Oxidative slow skeletal muscle contains carbonic anhydrase III in high concentration, but its primary function remains unknown. To determine whether its lack handicaps energy metabolism and/or acid elimination, we measured the intracellular pH and energy phosphates by (31)p magnetic resonance spectroscopy in hind limb muscles of wild-type and CA III knockout mice during and after ischemia and intense exercise (electrical stimulation). Thirty minutes of ischemia caused phosphocreatine (PCr) to fall and P-i to rise while pH and ATP remained constant in both strains of mice. PCr and P-i kinetics during ischemia and recovery were not significantly different between the two genotypes. From this we conclude that under neutral pH conditions resting muscle anaerobic metabolism, the rate of the creatine kinase reaction, intracellular buffering of protons, and phosphorylation of creatine by mitochondrial oxygen metabolism are not influenced by the lack of CA III. Two minutes of intense stimulation of the mouse gastrocnemius caused PCr, ATP, and pH to fall and ADP and P-i to rise, and these changes, with the exception of ATP, were all significantly larger in the CA III knockouts. The rate of return of pH and ADP to control values was the same in wild-type and mutant mice, but in the mutants PCr and Pi recovery were delayed in the first minute after stimulation. Because the tension decrease during fatigue is known to be the same in the two genotypes, we conclude that a lack of CA III impairs mitochondrial ATP synthesis.

To investigate the role of carbonic anhydrase in the recovery of skeletal muscle from anoxia, pH and cell phosphates were measured by (31)P-nuclear magnetic resonance in superfused newborn rabbit myotubes and cultured mouse soleus cells (H-2K(b)-ts a58) after approximately 2-3.5 h without superfusion. In control studies, pH and phosphocreatine fell and P(i) rose during anoxia and recovered within <10 min after reperfusion began. A carbonic anhydrase inhibitor, acetazolamide, and dimethylamiloride, an inhibitor of the Na(+)/H(+) antiporter NHE1, delayed the recoveries of pH, phosphocreatine, and P(i) for >10 min, but the rate of recovery, once initiated, was unchanged. In the presence of the inhibitors, after reperfusion started, the pH did not rise immediately, despite a large inwardly directed HCO(3)(-) gradient, suggesting that HCO(3)(-) movement was unimportant in acid elimination. Lactate, measured by its methyl protons, rose during anoxia and did not fall after 1 h of reperfusion and could not have eliminated protons by cotransport. We conclude that NHE1 is the major exporter of protons by skeletal muscle in recovery from a period of anoxia and that it is essential for functioning carbonic anhydrase to be attached to NHE1 to activate it. The mechanism of late recovery of pH could be the mobilization of another proton transporter or removal of the inhibition of the Na(+)/H(+) antiporter. Inhibition of carbonic anhydrase in skeletal muscle retards acid removal and modifies muscle metabolism significantly after anoxia.

H+ is maintained constant in the internal environment at a given body temperature independent of external environment according to Bernard’s principle of “milieu interieur”. But CO2 relates to ventilation and H+ to kidney. Hence, the title of the chapter. In order to do this, sensors for H+ in the internal environment are needed. The sensor–receptor is CO2/H+ sensing. The sensor–receptor is coupled to integrate and to maintain the body’s chemical environment at equilibrium. This chapter dwells on this theme of constancy of H+ of the blood and of the other internal environments. [H+] is regulated jointly by respiratory and renal systems. The respiratory response to [H+] originates from the activities of two groups of chemoreceptors in two separate body fluid compartments: (A) carotid and aortic bodies which sense arterial PO2 and H+; and (B) the medullary H+ receptors on the ventrolateral medulla of the central nervous system (CNS). The arterial chemoreceptors function to maintain arterial PO2 and H+ constant, and medullary H+ receptors to maintain H+ of the brain fluid constant. Any acute change of H+ in these compartments is taken care of almost instantly by pulmonary ventilation, and slowly by the kidney. This general theme is considered in Section 1.

The bicarbonate permeability of the plasma membrane of intact hagfish (Myxine glutinosa) red blood cells and the intracellular carbonic anhydrase activity of these cells were determined by applying the (18)O exchange reaction using a special mass spectrometric technique. When the macromolecular carbonic anhydrase inhibitor Prontosil-Dextran was used to suppress any extracellular carbonic anhydrase activity, the mean intracellular acceleration of the CO(2) hydration/HCO(3)(-) dehydration reaction over the uncatalyzed reaction (referred to as intracellular carbonic anhydrase activity A(i)) was 21 320+/-3000 at 10 degrees C (mean +/- s.d., N=9). The mean bicarbonate permeability of the red blood cell membrane (P(HCO3)-) was indistinguishable from zero. It can be concluded that CO(2) transport within hagfish blood does not follow the classical scheme of CO(2) transport in vertebrate blood. It is suggested that the combination of considerable intraerythrocytic carbonic anhydrase activity and low P(HCO3)- may serve to enhance O(2) delivery to the tissue in the exceptionally hypoxia-tolerant hagfish.

