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Interaction of catecholamines and ethanol on the kinetics of rat brain (Na+ + K+)-ATPase
European Journal o f Pharmacology, 70 (1981 ) 157--166
157
© Elsevier/North-Holland Biomedical Press
INTERACTION OF CATECHOLAMINES AND ETHANOL ON THE KINETICS OF RAT B R A I N (Na ÷ + K÷)-ATPase HAROLD KALANT * and NARAYAN RANGARAJ Department o f Pharmacology, University of Toronto, Toronto, Canada M5S 1A8 and Addiction Research Foundation of Ontario, Toronto, Canada M5S 2S1
Received 4 November 1980, accepted 26 November 1980
H. KALANT and N. RANGARAJ, Interaction of catecholamines and ethanol on the kinetics of rat brain (Na÷ + K+)-ATPase, European J. Pharmacol. 70 (1981) 157--166. The effects of catecholamines (CA) and ethanol (EtOH), singly and in combination, on the kinetics of rat brain (Na* + K÷)-ATPase were studied. Addition of 0.05 M EtOH alone did not change V m a x o r Km for K ÷, Na ÷, Mg2+ and ATP. Addition of 0.1 mM dopamine (DA) or noradrenaline (NA) alone stimulated the enzyme activity in presence of vanadium-containing ATP as substrate, but not with vanadium-free ATP except in the presence of high Mg2+ : ATP ratios. CA alone decreased the K m slightly for K ÷ and by about 50% for ATP, increased it for Mg2+ and did not change it for Na ÷. However, the combination of DA or NA + EtOH produced a marked inhibition which was competitive for K÷, and uncompetitive or mixed for Mg2+, Na ÷ and ATP. The inhibitory effect of NA + EtOH was abolished in 20 mM K ÷. These findings suggest that NA sensitizes the enzyme to EtOH inhibition at physiological K ÷ concentrations, by conformational change away from the outwardly facing K÷-binding E2P form to the inwardly facing Na÷-binding EIP form. (Na ÷ + K+)-ATPase
Rat brain
Ethanol
1. I n t r o d u c t i o n In p r e v i o u s p a p e r s (Rangaraj a n d K a l a n t , 1 9 7 8 , 1 9 7 9 ) , we r e p o r t e d t h a t c a t e c h o l a m i n e s (CA) sensitize t h e {Na ÷ + K÷)-ATPase (EC 3.6.1.3) o f r a t brain h o m o g e n a t e s t o inhibit i o n b y e t h a n o l ( E t O H ) . This a c t i o n o f CA was i n d e p e n d e n t o f t h e i r a c t i v a t i o n o f t h e A T P a s e in vitro, w h i c h has b e e n f o u n d b y m a n y investigators ( Y o s h i m u r a , 1 9 7 3 ; G o d f r a i n d et al., 1 9 7 4 ; L o g a n a n d O ' D o n o v a n , 1 9 7 6 ; Desaiah a n d H o , 1 9 7 7 ; H e x u m , 1 9 7 7 ; S u l a k h e et al., 1 9 7 7 ; L e e a n d Phillis, 1 9 7 7 ; R a n g a r a j a n d K a l a n t , 1 9 7 8 ; Phillis a n d Wu, 1 9 7 9 ; S c h a e f e r et al., 1 9 7 9 ; Wu a n d Phillis, 1 9 7 9 a ) . A c t i v a t i o n b y CA is w i d e l y believed t o result f r o m t h e i r ability to c h e l a t e v a n a d a t e * To whom correspondence should be sent: Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Canada M5S 1A8.
Catecholamines
Interaction
Kinetics
ions, p r e s e n t as t r a c e c o n t a m i n a n t s in c o m mercial p r e p a r a t i o n s o f h o r s e m u s c l e ATP. H o w e v e r , CA sensitize A T P a s e t o i n h i b i t i o n b y E t O H in t h e p r e s e n c e or a b s e n c e o f vanadiu m (Rangaraj and K a l a n t , 1 9 7 8 , 1979). T h e m e c h a n i s m o f i n h i b i t i o n o f r a t brain (Na ÷ + K÷)-ATPase b y E t O H a p p e a r s t o be an allosteric e f f e c t on t h e a f f i n i t y o f t h e K ÷b i n d i n g site ( K a l a n t et al., 1978). T h e e n z y m e a c t i v i t y is also highly d e p e n d e n t o n t h e relative c o n c e n t r a t i o n s o f Na*, K ÷, Mg 2÷ and A T P ( R o b i n s o n , 1 9 7 0 , 1 9 7 5 , 1977a, b; S k o u , 1 9 6 5 , 1 9 7 4 a , b, c; G a r a y a n d G a r r a h a n , 1 9 7 3 ; S c h w a r t z et al., 1 9 7 5 ; Albers, 1976}. Theref o r e it was o f i n t e r e s t t o d e t e r m i n e w h e t h e r t h e sensitization o f the e n z y m e t o E t O H b y CA was e x e r t e d via an e n h a n c e m e n t o f t h e aUosteric e f f e c t on t h e K ÷ site, or changes in o n e or m o r e o f t h e o t h e r ligand sites. In this p a p e r we r e p o r t t h e e f f e c t s o f varying K +, N a ÷, Mg 2÷ a n d A T P c o n c e n t r a t i o n s on
158
(Na ÷ + K÷)-ATPase activity in rat brain homogenates, in the presence and absence of CA and EtOH.
2. Materials and methods Male Wistar rats weighing 230--330 g were obtained from Biobreeding Laboratories (Ottawa, Canada) and Canadian Breeding Laboratories (Montreal, Canada). 'Old grade' Na2ATP (Sigma A-3127 and A-6144, containing vanadium) and vanadium-free Na2ATP (Sigma A-5394), L-arterenol HC1 and dopamine HC1 were purchased from Sigma Chemical Co. All other chemicals used were of ACS reagent grade. The rats were maintained on laboratory chow and water ad libitum. They were decapitated and the whole brain was removed immediately, wiped clean of blood, weighed and homogenized in 19 volumes of 0.32 M sucrose. The homogenate was further diluted 5-fold with cold distilled water for use in the ATPase assay. The usual assay mixture contained 30 mM imidazole, 30 mM glycyl glycine, 3 m M MgC12, 3 mM Na2ATP, 120 mM NaC1 and 5 mM KC1, plus 0.05 ml of diluted homogenate in a final volume of 1.3 ml at pH 7.4. In another tube, 1 mM ouabain was added and NaC1 and KC1 were ommited. Reaction was started by addition of the enzyme and the mixture was incubated for 20 min at 37°C in a shaking water bath. The reaction was stopped by addition of 0.5 ml of 1.2 M HC104. Inorganic phosphate (Pi) was estimated by a modified phosphomolybdate m e t h o d (Post and Sen, 1967). (Na÷+ K÷)ATPase activity was obtained by subtracting the activity in the presence of ouabain (Mg 2÷ATPase) from the total activity in the presence of Na ÷, K ÷ and Mg 2÷ and the absence of ouabaln. Mg2÷-ATPase activities in the various preparations ranged from 13.72 to 15.00 pmol of Pi per mg protein per h. The effects of various ligands were studied by varying their concentrations identically in
H. K A L A N T , N. R A N G A R A J
the assay mixtures for both total ATPase and Mg2÷-ATPase. The following final concentration ranges were studied: KC1 0.5--40 mM, MgCl2 0.5--12 mM, ATP 0.5--8 mM and NaC1 0--150 mM. Each ligand was varied individually, keeping the concentrations of the others constant. Since Na2ATP was used in all these experiments, the final concentrations of Na ÷ shown in the Results section included the contribution from the ATP. The studies of Na ÷, Mg2÷ and ATP kinetics were carried out at K ÷ concentrations of both 5 mM and 20 mM. The effects of 0.1 mM CA and 0.05 M EtOH separately and together were tested in all kinetic studies. All assays were run in duplicate. Protein was determined by the m e t h o d of Lowry et al. (1951), with bovine serum albumin as the standard. 3. Results 3.1. E f f ect o f D A on (Na÷ + K÷)-ATPase in presence and absence o f E t O H at various K ÷ concentrations
The effect of varying K ÷ on the enzyme activity with old grade ATP, in presence of 0.05 M EtOH and 0.1 mM DA added individually and in combination, is shown in fig. 1. The activity was stimulated by 0.1 mM DA alone, but inhibited by EtOH + DA at low concentrations of K ÷. The inhibition was partially reversible by high K ÷ (20 mM). A Lineweaver-Burk plot (fig. 1, insert) of the same data indicated a competitive type of inhibition by EtOH in presence of 0.1 mM DA, but n o t in its absence. Kr, for K ÷ increased 2.74 fold as in our earlier report (Rangaraj and Kalant, 1978). When vanadium-free ATP was used as substrate, 0.1 mM DA was no longer stimulatory, but the inhibitory effect of 0.05 M EtOH in combination with DA was similar to that seen with old grade ATP (fig. 2). Virtually identical results were also obtained, when the DA was replaced by 0.1 mM NA. The control activity of the enzyme at 5 mM K ÷ was about 28% higher with vanadiumfree ATP than with old grade ATP.
