INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Prodigiosins are a group of red pigments produced by microorganisms like Streptomyces and Serratia (1). They show a wide variety of biological activities involving selective inhibition of T cell proliferation induced by concanavalin A (2, 3), immunomodulation in immunized mice (4-6), and suppression of the bone resorption by osteoclasts (7). We reported previously that prodigiosin 25-C raises lysosomal pH and suppresses glycoprotein processing due to its uncoupling effect on vacuolar (lysosomal) H⁺-ATPase without showing apparent protonophoric activity (8). Although prodigiosin 25-C also affects mitochondrial F-ATPase and produces swelling of mitochondria besides Golgi, it did not affect the cellular ATP level, showing relatively specific perturbation of vacuolar pH when treated in vivo, which makes it a promising drug for the analysis of vacuolar function.

In the present paper, we show that H⁺/Cl symport activity is expressed even on liposomal membranes and present evidence that prodigiosins are a new group of H⁺/Cl symporters (or OH/Cl exchangers, in their equivalence) that uncouple (dissociate) proton translocation from otherwise coupled catalysis (for example, ATP hydrolysis) in such a way that they inhibit acidification but neither catalysis nor membrane potential formation. A preliminary account of this work was presented in the 22nd Meeting of the Japan Bioenergetics Group (9).

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Prodigiosin was prepared from the culture broth of Serratia marsescens as described (10) and metacycloprodigiosin and prodigiosin 25-C were obtained from Streptomyces hiroshimensis (11). Rats (Wistar, male) were obtained from Sankyo Labo Service (Tokyo, Japan). Fluorescein-labeled dextran (FD) was either synthesized according to Ref. 12 or supplied in the form of fluorescein isothiocyanate-dextran (M_r 70,000) from Sigma. Triton WR-1339 was obtained from Ruger Chemical Co. (Irvington, NJ). n-Octyl--D-thioglucoside was purchased from Dojin (Kumamoto, Japan). Protease inhibitors of microbial origin were obtained from the Peptide Research Institute (Osaka). Bafilomycin A₁ was kindly provided by Professor K. Altendorf (University of Osnabrück, Osnabrück, Germany). Phospholipids were obtained from Sigma (Sigma type II-S phosphatidylcholine from soybean phospholipids) and used without purification. Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid, trisodium salt, laser grade) was supplied from Eastman Kodak Co.(Rochester, NY), and bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol (Oxonol-V) and dipropylthiodicarbocyanine iodide (diS-C₃-(5)) were from Nippon Kanko Shikiso (Okayama). Chlorine 36 (³⁶Cl) was obtained from Amersham as sodium chloride solution (3.8 MBq/ml, 103 µCi/ml). Clearsol^TM was obtained from NEN Life Science Products Inc. (Wilmington, DE) through Dai-ichi radio isotope (Tokyo). Nigericin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, MES,¹ HEPES, tetramethylammonium hydroxide (TMAH), sodium gluconate, and glucono--lactone were supplied by Sigma. Other reagents were purchased as commercial products mostly from Sigma.

Preparation of Lysosomes (Dextranosomes) and Solubilization of Lysosomal V-ATPase-- Preparation of fluorescein-dextran loaded lysosomes (FD-dextranosomes) from rat liver was performed as described (13), with the exception that the Percoll washout procedure was omitted and the lysosomal layer in the Percoll gradient was used for the experiments. Bovine chromaffin granules were obtained as described (14). Lysosomal V-ATPase was solubilized from membrane ghosts of Triton-filled lysosomes (tritosomal membrane ghosts, TMG) with n-octyl--D-thioglucoside in the presence of asolectin (15).

