Intracellular pH of frog sartorius muscle

A weak base, morpholine, has been labelled with ³H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20°C in a solution of the same of ionic strength as frog Ringer was found to be 8.45 ± 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indicator. Intracellular pH was then measured by distribution of morpholine (6.985 ± 0.08), nicotine (6.915 ± 0.03) and the weak acid 5,5′-dimethyl-2,4-oxazolidinedione (7.10 ± 0.05) and with pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H⁺ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Observations on intracellular pH during cleavage of eggs of Xenopus laevis

Direct measurements of intracellular pH was made with recessed-tip pH microelectrodes in fertilized eggs of the frog, Xenopus laevis, from approximately 1 h after fertilization to mid-blastula. The intracellular pH just before first cleavage was 7.65 +/- 0.04 (SD; n = 9). By stage 5 to the middle of stage 6, average intracellular pH was 7.70 +/- 0.06 (SD; n = 16). A statistically significant alkalization of 0.18 +/- 0.03 pH unit (SD; n = 5) was observed beginning in early blastula. A cycle of less than or equal to 0.05 pH unit was occasionally observed during the pre-blastula period, but its significance is unknown. By exposing the early cleavage embryo to saline buffered with sodium propionate, pH 4.7-5.0, it was possible to lower intracellular pH with some degree of control. Apparently, normal cleavage continued to occur when intracellular pH had been forced as much as 0.3 unit below normal. We conclude that this implies no specific involvement of intracellular pH in mitosis and cytokinesis. If intracellular pH was lowered further, cell division ceased at about pH 7.2, and furrow regression began at about pH 7.0. Once furrow regression occurred, subsequent development was usually arrested or abnormal when the embryo was transferred back to normal saline.     

Approximating the isometric force-calcium relation of intact frog muscle using skinned fibers.

In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibrils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested predictions of this calculation using measurements of the potential across the boundary of skinned frog muscle fibers bathed in this solution. The average potential was -3.1 mV, close to that predicted from a simple Donnan equilibrium. The contribution of ATP hydrolysis to a diffusion potential was probably small because addition of 1 mM vanadate to the solution decreased the fiber actomyosin ATPase rate (measured by high-performance liquid chromatography) by at least 73% but had little effect on the measured potential. Using these solutions, we obtained force-pCa curves from mechanically skinned fibers at three different temperatures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temperature. The bath concentration of Ca2+ required for half-maximal activation of isometric force was 1.45 microM (22 degrees C, pH 7.18), 2.58 microM (16 degrees C, pH 7.25), and 3.36 microM (5 degrees C, pH 7.59). The [Ca2+] at the threshold of activation at 16 degrees C was approximately 1 microM, in good agreement with estimates of threshold [Ca2+] in intact frog muscle fibers.     

Chloride activity and its control in skeletal and cardiac muscle

Ion-selective microelectrodes have been used to compare the mechanisms controlling intracellular Cl- activity in skeletal and cardiac muscle. In frog Sartorius skeletal muscle fibres, Cl- levels are low (about 3 mM) and are determined mainly passively. The effect of any Cl- transport system will be quickly short-circuited through the high membrane Cl- conductance. In contrast, the sheep-heart Purkinje fibre, like other cardiac tissues, contains higher than passive levels of intracellular Cl- (20-30 mM). Many Cl- movements occur, not through Cl- channels (the permeability for Cl- is low), but by a Cl- -HCO3- countertransport system. High internal Cl- levels are achieved by an exchange of extracellular Cl- for intracellular HCO3-, which acidifies the fibre by 0.3 pH. Anion exchange in heart differs from that proposed for other excitable cells in that it is not specialized to compensate for an intracellular acidosis. Instead, it can prevent the fibres from becoming too alkaline by promoting a bicarbonate efflux and a chloride influx whenever internal bicarbonate levels rise. Possible reasons for this are briefly discussed.     

The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.     

Calcium mobilization and Na+/H+ antiport activation by endothelin in human skin fibroblasts

Endothelin (ET-1) has been shown to exert vasoconstrictor activity in vivo and mobilize Ca2+ in vascular smooth muscle cells in culture. In this paper we show that the human skin fibroblast exhibits specific receptors to ET-1 and that activation of these receptors results in increased intracellular Ca2+ (Ca2+i) and accelerated Na+/H+ antiport activity. ET-1 raised Ca2+i in a dose-response manner; the peak Ca2+i rise was from basal levels of 112.2 +/- 21.9 to 299.2 +/- 49.7 nM at 300 nM ET-1. This rise was attenuated by removal of extracellular Ca2+i0. Although ET-1 did not alter basal intracellular pH, it enhanced Na+/H+ antiport activity of acidified cells. Fibroblasts demonstrated 156 +/- 18 (mean +/- SE) ET-1 receptors per unit cell and an equilibrium dissociation constant of 203.4 +/- 35.6 pM. Inasmuch as ET-1 plays a role in the metabolism of cells such as the undifferentiated fibroblast, an important action of this peptide may be to act as a growth factor.     

