Gerolf Gros

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane

We report here the application of a previously described method to directly determine the CO2 permeability (PCO2) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1‐expressing Xenopus laevis oocytes. This method measures the exchange of 18O between CO2, HCO3–, and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pHS) transients caused by the dominant effect of entry of CO2 vs. HCO3– into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces PCO2 from the normal value of 0.15 ± 0.08 (SD; n85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases PCO2 2‐fold and doubles the alkaline pHS gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces PCO2 of RBCs solely by action on AQP1 as it has no effect in AQP1‐deficient RBCs. 3) PCO2 determinations of RBCs and pHS measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS‐sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high PCO2 of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total PCO2.—Endeward, V., Musa‐Aziz, R., Cooper, G. J., Chen, L., Pelletier, M. F., Virkki, L. V., Supuran, C. T., King, L. S., Boron, W. F., Gros, G. Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 20, 1974–1981 (2006)

RhAG protein of the Rhesus complex is a CO2 channel in the human red cell membrane

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 NH4Cl 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 NH4Cl/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.— Endeward, V., Cartron, J.‐P., Ripoche, P., and Gros, G. RhAG protein of the Rhesus complex is a CO2 channel in the human red cell membrane. FASEB J. 22, 64–73 (2008)

Protein Diffusion in Living Skeletal Muscle Fibers: Dependence on Protein Size, Fiber Type, and Contraction

Sarcoplasmic protein diffusion was studied under different conditions, using microinjection in combination with microspectrophotometry. Six globular proteins with molecular masses between 12 and 3700 kDa, with diameters from 3 to 30 nm, were used for the experiments. Proteins were injected into single, intact skeletal muscle fibers taken from either soleus or extensor digitorum longus (edl) muscle of adult rats. No correlation was found between sarcomere spacing and the sarcoplasmic diffusion coefficient (D) for all proteins studied. D of the smaller proteins cytochrome c (diameter 3.1 nm), myoglobin (diameter 3.5 nm), and hemoglobin (diameter 5.5 nm) amounted to only approximately 1/10 of their value in water and was not increased by auxotonic fiber contractions. D for cytochrome c and myoglobin was significantly higher in fibers from edl (mainly type II fibers) compared to fibers from soleus (mainly type I fibers). Measurements of D for myoglobin at 37 degrees C in addition to 22 degrees C led to a Q(10) of 1.46 for this temperature range. For the larger proteins catalase (diameter 10.5 nm) and ferritin (diameter 12.2 nm), a decrease in D to approximately 1/20 and approximately 1/50 of that in water was observed, whereas no diffusive flux at all of earthworm hemoglobin (diameter 30 nm) along the fiber axis could be detected. We conclude that 1) sarcoplasmic protein diffusion is strongly impaired by the presence of the myofilamental lattice, which also gives rise to differences in diffusivity between different fiber types; 2) contractions do not cause significant convection in sarcoplasm and do not lead to increased diffusional transport; and 3) in addition to the steric hindrance that slows down the diffusion of smaller proteins, diffusion of large proteins is further hindered when their dimensions approach the interfilament distances. This molecular sieve property progressively reduces intracellular diffusion of proteins when the molecular diameter increases to more than approximately 10 nm.

Mucus as a diffusion barrier to oxygen: Possible role in O2 uptake at low pH in carp (Cyprinus carpio) gills

Abstract 1. 1. The standard V o 2 of carp ( Cyprinus carpio ) is reduced at low pH, concurrently with the formation of mucus on the gills. 2. 2. The rate of diffusion of oxygen through mucus produced by carp in response to low pH was measured in a diffusion chamber and found to be 2.60 × 10 −5 cm 2 /min per atm. The diffusion constant for water measured with the same apparatus was 3.60 × 10 −5 cm 2 /min per atm. 3. 3. The inhibition of the diffusion of oxygen into the gill bloodstream, and the inhibition of the flow of respiratory water between the secondary lamellae, are calculated for a gill with assumed dimensions. At thicknesses of mucus on the gills up to 5 μm the effects are similar, but at greater thicknesses the effect of slowing the water flow predominates. We conclude that mucus on the gills contributes to the hypoxia observed in fish subjected to high acidities.

Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture.

The addition of cyclosporin A (500 ng ml−1) ‐ an inhibitor of the Ca2+‐calmodulin‐regulated serine/threonine phosphatase calcineurin ‐ to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 × 10−7m) was added to the medium, a partial fast‐to‐slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast‐to‐slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin‐regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin‐dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore‐ and electrostimulation‐induced fast‐to‐slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.

Myoglobin's old and new clothes: from molecular structure to function in living cells

SUMMARY Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers have surmounted technical challenges to measure Mb diffusion in the living cell. Their observations have stimulated a discussion about the relative contribution made by Mb-facilitated diffusion to the total oxygen flux. The calculation of the relative contribution, however, depends upon assumptions, the cell model and cell architecture, cell bioenergetics, oxygen supply and demand. The analysis suggests that important differences can be observed whether steady-state or transient conditions are considered. This article reviews the current evidence underlying the evaluation of the biophysical parameters of myoglobin-facilitated oxygen diffusion in cells, specifically the intracellular concentration of myoglobin, the intracellular diffusion coefficient of myoglobin and the intracellular myoglobin oxygen saturation. The review considers the role of myoglobin in oxygen transport in vertebrate heart and skeletal muscle, in the diving seal during apnea as well as the role of the analogous leghemoglobin of plants. The possible role of myoglobin in intracellular fatty acid transport is addressed. Finally, the recent measurements of myoglobin diffusion inside muscle cells are discussed in terms of their implications for cytoarchitecture and microviscosity in these cells and the identification of intracellular impediments to the diffusion of proteins inside cells. The recent experimental data then help to refine our understanding of Mb function and establish a basis for future investigation.

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres

1 In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3− buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+‐lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion‐selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2 Muscle fibres were exposed to 20 mm sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (ΔpHs, ΔpHi) of pH changes were quantified. From dpHi/dt, ΔpHi and the total buffer factor (BFtot) the lactate fluxes (mm min−1) and intracellular lactate concentrations ([lactate]i) were estimated. 3 BFtot was obtained as the sum of the non‐HCO3− buffer factor (BFnon‐HCO3) and the HCO3− buffer factor (BFHCO3). BFnon‐HCO3 was 35 ± 4 mmΔpH−1 for the EDL (n = 14) and 86 ± 16 mmΔpH−1 for the soleus (n = 14). 4 In soleus, 10 mm cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mm DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5 BZ (0.01 mm) and AZ (0.1 mm), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and ΔpHs by about 40 %‐150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %‐50 % and reduced [lactate]i by about 40 %. The membrane‐permeable CA inhibitors CZ (0.5 mm) and EZ (0.1 mm), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6 In soleus, 10 mm cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mm BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7 BZ (0.01 mm) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8 We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BFnon‐HCO3. The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span.

ABSTRACT Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

The diffusion of carbon dioxide in erythrocytes and hemoglobin solutions

SummaryThe CO2 diffusion constant (Kroghs diffusion constant) has been estimated from the CO2 flux across layers with defined thickness under steady state conditions.At 22°C and in hemoglobin solutions with a concentration of 33 g% the diffusion constant for CO2 was found to be 3.3×10−4 cm2 min−1 atm−1. This value is about 40% of the diffusion constant for CO2 in water. The relationship between the diffusion constant and the hemoglobin concentration was approximately linear in a concentration range of 10–40 g%. The temperature coefficient of the diffusion constant was −0.5%/°C both in water and hemoglobin solutions. At 38°C and in a hemoglobin solution with a concentration of 33 g%, the diffusion constant for CO2 was therefore 3.0×10−4 cm2 min−1 atm−1, the diffusion coefficient 11×10−6 cm2 s−1.A general theory for the diffusion of CO2 in hemoglobin solutions has been derived. According to this theory the diminution of the CO2 diffusion in hemoglobin solutions in comparison to water can be explained quantitatively by a reduction of the water space by the hemoglobin molecules.The diffusion constant for CO2 in layers of erythrocytes was insignificantly (0–3%) smaller than in hemoglobin solutions with the same hemoglobin concentration. It is concluded that the erythrocyte membrane does not offer a considerable resistance for the CO2 diffusion.