EVIDENCE THAT DIPHENYLHYDANTOIN DOES NOT AFFECT ADENOSINE TRIPHOSPHATASES FROM BRAIN* J. D. DEUPREE Department
of Pharmacology,
University
of Nebraska (Accepred
Medical
Center,
Omaha,
Nebraska
68105, U.S.A.
16 JU/J 1975)
Summary -The effects of diphenylhydantoin on brain sodium- and potassium-dependent adenosine triphosphatase (NaK ATPasc), ,I-nitrophenylphosphatase and the calcium-dependent-adenosinc triphosnhatase (Ca ATPasc) were examined. The results indicated that: (1) dinhenvlhvdantoin did not stimulate the NaK ATPase of beef brain sodium iodide-treated microsomes or rat brain synaptosomes at nonsaturating or saturating levels of sodium and potassium; (2) the apparent stimulation by diphcnylhydantoin under high sodium:potassium ratios rcportcd by other workers can bc explained by a pot’assium contamination in the diphenylhydantoin; (3) diphcnylhydantoin did not alter the Michaelis constant for ATP for the NaK ATPase: (4) diphenylhydantoin did not slowly interact with or alter the NaK ATPase: (5) diphcnylhydantoin did not alter the calcium inhibitton of the enzyme and (6) diphenylhydantoin did not alter the p-nitrophenylphosphatase or Ca ATPase. Based on these considerations, diphenylhydantoin must be exerting its pharmacological effect by altering a membrane constituent other than the NaK ATPase.
Diphenylhydantoin
(5.5-diphenyl-2,4-imidazolidene-
crone; DPH) is used extensively to treat major motor and psychomotor seizures. The anticonvulsant property is thought to be due to inhibition by DPH of the spread of seizure from the focal area (WOODBURY zrd KEMP, 1971); however, the mechanism by which DPH blocks the spread is not understood. WOOI>IIURY(1955) noted that DPH lowered the intracellular sodium concentration in rat brain. but a definite controversy exists concerning the mechanism of this effect. Suggestions include: (I) DPH inhibition of the influx of sodium into the nerve, which is supported by the results of SWANSONand CRANE (1972); PINCUS. GROVE, MARINO and GLASER (1970) and (2) DPH enhancement of the efflux of sodium from the nerve by active pumping of sodium out of the nerve by .he sodium- and potassium-dependent adenosine tri2hosphatase (NaK ATPase, EC 3.6.1.3) (SKOIJ, 1965). A number of effects of DPH on the NaK ATPase ‘lave been reported. The results indicate that DPH zither has no effect (FESTOFFand API’EL. 1968; SELL Ind GOODWIN. 1972; SPAIN and CHIDSI:Y, 1971; GmEONand HARRIS, 1969; SPEXELAKISand HENN, 1970), activates (FESTOFF and APPEL, 1968; WILENSKY and LOWDEN, 1972; SIEGEL and GOODWIN, 1972) or inhibits (RAWSON and PINCUS, 1968; WILENSKY and LO~DEN, 1972) the enzyme, depending on the relative saturation of the enzyme with sodium and potassium. If the anticonvulsant property of DPH is due to acti*The research was supported in part by a research starter grant from the Pharmaceutical Manufacturers’ Association Foundation, Inc., and from funds appropriated by the Nebraska Legislature for “Seed Money” research. A preliminary report appeared in Fedn Proc. Fedn Am. Sots exp. Bio/. (1974) 33, 438. 187
vation of the NaK ATPase, the NaK ATPase should be activated in vitro under the same ionic environment which exists normally in the brain. Accurate determinations of the effects of DPH on the kinetic parameters of the NaK ATPase require a precise enzyme assay. Since there are a number of different ATPases present in the brain, it becomes difficult to determine accurately the activity associated with only the NaK ATPase. Low activity calcium-dependent adenosine triphosphatase (Ca ATPase) and high activity magnesium-dependent adenosine triphosphatase (Mg ATPase) are present. and their activities are not affected by sodium, potassium, or ouabain. The NaK ATPase requires both sodium and potassium for enzymatic activity and is inhibited by ouabain. A multi-step reaction sequence has been proposed by ALBERS,KOVAL and SIEGEL(1968); POST, KUME, TOBIN. ORCUTT and SEN (!969) for the NaK ATPase, the last step being assayed as a potassiumdependent p-nitrophenylphosphatase. The NaK ATPase activity can be determined by measuring total ATPase activity in a medium containing sodium and potassium and subtracting the Mg ATPase activity assayed by omitting sodium and potassium from the medium (ALDRIDC;E,1962; SKOU, 1957) or by inhibiting the NaK ATPase with ouabain (UESUC;I,DULAK, DIXON, HEXUM, DAHL, PERDUE and HOKIN, 1971). Omitting sodium and potassium from the medium does change the ionic strength, but AHMED and THOMAS (1971) found that the addition of salts did not affect the Mg ATPase activity in rat brain microsomes. Adding ouabain has the advantage of maintaining constant ionic strength for both controls and test conditions and the disadvantage of possible competition of ouabain and DPH for the
188
J. D. DEUPREE
