Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na⁺/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na⁺ gradient. The uptake of l-carnitine was of high affinity (K_m=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na⁺/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H⁺ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [¹⁴C]TEA uptake, but unlabeled l-carnitine did not stimulate [¹⁴C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na⁺/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H⁺/organic cation antiporter.     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Determination of the Na(+)/glucose cotransporter (SGLT1) turnover rate using the ion-trap technique

The Na⁺/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of two Na⁺ ions and one glucose molecule using the so-called alternating access mechanism. According to this principle, each cotransporter molecule can adopt either of two main conformations: one with the binding sites accessible to the extracellular solution and one with the binding sites facing the intracellular solution. The turnover rate (TOR) is the number of complete cycles that each protein performs per second. Determination of the TOR has important consequences for investigation of the cotransport mechanism, as none of the rate constants involved in mediating transport in a given direction (conformational changes and binding and unbinding reactions) can be slower than the TOR measured under the same conditions. In addition, the TOR can be used to estimate the number of cotransporter molecules involved in generating a given ensemble activity. In this study, we obtain an independent estimation of the TOR for human SGLT1 expressed in Xenopus laevis oocytes applying the ion-trap technique. This approach detects the quantity of ions released in or taken up from the restricted space existing between the oocyte plasma membrane and the tip of a large ion-selective electrode. Taking advantage of the fact that hSGLT1 in the absence of Na⁺ can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s⁻¹ in the presence of 35 mM α-methyl-glucose at −150 mV (pH 5.5). For the same group of oocytes, a TOR of 13.3 ± 2.4 s⁻¹ was estimated under near-V_max conditions, i.e., in the presence of 90 mM Na⁺ and 5 mM α-methyl-glucose. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n = 14), and this activity was generated by an average of 3.6 ± 0.7 × 10¹¹ cotransporter molecules/oocyte.     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.     

Differential expression of Na<Superscript>+</Superscript>/<Emphasis Type="SmallCaps">d</Emphasis>-glucose cotransport in isolated cells of<Emphasis Type="Italic"> Marsupenaeus japonicus</Emphasis> hepatopancreas

d-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na⁺-dependent d-glucose transport (Na⁺/d-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na⁺/d-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na⁺/d-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na⁺/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na⁺/d-glucose cotransport system is still to be established.Communicated by: G. Heldmaier     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

The Actual Ionic Nature of the Leak Current through the Na/Glucose Cotransporter SGLT1

Expression of the Na⁺/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a “Na⁺ leak” and represents some 5-10% of the maximal Na⁺/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (V_R) of ≈−15 mV as observed for wt SGLT1. V_R showed only a modest negative shift when extracellular Na⁺ concentration ([Na⁺]_o) was lowered and it was completely insensitive to changes in extracellular Cl⁻. When extracellular pH (pH_o) was decreased from 7.5 to 6.5 and 5.5, V_R shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pH_o but other ions must be involved at pH_o 7.5. In the presence of 15 mM [Na⁺]_o (pH_o = 7.5), addition of 75 mM of either Na⁺, Li⁺, Cs⁺, or K⁺ generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na⁺/l-glutamate (l-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K⁺ and Cl^?. The presence of 100 mM K⁺ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K⁺ concentration and that in the absence of any sodium gradient, an outward K⁺ gradient was unable to influence the Na⁺/acidic amino acid transport system. It was also found that Cl^? could specifically activate the Na⁺-dependent l-glutamate (l-aspartate) uptake either in the presence or in the absence of K⁺. Also the effect of Cl^? was observed only in the presence of an inward Na⁺ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na⁺ gradient and in the presence of chloride gradient. l-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K⁺ or Cl^? did not show any translocation of net charge.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Taurine transport by rabbit kidney brush-border membranes: Coupling to sodium,chloride, and the membrane potential

Summary Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na⁺-d-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na⁺ gradient (K⁺ diffusion potential) and could be completely inhibited by other -amino acids. It displayed a specific anion requirement (Cl^–Br^–SCN^–>I^–>NO ₃ ^– ). At chemical Na⁺ equilibrium, Cl^– gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl^– gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl^– as well as Br^–. The Cl^– stoichiometry was found to be one, whereas taurine transport, in the presence of Cl^–, was sigmoidally related to the Na⁺ concentration, resulting in a coupling ratio of 2 to 3 Na⁺: 1 taurine. Upon Cl^– replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na⁺ concentration (up to 150mm). These results suggest a 2 to 3 Na⁺:1 Cl^–:1 taurine cotransport mechanism driven mainly by the Na⁺ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl^– appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

NAD+-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD+ hydrolysis

We previously demonstrated inhibition of Na⁺-dependent ³²P_i transport in canine renal brush-border membranes in association with NAD⁺-induced ADP ribosylation of membrane protein(s) and postulated that NAD⁺ inhibits P_i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD⁺ resulted in release of P_i which was prevented by EDTA. It was proposed that NAD⁺-mediated inhibition of ³²P_i transport might occur through this mechanism. To determine whether NAD⁺ inhibited ³²P_i transport by a mechanism other than or in addition to release of P_i, we compared Na⁺-dependent ³²P_i counterflow in brush-border membrane equilibrated with P_i or with P_i generated from NAD⁺. Release of P_i from NAD⁺ incubated with brush-border membrane was confirmed. The increased uptake of ³²P_i which was demonstrated in brush-border membrane equilibrated with P_i was not measured when intravesicular P_i was generated from a concentration of NAD⁺ which effected ADP-ribosylation of brush border membranes (100 μM NAD⁺). In contrast, increased uptake of ³²P_i was demonstrated when intravesicular P_i was generated from 1 μM NAD⁺ which did not effect ADP ribosylation. Mg²⁺-dependent ADP ribosylation of brush-border membrane incubated with NAD⁺ was demonstrated which persisted during the time interval of ³²P_i uptake measurements. Our findings are compatible with the hypothesis that NAD⁺-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P_i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg²⁺ and decreased ADP ribosylation.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.