Proton Pump Assay-- The formation of a pH gradient (inside acid) was measured by means of the fluorescence quenching of FD incorporated into lysosomes (intact lysosomes) (16-18) or the permeant basic dye acridine orange (chromaffin granules). The formation of a transmembrane potential gradient (inside positive) was measured by the recovery of fluorescence quenching of diS-C₃-(5) (intact lysosomes) (19). The assay buffer, unless otherwise indicated, contained 0.1 M KCl, 0.2 M sucrose, 20 mM HEPES-TMAH (pH 7.5), 0.5 mM MgCl₂, and 1 mM ATP-Na₂ in a final volume of 2.0 ml with or without the dyes. The concentrations of the dyes were 1 µM for acridine orange and 0.5 µM for diS-C₃-(5). Fluorescence was measured at 37 °C with a Hitachi 850 or F-4500 spectrofluorometer with excitation at 495 nm and emission at 520 nm for FD, 480 and 520 nm for acridine orange, and 620 and 670 nm for diS-C₃-(5), respectively, with a 5-nm slit width for both monochrometers. The initial velocity of acidification and membrane potential formation was defined by the rate of ATP-dependent quenching of fluorescence.

ATPase Assay-- ATPase (bafilomycin A₁-sensitive ATPase) assays were performed as described (15). The assay buffer contained 0.1 M KCl, 0.2 M sucrose, 40 mM HEPES-TMAH (pH 7.5), 0.5 mM MgCl₂, and 1 mM ATP-Na₂ in a final volume of 1.0 ml and the reaction mixture was incubated at 30 °C for 40 min. The liberated phosphate was estimated by the malachite green method (20). All data shown are the averages (with deviation of less than 5%) of duplicate experiments.

Preparation of Liposomes-- Liposomes were prepared from phosphatidylcholine essentially as described (21, 22): briefly, phosphatidylcholine (Sigma type II-S phospholipids, a so called asolectin) was suspended in a buffer (50 mg/ml, usually 2 ml), vortexed vigorously, and sonified for clarity at room temperature for 5 min in 20 mM HEPES-TMAH (pH 7.5) containing 25 mM of the desired salt, with or without (for anion requirement) 2.5 mM MgSO₄ and then applied onto a spin column (Sephadex G-25 column prepared in a 1-ml penicillin syringe) equilibrated with the assay buffer (e.g. 20 mM HEPES-TMAH (pH 7.5), 25 mM salt) and centrifuged at 2,000 rpm for 30 s just before use. The recovery of liposomes was 60 to 75% as monitored by A₅₅₀. In some cases, the liposome preparation thus prepared (2 ml) was sonified for 2 min (i.e. 1 min/ml) in a cycling mode (for 1 min followed by a 1-min interval) using a probe-type sonifier (Branson Cell Disrupter 200) equipped with a 7-mm titanium probe, with power output set at 5 (continuous mode) under an Argon atmosphere on ice to prepare liposomes composed mostly of small unilamellar vesicles according to Sarti et al. (23).

Fluorescent Assay of Intraliposomal pH and Transmembrane Potential-- Intraliposomal pH was monitored by the fluorescence of pyranine incorporated in the liposomes during their preparation (24, 25). Liposome suspensions for this experiment were prepared in a buffer containing 500 µM pyranine. The amount of liposome suspension added to the 2-ml buffer was usually 50 µl (final, 0.60 ~ 0.67 mg of phospholipids/ml). The fluorescence of pyranine was monitored at an excitation wavelength of 450 nm and emission wavelength of 510 nm with a 5.0-nm slit width for both monochrometers. Membrane potential formation was monitored either by the fluorescence quenching of diS-C₃-(5) for inside negative potential (19) or oxonol-V for inside positive potential (27), respectively, at excitation wavelengths of 620 and 580 nm and emission wavelengths of 670 and 620 nm, for diS-C₃-(5) and oxonol-V, respectively, with a 5.0-nm slit width for both monochrometers.