same site on the enzyme. An alternate approach for increasing the accuracy of the NaK ATPase activity is to remove 90% of the Mg ATPase activity from the microsomes with sodium iodide (NaI) (UESUGIet nl., 1971). This method reduces activity not due to NaK ATPase to a level where the inorganic phosphate (Pi) formed is due mainly to spontaneous hydrolysis of ATP. Variable effects of DPH may be due to the relative insolubility of DPH at pH 7.0. The solubility of DPH at pH 6.9 is 7.7 x 10m5 M, at pH 7.9 is 1.2 x 10-3~ and at pH 9.2 is 2.4 x 10A3M. Therefore stock solutions of DPH must contain a base (usually NaOH). The sodium added with DPH must be balanced by addition of sodium to control incubations to maintain identical ionic conditions. The variable effects of DPH may result from the assay conditions chosen for kinetic analysis. FESTOFF and APPEL (1968) reported that NaK ATPase of rat brain synaptosomes was not affected by DPH at saturating levels of sodium and potassium, was inhibited at low ratios of Na:K, and activated at Na:K ratios of 25:l or higher. Based on these results, DPH would activate the NaK ATPase only when the level of intracellular sodium was greater than 1OmM and the external potassium was less than 0.2m~. The NaK ATPase activity was measured as the difference between the ATPase activity in the presence of sodium, potassium and magnesium and the ATPase activity in the presence of magnesium and ouabain. Thus, the DPH effect may have been obscured by the high level of Mg ATPase present or altered by ouabain. SIEGEL and G~C~DWIN (1972) reported that optimal activation by DPH of NaK ATPase in NaI rat brain microsomes required a ratio of Na:K of 15O:l. The activity of NaK ATPase was measured in incubations without and with sodium and potassium. They suggested that DPH activated NaK ATPase by preventing the sodium inhibition of the enzyme which occurs at low levels of potassium. Alternative explanations of the results of others are possible. SIEGEL and GOODWIN (1972) did not rule out activation by traces of potassium in the DPH. Neither SIEGEL and GOODWIN (1972) or FESTOFFand APPEL (1968) described the pH of their DPH preparation, nor indicated whether DPH altered the pH of the assay, or whether DPH was soluble in their assay media. In addition, test and control incubations could have differed in sodium and solubilizer concentrations, as well as in DPH concentrations. The objective of this investigation was to reevaluate the reported effects of DPH on the kinetic parameters of the NaK ATPase isolated from beef and rat brain. Several assay methods and high purity reagents were used. The data indicate that DPH does not alter the kinetic properties of the NaK ATPase in in vitro assays. The data further show that the activation of the NaK ATPase reported by other workers could have been due to a potassium contamination in the DPH used. Thus, the effects of DPH on intracellular
sodium levels cannot be assigned to the NaK ATPase with certainty and may be the result of interaction with other membrane constituents. MATERIALS AND
METHODS
Chemiculs NaATP, Tris, ouabain, glycylglycine, Tris p-nitrophenylphosphate and EGTA (ethylene glycol-bis (P-amino-ethyl ether)N,N’-tetracetic acid) were obtained from Sigma Chemical Co. Diphenylhydantoin (free acid, 990: pure grade) was purchased from Aldrich Chemical Co.. Inc. An additional sample of DPH (free acid) was donated by Parke-Davis. Sodium hydroxide and potassium hydroxide were obtained from Fisher Scientific Co. Imidazole was obtained from Eastman Kodak Co. All other chemicals were obtained from Mallinckrodt. Quartz triple distilled water was used in all experiments. Reagents Tris ATP was prepared by passing NaATP through a Dowex-SOW resin (H+ form) and titrating to pH 7.0 with Tris. Diphenylhydantoin solution was prepared fresh each day by adding NaOH to a final concentration of 2 x lo-” M to a suspension of 1O-3 M DPH. The resulting clear solution had a pH of 10.0. Turbidity measurements at 400 and 600nm showed that DPH did not precipitate from the assay medium. The 10m3 M stock solution of DPH had a potassium concentration of 5.4 x 10mh M as determined by atomic absorption spectroscopy: 1.3 x IO-’ M potassium was present in the DPH and the balance was due to the potassium contamination in the NaOH used to solubilize the DPH. Because of the potassium contamination in the DPH solution, equivalent amounts of NaOH and KOH were added to the control tubes. This amount of NaOH was not sufficient to change the pH of the assay medium. Tissue sources cind preparotiorzs Beef brain microsomes Beef brains, donated by John Roth and Sons (Omaha. Nebraska), were kept on ice until used. The microsomes were isolated according to the procedure of HI’XLIM (1974) and stored at -70-C. This preparation contained both Ca ATPase and Mg ATPase activity, which are insensitive to ouabain. The average NaK ATPase specific activity of the microsomes was 25 prnol of Pi formed/hr per mg of protein. Sodium iodide-treuted beef bruin microsomes Sodium iodide micrbsomes were prepared according to the procedure of NAI\,r 01 ATP
wnl, cn,!mc
1 b
50 rn~ Trls. pH 8-C 50 rn~ Tns. pH X.0
: e I
SO rn~ Tns. pH 8.0 50 X0 50 rn~ Tin. pH X-O 50 IIIMTris. pH 8-O
: I :
50 30 IIIMTrn. Imidazolc, pH X.0 30 IIIMglycylglycine, 30 m*l Imldazole, 30 mM glycylglycine, 30 rn~ tn~ Imldazole, Imidarole, 30 30mM rn~ glycylglycine, glycylglycme, 30 mM Imidazole, 30 mM glycylglycme. 30 rnr.1Imldarole. 30 rn~ glycylplycine. 30 rn~ Imldatolc. 30 rn~ glycylglycmc. 30 rn~ Imida7olr. 30 rn~ gl)cylglycinr, 30 rn~ Inndarolc. 30 IIIMglyc~lglycmc. 30 rn~ Imidarole. 30 mhl glycylglycme. 30 rn~ hmdarole. pH 7 0 30 rn~ Im~darole, pH 7.0
I m n 0 P q r S Dctcrmmahon A B c D E H 1 J K L R S
+ P, 6
pH pH pH pH pH pH pH pH pH pH
6 h 6 6 6 6 7,5 7,5 7.5 75 75 7-5 7.5 75 l-5 7.5 6 6
7.4 7.4 7.4 7-4 74 74 74 74 74 7.4
