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Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum
Int. J. Biochem. Vol. 20, No. 12, pp. 1411-1415, 1988 Printed in Great Britain. All rights reserved
0020-711X/88 $3.00+0.00 Copyright © 1988 Pergamon Press pie
OUABAIN-INSENSITIVE Na-ATPase ACTIVITY IN THE BASOLATERAL M E M B R A N E FROM RAT JEJUNUM M. N. ORSENIGO 1, M. TOSCO 1, G. ESPOSITO2 and A. FAELLI 1. t Dipartimento di Fisiologia e Biochimica Generali, Universita' di Milano, via Celoria 26, 1-20133 Milano, Italy [Tel. 02-23 63 751] and 21stituto di Fisiologia Generale e Chimica Biologica, Facolt~i di Farmacia, Universitfi di Milano, via Saldini 50, 1-20133 Milano, Italy (Received 14 April 1988) A ~ t r a c t - - l . In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only
the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km= 15.7 + 2.3 mM and Vmax = 204 + 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km= 9.8 _ 1.2 mM and Vm,X= 640 _ 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.
INTRODUCTION
The (Na,K)-ATPase is an intrinsic plasma membrane protein that catalyzes transport of Na out of and K into cells, coupled with A T P hydrolysis. In addition to this enzyme, an N a - A T P a s e activity has been demonstrated in different tissues from various animal species. Gills from rainbow trout (Pfeiler and Kirschner, 1972), marine mussel (Howland and Faus, 1985) and sea bass (Borgatti et al., 1985; Ventrella et al., 1987) show a ouabain-insensitive Na-ATPase, which can be activated also by other monovalent cations and is present, at least in one case (Ventrella et al., 1987) in amounts similar to the (Na,K)-ATPase. The Na-ATPase was found also in the basolateral membrane of guinea-pig proximal tubular cells (Proverbio et al., 1975; Proverbio and Del Castillo, 1981; Del Castillo et al., 1982) and small intestinal epithelial cells of the same animal (Del Castillo and Robinson, 1985). In these mammalian tissues the Na-ATPase, present in a lesser quantity than the (Na,K)-ATPase, is always unaffected by ouabain, variably affected by furosemide, ethacrynic acid and triflocin, depending on the tissue and the animal (Proverbio et al., 1975; Proverbio and Del Castillo, 1981; Del Castillo et al., 1982; Del Castillo and Robinson, 1985) and modulated by Ca (Marin et al., 1985). It was found that both trypsin and sodium dodecylsulfate (SDS) treatments have different effects on the Na- and (Na,K)-ATPase activities of rat kidney basolateral plasma membrane (Proverbio et al., 1986) and only the Na-ATPase activity and the correspondent ouabain-insensitive N a pump are increased by a high Na diet (Matin et al., 1986a; Obando et al., 1987). The presence of the second N a *To whom correspondence should be addressed.
pump seems evident also from experiments performed on isolated enterocytes (Del Castillo and Whittembury, 1987). An ouabain-insensitive Na-ATPase, totally inhibited by furosemide, has been found in the intestinal mucosa from normal and diabetic rats; surprisingly both (Na,K)- and Na-ATPase activities were found distributed in many cellular structures, brush border included (Luppa and Mfiller, 1982). On the contrary Del Castillo and Robinson (1985) found that both (Na,K)- and Na-ATPase activities are only concentrated in the basolateral fraction. Since at least for the (Na,K)-ATPase the localization is known, we performed experiments only on basolateral membranes. In the present study, we have found an ouabain-insensitive Na-ATPase activity in the basolateral membrane of enterocytes obtained from the jejunal tract of rat intestine. The Na-ATPase has been compared with the well defined (Na,K)-ATPase and characterized by an enzymatic approach.
MATERIALS AND METHODS
Basolateral membrane separation Basolateral membranes from rat jejunum enterocytes were isolated and purified as described (Orsenigo et al., 1985). Briefly, 5 mM CaCI2 which preferentially aggregates all membranes except the brush border, was added to basolateral membranes, collected by self-orienting Percollgradient centrifugation. The pellets (basolateral membrane fraction) were washed and used for ATPase analyses. During the separation, phenylmethanesulfonylfluoride (PMSF) was 0.2 mM and 1,4-dithiothreitol was 1 mM. Specific activities and recoveries of marker enzymes in the basolateral membrane fraction are slightly better than those previously reported (Orsenigo et al., 1985). Only separated basolateral membranes with calculated enrichment factors of at least
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M . N . ORSENIGOet al.
ten were used. Basolateral membranes are usually present as a mixture of sealed fight side out (RSO) or inside out (IO) vesicles and as unsealed vesicles. From the determination of ATPase activities before and after SDS treatment (ratios mg protein:mg SDS = 1:0.25-0.30), we calculated that the proportions of unsealed to RSO to IO vesicles were 2:2:1 (Orsenigo et al., 1987). A TPase assays
To open the sealed vesicles, concentrated membrane suspensions obtained in 250 mM sorbitol, 0.2 mM PMSF, 20mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes)/Tris (pH 7.5) = Sol. 1, were either treated with SDS at the previously cited ratio and diluted with Sol. 1, or simply diluted with hypotonic solution (0.2mM PMSF, 20 mM Hepes/Tris buffer, pH 7.5). When ouabaininsensitive Na-ATPase was determined, membranes were preincubated for 15 rain at room temperature with 10 mM strophanthidin which acts as ouabain but is lipid soluble. Twenty #1 of diluted membrane suspension (0.2-0.3 mg protein/ml) were added to 180 #1 of incubating solution to start the reaction. The incubating solution usually contained 5 mM MgATP, different concentrations of salts and inhibitors and 20 mM Hepes/Tfis buffer, pH 7.5. After 20 min incubation at 28°C (some experiments were performed at 37~C) the inorganic phosphate released was detected by adding 0.6 ml of ice-cold 0.5 % ammonium heptamolybdate, 3% ascorbic acid, 3% SDS in 0.5 M HCI (protect this solution from light). The test tubes were kept on ice for 10 min and colour development was stopped by adding 1 ml of a solution of 2% sodium meta-arsenite, 2% sodium citrate and 2% acetic acid. After heating the test tubes at 37~C for 10 min, the colour is stable for hours. All samples were run in quadruplicate and the absorbance determined at 850nm (Perkin-Elmer, Lambda 5 model spectrophotometer). Blanks, in which the membrane suspension was added at the end of the incubation period, were run in parallel and the nmoles Pi per mg protein found were subtracted from enzyme activities expressed in nmoles Pi released per mg protein per min. Proteins were determined by the Coomassie Blue binding assay (Bradford, 1976). The Mg-ATPase is the ATPase determined in the presence of Mg. The Na-ATPase is the difference in activity between (Mg,Na)-ATPase and Mg-ATPase. The (Na,K)-ATPase is the difference in activity between (Mg, Na,K)-ATPase and (Mg,Na)-ATPase. Statistical significance was assessed by the Student's t-test. Materials
