Effect of plasmalemma ATPase inhibitors, diethylstilbestrol and orthovanadate, on fusicoccin-induced H+ extrusion in maize roots

Plant Science Letters, 21 (1981) 305--315

305

© Elsevier/North-Holland Scientific Publishers Ltd.

E F F E C T O F P L A S M A L E M M A ATPase INHIBITORS, D I E T H Y L S T I L B E S T R O L AND ORTHOVANADATE, ON FUSICOCCININDUCED H* EXTRUSION IN MAIZE ROOTS

ROBERTA COLOMBO, ALESSANDRA PIERA L A D O *

BONETTI, R A F F A E L L A

CERANA

and

Centro di Studio del C.N.R. per la Biologia Cellulare e Molecolare deUe Piante, Istituto di Scienze Botaniche, Universitd di Milano, V.G. Colombo, 60-1-20133 Milano (Italy)

(Received July 14th, 1980) (Revision received October 20th, 1980) (Accepted December 15th, 1980)

SUMMARY

In order to investigate the possibility t h a t fusicoccin(FC)-induced electrogenic H + extrusion and K ÷ uptake in higher plants depend on the activity of a plasmalemma ATPase, we have studied the effect of two nonmitochondrial membrane-bound ATPase inhibitors, diethylstilbestrol (DES) and vanadate, on the transport of H + and K ÷ in maize root segments. Both DES and vandate strongly inhibited the stimulating effect of FC on H ÷ extrusion and K + uptake. Furthermore, vanadate inhibited the FCinduced H + extrusion in the presence of a lipophilic cation, tributylbenzyla m m o n i u m (TBBA+), and in the absence of K ÷. The parallel determination of ATP and pyruvate levels showed t h a t in our experimental conditions the inhibiting effect of the two substances on transport did n o t seem to depend on an effect on energy metabolism. The results of this paper seem consistent with the view that FC-stimulated H ÷ extrusion and K + uptake depend on the activation of a plasmalemma ATPase, catalyzing an electrogenic H ÷ uniport, electrically coupled with K + influx.

INTRODUCTION T h e hypothesis that basal and FC-induced electrogenic H ÷ extrusion and *To whom correspondence should be sent. Abbreviations: CBT, Cercospora beticola toxin; DCCD, N,N'-dicyclohexilcarbodiimide; DES, diethylstilbestrol; FC, fusicoccin; HMT, Helmintosporium maydis race T toxin; MES, 2(n-morpholino)ethane sulfonic acid; PD, potential difference; TBBA ÷, tributylbenzylammonium.

306

K ÷ uptake in higher plants depend on the activity of a plasmalemma ATPase was suggested by the following in vivo evidence: (i) H ÷ extrusion, the parallel hyperpolarization of the transmembrane electric potential difference (PD) and K ÷ uptake are strongly inhibited by treatments lowering the intracellular ATP level [1--6] ; (ii) the stimulation of H ÷ extrusion and K + uptake by FC is accompanied by a significant dec}ease in ATP level, as expected if the H ÷ extruding system is fueled by ATP [3]. A stimulating effect of FC on the in vitro ATPase activity of microsomal preparations has been reported [ 7,8 ], b u t these results are u n f o r t u n a t e l y not conclusive due to their still unsatisfactory reproducibility [9,10]. Further support for the hypothesis that a plasmalemma ATPase is involved in H ÷ and K ÷ transport comes from data showing that in vitro inhibitors of the Mg 2÷, K÷-dependent microsomal ATPase {DES, N-N'-dicyclohexilcarbodiimide (DCCD), the toxins Cercospora beticola (CBT), zearalenone, Helmintosporium maydis race T (HMT) inhibit in vivo H ÷ extrusion [8,11--14], hyperpolarization of PD [4,14,15] and K ÷ uptake [8,12,13, 16,17]. The importance of these data, however, is weakened by the fact that most of these inhibitors also inhibit the mitochondrial activity. Thus, their inhibiting effect on transport in vivo might depend on a decreased energy supply. Recently Na-orthovanadate has been found to markedly inhibit the plasmalemma ATPase of Neurospora, while it does n o t affect the activity of mitochondrial ATPase [18]. In higher plants, t o o (radish seeds and maize coleoptiles [ 19,20], maize roots, pea and bean stems and carrot slices (L. Tognoli and M. Cocucci, unpublished data)), this substance has been demonstrated to inhibit the Mg 2÷, K÷-dependent ATPase at a pH-value of 6 of microsomal preparations, w i t h o u t affecting the mitochondrial ATPase and the phosphorylating activity of isolated mitochondria [ 19]. Some lines of evidence support the hypothesis that the Mg 2÷, K+-dependent ATPase at a pH-value of 6 of the microsomal preparations is localized on the plasmalemma [21], even though the problem is still debated and an unequivocal marker for plasmalemma is lacking [ 22]. Vanadate seems at present the most suitable inhibitor to investigate if in vivo H + extrusion and K ÷ uptake depend on the activity of non-mitochondrial, probably plasmalemma ATPase. An inhibiting effect of vanadate on H ÷ extrusion and K ÷ uptake has been reported in pea internode segments [ 19]. In this work we have studied the effect of t w o inhibitors of plasmalemma ATPase, vanadate and DES, on H ÷ extrusion and K ÷ uptake in maize roots. DES was chosen, in spite its inhibiting effect on mitochondrial activity, as its site of action on a partially solubilized ATPase from microsomat preparations seems different from that of vanadate [ 20]. We have determined in parallel their effects on the levels of ATP and of pyruvate, the latter taken as an indicator of changes of the rate of glycolysis c o n s e q u e n t to changes in energy charge [23,24]. In this investigation the effects of the inhibitors were studied n o t only