159
CATECHOLAMINES, ETHANOL AND ATPase KINETICS
20
25
20
..z: × c
2
o
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10
a..
0.4
T
::k
V~
>-_~r,o
a_ 1
0.4
&
-0.6
0
0.6
1.2
5
* K
-0.6
0
0.6 + K
1.2
1.8
2-4
1 (mM)
1.8
1 (raM) 0 I
I 10
I 20
i 30
410
KCI (raM)
I
J
10
210
3tO
40
KCI(mM)
Fig. 1. Effect of DA on (Na÷+ K+)-ATPase in presence and absence of EtOH at various K + concentrations, using vanadium-containing ATP as substrate. © Control, A 0.05 M EtOH, • 0.1 mM DA, • 0.1 mM DA + 0.05 M EtOH. Inset: Lineweaver-Burk plot o f the same data. V is the velocity of reaction expressed as p m o l Pi/mg protein X h. Each point is the average o f six separate experiments and each experiment included duplicate determinations. Vertical bars indicate S.E.M. for each point.
Fig. 2. Effect of 0.1 m M D A and 0.05 M E t O H added separately and in combination on (Na + + K+)-ATPase at various K + concentrations, using vanadium-free A T P as substrate. Inset: Lineweaver-Burk plot of the same data. © Control, A 0.05 M EtOH, • 0.1 m M DA, • 0.1 m M D A + 0.05 M EtOH. Each point represents the mean of six experiments, with duplicate determinations in each. Regression lines were calculated by the method of least squares. Vertical bars indicate S.E.M. for each point.
K+-- 5 mM
K÷=2OmM
20
3.2. E f f e c t o f D A and N A on (Na ÷ + K+) A T P a s e with and with'out E t O H at various Mg 2+ c o n c e n t r a t i o n s '
When Mg 2+ was varied from 0 . 0 5 mM to 12 mM t h e e n z y m e activity rose to a m a x i m u m at 3 mM under all c o n d i t i o n s and decreased at higher Mg 2+ (fig. 3). EtOH alone ( 0 . 0 5 M) had n o detectable effect o n the activity at a n y Mg 2÷ c o n c e n t r a t i o n . N A alone increased activity at all Mg 2÷ c o n c e n t r a t i o n s , while the c o m bination o f N A + EtOH reduced it b e l o w con-
.~
15
o
~0 a.
o 2
4
MgCI2
6 (mM)
8
10
12
2
4
6
8
MgCl:z (raM)
Fig. 3. Effect of 0.1 m M N A and 0.05 M E t O H added separately and in combination on (Na ÷ + K÷)-ATPase at 5 and 20 m M K + and various M g 2+ concentrations. o Control, ~ 0.05 M EtOH, • 0.1 m M NA, [] 0.1 m M N A + 0.05 M EtOH.
160
H. K A L A N T , N. R A N G A R A J
trol levels. Lineweaver-Burk plots of the ascending portions of these curves (fig. 4a) indicated an uncompetitive type of inhibition. DA alone had virtually the same effect as NA, except that stimulation of the enzyme was relatively greater with NA at Mg 2÷ concentrations below 2 mM, and with DA at higher Mg 2÷. Also, the inhibition produced by DA + EtOH appeared to be of a mixed rather than an uncompetitive type. This indicates decreases in both Vmax and Km for Mg :÷, in the presence of EtOH plus NA or DA, but no change with EtOH alone. Both CA alone increased Vmax, and DA increased the K~ for Mg 2÷. The kinetic constants (Kin and Vmax) are shown in table 1. When the K ÷ concentration was raised to 20 raM, EtOH alone still had no inhibitory effect and both CAs alone stimulated the activity, but the sensitization to EtOH was considerably less marked. Though the Km and Vm~x fell in the presence of EtOH + CA relative to those with CA alone, they did not fall below control values as had occurred with 5 mM K ÷. The Km for Mg 2÷ and the Vmax, as well as effects of NA and DA on these, were similar to those seen at
0151
K+= 5ram
(c~)
(b)
K+= 20ram
]~
~trol
: EtOH eNA EtOH+NA
OI
05
1'0 I Mg++(mM)
15
2'0
05
li0
1.5
2.0
Mg++{rnM)
Fig. 4. Lineweaver-Burk p l o t o f Mg 2+ kinetics s h o w n in fig. 3. (a) 5 m M K ÷, (b) 20 raM K ÷.
5 mM K ÷, but the inhibitory effect of EtOH + DA or NA, relative to DA or NA alone, was still present (fig. 4b and table 1). 3.3. Effect o f N A on (Na ÷ + K+)-ATPase with and w i t h o u t O.05 M E t O H at various A T P concentrations
For this study, vanadium-free ATP was used as substrate, at concentrations ranging
TABLE 1 Kinetic c o n s t a n t s for Mg 2+ and (Na ÷ + K+)-ATPase: e f f e c t o f 0.1 m M NA or D A and 0.05 M E t O H . Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
0.1 mM D A
0.1 mM DA + 0.05 M EtOH
0.37 + 0.03 (5) 16.1 + 0.27
0.38 +- 0.02 (5) 15.87 + 0.14
0.42 + 0.04 (4) 21.0 + 0.45 2
0.19 + 0.03 (4) 4 10.57 -+ 0.34 4
0.79 + 0.05 (4) 4 25.22 + 0.56 4
0.11 + 0.01 (4) 4 11.12 -+ 0.36 4
0.56 (1) 17.47
0.98 -+ 0.08 (4) 3 24.81 -+ 1.11 2
0.74 + 0.16 (3) 16.81 + 0.72
1.06 -+ 0.22 (3) 3 27.82 -+ 1.88 2
0.49 -+ 0.04 (3) 16.31 + 0.06
(a) K ÷ = 5 m M
Km 1 Vmax 1
(b) K + = 20 rnM
Km Vma x
0.57 + 0.05 (3) 17.74 -+ 0.37
1 Km is e x p r e s s e d in mM; Vmax e x p r e s s e d as p m o l Pi p r o d u c e d / h × mg p r o t e i n . Values are r e p o r t e d as m e a n + S.E.M. N u m b e r s in parentheses are n u m b e r o f e x p e r i m e n t s . Each e x p e r i m e n t was p e r f o r m e d in duplicate. Statistical significance of differences from corresponding c o n t r o l values, by t-test for paired data, is indicated as follows: 2 p < 0.02, 3 p < 0.01, 4 p < 0.001.
C A T E C H O L A M I N E S , E T H A N O L A N D ATPase K I N E T I C S
161
15
IO
/
c
2 0 mM K ÷
i,+" 5
O]
© control
5 mM K ÷
EtOH
3 E
eNA EtOH + NA
i
'
i
8
~TP]
-ram
Fig. 5. E f f e c t o f 0.1 mM N A and 0.05 M E t O H added separately and in c o m b i n a t i o n on (Na t + K÷)-ATPase at 5 and 20 mM K ÷ and various ATP concentrations.
from 0.5 to 8 mM. Activity increased with concentration of ATP, and attained a maximum at 4 mM, but at 8 mM the activity was decreased {fig. 5). Addition of NA alone, at low concentrations of ATP (less than 3 mM), increased the activity, but this effect disappeared at higher ATP concentrations. Addi-
0''+ 0.18
(a)
tion of 0.05 M EtOH alone had no effect on the activity, but the combination of EtOH + NA inhibited it at all concentrations of ATP above I mM. This inhibition was reversed by increasing K + concentration to 20 mM. Lineweaver-Burk plots of the same data showed that Km for ATP decreased about 50% with 0.1 mM NA or with NA + EtOH, but the Vmax was decreased by 35% only in the pres-
(b)
0.14
.~ 20
0.12
V ~ 0.110 /,A~ 0.08
a--lO
0.06
5
O.O4
K
5
o
0,02
0:5
1~
1
AT I~(rnM)
K+:SmM
K+=2OmM
K+= 20mM
M
,'5 -~o
o!6
,:0
1
/5
2'.o
AT P (raM)
Fig. 6. Lineweaver-Burk p l o t o f the data in fig. 5. (a) 5 mM K ÷, (b) 2 0 mM K ÷. © Control, A 0 . 0 5 M EtOH, • 0.1 m M N A , ~ 0.1 mM N A + 0.05 M EtOH.
40
80
120
NaCI (raM)
160
40
80
120
160
NoCI (raM)
Fig. 7. E f f e c t o f 0.1 mM N A and 0.05 M E t O H added separately and in c o m b i n a t i o n on (Na t + K÷)-ATPase at 5 and 20 mM K ÷ and various Na t concentrations. © Control, zk 0 . 0 5 M EtOH, • 0.1 mM N A , • 0.1 mM N A + 0.05 M EtOH.
162
H. KALANT, N. RANGARAJ
TABLE 2 Kinetic constants for ATP and (Na ÷ + K÷)-ATPase: effect of 0.1 mM NA, 0.05 M EtOH and NA + EtOH at 5 and 20 mM K÷.
