Water transport and homeostasis as a major function of erythrocytes.

Erythrocytes have long been known to change volumes and shapes in response to different salt concentrations. Aquaporin-1 (AQP1) was discovered in their membranes more than 20 yr ago. The physiological roles of volume changes and AQP1 expression, however, have remained unclear. We propose that rapid water exchange through AQP1 coupled with large capacity for volume change may allow erythrocytes to play an important role in water regulation. In this study, we showed that erythrocytes in situ gradually reduced their volumes by 39% in response to the hyperosmotic corticomedullary gradient within mouse kidneys. AQP1 knockout (KO) erythrocytes, however, displayed only minimal reduction. Constructing a microfluidic device resembling capillary flow with an extracellular fluorescent reporter demonstrated that water exchanges between erythrocytes and their hypotonic or hypertonic surroundings in vitro reached steady state in ~60 ms. AQP1 KO erythrocytes, however, did not show significant change. To simulate the water transport in circulation, we built basic units consisting of three compartments (i.e., erythrocyte, plasma, and interstitial fluid) using Kedem-Katchalsky equations for membrane transport, and connected multiple units to account for the blood flow. These simulations agreed with experimental results. Importantly, volume-changing erythrocytes in capillaries always "increase" the osmotic gradient between plasma and interstitial fluid, making them function as "micropumps" to speed up the regulation of local osmolarity. Trillions of these micropumps, mobile throughout the body, may further contribute to water homeostasis. These insights suggest that the enhanced exchange of water, in addition to O2 and CO2, may well be the third major function of erythrocytes. NEW & NOTEWORTHY Physiological roles of erythrocyte volume change and aquaporin-1 were proposed and investigated here. We conclude that fast water transport by aquaporin-1 coupled with large volume-change capacity allows erythrocytes to enhance water exchange with local tissues. Furthermore, their huge number and mobility allow them to contribute to body water homeostasis.

The mechanism underlying alpinetin-mediated alleviation of pancreatitis-associated lung injury through upregulating aquaporin-1.

Characterized by its acute onset, critical condition, poor prognosis, and high mortality rate, severe acute pancreatitis (SAP) can cause multiple organ failure at its early stage, particularly acute lung injury (ALI). The pathogenesis of ALI is diffuse alveolar damage, including an increase in pulmonary microvascular permeability, a decrease in compliance, and invasion of many inflammatory cells. Corticosteroids are the main treatment method for ALI; however, the associated high toxicity and side effects induce pain in patients. Recent studies show that the effective components in many traditional Chinese medicines can effectively inhibit inflammation with few side effects, which can decrease the complications caused by steroid consumption. Based on these observations, the main objective of the current study is to investigate the effect of alpinetin, which is a flavonoid extracted from Alpinia katsumadai Hayata, on treating lung injury induced by SAP and to explore the mechanism underlying the alpinetin-mediated decrease in the extent of ALI. In this study, we have shown through in vitro experiments that a therapeutic dose of alpinetin can promote human pulmonary microvascular endothelial cell proliferation. We have also shown via in vitro and in vivo experiments that alpinetin upregulates aquaporin-1 and, thereby, inhibits tumor necrosis factor-α expression as well as reduces the degree of lung injury. Overall, our study shows that alpinetin alleviates SAP-induced ALI. The likely molecular mechanism includes upregulated aquaporin expression, which inhibits tumor necrosis factor-α and, thus, alleviates SAP-induced ALI.

Increased Lung Ischemia-Reperfusion Injury in Aquaporin 1-Null Mice Is Mediated via Decreased Hypoxia-Inducible Factor 2α Stability.

Aquaporin (AQP) 1, a water channel protein expressed widely in vascular endothelia, has been shown to regulate cell migration, angiogenesis, and organ regeneration. Even though its role in the pathogenesis of lung ischemia-reperfusion (IR) injury has been defined, the functional role of AQP1 during long-term IR resolution remains to be clarified. Here, we found that AQP1 expression was increased at late time points (7-14 d) after IR and colocalized with endothelial cell (EC) marker CD31. Compared with IR in wild-type mice, IR in Aqp1(-/-) mice had significantly enhanced leukocyte infiltration, collagen deposition, and microvascular permeability, as well as inhibited angiogenic factor expression. AQP1 knockdown repressed hypoxia-inducible factor (HIF)-2α protein stability. HIF-2α overexpression rescued the angiogenic factor expression in pulmonary microvascular ECs with AQP1 knockdown exposed to hypoxia-reoxygenation. Furthermore, AQP1 knockdown suppressed cellular viability and capillary tube formation, and enhanced permeability in pulmonary microvascular ECs, which were partly rescued by HIF-2α overexpression. Thus, this study demonstrates that AQP1 deficiency delays long-term IR resolution, partly through repressing angiogenesis mediated by destabilizing HIF-2α. These results suggest that AQP1 participates in long-term IR resolution, at least in part by promoting angiogenesis.