MgATP, CTP Tris-salt, UTP Tris-salt, strophanthidin, furosemide and ethacrynic acid were from Sigma (St Louis, U.S.A.); GTP lithium-salt, ADP and AMP were from Boehringer (Mannheim, West Germany); bumetanide was a gift of Leo pharmaceutical products (Copenhagen, Denmark). RESULTS The two m e t h o d s for opening vesicles, i.e. SDS or h y p o t o n i c treatment, were r u n in parallel for some
experiments, a n d gave the same results in the A T P a s e assay. After that, only h y p o t o n i c t r e a t m e n t was used. A T P a s e activities of the basolateral m e m b r a n e in the presence of different inhibitors are presented in Table 1. Due to different yields from different preparations, these results are all for one basolateral m e m b r a n e preparation, but we repeated 3--4 times all the ATPase analyses with other preparations a n d the % changes with respect to the M g - A T P a s e were the same. M g - A T P a s e was e n h a n c e d by N a a n d further by N a + K + E G T A , i.e. the basolateral m e m b r a n e possessed b o t h the N a - A T P a s e and the (Na,K)ATPase a n d these enzymatic activities were present in different a m o u n t s (ratio 1:3.5). T h e ( N a , K ) - A T P a s e can be calculated either as o u a b a i n - i n h i b i t e d ATPase or as the K + E G T A effect on N a - A T P a s e ; the two values, 460 a n d 453 nmoles Pi/mg protein per min were the same. E G T A was a d d e d in the determ i n a t i o n of ( N a , K ) - A T P a s e because trace a m o u n t s of Ca m i g h t be present in o u r p r e p a r a t i o n a n d it is k n o w n t h a t ( N a , K ) - A T P a s e is inhibited by Ca. Proverbio a n d Del Castillo (1981) provided evidence t h a t M g - A T P a s e is unaffected by this chelating agent a n d N a - A T P a s e is partly inhibited. Mg-, Na- a n d (Na,K)ATPases behave differently in the presence o f the three substances tested. O u a b a i n inhibits only (Na,K)-ATPase, furosemide inhibits only NaA T P a s e (60%) a n d ethacrynate inhibits N a - A T P a s e almost totally ( > 9 0 % ) , ( N a , K ) - A T P a s e drastically (50%) a n d has practically no effect on Mg-ATPase. Bumetanide, a n o t h e r diuretic which has c o m m o n effects with furosemide, was also tested o n the three enzymatic activites. This possible inhibitor was added to the i n c u b a t i o n fluids, described in Table 1, in a wide range of concentrations, i.e. from 0.1/aM to 1 m M , but it was ineffective o n Mg-, N a - a n d ( N a , K ) - A T P a s e activities (results n o t reported in Table 1). The specificity for A T P of b o t h Na- a n d ( N a , K ) - A T P a s e was checked by replacing A T P with the o t h e r nucleotides U T P , CTP, G T P , A D P a n d A M P . F r o m the data in Tables 2 a n d 3, in which the results are reported, it seems t h a t the Pi released in the presence o f either N a or N a + K is always lower t h a n the Pi released in the presence o f ATP. Only C T P a n d G T P seem to activate the N a - A T P a s e partially. O n the contrary, for the (Na,K)-ATPase, A T P c a n n o t be substituted by other nucleotides (the % hydrolysis is always < 10%). The low activation of b o t h ATPases in the presence of A D P could be due to some A T P c o n t a m i n a n t in this substance. D a t a for the effect on N a - A T P a s e o f m o n o v a l e n t cations other t h a n N a and of a n i o n s o t h e r t h a n CI are reported in Table 4. It seems evident that all the
Table I. ATPase activities in the basolateral membrane ATPase ATPase ATPase Activating + 2.5 mM + 2.0 mM + 2.0 mM ions ATPase A ouabain A furosemide A ethacrynate A Mg 402 + 4 415 + 5* 400 + 3 395 -4-5 + 133 + 6 + 128 + 7 +57_ 6*** + 10_+6"** Mg + Na 535 + 4 543 + 5 457 _+5*** 405 + 4*** +453 + 7 -15 + 6*** +460 4- 7 +224 +_6*** Mg+Na+K 988+6 528+4*** 917+5"** 629+5*** Mean ATPase + SE in nmoles Pi/mg protein per min. A = Differencebetween left lower and left upper value. Number of determinations = 4. P values (*** = <0.001; * = <0.1; if no * = NS) vs the first value on the same line. The concentration of the activating ions was: 5 mM Mg (MgATP), 20 mM Na (NaCI), 5 mM K (KCI). When K was present also 0.2 mM EGTA was in the incubating fluid.
Ouabain-insensitive Na-ATPase in rat jejunum Table 2. Na-nucleotidase activities in the basolateral membrane Nucleotidase activities Nucleotide ATP UTP CTP GTP ADP AMP
Mg-
(Mg, Na)-
A
422 + 5 431 +_.4 358 _ 4 359 +_3 230+3 139 +- 2
543 +_6 436 +_4 396 +- 2 401 __.4 251 +_2 142 +_2
+ 121 __.8 +5 + 6
+ 36 +- 4 +42 +- 5 +21 +_4 +3 +_3
Mean nucleotidase -t- SE in nmoles Pi/mg protein per min. A = Na-nucleotidase = differences between the two values on the same line. Number of determinations = 4. The concentration of the activating inns was: 5 mM Mg (MgCI2), 20 mM Na (NaCI). The concentration of each nucleotide (Tris salt except GTP-2 Li)= 5raM. Membranes containing trace amounts of Ca, were pre-equilibrated with 10 mM strophanthidin. 2.5 mM ouabain was in the incubating fluid. cations tested can partially substitute Na: the highest a c t i v a t i o n ( 4 0 % ) is o b s e r v e d w i t h K a n d t h e l o w e s t (20%) with Cs; on the contrary, the Na-ATPase a c t i v i t y is u n a f f e c t e d w h e n CI is r e p l a c e d by t h e a n i o n s listed in T a b l e 4. All t h e s e r e s u l t s were o b t a i n e d at 2 8 ° C a n d in t h e p r e s e n c e o f 20 m M N a a n d 5 m M K . Q u a n t i t a t i v e l y h i g h e r v a l u e s ( n o t r e p o r t e d ) w e r e o b t a i n e d at 3 7 ° C in the presence of 100raM Na and 20mM K, but the behaviour of the ATPases was the same. F i g u r e 1 s h o w s t h e r a t e s o f A T P h y d r o l y s i s for b o t h N a - A T P a s e a n d ( N a , K ) - A T P a s e activities as functions of different Na concentrations. While the activation of the Na-ATPase follows a Michaelis-Menten kinetics, t h e ( N a , K ) - A T P a s e yields a s i g m o i d a l v e l o c i t y c u r v e . T h e k i n e t i c p a r a m eters t h a t c a n be e x t r a p o l a t e d f r o m t h e E a d i e H o f f s t e e p l o t f o r N a - A T P a s e a r e a Vmax v a l u e o f 204 + 1 9 n m o l e s P i / m g p r o t e i n p e r m i n a n d a Km value of 15.7+2.3mM (r = 0 . 9 0 ) . T h e k i n e t i c c h a r a c t e r i s t i c s o f ( N a , K ) - A T P a s e were a n a l y z e d b y t h e l o g a r i t h m i c f o r m o f t h e Hill e q u a t i o n , w h i c h g a v e a Hill coefficient ( o f c o o p e r a t i v i t y ) n = 1.76 + 0.15 ( m o r e t h a n o n e N a i n v o l v e d ) , a Vmax o f 640 _+ 15 n m o l e s P i / m g p r o t e i n p e r m i n a n d a K m v a l u e o f 9 . 8 _ 1 . 2 m M (r = 0.96). T h i s K m v a l u e is lower than those generally reported (Soltoff and M a n d e l , 1984; D e W e e r , 1985), b u t as s u g g e s t e d b y S o l t o f f a n d M a n d e l (1984), h i g h e r v a l u e s c a n be o b t a i n e d if h i g h e r K c o n c e n t r a t i o n s a r e u s e d . A s a m a t t e r o f fact, f r o m r e s u l t s o f t h e g r o u p o f e x p e r i m e n t s p e r f o r m e d in t h e p r e s e n c e o f 20 m M K (at
