Direct inhibitory effect of CCCP on the Clminus -H+ symporter of the guinea pig ileal brush-border membrane

Francisco Alvarado and Monique Vasseur

Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France

    ABSTRACT
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Abstract
Introduction
Methods
Results & Discussion
Appendix
References

The effect of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on Cl- uptake across the brush-border membrane (BBM) was quantified using 36Cl and BBM vesicles from guinea pig ileum. CCCP inhibited only partially both the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities present in these vesicles. In contrast, CCCP had no effect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the main action of CCCP does not consist of dissipating any imposed pH gradient but rather in inhibiting directly the pH gradient-activated Cl- uptake and Cl-/Cl- exchange activities characterizing the intestinal BBM. Because these two activities can be explained in terms of a single (homogeneous) random, nonobligatory two-site Cl--H+ symporter, in which Cl-/Cl- exchange occurs by counterflow [F. Alvarado and M. Vasseur. Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628, 1996], we developed a new, more general three-site symport model that fully explains the Cl- uptake inhibitions caused by CCCP. This new model postulates the existence of a third, allosteric, inhibitory CCCP-binding site separate from either of the two substrate-binding sites of the Cl--H+ symporter, the Cl--binding and the H+-binding sites. Finally, we show that, to explain the partial inhibitions observed, it is necessary to postulate that all the substrate-bound carrier complexes, =C-S, I=C-S, A=C-S, and IA=C-S, where C is carrier, I is inhibitor, S is substrate, and A is activator, can form and be translocated.

chloride transport; carbonyl cyanide-m-chlorophenylhydrazone; chloride ion; hydrogen ion

    INTRODUCTION
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Abstract
Introduction
Methods
Results & Discussion
Appendix
References

CARBONYL cyanide-m-chlorophenylhydrazone (CCCP) has long been classified as an uncoupler. By definition, uncouplers increase the proton permeability of the mitochondrial membrane, thereby preventing formation of the proton gradients postulated to be the energy source for ATP synthesis from ADP and phosphate, according to Mitchell's chemiosmotic coupling hypothesis for oxidative phosphorylation (7, 12). CCCP is a weak organic acid thought to act as a classic proton carrier, interacting with protons according to a monomolecular mechanism whereby CCCP and H+ cross the mitochondrial membrane in the neutral, undissociated acid form, CCCP-H. However true, these facts have been unduly generalized to other membrane systems, and this is why CCCP is presently generally regarded, without further evidence substantiating the generalization, as a pure protonophore capable of rapidly dissipating pH gradients across practically any biological membrane, provided, of course, that the appropriate counterions are present.

However, as first pointed out by Bakker et al. (4), the effectiveness of uncouplers may vary considerably, depending on the nature of the membrane, so that indiscriminate generalization of the CCCP effects on mitochondria to other membrane systems appears to be unwarranted.

The present paper concerns the effect of CCCP on pH gradient-activated Cl- uptake across the ileal brush-border membrane (BBM). As shown previously, this Cl- uptake involves a Cl--H+ symporter that is strongly inhibited by CCCP (3, 17). The observation that this inhibition occurs in both the absence and presence of short-circuiting conditions suggested that CCCP may act not only indirectly, by facilitating dissipation of an imposed pH gradient, but also, perhaps mainly, by directly inhibiting the Cl--H+ symporter (see Refs. 8 and 17). Up to now, a clear-cut explanation of the mechanism (or mechanisms) involved in CCCP inhibition has been lacking. The present work, specifically addressing this question, is based on the premise that a random, nonobligatory Cl--H+ symporter can adequately explain both the pH gradient-dependent Cl- uptake and the Cl-/Cl- exchange activities that characterize the BBM (see Ref. 3). If the main action of CCCP is to act directly on the Cl--H+ symporter, and not indirectly by dissipating an imposed pH gradient, then it should be expected that, in the absence of a pH gradient, both Cl- uptake and Cl-/Cl- exchange will be inhibited by CCCP. This proposal was investigated using BBM vesicles from guinea pig ileum. Cl- uptake was studied as a function of the extravesicular pH and the cis Cl- and CCCP concentrations, in both the absence and presence of trans Cl-. The results upheld the hypothesis that the main action of CCCP is to inhibit Cl- uptake directly.

A preliminary account of this work has been given (15).

    METHODS
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Abstract
Introduction
Methods
Results & Discussion
Appendix
References

Materials

H36Cl (0.4 mCi/mmol; Amersham, Arlington Heights, IL) was neutralized with tris(hydroxymethyl)aminomethane base before use. CCCP, valinomycin, and Triton X-100 were from Sigma (St. Louis, MO); tetramethylammonium hydroxide pentahydrate (TMA) was from Aldrich (Milwaukee, WI); and pyranine was from Eastman Kodak (Rochester, NY). All other chemicals were also of the highest purity available.

Membrane Vesicle Preparation and Transport Assay

After they were stunned, guinea pigs were killed by cervical dislocation, and BBM vesicles were prepared as described (14). Transport was measured using a rapid filtration technique (9), with 36Cl as the substrate. Initial uptake rate measurements (2 s) were performed using a short-time incubation apparatus (Innovativ Labor, Zürich, Switzerland) in a constant-temperature room at 23 ± 2°C, as described (17). Similar to valinomycin (17), CCCP dissolved in ethanol was allowed to evaporate to dryness before it was mixed with the membrane vesicle preparation.

Results are expressed (6) as either absolute uptakes (nmol/mg membrane protein) or absolute velocities (nmol · s-1 · mg membrane protein-1) and are presented as means ± SD of either representative experiments or of the pool of several experiments performed with two or more different membrane preparations. Uptake data were statistically compared by applying a global one-way analysis of variance (13). Uncorrected initial absolute entry rates as a function of the cis Cl- concentration were fitted by nonlinear least-squares regression analysis to an equation containing one saturable Michaelian transport system plus a diffusional component, as described (3). Details on the statistical evaluation of the kinetic results are given in Table 1. To test the fit of our data to equations derived from the general three-site symport model, we used commercial programs such as Multifit (Day Computing, Cambridge, UK). All calculations were performed using an Apple Macintosh microcomputer.

                              
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Table 1.   Quantitative evaluation of kinetic results in Fig.1

Spectrofluorometrical Studies

Proton fluxes were measured by monitoring changes in the fluorescence intensity of the pH-sensitive dye pyranine previously trapped within the vesicles (17).