Aquaporin-1 Deficiency Protects Against Myocardial Infarction by Reducing Both Edema and Apoptosis in Mice.

Many studies have determined that AQP1 plays an important role in edema formation and resolution in various tissues via water transport across the cell membrane. The aim of this research was to determine both if and how AQP1 is associated with cardiac ischemic injury, particularly the development of edema following myocardial infarction (MI). AQP1+/+ and AQP1-/- mice were used to create the MI model. Under physiological conditions, AQP1-/- mice develop normally; however, in the setting of MI, they exhibit cardioprotective properties, as shown by reduced cardiac infarct size determined via NBT staining, improved cardiac function determined via left ventricular catheter measurements, decreased AQP1-dependent myocardial edema determined via water content assays, and decreased apoptosis determined via TUNEL analysis. Cardiac ischemia caused by hypoxia secondary to AQP1 deficiency stabilized the expression of HIF-1α in endothelial cells and subsequently decreased microvascular permeability, resulting in the development of edema. The AQP1-dependent myocardial edema and apoptosis contributed to the development of MI. AQP1 deficiency protected cardiac function from ischemic injury following MI. Furthermore, AQP1 deficiency reduced microvascular permeability via the stabilization of HIF-1α levels in endothelial cells and decreased cellular apoptosis following MI.

Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling.

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with ß-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased ß-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with ß-catenin, glycogen synthase kinase 3ß, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased ß-catenin phosphorylation and increased ß-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.

Genetic deletion of aquaporin-1 results in microcardia and low blood pressure in mouse with intact nitric oxide-dependent relaxation, but enhanced prostanoids-dependent relaxation.

The water channels, aquaporins (AQPs) are key mediators of transcellular fluid transport. However, their expression and role in cardiac tissue is poorly characterized. Particularly, AQP1 was suggested to transport other molecules (nitric oxide (NO), hydrogen peroxide (H2O2)) with potential major bearing on cardiovascular physiology. We therefore examined the expression of all AQPs and the phenotype of AQP1 knockout mice (vs. wild-type littermates) under implanted telemetry in vivo, as well as endothelium-dependent relaxation in isolated aortas and resistance vessels ex vivo. Four aquaporins were expressed in wild-type heart tissue (AQP1, AQP7, AQP4, AQP8) and two aquaporins in aortic and mesenteric vessels (AQP1-AQP7). AQP1 was expressed in endothelial as well as cardiac and vascular muscle cells and co-segregated with caveolin-1. AQP1 knockout (KO) mice exhibited a prominent microcardia and decreased myocyte transverse dimensions despite no change in capillary density. Both male and female AQP1 KO mice had lower mean BP, which was not attributable to altered water balance or autonomic dysfunction (from baroreflex and frequency analysis of BP and HR variability). NO-dependent BP variability was unperturbed. Accordingly, endothelium-derived hyperpolarizing factor (EDH(F)) or NO-dependent relaxation were unchanged in aorta or resistance vessels ex vivo. However, AQP1 KO mesenteric vessels exhibited an increase in endothelial prostanoids-dependent relaxation, together with increased expression of COX-2. This enhanced relaxation was abrogated by COX inhibition. We conclude that AQP1 does not regulate the endothelial EDH or NO-dependent relaxation ex vivo or in vivo, but its deletion decreases baseline BP together with increased prostanoids-dependent relaxation in resistance vessels. Strikingly, this was associated with microcardia, unrelated to perturbed angiogenesis. This may raise interest for new inhibitors of AQP1 and their use to treat hypertrophic cardiac remodeling.

The role of aquaporin and tight junction proteins in the regulation of water movement in larval zebrafish (Danio rerio).

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K(u)) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 µM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²âº-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

Triazolothienopyrimidine inhibitors of urea transporter UT-B reduce urine concentration.

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

L-carnitine is an osmotic agent suitable for peritoneal dialysis.

