Biomedical subjects

M S Lipkowitz

Publications and source records attributed to M S Lipkowitz.

At least 19 recordsLinked to original sources

PubMed2001

Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells.

Recombinant protein produced from a cDNA cloned in our laboratory (UAT) functions in lipid bilayers as a urate transporter/channel. Because UAT is a galectin, a family of proteins presumed to be soluble, the localization and topology of UAT were assessed in living cells. UAT was targeted to plasma membrane in multiple epithelium-derived cell lines and, in polarized cells, was targeted to both apical and basolateral membranes. The amino and carboxy termini of UAT were both detected on the cytoplasmic side of plasma membranes, whereas cell surface biotinylation studies demonstrated that UAT is not merely a cytosolic membrane-associated protein but contains at least one extracellular domain. Madin-Darby canine kidney cells were shown both functionally and immunologically to contain an apparent homolog of UAT; however, transfection with UAT did not modify urate uptake. Because coimmunoprecipitation studies revealed that UAT is capable of forming both homo- and heteromultimers, it is proposed that monomers of endogenous channels are in part replaced by monomers of the protein expressed subsequent to transfection, thereby maintaining constancy of urate uptake at basal levels.

Animals↗

PubMed2001

Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter.

Elevated serum levels of uric acid have been associated with an increased risk for gout, hypertension, cardiovascular disease, and renal failure. The molecular mechanisms for the diminished excretion of urate in these disorders, however, remain poorly understood. Human galectin 9, which is highly homologous to the rat urate transporter rUAT, has been reported to be a secreted or cytosolic protein. We provide data that galectin 9 is hUAT, the first identified human urate transporter. hUAT is a highly selective urate ion channel when inserted in lipid bilayers. When expressed in renal epithelial cells it is an integral plasma membrane protein with at least two transmembrane domains. The gene for hUAT consists of 11 exons and is mapped to chromosome 17; a highly homologous gene, hUAT2, maps to a nearby region of chromosome 17 and is also likely to be a urate transporter. hUAT is expressed in a wide variety of tissues and is present in at least three isoforms; hUAT2 is less widely expressed at severalfold lower levels than hUAT. Further knowledge about the functions of hUAT, its isoforms, and hUAT2, as well as mutational analysis of hUAT1 and hUAT2 in individuals or families with hyperuricemia, should significantly improve our understanding of the molecular mechanisms of urate homeostasis.

Amino Acid Sequence↗

PubMed1999

Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Animals↗

PubMed1996

Deletion polymorphism of the angiotensin-converting enzyme gene is independently associated with left ventricular mass and geometric remodeling in systemic hypertension.

An insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with myocardial infarction, cardiomyopathy, and left ventricular (LV) hypertrophy. LV mass and geometry are related to cardiovascular morbidity and mortality. Two-dimensional directed M-mode echocardiograms and 24-hour ambulatory blood pressure monitoring were performed in 67 hypertensive subjects. Echocardiographic measurements were assessed in blinded fashion. LV mass index and relative wall thickness were calculated. ACE genotypes were determined by polymerase chain reaction amplification of deoxyribonucleic acid prepared from leukocytes, using primers that encompass the polymorphic segment. Systolic ambulatory blood pressure was higher in subjects with the II genotype. All other patient characteristics were similar across genotype groups. After adjustment for other covariables, the DD and ID genotypes were associated with significantly higher LV mass index than was the II genotype. Adjusted relative wall thickness was also higher in subjects with the DD genotype than in subjects with the ID and II genotypes. On multiple regression analysis, systolic ambulatory blood pressure, gender, body mass index, and the ACE genotype were each independently related to LV mass index (R2 = 0.53). Systolic ambulatory blood pressure, race, and ACE genotype were each independently related to relative wall thickness (R2 = 0.34). The ACE genotype explained an additional 3% and 4% of the variations of LV mass index and relative wall thickness, respectively. In conclusion, ACE polymorphism accounted for a small but statistically significant proportion of the variation in LV mass and geometry in our hypertensive subjects.

