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The fundamental body functions that determine maximal O2 uptake (VO2max) have not been studied in Aqp5-/- mice (aquaporin 5, AQP5). We measured VO2max to globally assess these functions and then investigated why it was found altered in Aqp5-/- mice. VO2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found VO2max reduced in Aqp5-/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5-/- mice. Lung function parameters as well as arterial O2 saturation were normal and identical between Aqp5-/- and WT mice, indicating that AQP5 does not contribute to pulmonary O2 exchange. The cause for the decreased VO2max thus might be found in decreased O2 consumption of an intensely O2-consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5-/- animals. We conclude that lack of AQP5 does not affect pulmonary O2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in VO2max under this condition. [ABSTRACT FROM AUTHOR]

We have studied the CO2 permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs 18 O-labelled CO2. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO3− permeability. For normal rat erythrocytes, we find at 37 °C a CO2 permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10−3 cm/s. We studied whether the rat erythrocyte membrane possesses protein CO2 channels similar to the human red cell membrane by applying the potential CO2 channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO2 permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO2 channels that are responsible for at least 50% of its CO2 permeability. [ABSTRACT FROM AUTHOR]

Oxygen on its transport route from lung to tissue mitochondria has to cross several cell membranes. The permeability value of membranes for O2 (PO2), although of fundamental importance, is controversial. Previous studies by mostly indirect methods diverge between 0.6 and 125 cm/s. Here, we use a most direct approach by observing transmembrane O2 fluxes out of 100 nm liposomes at defined transmembrane O2 gradients in a stopped-flow system. Due to the small size of the liposomes intra- as well as extraliposomal diffusion processes do not affect the overall kinetics of the O2 release process. We find, for cholesterol-free liposomes, the unexpectedly low PO2 value of 0.03 cm/s at 35 °C. This PO2 would present a serious obstacle to O2 entering or leaving the erythrocyte. Cholesterol turns out to be a novel major modifier of PO2, able to increase PO2 by an order of magnitude. With a membrane cholesterol of 45 mol% as it occurs in erythrocytes, PO2 rises to 0.2 cm/s at 35 °C. This PO2 is just sufficient to ensure complete O2 loading during passage of erythrocytes through the lung's capillary bed under the conditions of rest as well as maximal exercise. [ABSTRACT FROM AUTHOR]

Background/Aims: It has been described that cells in culture with very low oxidative metabolism possess a low CO2 membrane permeability, PCO2, of ∼ 0.01 cm/s. On the other hand, cardiomyocytes and mitochondria with extremely high rates of O2 consumption exhibit very high CO2 membrane permeabilities of 0.1 and 0.3 cm/s, repectively. To ascertain that this represents a systematic relationship, we determine here PCO2 of hepatocytes, which exhibit an intermediate rate of O2 consumption. Methods: We isolated intact hepatocytes with vitalities of ∼ 70% from rat liver and measured their CO2 permeability by the previously published mass spectrometric 18O exchange technique. Results: We find a PCO2 of hepatocytes of 0.03 cm/s in the presence of FC5-208A and verapamil. FC5-208A was necessary to inhibt extracellular carbonic anhydrase, and verapamil was necessary to inhibit intracellular uptake of FC5-208A by the organic cation transporter OCT1 of hepatocytes. Conclusion: Rat hepatocytes with their intermediate rate of oxygen consumption also possess an intermediate CO2 permeability. From pairs of data for five types of cells/organelles, we find an excellent positive linear correlation between PCO2 and metabolic rate, suggesting an adaptation of PCO2 to the rate of O2 consumption. [ABSTRACT FROM AUTHOR]

We summarize here, mainly for mammalian systems, the present knowledge of (a) the membrane CO2 permeabilities in various tissues; (b) the physiological significance of the value of the CO2 permeability; (c) the mechanisms by which membrane CO2 permeability is modulated; (d) the role of the intracellular diffusivity of CO2 for the quantitative significance of cell membrane CO2 permeability; (e) the available evidence for the existence of CO2 channels in mammalian and artificial systems, with a brief view on CO2 channels in fishes and plants; and, (f) the possible significance of CO2 channels in mammalian systems. [ABSTRACT FROM AUTHOR]