307 on basal b u t also on FC-induced H ÷ extrusion and K ÷ uptake. In fact, FC strongly and apparently specifically activates the system responsible for H ÷ extrusion and K ÷ uptake: thus, in its presence, any effect of inhibitors specifically acting on this system should appear more evident than when the rate of operation on the electrogenic H*/K ÷ exchange system is low. Finally, we have also investigated the effect of vanadate on FC-induced H ÷ extrusion in the presence of the lipophilic cation TBBA ÷ and in the absence of K ÷, in order to establish the relationship between the effect of the ATPase inhibitor on H ÷ extrusion and t h a t on K ÷ uptake. In fact, recent data suggest that FC-induced H ÷ extrusion does n o t strictly depend on the uptake of K ÷, as it can occur also when lipophilic cations replace K ÷ in the medium [ 25]. A preliminary report of some of these data has been published previously [26]. MATERIALS AND METHODS Maize (Zea mays L. cv. Dekalb XL 640) seeds, sterilized with 1% NaC10, were germinated in the dark at 27°C for 2 days on filter paper wetted with 0.5 mM CaSO4. Then the seedlings were transferred to germination trays fitted with a perforated plate and the roots were dipped into aerated 0.5 mM CaSO4 for 20 h. Roots segments (5 mm long), excised from the main root 2 m m below the apex, were kept in 0.5 mM CaSO4 for 30 min and then treated as described in every experiment. CaSO4 (0.5 mM) was present in all solutions. The tissue/volume ratio was 100 mg/5 ml. The experiments were run in a t h e r m o r e g u l a t e d water bath at 27°C with shaking (70 shakes/min). Proton extrusion measurements. Proton extrusion was measured on an aliquot of the incubation medium at the end of the treatment by back titration from the final to the initial pH, as previously described [11]. In pH-stat experiments (Fig. 1) the pH was maintained at the selected value (-+0.02 units) with appropriate additions of NaOH or HC1 by means of an automatic titrator (PANATROL mod. P 7000). K ÷ uptake assay. K ÷ uptake from 0.5 mM or 5 mM K2SO4 solution was measured by using 86Rb+ as a tracer. At the end of the incubation the tissue was briefly rinsed with 10 ml of ice-cold 0.5 mM CaSO4, washed for 5 min at 0°C with the unlabelled K2SO4 solution and finally rinsed again with distilled water. The samples were resuspended in Lumagel (Lumac) and the radioactivity was c o u n t e d with a Packard Tricarb Scintillator. S.E. did n o t exceed -+5% and -+3% for the values of H ÷ extrusion and K ÷ uptake, respectively. A TP and p yr u va te assays. The tissue was homogenized in a mortar with 0.8 N perchloric acid at 0°C. The homogenate was centrifuged for 5 min at 28 000 × g and the pellet washed with 0.8 N perchloric acid. The mixed supernatants were neutralized with KOH buffered with 120 mM triethanolamine--HC1/NaOH buffer at a pH-value of 7.6 and centrifuged. The enzymic assay of ATP and pyruvate was performed on the supernatant according to