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Table 4. Effects of different cations or different Na-salts on the Mg-ATPase of the basolateral membrane Activating substances
ATPase activities
A
Mg Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg +
410 + 4 546 + 6 447 + 5 438 _+4 465 + 6 449 + 6 436 + 7 555 +- 7 539 + 7 540 + 5 543 + 5 551 + 7 545 __.6
+ 136 + 7 + 37 + 6*** +28 + 6*** + 55 + 7*** + 39 + 7*** +26 + 8*** + 145 + 8 + 129 + 8 + 130 + 6 + 133 + 6 + 141 + 8 + 135 + 7
NaCI LiCI NH4CI KCI RbCI CsCI NaBr Na acetate Na gluconate Na ascorbate Na2SO 4 Na lactate
Mean ATPase + SE in nmoles Pi/mg protein per min. A= Differences between each value and the first value (=Mg-ATPase). P values (***= <0.001; if no * = NS) vs the first A value (= Na-ATPase activity). Number of determinations = 4. The basolateral membranes containing trace amounts of Ca were pre-equilibrated with 10raM strophanthidin. The concentration of the activating ions was: 5 mM Mg (MgATP), 20mM Na (NaC1 and other Na salts, Na2SO 4 = l0 mM), 20 mM Li = NH 4 = K = Rb = Cs (CI salts). 2.5 mM ouabain was in the incubating fluid. 28°C) we h a v e calculated a K m value of 15.0 _ 2 . 2 m M ( a n d Vm~x = 632 _ 18 n m o l e s P i / m g p r o t e i n p e r m i n ) . K ~ v a l u e s o f 7, 12 a n d 30 m M were o b t a i n e d in t h e k i d n e y w i t h K c o n c e n t r a t i o n s o f 5, 20 a n d 80 r e s p e c t i v e l y ( K i n s o l v i n g et al., 1963). DISCUSSION T h e m e t h o d u s e d f o r i n o r g a n i c p h o s p h a t e determ i n a t i o n w a s p r o p o s e d b y B a g i n s k i a n d Z a k (1960) a n d m o d i f i e d in v a r i o u s w a y s b y o t h e r s ( O t t o l e n g h i , 1975; F o r b u s h III, 1983; M a r i n et al., 1986b). W e u s e d relatively l a r g e v o l u m e s o f b o t h c o l o r develo p m e n t a n d s t o p s o l u t i o n s w i t h r e s p e c t to i n c u b a t i o n v o l u m e to a v o i d a n o n - s p e c i f i c l o w e r i n g o f t h e i n o r -
6O0 "~soo
i
40O
f
F __-------e--
Table 3. (Na, K)-nucleotidase activities in the basolateral membrane
--e--
Nucleotidase activities Nucleotide ATP UTP CTP GTP ADP AMP
(Mg, Na).
(Mg, Na, K)-
A
528+8 454 + 5 407+_5 423 +_4 280 _ 4 148 + 2
1002+_ 13 456 +_7 451 +_ 10 451 +_8 316 +- 5 145 +- 3
+474+ 15 +2 +_9 + 4 4 + 11 + 28 + 9 + 36 ___6 - 3 +- 4
Mean nucleotidase + SE in nmoles Pi/mg protein per min. A = (Na, K)-nucleotidase = differences between the two values on the same line. Number of determinations = 4. The concentration of the activating ions was: 5mM Mg (MgCI2), 20mM Na (NaCI), 5 mM K (KCI). Concentration of each nucleotide (Tris salt except GTP" 2 Li) = 5 mM. The incubating fluids contained 0.2 mM EGTA.
,o
2b
~
4b
~
mM
1~
Fig. 1. Effects of different Na concentrations (abscissa) on both the Na-ATPase (solid circles) and the (Na,K)-ATPase (open circles) activity, expressed in nmoles Pi released per m g protein and per min (ordinate). For Na-ATPase, membranes containing trace a m o u n t s of Ca were preequilibrated with 10 m M strophanthidin and 2.5 m M ouabain was in the incubating fluid; for (Na,K)-ATPase, the incubating fluid contained 0.2 m M E G T A and 5 m M KCI. For both, 5 m M M g A T P and 20 m M Hepes/Tris buffer, pH 7.5 were present. Mean v a l u e s + SE (=vertical bars, absent if lower than symbol dimension).
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M. N. ORSENIGO et al.
ganic phoshate found in the presence of even not very replacing ATP with other nucleotides. The Nahigh osmolar incubation fluids (e.g. with 150mM ATPase of rat intestine (our results) shows a lower sorbitol or 50 mM NaCI). specificity for ATP. On the contrary, the The results presented in this paper were obtained (Na,K)-ATPase behaves similarly in all tissues deat 28°C. Higher values but similar behaviour were scribed (with the exception of some activation by found at 37°C, in a few experiments not reported. GTP and ITP in the guinea-pig intestine). Moreover, The Na/K pump is activated in vivo by intracellular in all these tissues the stimulatory effect of Na is Na and extracellular K. For this reason, we used independent of the accompanying anions, while in the 20 mM Na and 5 mM K. The incubation temperature rat intestine Na seems not be replaceable by Li. For (28°C), lower than 37°C, was chosen after Na uptake (Na,K)-ATPase activity, different Km values are obexperiments with basolateral membrane vesicles, tained with different K concentrations (this work; made in parallel (results not reported in this paper). Kinsolving et al., 1963; Soltoff and Mandel, 1984) so At this temperature vesicles are more stable over it is difficult to compare Km values of (Na,K)- and time. Also the in vitro everted and perfused rat Na-ATPase even in the same preparation. By comintestine is more stable at this temperature (Faelli et paring our Km and Vma~ values with those obtained by al., 1976). Del Castillo et al. (1982) and Del Castillo and The basolateral membrane of the jejunal enterocyte Robinson (1985), it seems that our Na-ATPase activseems to have a Na-ATPase activity in addition to the ity shows lower affinity and higher capacity; on the well-known (Na,K)-ATPase (Table 1). The inhibitors contrary our (Na,K)-ATPase (data with 20mM K) tested affect the Na-ATPase and the (Na,K)-ATPase shows similar affinity and higher capacity. differently: ouabain inhibits the latter and has no By comparing our results with those obtained in effect on the former while the opposite is true for gills from different species we must take into account furosemide. Neither has any effect on Mg-ATPase. that these results were obtained in homogenates or in Ethacrynate inhibits Na-ATPase and (Na,K)- microsomal fractions. In the gills Na can be substiATPase to different extents. These results are more tuted by other monovalent cations (Howland and similar to those obtained for guinea-pig small intes- Faus, 1985; Ventrella et al., 1987); a