(a) K ÷ = 5
Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
0.88 -+ 0.048 17.54 -+ 0.41
0.87 + 0.07 17.44 -+ 0.50
0.47 + 0.035 2 17.10 + 0.24
0.45 -+ 0.014 2 11.18 + 0.75 3
0.78 - 0.15 20.34 -+ 1.2
0.59 -+ 0.09 20.36 + 0.98
0.74 -+ 0.12 20.89 + 1.01
mM
Km 1 Vmax 1
(b) K += 20 mM
Km
0.78 -+ 0.14 20.67 -+ 1.3
Vma x
1 Km is expressed in raM; Vma x expressed as pmol Pi produced/mg protein × h. Values are reported as mean -+ S.E.M. of 4 separate experiments. Each experiment was performed in duplicate. Statistical significance from corresponding control values, by t-test for paired data, is indicated as follows: 2p < 0.002, 3p < 0.001.
ence o f b o t h N A and E t O H (fig. 6a). T h e kinetic c o n s t a n t s are s h o w n in table 2. This e f f e c t was virtually abolished b y raising t h e K ÷ c o n c e n t r a t i o n t o 20 mM (fig. 6b). 3.4. E f f e c t o f 0.1 m M N A o n ( N a ÷ + K÷) A T P a s e w i t h a n d w i t h o u t O.05 M E t O H a t various N a ÷ c o n c e n t r a t i o n s
T h e effect o f various c o n c e n t r a t i o n s o f N a + ( 6 - - 1 5 6 mM) in presence o f 0.05 M E t O H a n d 0.1 mM N A a d d e d individually and in c o m b i n a t i o n is s h o w n in fig. 7. R e a c t i o n v e l o c i t y
(b)
K%5 mM
i
,10 f
J
K+=20mM control ,~EtOH • : EtOHt+ NA~ NA
(a)
was largely i n d e p e n d e n t o f Na ÷ c o n c e n t r a t i o n over a wide range, and was decreased signific a n t l y o n l y at the l o w e s t and highest Na ÷. N A a n d E t O H a d d e d separately did n o t have a n y e f f e c t on the activity, b u t in c o m b i n a t i o n t h e y inhibited t h e e n z y m e over the w h o l e range o f Na ÷ c o n c e n t r a t i o n . Kinetic c o n s t a n t s calculated f r o m L i n e w e a v e r - B u r k plots (fig. 8) are s h o w n in table 3. These c o n s t a n t s are o n l y a p p r o x i m a t i o n s because even the l o w e s t Na ÷ c o n c e n t r a t i o n used (6 mM) gave m o r e t h a n 50% o f t h e m a x i m u m activity in t h e c o n t r o l p r e p a r a t i o n . However, as n o t e d previously ( K a l a n t et al., 1978), t h e i n t e r a c t i o n b e t w e e n t h e Na ÷ and K ÷ binding sites renders the absolute Km values u n c e r t a i n in a n y case, and the i m p o r t a n t p o i n t in the present c o n t e x t is the c h a n g e p r o d u c e d b y E t O H and NA. N e i t h e r Vmax n o r K ~ f o r Na ÷ was a f f e c t e d b y E t O H o r N A alone, b u t b o t h were significantly decreased b y the c o m b i n a t i o n o f E t O H + NA. Again, the effect was abolished b y increasing K ÷ to 20 mM.
~ "06
4. Discussion
~/Na* (raM) Fig. 8. Lineweaver-Burk plot of the data of fig. 7. (a) 5 mM K ÷, (b) 20 mM K+.
T h e Km values d e t e r m i n e d in these experim e n t s f o r the various ligands (K ÷, Na ÷, Mg 2÷ a n d A T P ) fall w i t h i n t h e r e p o r t e d values. T h e r e is considerable variation in Km values in
163
CATECHOLAMINES, ETHANOL AND ATPase KINETICS
TABLE 3 Kinetic constants for Na + and (Na + + K+)-ATPase: effect of 0.1 mM NA, 0.05 M EtOH, and NA + EtOH at 5 and 20 mM K÷. Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
4.06 -+ 0.43 (7) 19.78 -+ 0.99
3.67 -+ 0.40 (7) 19.10 -+ 0.99
3.77 -+ 0.36 (7) 19.74 --4"0.90
1.75 -+ 0.37 (7) 2 13.63 + 0.88 2
(a) K + = 5 m M
Km 1 Vmax 1 (b) I C = 2 0 m M
Km Vma x
4.93 -+ 0.42 (5) 23.3 + 1.54
5.89 + 0.30 (5) 21.89 -+ 1.41
1 Km is expressed in mM; Vmax expressed as pmol Pi/mg protein × h. Values are reported as mean -+ S.E.M. Number of experiments indicated in parentheses. Each experiment was performed in duplicate. Statistical significance of differences from corresponding control values, by t-test for paired data, is indicated as follows: 2 p < 0.001.
t h e literature, d u e to the fact t h a t Km f o r (Na ÷ + K÷)-ATPase and a given ligand d e p e n d s o n m a n y factors such as relative c o n c e n t r a tions o f o t h e r ligands, pH, t e m p e r a t u r e , species and t y p e o f tissue. H o w e v e r , t h e similarity b e t w e e n t h e Km value f o r K ÷ f o u n d here and t h o s e o b t a i n e d in o u r o w n earlier studies ( K a l a n t et al., 1 9 7 8 ; Rangaraj and Kalant, 1978) suggests t h a t t h e e n z y m e p r e p a r a t i o n is comparable. A d d i t i o n o f 0.05 M E t O H had n o signific a n t e f f e c t o n Km f o r Na ÷, Mg 2+ and A T P and increased t h a t f o r K ÷ o n l y b y 10%. Changes in kinetic c o n s t a n t s have b e e n r e p o r t e d b y o t h e r s o n l y in presence o f E t O H c o n c e n t r a tions greater t h a n 0.22 M (Israel et al., 1 9 6 5 ; Sun and Samorajski, 1 9 7 0 ; Lin, 1 9 7 6 ; Sun, 1 9 7 6 ; K a l a n t et al., 1 9 7 8 ) . In t h e p r e s e n t s t u d y , D A (0.1 mM) stimulated the e n z y m e activity at all K ÷ c o n c e n t r a tions w h e n old grade A T P was used. This is similar t o o u r previous finding with NA (Rangaraj and Kalant, 1978). T h e degree o f s t i m u l a t i o n increased progressively w i t h rising K ÷ c o n c e n t r a t i o n Up to 4 mM, and t h e n r e m a i n e d c o n s t a n t at higher K ÷ values. This is in fairly g o o d a g r e e m e n t with results o b t a i n e d b y Desaiah and H o ( 1 9 7 7 ) , w h o r e p o r t e d t h a t D A s t i m u l a t i o n was seen o n l y in presence o f K + and r e a c h e d a plateau at 10 m M K ÷. T h e
s t i m u l a t i o n a p p e a r e d t o result f r o m increased a f f i n i t y f o r K* ( l o w e r Kin), with n o change in Vmax. Km f o r Mg 2+ was slightly increased in t h e presence o f NA and a l m o s t d o u b l e d in the presence o f D A (fig. 4 and table 1), suggesting t h a t CA decreased the a f f i n i t y o f the e n z y m e f o r Mg 2÷. T h e Km f o r Na ÷ did n o t change, b u t Km f o r A T P was decreased b y 50% at 5 mM K ÷, suggesting t h a t the a f f i n i t y f o r A T P is increased in presence o f NA. Thus, NA a l o n e h a d a d i f f e r e n t e f f e c t o n the binding o f each o f t h e principal ligands, while 0.05 M e t h a n o l had none. In contrast, t h e c o m b i n a t i o n o f 0.05 M E t O H plus 0.1 mM NA o r D A increased t h e Km f o r K ÷ b y m o r e t h a n t w o - f o l d irrespective o f the t y p e o f A T P used as substrate. Similar degrees o f increase in Km f o r K ÷ had b e e n observed with m u c h higher E t O H c o n c e n t r a t i o n s a l o n e (Israel et al., 1 9 6 5 ; Israel and Salazar, 1 9 6 7 ; Lin, 1 9 7 6 ; Sun, 1 9 7 6 ; K a l a n t et al., 1978). Therefore the effect of the combinat i o n on t h e K ÷ binding site m a y involve t h e same m e c h a n i s m as t h a t responsible f o r t h e e f f e c t o f a higher c o n c e n t r a t i o n o f E t O H alone. This m a y n o t be t r u e f o r t h e binding sites f o r ATP, Na ÷ and Mg 2+. O u r results indic a t e u n c o m p e t i t i v e o r m i x e d t y p e s o f inhibit i o n f o r Na ÷, A T P and Mg 2+ kinetics in the p r e s e n c e o f CA + E t O H t o g e t h e r . This differs
164
from the noncompetitive inhibition for ATP and Na ÷ (Sun and Samorajski, 1970; Lin, 1976) and competitive inhibition for Na ÷ (Sun, 1976) in presence of high concentrations (>0.5 M) of EtOH alone. Therefore the NA sensitization of the enzyme toward EtOH inhibition appears to be specific for the K ÷ site. The decrease of Vma x for Mg 2÷, Na ÷ and ATP kinetics by the combination of CA + EtOH may be due to retardation of any of the partial reactions of (Na ÷ + K÷)-ATPase, which are as follows (Albers, 1976): E1 + ATP + 3(Na÷)i ~ E1P(Na÷)3 + ADP E1P + Mg 2÷ ~ E2P + 3(Na+)0 E2P + 2(K÷)0 ~ E2P(K÷)2 H : O + E2P(K+)2 ~ E2(K+): + P E2(K÷)2 -~ El(K+)2 + Mg 2+ El(K+)2 ~ E 1 + 2K[ El and E2 are two conformations of the enzyme, facing the inside and outside of the membrane respectively, and E2 contains bound Mg 2÷. The subscripts i and o designate ions located respectively inside and outside the cell. Since the EtOH + CA combination decreases the Km for ATP, Mg2÷ and Na ÷, it m a y be facilitating the formation of E IP and possibly its conversion to E2P. However, the reduced affinity (higher Kin) for K ÷ would result in inhibition of the subsequent steps by which E2P is ultimately converted to El and hence in reduction of the overall reaction velocity. This is consistent with the increased affinity for Mg2÷, since Mg2÷ would be released during the conversion. The fact that increased K ÷ tends to overcome the inhibition and increase the Km for Mg 2÷ suggests again that the effect of EtOH + CA is the same as that of a higher concentration of EtOH alone. An incidental question concerns the mechanism of stimulation of ATPase activity by CA alone. Though this is not the major concern of our study, the findings may be of some relevance to a disagreement in the literature.