The search for the catalyst that liberates the body's CO2 from HCO– 3 from blood during its 1-s transit through the lung capillaries ended in an almost dead heat in 1932–33 between Norman U. Meldrum and Francis J. W. Roughton in Cambridge and William C. Stadie and Helen O'Brien at the University of Pennsylvania. Horace Davenport has written several pungent articles about the discoverers of carbonic anhydrase (Davenport, 1980, 1984) from his personal experiences at Oxford and Cambridge a little later. His tutor at Oxford was John Philpott who developed the ur-technique for the measurement of the rate of the reversible reactions of CO2/HCO– 3 as a student laboratory demonstration. Davenport spent a week in Cambridge with Vernon Booth, Roughton's colleague and assistant, in the course of his studies. I worked with Roughton in Philadelphia and in Cambridge in the 1950's on CO2 rapid reactions in blood, knew Stadie as a medical student and junior faculty member at Penn and through my family. I have continued an active interest in carbonic anhydrase (CA) ever since and as a result have gleaned a few facts to add to the story. Assembling the fine details of the search for the enzyme that accelerates CO2 reactions presents a lesson in humility, even seen through a retrospectroscope. Many very able investigators took what now appear to be circuitous routes to the truth and some never got there.

INVITED EDITORIALInvited Editorial on “Red cell distribution and the recruitment of pulmonary diffusing capacity”Robert E. ForsterRobert E. Forster Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085Published Online:01 May 1999https://doi.org/10.1152/jappl.1999.86.5.1458MoreSectionsPDF (69 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat the mammalian lung is an amazing creation in which a blood flow (in l/min) is conducted from vessels (in cm diameter at first) through ever smaller and more numerous branches until they are ∼7 μm in diameter, spreading a gossamer network enclosing air sacs 100 μm across, ventilated by airways that arborize down to 1/100th of the tracheal diameter, and carry gases that exchange with the red blood cells in a fraction of a second. The wonder is that blood and gas flows to all these alveolar capillary beds are uniform as a first approximation, which requires feedback control down to the capillary level. Nonuniformity is the greatest enemy of lung gas exchange and of the pulmonary diffusing capacity (Dl). In 1957, I discussed qualitatively the effects of all the types of nonuniformity that affected the measured value of Dl for CO (Dl CO) that I could conjure up (4), but I never thought of nonuniformity of red cell distribution along a single capillary, as shown by Hsia et al. (11).The rate of uptake of O2 by red blood cells in a single alveolar capillary cannot be measured by techniques available today, but extremely useful conclusions can be obtained by modeling, and a number of such studies have been reported over the past two decades. In 1977, Hellums (8) concluded from a cylindrical cell model that one-half the resistance to O2 transport in peripheral capillaries was in the capillaries themselves, but it was not until Federspiel’s paper in 1989 (3) that a model of O2 transport in red cell spheres within a cylindrical capillary was studied. Federspiel concluded that the discontinuous nature of red blood cells lowered membrane diffusing capacity. Frank et al. (6) extended this work and modeled O2 uptake of an individual red blood cell in a single pulmonary capillary assuming a parachute shape, probably the commonest shape of cells in the flowing stream, and demonstrated that not only was the flux of O2 through the pulmonary membrane to the surface of the cell closest to the endothelium important but also the gas was diffusing through the plasma between the cells, turning axially to deliver O2 to additional cell surface. This is a much longer path but more rapid than that through red cell cytoplasm because there is nothing to bind O2 and impede its diffusion. Thus a single red blood cell sufficiently distant from its nearest neighbors that this plasma diffusion path is not encroached on takes up O2 the most rapidly. As the number of cells in the capillary increases, the distance between cells decreases, and the path through the plasma becomes restricted, so that the rate of O2 uptake per cell goes down; but, of course, there are many more cells, so the total uptake in the capillary rises but less than proportionally. A cell shape that increases the mean diffusion path from the endothelium to all of the total red cell surface decreases the effective membrane diffusing capacity (3, 6).In their article in this issue of the Journal, Hsia et al. (11) report two-dimensional, finite-difference computations of CO diffusion across the pulmonary membrane to the surface of a fixed number of cylindrical red blood cells, distributed nonuniformly along the length of the capillary. The germinal idea is that when cells are unevenly distributed along the capillary the diffusion path through the plasma between those bunched together is restricted; CO uptake by these cells is reduced; and, of course, there is no CO uptake in the capillary space they vacate. Therefore, the recast uptake by the whole capillary is reduced. The greatest CO uptake occurs when the cells are evenly spaced. This effect of nonuniform spacing can be large and becomes worse as the capillary hematocrit decreases. A more uniform distribution of red blood cells in the capillary may explain part of the increase in Dl CO with exercise.Hsia et al. (10) chose values obtained by Holland (9) for the rate of CO uptake by red blood cells in milliliters per milliliter per millimeter Hg Pco at differentPO2 to describe CO transport inside the surface of the cell. These experimental rates are less than those obtained in dog red blood cells by using a continuous-flow, rapid-mixing apparatus (2), presumably because of stagnant layers in the stop-flow apparatus (1, 15). However, this does not alter the interesting new conclusion that nonuniforitiy of red blood cells in the alveolar capillary decreases Dl CO.Dl COmeasured in lungs perfused with hemolysate is reported to be greater than when