partial substitine (Del Castillo and Robinson, 1985), in which tution is also evident from our results (Table 4). The Na-ATPase was not totally inhibited by furosemide, possibility to replace ATP with other nucleotides, than for the guinea-pig kidney (Proverbio and Del evident in microsomes from sea bass gill (Borgatti et Castillo, 1981; Del Castillo et al., 1982) and rat al., 1985) is also present, even if to a lesser extent, in intestine (Luppa and Mfiller, 1982). Even though the our results (Table 2). In bass gill microsomes the concentration of furosemide used was high (2 mM), Na-ATPase and the (Na,K)-ATPase are present in only a half of the Na-ATPase activity was inhibited, corresponding amounts (Ventrella et al., 1987); in our so we tried a similar but more potent inhibitor, i.e. basolateral membranes the ratio is about 1: 3 and in bumetanide, but the Na-ATPase was still unaffected. the other mammalian basolateral membranes NaOuabain and strophanthidin are competitive in- ATPase is only 10% of the (Na,K)-ATPase. From hibitors which bind at the K site of the these results it seems that the Na-ATPase present in (Na,K)-ATPase; as K is usually present at mM the basolateral membrane of rat enterocyte is similar concentrations, we think that inhibitors concen- to the one found in the kidney and in the intestine of trations used (preincubation with 10mM stro- other mammals, but shares some features also with phanthidin and incubation with 2.5 mM ouabain) are the Na-ATPase found in the gills of sea bass and largely sufficient to block completely the (Na,K)- mussel. ATPase activity. Among tested substances only ouaFrom the results of this paper the distinction bain is a specific inhibitor; ethacrynate acts on sul- between Na-ATPase and (Na,K)-ATPase activities is phydryl groups and both furosemide and bumetanide always in terms of different behaviour. Del Castillo are known as inhibitors of the Na-K-2C1 cotransport and Robinson (1985) and Proverbio et al. (1986) suggest that Na-ATPase and (Na,K)-ATPase are system. Our results of both Na- and (Na,K)-ATPase activ- different molecular entities. Results from artificial ities in the basolateral membrane of the rat intestine membranes in which the supposed pure Na/K transcan be compared with the one found in rat intestine port system was reconstituted, show that Na can be (Luppa and Miiller, 1982) and in the guinea-pig pumped in the presence of ATP even if K is absent intestine (Del Castillo and Robinson, 1985) and (Goldin and Tong, 1974; Anner, 1980; Forgac and kidney (Del Castillo et al., 1982) only by assuming a Chin, 1982; Nagel et al., 1987); even if not always similar purity of the basolateral membrane prepara- specified, the implied assumption is that also this tion. Our values of both Na- and (Na,K)-ATPase K-independent, active Na transport is inhibited by activities are higher than those of Luppa and Mfiller ouabain. Furthermore the ATP hydrolysis-dependent (1982) even by considering that these results were Na Na exchange, which actively promotes an asymmetric and electrogenic transport of Na (Cornelius obtained at 37°C and in the presence of 100 mM Na. Our results show more than 4-5 times Na-ATPase in and Skou, 1985) cannot explain our data because of rat jejunum than in guinea-pig intestine and kidney the ouabain-sensitivity of this transport. To get more by taking into account that above cited investigators insight on this problem it would be useful to separate activated the Na-ATPase at 37°C and with 100 mM the two hypothesized molecular species by chemicoNa. On the contrary, (Na,K)-ATPase activities are physical methods or to discover a specific inhibitor of similar. There are some differences between our re- Na-ATPase. However the specific inhibitor is perhaps suits and those of Del Castillo et al. (1982) and Del not sufficient since it has been shown that cells Castillo and Robinson (1985) in the possibility of cultured in the presence of strophanthidin possess a
Ouabain-insensitive Na-ATPase in rat jejunum ouabain-insensitive ( N a , K ) - A T P a s e (Schulz a n d Cantley, 1988). N o w a d a y s more a n d m o r e new features are being ascribed to the mosaicism o f functions w h e n dealing with the N a p u m p or its enzymatic c o r r e s p o n d e n t (Na,K)-ATPase. T o sum up, it seems t h a t in rat intestine, as well as in o t h e r tissues a n d animals, there are o u a b a i n insensitive N a - A T P a s e s activities with similar properties. In these tissues, the N a - A T P a s e would constitute a ouabain°insensitive N a p u m p , which in rat intestine would also be present in a r e m a r k a b l e a m o u n t . The k n o w n difficulty of inhibiting the ( N a , K ) - A T P a s e of rat tissues by o u a b a i n m i g h t perhaps be due to the presence o f ouabain-insensitive N a - A T P a s e .
Acknowledgements--This research was supported by the Ministero della Pubblica Istruzione, Rome and the Consiglio Nazionale delle Ricerche, Rome, Italy. REFERENCES
Anner B. M. (1980) Reconstitution of the Na ÷, K ÷transport system in artificial membranes. Acta Physiol. Scand. Suppl. 481, 15-19. Baginski E. and Zak B. (1960) Micro-determination of serum phosphate and phospholipids. Clin. chim. Acta. 5, 834-838. Borgatti A. R., Trigari G., Pagliarani A. and Ventrella V. (1985) Ouabain-insensitive Na ÷ stimulation of a microsomal Mg2+-ATPase in gills of sea bass (Dicentrarchus labrax L.). Comp. Biochem. Physiol. 81A, 127-135. Bradford M. N. (1976) A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-252. Cornelius F. and Skou J. C. (1985) Na+-Na ÷ exchange mediated by (Na ÷ + K ÷ )-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP. Biochim. biophys. Acta 818, 211-221. Del Castillo J. R. and Robinson J. W. L. (1985) Na+-stimulated ATPase activities in basolateral plasma membranes from guinea pig small intestinal epithelial cells. Biochim. biophys. Acta 812, 413~122. Del Castillo J. R. and Whittembury G. (1987) Na ÷, K ÷ and C1- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na ÷ pump. Biochim. biophys. Acta 901, 209-216. Del Castillo J. R., Marin R., Proverbio T. and Proverbio F. (1982) Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes. Biochim. biophys. Acta 692, 61-68. De Weer P. (1985) Cellular sodium-potassium transport. In The Kidney: Physiology and Pathophysiology (Edited by Seldin D. W. and Giebisch G.), pp. 3148. Raven Press, New York. Faelli A., Esposito G. and Capraro V. (1976) Energy-rich phosphates and transintestinal transport in rat intestine incubated in vitro at different temperatures. Biochim. biophys. Acta 455, 759-766. Forbush B. III (1983) Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Analyt. Biochem. 128, 159-163.
