Robert E. Forster

Considerations of the physiological variables that determine the blood carboxyhemoglobin concentration in man.

were analyzed for CO concentrations with an infrared CO meter. This instrument has an error (SD) of 4-0.00004% CO and requires a 200-ml sample. These samples were col- lected during the summer of 1964. Smoking is prohibited in the areas where these samples were collected. We also measured the CO concentrations in air samples taken from smoke-filled conference rooms, a small nonventilated room that we purposely filled with smoke by burning cigarettes, and a rural area well away from automobile combustion. Diurnal changes in blood (COHb) were measured in one subject and compared with the changes in percentage of CO in his environment. Blood (COHb) was determined by a method in which gas extracted from a 2-ml blood sample is measured in the infrared CO meter. This method has an error (SD) of 40.03% (COHb)( 10).

Anti-oxidative response of carbonic anhydrase III in skeletal muscle

We propose that carbonic anhydrase III (CAIII) functions as an anti‐oxidant agent in skeletal muscle. To explore this hypothesis, we analyzed the gene expression profile of skeletal muscle in mice deficient in CAIII gene utilizing the murine genome U74Av2 set microarray. Pairwise comparison between CAIII knockout mice and their wild‐type littermates revealed that more than 500 of 12,000 genes in the array showed an altered level of transcription. Of particular note were transcriptional alterations among genes associated with the glutathione redox cycle, suggesting a possible involvement of CAIII in the glutathione‐mediated anti‐oxidant activity. We therefore investigated S‐glutathiolation and irreversible oxidation of the 2 reactive sulfhyryls of CAIII in skeletal muscle under oxidative stresses of ischemia, or exhaustive exercise. Analysis by isoelectric focusing followed by Western blot revealed that the two sulfhydryls were differentially and progressively oxidized. Brief ischemia of 10 ‐ 20 min provoked partial (one of the suflhydryls) modification of CAIII via reversible S‐glutathiolation. Protracted 60 min ischemia yielded equal amounts of both partially and completely (both sulfhydryls) modified CAIII due to irreversible oxidization. Twenty minutes of repetitive electrical stimulation, simulating exhaustive exercise, produced a mixed yield: partial modification by reversible S‐glutathiolation and complete modification by irreversible oxidation. Thus CAIII responds to oxidative stress with a distinctive sulfhydryl oxidation patterns reflecting duration and severity that may prove sensitive indices of extent and type of damage in muscle injury. IUBMB Life, 56: 343‐347, 2004

The Role of Carbonic Anhydrase in Hepatocyte Metabolism

The existence of carbonic anhydrase inside mammalian hepatic mitochondria has been suspected since 1959.8,23,25,28*29,33 Rossi” determined the carbonic anhydrase activity of mitochondria that had their inner membranes permeabilized by Triton X-100, but found no activity in intact mitochondria. He concluded that this was evidence for the existence of carbonic anhydrase inside the inner mitochondrial membrane. Work from two laboratories later showed that acetazolamide blocked the uptake of Ca2+ and HCO? by respiring mit~chondria;’~J~.~O these researchers concluded that carbonic anhydrase activity situated within the inner mitochondrial membrane was blocked by the drug. Subsequent work from our laboratory has shown that the hepatic mitochondrial carbonic anhydrase is a soluble matrix enzyme; we have quantitated its activity by monitoring the I8O disappearance from C18016010,18 with intact hepatic mitochondria. Separation of fractionated mitochondria into submitochondrial particles and matrix-derived soluble supernatant by ultracentrifugation resulted in finding that all carbonic anhydrase activity was in the supernatant fraction. Our first goal in this work was to confirm the existence of mitochondrial carbonic anhydrase and to quantitate its activity. Our initial evidence of its presence within intact hepatic mitochondria is given in FIGURE 1. When freezethawed mitochondria were added to the reaction chamber (Curve A) there was an immediate acceleration in the decrease of C160’s0 from NaHC03 (25 mM 2% labeled with I8O). This accelerated decrease was eliminated by the inclusion of 1 pM concentrations of acetazolamide in the solution. When intact mitochondria (Curve B) were added to the reaction chamber, the accelerated decrease in C180’60 was biphasic; this pattern indicates that the carbonic anhydrase is not in immediate contact with the reaction solution; that is, that it is contained within a membrane that is not freely permeable to HCO?.l8 We know from Rossi’s work initially33 and from our fractionation of the mitochondria1° that the carbonic anhydrase is contained within the inner mitochondrial membrane, not merely the outer. The outer membrane is known to be permeable to HCO,;’ our assay would give identical traces for intact and broken mitochondria if there were any carbonic anhydrase in the membrane space. We assayed for carbonic anhydrase activity with intact and cholate-treated mitochondria using a changing pH technique; we found activity by this method only with the broken mitochondria, not with the