Adult↗

PubMed1996

Molecular therapy for renal diseases.

The introduction of molecular therapy through the delivery of nucleic acids either as oligonucleotides or genetic constructs holds enormous promise for the treatment of renal disease. Significant barriers remain, however, before successful organ-specific molecular therapy can be applied to the kidney. These include the development of methods to target the kidney selectively, the definition of vectors that transduce renal tissue, the identification of appropriate molecular targets, the development of constructs that are regulated and expressed for long periods of time, the demonstration of efficacy in vivo, and the demonstration of safety in humans. As the genetic and pathophysiologic basis of renal disease is clarified, obvious targets for therapy will be defined, for example, polycystin in polycystic kidney disease, human immunodeficiency virus (HIV) type 1 in HIV-associated nephropathy, alpha-galactosidase A in Fabry's disease, insulin in diabetic nephropathy, and the "minor" collagen IV chains in Alport's syndrome. In addition, several potential mediators of progressive renal disease may be amenable to molecular therapeutic strategies, such as interleukin-6, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta(TGF-beta). To test the in vivo efficacy of molecular therapy, appropriate animal models for these disease states must be developed, an area that has received too little attention. For the successful delivery of genetic constructs to the kidney, both viral and nonviral vector systems will be required. The kidney has a major advantage over other solid organs since it is accessible by many routes, including intrarenal artery infusion, retrograde delivery through the uroexcretory pathways, and ex vivo during transplantation. To further restrict expression to the kidney, tropic vectors and tissue-specific promoters also must be developed. For the purpose of inhibition of endogenous or exogenous genes, current therapeutic modalities include the delivery of antisense oligodeoxynucleotides or ribozymes. For these approaches to succeed, we must gain a much better understanding of the nature of their transport into the kidney, requirements for specificity, and in vivo mechanisms of action. The danger of a rush to clinical application is that superficial approaches to these issues will likely fail and enthusiasm will be lost for an area that should be one of the most exciting developments in therapeutics in the next decade.

Animals↗

PubMed1996

Glycogen synthase polymorphism, insulin resistance and hypertension.

The A(2) allele of the human glycogen synthase gene may be associated with hypertension in diabetic and non-diabetic Finnish subjects. The prevalence of the A(2) allele was investigated in 64 non-diabetic hypertensive subjects with borderline hypertension or established hypertension. Ambulatory blood pressure was performed on all subjects. Insulin sensitivity index (S(I)) was determined in subjects with borderline hypertension. The DNA fragment containing the XBaI restriction site was amplified by the polymerase chain reaction, digested by the XBaI enzyme and compared by gel electrophoresis with a positive control from Finland. Mean age +/- SD for age, S(I) and ambulatory blood pressure were respectively: 39 +/- 10 yrs, 60 +/- 30 min(-1)(nmol/mL) and 132 +/- 7/ 83 +/- 6 mmHg. Sixteen of the subjects were insulin resistant as determined by S(I) <70.0 and they had significantly higher BP and BMI than insulin sensitive subjects. The A(2) allele of the glycogen synthase was not detected in any subject. This suggests that the relation between the XBaI polymorphism of the glycogen synthase gene, insulin resistance and elevated blood pressure may be restricted to a limited and genetically uniform Finnish population.

Adult↗

PubMed1994

Developmental changes of renal brushborder membrane ionic permeability.

