Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Na and Cl transport and short-circuit current in rabbit ileum

Summary Na and Cl fluxes and short-circuit current (I _sc) in rabbit ileum have been studied as a function of ionic concentrations in HCO₃-free solutions. Both net Na flux (J _net ^Na ) andI _sc show similar saturation functions of [Na] at fixed [Cl]. They show no significant difference between zero and 112mm Na but at 140mm NaI _sc is significantly greater than theJ _net ^Na . Net Cl transport, secretion, is observed only at 140mm Na and is approximately equivalent to the difference between theI _sc andJ _net ^Na . The transcellular mucosa-to-serosa Na fluxes measured at 140 and 70mm Na do not differ significantly from the correspondingI _sc. The net Cl flux varies with [Cl] at fixed [Na] whileI _sc is virtually not affected by [Cl]. These results suggest that the absorptive Na transport process is electrogenic and responsible for theI _sc and that the secretory fluxes of Na and Cl are coupled, require high [Na], vary with [Cl], and do not contribute toI _sc. K-free solution abolishes theI _sc after a prolonged lag. Finally, the effect of a low resistance shunt pathway on active Na absorption is examined with a four-compartment model.Deceased (October 16, 1974).     

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig

Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (I_sc) ranged from-15 to 220 A·cm^-2. Variations in basal I_sc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na⁺ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on I_sc was independent of Cl^- and HCO ₃ ^- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in I_sc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the I_sc. The decrease in I_sc due to 1 mM amiloride was dependent on both Cl^- and HCO ₃ ^- , and was attributed to reductions in the M-S and net Na⁺ fluxes as well as the M-S unidirectional Cl^- flux. Ion replacement experiments demonstrated that Cl^- substitution reduced the M-S and net Na fluxes, while replacement of HCO ₃ ^- with HEPES abolished net Cl^- absorption by reducing the M-S unidirectional Cl^- flux. From these data it can be concluded that: (1) Na⁺ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl^- absorption is completely HCO ₃ ^- -dependent (presumably mediated by Cl^-/HCO ₃ ^- exchange) and occurs independently of Na⁺ absorption.Abbreviations G_t tissue conductance - HEPES tris (hydroxymethyl) aminomethane - (tris) N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - I_sc short-circuit current - J_r residual flux - M-S mucosal-to-scrosal - S-M serosal-to-mucosal - TTX tetrodotoxin     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland

Summary Scatchard analysis of³H ouabain bound to isolated rectal gland cells as a function of increasing ouabain concentrations produced a concave curvilinear plot that was resolved into two specific sites with either a high (I) or low (II) affinity for ouabain. Cyclic cAMP/theophylline (±furosemide, 10^–4 m) increased the amount of³H ouabain bound to the high-affinity site I. Vanadate, a phosphate congener which promotes formation of the ouabain-binding state of the enzyme, mimicked the effects of cAMP/theophylline at low concentrations of ouabain, suggesting that cAMP/theophylline increases binding to site I by enhancing the rate of turnover of resident enzyme. Enhanced⁸⁶Rb uptake seen following cAMP/theophylline administration was primarily associated with increased flux through the high-affinity ouabain site, and this stimulation was not obliterated by the co-administration of furosemide. A model was presented which suggested the presence of two noninteracting pools of enzyme or isozymes which exhibit either a high or low affinity for ouabain. Cyclic AMP both stimulated turnover via site I, and modified the kinetics of binding of³H ouabain to site II. The (ave)K _d of³H ouabain for site II was increased from 3.6 m (controls) to 0.5 m (cAMP/theophylline) and the Hill coefficient was modified from 0.45 (controls) to 1.12 (caMP/theophylline), suggesting a transition from a negative- to a noncooperative binding state. While furosemide reversed the effects of cAMP/theophylline on site II kinetics, it did not obliterate cAMP/theophylline effects on site I. This suggests that cAMP may alter the intrinsic turnover rate of this particular pool of Na,K-ATPase in shark rectal gland.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

Summary The Na⁺/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na⁺/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of¹⁴C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na⁺-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na⁺/glucose cotransporter.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence,spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. Examination of the percentage enhancement and association constants (1.51×10⁶, 6.56×10⁶ and 4.17×10⁵ M^–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×10⁵, 1.3×10⁴, and 11.7×10⁵ M^–1 sec^–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10^–2, 83.3 sec^–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Cell Cl and transepithelial Na transport in toad urinary bladder

Relationships between short-circuit current (I _sc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30–60 min with an associated decrease in cell water content. Concomitantly, I _sc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and I _sc both recovered within 10 min. Serosal SITS (4 acetamido-4-isothiocyano-stilbene-2,2-disulfonate; 0.5 mm) plus bumetanide (0.1 mm), did not prevent the fall in I _sc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of I _sc in this medium. They also prevented the recovery of I _sc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO₃ exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/ HCO₃ exchange.This work was supported by a grant from the Medical Research Council of New Zealand. Purchase of equipment was made possible through grants from the Medical Research Council of New Zealand, the Medical Distribution Committee of the Lottery Board, the University Grants Committee, the Telford Trust, the New Zealand Neurological Foundation and the National Heart Foundation. The expert technical assistance of S. Zellhuber-McMillan is gratefully acknowledged.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (I_sc). Luminal ATP stimulated a transient increase in the release of mucins and of I _sc corresponding to a secretory Cl^– current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED₅₀ of approximately 40 m. Maximal peak I _sc after stimulation with ATP was approximately 35 A/cm² with an apparent ED₅₀ of about 0.4 mm. ATP-dependent I _sc was at least in part due to Cl^– secretion since removal of Cl^– from the medium reduced the peak I _sc by 40% and the I _sc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMPadenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P₂-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl^– channels. P₂-receptor-stimulated mucin and I _sc release was strongly inhibited by a 30 min preincubation with the classical K⁺ channel blockers quinine (1 mm), quinidine (1 mm), and Ba²⁺ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K⁺ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P₂ receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy.     

Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site

Summary We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 ×g × 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (K d=0.57±0.15 m vs.K d=3.3±0.7 m for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios>1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200mm salt, the high affinity bumetanide binding site sediments as a single band withS _20,w =8.8±0.8 S. This corresponds to a molecular weight 200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.