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

The oxidizing power of the glutathione thiyl radical as measured by its electrode potential at physiological pH

The oxidizing power of the thiyl radical (GS*) produced on oxidation of glutathione (GSH) was determined as the mid-point electrode potential (reduction potential) of the one-electron couple E(m)(GS*,H+/GSH) in water, as a function of pH over the physiological range. The method involved measuring the equilibrium constants for electron-transfer equilibria with aniline or phenothiazine redox indicators of known electrode potential. Thiyl and indicator radicals were generated in microseconds by pulse radiolysis, and the position of equilibrium measured by fast kinetic spectrophotometry. The electrode potential E(m)(GS*,H+/GSH) showed the expected decrease by approximately 0.06 V/pH as pH was increased from approximately 6 to 8, reflecting thiol/thiolate dissociation and yielding a value of the reduction potential of GS*=0.92+/-0.03 V at pH 7.4. An apparently almost invariant potential between pH approximately 3 and 6, with potentials significantly lower than expected, is ascribed at least in part to errors arising from radical decay during the approach to the redox equilibrium and slow electron transfer of thiol compared to thiolate.     

Simultaneous pressure and 19F NMR pH measurements of smooth muscle cells of intact hog carotid arteries at rest and during contractions with norepinephrine

Using 19F NMR we have measured the intracellular pH of the vascular smooth muscle cells of hog carotid arteries at rest and during contractions induced with norepinephrine. Experiments were performed on single, intact arteries closed at both ends, superfused from the lumen and loaded with the 19F NMR pH indicator alpha-difluoromethylalanine. At rest, luminal pressure was maintained at 100 +/- 2 mm Hg and intracellular pH was 7.12 +/- 0.04. Contractions elicited with 10(-5) M norepinephrine were associated with a pressure increase of 18 +/- 6 mm Hg and a decrease in pH of 0.04 +/- 0.02 units.     

Increase of intracellular pH in secreting frog gastric mucosa

A method of estimation of pH in frog gastric mucosa by measuring the apparent creatine kinase equilibrium was studied. In a resting, in vitro preparation of frog stomach the intracellular pH was found to increase linearly with an increase in the serosal pH. This increase was also accompanied by an increase in the apparent equilibrium constant of the creatine kinase reaction. A similar increase was found when the resting mucosa was stimulated with histamine plus theophylline. During this procedure the total content of adenine nucleotides and creatine plus creatine phosphate remained constant.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interstitial sodium nuclear magnetic resonance relaxation times in perfused hearts.

Separation of intracellular and extracellular sodium nuclear magnetic resonance (NMR) signals would enable nondestructive monitoring of intracellular sodium. It has been proposed that differences between the relaxation times of intracellular and extracellular sodium be used either directly or indirectly to separate the signal from each compartment. However, whereas intracellular sodium relaxation times have been characterized for some systems, these times were unknown for interstitial sodium. In this study, the interstitial sodium NMR relaxation times have been measured in perfused frog and rat hearts under control conditions. This was achieved by eliminating the NMR signal from the extracardiac (perfusate) sodium, and then quantifying the remaining cardiac signal. The intracellular signal was measured to be 8% (frog) or 22% (rat) of the cardiac signal and its subtraction was found to have a negligible effect on the cardiac relaxation times. Therefore this cardiac signal is considered to provide a good estimate of interstitial relaxation behavior. For perfused frog (rat) hearts under control conditions, this signal was found to have a T1 of 31.6 +/- 3.0 ms (27.3 +/- 1.6 ms) and a biexponential T2 of 1.9 +/- 1.0 ms (2.1 +/- 0.3 ms) and 25.2 +/- 1.3 ms (26.3 +/- 3.2 ms). Due to the methods used to separate cardiac signal from perfusate signal, it is possible that this characterized only a part of the signal from the interstitium. The short T2 component attributable to the interstitial signal indicates that separation of the NMR signals from each compartment on the basis of relaxation times alone may be difficult.     

Is the change in intracellular pH during fatigue large enough to be the main cause of fatigue?