    RESULTS AND DISCUSSION
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Methods
Results & Discussion
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Mixed-Type Inhibitory Effect of CCCP on the Kinetics of pH Gradient-Dependent Cl- Uptake

As shown previously, the kinetics of Cl- uptake in the presence of an alkaline-inside pH (pHin) gradient can be described by an equation involving a single Michaelian transport term plus a linear, nonsaturable component (17). To study the mechanism of CCCP inhibition, we began by comparing directly the kinetics of pH gradient-dependent Cl- uptake in either the absence or presence of CCCP. Because it is known that pH gradient-dependent Cl- uptake is noncompetitively inhibited by trans K+ (18), the necessary saturation curves were performed in absence of intravesicular K+.

The results (Fig. 1 and Table 1) indicate that the inhibition caused by CCCP is mixed; it involves both an inhibitory capacity effect and an inhibitory affinity effect, as indicated by the 69% drop in maximal velocity (Vmax) and the 167% increase in the apparent Michaelis constant (KT), respectively. If CCCP acted solely by facilitating dissipation of the pH gradient as, for example, trans K+ is known to do, Vmax would have been the only parameter affected (see Ref. 18). Therefore, the above findings strongly support the interpretation that the main action of CCCP does not consist of the dissipation of an imposed pH gradient. Rather, CCCP would act mainly by inhibiting directly the Cl--H+ symporter. Nevertheless, as such, these results cannot exclude the possibility that, on top of having a direct effect, CCCP could also have an indirect effect consisting of at least a partial diminishing of the pH gradient that constitutes the driving force for Cl- uphill transport under these conditions. Further work was therefore performed to address this question. The results support the conclusion that the main action of CCCP is indeed direct.


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Fig. 1.   Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) effects on kinetics of proton-coupled Cl- uptake by brush-border membrane (BBM) vesicles. Cl- saturation curves were performed using 0 trans outside Cl- concentration ([Cl-]out) values ranging from 4 to 84 mM in either absence (triangle ) or presence (black-triangle) of 300 µM CCCP. Both the extra- and intravesicular spaces contained a 20 mM HEPES-40 mM citric acid buffer supplemented with 200 mM Tris gluconate and adjusted with Tris base to give an initial outside pH/inside pH (pHout/pHin) gradient of 5.0/7.5. Results are means ± SD in nmol · s-1 · mg protein-1. Statistical analysis of results is given in Table 1.

Effect of CCCP on Cl- Influx Rate Across BBM Vesicles in Both Presence and Absence of Alkaline pHin Gradients

Effect of CCCP in absence of a pH gradient. At equilibrium [outside pH (pHout) = pHin = 5.5], there was a weak uptake of Cl- that nearly doubled (86% activation) when both the intra- and the extravesicular pH were increased by 2 pH units (compare Table 2, bottom set of data, lines 1 and 2). In the presence of 250 µM CCCP, both these uptakes were strongly inhibited by either 40% at pH = 5.5 or 66% at pH = 7.5. It should be emphasized that, in both cases, the total Cl- uptake dropped exactly to the same level (0.04 ± 0.01 nmol · s-1 · mg protein-1). Because diffusion is by definition insensitive to inhibition by "regular" effectors, this result appears to indicate that these uptakes correspond roughly to those expected from simple physical diffusion. If this were the case, CCCP inhibition would be complete and all mediated uptake would be inhibited by CCCP. Alternatively, however, the possibility exists and has demanded further study that the diffusion level lies below the line just defined, meaning that CCCP inhibition might be only partial. One way or another, at this point, the conclusion already seems inevitable that CCCP inhibits Cl- uptake both strongly and directly because, under the present equilibrated pH conditions, CCCP cannot possibly be said to act by dissipating a nonexisting pH gradient.

                              
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Table 2.   Effect of CCCP on initial Cl- uptake rates into brush-border membrane vesicles in either absence or presence of pH gradients and in either absence or presence of trans Cl- at equilibrated pH

Effect of CCCP in presence of an alkaline pHin gradient. In agreement with previous observations (17), when a pH gradient was superimposed, for instance, when pHout/pHin = 5.0/7.5 (Table 2, bottom set of data, line 3), Cl- uptake was strongly stimulated, respectively, by either 657 or 307%, depending on whether the reference, equilibrated pH, was either 5.5 or 7.5. Again, 250 µM CCCP was strongly inhibitory under these conditions, but a qualitatively quite meaningful difference became apparent. In contrast to the results obtained in the absence of a pH gradient, CCCP inhibition under pH gradient conditions was clearly partial. The total Cl- uptake rate observed could be decomposed into 64% of a CCCP-sensitive component and 36% of a noninhibitable component, and this last component was clearly greater than zero (0.19 nmol · s-1 · mg protein-1 vs. 0.04 nmol · s-1 · mg protein-1 under equilibrated pH conditions). There is no obvious explanation for the apparent lack of accord between these results, namely, why inhibition is either complete or partial in absence and presence, respectively, of a pH gradient. But, as we show, a closer analysis of the situation proves that CCCP inhibition is indeed partial under either condition.

Dose-dependent effects of CCCP on Cl- uptake. To confirm and extend the above observations, the experiment in Table 2 was repeated at variable CCCP concentrations. In both the presence and absence of a pH gradient (see Fig. 2), CCCP inhibited Cl- uptake in a concentration-dependent manner. In both cases, a distinct plateau greater than zero was attained, indicating the existence of partial inhibition. Again, however, the plateau attained in the presence of a pH gradient was about five times higher than that observed under equilibrated pH conditions. To quantify the fraction of Cl- uptake that is not inhibitable by CCCP, the results were linearized according to the Inui and Christensen (10) transformation. From the reciprocal of the y-axis intercept of the straight lines obtained (Fig. 2, inset), it was deduced that ~31 and 35% of the total Cl- uptake at Delta pH values of either 0 or 2.5, respectively (where Delta pH = pHin - pHout), are insensitive to inhibition by CCCP. From these results, an apparent kinetic diffusion constant (Kd; for an operational definition of this parameter, see Ref. 6) was also calculated equal to 13.3 and 63.0 nl · s-1 · mg protein-1 at Delta pH values of either 0 or 2.5, respectively. These Kd values are more than 2 and 10 times higher, respectively, than those estimated previously using a different approach (for further details, see Fig. 7). From the whole set of these results, we conclude that the apparent diffusion level, which is not constant, does not reflect the true Cl- physical permeability, confirming that the inhibition caused by CCCP is indeed partial. As shown next, two entirely different interpretations of these results can be given, depending on whether the transport system under investigation is homogeneous or heterogeneous (see Ref. 1).