Background/Aims: Across the mitochondrial membrane an exceptionally intense exchange of O2 and CO2 occurs. We have asked, 1) whether the CO2 permeability, PM,CO2, of this membrane is also exceptionally high, and 2) whether the mitochondrial membrane is sufficiently permeable to HCO3- to make passage of this ion an alternative pathway for exit of metabolically produced CO2. Methods: The two permeabilities were measured using the previously published mass spectrometric 18O exchange technique to study suspensions of mitochondria freshly isolated from rat livers. The mitochondria were functionally and morphologically in excellent condition. Results: The intramitochondrial CA activity was exclusively localized in the matrix. PM,CO2 of the inner mitochondrial membrane was 0.33 (SD ± 0.03) cm/s, which is the highest value reported for any biological membrane, even two times higher than PM,CO2 of the red cell membrane. PM,HCO3- was 2·10-6 (SD ± 2·10-6) cm/s and thus extremely low, almost 3 orders of magnitude lower than PM,HCO3- of the red cell membrane. Conclusion: The inner mitochondrial membrane is almost impermeable to HCO3- but extremely permeable to CO2. Since gas channels are absent, this membrane constitutes a unique example of a membrane of very high gas permeability due to its extremely low content of cholesterol. [ABSTRACT FROM AUTHOR]

Here we ask the following: 1) what is the CO2 permeability (PCO2) of unilamellar liposomes composed of L-α-phosphatidylcholine (PC)/L-α-phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane PCO2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric 18O-exchange measurements (Chol reduces PCO2 100-fold, AQP1 increases PCO2 10-fold) vs. from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a PCO2 = 0.01cm/s, and Chol-free vesicles exhibit ~1 cm/s, and 2) the PCO2 of 0.01 cm/s is increased ≥10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low PCO2 that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1. [ABSTRACT FROM AUTHOR]

Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases Here, we study the gas permeability of membranes for the case of CO2, using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO2 permeability (PCO2) of ~0.01 cm/s, which is 2 orders of magnitude lower than the PCO2 of pure planar phospholipid bilayers (~1 cm/s). Phosphollpid vesicles enriched with similar amounts of cholesterol also exhibit PCO2 ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane PEG2. This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in PEG2, respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in PCO2, indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability. [ABSTRACT FROM AUTHOR]

We have developed an experimental approach that allows us to quantify unstirred layers around cells suspended in stirred solutions. This technique is applicable to all types of transport measurements and was applied here to the 18O technique used to measure CO2 permeability of red cells . We measure in well-stirred red cell (RBC) suspensions of various viscosities adjusted by adding different amounts of 60 kDa dextran. Plotting vs. viscosity ν gives a linear relation, which can be extrapolated to ν= 0. Theoretical hydrodynamics predicts that extracellular unstirred layers vanish at zero viscosity when stirring is maintained, and thus this extrapolation gives us an estimate of the free from extracellular unstirred layer artifacts. The extrapolated value is found to be 0.16 cm s−1 instead of the experimental value in saline of 0.12 cm s−1 (+30%). This effect corresponds to an unstirred layer thickness of 0.5 μm. In addition, we present a theoretical approach modelling the actual geometrical and physico-chemical conditions of 18O exchange in our experiments. It confirms the role of an extracellular unstirred layer in the determination of . Also, it allows us to quantify the contribution of the so-called intracellular unstirred layer, which results from the fact that in these transport measurements – as in all such measurements in general – the intracellular space is not stirred. The apparent thickness of this intracellular unstirred layer is about 1/4–1/3 of the maximal intracellular diffusion distance, and correction for it results in a true of the RBC membrane of 0.20 cm s−1. Thus, the order of magnitude of this is unaltered compared to our previous reports. Discussion of the available evidence in the light of these results confirms that CO2 channels exist in red cell and other membranes, and that of red cell membranes in the absence of these channels is quite low. [ABSTRACT FROM AUTHOR]

We have determined CO2 permeabilities, PCO2, of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While PCO2 of normal red cells is ~0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rhnull) a significantly reduced PCO2 of 0.07 cm/s ±0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1null), suggesting that each of the Rh and AQP-1 proteins is responsible for ~1/2 of the normal CO2 permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO2 permeability. The CO2 pathway constituted by Rh proteins was inhibitable at pHe= 7.4 by NH4CI with an I50 of ~10 mM corresponding to an I50 for NH3 of ~0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH4CI/NH3. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal PCO2, while in contrast another AE1 inhibitor, DiBAC, does not inhibit PCO2, although it markedly reduces PHCO3-. We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO2 in addition to NH3. [ABSTRACT FROM AUTHOR]

