A quantitative study of bioenergetics in skeletal muscle lacking carbonic anhydrase III using 31P magnetic resonance spectroscopy.

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 P(i) 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.

Chemiluminescent measurements of nitric oxide pulmonary diffusing capacity and alveolar production in humans.

Measurements of nitric oxide (NO) pulmonary diffusing capacity (DL(NO)) multiplied by alveolar NO partial pressure (PA(NO)) provide values for alveolar NO production (VA(NO)). We evaluated applying a rapidly responding chemiluminescent NO analyzer to measure DL(NO) during a single, constant exhalation (Dex(NO)) or by rebreathing (Drb(NO)). With the use of an initial inspiration of 5-10 parts/million of NO with a correction for the measured NO back pressure, Dex(NO) in nine healthy subjects equaled 125 +/- 29 (SD) ml x min(-1) x mmHg(-1) and Drb(NO) equaled 122 +/- 26 ml x min(-1) x mmHg(-1). These values were 4.7 +/- 0.6 and 4.6 +/- 0.6 times greater, respectively, than the subject's single-breath carbon monoxide diffusing capacity (Dsb(CO)). Coefficients of variation were similar to previously reported breath-holding, single-breath measurements of Dsb(CO). PA(NO) measured in seven of the subjects equaled 1.8 +/- 0.7 mmHg x 10(-6) and resulted in VA(NO) of 0.21 +/- 0.06 microl/min using Dex(NO) and 0.20 +/- 0.6 microl/min with Drb(NO). Dex(NO) remained constant at end-expiratory oxygen tensions varied from 42 to 682 Torr. Decreases in lung volume resulted in falls of Dex(NO) and Drb(NO) similar to the reported effect of volume changes on Dsb(CO). These data show that rapidly responding chemiluminescent NO analyzers provide reproducible measurements of DL(NO) using single exhalations or rebreathing suitable for measuring VA(NO).

Hagfish (Myxine glutinosa) red cell membrane exhibits no bicarbonate permeability as detected by (18)O exchange.

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 effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane.

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.

Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-.

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.

Determination of production of nitric oxide by lower airways of humans--theory.

Exercise and inflammatory lung disorders such as asthma and acute lung injury increase exhaled nitric oxide (NO). This finding is interpreted as a rise in production of NO by the lungs (VNO) but fails to take into account the diffusing capacity for NO (DNO) that carries NO into the pulmonary capillary blood. We have derived equations to measure VNO from the following rates, which determine NO tension in the lungs (PL) at any moment from 1) production (VNO); 2) diffusion, where DNO(PL) = rate of removal by lung capillary blood; and 3) ventilation, where V A(PL)/(PB - 47) = the rate of NO removal by alveolar ventilation (V A) and PB is barometric pressure. During open-circuit breathing when PL is not in equilibrium, d/dt PL[V(L)/ (PB - 47)] (where V(L) is volume of NO in the lower airways) = VNO - DNO(PL) - V A(PL)/(PB - 47). When PL reaches a steady state so that d/dt = 0 and V A is eliminated by rebreathing or breath holding, then PL = VNO/DNO. PL can be interpreted as NO production per unit of DNO. This equation predicts that diseases that diminish DNO but do not alter VNO will increase expired NO levels. These equations permit precise measurements of VNO that can be applied to determining factors controlling NO production by the lungs.

Rate of uptake of CO by hemoglobin in pig erythrocytes as a function of PO2.

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.

Continuous measurement of 13C16O2 production from [13C]pyruvate by intact liver mitochondria: effect of HCO3-.

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.

31P-NMR spectroscopy of isolated perfused rat lung.

We obtained 202.5-MHz 31P-nuclear magnetic resonance (NMR) spectra of isolated perfused rat lungs, degassed and inflated, and of lung extract. The spectra included those of ATP, ADP, phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters, phosphodiesters, and a broad component due to the membrane phospholipids. The line width at one-half peak height for beta-ATP was 1.0 ppm for the degassed lung and 1.2 ppm for the inflated lung. This suggests that the air-water interfaces in inflated lung, which produce proton NMR line broadening, do not act prominently in 31P-NMR spectroscopy. In a degassed lung, when perfusion was stopped for up to 30 min, PCr and ATP peaks decreased progressively with time while Pi and phosphomonoester peaks increased. On return of flow, these changes reversed. The intracellular pH values calculated from the difference in magnetic field between PCr and Pi peaks of inflated and degassed lungs were 7.16 +/- 0.10 (SD; n = 4) and 6.99 +/- 0.10 (n = 4), respectively. The change of intracellular pH caused by 30 min of ischemia was -0.2 pH units. Our findings indicate that air-water interfaces should not broaden lung 31P peaks in vivo.

Carbonic anhydrase in the membrane of the endoplasmic reticulum of male rat liver.

We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. & Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with 18O exchange between CO2/HCO3- and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 mumol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10(-9) M. The Km for CO2 was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.