Renal brush-border membrane vesicles (BBMV) show an age-dependent increase in height of the Na(+)-gradient driven overshoot for glucose and proline uptake. Conversely, early uptake of 22Na+ is more rapid in BBMV from kidney of 7-day-old vs. adult rats. To understand the mechanisms responsible for these observations, ionic permeability characteristics of BBMV from different aged animals were determined using an electrical potential sensitive fluorescent dye, diS-C3(5). Absolute and relative ionic permeabilities were determined after a 3-h incubation in 100 mM KCl. Intravesicular K+ ([K+]in), a measure of absolute K+ permeability, was calculated from the extravesicular K+ at which valinomycin produced no potential difference (PD). [K+]in was significantly lower in vesicles from 7-day, compared to adult (P < 0.01). While Cl- permeability, relative to that of K+ was similar, PNa+/PK+ decreased significantly with age (P < 0.05, 7 day vs. adult). In the presence of an inwardly directed NaCl gradient, the lower PNa+ relative to PCl- of the adult vesicles would result in a less positive intravesicular charge, which would therefore augment Na(+)-solute co-transport. Fluorescence polarization studies also show that lipids from BBM vesicles of 7-day-old rats are more fluid than those from adult. These differences are likely due to developmental lipid compositional changes, which influence membrane transport and permeability characteristics. These findings would explain, in part, the age-dependent alterations of renal BBMV solute transport.

Aging↗

PubMed1994

Isolation and immunolocalization of a rat renal cortical membrane urate transporter.

Two modalities of urate transport have been reported in rat kidney, a urate/anion exchanger and a potential sensitive, uricase-like uniporter. As an initial attempt to isolate and characterize the responsible transport protein(s), rat renal cortical membranes were harvested, solubilized, and subjected to affinity chromatography with urate or xanthine as the affinity ligand. Pig liver peroxisomal uricase was purified with the same system, and the enzymatically active protein was used to generate polyclonal antibodies in rabbit. Silver stain of SDS-polyacrylamide gel electrophoresis gels of the eluted fraction containing the affinity-purified renal membrane protein(s) demonstrated bands at 25, 32, 36, and 41 kDa. On Western blot, two of these bands (32 and 36 kDa) were immunoreactive to the polyclonal antibody to pig liver uricase. In 6 of 10 studies, the affinity-purified renal membrane protein(s) also oxidized urate. Anti-pig liver uricase produced a selective and dose-dependent inhibition of the uricase-like urate uniporter in renal membrane vesicles, but did not affect the urate/anion exchanger or the sodium-dependent glucose transporter. Immunocytochemical studies of rat renal cortex with the same antibody indicated that the immunoreactivity was localized to proximal tubules. These studies demonstrate that the renal cortical plasma membranes contain urate-binding proteins, which have some functional and immunological homology to the hepatic peroxisomal core protein, uricase. Within the renal cortex, these proteins are localized to proximal tubules, the site of urate transport. Since the antibody that reacts with the affinity-purified urate-binding proteins on Western blot selectively inhibits urate transport in intact membrane vesicles, it is concluded that at least one of the affinity-purified urate-binding proteins is a uricase-like urate transporter.

Animals↗

PubMed1993

Hormonal modulation of ionic permeability in human red blood cells.

It has previously been reported that both exogenous adenosine cAMP analogs and forskolin-induced elevations in intracellular cAMP concentrations selective increase relative ionic chloride permeability in normal human red blood cells (RBC). A similar selectively increase in relative ionic chloride permeability was observed in untreated uremic subjects in whom endogenous RBC cAMP concentrations are chronically elevated. To detect which hormones might modulate RBC cAMP and ionic permeabilities, RBC were exposed to norepinephrine, epinephrine, and parathyroid hormone. Thereafter, RBC cAMP concentrations were measured by RIA and relative ionic permeabilities were determined in human RBC ghosts with the potential sensitive fluorescent probe diS-C3-(5). In ghosts prepared from normal RBC, norepinephrine and epinephrine significantly increased intracellular cAMP concentrations; in these ghosts, relative ionic chloride permeability (permeability of chloride/permeability of potassium (PCI/PK)), but not PNa/PK (permeability of sodium/permeability of potassium), was significantly increased. In contrast, exposure to parathyroid hormone did not affect either cAMP concentrations or relative ionic permeabilities. These results are consistent with the presence of adrenergic receptors and the absence of parathyroid hormone receptors in RBC. These studies demonstrate that hormonally induced changes in cAMP can modulate RBC relative ionic chloride permeability and suggest that, in uremic RBC, increased relative ionic chloride permeability could be consequent to elevated plasma levels of epinephrine or norepinephrine.