Excessive intraperitoneal absorption of glucose during peritoneal dialysis has both local cytotoxic and systemic metabolic effects. Here we evaluate peritoneal dialysis solutions containing L-carnitine, an osmotically active compound that induces fluid flow across the peritoneum. In rats, L-carnitine in the peritoneal cavity had a dose-dependent osmotic effect similar to glucose. Analogous ultrafiltration and small solute transport characteristics were found for dialysates containing 3.86% glucose, equimolar L-carnitine, or combinations of both osmotic agents in mice. About half of the ultrafiltration generated by L-carnitine reflected facilitated water transport by aquaporin-1 (AQP1) water channels of endothelial cells. Nocturnal exchanges with 1.5% glucose and 0.25% L-carnitine in four patients receiving continuous ambulatory peritoneal dialysis were well tolerated and associated with higher net ultrafiltration than that achieved with 2.5% glucose solutions, despite the lower osmolarity of the carnitine-containing solution. Addition of L-carnitine to endothelial cells in culture increased the expression of AQP1, significantly improved viability, and prevented glucose-induced apoptosis. In a standard toxicity test, the addition of L-carnitine to peritoneal dialysis solution improved the viability of L929 fibroblasts. Thus, our studies support the use of L-carnitine as an alternative osmotic agent in peritoneal dialysis.

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models.

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.

In situ fluorescence measurement of tear film [Na+], [K+], [Cl-], and pH in mice shows marked hypertonicity in aquaporin-5 deficiency.

PURPOSE: Tear film composition depends on water and ion transport across ocular surface epithelia and on fluid secretion by lacrimal glands. The purpose of this study was to establish in situ fluorescence methods to measure tear film ionic concentrations and pH in mice and to determine whether tear film composition is sensitive to deficiency of the major ocular surface aquaporin water channels. METHODS: Tear film ionic concentrations and pH were measured in anesthetized mice by ratio imaging fluorescence microscopy after topical application of ion/pH-sensing, dual-wavelength fluorescent indicators. [Na(+)], [K(+)], and [Cl(-)] were measured with membrane-impermeant indicators developed by our laboratory, and pH was measured with bis(carboxyethyl)-carboxyfluorescein fluorescence-conjugated dextran. Measurements were performed on wild-type mice and on knockout mice lacking aquaporins AQP1, AQP3, and AQP5. RESULTS: In wild-type mice, tear film [Na(+)] was 139 +/- 8 mM, [K(+)] was 48 +/- 1 mM, [Cl(-)] was 127 +/- 4 mM, and pH was 7.59 +/- 0.2 (SE; n = 5-8). pH did not differ significantly in the AQP knockout mice. [Na(+)] was increased by approximately twofold in AQP5 null mice (230 +/- 20 mM) and was greatly reduced after exposure of the ocular surface to a humidified atmosphere. [K(+)] was mildly reduced in AQP1 null mice. CONCLUSIONS: These results establish an in situ optical methodology to measure tear film [Na(+)], [K(+)], [Cl(-)], and pH in living mice, without the need for fluid sampling. Tear film hypertonicity in AQP5 deficiency is likely caused by reduced transcorneal water secretion in response to evaporative water loss.

Antenatal Bartter's syndrome: why is this not a lethal condition?

There are four themes in this teaching exercise for Professor McCance. The first challenge was to explain how a premature infant with Bartter's syndrome could survive despite having such a severe degree of renal salt wasting. Second, the medical team wanted to know why there was such a dramatic decrease in the natriuresis in response to therapy, despite the presence of a permanent molecular defect that affected the loop of Henle. Third, Professor McCance was asked why this patient seemed to have a second rare disease, AQP2 deficiency type of nephrogenic diabetes insipidus. The fourth challenge was to develop a diagnostic test to help the parents of this baby titrate the dose of indomethacin to ensure an effective dose while minimizing the likelihood of developing nephrotoxicity. The missing links in this interesting story emerge during a discussion between the medical team and its mentor.

Water channel activity of plasma membrane affects chondrocyte migration and adhesion.