308

Lamprecht and Trautschold [27] and to Biicher et -1. [28], respectively. S.E. did not exceed -+4% and -+10% for the values of ATP and pyruvate, respectively. All the values reported are the mean of three or more experiments in triplicate. Chemicals. Stock solution of DES was in absolute ethanol. 3 X 10 -2 M ethanol was present in all samples. RESULTS

Effects o f DES A TP and pyruvate levels. DES preferentially inhibits plasmalemma ATPase without significantly affecting mitochondrial ATPase [29]. However it decreases the phosphorylating activity of isolated mitochondria [ 29] presumably by inhibiting electron transport chain as'shown for heart mitochondria [30]. Thus, we have determined the changes of ATP level in the tissue as a function of DES concentration and of time of treatment to find the condition of minimal damage to the mitochondrial activity. As a further test of possible effects of the inhibitor on the availability of ATP in the cytoplasm, we have also measured the pyruvate level. In fact, it is generally accepted that even small decreases of energy charge value induce an increase of the pyruvate level as a consequence of the acceleration of the glycolysis rate [23,24]. Table I shows that in the absence of FC the ATP level is unaffected by 10 ~M DES even after 35 rain of incubation. With 50 gM DES the ATP level is unaffected after 10 min, while it is reduced by 16% after 35 min of treatment. These data are in agreement with those reported by Balke and Hodges in oat roots [16]. I n the FC-treated samples, 10/~M DES is nearly ineffective on the ATP level, while 50 ~M DES reduces the ATP level by 14% and by 27% after 10 min and 35 rain of treatment, respectively. The data of Table II TABLE I CHANGES

IN A T P L E V E L S IN T H E P R E S E N C E

OF DES

The segments were incubated for 10 rain or 35 rain in 5 mM K,SO4 ± 20 #M FC ± DES (10 or 50 #M). The values are expressed as nmol x g fresh Wty'. DES

Control 10 min 35 rain FC 10 min 35 rain

10 ~M

50 ~M

158 153

-146 (--5%)

149 (--6%) 129 (--16%)

153 133

-123 (--8%)

131 (--14%) 97 (--27%)

309 TABLE II CHANGES IN PYRUVATELEYELS IN THE PRESENCE OF DES (50 uM) The segments were incubated for 10 minor 35 m i n i n 5 mM K2SO4 ~ 20 ,M FC -+ 50 ,M DES. The values are expressed as nmol x g fresh wt.- '. 0

35

10 rain

Control FC

min

--

DES

Effect of DES

--

DES

Effect of DES

79 112

98 150

+24% +34%

73 115

87 159

+19% +38%

show t h a t DES induces a relatively small increase in the level of p y r u v a t e b o t h in th e absence and in t he presence o f FC, thus suggesting t h a t the observed decrease o f ATP level c or r e s ponds t o a slight decrease o f the energy charge in th e cytoplasm. T h e increase o f p y m v a t e level in the FC-treated samples m a y be i n t e r p r e t e d as a c o n s e q u e n c e o f either t he small decrease o f ATP level in these samples [3] or the increase o f malate c o n t e n t , correlated t o th e FC-induced activation of H + extrusion [31, 32]. On the basis o f these results, in the following e x p e r i m e n t s we have tested t h e e f f e c t o f 50/~M DES on H + extrusion and K ÷ u p t a k e during 10 min o f treatment. I-I + e x t r u s i o n . Fig. 1 (A,B) shows in a pH-stat e x p e r i m e n t t h e effect o f DES on H ÷ e x t r u s i o n o f samples p r e t r e a t e d f or 10 min with or w i t h o u t FC (controls). In the controls, ne t H + extrusion is already depressed 3 rain and c o m p l e t e l y b lo ck ed (namely an a p p a r e n t O H - extrusion is observed) 10 rain after the addition o f DES (Fig. 1A). In the FC-treated samples DES strongly C

3OOoo~ FC

• lOOfOoo ° / e.

20~

~0000

200 -

100-

mm

100-

e

DES

Fc

Oooo

1 ---...

0-

O"

o minutes

minutes

minutes

Fig. 1. Effect of D E S on basal and FC-stimuhted H ÷ extrusion. The segments were preincubated in 0.5 m M CaSO4 for 10 rain, then transferred (zero time) into 5 m M K2SO4 with 20 ~ M F C (B) or without F C (A,C). At the time indicated by arrows, D E S was added at the final concentration of 50 ~M. (C) 20 . M F C was added after DES. The p H of the incubation m e d i u m ~ maintained at a pH-value of 6 (~ 0.02 units) by continuous additions of N a O H oJc~ICl. C~utrols, o,.; FC, [],=;Open s y m b ~ Without DES. Closed symbols: with DES.