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Fig. 2.   Inhibition of Cl- influx into BBM vesicles as a function of CCCP concentration ([CCCP]). Extra- and intravesicular spaces contained a 20 mM HEPES-40 mM citric acid buffer supplemented with 200 mM Tris gluconate and adjusted with Tris base to give pHout/pHin gradients of either 7.5/7.5 (black-square) or 5.0/7.5 (black-triangle). Cl- uptake was determined with 3 mM 0 trans 36Cl as substrate and the indicated [CCCP]. Initial Cl- entry rates are means ± SD in nmol · s-1 · mg protein-1; n = 6-12 determinations per point. In inset, same data are plotted according to Inui and Christensen (10). Vo and Vi, initial velocities in absence and presence of indicated inhibitor concentrations, respectively.

First, if Cl- uptake were heterogeneous, the partial inhibitions observed could warrant the interpretation that there are two distinct Cl- transport systems or pathways, one that is fully inhibited and one that is totally unaffected by CCCP. In accordance with this proposal, the results in Fig. 2 permit the calculation that the relative proportions of each of these pathways are 60-70 and 30-40% for the inhibitable and the noninhibitable systems, respectively, independent of the absolute pH value. In principle, however, this interpretation should be rejected, because all of the evidence available to us at present indicates that, after correction for the diffusion component, Cl- transport across the BBM involves a single carrier that is, by definition, homogeneous (3, 17, 18).

Second, if we admit to the contrary that only one Cl- transport system exists in these vesicles, then it can be postulated that CCCP inhibits allosterically (for an operational definition of this term, see Ref. 2). Such an explanation would require postulation of the existence of an additional specific inhibitory CCCP-binding site. By binding to this site, CCCP could induce a conformational change in the carrier whereby a change in either Vmax (capacity-type inhibition), KT (affinity-type inhibition), or a mixture of both (mixed-type inhibition) can be expected to result. The kinetic results previously described (see Mixed-Type Inhibitory Effect of CCCP on the Kinetics of pH Gradient-Dependent Cl- Uptake and Table 1) are fully compatible with this last possibility.

Effect of CCCP on Cl-/Cl- Exchange Activity of BBM Vesicles

Having established that CCCP does directly inhibit the Cl--H+ symporter, we studied its effect on the Cl-/Cl- exchange activity also present in these vesicles. If a random, nonobligatory Cl--H+ symporter can explain both the pH gradient-dependent Cl- uptake and the Cl-/Cl- exchange activities in terms of a single "mobile carrier" where exchange occurs by counterflow (3), then it can be predicted that CCCP should also inhibit the Cl-/Cl- exchange activity. The results confirm the inhibitory effect of CCCP at Delta pH = 0 (Fig. 3, lower 2 curves). These effects were practically instantaneous, because the initial (2 s) Cl- entry rates were significantly decreased by ~65% under either condition. Furthermore, the inhibitions were quantitatively equivalent during the first 2-20 s in both the absence and presence of trans Cl-.


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Fig. 3.   CCCP effect on Cl-/Cl- exchange activity of BBM vesicles under equilibrated pH conditions. Cl- uptake was determined with 14 mM 36Cl as substrate in both absence and presence of 200 mM cold intravesicular Cl-. Extra- and intravesicular spaces contained a 20 mM HEPES-40 mM citric acid buffer (pH 7.5) supplemented with a 200 mM K+ salt of either gluconate or Cl- to obtain outside Cl- concentration/inside Cl- concentration ([Cl-]out/[Cl-]in) gradients of either 14/0 mM (square , black-square) or 14/200 mM (triangle , black-triangle). Valinomycin (10 µg/mg membrane protein) was present throughout. When present, CCCP was at 250 µM (black-square, black-triangle). Absolute Cl- uptakes are means ± SD in nmol/mg protein; n = 6-12 determinations per point.

Similar to those observed on zero-trans Cl- uptake, the CCCP effects on Cl-/Cl- exchange were also partial. Finally, Fig. 3 further illustrates that identical uptakes at equilibrium were obtained; i.e., all four curves in this figure converged after a 2-h incubation period, indicating that the apparent vesicular volume (or "functional vesicle yield," see Ref. 5) is not affected by CCCP.

To complement the preceding observations, the experiment in Fig. 3 was repeated at variable trans Cl- concentrations. The relevant results (Table 2, bottom set of data) confirm that the rate of Cl- uptake increases as the trans Cl- concentration increases (see Ref. 17). They further indicate that CCCP inhibits to the same extent (~68%) all of the Cl-/Cl- exchange activities observed. As a consequence, Cl- uptake in the presence of 250 µM CCCP increases as the trans Cl- concentration increases. By dividing this uptake by the substrate concentration (14 mM), apparent Kd values were calculated equal to 13.6, 21.4, and 37.9 nl · s-1 · mg protein-1 at 0, 75, and 200 mM trans Cl-, respectively. The fact that the apparent Kd is not constant confirms that CCCP inhibition is partial even when Delta pH = 0.

Effect of CCCP on Cl- Efflux at Equilibrated pH

To further test whether CCCP interacts directly with the Cl--H+ symporter, we next investigated its effect on Cl- efflux at equilibrated pH values of either 7.5 or 5.5. In the presence of an outside-directed Cl- gradient, the intravesicular Cl- content decreased with time, indicating Cl- efflux (Fig. 4). Similar to the Cl- influx results described above (Table 1), the Cl- efflux rate was slower at pH 5.5 than at pH 7.5. Furthermore, efflux was inhibited by CCCP to give efflux rates that were essentially the same at either pH value (Fig. 4, bottom curve). Such a result indicates strongly that Cl- influx and efflux both involve the same CCCP-inhibitable pathway, entirely in accord with the interpretation that a single reversible carrier system is involved in all forms of Cl- transport observed across the intestinal BBM.