2 2 2
2 4 4
_
+ + + + + + +
_ _ _ + + + + + + + + + + _
_ _ _ _ _ _
_ _ _
_ _ _ _ _
_ _
_
+ + + + + + + + + + + +
+ + + + + + + + + + + +
_ _ + + _ _ _ + + + + + _
of total ATPare 50 rn~ 7115, pH X.0 50 HIMTrls, pH 8.0 50 rn~ Tns. pH 8 0 50 rn~ Tns, pH 8-O 50 mha Tns. pH 8 0 30 rn~ Imh~olc. 30 IIIMglycyl~lycmc. 30 rn~ Im~darolc. 30 rn~ &c~l~lycmc. 30 “IM Im,datole. 30 rn~ &~ylglycme. 30 rn~ Im~dazole. 30 rn~ &c~l~l~cme. 30 rn~ Imldazole, 30 rn~ glycylglycine. 30 rn~ Imidarole, pH 7.0 30 rn~ Imldazole. pH 7.0
pH PH pH PH pH
6 6 6 6 6 1.5 7.1 71 75 75 6 6
74 7.4 7-4 7.4 7.4
Determination of NaK ATPase = Total ATPasee hydrolysis of ATP). NaK ATPase in absence of DPH calculated as: VS
NaK ATPase in presence of DPH calculated
as:
(Mg ATPase B-b D-d
+ + + + +
_ _
+ Pi associated A-a
A-f
_ + + + + + _
I-i
with protein K-k
I-n
K-p
_ _ _ _
_ _ _
+ Pi due to spontaneous H-h
H-h H-m H-m
R-r
J. D. DEUPRE~
190
amount of Pi formed was determined as described under Mg ATPase assay. The NaK ATPase was assayed above its optimum pH in some cases, since DPH was more soluble at the higher pH. Diphenylhydantoin did not interfere with the measurement of Pi. Poro\sizfm-t/[~perltlr,lr p-nirroph~r7~~lpho.spl7utL7.tr L/.S.\I7)‘. The potassium-dependent p-nitrophenylphosphatasc activity was calculated as the difference in rate of hydrolysis observed in the presence and absence of KCI. The potassium-dependent p-nitrophenylphosphatase activity was determined by pre-incubating the NaI microsomes at 37’C for 10min in 1 ml containing 50 mM Tris, pH 7%: 5 mM MgCl,: with and without 15 mM KCl; with and without DPH. The 10min reaction was initiated by the addition of 6,~mol p-nitrophenylphosphate and terminated by the addition of 1 ml 1.0~ NaOH. The tubes were centrifuged for 10 min at 1000 9 to remove the cloudy material. The centrifugation did not pellet any p-nitrophenol. The amount of p-nitrophenol released was determined by measuring the absorption at 400nm. Ca ATPase assay. The Ca ATPase activity was calculated as the difference in activity obtained in the presence and in the absence of calcium. The test conditions contained DPH solubilized with NaOH. The assay was conducted by pre-incubating the microsomes at 37 C for 10 min in 1 ml containing 67 mM glycylglycine adjusted to pH 7.4 with imidazole, 5 mM MgCl,, 0.125mM EGTA, 10m4~ ouabain, with and without 0.2rn~ CaCl,, with and without DPH. The 5 min reaction was started by the addition of 1.5 /*mol NaATP. The reaction was stopped by the addition. with immediate mixing, of cold 5”,, trichloroacetic acid in chloroform:methanol (1 :l), and the amount of Pi formed was determined as described under Mg ATPase assay. Protein determination. Protein was determined by the method of LOWRY, ROSENBROUGH, FARR and RANDALL (1951) using bovine serum albumin as a standard. Statist ical analysis
Table 2. Effects of DPH on Mg ATPase present somes isolated from beef brain DPH (hll
in micro-
Mvlg ATPar (pmobhr per mgl
Microsomes were assayed using conditions b vs c in Table 1. The Mg ATPase was calculated as the difference between activity in the presence of magnesium and the activity in the absence of magnesium. Values are the mean of 6 determinations i SE.
extreme variation in this essential correction factor, the microsomal enzyme preparation was not used for this study. Sodium iodide microsomes were used for further studies since the Nal treatment removes 902, of the Mg ATPase activity. Eflkcts of DPH on NaK rnicrosomes
ATPase
in beef brain NaI
In the studies reported in this paper ouabain was not used in the NaK ATPase assay since ouabain and DPH may have been acting at the same site on the enzyme. Rather, the NaK ATPase was measured as the difference between activity in the presence and the absence of sodium and potassium. The Mg ATPase activity in the presence of ouabain and a high salt concentration (120mM NaCl + 30m~ KCI) was the same as the activity in the absence of NaCl and KC1 (Table 3). Thus, Mg ATPase activity was not affected by ionic strength. Since the NaK ATPase activity was higher in the presence of the chelator, and heavy metals have been shown to inhibit the enzyme (SPITHT and Ront~~so~. 1973) (see Table 3). EGTA was used in all assays. Diphenylhydantoin solubilizcd with NaOH had no effect on the NaK ATPase when assayed at pH 8.0 in the presence of 50miv1 NaCl and KC1 at concentrations ranging from 0.2 to 1Om~ (Fig. 1). In addition. DPH did not alter NaK ATPasc activity obtained in the presence of 2 or @2m~ KC1 with
The data were analyzed using the Student t-test. A P value greater than 0.05 was considered not significant. RESULTS
Effbcts
ofDPH on My ATPuse
Since one method of determining NaK ATPase was to subtract Mg ATPase activity from the total ATPase, it was necessary to ascertain the effects of DPH on the Mg ATPase in the assay system used. Diphenylhydantoin has been reported to inhibit the Mg ATPase (WILENSKY and LOWDEN, 1972). Diphenylhydantoin at concentrations between lo-* and 10m4 M did not inhibit the Mg ATPase present in microsomal fractions (Table 2). Because of the
i
20 t-t
p,,,,,,,,,, 2
4 6 KCl(mM)
8
IO
Fig. 1, Effects of DPH solubilized with NaOH on the NaK ATPase assayed at high levels of sodium and varying levels of potassium. The NaI microsomes from beef brain were assayed using conditions B-b vs C-c in Table 1.