310

inhibits net H + extrusion (85 nmol × g fresh w t ; ' × min -1 vs. 180 nmol × g fresh wt. -~ X min -~) (Fig. 1B). The inhibiting effect of DES on H* extrusion is the same when FC is added 5 min after the addition of the inhibitor (Fig. 1C). Also the stimulating effect of FC on H ÷ extrusion (+110 nmol × g fresh w t _ ~ X rain -I ) is inhibited by DES (+70 nmol × g fresh wt_ j × min -~) (Fig. 1C). The experiments described above have been performed with 10 mM K ÷ in the medium; a quite similar picture is obtained with 1 mM K +. A comparison on a percent basis between the inhibiting effects of DES on basal and FC-induced H ÷ extrusion is n o t feasable, since the measured proton extrusion presumably corresponds to only a fraction of the H ÷ extruded by the hypothesized H ÷ pump. In fact, it is generally accepted that a part of the extruded protons re-enter the cell both by passive diffusion and by s y m p o r t or antiport with other solutes [32]. Hence, as the e x t e n t of H ÷ re-entering the cell is presumably different in the controls and in the FCtreated samples, the percent inhibition of net proton extrusion by DES does n o t represent the actual inhibition of the proton pump. The main conclusion from these experiment seems t h a t DES clearly inhibits b o t h basal and FC-induced H ÷ extrusion. K ÷ uptake. Table III shows that, in the absence of FC, DES strongly inhibits (67%) K ÷ uptake from 1 mM K+; with 10 mM K ÷ in the medium the inhibition is reduced to 11%, thus confirming the data of Baike and Hodges [16]. In the presence of FC the inhibiting effect of DES on K ÷ influx is even more marked, namely 82% and 50% with 1 mM and 10 mM K ÷ respectively. As already observed for the effect of FC on H ÷ extrusion, also the effect of FC (A FC) on K ÷ uptake is severely reduced but n o t suppressed by DES. E f f e c t o f vanadate A T P and pyruvate levels. Table IV shows t h a t the ATP level is unchanged after 1 h of treatment with vanadate (200--500 pM), as previously reported for germinating radish seeds [19]. Also pyruvate level is unaffected, thus

T A B L E III I N H I B I T I N G E F F E C T O F DES O N B A S A L A N D F C - S T I M U L A T E D K+ U P T A K E T h e s e g m e n t s w e r e i n c u b a t e d for 10 rain in 1 m M 2 ( n - m o r p h o l i n o ) e t h a n e s u l f o n i c acid ( M E S ) ( p H 6), c o n t a i n i n g 0.5 m M or 5 m M K2SO4 ± 20 u M FC -+ 50 ~M DES. T h e values are e x p r e s s e d as u tool x g fresh wt.- ~ x 10 r a i n - '. lmMK

Control FC A FC

÷

10 m M K+

--

DES

--

DES

0.3 1.1 0.8

0.1 ( - 6 7 % ) 0.2 ( - 8 2 % ) 0.1

0.9 2.6 1.7

0.8 (--11%) 1,3 (--50%) 0.5

311 TABLE IV CHANGES IN ATP AND PYRUVATE LEVELS IN THE PRESENCE OF VANADATI~ ,' The segments were incubated for 1 h in 3 mM MES (pH 6) containing 0.5 mM K2SO4 + 20 uM FC +- vanadate (200 or 500 pM). The values are expressed as nmol × g fresh wt.-' Vanadate