The relationships between extracellular potassium elevation and EMG variables in relation to muscle fatigue were investigated during handgrip exercise in humans. Acid–base state, lactate, potassium ([K +]v) and sodium in venous plasma, as well as variables of surface voluntary and evoked (M-wave) EMG were determined during repeated dynamic (DE) and static (SE) exercise (1 min exercise, 4 min rest). The different rises of [K +]v were induced by randomly varied workloads. After 15 min of warming up, the M-wave area increased to 124.9 ± 19.6% ( P < 0.001) in comparison with the control value. Simultaneously, the [K +]v decreased from 4.1 ± 0.3 to 3.6 ± 0.3 mmol l−1 ( P < 0.01). During both SE and DE, there were marked intensity-dependent signs of fatigue. The [K +]v correlated with changes of the integrated EMG ( r= 0.87, P < 0.001 for both DE and SE). Changes in the M-wave area during the exercise bouts correlated inversely with the [K +]v ( r=−0.73, P < 0.001). The M-wave area did not decrease below the control value at any intensity. The median frequency of the EMG decreased during exercise, depending on the exercise intensity ( r=−0.73 for SE, r=−0.47 for DE, P < 0.001) with a maximal decrease to about 80% after SE with the maximal workload. The muscle action potential propagation velocity changed in the range of about ±2%. For the first time, a negative relationship between venous potassium and M-wave area was shown during voluntary exercise. However, there was no evidence that the decrease in muscle performance was mainly caused by a decrease in sarcolemmal excitability resulting from a high extracellular [K +]. [ABSTRACT FROM AUTHOR]

We show here that luminal mucus from the colon and the stomach of guinea pigs, mice and humans exhibits substantial carbonic anhydrase (CA) activity, by which the velocity of the CO2 hydration reaction is accelerated 1000–2000-fold, approximately 1/10 of what is found in the red cell. Although this CA shares several properties with CA II, studies with CA II-deficient mice show that gastrointestinal mucus CA is not affected in these animals and thus does not appear to be CA II. We speculate that the mucus layer covering the luminal surface of gastrointestinal epithelium can, due to the presence of CA, maintain a normal tissue pCO2 in the epithelium, even when the pCO2 values in the lumen are much higher, as is known for stomach and colon. To test this hypothesis, we have developed a mathematical model which describes (a) diffusion of CO2 and HCO3− across the mucus layer and (b) H+ transport mediated by continuous secretion of mucus, which due to its high H+ buffer capacity transports H+ by convection towards the lumen. The model predicts that continuous transport of the reaction products of CO2 towards the lumen, by diffusion and convection, protects the epithelium against high CO2 partial pressures in the lumen. [Copyright &y& Elsevier]

The fundamental body functions that determine maximal O 2 uptake (V̇o 2max ) have not been studied in Aqp5 -/- mice (aquaporin 5, AQP5). We measured V̇o 2max to globally assess these functions and then investigated why it was found altered in Aqp5 -/- mice. V̇o 2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o 2max reduced in Aqp5 -/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5 -/- mice. Lung function parameters as well as arterial O 2 saturation were normal and identical between Aqp5 -/- and WT mice, indicating that AQP5 does not contribute to pulmonary O 2 exchange. The cause for the decreased V̇o 2max thus might be found in decreased O 2 consumption of an intensely O 2 -consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5 -/- animals. We conclude that lack of AQP5 does not affect pulmonary O 2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o 2max under this condition.

We have studied the CO 2 permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs 18  O-labelled CO 2 . The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO 3 - permeability. For normal rat erythrocytes, we find at 37 °C a CO 2 permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10 -3  cm/s. We studied whether the rat erythrocyte membrane possesses protein CO 2 channels similar to the human red cell membrane by applying the potential CO 2 channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO 2 permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO 2 channels that are responsible for at least 50% of its CO 2 permeability.

We present here a compilation of membrane CO 2 permeabilities (Pco 2 ) for various cell types from the literature. Pco 2 values vary over more than two orders of magnitude. Relating Pco 2 to the cholesterol content of the membranes shows that, with the exception of red blood cells, it is essentially membrane cholesterol that determines the value of Pco 2 . Thus, the observed strong modulation of Pco 2 in the majority of membranes is caused by cholesterol rather than gas channels.

Here we ask the following: 1) what is the CO2 permeability (Pco2) of unilamellar liposomes composed of l-α-phosphatidylcholine (PC)/l-α-phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric (18)O-exchange measurements (Chol reduces Pco2 100-fold, AQP1 increases Pco2 10-fold) vs. from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a Pco2 = 0.01cm/s, and Chol-free vesicles exhibit ∼1 cm/s, and 2) the Pco2 of 0.01 cm/s is increased ≥ 10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco2 that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1., (© FASEB.)

Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO(2), using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.