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.

The water permeability of erythrocytes

Abstract 1. 1. We have used a stopped-flow rapid-reaction apparatus to record rapid changes in light transmission of cell suspensions following abrupt alterations in osmolality. These changes in osmolality can be related to average cell volume to give the kinetics of water transfer. 2. 2. The sudden cessation of forward movement of a cell suspension in the apparatus, in the absence of any change in osmolality, produced a consistent pattern of variation in light transmission which we reduced in magnitude but could not eliminate completely by the introduction of glass capillaries into the observation tube. Therefore in each experiment a control record following the mixing of the cell suspension under study with isosmotic fluid was obtained and subtracted from the record following a change in osmolality. 3. 3. Water permeability, P w , of human red cells at room temperature averaged 0.30 cm 4 /osm·sec during exosmosis and 0.36 cm 4 /osm·sec during endosmosis. P w decreased with increasing extracellular osmolality according to the regression, P w = 0.41 – 0.00029 mosm/l. P w did not vary with changes in intracellular osmolality although a slight dependency could not be ruled out. 4. 4. P w for horse red cells: −0.28 − 0.00026 × extracellular osmolality (mosm/l). P w for nucleated eel and chicken cells averaged 0.054 cm 4 /osm·sec, one-fifth of that for human cells, and in the case of the eel cells only, did not vary with extracellular osmolality. 5. 5. From arguments based in part on measurements of the rates of gas exchange of red cells, we conclude that any stagnant layer of fluid must be less than 1 μ thick. 6. 6. If the cell membranes are heterogeneous in the direction of water flux and water permeability varies with osmolality to a different degree in the different layers, our experimental results are explicable by classical diffusion theory.

Erythrocyte water permeability. The effects of anesthetic alcohols and alterations in the level of membrane cholesterol

1. Treatment of human erythrocytes with anesthetic n-alkanols (pentanol, hexanol and hepatanol) results in a decrease in the osmotic water permeability of the red cell membrane. 2. The alcohol-induced changes in osmotic water permeability are proportional to the alcohol concentration and roughly parallel diphenylhexatriene that are induced by the alcohols. 3. Enrichment of the red cell membrane in cholesterol also results in a decrease in the osmotic water permeability. 4. Moderate depletion (9% or 40%) of membrane cholesterol is without effect on the osmotic water permeability, even though this treatment results in a significant increase in the rotational mobility of diphenylhexatriene in the membrane lipids.

The value of inherited deficiencies of human carbonic anhydrase isozymes in understanding their cellular roles.

Very little light has been shed on the role of the low-activity CA I isozyme in humans by studies on CA I-deficient individuals. On the other hand, CA II-deficient individuals exhibit abnormalities of bone, kidney and brain, implicating a functional role for the high-activity CA II isozyme in cells from these tissues and organs. It also appears that the CA II-deficient red cell is capable of normal respiratory function under unstressed conditions. In addition, there is some preliminary evidence that those organs such as the eye which primarily contain the CA II isozyme, may be able to function effectively in the absence of CA II.

The role of exchange transfusion in the management of low-birth-weight infants with and without severe respiratory distress syndrome. I. Initial observations.