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Fig. 4.   Effect of CCCP on Cl- efflux from Cl--loaded BBM vesicles under equilibrated pH conditions. Extra- and intravesicular spaces contained a HEPES-citric acid-Tris gluconate buffer adjusted with Tris base to give pHout/pHin ratios of either 7.5/7.5 (square , black-square) or 5.5/5.5 (triangle , black-square). Cl- efflux was determined as described by Vasseur et al. (18) after charging the vesicles with buffers containing 5 mM 36Cl, followed by mixing the loaded vesicles with buffers of the same composition without added Cl-. Because of carryover of a fixed quantity of 36Cl from the preincubation to incubation media (proportion of 1/20), the imposed, initial [Cl-]out/[Cl-]in ratios = 0.25/5 mM. When present, CCCP was at 250 µM. Cl- efflux was calculated, in percent, as difference between intravesicular 36Cl content (in nmol/mg protein) before and after incubation for the indicated time periods. Because statistically indistinguishable results were obtained in the presence of CCCP, independent of pH, relevant results have been pooled and are illustrated under the same symbol (black-square); n = 3-12 determinations per point.

Absence of Effect of CCCP on Initial Decay Rate of Alkaline pHin Gradients Across the BBM: Pyranine Experiments

Vesicles charged with the pH-sensitive fluorophore pyranine were used to assay for H+ fluxes in the presence and absence of CCCP under appropriate conditions (see Ref. 17 for rationale of technique). In a first series of experiments (Fig. 5, curves a-h), vesicles charged with a pH 7.5 buffer supplemented with 200 mM TMA gluconate were used. The extravesicular medium contained the same buffer at pH 6.0, but the TMA gluconate was substituted or not with other salts, as discussed below. The time-dependent drop in pyranine fluorescence (intravesicular acidification) was used to monitor the rate of proton gradient decay. Typically, all decay curves were characterized by a rapid drop from the initial pH 7.5 value to a lower level (pH 6.8 in experiments in Fig. 5), representing the practically instantaneous neutralization of extravesicular pyranine bound to the outer vesicle surface. This rapid initial phase was then followed by the true pH gradient decay, consisting of a slower fluorescence decrease toward the limiting equilibrium value (pHin = pHout) determined at the end of each run by lysing the vesicles with Triton X-100.


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Fig. 5.   Effect of certain ions and CCCP on decay of alkaline pHin gradients according to the pyranine method. BBM vesicles were loaded with pyranine in presence of a 20 mM HEPES-40 mM MES buffer supplemented with either 200 mM tetramethylammonium hydroxide pentahydrate (TMA) gluconate (curves a-h) or 200 mM potassium gluconate (curve i) and adjusted with Tris base to pHin = 7.5. At time 0, vesicles were mixed with the same buffer, which was adjusted with Tris base to pHout = 6.0, contained 200 mM of the salts indicated at bottom of figure, and was supplemented with either no CCCP (curves a, c, e, and g) or CCCP at 72 µM final concentration (curves b, d, f, and h). Because CCCP has no statistically significant effect in presence of trans K+, relevant results have been pooled into a single curve (i). At 180 s, Triton X-100 was added to lyse vesicles (18). First 8 s of decay curves are illustrated in inset. Fluorescence intensity results were transformed into pHin (negative logarithm of [H]i in mol/mg protein) calculated according to Eq. 1 of Vasseur et al. (18). See text for further explanations.

The rate of spontaneous proton gradient decay is given in Fig. 5, curves c and d, where the vesicles were equilibrated with TMA gluconate. This rate strongly increased when the external gluconate was substituted by Cl- (curves g and h), as expected from the known existence of Cl--H+ symport activity in these membranes. In contrast, when the external TMA was substituted by K+, the decay rate slowed down significantly (curves a and b), indicating operation of an already described K+/H+ antiport activity (17). When the TMA gluconate was substituted by KCl, an intermediate result was obtained (curves e and f) in accord with the previous observation that the effects of K+ (intravesicular alkalinization) and Cl- (acidification) tend to cancel each other, although the effect of Cl- is stronger than that of K+ (17).

A statistical analysis of these results indicates that CCCP does not significantly modify the rates of proton gradient decay just described, because for each pair of curves (a vs. b and so on), the results were indistinguishable during the first 30 s. It was only at longer times that significant accelerations caused by CCCP on the proton decay curves became apparent, namely, after either 30 s (TMA chloride), 60 s (TMA gluconate and KCl), or 110 s (potassium gluconate). From these data, we conclude that, although CCCP may behave as a protonophore in these vesicles, its effects are weak and rather slow in the conditions of our experiments. On the other hand, the results confirm our earlier conclusion (17) that the Cl--H+ symport and K+/H+ antiport activities characterizing the intestinal BBM are both electroneutral.

One comment appears to be necessary here to explain why, contrary to widespread belief, CCCP does not behave as a strong protonophore in the present experiments. In principle, the 72 µM CCCP concentration used is appropriate because it is well above the level at which the CCCP inhibitory effect on Cl- uptake reaches its maximum (see Fig. 2). One explanation for this weakness might be the fact that to collapse a pH gradient, CCCP requires the presence of a permeable counterion, such as a cation in the trans side or an anion in the cis side of the membrane. The ions used in our experiments are not effective in this regard, probably due to the low ionic permeability characterizing the BBM (17).

Dwelling further on this question, we performed the following experiment (Fig. 5, curve i). Pyranine-loaded vesicles were prepared in which the intravesicular TMA gluconate had been substituted by potassium gluconate. It was expected that trans K+ would act as a counterion for H+, thereby permitting demonstration of the dissipation of the imposed pH gradient by CCCP. But the experiment proved to be inconclusive, due to the fact that intravesicular K+ causes strong intravesicular acidification via the K+/H+ antiport activity previously demonstrated (18). The proton gradient decay rate taking place under such conditions is so large that no further acceleration could be observed on addition of CCCP.

In summary, independent of the possible effectiveness of CCCP as a protonophore in isolated intestinal brush-border vesicles, our experiments indicate that in the short time intervals (2 s) used in the present study of Cl- uptake kinetics, CCCP does not significantly modify the imposed pH gradient. Therefore, CCCP does not act indirectly by dissipating any pH gradient but rather acts as a direct inhibitor of the Cl--H+ symport activity of these membranes.

Is Cl- Uptake Inhibition by CCCP Partial or Total?