1. Recent studies indicate that the aquaporin-1 (AQP1) water channel is expressed in human and equine articular chondrocytes. The role of AQP1 in chondrocyte function has not been characterized. In the present study, we investigated the expression of the AQP1 water channel in cultured articular chondrocytes from wild-type (AQP1(+/+)) and AQP1-knockout (AQP1(-/-)) mice and characterized its function in chondrocyte proliferation, migration and adhesion. 2. Expression of AQP1 mRNA and protein was identified in freshly isolated neonatal AQP(+/+) chondrocytes. Immunofluorescence localized the AQP1 protein to the plasma membrane of AQP(+/+) chondrocytes in primary cultures. Relative plasma membrane water permeability of AQP1(+/+) chondrocytes was approximately 1.6-fold higher than that of AQP1(-/-) chondrocytes. 3. The chondrocyte proliferation rate was not affected by AQP1 deletion. However, the serum-induced transwell migration rate of AQP1(-/-) chondrocytes was markedly reduced compared with AQP1(+/+) chondrocytes (16.2 +/- 0.2 vs 27.1 +/- 0.3%, respectively; P < 0.01). Cell adhesion to type II collagen-coated plates was also significantly reduced in AQP1(-/-) chondrocytes compared with AQP1(+/+) chondrocytes (38.1 +/- 0.3 vs 51 +/- 1%, respectively; P < 0.01). 4. The results provided direct evidence that AQP1-mediated plasma membrane water permeability plays an important role in chondrocyte migration and adhesion.

Novel role of AQP-1 in NO-dependent vasorelaxation.

Nitric oxide (NO) produced by endothelial cells diffuses to vascular smooth muscle cells to cause dilatation of the renal vasculature and other vessels. Although it is generally assumed that NO moves from cell to cell by free diffusion, we recently showed that aquaporin-1 (AQP-1) transports NO across cell membranes. AQP-1 is expressed in endothelial and vascular smooth muscle cells. We hypothesized that diffusion of NO into vascular smooth muscle cells and out of endothelial cells is facilitated by AQP-1, and transport of NO by AQP-1 is involved in endothelium-dependent relaxation. In intact aortic rings from AQP-1 -/- mice, vasorelaxation induced by acetylcholine (which increases endogenous NO) was reduced (P < 0.0001 vs. control). No differences were found in the relaxation caused by intracellular delivery of NO or intracellular cGMP between strains. In endothelium-denuded aortic rings from AQP-1 -/- mice, the vasorelaxant capability of NO released in the extracellular space was reduced (P < 0.0001 vs. control). Influx of NO (5 microM) into vascular smooth muscle cells was 0.17 +/- 0.02 f.u./s for control and 0.07 +/- 0.01 f.u./s for AQP-1 -/- mice, 62% lower (P < 0.002). NO released by endothelial cells in response to 1 microM acetylcholine was 96.2 +/- 17.7 pmol NO/mg for control and 41.9 +/- 13.4 pmol NO/mg for AQP-1 -/- mice, 56% reduction (P < 0.04). NOS3 expression was 1.33 +/- 0.29 O.D. units for control and 3.84 +/- 0.76 O.D. units for AQP-1 -/- mice, 188% increase (P < 0.01). We conclude that 1) AQP-1 facilitates NO influx into vascular smooth muscle cells, 2) AQP-1 facilitates NO diffusion out of endothelial cells, and 3) transport of NO by AQP-1 is required for full expression of endothelium-dependent relaxation.

Accelerated cataract formation and reduced lens epithelial water permeability in aquaporin-1-deficient mice.

PURPOSE: To investigate the involvement of aquaporin (AQP)-1 in lens epithelial cell water permeability and maintenance of lens transparency in experimental models of cataract formation. METHODS: Comparative studies were performed on wild-type versus AQP1-null mice. Osmotic water permeability was measured in calcein-stained epithelial cells in intact lenses from fluorescence changes in response to osmotic gradients. Lens water content was measured by gravimetry using kerosene-bromobenzene density gradients, and from wet/dry weight measurements. Lens transparency was measured by contrast analysis of transmitted grid images. Cataract formation was induced in vitro by incubation in high-glucose solutions and in vivo by acetaminophen toxicity. RESULTS: Immunofluorescence showed AQP1 expression in wild-type mice in epithelial cells covering the anterior surface of the lens. AQP1 deletion did not alter baseline lens morphology or transparency, though basal water content was approximately 3% greater (P < 0.001). AQP1 deficiency reduced plasma membrane water permeability in lens epithelium by 2.8 +/- 0.3-fold (P < 0.0001). Loss of lens transparency was accelerated by more than 50-fold in AQP1-null lenses bathed in a 55-mM glucose solution for 18 hours. At 4 hours after acetaminophen administration in 3-methylcholantrene-treated mice, lens opacification was seen in none of the six wild-type mice and in six of six AQP1-null mice. CONCLUSIONS: Lens AQP1 facilitates the maintenance of transparency and opposes cataract formation.

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force.

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.

To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and Rhag-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag-/- and AQP1-/-. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions.

Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.