Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

Vitamin analysis of five planktonic microalgae and one macroalga

Vitamin analysis was carried out on five microalgae used in aquaculture:Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum andChaetoceros calcitrans and one macroalga,Sargassum muticum, which is invasive on the Atlantic shores of France. Both liposoluble (provitamin A, E, K) and hydrosoluble (B₁, B₂, B₆, B₁₂, C, PP) vitamins were quantified. For most of them, greater amounts were obtained in the algal products than in the usual sources. On a dry weight basis,Tetraselmis suecica contained 4280 μg g^?1 provitamin A and 6323 μg g^?1 vitamin E,Pavlova lutheri 1162 μg g^?1 vitamin B₁₂ and 837 μg g^?1 vitamin C,Isochrysis galbana 2690 μg g^?1 vitamin PP and 183 μg g^?1 vitamin B₆, andSkeletonema costatum 710 μg g^?1 vitamin B₁.     

Characterization of Phase-Based Methods Used for Transmission Field Uniformity Mapping: A Magnetic Resonance Study at 3.0 T and 7.0 T

Knowledge of the transmission field (B₁ ⁺) of radio-frequency coils is crucial for high field (B₀ = 3.0 T) and ultrahigh field (B₀≥7.0 T) magnetic resonance applications to overcome constraints dictated by electrodynamics in the short wavelength regime with the ultimate goal to improve the image quality. For this purpose B₁ ⁺ mapping methods are used, which are commonly magnitude-based. In this study an analysis of five phase-based methods for three-dimensional mapping of the B₁ ⁺ field is presented. The five methods are implemented in a 3D gradient-echo technique. Each method makes use of different RF-pulses (composite or off-resonance pulses) to encode the effective intensity of the B₁ ⁺ field into the phase of the magnetization. The different RF-pulses result in different trajectories of the magnetization, different use of the transverse magnetization and different sensitivities to B₁ ⁺ inhomogeneities and frequency offsets, as demonstrated by numerical simulations. The characterization of the five methods also includes phantom experiments and in vivo studies of the human brain at 3.0 T and at 7.0 T. It is shown how the characteristics of each method affect the quality of the B₁ ⁺ maps. Implications for in vivo B₁ ⁺ mapping at 3.0 T and 7.0 T are discussed.     

Folate chemotactic receptors in Dictyostelium discoideum II. Guanine nucleotides alter the rates of interconversion and the proportioning of four receptor states

The ligand binding properties of folate chemotactic receptors on isolated membranes of Dictyostelium discoideum were analyzed. Three out of the four receptor states (B^F, B^S and B^SS) were detected, showing rate constants and K_d values similar to those obtained for intact cells. Guanine nucleotides changed the proportioning of the receptor states as well as the rates of several conversions. (i) The transformation of B^F into B^S was inhibited by GDP but not by guanylyl imidodiphosphate (GuaPP[NH]P) or GTP. (ii) The number of B^S sites was lowered by GTP and GuaPP[NH]P. (iii) The binding to B^SS was lowered by GTP and GDP, but increased by GuaPP[NH]P. (iv) The rate of disappearance of B^SS was increased by GTP, but not by GuaPP[NH]P. Effects of guanine nucleotides were not observed after treatment of the membrane preparations with 15 mg/ml bovine serum albumin. This treatment caused the detection of a binding type different from the types described previously. The affinity of this binding site was extremely high (K_d ≤ 0.2 nM for N₁₀-methylfolic acid), while the dissociation was relatively slow (k₋₁ ≤ 3·10⁻⁴ s⁻¹). It is proposed that bovine serum albumin uncouples the folate receptor from a guanine nucleotide regulatory (G) protein in an irreversible manner. A model is presented in which the four receptor states correspond to distinct interactions with a G protein and GDP or GTP.     