Diphenylhydantoin
I91
and ATPases
Table 3. Effecrs of ionic strength, EGTA and EDTA on ATPase activity TotalATPasc I” p’cscnce of Na + K (umts,mg)
Additions NDX Ouaham EGTA EGTA + ouaham tDTA EDTA + ouahsm
23-7 23 2 ‘I I 21 5 22.9 23.7
40 5 234 444 21.5 42.0 23.9
IfIX: 17 lb 23 7: 90 190:
21-X* Jl.lt Ii).?*
23
I9hi
?09* ?? 3-1
l8.m‘
* Mg ATPase = (Total ATPase in absence of Na + K) - (Pi associated with microsomes + Pi from spontaneous hydrolysis of ATP). ‘r Mg ATPase = (Total ATPase in presence of Na, K and ouabain) - (PI associated with microsomes + Pi from spontaneous hydrolysis of ATP). : NaK ATPase = (Total ATPase in presence of Na + K) - (Total ATPase in absence of Na + K). NaK ATPase = (Total ATPase in presence of Na + K) ~ (Total ATPase in presence of Na. K and ouabam). Microsomes isolated from beef brain were assayed for ATPase activity in the presence of 50m~ Tris, pH 8.0, for 5 min at 37-C, and the Pi produced was determined as described in the Methods. Total ATPase was determined by adding to the incubation mixture 6rn~ MgCI,, 4rn~ Tris ATP with or without l30m~ NaCl and 20m~ KCI. The Pi associated with the enzyme was determined by omitting Mg, Na, K and ATP from the assay medium. The spontaneous hydrolysis of ATP was determined by omitting enzyme from the assay medium. The concentrations of ouabain, EGTA and EDTA were 10m4.~.
PJaCl at concentrations varying between 2.5 and 12Om~ NaCl (Fig. 2). Likewise, lo-’ to 3 x 10m4 M I>PH did not significantly (P > 0.05) alter the NaK ATPase assayed in the presence of 50m~ NaCl and C1.2rn~ KC1 (not shown). In these experiments the NaK ATPase activity in the absence of DPH was compared with the NaK ATPase activity in the presence of DPH (Table 1, B-b compared with C-c). The only difference between test conditions and control c,onditions was the amount of DPH, since the same amount of NaOH and contaminating potassium was added to the control conditions as was added to the test conditions. Changing the pH of the assay medium did not result in activation of the NaK ATPase by DPH. The ?\JaK ATPase activity was not altered by DPH at pH 7.0 or 7.4 when assayed at 50m~ NaCl and
0
20
I 40
I NaCl
I 60
I
I 80
0.2 mM KC1 (l-i compared with J-j, and R-r compared with S-s as listed in Table 1). If DPH was solubilized with KOH, and KOH was added to the control conditions to balance the KOH in the test conditions, there was no apparent stimulation of the NaK ATPase by DPH in assay media containing a high ratio of sodium to potassium (media D-d vs E-e, K-k vs L-l, and K-p vs L-q in Table 1). However, if KOH was not added to the control tubes (A-a vs E-e and H-h vs L-l as in Table 1) a two-fold stimulation of the NaK ATPase by DPH was obtained (Fig. 3). Thus, activation under these conditions would have been due to the potassium and not the DPH.
l - DPH n-t DPH
l -DPH
I
I 100
I 120
(rn~)
I
I
I
I
20
40
60
80
NaCl
l?g. 2. Effects of DPH solubilized with NaOH on the NaK ATPase assayed at low potassium and varying levels of 1. The NaI microsomes from beef brain were assayed using conditions B-b vs C-c in Table 1.
I
I
100
120
(mM)
Fig. 3. Effects of DPH solubilized with KOH on the NaK ATPase assayed at low levels of potassium and varying levels of sodium. The NaI microsomes from beef brain were assayed using conditions A-a vs E-e in Table 1.
192
J. D. DEUPREE
Effects of DPH on NuK ATPase in synaptosomes from rat brain
Interaction of DPH and the NaK ATPase ence of various substrates of the enzyme
To make our conditions as comparable as possible to those reported by FESTOFF and APPEL (1968), synaptosomes were prepared from rat brain using procedures similar to those reported by FESTOFF and APPEL (1968). The assay method was also similar to theirs except the protein concentration in the assay medium was reduced to a level at which the assay was linear for 30min and the modified Fiske-SubbaRow method (CLARK, 1964) was used to determine Pi. The Mg ATPase activity was determined by either omitting sodium and potassium from the assay media or by omitting sodium and potassium and adding ouabain. The NaK ATPase activity was measured as I-i vs J-j, K-k vs L-l, I-n vs J-o, K-p vs L-q, H-h vs K-k, H-h vs L-l, H-m vs L-q, and H-m vs K-p (Table 1). If DPH was solubilized with NaOH, and NaOH was added to control tubes, no activation of the NaK ATPase was seen, and results similar to those in Figures 1 and 2 were obtained. Activation of NaK ATPase, similar to that shown in Figure 3. was obtained only when DPH was solubilized with KOH and KOH was omitted from the control tubes. Thus, apparent stimulation by DPH could be accounted for by an increase in potassium, resulting in a change in the Na:K ratio.
The NaK ATPase appears to take on a number of different conformation states depending on the cations bound to it (BANCRJE~.WOXG and SEN, 1972). It is possible that DPH binds to only one conformation state of the enzyme, or that DPH takes a prolonged time to react with the NaK ATPase. To test this possibility, the NaK ATPase present in NaI microsomes (9 mg protein/ml) isolated from beef brain was incubated at 4’C in the presence of 10m4 M DPH containing 2 x 1O-4 M NaOH, or in 2 x 10m4~ NaOH without the DPH. The following additions were made as indicated: 50m1~1 NaCl, 0.15 mM KCl, 3.3 mM Tris ATP, 4 mM MgClz. After 24 hr the NaI microsomes were diluted and assayed using conditions B-b vs C-c in Table 1. Diphenylhydantoin did not have any significant effect on the NaK ATPase on prolonged incubation with various cations (not shown).