Cell Membrane Permeability↗

PubMed1992

Altered membrane ionic permeability in a rat model of chronic renal failure.

Acute elevations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentrations are known to increase ionic chloride permeability in diverse tissues. To determine if chronic endogenous increases in cAMP are associated with sustained alterations in membrane ionic permeabilities, renal cortical brush border membrane vesicles (BBMV) were prepared and red blood cells were harvested in a model of chronic renal failure, the 75% nephrectomized rat. Relative ionic permeabilities were determined using the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. These studies demonstrate that renal cortical homogenate and RBC cAMP concentrations are increased in chronic renal failure animals. In the same animals relative ionic chloride permeability (PCl/PK) was significantly increased in renal cortical BBMV and RBC ghosts: PNa/PK was not affected. This selective change in permeability results in a significant increase in PCl/PNa and hyperpolarization of BBMV of sufficient magnitude to stimulate Na(+)-dependent glutamine transport. The change in glutamine uptake was not consequent to an alteration in the kinetics of glutamine transport or delayed dissipation of the inward Na+ gradient. Renal hypertrophy per se did not effect renal homogenate cAMP concentration or relative ionic permeability of renal cortical BBMV prepared from kidneys of uninephrectomized animals fed a 40% protein diet. These studies demonstrate that relative ionic chloride permeability and tissue [cAMP] are chronically increased in diverse cells (renal proximal tubule and RBCs) in a rat model of renal failure. These findings suggest that membrane ionic permeability may be altered and electrogenic transport secondarily perturbed in renal failure in association with hormonally-induced chronic elevations of intracellular cAMP concentrations.

Animals↗

PubMed1992

Hormonal regulation of rat renal proximal tubule brush-border membrane ionic permeability.

The effects on ionic permeability of toxins and hormones that activate or deactivate the guanine nucleotide regulatory (G) proteins that govern adenylate cyclase activity were examined in rat renal proximal tubule cell brush-border membranes. These studies demonstrate that activation of stimulatory G (Gs) proteins by cholera toxin or parathyroid hormone and deactivation of inhibitory (G (Gi) proteins by pertussis toxin result in a selective increase in Cl- permeability relative to that of K+ as determined with the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. In contrast, activation of Gi by angiotensin II significantly decreases relative Cl- permeability. The selective increase in relative Cl- permeability induced by parathyroid hormone results in an inside-negative potential in membrane vesicles exposed to an inward NaCl gradient that is of sufficient magnitude to stimulate electrogenic, Na(+)-dependent glucose transport. These data suggest that the relative ionic permeabilities of brush-border membranes are tonically regulated by the opposing effects of hormones that act via Gs or Gi proteins. Changes in membrane potential resulting from this regulation may play an important role in modifying transport in the proximal tubule.

Adenylate Cyclase Toxin↗

PubMed1991

Ontogeny of Na/H antiporter activity in rabbit renal brush border membrane vesicles.

The development of the Na/H antiporter was studied in renal brush border membrane vesicles (BBMV) from fetal and adult rabbits using isotopic and fluorescent techniques. The kinetics of the antiporter studied by 22Na+ uptake revealed that the Vmax was only 25% of that in the adult; however, the Km's for Na+ were not significantly different. These data were confirmed by a fluorescent assay using the pH-sensitive probe, acridine orange: the Vmax was significantly lower in the fetal BBMV. Conductive Na+ movement was estimated from amiloride-insensitive 22Na+ uptake and the rate of alkalinization induced by K+, an ion whose relative conductance was found to be similar to that of Na+. Although relative Na+ conductance was significantly greater in fetal BBMV, the lower Vmax in fetal vesicles could not be ascribed to this factor. Maternal administration of betamethasone (50 micrograms/kg intramuscularly) for 2 d before delivery significantly increased the Vmax of the antiporter to levels observed in the adult; Km was unaffected. Na/K ATPase activity increased fourfold after betamethasone, but the specific activities of four brush border marker enzymes and the kinetics of Na(+)-glucose cotransport were unchanged. These data indicate that there is a developmental increase in brush border Na/H exchange which is the result of an increase in the number and/or the turnover number of the carriers. Further, these data suggest that the postnatal increase in antiporter activity may be related to the surge in glucocorticoid concentration that occurs perinatally.