Forgac M. and Chin G. (1982) Na ÷ transport by the (Na÷)-stimulated adenosine triphosphatase. J. biol. Chem. 257, 5652-5655. Goldin S. M. and Tong S. W. (1974) Reconstitution of active transport catalyzed by the purified sodium and
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potassium ion-stimulated adenosine triphosphatase from canine renal medulla. J. biol. Chem. 249, 5907-5915. Howland J. L. and Faus I. (1985) Cation-sensitive ATPase from gills of the salt water mussel, Mitilus edulis. Comp. Biochem. Physiol. 81B, 551-553. Kinsolving C. R., Post R. L. and Beawer D. L. (1963) Sodium plus potassium transport adenosine triphosphatase activity in kidney. J. cell. comp. Physiol. 62, 85-93. Luppa D. and Miiller F. (1982) Nachweis einer Na ÷aktivierbaren, durch Furosemid hemmbaren ATPaseAktivit/it in der Diinndarmmukosa der Ratte. Acta biol. med. germ. 41, 1037-1043. Marin R., Proverbio T. and Proverbio F. (1985) Effect of Ca 2÷ on the ouabain-insensitive, active Na ÷ uptake in inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells. Biochim. biophys. Acta 817, 299-306. Marin R., Obando M. A., Proverbio T. and Proverbio F. (1986a) Effect of a high NaC1 diet on the active mechanisms of Na ÷ extrusion in rat kidney. Kidney Int. 30, 518-523. Marin R., Proverbio T. and Proverbio F. (1986b) Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular ceils. Biochim. biophys. Acta 858, 195-201. Nagel G., Fendler K., Grell E. and Bamberg E. (1987) Na + currents generated by the purified ( N a ÷ + K +)-ATPase on planar lipid membranes. Biochim. biophys. Acta 901, 239-249. Obando M. A., Marin R., Proverbio T. and Proverbio F. (1987) High sodium diet and Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells. Biochem. Pharmac. 36, 7-11. Orsenigo M. N., Tosco M., Esposito G. and Faelli A. (1985) The basolateral membrane of rat enterocyte: its purification from brush border contamination. Analyt. Biochem. 144, 577-583. Orsenigo M. N., Tosco M., Esposito G. and Faelli A. (1987) Sodium transport in basolateral membrane vesicles from rat enterocytes. Arch. int. Physiol. Biochim. 95, 57-66. Ottolenghi P. (1975) The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem. J. 151, 61-66. Pfeiler E. and Kirschner L. B. (1972) Studies on gill ATPase of rainbow trout (Salmo gairdneri). Biochim. biophys. Acta 282, 301 310. Proverbio F. and Del Castillo J. R. (1981) Na+-stimulated ATPase activities in kidney basal-lateral plasma membranes. Biochim. biophys. Acta 646, 9%108. Proverbio F., Condrescu-Guidi M. and Whittembury G. (1975) Ouabain-insensitive Na ÷ stimulation of an Mg2+-dependent ATPase in kidney tissue. Biochim. biophys. Acta 394, 281-292. Proverbio F., Proverbio T. and Marin R. (1986) Na ÷ATPase is a different entity from the (Na ÷ + K + ) ATPase in rat kidney basolateral plasma membranes. Biochim. biophys. Acta 858, 202-205. Schulz J. T. and Cantley L. C. (1988) CV-I cell recipients of the mouse ouabain resistance gene express a ouabaininsensitive Na,K-ATPase after growth in cardioactive steroids. J. biol. Chem. 263, 624-632. Soltoff S. P. and Mandel L. J. (1984) Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump. J. gen. Physiol. 84, 623-642. Ventrella V., Pagliarani A., Trigari G., Trombetti F. and Borgatti A. R. (1987) Na+-like effect of monovalent cations in the stimulation of the sea bass gill Mg 2+dependent Na÷-stimulated ATPase. Comp. Biochem. Physiol. 88B, 691-695.
0020-711X/88 $3.00+0.00 Copyright © 1988 Pergamon Press pie
OUABAIN-INSENSITIVE Na-ATPase ACTIVITY IN THE BASOLATERAL M E M B R A N E FROM RAT JEJUNUM M. N. ORSENIGO 1, M. TOSCO 1, G. ESPOSITO2 and A. FAELLI 1. t Dipartimento di Fisiologia e Biochimica Generali, Universita' di Milano, via Celoria 26, 1-20133 Milano, Italy [Tel. 02-23 63 751] and 21stituto di Fisiologia Generale e Chimica Biologica, Facolt~i di Farmacia, Universitfi di Milano, via Saldini 50, 1-20133 Milano, Italy (Received 14 April 1988) A ~ t r a c t - - l . In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only
the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km= 15.7 + 2.3 mM and Vmax = 204 + 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km= 9.8 _ 1.2 mM and Vm,X= 640 _ 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.
INTRODUCTION
The (Na,K)-ATPase is an intrinsic plasma membrane protein that catalyzes transport of Na out of and K into cells, coupled with A T P hydrolysis. In addition to this enzyme, an N a - A T P a s e activity has been demonstrated in different tissues from various animal species. Gills from rainbow trout (Pfeiler and Kirschner, 1972), marine mussel (Howland and Faus, 1985) and sea bass (Borgatti et al., 1985; Ventrella et al., 1987) show a ouabain-insensitive Na-ATPase, which can be activated also by other monovalent cations and is present, at least in one case (Ventrella et al., 1987) in amounts similar to the (Na,K)-ATPase. The Na-ATPase was found also in the basolateral membrane of guinea-pig proximal tubular cells (Proverbio et al., 1975; Proverbio and Del Castillo, 1981; Del Castillo et al., 1982) and small intestinal epithelial cells of the same animal (Del Castillo and Robinson, 1985). In these mammalian tissues the Na-ATPase, present in a lesser quantity than the (Na,K)-ATPase, is always unaffected by ouabain, variably affected by furosemide, ethacrynic acid and triflocin, depending on the tissue and the animal (Proverbio et al., 1975; Proverbio and Del Castillo, 1981; Del Castillo et al., 1982; Del Castillo and Robinson, 1985) and modulated by Ca (Marin et al., 1985). It was found that both trypsin and sodium dodecylsulfate (SDS) treatments have different effects on the Na- and (Na,K)-ATPase activities of rat kidney basolateral plasma membrane (Proverbio et al., 1986) and only the Na-ATPase activity and the correspondent ouabain-insensitive N a pump are increased by a high Na diet (Matin et al., 1986a; Obando et al., 1987). The presence of the second N a *To whom correspondence should be addressed.
pump seems evident also from experiments performed on isolated enterocytes (Del Castillo and Whittembury, 1987). An ouabain-insensitive Na-ATPase, totally inhibited by furosemide, has been found in the intestinal mucosa from normal and diabetic rats; surprisingly both (Na,K)- and Na-ATPase activities were found distributed in many cellular structures, brush border included (Luppa and Mfiller, 1982). On the contrary Del Castillo and Robinson (1985) found that both (Na,K)- and Na-ATPase activities are only concentrated in the basolateral fraction. Since at least for the (Na,K)-ATPase the localization is known, we performed experiments only on basolateral membranes. In the present study, we have found an ouabain-insensitive Na-ATPase activity in the basolateral membrane of enterocytes obtained from the jejunal tract of rat intestine. The Na-ATPase has been compared with the well defined (Na,K)-ATPase and characterized by an enzymatic approach.