We have seen that the Cl- uptake inhibitions caused by CCCP in the presence of a pH gradient are clearly partial. But, in the absence of such a gradient, the results were less clear, even when arguments in favor of partial inhibition seemed both possible and sound. The reason for this uncertainty is that, in the absence of a pH gradient, Cl- uptake is quite weak, so it is difficult to know with precision whether or not the limiting values of the uptake curves in the presence of saturating CCCP concentrations are either equal to or higher than zero. This difficulty is compounded by the fact that, in practice, a level of zero cannot be expected, because some Cl- uptake will always remain, taking place through simple physical diffusion. Therefore, it appeared necessary to establish whether the observed limiting uptake values at high CCCP concentration ([CCCP]) can in fact be decomposed into two distinct levels, corresponding to diffusion and to a hypothetical CCCP-insensitive transport component. To estimate the limiting uptake value at high [CCCP], we have used as a first approximation the Inui and Christensen (10) transformation (see Fig. 2). However, this procedure proved to be insufficient to achieve the desired splitting, and a more sophisticated approach was clearly necessary.

This new approach was found, as we describe. It consisted in developing a kinetic model and equations that, by fitting to our data by nonlinear regression analysis, have allowed for a quantitative distinction between the physical diffusion level and a CCCP-insensitive Cl- uptake component. The argument is that, if this level was found to be higher than that of the diffusion, then the conclusion would be warranted that CCCP inhibition was indeed partial. This would mean that all of our results are open to rationalization in terms of a single, coherent theoretical model valid in both the absence and presence of a pH gradient.

This new kinetic model is schematized in Fig. 6. In the remainder of the RESULTS AND DISCUSSION section, we quantitatively test our experimental data using equations arising from this model, the kinetic implications of which are fully developed in the APPENDIX.


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Fig. 6.   Two-dimensional representation of the 3-site symport model that includes an additional allosteric inhibitor-binding site. This 3-site model is based directly on the 2-site model (see Ref. 3), which is shown as forming a "core" (heavy lines) over which those interactions involving the inhibitor have been superimposed (thin lines). For further details, see text.

Definition and Testing of a Three-Site Symport Model That Can Fully Explain the Partial Inhibitions of Cl- Uptake Caused by CCCP

As shown, all the experimental evidence available so far indicates strongly that CCCP acts as an allosteric inhibitor of Cl- uptake across the intestinal BBM. Given that this uptake involves a homogeneous carrier system consisting of a random, nonobligatory Cl--H+ symporter (3), the next logical step was to put all of these ideas together. If the Cl--H+ symporter involves two distinct, specific binding sites, the Cl--binding and the H+-binding sites, what was needed was to develop a new three-site model consisting of a carrier with these two substrate-binding sites plus an additional inhibitory CCCP-binding site.

This model gives rise to a general equation (Eq. A1 in APPENDIX) that, in the absence of any restriction, corresponds to the full, random, nonobligatory model (Table 4, submodel 1), in which all the rate constants involved are assumed to have values greater than zero. In addition to this general model, other submodels can be defined that are also capable of fitting our results, as discussed in the APPENDIX (e.g., Table 4, submodels 3 and 4). But these are special cases, the possible existence of which does not modify the fact that submodel 1 is the simplest imaginable and suffices to fully explain our results. The key point is that of all possible substrate (S)-bound carrier (C) complexes (i.e., all those complexes giving rise to transport), the inhibitor (I)-bound ternary (I=C-S) and quaternary (IA=C-S, where A is activator) complexes must be postulated to both be able to form and be mobile, meaning that the rate constants p and q both need to be greater than zero. As explained in the APPENDIX, the possible formation and translocation of other I-bound complexes have no relevance to the question posed in this article, the mechanism of CCCP inhibition.

To verify the agreement of our results with the preceding postulates, we performed a nonlinear regression analysis of the data in Fig. 2 to test the fit of Eq. A1 to our CCCP inhibition results. The procedure used is described in Table 3, in which the resulting kinetic parameters are listed. To facilitate the iteration procedure, before each run was performed, the apparent Kd was fixed to a reasonable value, namely, 6 nl · s-1 · mg protein-1, which is the average value of a series of Kd control measurements (see Ref. 3) estimated from the limiting slope of Cl- saturation curves performed with different batches but the same type of guinea pig BBM vesicles used in the present work. With the use of these parameters and Eq. A1, the theoretical curves in Fig. 7 were computed. The fits are excellent, which is both evident to the naked eye and supported by the correlation coefficient values that are practically equal to one for both pH gradient conditions studied (Table 3). We conclude that the three-site model in Fig. 6 (and, in particular, submodel 1 in Table 4) fully explains our CCCP results.

                              
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Table 3.   Kinetic parameters used to calculate theoretical curves in Fig. 7 according to the three-site symport model


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Fig. 7.   Fit of CCCP inhibition results according to 3-site symport model. Results in Fig. 2 have been redrawn to permit illustration of theoretical fits obtained by applying Eq. A1 of the full, nonobligatory, 3-site symport model to each of the 2 sets of data. Relevant parameters and procedure used to perform fits are given in Table 3. Eq. A1 has been given the form curve ct = curve c1 + curve c2 + (Kd · [S]), where curve c1 corresponds to the first, Michaelian term; c2 is the second, convex term; and Kd · [S] is the third, diffusional term. Heavy line at top (cta) shows overall fit of results in presence of a pH gradient. Components c1a and c2a are shown as wavy lines, and diffusion level is shown as thin line at bottom. To permit a direct comparison of results without overcrowding, data in absence of a pH gradient are illustrated only as the overall curve ctb. In inset, splitting of curve ctb into its 3 components is illustrated. Further details are given in text.

                              
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Table 4.   Kinetic criteria to distinguish between various three-site symport submodels considered in text

Further comment on the meaning of the theoretical curves in Fig. 7 seems warranted here. The two components of Eq. A1 can be regarded as representing two distinct pathways for Cl- uptake, even when both involve the same molecular entity, the three-site symport carrier. We begin by considering the second pathway, represented by the non-Michaelian, convex term of the equation. Its net rate is highest in the absence of CCCP but decreases hyperbolically to approach zero at saturating [CCCP]. Thus CCCP behaves here as a full inhibitor.

In contrast, as concerns the first pathway, CCCP looks like an activator here because, in the absence of CCCP, the reaction rate is zero but increases hyperbolically as [CCCP] increases to reach a constant, limiting value equal to V1I,o. For submodels 1, 3, and 4 (Table 4), the V1I,o parameter is by definition greater than zero (see Eq. A2). This is, of course, the reason why CCCP inhibition is partial. At saturating [CCCP], the overall Cl- transport rate cannot equal zero.