Uptake of [³⁶Cl] into Liposomes-- Uptake experiments of [³⁶Cl] into liposomes were performed by the rapid gel filtration method (28). Briefly, 250 µl of the liposome preparation (A₅₅₀ = 0.24; 28 mg of phosphatidylcholine/ml) containing pyranine prepared in 20 mM HEPES-TMAH (pH 7.5), 5 mM potassium gluconate, and 2.5 mM MgSO₄, and filtered through Sephadex G-25 equilibrated with the assay buffer with gluconate in place of chloride (20 mM MES-TMAH (pH 6.0), 5 mM potassium gluconate) was incubated in the same volume (250 µl) of the buffer (20 mM MES-TMAH (pH 6.0), 5 mM NaCl, containing Na[³⁶Cl] (0.1 µCi/ml)) at 25 or 37 °C, inhibitor added (tributyltin chloride (TBT) or one of the prodigiosins) at 1 min and the mixture further incubated for the indicated period. After incubation, 400 µl of the incubation mixture was applied onto Dowex 1-X8 ion-exchanging resin (Cl form, prepared in a 1-ml syringe) equilibrated with the assay buffer, centrifuged at 2,000 rpm for 3 min, and the radioactivity of [³⁶Cl] in 200 µl of the eluate was measured in a liquid scintillation counter (Aloka) in a vial containing 10 ml of Clearsol^TM. All data are the averages (mean ± S.D.) of triplicate experiments.

Other Analytical Methods-- Protein level was determined by the Amido Black/solid phase method of Schaffner and Weissmann (29) or Coomassie Brilliant Blue/liquid phase method of Bradford (30) using the Bio-Rad protein assay kit according to the manufacture's instruction manual, with bovine serum albumin as the standard.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Prodigiosins Inhibit Acidification, but Affect Neither ATP Hydrolysis nor Membrane Potential Formation Mediated by V-ATPase

Prodigiosins have a general structure of methoxybipyrrolopyrromethene with hydrocarbon side chain(s) attached to the "right-hand" pyrrole ring. In this study, we used prodigiosin, metacycloprodigiosin, and prodigiosin 25-C (Fig. 1) but present the results of metacycloprodigiosin in most cases because the results were almost the same among the three prodigiosins. In the previous paper (8), we showed that prodigiosin 25-C increased lysosomal pH in cultured cells as a result of its uncoupling activity on the lysosomal H⁺-ATPase (V-ATPase), although prodigiosin 25-C uncoupled F-ATPases, too. Fig. 2A shows the dose responses of the effect of metacycloprodigiosin, as compared with bafilomycin A₁, on the acidification, bafilomycin A₁-sensitive ATPase, and membrane potential formation of lysosomal V-ATPase. Metacycloprodigiosin (and prodigiosin), like prodigiosin 25-C, inhibited acidification with an IC₅₀ of 5 ~ 20 nM (at 170 µg of protein/ml; corresponding to 30 ~ 120 pmol/mg of protein) but hardly affected ATPase activity up to ~1 µM (>50 ~ 100 times that of the IC₅₀ acidification inhibition). This contrasts with the effect of bafilomycins which inhibit both acidification and ATPase activities with an IC₅₀ of about 1 nM (Fig. 2B; for bafilomycin A₁). Furthermore, prodigiosins did not affect the membrane potential formation of V-ATPase either (Fig. 2A, for metacycloprodigiosin), which also contrasts with the effect of bafilomycins (IC₅₀ inhibition of membrane potential formation ~1 nM) (Fig. 2B; for bafilomycin A₁). This feature is unique to prodigiosins and not shared by the other basic substances tested so far in our laboratory. The insensitivity to prodigiosins of ATPase and membrane potential formation was shared also with F-ATPases (mitochondrial and bacterial H⁺-ATPases).²

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structures of prodigiosins (prodigiosin, metacycloprodigiosin, and prodigiosin 25-C).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of metacycloprodigiosin (A) as compared with bafilomycin A1 (B) on acidification, bafilomycin A1-sensitive ATPase, and membrane potential formation of isolated lysosomes. Acidification and membrane potential formation of lysosomes were measured using dextranosomes loaded with FD as described under "Experimental Procedures" in buffers (20 mM HEPES-TMAH (pH 7.5), 0.1 M KCl, 0.2 M sucrose, 0.5 mM MgCl2, and diS-C3-(5)) with or without the indicated concentrations of inhibitors (1% Me2SO as solvent control) added 1 min before the addition of 1 mM ATP-Na2. Bafilomycin A1-sensitive ATPase activity of lysosomes was measured using TMG solubilized with n-octyl--D-thioglucoside (15, 27) as described under "Experimental Procedures" in buffers with or without the indicated concentrations of inhibitors (1% Me2SO as solvent control). The ATPase reaction (for 40 min at 30 °C) was started by the addition of enzyme.