H. KALANT, N. RANGARAJ
Many authors have reported that stimulation occurs only when the ATP substrate is contaminated with vanadate or some other metallic inhibitor which can be chelated by CA (Cantley et al., 1977). In contrast, Wu and Phillis (1978, 1979a, 1979b) and Phillis and Wu (1979) have reported that NA stimulates the activity by a receptor-specific mechanism regardless of the ATP used. We observed that, even with V-free ATP, NA did stimulate the enzyme at low concentrations of ATP (<3 mM) and a fixed concentration (3 mM) of Mg 2+. When ATP exceeded 3 mM, there was no stimulation. These findings are compatible with the idea that an excessive ratio of Mg 2÷ : ATP is inhibitory, and that NA produces activation under these conditions by chelating the excess Mg 2÷ (Rajan et al., 1972; Schaefer et al., 1979). However, Phillis and Wu (personal communication) have found activation by NA over a wide range of Mg : ATP ratios, and the question is still unresolved. In summary, our present findings suggest that NA sensitizes the enzyme to inhibition by EtOH at physiological concentrations of K ÷, by facilitating an EtOH-induced conformational change that impairs K ÷ binding to the outward-facing form and prevents its dephosphorylation and conversion to the inward-facing form (Kalant et al., 1980). Further support of this interpretation is provided by NA + EtOH effects on Arrhenius plots of ATPase activity, to be reported separately.
Acknowledgments The authors are indebted to Mr. Gregory Ho and Mrs. Anita Chau for preparation of the illustrations.
References Albers, R.W., 1976, The (sodium plus potassium) transport ATPase, in: The Enzymes of Biological Membranes, Vol. 3, ed. A. Martonosi (Plenum Press, New York) p. 283. Cantley, L.D., Jr., L. Josephson, R. Warner, M. Yanagisawa, C. Lechene and G. Guidotti, 1977, Vana-
CATECHOLAMINES, ETHANOL AND ATPase KINETICS date is a potent inhibitor found in ATP derived from muscle, J, Biol. Chem. 252, 7421. Desaiah, D. and I.K. Ho, 1977, Kinetics of catecholamine sensitive Nat-K ÷ ATPase activity in mouse brain synaptosomes, Biochem. Pharmacol. 26, 2029. Garay, R.P. and P.G. Garahan, 1973, The interaction of sodium and potassium with sodium pump in red cells, J. Physiol. 231,297. Godfraind, T., M.C. Koch and N. Verbeke, 1974, The action of EGTA on the catecholamine stimulation of rat brain Na-K ATPase, Biochem. Pharmacol. 23, 3505. Hexum, T.D., 1977, The effects of catecholamine on transport (Na, K) adenosine triphosphatase, Biochem. Pharmacol. 26, 1221. Israel, Y. and I. Salazar, 1967, Inhibition of brain microsomal adenosine triphosphatase by general depressants, Arch. Biochem. Biophys. 122, 310. Israel, Y., H. Kalant and I. Laufer, 1965, Effects of ethanol on Na, K, Mg-stimulated microsomal ATPase activity, Biochem. Pharmacol. 14, 1803. Kalant, H., N. Rangaraj, N. Woo and L. Endrenyi, 1980, Effects of ethanol on neuronal membrane transport systems, in: Progress in Neurotoxicology (Proc. 1st Int. Congr. Neurotoxicol., Varese, 1979), eds. L. Manzo, N. Lery, Y. Lacasse and L. Roche (Pergamon Press, Oxford) p. 91. Kalant, H., N. Woo and L. Endrenyi, 1978, Effect of ethanol on the kinetics o f rat brain (Nat+ K+)ATPase and K-dependent phosphatase with different alkali ions, Biochem. Pharmacol. 27, 1353. Lee, S.L. and J.W. Phillis, 1977, Stimulation of cerebral cortical synaptosomal Na-K ATPase by biogenic amines, Can. J. Physiol. Pharmacol. 55,961. Lin, D.C., 1976, Effects of ethanol on the kinetic parameters of brain (Na t + K ÷) adenosine triphosphatase, Ann. N.Y. Acad. Sci. 273, 331. Logan, J.G. and D.J. O'Donovan, 1976, The effects of ouabain and the activation of neural membrane ATPase by biogenic amines, J. Neurochem. 27, 185. Lowry, O.H., M.J. Rosebrough, A.C. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193,265. Phillis, J.W. and P.H. Wu, 1979, Failure of vanadate contamination in ATP to account for noradrenaline .stimulation of Na +, K+-ATPase in rat brain homogenates, J. Pharm. Pharmacol. 31,556. Post, R.L. and A.K. Sen, 1967, Sodium and potassium stimulated ATPase, in: Methods in Enzymology, Vol. X, eds. R.E. Estabrook and M.E. Pullman (Academic Press, New York) p. 762. Rajan, K.S., J.M. Davis, R.W. Colburn and F.H. Jarke, 1972, Metal chelates in the storage and transport of neurotransmitters: Formation of mixed ligands of Mg2+-ATP with biogenic amines, J. Neurochem. 19, 1099.
165 Rangaraj, N. and H. Kalant, 1978, Effects of ethanol withdrawal, stress and amphetamine on rat brain (Na t + K÷)-ATPase, Biochem. Pharmacol. 27, 1139. Rangaraj, N. and H. Kalant, 1979, Interaction of ethanol and catecholamine on rat brain (Na t + K÷)ATPase, Can. J. Physiol. Pharmacol. 57, 1098. Robinson, J.D., 1970, Interactions between monovalent cations and the Na t, K÷-dependent adenosine-triphosphatase, Arch. Biochem. Biophys. 139, 17. Robinson, J.D., 1975, Functionally different classes of K ÷ sites on the (Nat+ K÷)-dependent ATPase, Biochim. Biophys. Acta 380, 250. Robinson, J.D., 1977a, K ÷ stimulation of ADP/ATP exchange catalyzed by the ( N a t + K÷)-dependent ATPase, Biochim. Biophys. Acta 484, 161. Robinson, J.D., 1977b, Na t sites of the ( N a t + K÷)dependent ATPase, Biochim. Biophys. Acta 482, 427. Schaefer, A., M. Komlos and A. Seregi, 1979, Studies on the effects of catecholamines and chelating agents on the synaptic membrane Na t, K ÷ ATPase activity in the presence and absence of hydroxylamine, Biochem. Pharmacol. 28, 2307. Schwartz, A., G.E. Lindenmayer and J.C. Allen, 1975, The sodium potassium adenosine triphosphatase. Pharmacological, physiological and biochemical aspects, Pharmacol. Rev. 27, 3. Skou, J.C., 1965, Enzymatic basis for active transport of Na t and K ÷ across cell membrane, Physiol. Rev. 45, 596. Skou, J.C., 1974a, Effects of ATP on the intermediary steps of the reaction of the ( N a t + K÷)dependent enzyme system. I. Studied by the use of N-ethylmaleimide inhibition as a tool, Biochim. Biophys. Acta 339,234. Skou, J.C., 1974b, Effect of ATP on the intermediary steps of the reaction of the (Na t + K÷)-dependent enzyme system. II. Effects of a variation in the ATP/Mg 2+ ratio, Biochim. Biophys. Acta 339, 246. Skou, J.C., 1974c, Effect of ATP on the intermediary steps of the reaction of the (Na t + K÷)-dependent enzyme system. III. Effect on the p-nitrophenylphosphatase activity of the system, Biochim. Biophys. Acta 339,258. Sulakhe, P.V., S.H. Jean and S.J. Sulakhe, 1977, Studies on the stimulation of (Nat+ K÷)-ATPase of neural tissues by catecholamines, Gen. Pharmacol. 8, 37. Sun, A.Y., 1976, Alcohol-membrane interaction in the brain. I. Reversibility, Ann. N.Y. Acad. Sci. 273,295. Sun, A.Y. and T. Samorajski, 1970, Effects of ethanol on the activity of adenosine triphosphatase and acetyleholine esterase in synaptosomes isolated from guinea pig brain, J. Neurochem. 17, 1365.