An identical conclusion is reached by considering the results in terms of individual carrier-substrate complexes. The quantitative participation of each of the two pathways to the total Cl- influx rate will depend on the outside inhibitor concentration ([I]o) at each given value of outside and inside hydrogen ion concentration ([H]o and [H]i, respectively) and outside and inside substrate concentration ([S]o and [S]i, respectively). Thus, for instance, Cl- uptake via both the =C-S and the A=C-S complexes will predominate in the absence of [I]o (see Eqs. A1 and A3). As [I]o increases, fluxes via these two complexes will decrease (Fig. 7, curve c2), whereas fluxes via the I=C-S- and IA=C-S- complexes will increase (curve c1). It is at high [I]o that fluxes via these last two complexes will predominate (Eqs. A1 and A2). Again, the quantitative participation of the various fluxes to either pathway, involving either the simple Michaelian or the non-Michaelian components, will depend on the relative values of [H]o and [S]i. At pHout = 7.5, fluxes via either the =C-S (curve c2) or I=C-S- pathways (curve c1) will predominate [see Eq. A3 as outside activator concentration ([A]o) tends to 0 and Eq. A6, respectively]. In contrast, at an acidic pHout of 5.0, fluxes via either the A=C-S (curve c2) or the IA=C-S- (curve c1) will predominate (see Eq. A3 as [A]o tends to infinity and Eq. A7, respectively). Finally, Fig. 7 illustrates how, as [H]o increases, the overall reaction rate increases, in agreement with the corresponding increases experienced by each V1I,o and V2I,o (see Table 3).

Concluding Remarks

The results presented here permit the conclusion that the mixed-type inhibition of pH gradient-activated Cl- uptake caused by CCCP cannot be explained in terms of a CCCP-induced increase in proton conductance that would cause total or partial dissipation of the pH gradient acting as the driving force for Cl- uphill transport across the intestinal BBM. Rather, in accord with an earlier proposal of Liedtke and Hopfer (11), a direct interaction of CCCP with the Cl--H+ symporter appears to be involved, particularly because, even at equilibrated pH, CCCP inhibits both Cl- efflux and Cl- influx independent of the absence or presence of trans Cl-.

    APPENDIX
Top
Abstract
Introduction
Methods
Results & Discussion
Appendix
References

General Cl--H+ Symport Model

The present analysis is based on the random, nonobligatory Cl--H+ symport model recently proposed to explain pH gradient-activated Cl- uptake across the guinea pig ileal BBM. This model (see Ref. 3) consists of a classic mobile carrier existing in two mutually exclusive conformations, Co and Ci, in which o (out, cis) and i (in, trans) represent the outer and the inner sides of the membrane, respectively. The carrier has two substrate-binding sites, one for Cl- and another for H+. It is entirely symmetrical, so that, by definition, Cl- and H+ are both substrates (cosubstrates) and allosterically activate each other's binding to their respective sites. Nevertheless, for practical purposes, we use here the convention that Cl- is the substrate (S), and H+ is an allosteric modifier acting as an activator (A). This is clearly a warranted simplification of a more general allosteric model (see Ref. 2), in which A may be thought to act either as an activator or an inhibitor or be inert. If -C- is the two-site carrier, the existence of three carrier-substrate complexes can be envisaged: two binary complexes, -C-S and A-C-, and a ternary complex, A-C-S. The symport model is general, and, as such, it considers that the three carrier-substrate complexes as well as the empty carrier are all mobile. By definition, the model is random (nonordered) and nonobligatory, meaning that all the rate constants governing the translocation of the binary complexes are greater than zero. To use well-established jargon, the model includes "slippage." For a complete kinetic development and further details, see Ref. 3.

Symport Model With an Additional, Inhibitory CCCP-Binding Site

Symport models have already been developed that explicitly consider inhibition. For instance, Turner and Silverman (16) have defined a random, nonobligatory symport model in which binding of the inhibitor and the substrate are mutually exclusive; i.e., they both compete for the same binding site. Furthermore, both the binary complex (I-C-) and the ternary complex (I-C-A) have been assumed not to be mobile. But such a model (that nevertheless can be assimilated to one of the submodels of our general model, e.g., Table 4, submodel 8) is not useful for our present purposes for two main reasons. As defined by Turner and Silverman, I is a competitive inhibitor, and therefore it can cause full inhibition. In contrast, in our BBM Cl- transport experiments, CCCP inhibition is mixed type and, more importantly, partial. To explain partial inhibition within a homogeneous carrier system, the existence of an allosteric site specific for CCCP is necessary (see Ref. 1).

To meet this need, we have developed the more general, three-site symport model illustrated in Fig. 6. The key difference between the two models is that, on top of the S- and A-binding sites, the new model postulates the existence of a third, CCCP-specific site. Nevertheless, the new model is formally identical to the original one of Alvarado and Mahmood (2), in which an allosteric modifier can be imagined to act either as an activator (A in the present model, which we have previously defined to be H+) or an inhibitor (I), which is CCCP.

Seven instead of three specific complexes are therefore possible, namely, three binary complexes, A=C-, I=C-, and =C-S; three ternary complexes, A=C-S, I=C-S, and IA=C-; plus a quaternary complex, IA=C-S. To simplify the model's representation and the writing of equations, the three-site carrier (=C-) is drawn only in one dimension, as shown. In effect, to avoid a three-dimensional representation, at the same time indicating that A and I do not bind to the same site, the symbol "=" (which for simplicity is not illustrated in Fig. 6) is used here to suggest the existence of two independent binding sites for A and I, respectively, on the left side of the symbol C. As was the case with the two-site symport model, we assume that all carrier-bound complexes are mobile, meaning that the three-site model remains, by definition, random and nonobligatory.