Acridine Orange↗

PubMed1989

Differential permeabilities of rat renal brush-border and basolateral membrane vesicles.

Potassium chloride permeability and relative ionic conductances of rat renal cortical brush-border (BBMV) and basolateral (BLMV) membrane vesicles were examined using the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. Vesicles were simultaneously isolated and separated by free-flow electrophoresis. These studies demonstrated that neither BBMV nor BLMV equilibrated in 100 mM KCl despite prolonged incubation. In both, an inwardly directed KCl gradient was sustained for 3 h. The low intravesicular KCl concentration of BLMV was confirmed utilizing the response of electrogenic Na+-dependent [3H]glutamine transport to variations in the membrane potential. Chloride conductance was significantly less than potassium conductance in BBMV and BLMV. Consequently, an inside-positive potential was maintained across both membranes. BLMV were significantly more fluid, less permeable, had a lower relative chloride conductance, and maintained a greater inside-positive potential than BBMV. The KCl permeabilities of BBMV and BLMV were inversely related to endogenous membrane copper content and were significantly reduced by exogenous copper. Permeability did not correlate with membrane magnesium content, nor was it affected by exogenous magnesium. These studies suggest that endogenous copper as well as intracellular factors may regulate the permeabilities of the brush-border and basolateral membranes of proximal tubule cells in vivo.

Algorithms↗

PubMed1989

Modulation of the ionic permeability of renal cortical brush-border membranes by cAMP.

The effects of adenosine 3',5'-cyclic monophosphate (cAMP) on potassium chloride permeability, relative ionic permeabilities, and Na+-dependent glucose transport were examined in rat renal cortical membrane vesicles. Brush-border membrane vesicles were prepared from paired control and cAMP- or forskolin-exposed homogenates by use of a magnesium aggregation technique. These studies demonstrate that exposure to exogenous cAMP or increases in endogenous cAMP significantly increase KCl permeability and ionic chloride permeability relative to that of potassium (PCl/PK) as determined with the fluorescent potential-sensitive probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)] and 36Cl uptake. Because PNa/PK did not change, PCl/PNa was also significantly increased by cAMP. These changes in ionic permeabilities were associated with a significant stimulation of Na+-dependent glucose transport that was unassociated with either an alteration in the kinetics of glucose transport or delayed dissipation of the inwardly directed Na+ gradient. These findings indicate that the cAMP-induced stimulation of glucose transport resulted from hyperpolarization of the vesicles secondary to the increase in PCl/PNa. These studies suggest that in vivo variations in intracellular cAMP concentration may modulate electrogenic transport processes by altering relative ionic permeabilities and membrane potential in renal proximal tubule cells.

Animals↗

PubMed1989

Modulation of ionic permeability in a nonpolarized cell: effect of cAMP.

To evaluate whether adenosine 3',5'-cyclic monophosphate (cAMP) modulates ionic permeabilities of nonpolarized cells, as reported in diverse polarized epithelia, relative ionic permeabilities were determined in human red cell ghosts by means of the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide. Relative ionic chloride permeability (PCl/PK), but not PNa/PK, was significantly increased in ghosts prepared from normal red blood cells (RBCs) exposed to cAMP analogues or forskolin, with the latter at a concentration that significantly increased intracellular cAMP concentration. As basal RBC cAMP concentrations of untreated uremic subjects were also increased, relative permeabilities of ghosts and unstimulated RBC cAMP concentrations were compared in normal, uremic, and dialyzed subjects, PCl/PK was significantly increased in uremic compared with normal subjects; PNa/PK was not altered. PCl/PK and RBC cAMP concentrations were indistinguishable in normal and dialyzed subjects. Neither the kinetics nor number of Cl(-)-HCO3- antiporters, assessed with the pH-sensitive probe acridine orange and the disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, respectively, were altered in uremic cells. These studies suggest that cAMP modulates ionic chloride permeability via increased chloride conductance of each Cl(-)-HCO3- antiporter or by activation/opening of new or existing channels in RBC membranes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1988

Characterization of the fetal glucose transporter in rabbit kidney. Comparison with the adult brush border electrogenic Na+-glucose symporter.