166 Wu, P.H. and J.W. Phillis, 1978, Effects of a and /3 adrenergic blocking agents on the biogenic amine stimulated (Na ÷ + K÷)ATPase ~of rat cerebral cortical synaptosomal membrane, Gen. Pharmacol. 9, 421. Wu, P.H. and J.W. Phillis, 1979a, Receptor-mediated noradrenaline stimulation of (Na ÷ + K+)-ATPase in rat brain cortical homogenates, Gen. Pharmacol. 10,189.
H. KALANT, N. RANGARAJ Wu, P.H. and J.W. Phillis, 1979b, Effects of vanadate on brain (Na*+ K+)-ATPase and p-nitrophenylphosphatase-interactions with mono and divalent ions and with noradrenaline, Int. J. Biochem. 10, 629. Yoshimura, K., 1973, Activation of Na-K-activated ATPase in rat brain by catecholamine, J. Biochem. 74, 389.
157
© Elsevier/North-Holland Biomedical Press
INTERACTION OF CATECHOLAMINES AND ETHANOL ON THE KINETICS OF RAT B R A I N (Na ÷ + K÷)-ATPase HAROLD KALANT * and NARAYAN RANGARAJ Department o f Pharmacology, University of Toronto, Toronto, Canada M5S 1A8 and Addiction Research Foundation of Ontario, Toronto, Canada M5S 2S1
Received 4 November 1980, accepted 26 November 1980
H. KALANT and N. RANGARAJ, Interaction of catecholamines and ethanol on the kinetics of rat brain (Na÷ + K+)-ATPase, European J. Pharmacol. 70 (1981) 157--166. The effects of catecholamines (CA) and ethanol (EtOH), singly and in combination, on the kinetics of rat brain (Na* + K÷)-ATPase were studied. Addition of 0.05 M EtOH alone did not change V m a x o r Km for K ÷, Na ÷, Mg2+ and ATP. Addition of 0.1 mM dopamine (DA) or noradrenaline (NA) alone stimulated the enzyme activity in presence of vanadium-containing ATP as substrate, but not with vanadium-free ATP except in the presence of high Mg2+ : ATP ratios. CA alone decreased the K m slightly for K ÷ and by about 50% for ATP, increased it for Mg2+ and did not change it for Na ÷. However, the combination of DA or NA + EtOH produced a marked inhibition which was competitive for K÷, and uncompetitive or mixed for Mg2+, Na ÷ and ATP. The inhibitory effect of NA + EtOH was abolished in 20 mM K ÷. These findings suggest that NA sensitizes the enzyme to EtOH inhibition at physiological K ÷ concentrations, by conformational change away from the outwardly facing K÷-binding E2P form to the inwardly facing Na÷-binding EIP form. (Na ÷ + K+)-ATPase
Rat brain
Ethanol
1. I n t r o d u c t i o n In p r e v i o u s p a p e r s (Rangaraj a n d K a l a n t , 1 9 7 8 , 1 9 7 9 ) , we r e p o r t e d t h a t c a t e c h o l a m i n e s (CA) sensitize t h e {Na ÷ + K÷)-ATPase (EC 3.6.1.3) o f r a t brain h o m o g e n a t e s t o inhibit i o n b y e t h a n o l ( E t O H ) . This a c t i o n o f CA was i n d e p e n d e n t o f t h e i r a c t i v a t i o n o f t h e A T P a s e in vitro, w h i c h has b e e n f o u n d b y m a n y investigators ( Y o s h i m u r a , 1 9 7 3 ; G o d f r a i n d et al., 1 9 7 4 ; L o g a n a n d O ' D o n o v a n , 1 9 7 6 ; Desaiah a n d H o , 1 9 7 7 ; H e x u m , 1 9 7 7 ; S u l a k h e et al., 1 9 7 7 ; L e e a n d Phillis, 1 9 7 7 ; R a n g a r a j a n d K a l a n t , 1 9 7 8 ; Phillis a n d Wu, 1 9 7 9 ; S c h a e f e r et al., 1 9 7 9 ; Wu a n d Phillis, 1 9 7 9 a ) . A c t i v a t i o n b y CA is w i d e l y believed t o result f r o m t h e i r ability to c h e l a t e v a n a d a t e * To whom correspondence should be sent: Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Canada M5S 1A8.
Catecholamines
Interaction
Kinetics
ions, p r e s e n t as t r a c e c o n t a m i n a n t s in c o m mercial p r e p a r a t i o n s o f h o r s e m u s c l e ATP. H o w e v e r , CA sensitize A T P a s e t o i n h i b i t i o n b y E t O H in t h e p r e s e n c e or a b s e n c e o f vanadiu m (Rangaraj and K a l a n t , 1 9 7 8 , 1979). T h e m e c h a n i s m o f i n h i b i t i o n o f r a t brain (Na ÷ + K÷)-ATPase b y E t O H a p p e a r s t o be an allosteric e f f e c t on t h e a f f i n i t y o f t h e K ÷b i n d i n g site ( K a l a n t et al., 1978). T h e e n z y m e a c t i v i t y is also highly d e p e n d e n t o n t h e relative c o n c e n t r a t i o n s o f Na*, K ÷, Mg 2÷ and A T P ( R o b i n s o n , 1 9 7 0 , 1 9 7 5 , 1977a, b; S k o u , 1 9 6 5 , 1 9 7 4 a , b, c; G a r a y a n d G a r r a h a n , 1 9 7 3 ; S c h w a r t z et al., 1 9 7 5 ; Albers, 1976}. Theref o r e it was o f i n t e r e s t t o d e t e r m i n e w h e t h e r t h e sensitization o f the e n z y m e t o E t O H b y CA was e x e r t e d via an e n h a n c e m e n t o f t h e aUosteric e f f e c t on t h e K ÷ site, or changes in o n e or m o r e o f t h e o t h e r ligand sites. In this p a p e r we r e p o r t t h e e f f e c t s o f varying K +, N a ÷, Mg 2÷ a n d A T P c o n c e n t r a t i o n s on
158
(Na ÷ + K÷)-ATPase activity in rat brain homogenates, in the presence and absence of CA and EtOH.
2. Materials and methods Male Wistar rats weighing 230--330 g were obtained from Biobreeding Laboratories (Ottawa, Canada) and Canadian Breeding Laboratories (Montreal, Canada). 'Old grade' Na2ATP (Sigma A-3127 and A-6144, containing vanadium) and vanadium-free Na2ATP (Sigma A-5394), L-arterenol HC1 and dopamine HC1 were purchased from Sigma Chemical Co. All other chemicals used were of ACS reagent grade. The rats were maintained on laboratory chow and water ad libitum. They were decapitated and the whole brain was removed immediately, wiped clean of blood, weighed and homogenized in 19 volumes of 0.32 M sucrose. The homogenate was further diluted 5-fold with cold distilled water for use in the ATPase assay. The usual assay mixture contained 30 mM imidazole, 30 mM glycyl glycine, 3 m M MgC12, 3 mM Na2ATP, 120 mM NaC1 and 5 mM KC1, plus 0.05 ml of diluted homogenate in a final volume of 1.3 ml at pH 7.4. In another tube, 1 mM ouabain was added and NaC1 and KC1 were ommited. Reaction was started by addition of the enzyme and the mixture was incubated for 20 min at 37°C in a shaking water bath. The reaction was stopped by addition of 0.5 ml of 1.2 M HC104. Inorganic phosphate (Pi) was estimated by a modified phosphomolybdate m e t h o d (Post and Sen, 1967). (Na÷+ K÷)ATPase activity was obtained by subtracting the activity in the presence of ouabain (Mg 2÷ATPase) from the total activity in the presence of Na ÷, K ÷ and Mg 2÷ and the absence of ouabaln. Mg2÷-ATPase activities in the various preparations ranged from 13.72 to 15.00 pmol of Pi per mg protein per h. The effects of various ligands were studied by varying their concentrations identically in
H. K A L A N T , N. R A N G A R A J
the assay mixtures for both total ATPase and Mg2÷-ATPase. The following final concentration ranges were studied: KC1 0.5--40 mM, MgCl2 0.5--12 mM, ATP 0.5--8 mM and NaC1 0--150 mM. Each ligand was varied individually, keeping the concentrations of the others constant. Since Na2ATP was used in all these experiments, the final concentrations of Na ÷ shown in the Results section included the contribution from the ATP. The studies of Na ÷, Mg2÷ and ATP kinetics were carried out at K ÷ concentrations of both 5 mM and 20 mM. The effects of 0.1 mM CA and 0.05 M EtOH separately and together were tested in all kinetic studies. All assays were run in duplicate. Protein was determined by the m e t h o d of Lowry et al. (1951), with bovine serum albumin as the standard. 3. Results 3.1. E f f ect o f D A on (Na÷ + K÷)-ATPase in presence and absence o f E t O H at various K ÷ concentrations
The effect of varying K ÷ on the enzyme activity with old grade ATP, in presence of 0.05 M EtOH and 0.1 mM DA added individually and in combination, is shown in fig. 1. The activity was stimulated by 0.1 mM DA alone, but inhibited by EtOH + DA at low concentrations of K ÷. The inhibition was partially reversible by high K ÷ (20 mM). A Lineweaver-Burk plot (fig. 1, insert) of the same data indicated a competitive type of inhibition by EtOH in presence of 0.1 mM DA, but n o t in its absence. Kr, for K ÷ increased 2.74 fold as in our earlier report (Rangaraj and Kalant, 1978). When vanadium-free ATP was used as substrate, 0.1 mM DA was no longer stimulatory, but the inhibitory effect of 0.05 M EtOH in combination with DA was similar to that seen with old grade ATP (fig. 2). Virtually identical results were also obtained, when the DA was replaced by 0.1 mM NA. The control activity of the enzyme at 5 mM K ÷ was about 28% higher with vanadiumfree ATP than with old grade ATP.