The intracellular pH of frog sartorius muscles exposed to an extracellular pH 8.0 (25 mM HCO3-, 1% CO2) was 6.9-7.1. Following a fatiguing stimulation period (one tetanic contraction per second for 3 min), the intracellular pH was 6.5-6.7. When similar experiments were repeated with frog sartorius muscles exposed to pH 6.4 (2mM HCO3-, 1% CO2), the intracellular pH was 6.8-6.9 at rest and 6.3-6.4 following fatigue. So, in both experiments the intracellular pH decreased by 0.4-0.5 pH unit during fatigue. When the CO2 concentration of the bathing solution was increased from 1 to 30%, the intracellular pH of resting muscles decreased from 7.0 to 6.2-6.3. Although the effect of CO2 on the intracellular pH was greater than the fatigue effect, the decrease in tetanic force with CO2 was less than 40%, while during fatigue the tetanic force decreased by at least 70%. Therefore in frog sartorius muscle the decrease in tetanic force during fatigue exceeds the decrease that is expected from just a change in intracellular pH.     

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

The simultaneous determination of intracellular pH and cell energy status

The relationship between the apparent equilibrium constant of creatine kinase and intracellular pH was evaluated in CHO and murine FSaII tumor cells. The apparent equilibrium constant, K' = [ATP][Cr]/[ADP][PCr], was determined from acid extracts at variable pH. Intracellular pH (pHi) was determined from the intracellular/extracellular distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. Over the intracellular pH range of 7.2 to 6.1, K' increased by a factor of approximately 10. Intracellular pH was related to the apparent equilibrium constant by the equation pHi = -log K' + log K, where the value of the constant log (log[K'/H+]) was 8.09. Over the same pH range, the concentration of phosphocreatine decreased with pH. Essentially identical results were obtained in CHO and FSaII tumor cells. The similar apparent equilibrium constants in CHO and FSaII cells suggest that assessment of the creatine kinase metabolites will be useful not only for determination of cell energy status but also for the determination of intracellular pH. This information may be useful for the design of therapeutic strategies which are influenced by pH or energy status such as hyperthermia, and drugs which are weak acids or bases, including hypoxic cell radiosensitizers.     

Evidence for poliovirus-induced cytoplasmic alkalinization in HeLa cells

During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.     

Standard electromotive force of the H2-AgCl;Ag cell in 30, 40, and 50 mass% glycerol/water from -20 to 25 degrees C: pK2 and pH values for a standard "mops" buffer in 50 mass% glycerol/water

Data are presented regarding the establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from -20 to 25 degrees C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H2(g, 1 atm) / Mops, Na Mopsate, NaCl / AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Mops) +/- in equilibrium with (Mopsate)- + H+. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures.     

Weak bases inhibit cleavage and embryogenesis in amphibians and echinoderms

The action of weak bases was studied on the early embryonic development of a number of species. Gastrulation was disrupted in the frog, Xenopus laevis, the newt, Pleurodeles watlii, the sea urchins, Paracentrotus lividus and Sphaerechinus granularis and the starfish, Asterias rubens. This required only submillimolar amounts of either NH+4 (pH 9.0) or procaine (pH 8.2). At higher concentrations even early cell division was inhibited in all the species with furrow regression particularly noticeable in Xenopus eggs. A similar action of the weak bases on early development, the lack of any action at lower extracellular pH, and the counteracting action of NH+4 on acidity-induced disruption of sea urchin development, all implicate an elevation of intracellular pH. However, a more direct intracellular action of the weak bases cannot be ruled out.     

Cellular functions of a cell in a metastable equilibrium state

A unifying hypothesis which might replace some of the many ion pumps which are invoked to describe distribution of ions across living cell membranes is developed quantitatively. Resting cells are assumed to be in a metastable state such that ions are in equilibrium between an extracellular aqueous phase, in which water has the properties of the bulk liquid, and an intracellular aqueous phase in which water has enhanced structure and strongly modified solvent properties. Partition coefficients or medium effects for Na⁺, K⁺ and Cl⁻ are calculated for several cell types. It is shown that in such a hypothetical cell, possessing no ion pumps there is an amplified Donnan potential between the two phases, its sign determined by the net charge on intracellular proteins, and its magnitude increased by a separation of ions induced by the difference in solvent properties of the water in the two phases. It is shown that a cell in such a metastable state is excitable and can generate an action potential with an inward surge of Na+ followed by an outward surge of K+. An explanation is offered for the transient release of Caa+ from the sarcoplasmic reticulum following excitation of a muscle fibre. Regulation of cellular volume is shown to be a necessary result of the presence in the extracellular solution of a high concentration of Na+, an ion with a very low affinity for intracellular water. It is concluded that the principal cellular functions that are commonly attributed to the sodium pump are also a feature of a cell in a metastable equilibrium state.     

Carbon dioxide permeability of aquaporin-1 measured in erythrocytes and lung of aquaporin-1 null mice and in reconstituted proteoliposomes

Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.