perfused with a cell suspension (7); from this it has been concluded that there is a significant diffusion resistance outside the red blood cells in the plasma (stagnant layer). This is not necessarily a helpful concept. First, if the red bood cells in a pulmonary capillary are replaced by an Hb solution and all other conditions remain unchanged, the rate of formation of HbCO in the capillaries will increase by the physical laws of simultaneous chemical reaction and diffusion. This is because, while the volume in which HbCO is formed increases, the rate of HbO2formation per unit volume does not decrease proportionally. As an example, the red blood cell was modeled as a layer of Hb by Nicholson and Roughton (12), and an analytic solution for the initial rate of HbCO was obtainedd[HbCO]/dt=[CO]×b−1(dl′[Hb])1/2 ×factor of∼1Equation 1where d[HbCO]/dt is the rate of change of HbCO concentration per cm3 in the layer (the red cell cytoplasm), [CO] is the concentration of CO at the surface of the layer, b is the half thickness of the layer in cm, d is the diffusion coefficient of O2 in the layer,l′ is the bimolecular reaction velocity constant of CO reacting with Hb, and [Hb] is the concentration of unliganded Hb in the layer (all concentrations are in mol/cm3). Because red blood cells approximately fill the capillary lumen,b should remain about the same in hemolysate, but [Hb] would decrease. Federspiel (3) used a relationship analogous to that above to obtain the O2 uptake rate in cells. With hemolysate in the capillaries, the volume in which HbCO is being formed is greater by the factor 1/hematocrit, [Hb] would decrease by its reciprocal, the hematocrit, that according toEq. 1 would decrease d[HbCO]/dt, the rate per cm3 by (hematocrit)1/2. Thus the rate of HbCO formation in the whole capillary would increase by (hematocrit)−1/2, which is >1. Although considering the red blood cell and the capillary as semi-infinite layers is a crude approximation of their shape, this same model has been used in several computations (6, 11), and the principle that the rate of HbCO formed in a capillary perfused with hemolysate is not decreased in proportion to the dilution of intracellular Hb holds regardless of shape. This principle can be seen in a three-dimensional finite-difference calculation of O2 entrance into a discoidal red blood cell. (5).Second, and this is an intuitive observation, hemolysate fills the volume between red blood cells, exposing Hb solution to a greater endothelial surface and reducing the mean diffusion path from endothelium to Hb molecule, which increases the total capillary CO uptake rate. If one chooses to consider the diffusion path through plasma between cells as a stagnant layer, this path certainly disappears when the perfusate is hemolysate, but this same path also facilitates gas exchange of individual cells.This new form of nonuniformity in the alveolar capillaries may be important in many clinical conditions, such as blood dyscrasies (e.g., anemia) and alterations in red cell adhesiveness as well as in abnormalities of pulmonary blood flow.REFERENCES1 Coin J. T., Olson J. S.The rate of oxygen uptake by human red blood cells.J. Biol. Chem.254197911781190Crossref | PubMed | ISI | Google Scholar2 Crapo R. O., Bitterman N., Berlin S. L., Forster R. E.Rate of CO uptake by canine erythrocytes as a function of PO2.J. Appl. Physiol.67198922652268Link | ISI | Google Scholar3 Federspiel W. J.Pulmonary diffusing capacity: implications of two-phase blood flow in capillaries.Respir. Physiol.771989119134Crossref | Google Scholar4 Forster R. E.Exchange of gases between alveolar air and pulmonary capillary blood: pulmonary diffusing capacity.Physiol. Rev.111957290302Google Scholar5 Forster R. E.Rate of gas uptake by red cells.Handbook of Physiology. Respiration.I1964Am. Physiol. Soc.Wahington, DC, chapt. 33, p. 839–872.Google Scholar6 Frank A. O., Charles-Chuong C. J., Johnson R. L.A finite-element model of oxygen diffusion in the pulmonary capillaries.J. Appl. Physiol.82199720362044Link | ISI | Google Scholar7 Geiser W. J., Betticher D. C.Gas transfer in isolated lungs perfuse with red cell suspension or hemoglobin solution.Respir. Physiol.7719893140Crossref | Google Scholar8 Hellums J. D.The resistance to oxygen transport in the capillaries relative to that in the surrounding tissue.Microvasc. Res.131977131136Crossref | PubMed | ISI | Google Scholar9 Holland R. A. B.Rate at which CO replaces O2 from O2Hb in red cells of different species.Respir. Physiol.719694363Crossref | Google Scholar10 Hsia C. C. W., Chuong C. J. C., Johnson R. L.Critique of conceptual basis of diffusing capacity estimates: a finite element analysis.J. Appl. Physiol.79199510391047Link | ISI | Google Scholar11 Hsia C. C. W., Johnson R. L., Shah D.Red cell distribution and the recruitment of pulmonary diffusing capacity.J. Appl. Physiol.86199914601467Link | ISI | Google Scholar12 Nicholson P., Roughton F. J. W.Influence of diffusion and chemical reaction velocity on the rate of exchange of CO and O2 between red blood corpuscle and surrounding fluid.Proc. R. Soc. Lond. B Biol. Sci.1381951241264Crossref | ISI | Google Scholar13 Sarelius I. H.Invited editorial on “Effect of RBC shape and deformability on pulmonary O2 diffusing capacity and resistance to flow in rabbit lungs”J. Appl. Physiol.781995778783Link | Google Scholar14 Wang C.-H., Popel A. S.Determinants of red blood cell shape on oxygen transport in capillaries.Math. Biosci.116199389110Crossref | PubMed | ISI | Google Scholar15 Yamaguchi K., Nguyen-Phu D., Scheid P., Piiper J.Kinetics of O2 uptake and release by human erythrocytes studied by a stopped-flow technique.J. Appl. Physiol.58198512151224Link | ISI | Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated ArticlesThis Month in the Journal 01 May 1999Journal of Applied PhysiologyCited ByTheoretical analysis of the determinants of lung oxygen diffusing capacityJournal of Theoretical Biology, Vol. 351 More from this issue > Volume 86Issue 5May 1999Pages 1458-1459 Copyright & PermissionsCopyright © 1999 the American Physiological Societyhttps://doi.org/10.1152/jappl.1999.86.5.1458PubMed10233104History Published online 1 May 1999 Published in print 1 May 1999 Metrics

