INTRODUCTION

Top 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

Top Abstract Introduction Methods Results & Discussion Appendix References

Materials

Membrane Vesicle Preparation and Transport Assay

Results are expressed (6) as either absolute uptakes (nmol/mg membrane protein) or absolute velocities (nmol · s¹ · mg membrane protein¹) 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.

Spectrofluorometrical Studies

	RESULTS AND DISCUSSION

Top Abstract Introduction Methods Results & Discussion Appendix References

Mixed-Type Inhibitory Effect of CCCP on the Kinetics of pH Gradient-Dependent Cl Uptake

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 (V_max) and the 167% increase in the apparent Michaelis constant (K_T), respectively. If CCCP acted solely by facilitating dissipation of the pH gradient as, for example, trans K⁺ is known to do, V_max 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.

View larger version (11K): [in this window] [in a new window]   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 () or presence () 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 · s1 · mg protein1. 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 pH_in Gradients

Effect of CCCP in absence of a pH gradient. At equilibrium [outside pH (pH_out) = pH_in = 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 · s¹ · mg protein¹). 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.

View this table: [in this window] [in a new window]   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 pH_in gradient. In agreement with previous observations (17), when a pH gradient was superimposed, for instance, when pH_out/pH_in = 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 · s¹ · mg protein¹ vs. 0.04 nmol · s¹ · mg protein¹ 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 pH values of either 0 or 2.5, respectively (where pH = pH_in  pH_out), are insensitive to inhibition by CCCP. From these results, an apparent kinetic diffusion constant (K_d; for an operational definition of this parameter, see Ref. 6) was also calculated equal to 13.3 and 63.0 nl · s¹ · mg protein¹ at pH values of either 0 or 2.5, respectively. These K_d 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).

View larger version (15K): [in this window] [in a new window]   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 () or 5.0/7.5 (). 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 · s1 · mg protein1; 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.

Mixed-Type Inhibitory Effect of CCCP on the Kinetics of pH Gradient-Dependent Cl Uptake

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

View larger version (13K): [in this window] [in a new window]   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 (, ) or 14/200 mM (, ). Valinomycin (10 µg/mg membrane protein) was present throughout. When present, CCCP was at 250 µM (, ). 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 K_d values were calculated equal to 13.6, 21.4, and 37.9 nl · s¹ · mg protein¹ at 0, 75, and 200 mM trans Cl, respectively. The fact that the apparent K_d is not constant confirms that CCCP inhibition is partial even when pH = 0.

Effect of CCCP on Cl Efflux at Equilibrated pH

View larger version (11K): [in this window] [in a new window]   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 (, ) or 5.5/5.5 (, ). 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 (); n = 3-12 determinations per point.

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

View larger version (32K): [in this window] [in a new window]   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?

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.

View larger version (30K): [in this window] [in a new window]   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

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 K_d was fixed to a reasonable value, namely, 6 nl · s¹ · mg protein¹, which is the average value of a series of K_d 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.

View larger version (22K): [in this window] [in a new window]   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.

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 V_1I,o. For submodels 1, 3, and 4 (Table 4), the V_1I,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 pH_out = 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 pH_out 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 V_1I,o and V_2I,o (see Table 3).

Concluding Remarks