Effects of DPH on NaK ATPase in Nal microsomes from beef brain under conditions qf saturating sodium and potassium Diphenylhydantoin at concentrations between 10d8 and 2 x 10m4 M did not have any significant effect on the NaK ATPase in the presence of saturating levels of sodium and potassium (not shown). This was demonstrated by assaying NaK ATPase present in NaI microsomes prepared from beef brain in the presence of 120m~ NaCl, 20m~ KC1 and assay conditions B-b vs C-c in Table 1. Sodium hydroxide was added to both test and control conditions. Eflect of DPH on the Michaelis constant for ATP $v the NaK ATPase in Nal microsomes from beef bruin Most experiments testing for an effect of DPH on NaK ATPase have been conducted at saturating levels of ATP and would not indicate whether DPH was altering the binding of ATP to the enzyme. The effects of DPH on the ATP binding to the Na ATPase were estimated by determining the Michaelis constant (K,) for ATP in both the presence and absence of DPH. A K, of 0.24 mM for ATP for the NaK ATPase in NaI microsomes isolated from beef brain was obtained using the method of Lineweaver and Burk. This agrees with the K, values previously reported in the literature (KLINE, HEXUM, DAHL and HOKIN, 1971). The K, for ATP was not affected by 10m4h.1 DPH solubilized with 10e4 M NaOH (not shown). Assay conditions B-b vs C-c were used. A MgCI, to ATP ratio of 2 was maintained at all levels of ATP.
in the pres-
Ef,Cect ok’ DPH on the potussiunz-cieperztlent p-nitrophen!,/pho.sphatLIsr
It is possible that DPH only alters one of the steps in the NaK ATPase reaction. Since a potassium-dependent p-nitrophenylphosphatase reaction is thought to reflect the last step in the NaK-dependent ATP hydrolysis (ASKARI and KOYAL, 1968), the effects of DPH on this reaction were measured. Diphenylhydantoin in concentrations of lo-’ to 10m4M had no effect on the potassium-dependent p-nitrophenylphosphatase present in beef brain NaI microsomes assayed as described in the Methods (not shown). In addition, 10e4M DPH did not affect the potassium-dependent p-nitrophenylphosphatase at K, levels of potassium (not shown). Eflect of DPH and calcium on NaK ATPase microsomes from beef brain
in Nal
Since it has been postulated that DPH displaces calcium from the membrane (PINCUS and LEE, 1973; PINCUS et al., 1970), it is conceivable that DPH could activate the NaK ATPase by removing calcium from the enzyme. To test this hypothesis, the NaK ATPase was incubated with varying concentrations of calcium in the presence and absence of DPH. Increasing concentrations of calcium inhibited the NaK ATPase as has been reported by others (SKOU, 1962), but DPH did not reverse the inhibition by calcium (Table 4).’ EfSrct of DPH on Ca ATPase in microsomes from beef brain To determine if DPH was inhibiting the active transport of calcium, the effect of DPH on the Ca ATPase was studied. The Ca ATPase present in beef brain microsomes was not significantly (P > 0.05) inhibited by DPH when measured as described in the Methods (not shown).
Diphenylhydantoin Table 4. Effects of calcium and DPH on NaK ATPase present in Nal microsomes isolated from beef brain
-
Cdlctum
Wathout
(NI
li~rni,i hr
0
-
10 6 IO 5 ,i, 4 IO ’
NaK DPH per mg,
ATPew
WithDPH (pmal hr per mg)
54x5 k 035 5665 f 1.65 56.70i 0 20 .lj55 f 095
1335 i I35
5665 - 0.65 5X.25 + 0.65 575 + O-20 46.1? 0.50 13.15+ 005
P
“S “S “.S “_S. n.5.
‘The NaI microsomes were assayed using conditions B-b vs C-c in Table 1 with 120 mM NaCl and 20m~ KCI. T,e values are expressed as the mean of 2 experiments rln in duplicate f SD.
DISCUSSION
In most of these experiments the ouabain-inhibited assay used by FESTOFFand APPEL (1968) was not used bN:cause of possible competition in binding between olabain and DPH. Rather, we tried to reaffirm the results of other investigators using an assay system that was more generally applicable to studying the ell’ects of drugs. In addition, the NaK ATPase was assayed at pH 8.0, which was above its optimum pH, to further assure that DPH was in solution during the enzyme assay. No effects of DPH on kinetic parameters were seen under these conditions or at pH 7 0 and 7.4. Under conditions which should closely approach those used by SIEGEL and GWDWIN (1972) there was no stimulation or inhibition of the NaK ATPase incubated with DPH solubilized with either NaOH or KOH provided the corresponding cation was also added to the control tubes. As seen in Figure 3, activation of the NaK ATPase occurred only when the trace amount of potassium added along with the DPH was not balanced by adding potassium to the control tube. A 2 x 10e5 M potassium contamination would account for their apparent two-fold stimulation (see Fig. 2 in SIEGEL and GOODWIN, 1972). This strongly suggests that the stimulation of NaK ATPase ty DPH at high Na:K ratio was due to a potassium c,ontamination in the DPH, thereby causing an imbalance in potassium levels in the test and control conditions. In addition, the DPH used in the results reported here was determined to be 99’;:, pure (infrared spectrometery by Aldrich Chemical Co.). Pure DPH (free acid) donated by Parke-Davis and Company and solubilized with NaOH, did not activate the NaK ATPase at a high Na:K ratio. These results indicated that lack of DPH activation was not due lo a single lot of DPH. Using a synaptosomal preparation similar to that reported by FESTOFF and APPEL (1968), stimulation of the NaK ATPase by DPH at a high ratio of Na:K was not observed unless DPH solubilized with KOH was used and a potassium imbalance existed between test conditions and control conditions. The lack of etfect of DPH on NaK ATPase was noted when Mg ATPase was estimated, using either ouabain inhibi-
and ATPases
193