The inability of prodigiosins to inhibit membrane potential formation suggests that they functionally: 1) inhibit transformation of (driven by V-ATPase) into pH (31); 2) change the participating ion from H⁺ to another cation, either through ionophoric activity (of say nigericin, a H⁺/K⁺ exchange ionophore) or by changing the ion specificity of the ATPase (like (V or F)-type Na⁺-ATPase or respiratory Na⁺ pump) (32-34); or 3) change the participating ion from H⁺ (or OH) to another anion either through ionophoric activity (for example, TBT, a OH/Cl exchanger (35)), or by changing the ion specificity of the ATPase (like the relationship between halorhodopsin and bacteriorhodopsin (36)). But, the prodigiosins did not show any ionophoric activity against H⁺ and/or K⁺ (8). Therefore, H⁺/Cl symport (or OH/Cl exchange) activity was suspected. In fact, their activity to inhibit vesicular acidification required chloride (with no obvious saturation), even in the presence of K⁺-valinomycin added to the assay buffer to dissipate and to eliminate possible participation of chloride channel(s).^2,3 On the basis of these observations, we next tested if prodigiosins show H⁺/Cl symport (or OH/Cl exchange) activity on phospholipid membranes using liposomes that are devoid of proton pump protein.

H⁺/Cl^- Symport (OH^-/Cl^- exchange) Activity of Prodigiosins on Liposomes

Prodigiosins Produced Chloride Gradient-dependent Perturbation of Intraliposomal pH-- Depending on the preliminary observations, we performed a direct assay of the H⁺/Cl symport activity of prodigiosins by measuring the effect of prodigiosins on the chloride-dependent change of internal pH of the liposomes (Fig. 3). In Fig. 3A, liposomes containing the pH-sensitive fluorescent probe pyranine (24) prepared in KCl were diluted 40-fold in external buffer containing either gluconate (sodium gluconate) or chloride (NaCl). In control experiments, Me₂SO or EtOH (solvent control), added instead of prodigiosins or TBT, did not produce significant change in the fluorescence, and the addition of MES-TMAH produced a rapid decrease in fluorescence to a certain extent (indicative of the presence of non-trapped extraliposomal pyranine) followed by a slow gradual decrease of fluorescence (indicating acidification of the liposome interior), suggesting limited permeability of liposomal membranes to protons (or hydroxyl anions). On the other hand, metacycloprodigiosin (1 µM), like TBT (100 nM), produced a rapid increase in the pyranine fluorescence (indicating internal alkalinization) in gluconate buffer but not in chloride buffer. However, the addition of MES-TMAH (pH 6.0) produced a marked decrease of pyranine fluorescence (indicating intraliposomal acidification) in prodigiosin- or TBT-treated liposomes relative to control (Me₂SO or EtOH), and the rate of this decrease was greater in chloride than in gluconate buffer.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Chloride gradient-dependent changes of intraliposomal pH produced by metacycloprodigiosin and TBT. Liposomes containing pyranine, prepared in a buffer (20 mM HEPES-TMAH, pH 7.5) containing either (A) KCl (25 mM KCl, 2.5 mM MgCl2) (Cl--containing liposomes) or (B) potassium gluconate (25 mM potassium gluconate, 2.5 mM MgSO4) (Cl--free liposomes) were diluted 40-fold in external buffer (20 mM HEPES-TMAH, pH 7.5) containing either 25 mM sodium gluconate or NaCl and the pyranine fluorescence was recorded as described under "Experimental Procedures." A, Cl--containing liposomes. MES-TMAH (pH 6.0), 50 mM; TX-100, 0.2% Triton X-100; Meta, 100 nM metacycloprodigiosin; TBT, 1 µM; DMSO, 1% Me2SO (solvent control for metacycloprodigiosin) in sodium gluconate buffer (dashed line); EtOH, 1% EtOH (solvent control for TBT) in sodium gluconate buffer (dashed line). B, Cl--free liposomes. MES-TMAH (pH 6.0), 50 mM; TX-100, 0.2% Triton X-100; Meta, 1 µM metacycloprodigiosin; TBT, 100 nM; DMSO, 1% Me2SO (solvent control for metacycloprodigiosin) in NaCl buffer (dashed line); EtOH, 1% EtOH (solvent control for TBT) in NaCl buffer (dashed line).