ATP Control FC Pyruvate Control FC

200 pM

500 aM

167 168

176 171

184 160

117 175

106 169

112 160

i n d i c a t i n g t h a t t h e e n e r g y c h a r g e o f t h e a d e n y l a t e p o o l in t h e c y t o p l a s m has n o t u n d e r g o n e such a c h a n g e t o i n f l u e n c e t h e rate o f glycolysis. H ÷e x t r u s i o n . T a b l e V s h o w s t h e e f f e c t s o f 2 0 0 pM v a n a d a t e o n H ÷ ext r u s i o n , m e a s u r e d as t i t r a t a b l e a c i d i t y o f t h e m e d i u m . As 2 0 0 pM v a n a d a t e is a w e a k b u f f e r a n d as H ÷ e x t r u s i o n is i n f l u e n c e d b y e x t e r n a l p H [ 3 3 ] , we a d d e d m t h e u n t r e a t e d samples 2 0 0 pM p h o s p h a t e , w h o s e b u f f e r i n g c a p a c i t y is similar t o t h a t o f v a n a d a t e in t h e p H range o f o u r e x p e r i m e n t (pH 6 . 3 - - 5 . 0 ) . t.n t h e s e e x p e r i m e n t a l c o n d i t i o n s v a n a d a t e has n o e f f e c t in t h e absence o f FC. Also in t h e F C - t r e a t e d samples v a n a d a t e d o e s n o t i n f l u e n c e the small H ÷ e x t r u s i o n o c c u r r i n g in t h e a b s e n c e o f e x o g e n o u s l y a d d e d cations. O n t h e c o n t r a r y , v a n a d a t e m a r k e d l y inhibits F C - i n d u c e d H ÷ e x t r u s i o n in t h e p r e s e n c e o f b o t h 1 m M a n d 10 m M K ÷. T h e r e f o r e , these e x p e r i m e n t s d e m o n s t r a t e a clear i n h i b i t i n g e f f e c t o f v a n a d a t e o n F C - i n d u c e d H ÷ e x t r u s i o n in t h e p r e s e n c e o f K ÷. K ÷ u p t a k e . T h e e f f e c t s o f v a n a d a t e o n K + u p t a k e were investigated in c o n d i t i o n s s o m e w h a t d i f f e r e n t (high b u f f e r c o n c e n t r a t i o n ) f r o m t h o s e TABLE V EFFECT'OF VANADATE ON BASAL AND FC-STIMULATED H + EXTRUSION T h e s e g m e n t s w e r e p r e i n c u b a t e d for 90 rain in 0.5 mM CaSO, -+ 20 ,M FC, then treated

for 1 h in 200 ,M Na phosphate or Na vanadate ± 0.5 mM or 5 mM K~SO3 ± 20 ,M FC. The values are expressed as pmol × g fresh wt. -~ × h -j. Control

Nil 1 mM K*

10 mM K ÷

FC

Phosphate

Vanadate

Phosphate

Vanadate

-0.68 --0.21 --0.11

-0.35 --0.15 --0.10

0.42 5.25 9.20

0.50 2.90 5,25

312 T A B L E VI INHIBITING E F F E C T OF V A N A D A T E ON BASAL AND FC-STIMULATED K ÷ UPTAKE T h e s e g m e n t s w e r e p r e i n c u b a t e d f o r 90 m i n in 0.5 m M CaSO4 -+ 20 #M FC, then treated for 1 h in 5 m M M E S b u f f e r (pH 6) c o n t a i n i n g 0.5 m M o r 5 m M K2SO4 ± 20 ~M FC ± 200 # M v a n a d a t e . T h e values are e x p r e s s e d as # m o l × g f r e s h w t . - ' × h - ' . 1 mM

Control FC FC

4.3 11.1 6.8

K *

10 m M

K ÷

Vanadate

--

Vanadate

3.1 ( - 2 8 % ) 7.1 ( - - 3 6 % ) 4.0

5.0 15.0 10.0

4.0 ( - 2 0 % ) 8.0 ( - - 4 7 % ) 4.0

adopted, as most suitable, for the s t u d y of the effects on H ÷ extrusion (low buffer concentration). It is well known, in fact, that K ÷ uptake is strongly influenced b y pH of the medium [34]. Thus, at low buffer concentration, the evidence for an effect of vanadate on K ÷ uptake could be disturbed b y the interference of the changes in pH of the medium due to vanadate inhibition of H + extrusion. Table VI shows that vanadate markedly inhibits K + uptake both i~1 the controls and in the FC-treated samples. The extent of the inhibition is nearly the same with 1 mM and 10 mM K ÷ in the incubation medium, unlike the results obtained with DES. As for H + extrusion, the effect of vanadate on K + influx does n o t significantly change by increasing the concentration of the inhibitor up to 500 ~M (data n o t shown). H +e x t r u s i o n in the presence o f T B B A +. FC p r o m o t e s H + extrusion n o t only in the presence of K + (or of Na ÷ at high concentrations), b u t also in the presence of some lipophilic cations [25]. This finding supports the T A B L E VII EFFECT OF VANADATE ON BASAL AND PRESENCE OF 5 mM TBBA ÷

FC-INDUCED H + EXTRUSION

IN T H E

T h e s e g m e n t s w e r e p r e i n c u b a t e d f o r 90 m i n in 0.5 mM CaSO4 ± 20 #M FC. T h e n t h e s e g m e n t s w e r e i n c u b a t e d f o r 1 h in 200 #M Na p h o s p h a t e or Na v a n a d a t e -+ 20 #M FC + 5 m M T B B A ÷. T h e values are e x p r e u e d as ~ m o l × g f r e s h wt. -~ × h -~ .