tion or omission of sodium and potassium from the medium. The activation reported by FESTOFF and APPEL (1968) could be explained by a 3 x 10e5 M potassium contamination in DPH. Their solutions reportedly contained less than lo-‘M sodium and potassium, but no description was given of the procedure used to solubilize DPH. A 10e3 M DPH solution requires 10 -3 M NaOH or KOH for solubilization, and the DPH must have contained some sodium or potassium. In addition, FESTOFFand APPEL (1968) used an enzyme preparation which contained a high concentration of Mg ATPase. Extreme variability among assays was observed when DPH was added to the Mg ATPase (Table 2). Although FESTOFF and APPEL (1968) did not report the effects of DPH on the Mg ATPase, that variability could account for the stimulation of the NaK ATPase observed when very low activities of enzyme were measured at high ratios of sodium to potassium. LEWIN and BLECK (1971) injected DPH into rats, sacrificed the rats, isolated the microsomal fractions and assayed the NaK ATPase. In the process of isolating the microsomal fraction, the DPH should have been substantially diluted, and since DPH was not added to the assay medium, it becomes questionable as to whether the reported activation was directly related to DPH. The results reported here, as well as by SIEGEL and GWDWIN (1972), SPAIN and CHIDSEY (1971), GIBSON and HARRIS (1969), SPERELAKISand HENN (1970), indicated that DPH did not affect the maximal velocity of. the NaK ATPase since the drug did not significantly alter the rate of reaction at saturating sodium, potassium, and MgATP levels. FESTOFF and APPEL (1968) reported a less than 10% inhibition of the NaK ATPase at 50 mM sodium and 1Om~ potassium, which may have been within experimental error of the assay. However, RAWSON and PINCUS (1968) reported a 20% inhibition (statistical significance was not given) of the enzyme under these conditions. WILENSKYand LOWDEN (1972) reported an 18% activation of the NaK ATPase at 10m4M DPH and a 36’% inhibition at 2 x 10e4 M DPH (statistical significance was not given) and a statistically significant inhibition of the Mg ATPase with 1O-4 M DPH. These variations could have been due to the high concentration of Mg ATPase in comparison with the NaK ATPase in the microsomal preparation, thus obscuring the effects of DPH on the NaK ATPase. Since DPH did not alter the K, for ATP, this would indicate that the anticonvulsant did not interfere with the binding of ATP to the NaK ATPase. Nor did DPH appear to slowly activate the NaK ATPase, since the NaK ATPase was not activated following a 24 hr incubation of the NaK ATPase with DPH. The presence of specific ligands (Mg, ATP, Na and K) was not important since DPH did not activate the NaK ATPase when they were present. Diphenylhydantoin did not activate the third step in the NaK ATPase reaction since it had no effect
194
J. D. DEUPREE
on the potassium-dependent p-nitrophenylphosphatase reaction. LEWIN, WALKER and BLECK (1973) reported that potassium-dependent p-nitrophenylphosphatase activity was the same in microsomes isolated from the brains of rats receiving DPH as compared with the enzyme from rats not treated with DPH. In addition, DPH did not affect the K, for potassium for this reaction, indicating that DPH did not compete with potassium in the reaction. There is some indication that DPH may have been blocking nerve conduction in a manner similar to that of local anaesthetics (PINCUS and LEE, 1973). It has been postulated that local anaesthetics and DPH displaced calcium from the membrane (HASBANI, PINCUS and LEE, 1974) and that the NaK ATPase may have been slightly inhibited in riro by calcium (TOBIN, AKERA, BASKIN and BRODY. 1973). It is not likely that DPH activated the NaK ATPase by displacing calcium from the enzyme since DPH did not reverse the inhibition of the NaK ATPase by calcium. In addition. FESTOFF and APPEL (1968) reported activation of the NaK ATPase under conditions where calcium would have been chelated by EGTA. Although DPH may have blocked calcium uptake into the nerve (HASBANI~‘1al., 1974; PINCUS and LEE, 1973), the anticonvulsant probably did not affect the active transport of calcium. since DPH did not inhibit the Ca ATPase. If a drug exerts a direct effect on an enzyme, then some parameter of enzyme activity (either the K, of one or more of the substrates or the V,,,, for the enzyme) should be altered by the drug under in vitro conditions. The results reported here show that DPH did not alter the velocity of the enzyme at saturating and non-saturating levels of sodium or potassium; did not affect the K,, for ATP; did not slowly affect the enzyme; did not affect the enzyme in the presence of specific ligands; did not affect the partial reaction of the NaK ATPase, as measured by p-nitrophenylphosphatase activity: and did not relieve the calcium inhibition of the NaK ATPase. In addition, the reported activation of NaK ATPase at a high ratio of Na:K by DPH appeared to be due to potassium contamination of the DPH. Therefore, I conclude that DPH does not affect the NaK ATPase from rat or beef brain, and the pharmacological action of DPH must be due to the interaction at sites other than the NaK ATPase. Ackno~lrd~rment.~~K~~~~ SORIA provided excellent techn~cal assistance and KII 761SIILI\~ONS performed the atomic absorption spectrum analysis.
REFERENCES AHMED.K. and THOLIAS.B. S. (1971). The effects of long chain fatty acids on sodium plus potassium ion-stimulated adenosine triphosphatase of rat brain. J. hid. Chem. 246: 103 -109.
ALBERS,R. W., KOVAL, G. J. and SIEGEL, G. J. (1968). Studies on the interaction of ouabain and other cardioactive steroids with sodium-potassium-activated adenosine triphosphatase. Mo/ec. Pharmuc. 4: 324336. ALDRIDGE,W. N. (1962). Adenosine triphosphatase in the microsomal fraction from rat brain. Biochem. J. 83: 527-533. ASKARI,A. and KOYAL, D. (1968). Different oligomycin sensitivities of the Na+ + K+-activated adenosinetriphosphatase and its partial reactions. Biochem. biophys. Res. Commun. 32: 227-232.
AUTILIO,L. A., APPEL, S. H.. PETTIS, P. and GAMBETTI, P. (1968). Biochemical studies of synapses in citr+I. Protein synthesis. Biochemistry 7: 2615-2622. BANERJEE,S. P., WONG, S. M. E. and SEN, A. K. (1972). Inhibition of sodium- and potassium-dependent adenosine triphosphatase by N-ethylmaleimide-II, Effects on sodium-activated transphorphorylation. Molec. Phurmuc. 8: 18-29. CLARK,J. M., JR. (Ed.) (1964). Experimental Biochemistry. W. H. Freedman and Co., San Francisco. FESTOFF,B. W. and APPEL,S. H. (1968). Effect of diphenylhydantoin on synaptosome sodium-potassium-ATPase. J. c/in. Inocst. 47: 2752~-2758. GIIISON.K. and HARRIS.P. (1969). Diphenylhydantoin and human myocardial microsomal (Na+. K’)-ATPase. Bio&em. hiophys. Res. Commun. 35: 75-78.