MATERIALS AND METHODS
Basolateral membrane separation Basolateral membranes from rat jejunum enterocytes were isolated and purified as described (Orsenigo et al., 1985). Briefly, 5 mM CaCI2 which preferentially aggregates all membranes except the brush border, was added to basolateral membranes, collected by self-orienting Percollgradient centrifugation. The pellets (basolateral membrane fraction) were washed and used for ATPase analyses. During the separation, phenylmethanesulfonylfluoride (PMSF) was 0.2 mM and 1,4-dithiothreitol was 1 mM. Specific activities and recoveries of marker enzymes in the basolateral membrane fraction are slightly better than those previously reported (Orsenigo et al., 1985). Only separated basolateral membranes with calculated enrichment factors of at least
1411
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M . N . ORSENIGOet al.
ten were used. Basolateral membranes are usually present as a mixture of sealed fight side out (RSO) or inside out (IO) vesicles and as unsealed vesicles. From the determination of ATPase activities before and after SDS treatment (ratios mg protein:mg SDS = 1:0.25-0.30), we calculated that the proportions of unsealed to RSO to IO vesicles were 2:2:1 (Orsenigo et al., 1987). A TPase assays
To open the sealed vesicles, concentrated membrane suspensions obtained in 250 mM sorbitol, 0.2 mM PMSF, 20mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes)/Tris (pH 7.5) = Sol. 1, were either treated with SDS at the previously cited ratio and diluted with Sol. 1, or simply diluted with hypotonic solution (0.2mM PMSF, 20 mM Hepes/Tris buffer, pH 7.5). When ouabaininsensitive Na-ATPase was determined, membranes were preincubated for 15 rain at room temperature with 10 mM strophanthidin which acts as ouabain but is lipid soluble. Twenty #1 of diluted membrane suspension (0.2-0.3 mg protein/ml) were added to 180 #1 of incubating solution to start the reaction. The incubating solution usually contained 5 mM MgATP, different concentrations of salts and inhibitors and 20 mM Hepes/Tfis buffer, pH 7.5. After 20 min incubation at 28°C (some experiments were performed at 37~C) the inorganic phosphate released was detected by adding 0.6 ml of ice-cold 0.5 % ammonium heptamolybdate, 3% ascorbic acid, 3% SDS in 0.5 M HCI (protect this solution from light). The test tubes were kept on ice for 10 min and colour development was stopped by adding 1 ml of a solution of 2% sodium meta-arsenite, 2% sodium citrate and 2% acetic acid. After heating the test tubes at 37~C for 10 min, the colour is stable for hours. All samples were run in quadruplicate and the absorbance determined at 850nm (Perkin-Elmer, Lambda 5 model spectrophotometer). Blanks, in which the membrane suspension was added at the end of the incubation period, were run in parallel and the nmoles Pi per mg protein found were subtracted from enzyme activities expressed in nmoles Pi released per mg protein per min. Proteins were determined by the Coomassie Blue binding assay (Bradford, 1976). The Mg-ATPase is the ATPase determined in the presence of Mg. The Na-ATPase is the difference in activity between (Mg,Na)-ATPase and Mg-ATPase. The (Na,K)-ATPase is the difference in activity between (Mg, Na,K)-ATPase and (Mg,Na)-ATPase. Statistical significance was assessed by the Student's t-test. Materials
MgATP, CTP Tris-salt, UTP Tris-salt, strophanthidin, furosemide and ethacrynic acid were from Sigma (St Louis, U.S.A.); GTP lithium-salt, ADP and AMP were from Boehringer (Mannheim, West Germany); bumetanide was a gift of Leo pharmaceutical products (Copenhagen, Denmark). RESULTS The two m e t h o d s for opening vesicles, i.e. SDS or h y p o t o n i c treatment, were r u n in parallel for some
experiments, a n d gave the same results in the A T P a s e assay. After that, only h y p o t o n i c t r e a t m e n t was used. A T P a s e activities of the basolateral m e m b r a n e in the presence of different inhibitors are presented in Table 1. Due to different yields from different preparations, these results are all for one basolateral m e m b r a n e preparation, but we repeated 3--4 times all the ATPase analyses with other preparations a n d the % changes with respect to the M g - A T P a s e were the same. M g - A T P a s e was e n h a n c e d by N a a n d further by N a + K + E G T A , i.e. the basolateral m e m b r a n e possessed b o t h the N a - A T P a s e and the (Na,K)ATPase a n d these enzymatic activities were present in different a m o u n t s (ratio 1:3.5). T h e ( N a , K ) - A T P a s e can be calculated either as o u a b a i n - i n h i b i t e d ATPase or as the K + E G T A effect on N a - A T P a s e ; the two values, 460 a n d 453 nmoles Pi/mg protein per min were the same. E G T A was a d d e d in the determ i n a t i o n of ( N a , K ) - A T P a s e because trace a m o u n t s of Ca m i g h t be present in o u r p r e p a r a t i o n a n d it is k n o w n t h a t ( N a , K ) - A T P a s e is inhibited by Ca. Proverbio a n d Del Castillo (1981) provided evidence t h a t M g - A T P a s e is unaffected by this chelating agent a n d N a - A T P a s e is partly inhibited. Mg-, Na- a n d (Na,K)ATPases behave differently in the presence o f the three substances tested. O u a b a i n inhibits only (Na,K)-ATPase, furosemide inhibits only NaA T P a s e (60%) a n d ethacrynate inhibits N a - A T P a s e almost totally ( > 9 0 % ) , ( N a , K ) - A T P a s e drastically (50%) a n d has practically no effect on Mg-ATPase. Bumetanide, a n o t h e r diuretic which has c o m m o n effects with furosemide, was also tested o n the three enzymatic activites. This possible inhibitor was added to the i n c u b a t i o n fluids, described in Table 1, in a wide range of concentrations, i.e. from 0.1/aM to 1 m M , but it was ineffective o n Mg-, N a - a n d ( N a , K ) - A T P a s e activities (results n o t reported in Table 1). The specificity for A T P of b o t h Na- a n d ( N a , K ) - A T P a s e was checked by replacing A T P with the o t h e r nucleotides U T P , CTP, G T P , A D P a n d A M P . F r o m the data in Tables 2 a n d 3, in which the results are reported, it seems t h a t the Pi released in the presence o f either N a or N a + K is always lower t h a n the Pi released in the presence o f ATP. Only C T P a n d G T P seem to activate the N a - A T P a s e partially. O n the contrary, for the (Na,K)-ATPase, A T P c a n n o t be substituted by other nucleotides (the % hydrolysis is always < 10%). The low activation of b o t h ATPases in the presence of A D P could be due to some A T P c o n t a m i n a n t in this substance. D a t a for the effect on N a - A T P a s e o f m o n o v a l e n t cations other t h a n N a and of a n i o n s o t h e r t h a n CI are reported in Table 4. It seems evident that all the
Table I. ATPase activities in the basolateral membrane ATPase ATPase ATPase Activating + 2.5 mM + 2.0 mM + 2.0 mM ions ATPase A ouabain A furosemide A ethacrynate A Mg 402 + 4 415 + 5* 400 + 3 395 -4-5 + 133 + 6 + 128 + 7 +57_ 6*** + 10_+6"** Mg + Na 535 + 4 543 + 5 457 _+5*** 405 + 4*** +453 + 7 -15 + 6*** +460 4- 7 +224 +_6*** Mg+Na+K 988+6 528+4*** 917+5"** 629+5*** Mean ATPase + SE in nmoles Pi/mg protein per min. A = Differencebetween left lower and left upper value. Number of determinations = 4. P values (*** = <0.001; * = <0.1; if no * = NS) vs the first value on the same line. The concentration of the activating ions was: 5 mM Mg (MgATP), 20 mM Na (NaCI), 5 mM K (KCI). When K was present also 0.2 mM EGTA was in the incubating fluid.