Dependence of the Prodigiosin-mediated Liposomal Acidification on External Chloride Ion-- In Fig. 4A, pyranine-containing liposomes prepared in potassium gluconate were diluted 40-fold in external buffer consisting of different ratios (1:0, 1:1, 1:3, 1:7, and 0:1) of NaCl and sodium gluconate. The results clearly show that both metacycloprodigiosin and TBT acidified intraliposomal pH almost linearly with no obvious saturation dependent on the chloride concentration as summarized in Fig. 4B: again note that the rate of the acidification in gluconate was greater in prodigiosin- than TBT-treated liposomes. This result is also consistent with the ionophoric nature of intraliposomal acidification driven by prodigiosins. In order of effectiveness, the inhibitors ranked as follows: TBT > prodigiosin, metacycloprodigiosin > prodigiosin 25-C: the effective concentrations in chloride buffer for 50% of maximum fluorescence quenching in 10 s were 1.0, 0.7, and 9.0 nmol/mg phospholipid for prodigiosin, metacycloprodigiosin, and prodigiosin 25-C, respectively, as compared with 23 pmol/mg phospholipid for TBT.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Chloride dose responses of intraliposomal acidification mediated by metacycloprodigiosin and TBT. Liposomes containing pyranine, prepared in 20 mM HEPES-TMAH (pH 7.5), 25 mM potassium gluconate, and 2.5 mM MgSO4 were diluted 50-fold in external buffer (20 mM HEPES-TMAH (pH 7.5) containing 25 mM NaCl or sodium gluconate (pH 7.5), alone or in combination at ratio of 1:0, 1:1, 1:3, 1:7, or 0:1, respectively) and the pyranine fluorescence was recorded as described under "Experimental Procedures." At the times indicated (arrows), 100 nM of either metacycloprodigiosin (1% M2SO as solvent control) (top) or TBT (1% EtOH as solvent control) (bottom) was added. A, fluorescence trace. Meta, 100 nM metacycloprodigiosin; TBT, 100 nM; TX-100, 0.2% Triton X-100; DMSO, 1% Me2SO in NaCl buffer; EtOH, 1% EtOH in NaCl buffer. B, chloride dose-response. Rates of acidification (% of maximum values) are plotted against mol % of Cl in Cl/gluconate buffer.

Electroneutral Nature of the Prodigiosin-catalyzed H⁺/Cl^- Symport-- In Fig. 5, pyranine-containing liposomes prepared in KCl (and MgCl₂) at pH 6.0 were first diluted 40-fold in external buffer of either sodium gluconate or potassium gluconate (pH 7.5), then added with 5 µM valinomycin followed by prodigiosin (1 µM) or TBT (100 nM) to see the effect of inside-negative membrane potential generated by the addition of valinomycin on the prodigiosin- and TBT-induced liposomal alkalinization. The rate of increase of fluorescence after the addition of prodigiosin was slightly accelerated by the presence of valinomycin (Fig. 5A). However, this occurred irrespective of the cation species (sodium or potassium) (with the relative extent of the acceleration being unchanged), similarly to TBT (Fig. 5IB) the electroneutrality of whose action has been well established (37, 38). The membrane potential measured by the fluorescence of diS-C₃-(5) and oxonol-V also suggested little membrane potential difference produced by prodigiosins. These results suggest that the H⁺/Cl symport (or OH/Cl antiport) mediated by prodigiosin is electroneutral in essence.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Electroneutrality of intraliposomal alkalinization induced by metacycloprodigiosin (A) and TBT (B). Potassium-loaded liposomes containing pyranine were prepared from phosphatidylcholine (100 mg) in 2.0 ml of 25 mM KCl, 20 mM HEPES-TMAH (pH 7.5), and 500 µM pyranine, diluted 50-fold into 2 ml of assay buffer consisting of 20 mM HEPES-TMAH (pH 7.5) and 25 mM solution of either sodium gluconate or potassium gluconate, and the pyranine fluorescence was recorded as described under "Experimental Procedures" in the presence or absence of valinomycin. Meta, 1 µM metacycloprodigiosin; TBT, 100 nM; TX, 0.2% Triton X-100; Val, 5 µM valinomycin; DMSO, 1% Me2SO (solvent control for metacycloprodigiosin) in sodium gluconate buffer; EtOH, 1% EtOH (solvent control for TBT) in sodium gluconate buffer.