GRAY, E. F. and WHITTAKER,V. P. (1962). The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96: 79-87. HASBANI, M., PINTUS, J. H. and LEE, S. H. (1974). Diphenylhydantoin and calcium movement in lobster nerves. Arch. Neural. 31: 25&254. HEXUM,T. D. (1974). Studies on the reaction catalyzed by transport (Na, K) adenosine triphosphatase-I. Effects of divalent metals. Biochem. Pharmac. 25: 3441-3447. KLINE, M. H., HEXUM,T. D., DAHL, J. L. and HOKIN, L. E. (1971). Studies on the characterization of the sodiumpotassium transport adenosine triphosphatace_VII. Comparison of the properties of the membrane-bound and partially purified soluble and insoluble forms of the enzyme. Ads Biochem. Biophys. 147: 781-787. LEWIN,E. and BLECK,V. (1971). The effect of diphenylhydantoin administration on sodium-potassium-activated ATPase in cortex. Neurology 21: 647-651. LEWIN,E., WALKER,J. E. and BLECK,V. (1973). Lack of effect of diphenylhydantoin administration on cortex potassium-activated p-nitrophenylphosphatase. Neuropharmacology 12: 577-581. Lomu, 0. H., ROSENBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951). Protein measurement with folin phenol reagent. J. hid. Chem. 193: 265-275. NAKAO, T., TASHIMA,Y., NAGANO, K. and NAKAO, M. (1965). Highly specific sodium-potassium-activated adenosine tiiphosphatase from various tissues of rabbit. Biochem. bionhvs. Res. Commun. 19: 755-758. PINCUS,J. H. and LEE, S. (1973). Diphenylhydantoin and calcium. Relation to norepinephrine release from brain slices. Arch. Neural. 29: 239-244. PINCUS, J. H., GROVE,I., MARINO,B. B. and GLASER,G. E. (1970). Studies on the mechanism of action of diphenylhydantoin. Arch. Neural. 22: 566-571. POST. R. L., KUME, S., TOBIN, T., ORCUTT, B. and SEX, A. K. (1969). Flexibility of an active center in sodiumplus-potassium adenosine triphosphatase. J. yen. Physiol. 54: 306~~326s. RAWSON, J. D.
and PINCUS, J. H. (1968). The effect of diphenylhydantoin on sodium. potassium, magnesiumactivated adenosine triphosphatase in microsomal fractions of rat and guinea pig brain and on whole homogenatcs of human brain. Biochcm. Pharmuc. 17: 573~ 579.
Diphenylhydantoin SIEGEL,G. J. and
GOODWIN, B. B. (1972). Sodium-potassium-activated adenosine triphosphatase of brain microsomes: modification of sodium inhibition by diphenylhydantoin. .r. clin. Invest. 51: 1165-1169. SKOU, J. C. (1957). The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biodim. biophys. Acta 23: 394401. SKOU, J. C. (1962). Preparation from mammalian brain and kidney of the enzyme system involved in active transport of Na+ and K+. Biochim. biophys. Acta 58: 314325. SKOU, J. C. (1965). Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol. Rec. 45: 596617. SPAIN, R. C. and CHIDSEY,C. A. (1971). Myocardial Na/K adenosine triphosphatase activity during reversal of ouabain toxicity with diphenylhydantoin. J. Pharmac. exp. Ther. 179: 594598. SPECHT,S. C. and ROBINSON, J. D. (1973). Stimulation of the (Naf + K+)-dependent adenosine triphosphatase by amino acids and phosphatidylserine: chelation of trace metal inhibitors, Archs Biochem. Biophgs. 154: 314323. ~PWELAKIS, N. and HENN, F. A. (1970). Effect of diphenylhydantoin on membrane potentials and Na-K-ATPase of cultured chick heart cells. Am. .I. Phrsiol. 218: 12241227.
and ATPases
195
SWANSON, P. D. and CRANE, P. 0. (1972). Diphenylhydantoin and movements of radioactive sodium into electrically stimulated cerebral slices. Biochem. Pharmac. 21: 2899-2905. TOBIN, T., AKERA, T., BASKIN, S. I. and BRODY, T. M. (1973). Calcium ion and sodium- and potassium-dependent adenosine triphosphatase: its mechanism of inhibition and identification of the Et-P intermediate. Molec. Pharmac. 9: 336-349. UESUGI, S., DULAK, N. C., DIXON, J. F., HEXUM, T. D., DAHL, J. L., PERDUE, J. F. and HOKIN, L. E. (1971). Studies on the characterization of the sodium-potassium transport adenosine triphosphatase_VI. Large scale partial purification and properties of a lubrol-solubilized bovine brain enzyme. J. biol. Chem. 246: 531-543. WILENSKY. A. J. and LOWDEN, J. A. (1972). The inhibitory effect of diphenylhydantoin on microsomal ATPases. Life Sci. 11: 319-327. WOODBURY, D. M. (1955). Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats. J. Pharmuc. exp. Ther. 115: 74-95. WOODBURY, D. M. and KEMP, J. W. (1971). Pharmacology and mechanism of action of diphenylhydantoin. Pspchiar. Neural. Neurochir. 74: 91-115.
of Pharmacology,
University
of Nebraska (Accepred
Medical
Center,
Omaha,
Nebraska
68105, U.S.A.