159
CATECHOLAMINES, ETHANOL AND ATPase KINETICS
20
25
20
..z: × c
2
o
0.6 .5 1
10
a..
0.4
T
::k
V~
>-_~r,o
a_ 1
0.4
&
-0.6
0
0.6
1.2
5
* K
-0.6
0
0.6 + K
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2-4
1 (mM)
1.8
1 (raM) 0 I
I 10
I 20
i 30
410
KCI (raM)
I
J
10
210
3tO
40
KCI(mM)
Fig. 1. Effect of DA on (Na÷+ K+)-ATPase in presence and absence of EtOH at various K + concentrations, using vanadium-containing ATP as substrate. © Control, A 0.05 M EtOH, • 0.1 mM DA, • 0.1 mM DA + 0.05 M EtOH. Inset: Lineweaver-Burk plot o f the same data. V is the velocity of reaction expressed as p m o l Pi/mg protein X h. Each point is the average o f six separate experiments and each experiment included duplicate determinations. Vertical bars indicate S.E.M. for each point.
Fig. 2. Effect of 0.1 m M D A and 0.05 M E t O H added separately and in combination on (Na + + K+)-ATPase at various K + concentrations, using vanadium-free A T P as substrate. Inset: Lineweaver-Burk plot of the same data. © Control, A 0.05 M EtOH, • 0.1 m M DA, • 0.1 m M D A + 0.05 M EtOH. Each point represents the mean of six experiments, with duplicate determinations in each. Regression lines were calculated by the method of least squares. Vertical bars indicate S.E.M. for each point.
K+-- 5 mM
K÷=2OmM
20
3.2. E f f e c t o f D A and N A on (Na ÷ + K+) A T P a s e with and with'out E t O H at various Mg 2+ c o n c e n t r a t i o n s '
When Mg 2+ was varied from 0 . 0 5 mM to 12 mM t h e e n z y m e activity rose to a m a x i m u m at 3 mM under all c o n d i t i o n s and decreased at higher Mg 2+ (fig. 3). EtOH alone ( 0 . 0 5 M) had n o detectable effect o n the activity at a n y Mg 2÷ c o n c e n t r a t i o n . N A alone increased activity at all Mg 2÷ c o n c e n t r a t i o n s , while the c o m bination o f N A + EtOH reduced it b e l o w con-
.~
15
o
~0 a.
o 2
4
MgCI2
6 (mM)
8
10
12
2
4
6
8
MgCl:z (raM)
Fig. 3. Effect of 0.1 m M N A and 0.05 M E t O H added separately and in combination on (Na ÷ + K÷)-ATPase at 5 and 20 m M K + and various M g 2+ concentrations. o Control, ~ 0.05 M EtOH, • 0.1 m M NA, [] 0.1 m M N A + 0.05 M EtOH.
160
H. K A L A N T , N. R A N G A R A J
trol levels. Lineweaver-Burk plots of the ascending portions of these curves (fig. 4a) indicated an uncompetitive type of inhibition. DA alone had virtually the same effect as NA, except that stimulation of the enzyme was relatively greater with NA at Mg 2÷ concentrations below 2 mM, and with DA at higher Mg 2÷. Also, the inhibition produced by DA + EtOH appeared to be of a mixed rather than an uncompetitive type. This indicates decreases in both Vmax and Km for Mg :÷, in the presence of EtOH plus NA or DA, but no change with EtOH alone. Both CA alone increased Vmax, and DA increased the K~ for Mg 2÷. The kinetic constants (Kin and Vmax) are shown in table 1. When the K ÷ concentration was raised to 20 raM, EtOH alone still had no inhibitory effect and both CAs alone stimulated the activity, but the sensitization to EtOH was considerably less marked. Though the Km and Vm~x fell in the presence of EtOH + CA relative to those with CA alone, they did not fall below control values as had occurred with 5 mM K ÷. The Km for Mg 2÷ and the Vmax, as well as effects of NA and DA on these, were similar to those seen at
0151
K+= 5ram
(c~)
(b)
K+= 20ram
]~
~trol
: EtOH eNA EtOH+NA
OI
05
1'0 I Mg++(mM)
15
2'0
05
li0
1.5
2.0
Mg++{rnM)
Fig. 4. Lineweaver-Burk p l o t o f Mg 2+ kinetics s h o w n in fig. 3. (a) 5 m M K ÷, (b) 20 raM K ÷.
5 mM K ÷, but the inhibitory effect of EtOH + DA or NA, relative to DA or NA alone, was still present (fig. 4b and table 1). 3.3. Effect o f N A on (Na ÷ + K+)-ATPase with and w i t h o u t O.05 M E t O H at various A T P concentrations
For this study, vanadium-free ATP was used as substrate, at concentrations ranging
TABLE 1 Kinetic c o n s t a n t s for Mg 2+ and (Na ÷ + K+)-ATPase: e f f e c t o f 0.1 m M NA or D A and 0.05 M E t O H . Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
0.1 mM D A
0.1 mM DA + 0.05 M EtOH
0.37 + 0.03 (5) 16.1 + 0.27
0.38 +- 0.02 (5) 15.87 + 0.14
0.42 + 0.04 (4) 21.0 + 0.45 2
0.19 + 0.03 (4) 4 10.57 -+ 0.34 4
0.79 + 0.05 (4) 4 25.22 + 0.56 4
0.11 + 0.01 (4) 4 11.12 -+ 0.36 4
0.56 (1) 17.47
0.98 -+ 0.08 (4) 3 24.81 -+ 1.11 2
0.74 + 0.16 (3) 16.81 + 0.72
1.06 -+ 0.22 (3) 3 27.82 -+ 1.88 2
0.49 -+ 0.04 (3) 16.31 + 0.06
(a) K ÷ = 5 m M
Km 1 Vmax 1
(b) K + = 20 rnM
Km Vma x
0.57 + 0.05 (3) 17.74 -+ 0.37
1 Km is e x p r e s s e d in mM; Vmax e x p r e s s e d as p m o l Pi p r o d u c e d / h × mg p r o t e i n . Values are r e p o r t e d as m e a n + S.E.M. N u m b e r s in parentheses are n u m b e r o f e x p e r i m e n t s . Each e x p e r i m e n t was p e r f o r m e d in duplicate. Statistical significance of differences from corresponding c o n t r o l values, by t-test for paired data, is indicated as follows: 2 p < 0.02, 3 p < 0.01, 4 p < 0.001.