Prodigiosin-mediated Chloride Transport in Liposomes Measured using [³⁶Cl]-- To obtain direct evidence that prodigiosins possess activity to promote the movement of chloride across membranes, we first tried to extract chloride with organic solvent. Like TBT and manganese porphyrin (a recently reported chloride ionophore (39, 40)), prodigiosins showed some, albeit weak, activity to mediate the movement of chloride into organic solvent, notably at acidic pH (data not shown). However, the phase transfer activity of prodigiosins does not necessarily explain their "catalytic activity" to promote transport of Cl across membranes coupled with H⁺ (or in exchange with OH). So, we next measured [³⁶Cl] movement in parallel with the change in intraliposomal pH. As shown in Fig. 6, all three prodigiosins tested, like TBT, mediated dose- and time-dependent uptake of chloride into liposomes coupled with concomitant intraliposomal acidification (indicating uptake of H⁺ or extrusion of OH). Among the prodigiosins tested, prodigiosin and metacycloprodigiosin are the most potent with prodigiosin 25-C having about half their activity. Fig. 7 shows the pH dependence of intraliposomal uptake of [³⁶Cl] and H⁺ promoted by metacycloprodigiosin and TBT. The results show an accelerated uptake of both [³⁶Cl] and H⁺ at acidic pH for both metacycloprodigiosin and TBT, although slight differences are noted between metacycloprodigiosin and TBT in pH dependence. The temperature dependence of the uptake of [³⁶Cl] into liposomes indicated an activation energy of about 15 ~ 16 kcal/mol for both metacycloprodigiosin and TBT which matched with the value reported for TBT (41).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Concomitant uptake of chloride [36Cl] (A) and proton (or extrusion of hydroxyl ion) (B) into liposomes mediated by prodigiosins and TBT. Uptake of [36Cl] into liposomes was performed according to "Experimental Procedures." Briefly, potassium-loaded liposomes (50 mg/ml) containing 500 µM pyranine prepared in 20 mM HEPES-TMAH (pH 7.5), 5 mM potassium gluconate and 2.5 mM MgSO4 were centrifuged through Sephadex G-25 equilibrated with 20 mM MES-TMAH (pH 6.0) and 5 mM KCl and the eluate was used for the experiments as liposome suspension (28 mg of phospholipids/ml). The assay buffer contained 20 mM MES-TMAH (pH 6.0), 5 mM KCl, and Na[36Cl] (0.1 µCi/ml). 250 µl each of the above liposome suspension and assay buffer were mixed and the reaction started at 37 °C. At 1 min, 5 µl of either prodigiosins (1% Me2SO as solvent control) or TBT (1% EtOH as solvent control) was added to the assay buffer to attain the indicated final concentrations. Mean ± S.D. of triplicates are plotted. Fluorescence assays were performed as described under Fig. 3, after dilution of 40 µl of the above liposome suspension into the above assay buffer. DMSO, 1% Me2SO; EtOH, 1% EtOH; PG, prodigiosin; 25-C, prodigiosin 25-C; Meta, metacycloprodigiosin; TX-100, 0.2% Triton X-100.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   pH dependence of the uptake of (A) chloride [36Cl] and (B) proton into liposomes mediated by metacycloprodigiosin and TBT. Preparation of pyranine-containing, potassium-loaded liposomes and the uptake assays of [36Cl] and proton were as described in the legend to Fig. 6, except that the pH of the assay buffer was either 6.0 (20 mM MES-TMAH), 7.0 (20 mM HEPES-TMAH) or 8.0 (20 mM Bicine-TMAH). Mean ± S.D. of triplicates are plotted. Meta, 1 µM metacycloprodigiosin; TBT, 1 µM TBT; DMSO, 1% Me2SO (solvent control for metacycloprodigiosin); EtOH, 1% EtOH (solvent control for TBT).