Glucose transport was characterized in rabbit renal brush border membrane vesicles (BBMV) of the fetus late in gestation. Highly purified, osmotically reactive fetal BBMV contained a glucose transporter that was qualitatively indistinguishable from that in the adult: both are concentrative, Na+ dependent, electrogenic, stereospecific, and sensitive to phlorizin. Although the apparent Km for glucose is similar in the fetus and adult, the Vmax is significantly higher in the adult. When the membrane potential was clamped with a protonophore, this difference diminished; however, Vmax remained significantly higher in adult BBMV. This postnatal increase in Vmax was paralleled by a similar increase in the number of phlorizin binding sites. These findings indicate that the maturational increase in glucose transport is, in part, consequent to a more favorable electrical potential for Na+-dependent glucose transport and, in part, the result of the insertion of new transporters. The homogenate activity of several brush border enzymes also demonstrated significant maturational increases. The magnitude of these changes was variable and enzyme dependent. These combined observations suggest that mature expression of membrane proteins (transporters and enzymes) occurs at different stages of development of renal proximal tubule cells.

Aging↗

PubMed1987

Ionic permeabilities of rat renal cortical brush-border membrane vesicles.

It is generally assumed that electrolytes equilibrate readily across renal cortical brush-border membrane vesicles (BBMV). This assumption was tested by use of two new methods in rat BBMV prepared with free-flow electrophoresis (FFE), Mg aggregation, or Ca aggregation. Intravesicular KCl and RbCl concentrations, as well as the conductance of Cl relative to K (GCl/GK) and GNa/GK were determined with the fluorescent, potential-sensitive probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]; intravesicular KCl concentration was also approximated utilizing the response of Na-dependent [3H]glucose uptake to variations in the membrane potential. These studies demonstrated that KCl fails to attain electrochemical equilibrium in BBMV prepared by the three methods, despite prolonged incubation at 22 degrees C; a significant, inwardly directed electrolyte gradient was sustained in all cases. The intravesicular electrolyte concentration was lower in BBMV prepared with FFE than in those prepared with Mg or Ca. GCl/GK was lowest in BBMV prepared with FFE and highest in those prepared with Ca; GNa/GK was comparable in all preparations. The apparent impermeance of BBMV may impact significantly in interpreting data from studies that require knowledge of the precise concentration of intravesicular electrolytes.

Animals↗

PubMed1987

Cl-/HCO-3 antiporter in red cell ghosts: a kinetic assessment with fluorescent probes.

The pH sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe acridine orange and membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide were used to evaluate the Cl-/HCO-3 antiporter and proton and potassium conductances, respectively, in human red blood cell ghosts. Acidic, chloride-loaded ghosts alkalinized rapidly in pH 8.5 chloride-free media. Alkalinization could not be ascribed to conductive proton efflux with either depolarizing potassium influx or chloride efflux. Alkalinization was consequent to flux on the Cl-/HCO-3 antiporter: this process displayed saturation kinetics, competitive inhibition by external chloride, and inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The mean Kms for internal chloride, external bicarbonate, and external chloride were 2.19, 0.24, and 0.44 mM, respectively. These studies confirm both the asymmetry of this carrier and the high affinity for external HCO-3; however, the affinities for internal and external chloride are significantly greater than prior estimates. The Km for internal chloride (2.19 mM) was considerably lower than levels previously reported (20-65 mM) unless external (trans) chloride was raised above 2 mM. The present studies thus demonstrate and emphasize the critical importance of trans substrate concentration in assessing the kinetics of a carrier whose mobility is faster in the loaded than in the unloaded state.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