Estimation of leaf area at the level of branch,tree and stand inCryptomeria japonica

The frequency distribution of diameter (x) in foliage shoot segments, ø(x), was examined in 18 branches at different height levels of three trees in a 25-yr-old sugi (Cryptomeria japonica D. Don) plantation. The ø-x relationships were approximated by power-form equations, in which the exponent differed among the branches from ?0.6 to ?4.2. Leaf area (S _B) and leaf weight (W _B) of a branch were estimated on the basis of the ø-x relationship, and the dependency of specific leaf area (SLA ₀) and density (ρ ₀) of a foliage shoot segment on itsx. SLA _B value of a branch defined byS _B/W _B ranged from 27 to 80 cm² g. d.w.^?1 according to the exponent in the function of ø(x). Total leaf area (u) and leaf weight (wl) of a tree were estimated by summation ofS _B andW _B for seven sample trees. TheSLA _T value of a tree defined byu/wl ranged from 65 to 76 cm² g d.w.^?1 and increased with stem diameter at clear length (D _B). By use of the allometric equations betweenu andD _B,LAI of the plot was estimated to be 17.3 ha ha^?1 (half of the total surface area of needles). By a process similar to that used for calculatingLAI, the amount of woody tissues included in sugi foliage was evaluated to be about 10% of the stand foliage biomass.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Transport of vitamin B12 into mouse leukemia cells.

Transport of [⁵⁷Co]cyanocobalamin (vitamin B₁₂) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B₁₂-transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K_m and V values are 180 pM and 5.9 pmol/min/10⁹ cells, respectively. This step is relatively insensitive to temperature, has an apparent pK_a of 6.0, and is inhibited by EDTA; Ca²⁺ or Mg²⁺ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10⁹ cells. Temperature and pH optima are 30 ° and 6.5?7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p-chloromercuriphenylsulfonate and dinitrophenol. Labeled B₁₂ in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B₁₂-free medium; (b) addition of unlabeled B₁₂-transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B₁₂-transcobalamin-II.     

Levels and turnover of the proteinase B inhibitors in yeast

The ratio of the proteinase B inhibitors I₁^B and I₂^B from baker'sd yeast was shown to depend on the yeast strain by specific immunoprecipitation from boiled yeast extract and subsequent electrophoresis of the heat-dissociated precipitates on polyacrylamide gels. Both I₁^B and I₂^B were found, I₂^B being by far predominant. Saccharomyces carlsbergensis NCYC 74 contained I₁^B, whereas in Saccharomyces cerevisiae X 2180 only I₂^B was present. When cells of the latter strain were labelled with [¹⁴C] leucine from the beginning of growth and pulsed with [³H] leucine during the stationary phase, no short-lived I₁^B could be detected. However, the peak of I₂^B resolved on the gel showed an increased [³H/¹⁴C ration in comparison to the majority of the other cellular proteins. The increased ³H/¹⁴C ratio was found to be the result of catabolite repression of inhibitor synthesis during exponential growth: cells growing on glucose as carbon source contain high inhibitor levels only during the stationary phase of growth, whereas during growth on acetate high amounts of inhibitor are present even in exponentially growing cells. During the stationary phase of growth the inhibitor is degraded with the same half-life as the total cellular proteins (about 50 h).     

Axial ligation constants of [5,10,15,20-tetraphenylporphyrinato(2-)] cobalt

Axial ligation constants (log K^B) of bases for [5, 10,15,20-tetraphenylporphyrinato(2-)]cobalt ([Co^II (tpp)]) are reported. The log K^B values of pyridine derivatives except for 4-cyanopyridine show a good linear relationship in a plot of log K^B vs. pK_a of the axial ligand. 4-Cyanopyridine gives a larger log K^B than expected. The log K^B value of 1-methylimidazole for [Co^II(tpp)] is almost the same as that for tetrakis(p-methoxyphenyl)porphyrinatocobalt(II) ([Co((p-CH₃O)tpp)]), althoug the other log K^B values for [Co^II(tpp)] are always slightly larger than those for [Co((p-CH₃O)tpp)]. These results are discussed on the basis of the σ- and π-bonding abilities of the bases, and the solvent effects on the log K^B values. The lower base affinities of cobalt(II) capped porphyrins are also discussed.     

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q^⨪_B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential (E_h) to 200–300 mV is the result of increasing the lifetime of Q^⨪_B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q^⨪_B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q^⨪_B, the flash-induced redox changes of Q^⨪_B were observed at low E_h values unless E_h reached a value at which Q_B underwent reduction at equilibrium to form Q_BH₂. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q_BH₂) of the secondary acceptor.     

