Coordinate expression of piretanide receptors and Na+,K+,Cl- cotransport activity in Madin-Darby canine kidney cell mutants

Two receptor sites for [3H]piretanide, a sulfamoylbenzoic acid loop diuretic, have been identified in intact Madin-Darby canine kidney cells, an epithelial cell line derived from dog kidney. The two receptor sites differed in their affinity for piretanide (KD1 = 2.1 +/- 1.4 nM and KD2 = 264 +/- 88 nM) and the maximal number of sites (Bmax1 = 11 +/- 4 and Bmax2 = 120 +/- 80 fmol/mg of protein). Madin-Darby canine kidney cells are known to possess a tightly coupled and highly cooperative Na+,K+,Cl- cotransporter which is sensitive to loop diuretics. Under ionic conditions identical to those used to study piretanide binding (30 mM Na+, 30 mM K+, 30 mM Cl-), the Ki for inhibition of the initial rate of 86Rb+ uptake by piretanide was 333 +/- 92 nM, a value not significantly different from the KD of the low affinity receptor site. [3H]Piretanide binding to three low K+-resistant mutants derived from this cell line was also studied. These mutants had been previously characterized as being partially or completely defective in Na+,K+,Cl- cotransport activity (McRoberts, J. A., Tran, C. T., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 12320-12326). One of these mutants had undetectable levels of Na+,K+,Cl- cotransport activity and low to undetectable levels of specific piretanide binding. The second mutant had low but measurable levels of cotransport activity (11% of the wild-type levels) and displayed very low affinity (KD approximately 8000 nM) specific piretanide binding. In the third mutant, expression of Na+,K+,Cl- cotransport activity and both piretanide receptors was cell density-dependent. Subconfluent to just-confluent cultures of this mutant lacked detectable cotransport activity as well as specific piretanide binding, whereas very dense cultures displayed both piretanide receptors and had intermediate to nearly normal levels of cotransport activity. These results demonstrate that the Na+,K+,Cl- cotransporter is a receptor for loop diuretics, but they also raise questions about the functional significance of the two piretanide receptor sites.     

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.     

Role of cyclic GMP in atrial natriuretic factor stimulation of Na+,K+,Cl- cotransport in vascular smooth muscle cells

Atrial natriuretic factor (ANF) has been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an increase in intracellular cyclic GMP (cGMP) content. We have recently demonstrated that a prominent Na+,K+,Cl- cotransport system is present in VSMC and that a permeable cGMP analog (8-bromo-cGMP) stimulates activity of the cotransporter. We have also shown that the ANF peptide, rat atriopeptin III, stimulates Na+,K+,Cl- cotransport and elevates intracellular cGMP levels in VSMC. In the present study, we tested the hypothesis that ANF stimulation of Na+,K+,Cl- cotransport occurs via an increase in cGMP levels. When the quinolinedione, 6-anilo-5,8-quinolinedione (LY83583) (10 microM), was used to block formation of cGMP in VSMC from primary cultures of rat thoracic aorta, it was found that both basal and rat atriopeptin III (100 nM)-stimulated Na+,K+,Cl- cotransport were significantly inhibited. The effect of LY83583 was dose-dependent and the half-maximal inhibitory concentration was 0.5 microM. LY83583 also inhibited cotransport in the presence of a maximal concentration of 8-bromo-cGMP. However, this inhibition was not seen in cells also treated with 2-O-propoxyphenyl-8-azapurin-6-one (M&B 22,948), an inhibitor of cGMP phosphodiesterase. M&B 22,948 alone also increased levels of cotransport. Since inhibition of cGMP formation blocks ANF-stimulated Na+,K+,Cl- cotransport and inhibition of cGMP breakdown enhances Na+, K+, Cl- cotransport, we conclude that ANF stimulation of Na+,K+,Cl- cotransport in VSMC is mediated via increase in intracellular cGMP levels.     

Characterization of subclones of Madin-Darby canine kidney renal epithelial cell line

Heterogeneity in Madin-Darby canine kidney (MDCK) epithelial cells has been reported, however, its details have not been well described. In the present study, we show that subclones obtained from a MDCK cell line could be divided into two morphologically and biochemically distinct cell types with different hormonal responsiveness. Clones of the first type, motile clones, which had extended and flattened cytoplasm, were devoid of carbonic anhydrase activity. Clones of the second type, nonmotile clones, formed colonies of cuboidal cells and showed carbonic anhydrase activity. Motile clones synthesized cAMP in response to arginine vasopressin, prostaglandin E1, and isoproterenol but not glucagon. In contrast, nonmotile clones responded to all of these hormones. These findings suggest MDCK cells have multiple cellular origins. The motile clones have characteristics similar to the principal cells of the collecting system, whereas the nonmotile clones may be derived from the thick ascending limb or the intercalated cell. Our studies also demonstrate a significant influence of culture condition on MDCK cellular behavior (carbonic anhydrase activity, Na+/K+-ATPase activity and vasopressin responsiveness). Therefore, physiologic and biochemical experiments with MDCK cells must be interpreted with reservations about cellular heterogeneity as well as differences induced by culture conditions.     

Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery

Madin-Darby canine kidney cells were used to study events in the postsynthetic processing and cell surface delivery of Na,K-ATPase. The photoactivable 2-nitro-5-azidobenzoyl (NAB) derivative of ouabain and an anti-ouabain antibody were employed in experiments designed to determine the time intervals required for newly synthesized Na,K-ATPase to achieve the capacity to bind ouabain and to arrive at the cell surface. Ouabain-binding capacity was assessed in Madin Darby canine kidney cells which were pulse-labeled with [35S]methionine. At various chase intervals cells were disrupted by probe sonication and the resultant vesicles were permeabilized. Vesicles were incubated with NAB-ouabain and, following UV photolysis, solubilized and subjected to immunoprecipitation with an anti-ouabain antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates revealed that newly synthesized Na,K-ATPase can carry out type II (Mg2+ and Pi supported) ouabain binding throughout the course of its postsynthetic processing. In contrast, the ability to carry out type I (Na+, Mg2+, and ATP-supported) ouabain binding is not attained until 10 min after the completion of the sodium pump's synthesis. Experiments in which intact pulse-labeled cells were incubated with NAB-ouabain revealed that the Na,K-ATPase arrives at the cell surface as soon as 50 min after its synthesis. These results suggest that postsynthetic processing is required before the newly synthesized Na,K-ATPase can display its full repertoire of catalytic functions. This processing seems to be complete prior to the newly synthesized sodium pump's arrival at the cell surface.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.     

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.     

Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla.

Specific and ouabain-sensitive potassium binding to Na⁺, K⁺-ATPase was directly observed by centrifugation method with the purified enzyme and ⁴²K. The specific binding reached to saturation level at concentrations more than 0.2 mM KCl and the level was 6.2 nmol per mg ATPase with specific activity of 1470 μmol Pi/h·mg. The binding level, however, was proportional to the enzyme unit used. Simultaneous determination of ⁴²K binding and [³H]ouabain binding showed that two mol of potassium binding were blocked by one mol of ouabain binding per 3.2×10⁵ g enzyme. Although the apparent dissociation constant of the specific potassium binding was estimated at about 50 μM, Scatchard plot of the binding revealed non-linear relationship suggesting that the two potassium sites existed on one catalytic unit of enzyme would be not equivalent but cooperative.     

Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia

We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.     

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

Regulation of ion concentration by Drosophila hydei Na+, K+, Ca2+, and Cl-

The ionic composition of the haemolymph of osmotically unencumbered larvae of Drosophila hydei shows a pattern that is typical (Bone, 1944) for highly developed phytophagous insect larvae: 36 mval/l. Cl^?; 56 mval/l. Na⁺; 31 mval/l. K⁺; approximately 18 mval/l. Ca²⁺ at an osmolality of 299 mOsmol/l.The larvae are able to maintain their most favourable ionic concentrations in the haemolymph after experimental osmotic stress in hypertonic as well as in hypotonic media. The reactions are most distinct with an increase or decrease of Cl^? concentration of the external medium. Characteristic regulating processes begin, and the Cl^? concentration of the haemolymph adjusts to the ‘standard’ again. The principal lapse shows the functional representation of an intensely suppressed oscillation. There seems to be a two-point regulation which requires the existence of a Cl^? ion depot and the existence of Cl^?-sensitive receptors.     

Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

The glycosphingolipids (GSLs) of two sublines of Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, were characterized by t.l.c., antibody overlay and mass spectrometry. The major characteristic which distinguishes the two MDCK cell strains is their trans-epithelial electrical resistance which is typically of the order of 3000 ohm.cm2 for strain I and 100 ohm.cm2 for strain II cells. Strain I and II cells were equally rich in glycolipids, the cellular GSL/phospholipid ratio being 0.04. However, while the phospholipid patterns were identical, the GSLs showed striking differences, and each cell strain expressed appreciable amounts of GSLs that were not found in the other strain. Both cell types possessed neutral GSLs with one, two or three carbohydrate moieties. The monoglycosylceramide accounted for 50% of the total GSLs in each strain. However, while in strain I cells over 90% of this monoglycosylceramide was monoglucosylceramide, in strain II cells over 90% consisted of monogalactosylceramide. In addition, MDCK strain II cells selectively expressed GSLs belonging to the globo series (26% of its neutral GSLs), including globoside and Forssman antigen, a globoside derivative. MDCK strain I cells, on the other hand, expressed another series of GSLs with 4-7 carbohydrate moieties characterized by the common sequence Hex-HexNAc-Hex-Hex-Cer. The presence of two fucosylated GSLs in these series was established. Both MDCK strain I and II cells contained negatively charged GSLs, the major component of which was the ganglioside GM3. MDCK strain II cells in addition expressed sulfatide, the sulfated derivative of galactosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats.

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.