Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of sendai virus as fusogen

Bumetanide is a potent diuretic drug which has some structural features in common with furosemide. The steady-state exchange of K⁺ and Cl^? was investigated in Ehrlich ascites tumor cells treated with bumetanide. This agent did not alter the cellular content of K⁺ or Cl^? but the self-exchange of both ions was depressed. K⁺ self-exchange was inhibited by 55% at bumetanide concentrations as low as 10^?6 M. Cl^? self-exchange was less sensitive to this drug but at low concentrations (between 10^?6 and 10^?3 M) bumetanide was a more effective inhibitor of Cl^? transfer than furosemide. The steady-state K⁺ flux of cells equilibrated in NO₃^? media was compared with the K⁺ flux in cells treated with 10^?4 or 10^?3 M bumetanide; the Cl^? -sensitive K⁺ exchange was equivalent to the bumetanide-sensitive K⁺ exchange. Since the results suggested that a bumetanide-sensitive (Cl^?, K⁺) cotransport could be operative in steady-state cells, the stoichiometry of the bumetanide-sensitive fluxes was determined by measuring Cl^? and K⁺ fluxes simultaneously in the same cell suspension. At $\text{5 · 10}{}^{\mathrm{?4}}$ and 10^?3 M bumetanide concentrations, the ratio of these fluxes was $\text{0.98 ? 0.07 (}\text{S.E.}\text{)}\text{and}\text{1.04 ? 0.06}$, respectively, consistent with the postulated cotransport mechanism. At 10^?4 and 10^?5 M, however, the ratio of the bumetanide-sensitive Cl^?/K⁺ flux was significantly less than 1.0. Since the magnitude of the bumetanide-sensitive K⁺ flux at 10^?4 M was close to that of the Cl^?-sensitive flux, a ratio of less than 1.0 at this drug level indicates that Cl^? sensitivity and drug sensitivity may not reflect inhibition of the same process under all circumstances.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

Peculiarities of the Na+/d-glucose cotransport system in necturus renal tubules

The effects of d-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d-glucose produces $\text{10.5}\text{mV}\text{± 1.1}$ (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to $\text{2.1 ± 0.1}\text{mV}$. (3) The glucose-evoked depolarization, $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$, does not alter the intracellular K⁺ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na⁺ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li⁺ substituting for Na⁺) does not change the magnitude of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$; complete removal of sodium from the lumen lowers the value of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$ ($\text{3.2 ± 0.2}\text{mV}$) but the response is not abolished. This observation suggests that the d-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.     

The overshoot phenomenon in cotransport

Based on simplified equations, the overshoot curve experimentally observed with Na⁺-linked cotransport of neutral substrate (sugars or amino acids) has been simulated by computer. The approach is in principle similar to that of previous approaches (Weiss, S.D., McNamara, P.D. and Segal, S. (1981) J. Theor. Biol. 91, 597–608), but more general; in particular, it includes the effect of electrical membrane potential difference, and the quantitative relationship between height of peak and certain transport parameters, such as maximum rate, dissociation constant of ternary complex, electric charge of translocator, respectively. In addition, it tests two alternative models with respect to the rate-determining step: the translocation, on the one hand, and the association/dissociation of the ligands at the translocator site, on the other. The major findings are the following: (1) An overshoot can be obtained similar to that usually found experimentally, provided that maximum rate and affinity between translocator and transport of solute exceed certain minimum values. (2) The overshoot effect with Na-linked cotransport is enhanced by a negative membrane potential (inside relative to outside) and decreased by a positive potential. In the first case, the peak is higher and occurs faster. In the latter case, the peak is lower and delayed. (3) The effect of an electric potential difference on the overshoot curve does not depend appreciably on the charge of the empty translocator, except if the translocation of the latter is strongly rate-limiting. (4) To obtain an overshoot curve, it is not necessary that the translocation step be rate-limiting, contrary to what has been postulated previously (Läuger, P. (1980) J. Membrane Biol. 57, 163–178).     

Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves

Phlorizin (1-[2-(β- d -glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone) is a well-known non-transported inhibitor of glucose uptake in animal cells. The effects of this compound were studied on the transmembrane potential difference (PD) of broad bean ( Vicia faba L. cv. Aguadulce) mesophyll cells, and on the uptake of amino acids and sugars by the leaf tissues. Phlorizin (5 m M ) induced a marginal depolarization (7 to 10 mV) of the normal PD (-140 mV), and a slight decrease in the uptake of glycine and serine. By contrast, phlorizin induced a stronger inhibition of the uptake of glucose and 3–O-methylglucose, and more particularly of sucrose uptake and phloem loading. In tissues aged for 12 h, 5 m M phlorizin inhibited the absorption of sucrose from a 1 m M solution by 70%. Kinetic experiments demonstrated that phlorizin acted as a competitive inhibitor for the sucrose carrier and for the hexose carrier. Efflux experiments showed that the counter-exchange of sucrose and of 3–O-methylglucose was also phlorizin-sensitive. Overall, the data show that phlorizin is recognized by the hexose carrier and, more efficiently, by the sucrose carrier of the material investigated, but that it is not transported across the membrane.     

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K⁺ efflux and Cl⁻ loss. When human erythrocytes were suspended in a physiological NaCl solution (pH _o = 7.4), the measured pH _i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH _i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl⁻ content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H⁺ influx with the measured unidirectional K⁺ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H⁺ influx and a K⁺ efflux in LIS solution of 108.2 ± 20.4 mmol (l _cells hr)⁻¹ and 98.7 ± 19.3 mmol (l _cells hr)⁻¹, respectively. For bovine and porcine erythrocytes, in LIS media, H⁺ influx and K⁺ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K⁺(Na⁺)/H⁺ exchanger, inhibited the K⁺ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K⁺(Na⁺)/H⁺ exchanger in the human erythrocyte membrane. Received: 22 December 1999/Revised: 10 April 2000     

Leucine transport in Xenopus laevis oocytes: Functional and morphological analysis of different defolliculation procedures

l-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, l-leucine uptake was reduced by 67.5%±5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5%±6.4 after mechanical defolliculation. The Na⁺-dependent uptake of 0.1 mM l-leucine was 18.6±4.6 pmol oocyte⁻¹ 40 min⁻¹ in folliculated oocytes and 5.6±1.9 in collagenase defolliculated oocytes (means±SE). l-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled l-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different l-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific l-leucine binding to membranes. l-leucine kinetics showed that the l-leucine V_max and K_m values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The V_max and K_m values of Na⁺-dependent l-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16±1.5 pmol oocyte⁻¹ 40 min⁻¹ and 57±21 μmol (mean±SD). The Na⁺-activation curve of 0.1 mM l-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of l-leucine V_max, and microscopic observation showing recovery of the microvillar apparatus.     

Adenosine Receptors in Myelin Fractions and Subtractions: The Effect of the Agonist (R)-Phenylisopropyladenosine on Myelin Membrane Microviscosity

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.