Rate Equation as a Function of [I]o

We have applied a series of assumptions, similar to those defined earlier for the two-site symport model (3), to obtain a relatively simple set of kinetic equations describing the general three-site symport model in Fig. 6. The key equation concerns the initial rate of S influx (v) as a function of the cis inhibitor concentration, [I]o
<IT>v</IT> = <FR><NU>[(<IT>V</IT><SUB>1I,o</SUB> ⋅ [I]<SUB>o</SUB>) + (<IT>V</IT><SUB>2I,o</SUB> ⋅ <IT>K</IT><SUB>I,o</SUB>)]</NU><DE>([I]<SUB>o</SUB> + <IT>K</IT><SUB>I,o</SUB>)</DE></FR> (A1)
where the maximal velocities of S transport when [I]o tends to either infinity or zero, V1I,o and V2I,o, respectively, are defined as
<IT>V</IT><SUB>1I,o</SUB> = <FR><NU>C<SUB>T</SUB> ⋅ &agr; ⋅ [ <IT>p</IT><SUB>o</SUB> + (<IT>q</IT><SUB>o</SUB> ⋅ [A]<SUB>o</SUB>/<IT>K</IT><SUB>sxa,o</SUB>)]</NU><DE><IT>D</IT> + {([A]<SUB>o</SUB>/<IT>K</IT><SUB>sxa,o</SUB>) ⋅ <IT>E</IT>}</DE></FR> (A2)
<IT>V</IT><SUB>2I,o</SUB> = <FR><NU>C<SUB>T</SUB> ⋅ &agr; ⋅ [ <IT>f</IT><SUB>o</SUB> + ( <IT>g</IT><SUB>o</SUB> ⋅ [A]<SUB>o</SUB>/<IT>K</IT><SUB>sa,o</SUB>)]</NU><DE><IT>B</IT> + {([A]<SUB>o</SUB>/<IT>K</IT><SUB>sa,o</SUB>) ⋅ <IT>C</IT>}</DE></FR> (A3)
and the apparent affinity constant for the external inhibitor (KI,o) is given by
<IT>K</IT><SUB>I,o</SUB> = <FR><NU><IT>K</IT><SUB>sx,o</SUB> ⋅ [<IT>B</IT> + {([A]<SUB>o</SUB>/<IT>K</IT><SUB>sa,o</SUB>) ⋅ <IT>C</IT>}]</NU><DE><IT>D</IT> + {([A]<SUB>o</SUB>/<IT>K</IT><SUB>sxa,o</SUB>) ⋅ <IT>E</IT>}</DE></FR> (A4)
where CT is the total number of carrier forms and

&agr; = <IT>k</IT><SUB>i</SUB> + (<IT>h</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>a,i</SUB>) + {([I]<SUB>i</SUB>/<IT>K</IT><SUB>x,i</SUB>) ⋅ [<IT>m</IT><SUB>i</SUB> + (<IT>l</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>xa,i</SUB>)]}
+ {([S]<SUB>i</SUB>/<IT>K</IT><SUB>s,i</SUB>) ⋅ [ <IT>f</IT><SUB>i</SUB> + (<IT>g</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>sa,i</SUB>) + {[I]<SUB>i</SUB>/<IT>K</IT><SUB>sx,i</SUB> ⋅ [ <IT>p</IT><SUB>i</SUB> + ( <IT>q</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>sxa,i</SUB>)]}]} (A5a)
&bgr; = 1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>a,i</SUB>) + {([I]<SUB>i</SUB>/<IT>K</IT><SUB>x,i</SUB>) ⋅ [1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>xa,i</SUB>)]} + {([S]<SUB>i</SUB>/<IT>K</IT><SUB>s,i</SUB>) ⋅ [1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>sa,i</SUB>) + {([I]<SUB>i</SUB>/<IT>K</IT><SUB>sx,i</SUB>) ⋅ [1 + ([A]<SUB>i</SUB><IT>K</IT><SUB>sxa,i</SUB>)]}]} (A5b)
<IT>B</IT> = (&agr; ⋅ &khgr; + &bgr; ⋅ &dgr;)  <IT>C</IT> = (&agr; ⋅ &egr; + &bgr; ⋅ &phgr;)  <IT>D</IT> = (&agr; ⋅ &khgr;′ + &bgr; ⋅ &dgr;′)  <IT>E</IT> = (&agr; ⋅ &egr;′ + &bgr; ⋅ &phgr;′)
&khgr; = 1 + (<IT>K</IT><SUB>s,o</SUB>/[S]<SUB>o</SUB>)  &dgr; = <IT>f</IT><SUB>o</SUB> + (<IT>k</IT><SUB>o</SUB> ⋅ <IT>K</IT><SUB>s,o</SUB>/[S]<SUB>o</SUB>)  &egr; = 1 + (<IT>K</IT><SUB>as,o</SUB>/[S]<SUB>o</SUB>)
&phgr; = <IT>g</IT><SUB>o</SUB> + (<IT>h</IT><SUB>o</SUB> ⋅ <IT>K</IT><SUB>as,o</SUB>/[S]<SUB>o</SUB>)  &khgr;′ = 1 + (<IT>K</IT><SUB>sx,o</SUB>/[S]<SUB>o</SUB>)  &dgr;′ = <IT>p</IT><SUB>o</SUB> + (<IT>m</IT><SUB>o</SUB> ⋅ <IT>K</IT><SUB>xs,o</SUB>/[S]<SUB>o</SUB>)
&egr;′ = 1 + (<IT>K</IT><SUB>xas,o</SUB>/[S]<SUB>o</SUB>)  &phgr;′ = <IT>q</IT><SUB>o</SUB> + (<IT>l</IT><SUB>o</SUB> ⋅ <IT>K</IT><SUB>xas,o</SUB>/[S]<SUB>o</SUB>) (A5c)

In Eq. A1, v represents the sum of two hyperbolas, namely, one that is Michaelian (concave) and involves the parameters V1I,o and KI,o and a second (convex) that involves V2I,o and KI,o. As can be seen, Eq. A1 explains fully why CCCP inhibition is partial. Because as [I]o increases, v will tend to V1I,o and the limiting value of v can never be zero.

Nevertheless, for completeness, a series of possible variants of Eq. A1 have been considered and are listed in Table 4. The most general case is represented by the full, nonobligatory model (submodel 1), in which the ternary (I=C-S-) and the quaternary (IA=C-S-) complexes are both mobile (p and q both >0). Similar kinetic behavior will be exhibited by those submodels in which p and/or q have positive values independent of the absence or presence of the I=C- and IA=C- complexes and with or without slippage (l and m both >=  0). This is because here the numerator (N) in V1I,o has a finite value (Table 4, submodels 3, 4, and 9 - 14). To the contrary, in models in which the p and q constants equal zero, the N parameter will be zero (and therefore V1I,o = 0), so that Eq. A1 will simplify to a (simple) convex hyperbola. Thus, for the obligatory model and its four possible submodels (Table 4, submodels 5 - 8), the inhibition will be total, because when V1I,o = 0, v will tend to zero as [I]o tends to infinity.