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

In recent years, there have been several important findings regarding the mechanism of energy transduction, some relating to structural biology (for F₀F₁-ATPase (42) and cytochrome oxidase complexes (43, 44)), others to combined molecular fluorescence micromanipulation technology (45) (showing molecular spin of H⁺-ATPase molecules). Nevertheless, one important aspect of energy transduction, "energy coupling," remains to be clarified. From a biochemical point of view, specific modifiers of energy transduction would represent a breakthrough, just as uncouplers opened the way to the chemiosmotic hypothesis of Mitchell in 1961.

Prodigiosins were initially thought of as candidates of such modifiers to help elucidate the energy transduction mechanism: prodigiosins inhibited the acidification mediated by various H⁺-translocating ATPases without inhibiting ATP hydrolysis nor membrane potential formation (8) (Fig. 2), unlike ordinary uncouplers of oxidative phosphorylation or ATPase inhibitors. However, evidence has been accumulating that prodigiosins have an ionophoric nature and we demonstrated in the present study that prodigiosins act as a H⁺/Cl symporter (or OH/Cl antiporter, in its equivalence) on liposomes, by showing: 1) alkalinization and acidification of intraliposomal pH depending on the outward and inward chloride gradient, respectively; 2) the electroneutrality of their effects; and finally, 3) the pH gradient (outside acid)-dependent uptake of radioactive chloride into liposomes in parallel with intraliposomal acidification.

These results on the whole favor the idea that the uncoupling effect of prodigiosins is due to their H⁺/Cl symport activity across biological membranes. In fact, their effective concentrations for H⁺/Cl symporting activity on liposomes decreased in parallel with the amount of phospholipid used, and were comparable to those on organellar membranes (8) when expressed on the basis of the amount of vesicles used (in the order of nmol/mg: for 50% maximum pH change in 10 s).² The possibility of specific interaction of prodigiosins with H⁺-translocators is, however, not completely excluded, especially at higher concentrations where they inhibited catalytic (ATPase) activity. Studies are now in progress to unravel the mode of direct interaction between prodigiosins and the enzymes. The reason for the much higher effectiveness of TBT on liposomes (Figs. 4 and 6)^2,3 is not clear. It may reflect difference of permeability against the ionophore between liposomes and organellar membranes due to compositional differences (for example, absence or presence of proteins).

The H⁺/Cl symport (or OH/Cl antiport) activity of prodigiosins also explains several previous findings: 1) that the ATP level was hardly affected by prodigiosin 25-C; 2) mitochondrial swelling induced by prodigiosin 25-C, and so on. As prodigiosins did not affect the membrane potential formation, they are not expected to affect ATP formation in mitochondria where the proton motive force is stored mainly in the form of transmembrane potential. In this regard, prodigiosins are quite similar to the traditional OH/Cl exchangers, triorganotins (for example, TBT), which inhibit vesicular acidification but hardly affect membrane potential formation in reconstituted cytochrome oxidase proteoliposomes (46) nor ATP formation in the photophosphorylation driven by halorhodopsin (47). Like prodigiosins, triorganotins induce mitochondrial swelling (48, 49), which is explained by the uptake of osmotically active Cl in exchange for respiration-induced OH (equivalent to coupled transport of Cl with respiration-induced H⁺). However, triorganotins act, at the same time, as uncouplers of oxidative phosphorylation and inhibit mitochondrial ATP formation, because they inhibit the F-ATPase molecules themselves through SH-protecting reagent (for example, N-ethylmaleimide)-sensitive binding (46, 50-54)). In fact, the effect of TBT on the acidification by H⁺-ATPases was not suppressed by the presence of large amounts of liposomes, contrary to the effect of prodigiosins.⁴ Golgi swelling might be due to pH-induced accumulation of osmotically active protonated weak bases (prodigiosins) within the Golgi apparatus. Golgi, rather than lysosomes swell possibly because Golgi membranes are less permeable to K⁺ (19, 55), resulting in more Cl uptake than K⁺ extrusion.