Exchange transfusion, as a form of therapy, was contrasted with the use of fresh frozen plasma or conventional supportive care alone in the management of 19 infants with birth weights of less than 1,000 gm, without severe respiratory distress, and in the management of 82 infants, birth weights less than 2,000 gm, with severe respiratory distress whose disease manifested itself within the first 24 hours of life. Survival for more than five days was similar, regardless of therapy, in infants weighing less than 1,000 gm without severe RDS. In contrast, the use of exchange transfusion significantly decreased the case fatality rate of infants with severe RDS. In the groups receiving exchange transfusion, the mortality rate was 41%, whereas the groups receiving either plasma or supportive care alone the mortality was 80%. Study of coagulation factors and red cell concentrations of fetal hemoglobin and of 2,3-DPG failed to demonstrate any relationship between either improvement in coagulation or oxygen unloading and the improved survival of infants receiving exchange transfusion. Following exchange transfusion there was a significant decrease in the ratio of FIO2 to PaO2, suggesting that pulmonary perfusion and/or ventilation was improved by the procedure.

Rate of disappearance of labeled carbon dioxide from the lungs of humans during breath holding: a method for studying the dynamics of pulmonary CO2 exchange

The dynamics of CO(2) exchange in the lungs of man was studied by observing the rate of disappearance of a stable isotope of CO(2) ((13)CO(2)) from the alveolar gas during breath holding. Over 50% of the inspired isotope disappeared within the first 3 sec followed by a moderately rapid logarithmic decline in which one-half of the remaining (13)CO(2) disappeared every 10 sec. The large initial disappearance of (13)CO(2) indicated that alveolar (13)CO(2) equilibrated in less than 3 sec with the CO(2) stored in the pulmonary tissues and capillary blood. The volume of CO(2) in the pulmonary tissues calculated from this initial disappearance was 200 ml or 0.33 ml of CO(2) per milliliter of pulmonary tissue volume. The alveolar to end-capillary gradient for (13)CO(2) was calculated by comparing the simultaneous disappearance rates of (13)CO(2) and acetylene. At rest and during exercise this gradient for (13)CO(2) was either very small or not discernible, and diffusing capacity for CO(2) (D(LCO2)) exceeded 200 ml/(min x mm Hg). After the administration of a carbonic anhydrase inhibitor the rate of disappearance of (13)CO(2) decreased markedly. D(LCO2) fell to 42 ml/(min x mm Hg) and at least 70% of the exchange of (13)CO(2) with the CO(2) stores in the pulmonary tissues and blood was blocked by the inhibitor. These changes were attributed to impairment of exchange of (13)CO(2) with the bicarbonate in the pulmonary tissues and blood. The pH of the pulmonary tissues (V(tis)) was determined by a method based on the premise that the CO(2) space in the pulmonary tissues blocked by the inhibitor represented total bicarbonate content. At an alveolar P(CO2) of 40 mm Hg pH of V(tis) equalled 6.97 +/- 0.09.

The Transport of Water in Erythrocytes

Publisher Summary This chapter focuses on the considerations of water movements of red blood cells, particularly to the explanation of existing experimental results. Investigations of the water permeability of the red blood cell not only have intrinsic value but have made substantial contributions to the understanding of the function and even the structure of the plasma membrane. These cells are also convenient biological material, easily available, and can be maintained in essentially in vivo conditions in vitro. Red blood cells have a small volume in relation to their water permeability, however, so that water exchanges are complete in several seconds at physiological temperatures. Therefore, special rapid-reaction apparatus is required to follow the volume changes of the cells. Although there are errors in the experimental techniques used to measure rapid exchanges across the red blood cell membrane, even with proper rapid-reaction techniques, the influence of such phenomena as unstirred layers outside or inside the membrane should not be great enough to influence the general conclusions. The unidirectional diffusion flux across the thin red blood cell membrane is so much greater than any net osmotic volume flux, yet produced so as to render experimentally undetectable at present any possible drag of volume flux on diffusing molecules. The overall results indicate that there is an exchange of momentum between water and water-soluble solutes within the membrane. At the same time, there is no unambiguous evidence for bulk flow, hydrodynamic flow, or the necessary presence of continuous water-filled channels in the membrane.