Synthesis and characterisation of the halogenated azanonaboranes [(PrNH2)B8H10XNHPr] (X=Cl, Br, I) and their unexpected hydrolysis to hypho-[B5H10(μ-NHPr)]

Treatment of [(ⁱPrNH₂)B₈H₁₁NHⁱPr] with elemental halogen affords the 8-exo-halogen-substituted derivatives [(ⁱPrNH₂)B₈H₁₀XNHⁱPr] (X=Cl, Br, I). The structures of all three compounds are confirmed by NMR spectroscopy and mass spectrometry, and (for X=Br) by an X-ray diffraction study. The bromoazanonaborane undergoes hydrolytic decomposition to the new five-vertex compound [B₅H₁₀(μ-NHⁱPr)] of hypho-type structure.     

Isolation,structural and toxicological characterization of three new mycotoxins produced by the fungusAureobasidium pullulans

3 substances, B₁, B₂, and E₁ were isolated from culture medium extracts ofAureobasidium pullulans by reversed phase liquid chromatography and subsequent liquid chromatographic purification steps on silica gel. The 3 compounds inhibited the metabolism ofSaccharomyces cerevisiae and showed toxic effects in the growth inhibition test toEscherichia coli andBacillus subtilis. Elementary analysis and mass spectroscopical methods revealed sum formulas of C₂₃H₂₂O₆, C₂₂H₂₀O₆ and C₂₄H₂₈O₃ for B₁ B₂, and E₁ and molecular weights of 394, 380, and 364, respectively. Mass spectroscopical, UV-, IR-,¹³C-NMR, and¹H-NMR-spectroscopical investigations revealed polycyclic, non-aromatic compounds containing several carbonyl functions and double bonds and, most notably, spiroepoxy-functions, in the case of B₁ and B₂.     

The iron-quinone acceptor complex in Rhodospirillum rubrum chromatophores studied by EPR

A new EPR signal is reported in Rhodospirillum rubrum chromatophores. The signal is attributed to Q⁻_BFe²⁺, the semiquinone-iron complex of the secondary quinone electron acceptor, on the basis of the following observations. (1) It is induced by a single laser flash given a room temperature and is stable. (2) It is present after odd-numbered flashes and absent after even-numbered flashes when a series of flashes is given. (3) When it is already present, low-temperature illumination results in the disappearance of the signal due to formation of the Q⁻_AFe²⁺Q⁻_B state. (4) Its formation is inhibited by the presence of orthophenanthroline at normal values of pH. The Q⁻_BFe²⁺ signal has two main features, one at g = 1.93 and the other at g = 1.82. The two features have different microwave power and temperature dependences, with the g = 1.82 signal being more difficult to saturate and requiring lower temperatures to be observable. Raising the pH leads to an increase in the g = 1.82 feature, while the g = 1.93 signal decreases in amplitude. It is suggested that the two parts of the signal may represent two EPR forms due to structural heterogeneity. The low-field feature of the Q⁻_BFe²⁺ signal shifts to lower field as the pH is raised and a pK for this change seems to occur at pH 9.4. The Q⁻_AFe²⁺ signal at g = 1.88 also shifts as the pH is increased; however, the shift is less marked than that seen for Q⁻_BFe²⁺, the shift is to higher field and the range over which it occurs is wider and depends upon the temperature of Q⁻_AFe²⁺ formation. This effect may be due to a pK on a protein group being shifted to higher pH by the presence of Q⁻_A. ortho-Phenanthroline broadens and shifts the Q⁻_AFe²⁺ signal. The inhibition of electron transfer between Q⁻_A and Q_B by ortho-phenanthroline becomes less effective at high pH. The new Q⁻_BFe²⁺ signal is unlike other semiquinone-iron signals reported in the literature in bacteria; however, it is remarkably similar to the Q⁻_BFe²⁺ signal reported in Photosystem II.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

Subunits of laminin are differentially synthesized in mouse eggs and early embryos

Synthesis of laminin by mouse preimplantation embryos was examined by specific immunoprecipitation of [³⁵S]methionine-labeled cell lysates. The three polypeptide subunits of laminin, A, B₁, and B₂, are not necessarily synchronously expressed during development. Oocytes and eggs synthesize only one immunoprecipitable polypeptide, which comigrates with the intracellular B₁ chains made by PYS cells and contains N-linked oligosaccharide residues which are sensitive to endo-β-N-acetylglucosaminidase H. From the 4- to 8-cell stage, only B₁ and B₂ polypeptides are synthesized, whereas from the 16-cell stage onwards all three laminin polypeptides are made.     