The nature of prodigiosins as H⁺/Cl symporters also suggests that there are additional translocators or biological activities affected by prodigiosins: they will include respiratory proton pumps, vacuolar H⁺-translocating pyrophosphatases, ATP-dependent transhydrogenases of NADP⁺ and NADH, H⁺- or Cl-transporting bacterio/halo-rhodopsins, H⁺(OH)- or Cl-coupled symporters or antiporters, and Cl-pumps. Prodigiosins may prove useful for the clarification of ion transport mechanisms.

What is the mechanism of H⁺/Cl symport (or OH/Cl antiport) of prodigiosins? Organotins are considered to support the exchange of halides with OH across membranes due to their covalent bonding (co-ordination) with halides on one side of the membrane accompanied by their hydrolyses on the other side (41, 57, 58). Prodigiosins, on the other hand, probably bind with halides electrostatically in accordance with Hofmeister series (possibly helped by the stabilizing effect of hydrogen-bonding and/or charge transfer (59, 60) interactions of protonated nitrogen in prodigiosins), resulting in the formation of lipophilic ion pairs which facilitate proton-coupled transmembrane transport of halides as do phase transfer catalysts (61, 62). In this sense, prodigiosins are probably H⁺/Cl^- symporters rather than OH/Cl exchangers: protonated prodigiosins are less likely to bind OH, especially at acidic pH. In addition, prodigiosins show anion specificity similar to that of TBT but with some important differences: for example, gluconate prefers prodigiosins to TBT (Figs. 3 and 4). Studies on the crystallographic structure of prodigiosin salts as well as on the structure-activity relationship will help clarify the essential structures and the action mechanism of these new H⁺/Cl symporters.

Several other new H⁺/Cl symporters (or OH/Cl exchangers) have been reported recently. They include thallium chloride (Tl³⁺, e.g. TlCl₃) (63), bepridil (64), Hg²⁺ and Cu⁺ (65), sapphyrin (66), and cryptate (67). Also, the number of new Cl sensing ionophores has been accumulating: they include Mn²⁺-porphyrin (39, 40), pamamycin (68), mercury organic compounds (ETH 9018 (69)), besides methyltributylammonium chloride, tetrabutylammonium chloride, and tridodecylmethylammonium chloride, some of which may exhibit H⁺/Cl symporting (OH/Cl antiporting) activity, too.

The activity of prodigiosins presented in this article suggests that these anion-exchanging compounds constitute a new group of probes for the analysis of vacuolar function, as their effects are relatively selective to vacuolar pH in vivo, hardly affecting the cellular ATP level. Namely, we can expect that they affect all aspects of cellular functions involving V-ATPase, including endocytosis, exocytosis, and intracellular trafficking as well as cell growth, cell differentiation, and cell death (apoptosis). In fact, we have already reported suppressive activity of prodigiosins on various aspects of immunity and bone resorption (2-7). Also, we can expect prodigiosin-specific reactions, because prodigiosins do not seriously affect membrane potential formation: for example, they selectively inhibit pH-dependent uptake of certain neurotransmitters (like dopamine or histamine) without affecting dependent uptake of other neurotransmitters (like glutamate) into synaptic vesicles (70). In summary, we have now three different types of vacuolar perturbators in hand: 1) weak bases and acidic ionophores which raise the pH and induce swelling of the vacuolar system; 2) prodigiosin group H⁺/Cl symporters that affect only vacuolar pH; and 3) bafilomycin group antibiotics (including concanamycins and destruxins) that affect both pH and membrane potential formation. By proper application of these probes, we will be able to clarify much more extensively the unknown functions of the vacuolar system. Furthermore, the H⁺/Cl symport activity of the compounds reported in this study may actually be a sort of "missing link" and should be taken seriously, especially in regard to therapeutic drugs.

Finally, prodigiosins are also interesting from the point of view of organic synthesis, because they are small enough for chemical modifications aimed at providing more active and less toxic compounds. Although their synthetic history is long (71-74), a simple and elegant way to synthesize prodigiosins was reported recently (75) which will help in the synthesis of new active derivatives.