Ouabain-insensitive Na-ATPase in rat jejunum Table 2. Na-nucleotidase activities in the basolateral membrane Nucleotidase activities Nucleotide ATP UTP CTP GTP ADP AMP
Mg-
(Mg, Na)-
A
422 + 5 431 +_.4 358 _ 4 359 +_3 230+3 139 +- 2
543 +_6 436 +_4 396 +- 2 401 __.4 251 +_2 142 +_2
+ 121 __.8 +5 + 6
+ 36 +- 4 +42 +- 5 +21 +_4 +3 +_3
Mean nucleotidase -t- SE in nmoles Pi/mg protein per min. A = Na-nucleotidase = differences between the two values on the same line. Number of determinations = 4. The concentration of the activating inns was: 5 mM Mg (MgCI2), 20 mM Na (NaCI). The concentration of each nucleotide (Tris salt except GTP-2 Li)= 5raM. Membranes containing trace amounts of Ca, were pre-equilibrated with 10 mM strophanthidin. 2.5 mM ouabain was in the incubating fluid. cations tested can partially substitute Na: the highest a c t i v a t i o n ( 4 0 % ) is o b s e r v e d w i t h K a n d t h e l o w e s t (20%) with Cs; on the contrary, the Na-ATPase a c t i v i t y is u n a f f e c t e d w h e n CI is r e p l a c e d by t h e a n i o n s listed in T a b l e 4. All t h e s e r e s u l t s were o b t a i n e d at 2 8 ° C a n d in t h e p r e s e n c e o f 20 m M N a a n d 5 m M K . Q u a n t i t a t i v e l y h i g h e r v a l u e s ( n o t r e p o r t e d ) w e r e o b t a i n e d at 3 7 ° C in the presence of 100raM Na and 20mM K, but the behaviour of the ATPases was the same. F i g u r e 1 s h o w s t h e r a t e s o f A T P h y d r o l y s i s for b o t h N a - A T P a s e a n d ( N a , K ) - A T P a s e activities as functions of different Na concentrations. While the activation of the Na-ATPase follows a Michaelis-Menten kinetics, t h e ( N a , K ) - A T P a s e yields a s i g m o i d a l v e l o c i t y c u r v e . T h e k i n e t i c p a r a m eters t h a t c a n be e x t r a p o l a t e d f r o m t h e E a d i e H o f f s t e e p l o t f o r N a - A T P a s e a r e a Vmax v a l u e o f 204 + 1 9 n m o l e s P i / m g p r o t e i n p e r m i n a n d a Km value of 15.7+2.3mM (r = 0 . 9 0 ) . T h e k i n e t i c c h a r a c t e r i s t i c s o f ( N a , K ) - A T P a s e were a n a l y z e d b y t h e l o g a r i t h m i c f o r m o f t h e Hill e q u a t i o n , w h i c h g a v e a Hill coefficient ( o f c o o p e r a t i v i t y ) n = 1.76 + 0.15 ( m o r e t h a n o n e N a i n v o l v e d ) , a Vmax o f 640 _+ 15 n m o l e s P i / m g p r o t e i n p e r m i n a n d a K m v a l u e o f 9 . 8 _ 1 . 2 m M (r = 0.96). T h i s K m v a l u e is lower than those generally reported (Soltoff and M a n d e l , 1984; D e W e e r , 1985), b u t as s u g g e s t e d b y S o l t o f f a n d M a n d e l (1984), h i g h e r v a l u e s c a n be o b t a i n e d if h i g h e r K c o n c e n t r a t i o n s a r e u s e d . A s a m a t t e r o f fact, f r o m r e s u l t s o f t h e g r o u p o f e x p e r i m e n t s p e r f o r m e d in t h e p r e s e n c e o f 20 m M K (at
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Table 4. Effects of different cations or different Na-salts on the Mg-ATPase of the basolateral membrane Activating substances
ATPase activities
A
Mg Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg + Mg +
410 + 4 546 + 6 447 + 5 438 _+4 465 + 6 449 + 6 436 + 7 555 +- 7 539 + 7 540 + 5 543 + 5 551 + 7 545 __.6
+ 136 + 7 + 37 + 6*** +28 + 6*** + 55 + 7*** + 39 + 7*** +26 + 8*** + 145 + 8 + 129 + 8 + 130 + 6 + 133 + 6 + 141 + 8 + 135 + 7
NaCI LiCI NH4CI KCI RbCI CsCI NaBr Na acetate Na gluconate Na ascorbate Na2SO 4 Na lactate
Mean ATPase + SE in nmoles Pi/mg protein per min. A= Differences between each value and the first value (=Mg-ATPase). P values (***= <0.001; if no * = NS) vs the first A value (= Na-ATPase activity). Number of determinations = 4. The basolateral membranes containing trace amounts of Ca were pre-equilibrated with 10raM strophanthidin. The concentration of the activating ions was: 5 mM Mg (MgATP), 20mM Na (NaC1 and other Na salts, Na2SO 4 = l0 mM), 20 mM Li = NH 4 = K = Rb = Cs (CI salts). 2.5 mM ouabain was in the incubating fluid. 28°C) we h a v e calculated a K m value of 15.0 _ 2 . 2 m M ( a n d Vm~x = 632 _ 18 n m o l e s P i / m g p r o t e i n p e r m i n ) . K ~ v a l u e s o f 7, 12 a n d 30 m M were o b t a i n e d in t h e k i d n e y w i t h K c o n c e n t r a t i o n s o f 5, 20 a n d 80 r e s p e c t i v e l y ( K i n s o l v i n g et al., 1963). DISCUSSION T h e m e t h o d u s e d f o r i n o r g a n i c p h o s p h a t e determ i n a t i o n w a s p r o p o s e d b y B a g i n s k i a n d Z a k (1960) a n d m o d i f i e d in v a r i o u s w a y s b y o t h e r s ( O t t o l e n g h i , 1975; F o r b u s h III, 1983; M a r i n et al., 1986b). W e u s e d relatively l a r g e v o l u m e s o f b o t h c o l o r develo p m e n t a n d s t o p s o l u t i o n s w i t h r e s p e c t to i n c u b a t i o n v o l u m e to a v o i d a n o n - s p e c i f i c l o w e r i n g o f t h e i n o r -
6O0 "~soo
i
40O
f
F __-------e--
Table 3. (Na, K)-nucleotidase activities in the basolateral membrane
--e--
Nucleotidase activities Nucleotide ATP UTP CTP GTP ADP AMP
(Mg, Na).
(Mg, Na, K)-
A
528+8 454 + 5 407+_5 423 +_4 280 _ 4 148 + 2
1002+_ 13 456 +_7 451 +_ 10 451 +_8 316 +- 5 145 +- 3
+474+ 15 +2 +_9 + 4 4 + 11 + 28 + 9 + 36 ___6 - 3 +- 4
Mean nucleotidase + SE in nmoles Pi/mg protein per min. A = (Na, K)-nucleotidase = differences between the two values on the same line. Number of determinations = 4. The concentration of the activating ions was: 5mM Mg (MgCI2), 20mM Na (NaCI), 5 mM K (KCI). Concentration of each nucleotide (Tris salt except GTP" 2 Li) = 5 mM. The incubating fluids contained 0.2 mM EGTA.
,o
2b
~
4b
~
mM
1~
Fig. 1. Effects of different Na concentrations (abscissa) on both the Na-ATPase (solid circles) and the (Na,K)-ATPase (open circles) activity, expressed in nmoles Pi released per m g protein and per min (ordinate). For Na-ATPase, membranes containing trace a m o u n t s of Ca were preequilibrated with 10 m M strophanthidin and 2.5 m M ouabain was in the incubating fluid; for (Na,K)-ATPase, the incubating fluid contained 0.2 m M E G T A and 5 m M KCI. For both, 5 m M M g A T P and 20 m M Hepes/Tris buffer, pH 7.5 were present. Mean v a l u e s + SE (=vertical bars, absent if lower than symbol dimension).