Formation of nonquasineutral vortex plasma structures with a zero net current

A nonquasineutral vortex structure with a zero net current is described that arises as a result of electron drift in crossed magnetic and electric fields, the latter being produced by charge separation on a spatial scale of about the magnetic Debye radius r _B = |B|/(4πen _e ). In such a structure with a radius of r ～ r _B , the magnetic field is maintained by a drift current on the order of the electron Alfvén current J _Ae = m _e c ³/(2e) and can become as strong as B ? m _e c ²/(er). Estimates show that, in a plasma with a density of n _e = 10²¹?10²³ cm^?3 and with nonzero electron vorticity driven by high-power laser radiation on a time scale on the order of θ _pe ^?1 , magnetic fields with a strength of B ～ 10⁸?10⁹ G are generated on micron and submicron scales. The system with closed current that is considered in the present paper can also serve as a model of hot spots in the channel of a Z-pinch.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.KEY WORDS: ascorbic acid, cyanocobalamin, degradation, hydroxocobalamin, kinetics, two-component spectrometry     

Hydrochlorothiazide enhances the apical Cl− backflux in rabbit gallbladder epithelium: Radiochemical analysis

Hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^? symport and to depolarize the apical membrane potential in the rabbit gallbladder epithelium. The depolarization was likely related to the opening of a Cl^? conductance. To better understand whether an apical Cl^? leak is involved in the mechanism of action of HCTZ, the transapical Cl^? backflux was measured radiochemically by the washout technique. The gallbladder wall, pretreated with pronase on the serosal side to homogenize the subepithelium, was loaded with ³⁶Cl^? on the luminal side; mucosal and serosal ³⁶Cl^? effluxes (J _m , J _s ) were then measured every 2 min. The pretreatment with pronase did not alter the membrane potentials and the selectivity of the epithelium. Under control conditions and the tissue in steady-state, J _m and J _s time courses were each described by two exponential decays (A,B); the rate constants, k ^A and k ^B , were 0.71 ±0.03 and 0.16±0.01 min^?1, respectively, and correspondingly the half-times (t ₁ ^2A , t ₁ ^2B ) were 1.01±0.05 and 5.00±0.44 min (n=10); these parameters were not significantly different for J _m and J _s time courses. J _s was always greater than J _m (J _s /J _m =2.02±0.22 and 1.43 ±0.17 for A and B decays). Under SCN^? treatment in steady-state conditions, both J _m and J _s time courses were described by only one exponential decay, the component B being abolished. Moreover t ₁ ^2A was similar to that predictable for the subepithelium. It follows that it is the component B which exits the epithelial compartment. Based on the intracellular specific activity and ³⁶Cl^? J _m ^B at 0 min time of the washout experiment, the cell-lumen Cl^? backflux in steady-state was calculated to be equal to about 2 μmol cm^?2hr^?1, in agreement with the value indirectly computable by other techniques. The experimental model was well responsive to different external challenges (increases in media osmolalities; luminal treatment with nystatin). HCTZ (2.5 · 10^?4 m) largely increased ³⁶Cl^? J _m ^B . The increase was abolished by luminal treatment with 10^?4 m SITS, which not only brought back the efflux time courses to the ones observed under control conditions but even increased J _s /J _m of the cellular component, an indication of a reduced J _m ^B . It is concluded that HCTZ opens an apical, SITS-sensitive Cl^? leak, which contributes to dissipate the intracellular Cl^? accumulation and to inhibit the NaCl transepithelial transport. Moreover, the drug is likely to reduce the basal electroneutral Cl^? backflux supported by Na⁺-Cl^? cotransport, in agreement with the inhibition of the cotransport itself.