C A T E C H O L A M I N E S , E T H A N O L A N D ATPase K I N E T I C S
161
15
IO
/
c
2 0 mM K ÷
i,+" 5
O]
© control
5 mM K ÷
EtOH
3 E
eNA EtOH + NA
i
'
i
8
~TP]
-ram
Fig. 5. E f f e c t o f 0.1 mM N A and 0.05 M E t O H added separately and in c o m b i n a t i o n on (Na t + K÷)-ATPase at 5 and 20 mM K ÷ and various ATP concentrations.
from 0.5 to 8 mM. Activity increased with concentration of ATP, and attained a maximum at 4 mM, but at 8 mM the activity was decreased {fig. 5). Addition of NA alone, at low concentrations of ATP (less than 3 mM), increased the activity, but this effect disappeared at higher ATP concentrations. Addi-
0''+ 0.18
(a)
tion of 0.05 M EtOH alone had no effect on the activity, but the combination of EtOH + NA inhibited it at all concentrations of ATP above I mM. This inhibition was reversed by increasing K + concentration to 20 mM. Lineweaver-Burk plots of the same data showed that Km for ATP decreased about 50% with 0.1 mM NA or with NA + EtOH, but the Vmax was decreased by 35% only in the pres-
(b)
0.14
.~ 20
0.12
V ~ 0.110 /,A~ 0.08
a--lO
0.06
5
O.O4
K
5
o
0,02
0:5
1~
1
AT I~(rnM)
K+:SmM
K+=2OmM
K+= 20mM
M
,'5 -~o
o!6
,:0
1
/5
2'.o
AT P (raM)
Fig. 6. Lineweaver-Burk p l o t o f the data in fig. 5. (a) 5 mM K ÷, (b) 2 0 mM K ÷. © Control, A 0 . 0 5 M EtOH, • 0.1 m M N A , ~ 0.1 mM N A + 0.05 M EtOH.
40
80
120
NaCI (raM)
160
40
80
120
160
NoCI (raM)
Fig. 7. E f f e c t o f 0.1 mM N A and 0.05 M E t O H added separately and in c o m b i n a t i o n on (Na t + K÷)-ATPase at 5 and 20 mM K ÷ and various Na t concentrations. © Control, zk 0 . 0 5 M EtOH, • 0.1 mM N A , • 0.1 mM N A + 0.05 M EtOH.
162
H. KALANT, N. RANGARAJ
TABLE 2 Kinetic constants for ATP and (Na ÷ + K÷)-ATPase: effect of 0.1 mM NA, 0.05 M EtOH and NA + EtOH at 5 and 20 mM K÷.
(a) K ÷ = 5
Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
0.88 -+ 0.048 17.54 -+ 0.41
0.87 + 0.07 17.44 -+ 0.50
0.47 + 0.035 2 17.10 + 0.24
0.45 -+ 0.014 2 11.18 + 0.75 3
0.78 - 0.15 20.34 -+ 1.2
0.59 -+ 0.09 20.36 + 0.98
0.74 -+ 0.12 20.89 + 1.01
mM
Km 1 Vmax 1
(b) K += 20 mM
Km
0.78 -+ 0.14 20.67 -+ 1.3
Vma x
1 Km is expressed in raM; Vma x expressed as pmol Pi produced/mg protein × h. Values are reported as mean -+ S.E.M. of 4 separate experiments. Each experiment was performed in duplicate. Statistical significance from corresponding control values, by t-test for paired data, is indicated as follows: 2p < 0.002, 3p < 0.001.
ence o f b o t h N A and E t O H (fig. 6a). T h e kinetic c o n s t a n t s are s h o w n in table 2. This e f f e c t was virtually abolished b y raising t h e K ÷ c o n c e n t r a t i o n t o 20 mM (fig. 6b). 3.4. E f f e c t o f 0.1 m M N A o n ( N a ÷ + K÷) A T P a s e w i t h a n d w i t h o u t O.05 M E t O H a t various N a ÷ c o n c e n t r a t i o n s
T h e effect o f various c o n c e n t r a t i o n s o f N a + ( 6 - - 1 5 6 mM) in presence o f 0.05 M E t O H a n d 0.1 mM N A a d d e d individually and in c o m b i n a t i o n is s h o w n in fig. 7. R e a c t i o n v e l o c i t y
(b)
K%5 mM
i
,10 f
J
K+=20mM control ,~EtOH • : EtOHt+ NA~ NA
(a)
was largely i n d e p e n d e n t o f Na ÷ c o n c e n t r a t i o n over a wide range, and was decreased signific a n t l y o n l y at the l o w e s t and highest Na ÷. N A a n d E t O H a d d e d separately did n o t have a n y e f f e c t on the activity, b u t in c o m b i n a t i o n t h e y inhibited t h e e n z y m e over the w h o l e range o f Na ÷ c o n c e n t r a t i o n . Kinetic c o n s t a n t s calculated f r o m L i n e w e a v e r - B u r k plots (fig. 8) are s h o w n in table 3. These c o n s t a n t s are o n l y a p p r o x i m a t i o n s because even the l o w e s t Na ÷ c o n c e n t r a t i o n used (6 mM) gave m o r e t h a n 50% o f t h e m a x i m u m activity in t h e c o n t r o l p r e p a r a t i o n . However, as n o t e d previously ( K a l a n t et al., 1978), t h e i n t e r a c t i o n b e t w e e n t h e Na ÷ and K ÷ binding sites renders the absolute Km values u n c e r t a i n in a n y case, and the i m p o r t a n t p o i n t in the present c o n t e x t is the c h a n g e p r o d u c e d b y E t O H and NA. N e i t h e r Vmax n o r K ~ f o r Na ÷ was a f f e c t e d b y E t O H o r N A alone, b u t b o t h were significantly decreased b y the c o m b i n a t i o n o f E t O H + NA. Again, the effect was abolished b y increasing K ÷ to 20 mM.
~ "06
4. Discussion
~/Na* (raM) Fig. 8. Lineweaver-Burk plot of the data of fig. 7. (a) 5 mM K ÷, (b) 20 mM K+.
T h e Km values d e t e r m i n e d in these experim e n t s f o r the various ligands (K ÷, Na ÷, Mg 2÷ a n d A T P ) fall w i t h i n t h e r e p o r t e d values. T h e r e is considerable variation in Km values in
163
CATECHOLAMINES, ETHANOL AND ATPase KINETICS
TABLE 3 Kinetic constants for Na + and (Na + + K+)-ATPase: effect of 0.1 mM NA, 0.05 M EtOH, and NA + EtOH at 5 and 20 mM K÷. Control
0.05 M EtOH
0.1 mM NA
0.1 mM NA + 0.05 M EtOH
4.06 -+ 0.43 (7) 19.78 -+ 0.99
3.67 -+ 0.40 (7) 19.10 -+ 0.99
3.77 -+ 0.36 (7) 19.74 --4"0.90
1.75 -+ 0.37 (7) 2 13.63 + 0.88 2
(a) K + = 5 m M
Km 1 Vmax 1 (b) I C = 2 0 m M
Km Vma x
4.93 -+ 0.42 (5) 23.3 + 1.54
5.89 + 0.30 (5) 21.89 -+ 1.41
1 Km is expressed in mM; Vmax expressed as pmol Pi/mg protein × h. Values are reported as mean -+ S.E.M. Number of experiments indicated in parentheses. Each experiment was performed in duplicate. Statistical significance of differences from corresponding control values, by t-test for paired data, is indicated as follows: 2 p < 0.001.