Control

Phosphate Vanadate A Vanadate

-0.68 -0.40 +0.28

FC TBBA *

Effect of TBBA ÷

--

TBBA ÷

Effect of TBBA +

-0.53 -0.30 +0.23

+0.15 +0.10 --0.05

0.38 0.50 +0.12

2.60 1.55 -1.05

+2.22 +1.05 -1.17

313 hypothesis that FC-induced H ÷ extrusion is not obligatorily coupled (chemical coupling) to K ÷ uptake and that F C activates an electrogenic H ÷ uniport. O n the basis of this interpretation, it seemed interesting to investigate the effect of vanadate on H ÷ extrusion in the presence of T B B A ÷, one of the most active lipophilic cations in enhancing H + extrusion. The data of Table VII show that, as already reported [25], T B B A ÷ can partially replace K ÷ in stimulating H ÷ extrusion, this effect being m u c h more signifieant in the presence of FC. Vanadate inhibits FC-induced H ÷ extrusion in the presence of T B B A + practically at the same extent as in the presence of K ÷. This result can be easily interpreted as indicating a direct effect of vanadate on the electrogenic H ÷ uniport, independent of possible effects on K + uptake.

DISCUSSION FC-induced H ÷ extrusion and K ÷ uptake

The results of this paper show t h a t two inhibitors of non-mitochondrial, membrane-bound ATPase, DES and vanadate, inhibit FC-induced H ÷ extrusion and K + uptake. Parallel determinations of ATP and pyruvate levels show t h a t DES induces an early decrease of ATP and an increase of pyruvate. These effects, however, are relatively small within the first 10 rain of treatment, wheri the inhibition of H ÷ and K ÷ transport is quite marked. This suggests t h a t the effect of this inhibitor on H + and K ÷ transport depends on its capacity of inhibiting a membrane-bound ATPase rather than on an effect on % P energy supply. The same conclusion seems even more legitimate for vanadate, which does n o t induce in vivo detectable changes in the levels of either ATP or pyruvate and does n o t influence in vitro mitochondrial oxidative phosphorylation. The possibility t h a t the effects on H + and K ÷ transport depend on collateral effects of DES and vanadate, seems improbable as the effects of the inhibitors on m e m b r a n e ATPases seem mediated by different mechanisms [20,35]. Our results are thus consistent with the view that the electrogenic H÷/K + exchange, activated by FC, is mediated by a Mg2+,K+-dependent membrane ATPase, probably located at the plasmalemma. This interpretation is strengthened also by the fact that DES and vanadate have been shown to inhibit the hyperpolarizing effect of FC on PD [4]. Furthermore, the fact that vanadate inhibits TBBA+
314 effect o f FC on microsomal ATPase from maize tissues and spinach leaves has been shown [7,8]. Basal H ÷ e x t r u s i o n

While DES and vanadate show the same effect on FC-induced H ÷ extrusion, they differently affect basal H ÷ extrusion, strongly inhibited by DES, unaffected or even slightly stimulated b y vanadate. This might suggest that basal and FC-induced H ÷ extrusion depend on the activity of t w o distinct mechanisms, differently affected by the inhibitors. This hypothesis has been formulated by other authors [8]. However, a simpler interpretation can be proposed. The H ÷ extruding system has a relatively small basal rate of operation and is probably very sensitive to endogenous and exogenous factors, to which it can react with small rapid changes of activity. This legitimates the assumption that the system can differently respond to inhibitors active with different mechanisms. On the contrary, in the presence of FC the H ÷ extruding system is so strongly activated that it can partially escape from physiological regulation. For example, FC-induced net proton extrusion seems relatively insensitive to changes in the external pH, which, on the other hand, markedly influence the basal activity of this process [ 33 ]. ACKNOWLEDGEMENTS

The authors thank Professor E. Marr~ for helpful advice and criticism during this work. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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