for the medium, which may be used to quantify the flux of nonvolatile acid or base added to the venous effluent during tissue perfusions.     

Possible role of carbonic anhydrase, V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion transport across the gills of the euryhaline crab Chasmagnathus granulatus

We studied the participation of carbonic anhydrase (CA), V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion absorption through the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2 per thousand, 10 per thousand, 30 per thousand (about seawater), and 45 per thousand salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from crabs acclimated to either 2 per thousand or 10 per thousand with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2 per thousand and perfused with 20 per thousand saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (V(te)) by 47%, further addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30 per thousand saline (iso-osmotic for seawater acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V-H(+)-ATPase and Cl(-)/HCO3-) reduced V(te) by 15-16% in gills perfused with normal 20 per thousand saline, and by 77% and 45%, respectively when they were applied in Na-free 20 per thousand saline, suggesting the participation of those transporters and cytosolic CA in electrogenic ion absorption.     

Mechanisms of interaction between oxygen and granulocytes in hyperoxic lung injury

Hyperoxia and infused granulocytes act synergistically in producing a nonhydrostatic high-permeability lung edema in the isolated perfused rabbit lung within 4 h, which is substantially greater than that seen with hyperoxia alone. We hypothesized that the interaction between hyperoxia and granulocytes was principally due to a direct effect of hyperoxia on the lung itself. Isolated perfused rabbit lungs that were preexposed to 2 h of hyperoxia (95% O2-5% CO2) prior to the infusion of unstimulated granulocytes (under normoxic conditions) developed significant nonhydrostatic lung edema (P = 0.008) within 2 h when compared with lungs that were preexposed to normoxia (15% O2-5% CO2) prior to granulocyte perfusion. The edema in the hyperoxic-preexposed lungs was accompanied by significant increases in bronchoalveolar lavage (BAL) protein, BAL granulocytes, BAL thromboxane and prostacyclin levels, perfusate chemotactic activity, and lung lipid peroxidation. These findings suggest that the synergistic interaction between hyperoxia and granulocytes in producing acute lung injury involves a primary effect of hyperoxia on the lung itself.     

The role of the blood in metabolism of prostaglandin E1 in the cat lung.

We found that when 15-keto-PGE1 was added to cat blood, it was converted to 13, 14-dihydro-15-keto-PGE1 (dihydro-keto-PGE1) by a NADH-dependent enzyme associated with some formed element(s) in the blood. When PGE1 was injected into the pulmonary artery of blood-perfused lungs, the only metabolite detectable in the pulmonary venous blood was the dihydro-keto-PGE1. However, when the lungs were perfused with an artificial perfusate containing no blood cells, a small amount of 15-keto-PGE1 was detected in the venous effluent. Therefore it would appear that a blood-borne delta13 reductase was partially responsible for the conversion of PGE1 to dihydro-keto-PGE1 on passage through blood-perfused cat lungs.     

The role of the blood in metabolism of prostaglandin E1 in the cat lung

We found that when 15-keto-PGE1 was added to cat blood, it was converted to 13,14-dihydro-15-keto-PGE1 (dihydro-keto-PGE1) by a NADH-dependent enzyme associated with some formed element(s) in the blood. When PGE1 was injected into the pulmonary artery of blood-perfused lungs, the only metabolite detectable in the pulmonary venous blood was the dihydro-keto-PGE1. However, when the lungs were perfused with an artificial perfusate containing no blood cells, a small amount of 15-keto-PGE1 was detected in the venous effluent. Therefore it would appear that a blood-borne Δ13 reductase was partially responsible for the conversion of PGE1 to dihydro-keto-PGE1 on passage through blood-perfused cat lungs.