t h e literature, d u e to the fact t h a t Km f o r (Na ÷ + K÷)-ATPase and a given ligand d e p e n d s o n m a n y factors such as relative c o n c e n t r a tions o f o t h e r ligands, pH, t e m p e r a t u r e , species and t y p e o f tissue. H o w e v e r , t h e similarity b e t w e e n t h e Km value f o r K ÷ f o u n d here and t h o s e o b t a i n e d in o u r o w n earlier studies ( K a l a n t et al., 1 9 7 8 ; Rangaraj and Kalant, 1978) suggests t h a t t h e e n z y m e p r e p a r a t i o n is comparable. A d d i t i o n o f 0.05 M E t O H had n o signific a n t e f f e c t o n Km f o r Na ÷, Mg 2+ and A T P and increased t h a t f o r K ÷ o n l y b y 10%. Changes in kinetic c o n s t a n t s have b e e n r e p o r t e d b y o t h e r s o n l y in presence o f E t O H c o n c e n t r a tions greater t h a n 0.22 M (Israel et al., 1 9 6 5 ; Sun and Samorajski, 1 9 7 0 ; Lin, 1 9 7 6 ; Sun, 1 9 7 6 ; K a l a n t et al., 1 9 7 8 ) . In t h e p r e s e n t s t u d y , D A (0.1 mM) stimulated the e n z y m e activity at all K ÷ c o n c e n t r a tions w h e n old grade A T P was used. This is similar t o o u r previous finding with NA (Rangaraj and Kalant, 1978). T h e degree o f s t i m u l a t i o n increased progressively w i t h rising K ÷ c o n c e n t r a t i o n Up to 4 mM, and t h e n r e m a i n e d c o n s t a n t at higher K ÷ values. This is in fairly g o o d a g r e e m e n t with results o b t a i n e d b y Desaiah and H o ( 1 9 7 7 ) , w h o r e p o r t e d t h a t D A s t i m u l a t i o n was seen o n l y in presence o f K + and r e a c h e d a plateau at 10 m M K ÷. T h e
s t i m u l a t i o n a p p e a r e d t o result f r o m increased a f f i n i t y f o r K* ( l o w e r Kin), with n o change in Vmax. Km f o r Mg 2+ was slightly increased in t h e presence o f NA and a l m o s t d o u b l e d in the presence o f D A (fig. 4 and table 1), suggesting t h a t CA decreased the a f f i n i t y o f the e n z y m e f o r Mg 2÷. T h e Km f o r Na ÷ did n o t change, b u t Km f o r A T P was decreased b y 50% at 5 mM K ÷, suggesting t h a t the a f f i n i t y f o r A T P is increased in presence o f NA. Thus, NA a l o n e h a d a d i f f e r e n t e f f e c t o n the binding o f each o f t h e principal ligands, while 0.05 M e t h a n o l had none. In contrast, t h e c o m b i n a t i o n o f 0.05 M E t O H plus 0.1 mM NA o r D A increased t h e Km f o r K ÷ b y m o r e t h a n t w o - f o l d irrespective o f the t y p e o f A T P used as substrate. Similar degrees o f increase in Km f o r K ÷ had b e e n observed with m u c h higher E t O H c o n c e n t r a t i o n s a l o n e (Israel et al., 1 9 6 5 ; Israel and Salazar, 1 9 6 7 ; Lin, 1 9 7 6 ; Sun, 1 9 7 6 ; K a l a n t et al., 1978). Therefore the effect of the combinat i o n on t h e K ÷ binding site m a y involve t h e same m e c h a n i s m as t h a t responsible f o r t h e e f f e c t o f a higher c o n c e n t r a t i o n o f E t O H alone. This m a y n o t be t r u e f o r t h e binding sites f o r ATP, Na ÷ and Mg 2+. O u r results indic a t e u n c o m p e t i t i v e o r m i x e d t y p e s o f inhibit i o n f o r Na ÷, A T P and Mg 2+ kinetics in the p r e s e n c e o f CA + E t O H t o g e t h e r . This differs
164
from the noncompetitive inhibition for ATP and Na ÷ (Sun and Samorajski, 1970; Lin, 1976) and competitive inhibition for Na ÷ (Sun, 1976) in presence of high concentrations (>0.5 M) of EtOH alone. Therefore the NA sensitization of the enzyme toward EtOH inhibition appears to be specific for the K ÷ site. The decrease of Vma x for Mg 2÷, Na ÷ and ATP kinetics by the combination of CA + EtOH may be due to retardation of any of the partial reactions of (Na ÷ + K÷)-ATPase, which are as follows (Albers, 1976): E1 + ATP + 3(Na÷)i ~ E1P(Na÷)3 + ADP E1P + Mg 2÷ ~ E2P + 3(Na+)0 E2P + 2(K÷)0 ~ E2P(K÷)2 H : O + E2P(K+)2 ~ E2(K+): + P E2(K÷)2 -~ El(K+)2 + Mg 2+ El(K+)2 ~ E 1 + 2K[ El and E2 are two conformations of the enzyme, facing the inside and outside of the membrane respectively, and E2 contains bound Mg 2÷. The subscripts i and o designate ions located respectively inside and outside the cell. Since the EtOH + CA combination decreases the Km for ATP, Mg2÷ and Na ÷, it m a y be facilitating the formation of E IP and possibly its conversion to E2P. However, the reduced affinity (higher Kin) for K ÷ would result in inhibition of the subsequent steps by which E2P is ultimately converted to El and hence in reduction of the overall reaction velocity. This is consistent with the increased affinity for Mg2÷, since Mg2÷ would be released during the conversion. The fact that increased K ÷ tends to overcome the inhibition and increase the Km for Mg 2÷ suggests again that the effect of EtOH + CA is the same as that of a higher concentration of EtOH alone. An incidental question concerns the mechanism of stimulation of ATPase activity by CA alone. Though this is not the major concern of our study, the findings may be of some relevance to a disagreement in the literature.
H. KALANT, N. RANGARAJ
Many authors have reported that stimulation occurs only when the ATP substrate is contaminated with vanadate or some other metallic inhibitor which can be chelated by CA (Cantley et al., 1977). In contrast, Wu and Phillis (1978, 1979a, 1979b) and Phillis and Wu (1979) have reported that NA stimulates the activity by a receptor-specific mechanism regardless of the ATP used. We observed that, even with V-free ATP, NA did stimulate the enzyme at low concentrations of ATP (<3 mM) and a fixed concentration (3 mM) of Mg 2+. When ATP exceeded 3 mM, there was no stimulation. These findings are compatible with the idea that an excessive ratio of Mg 2÷ : ATP is inhibitory, and that NA produces activation under these conditions by chelating the excess Mg 2÷ (Rajan et al., 1972; Schaefer et al., 1979). However, Phillis and Wu (personal communication) have found activation by NA over a wide range of Mg : ATP ratios, and the question is still unresolved. In summary, our present findings suggest that NA sensitizes the enzyme to inhibition by EtOH at physiological concentrations of K ÷, by facilitating an EtOH-induced conformational change that impairs K ÷ binding to the outward-facing form and prevents its dephosphorylation and conversion to the inward-facing form (Kalant et al., 1980). Further support of this interpretation is provided by NA + EtOH effects on Arrhenius plots of ATPase activity, to be reported separately.
Acknowledgments The authors are indebted to Mr. Gregory Ho and Mrs. Anita Chau for preparation of the illustrations.
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165 Rangaraj, N. and H. Kalant, 1978, Effects of ethanol withdrawal, stress and amphetamine on rat brain (Na t + K÷)-ATPase, Biochem. Pharmacol. 27, 1139. Rangaraj, N. and H. Kalant, 1979, Interaction of ethanol and catecholamine on rat brain (Na t + K÷)ATPase, Can. J. Physiol. Pharmacol. 57, 1098. Robinson, J.D., 1970, Interactions between monovalent cations and the Na t, K÷-dependent adenosine-triphosphatase, Arch. Biochem. Biophys. 139, 17. Robinson, J.D., 1975, Functionally different classes of K ÷ sites on the (Nat+ K÷)-dependent ATPase, Biochim. Biophys. Acta 380, 250. Robinson, J.D., 1977a, K ÷ stimulation of ADP/ATP exchange catalyzed by the ( N a t + K÷)-dependent ATPase, Biochim. Biophys. Acta 484, 161. Robinson, J.D., 1977b, Na t sites of the ( N a t + K÷)dependent ATPase, Biochim. Biophys. Acta 482, 427. Schaefer, A., M. Komlos and A. Seregi, 1979, Studies on the effects of catecholamines and chelating agents on the synaptic membrane Na t, K ÷ ATPase activity in the presence and absence of hydroxylamine, Biochem. Pharmacol. 28, 2307. Schwartz, A., G.E. Lindenmayer and J.C. Allen, 1975, The sodium potassium adenosine triphosphatase. Pharmacological, physiological and biochemical aspects, Pharmacol. Rev. 27, 3. Skou, J.C., 1965, Enzymatic basis for active transport of Na t and K ÷ across cell membrane, Physiol. Rev. 45, 596. Skou, J.C., 1974a, Effects of ATP on the intermediary steps of the reaction of the ( N a t + K÷)dependent enzyme system. I. Studied by the use of N-ethylmaleimide inhibition as a tool, Biochim. Biophys. Acta 339,234. Skou, J.C., 1974b, Effect of ATP on the intermediary steps of the reaction of the (Na t + K÷)-dependent enzyme system. II. Effects of a variation in the ATP/Mg 2+ ratio, Biochim. Biophys. Acta 339, 246. Skou, J.C., 1974c, Effect of ATP on the intermediary steps of the reaction of the (Na t + K÷)-dependent enzyme system. III. Effect on the p-nitrophenylphosphatase activity of the system, Biochim. Biophys. Acta 339,258. Sulakhe, P.V., S.H. Jean and S.J. Sulakhe, 1977, Studies on the stimulation of (Nat+ K÷)-ATPase of neural tissues by catecholamines, Gen. Pharmacol. 8, 37. Sun, A.Y., 1976, Alcohol-membrane interaction in the brain. I. Reversibility, Ann. N.Y. Acad. Sci. 273,295. Sun, A.Y. and T. Samorajski, 1970, Effects of ethanol on the activity of adenosine triphosphatase and acetyleholine esterase in synaptosomes isolated from guinea pig brain, J. Neurochem. 17, 1365.
166 Wu, P.H. and J.W. Phillis, 1978, Effects of a and /3 adrenergic blocking agents on the biogenic amine stimulated (Na ÷ + K÷)ATPase ~of rat cerebral cortical synaptosomal membrane, Gen. Pharmacol. 9, 421. Wu, P.H. and J.W. Phillis, 1979a, Receptor-mediated noradrenaline stimulation of (Na ÷ + K+)-ATPase in rat brain cortical homogenates, Gen. Pharmacol. 10,189.
H. KALANT, N. RANGARAJ Wu, P.H. and J.W. Phillis, 1979b, Effects of vanadate on brain (Na*+ K+)-ATPase and p-nitrophenylphosphatase-interactions with mono and divalent ions and with noradrenaline, Int. J. Biochem. 10, 629. Yoshimura, K., 1973, Activation of Na-K-activated ATPase in rat brain by catecholamine, J. Biochem. 74, 389.