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3- and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3- (Pm,HCO-3). To test this technique, we added sufficient 4, 4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) to inhibit all the HCO3-/Cl- transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO-3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3-, we found that DIDS inhibited both Pm,HCO-3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.

We have measured continuously the production of mass 45 CO2(13C16O2) from 13C-labeled pyruvate in a guinea pig liver mitochondrial suspension and simultaneously the O2 consumption at 37 degrees C and pH 7.4. The reactions took place in a closed 3-ml volume, stirred, thermoregulated chamber separated from the ion source of a mass spectrometer by a gas-permeable membrane that permitted recording the mass peaks of any gas dissolved in the reaction mixture with a response time as fast as 3 s. If the pyruvate was labeled on C-2, no 13C16O2 was formed, even after 1 h, indicating that C-2 and C-3 were not metabolized in the citric acid cycle. We found that production of 13C16O2 was five times greater in the presence of 25 mM HCO3- than in its absence. A probable mechanism of this CO2/HCO3- effect is carboxylation of pyruvate to oxaloacetate, which would react with acetyl CoA to form citrate and with NADH to form malate, thus removing two major inhibitors of pyruvate dehydrogenase. We conclude that CO2/HCO3- has a potent and hitherto unappreciated regulatory effect on liver pyruvate dehydrogenase.

This study was initiated to obtain data on the rate of carbon monoxide (CO) uptake (theta CO) by hemoglobin in pig erythrocytes to derive, in a later study, the pulmonary capillary blood volume (Qc) in pigs from the Roughton-Forster relationship. Blood from five different female pigs was used. The theta CO, the milliliters of CO taken up by 1 ml of whole blood per minute per Torr CO tension, was determined on each blood sample with a continuous-flow rapid-mixing apparatus and double-beam spectrophotometry at 37 degrees C and pH 7.4 at four or five different PO2 values. Because the individual regression lines of theta CO vs. PO2 were not significantly different, a common regression equation was calculated: 1/theta CO = 0.0084 PO2 + 0.63. The slope of this regression line is significantly steeper than the reported slopes of regression lines for human and dog erythrocytes measured under the same conditions. Our results revealed that calculation of Qc in pigs by using theta CO values for human or dog erythrocytes would result in an underestimation of 51 and 50%, respectively.