It seems evident from the above considerations that partial inhibition is the necessary consequence of the presence of a Michaelian component in Eq. A1 whose limiting value (as [I]o right-arrow infinity ) is by definition greater than zero. In effect, if we assume that [I]o right-arrow infinity , it is clear that the function v = f([I]o) will tend to V1I,o, which itself depends on po and/or qo, and, furthermore, appears to be a complex function of each [A]o, [A]i, [I]i, and [S]i (see Eqs. A5a-A5c). However, among those models in which Eq. A1 applies, two submodels can be readily rejected (see Table 4, submodels 13 and 14) because, in both cases, V1I,o is independent of [A]o. This result is incompatible with our data indicating the limiting value of Cl- uptake at very high CCCP concentration to increase as the pHout decreases from 7.5 to 5.0 in presence of a constant pHin of 7.5 (Fig. 2).

Another question worthy of consideration here is whether one or both of the rate constants in Eq. A2 have positive values. To answer that question, we derived the limiting values of V1I,o when [A]o and/or [S]i tend to either zero or infinity. One difficulty could arise from the fact that [A]o represents a proton concentration that cannot have values of either zero or infinity. Nevertheless, mathematically speaking, the approximation is warranted that, in the present experiments, zero and infinity [H+] can be thought to correspond in practice to pH values of 7.5 and 5.0, respectively (3). For the general three-site model, the limiting values of V1I,o as [A]o tends to either zero or infinity are given by
<IT>V</IT><SUB>1I,o([A]<SUB>0</SUB> → 0)</SUB> = C<SUB>T</SUB> ⋅ &agr; ⋅ <IT>p</IT><SUB>o</SUB>/<IT>D</IT> (A6)
and
<IT>V</IT><SUB>1I,o([A]<SUB>o</SUB> → ∞)</SUB> = C<SUB>T</SUB> ⋅ &agr; ⋅ q<SUB>o</SUB>/<IT>E</IT> (A7)
This is the result to be expected from the premises of the full three-site symport model and/or those submodels in which the rate constants p and q have positive values (Table 4, submodels 1, 3, and 4). Clearly, V1I,o will attain two different limiting values as [A]o tends to either zero or infinity. The result will be the same whether the ternary complex I=C-S- is or is not mobile (po = 0). From this restriction, it follows that the inhibition will be total as [A]o tends to zero (in practice, as mentioned, at pHout >=  7.5) independent of the absence or presence of [S]i. However, the results show that partial inhibition also occurred at a pHout of 7.5 in the presence of trans Cl- (Table 2B). By assuming that po > 0, we reach the conclusion that <IT>V</IT><SUB>1I,o([A]<SUB>o</SUB>→ 0)</SUB> will attain two different limiting values as [S]i tends to either zero or infinity, namely
<IT>V</IT><SUB>1I,o([A]<SUB>o</SUB> → 0,[S]<SUB>i</SUB> → 0)</SUB> = C<SUB>T</SUB> ⋅ &agr;<SUB>1</SUB> ⋅ <IT>p</IT><SUB>o</SUB>/(&agr;<SUB>1</SUB> ⋅ &khgr;′ + &bgr;<SUB>1</SUB> ⋅ &dgr;′) (A8)
<IT>V</IT><SUB>1I,o([A]<SUB>o</SUB> → 0, [S]<SUB>i</SUB> → ∞)</SUB> = C<SUB>T</SUB> ⋅ &agr;<SUB>2</SUB> ⋅ <IT>p</IT><SUB>o</SUB>/(&agr;<SUB>2</SUB> ⋅ &khgr;′ + &bgr;<SUB>2</SUB> ⋅ &dgr;′) (A9)
where
&agr;<SUB>1</SUB> = <IT>k</IT><SUB>i</SUB> + (<IT>h</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>a,i</SUB>) + {([I<SUB>i</SUB>]/<IT>K</IT><SUB>x,i</SUB>) ⋅ [<IT>m</IT><SUB>i</SUB> + (<IT>l</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>xa,i</SUB>)]}
&agr;<SUB>2</SUB> = <IT>f</IT><SUB>i</SUB> + (<IT>g</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>sa,i</SUB>) + {([I]<SUB>i</SUB>/<IT>K</IT><SUB>sx,i</SUB>) ⋅ [<IT> p</IT><SUB>i</SUB> + (<IT>q</IT><SUB>i</SUB> ⋅ [A]<SUB>i</SUB>/<IT>K</IT><SUB>sxa,i</SUB>)]}
&bgr;<SUB>1</SUB> = 1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>a,i</SUB>) + {([I]<SUB>i</SUB>/<IT>K</IT><SUB>x,i</SUB>) ⋅ [1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>xa,i</SUB>)]}
&bgr;<SUB>2</SUB> = 1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>sa,i</SUB>) + {([I]<SUB>i</SUB><IT>K</IT><SUB>sx,i</SUB>) ⋅ [1 + ([A]<SUB>i</SUB>/<IT>K</IT><SUB>sxa,i</SUB>)]} (A10)
From the above set of considerations, the conclusion is warranted that formation and translocation of the complexes I=C-S and IA=C-S are both necessary to explain the partial inhibitory effect of CCCP on Cl- uptake. Whether or not the I=C- and IA=C- complexes do form and can be translocated (l and m being >= 0), the CCCP effect will be the same. However, in the absence of further information, the full nonobligatory model appears to be the simplest, and hence the best, solution to explain the CCCP transport inhibition data subject of the present paper.

    ACKNOWLEDGEMENTS

We thank Michèle Caüzac and Régine Frangne for excellent technical assistance.

    FOOTNOTES

This work was supported in part by the Institut National de la Santé et de la Recherche Médicale, by the Fondation pour la Recherche Médicale, Paris, France, and by the INCO Program of the European Economic Community (grant ERB 3514 PL 950019).

Address for reprint requests: F. Alvarado, INSERM, Faculté de Pharmacie, Université de Paris XI, 5, rue J.-B. Clément, 92296 Châtenay-Malabry, France.

Received 30 June 1997; accepted in final form 3 November 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results & Discussion
Appendix
References

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