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M. N. ORSENIGO et al.
ganic phoshate found in the presence of even not very replacing ATP with other nucleotides. The Nahigh osmolar incubation fluids (e.g. with 150mM ATPase of rat intestine (our results) shows a lower sorbitol or 50 mM NaCI). specificity for ATP. On the contrary, the The results presented in this paper were obtained (Na,K)-ATPase behaves similarly in all tissues deat 28°C. Higher values but similar behaviour were scribed (with the exception of some activation by found at 37°C, in a few experiments not reported. GTP and ITP in the guinea-pig intestine). Moreover, The Na/K pump is activated in vivo by intracellular in all these tissues the stimulatory effect of Na is Na and extracellular K. For this reason, we used independent of the accompanying anions, while in the 20 mM Na and 5 mM K. The incubation temperature rat intestine Na seems not be replaceable by Li. For (28°C), lower than 37°C, was chosen after Na uptake (Na,K)-ATPase activity, different Km values are obexperiments with basolateral membrane vesicles, tained with different K concentrations (this work; made in parallel (results not reported in this paper). Kinsolving et al., 1963; Soltoff and Mandel, 1984) so At this temperature vesicles are more stable over it is difficult to compare Km values of (Na,K)- and time. Also the in vitro everted and perfused rat Na-ATPase even in the same preparation. By comintestine is more stable at this temperature (Faelli et paring our Km and Vma~ values with those obtained by al., 1976). Del Castillo et al. (1982) and Del Castillo and The basolateral membrane of the jejunal enterocyte Robinson (1985), it seems that our Na-ATPase activseems to have a Na-ATPase activity in addition to the ity shows lower affinity and higher capacity; on the well-known (Na,K)-ATPase (Table 1). The inhibitors contrary our (Na,K)-ATPase (data with 20mM K) tested affect the Na-ATPase and the (Na,K)-ATPase shows similar affinity and higher capacity. differently: ouabain inhibits the latter and has no By comparing our results with those obtained in effect on the former while the opposite is true for gills from different species we must take into account furosemide. Neither has any effect on Mg-ATPase. that these results were obtained in homogenates or in Ethacrynate inhibits Na-ATPase and (Na,K)- microsomal fractions. In the gills Na can be substiATPase to different extents. These results are more tuted by other monovalent cations (Howland and similar to those obtained for guinea-pig small intes- Faus, 1985; Ventrella et al., 1987); a partial substitine (Del Castillo and Robinson, 1985), in which tution is also evident from our results (Table 4). The Na-ATPase was not totally inhibited by furosemide, possibility to replace ATP with other nucleotides, than for the guinea-pig kidney (Proverbio and Del evident in microsomes from sea bass gill (Borgatti et Castillo, 1981; Del Castillo et al., 1982) and rat al., 1985) is also present, even if to a lesser extent, in intestine (Luppa and Mfiller, 1982). Even though the our results (Table 2). In bass gill microsomes the concentration of furosemide used was high (2 mM), Na-ATPase and the (Na,K)-ATPase are present in only a half of the Na-ATPase activity was inhibited, corresponding amounts (Ventrella et al., 1987); in our so we tried a similar but more potent inhibitor, i.e. basolateral membranes the ratio is about 1: 3 and in bumetanide, but the Na-ATPase was still unaffected. the other mammalian basolateral membranes NaOuabain and strophanthidin are competitive in- ATPase is only 10% of the (Na,K)-ATPase. From hibitors which bind at the K site of the these results it seems that the Na-ATPase present in (Na,K)-ATPase; as K is usually present at mM the basolateral membrane of rat enterocyte is similar concentrations, we think that inhibitors concen- to the one found in the kidney and in the intestine of trations used (preincubation with 10mM stro- other mammals, but shares some features also with phanthidin and incubation with 2.5 mM ouabain) are the Na-ATPase found in the gills of sea bass and largely sufficient to block completely the (Na,K)- mussel. ATPase activity. Among tested substances only ouaFrom the results of this paper the distinction bain is a specific inhibitor; ethacrynate acts on sul- between Na-ATPase and (Na,K)-ATPase activities is phydryl groups and both furosemide and bumetanide always in terms of different behaviour. Del Castillo are known as inhibitors of the Na-K-2C1 cotransport and Robinson (1985) and Proverbio et al. (1986) suggest that Na-ATPase and (Na,K)-ATPase are system. Our results of both Na- and (Na,K)-ATPase activ- different molecular entities. Results from artificial ities in the basolateral membrane of the rat intestine membranes in which the supposed pure Na/K transcan be compared with the one found in rat intestine port system was reconstituted, show that Na can be (Luppa and Miiller, 1982) and in the guinea-pig pumped in the presence of ATP even if K is absent intestine (Del Castillo and Robinson, 1985) and (Goldin and Tong, 1974; Anner, 1980; Forgac and kidney (Del Castillo et al., 1982) only by assuming a Chin, 1982; Nagel et al., 1987); even if not always similar purity of the basolateral membrane prepara- specified, the implied assumption is that also this tion. Our values of both Na- and (Na,K)-ATPase K-independent, active Na transport is inhibited by activities are higher than those of Luppa and Mfiller ouabain. Furthermore the ATP hydrolysis-dependent (1982) even by considering that these results were Na Na exchange, which actively promotes an asymmetric and electrogenic transport of Na (Cornelius obtained at 37°C and in the presence of 100 mM Na. Our results show more than 4-5 times Na-ATPase in and Skou, 1985) cannot explain our data because of rat jejunum than in guinea-pig intestine and kidney the ouabain-sensitivity of this transport. To get more by taking into account that above cited investigators insight on this problem it would be useful to separate activated the Na-ATPase at 37°C and with 100 mM the two hypothesized molecular species by chemicoNa. On the contrary, (Na,K)-ATPase activities are physical methods or to discover a specific inhibitor of similar. There are some differences between our re- Na-ATPase. However the specific inhibitor is perhaps suits and those of Del Castillo et al. (1982) and Del not sufficient since it has been shown that cells Castillo and Robinson (1985) in the possibility of cultured in the presence of strophanthidin possess a
Ouabain-insensitive Na-ATPase in rat jejunum ouabain-insensitive ( N a , K ) - A T P a s e (Schulz a n d Cantley, 1988). N o w a d a y s more a n d m o r e new features are being ascribed to the mosaicism o f functions w h e n dealing with the N a p u m p or its enzymatic c o r r e s p o n d e n t (Na,K)-ATPase. T o sum up, it seems t h a t in rat intestine, as well as in o t h e r tissues a n d animals, there are o u a b a i n insensitive N a - A T P a s e s activities with similar properties. In these tissues, the N a - A T P a s e would constitute a ouabain°insensitive N a p u m p , which in rat intestine would also be present in a r e m a r k a b l e a m o u n t . The k n o w n difficulty of inhibiting the ( N a , K ) - A T P a s e of rat tissues by o u a b a i n m i g h t perhaps be due to the presence o f ouabain-insensitive N a - A T P a s e .
Acknowledgements--This research was supported by the Ministero della Pubblica Istruzione, Rome and the Consiglio Nazionale delle Ricerche, Rome, Italy. REFERENCES
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