16 JU/J 1975)
Summary -The effects of diphenylhydantoin on brain sodium- and potassium-dependent adenosine triphosphatase (NaK ATPasc), ,I-nitrophenylphosphatase and the calcium-dependent-adenosinc triphosnhatase (Ca ATPasc) were examined. The results indicated that: (1) dinhenvlhvdantoin did not stimulate the NaK ATPase of beef brain sodium iodide-treated microsomes or rat brain synaptosomes at nonsaturating or saturating levels of sodium and potassium; (2) the apparent stimulation by diphcnylhydantoin under high sodium:potassium ratios rcportcd by other workers can bc explained by a pot’assium contamination in the diphenylhydantoin; (3) diphcnylhydantoin did not alter the Michaelis constant for ATP for the NaK ATPase: (4) diphenylhydantoin did not slowly interact with or alter the NaK ATPase: (5) diphcnylhydantoin did not alter the calcium inhibitton of the enzyme and (6) diphenylhydantoin did not alter the p-nitrophenylphosphatase or Ca ATPase. Based on these considerations, diphenylhydantoin must be exerting its pharmacological effect by altering a membrane constituent other than the NaK ATPase.
Diphenylhydantoin
(5.5-diphenyl-2,4-imidazolidene-
crone; DPH) is used extensively to treat major motor and psychomotor seizures. The anticonvulsant property is thought to be due to inhibition by DPH of the spread of seizure from the focal area (WOODBURY zrd KEMP, 1971); however, the mechanism by which DPH blocks the spread is not understood. WOOI>IIURY(1955) noted that DPH lowered the intracellular sodium concentration in rat brain. but a definite controversy exists concerning the mechanism of this effect. Suggestions include: (I) DPH inhibition of the influx of sodium into the nerve, which is supported by the results of SWANSONand CRANE (1972); PINCUS. GROVE, MARINO and GLASER (1970) and (2) DPH enhancement of the efflux of sodium from the nerve by active pumping of sodium out of the nerve by .he sodium- and potassium-dependent adenosine tri2hosphatase (NaK ATPase, EC 3.6.1.3) (SKOIJ, 1965). A number of effects of DPH on the NaK ATPase ‘lave been reported. The results indicate that DPH zither has no effect (FESTOFFand API’EL. 1968; SELL Ind GOODWIN. 1972; SPAIN and CHIDSI:Y, 1971; GmEONand HARRIS, 1969; SPEXELAKISand HENN, 1970), activates (FESTOFF and APPEL, 1968; WILENSKY and LOWDEN, 1972; SIEGEL and GOODWIN, 1972) or inhibits (RAWSON and PINCUS, 1968; WILENSKY and LO~DEN, 1972) the enzyme, depending on the relative saturation of the enzyme with sodium and potassium. If the anticonvulsant property of DPH is due to acti*The research was supported in part by a research starter grant from the Pharmaceutical Manufacturers’ Association Foundation, Inc., and from funds appropriated by the Nebraska Legislature for “Seed Money” research. A preliminary report appeared in Fedn Proc. Fedn Am. Sots exp. Bio/. (1974) 33, 438. 187
vation of the NaK ATPase, the NaK ATPase should be activated in vitro under the same ionic environment which exists normally in the brain. Accurate determinations of the effects of DPH on the kinetic parameters of the NaK ATPase require a precise enzyme assay. Since there are a number of different ATPases present in the brain, it becomes difficult to determine accurately the activity associated with only the NaK ATPase. Low activity calcium-dependent adenosine triphosphatase (Ca ATPase) and high activity magnesium-dependent adenosine triphosphatase (Mg ATPase) are present. and their activities are not affected by sodium, potassium, or ouabain. The NaK ATPase requires both sodium and potassium for enzymatic activity and is inhibited by ouabain. A multi-step reaction sequence has been proposed by ALBERS,KOVAL and SIEGEL(1968); POST, KUME, TOBIN. ORCUTT and SEN (!969) for the NaK ATPase, the last step being assayed as a potassiumdependent p-nitrophenylphosphatase. The NaK ATPase activity can be determined by measuring total ATPase activity in a medium containing sodium and potassium and subtracting the Mg ATPase activity assayed by omitting sodium and potassium from the medium (ALDRIDC;E,1962; SKOU, 1957) or by inhibiting the NaK ATPase with ouabain (UESUC;I,DULAK, DIXON, HEXUM, DAHL, PERDUE and HOKIN, 1971). Omitting sodium and potassium from the medium does change the ionic strength, but AHMED and THOMAS (1971) found that the addition of salts did not affect the Mg ATPase activity in rat brain microsomes. Adding ouabain has the advantage of maintaining constant ionic strength for both controls and test conditions and the disadvantage of possible competition of ouabain and DPH for the
188
J. D. DEUPREE
same site on the enzyme. An alternate approach for increasing the accuracy of the NaK ATPase activity is to remove 90% of the Mg ATPase activity from the microsomes with sodium iodide (NaI) (UESUGIet nl., 1971). This method reduces activity not due to NaK ATPase to a level where the inorganic phosphate (Pi) formed is due mainly to spontaneous hydrolysis of ATP. Variable effects of DPH may be due to the relative insolubility of DPH at pH 7.0. The solubility of DPH at pH 6.9 is 7.7 x 10m5 M, at pH 7.9 is 1.2 x 10-3~ and at pH 9.2 is 2.4 x 10A3M. Therefore stock solutions of DPH must contain a base (usually NaOH). The sodium added with DPH must be balanced by addition of sodium to control incubations to maintain identical ionic conditions. The variable effects of DPH may result from the assay conditions chosen for kinetic analysis. FESTOFF and APPEL (1968) reported that NaK ATPase of rat brain synaptosomes was not affected by DPH at saturating levels of sodium and potassium, was inhibited at low ratios of Na:K, and activated at Na:K ratios of 25:l or higher. Based on these results, DPH would activate the NaK ATPase only when the level of intracellular sodium was greater than 1OmM and the external potassium was less than 0.2m~. The NaK ATPase activity was measured as the difference between the ATPase activity in the presence of sodium, potassium and magnesium and the ATPase activity in the presence of magnesium and ouabain. Thus, the DPH effect may have been obscured by the high level of Mg ATPase present or altered by ouabain. SIEGEL and G~C~DWIN (1972) reported that optimal activation by DPH of NaK ATPase in NaI rat brain microsomes required a ratio of Na:K of 15O:l. The activity of NaK ATPase was measured in incubations without and with sodium and potassium. They suggested that DPH activated NaK ATPase by preventing the sodium inhibition of the enzyme which occurs at low levels of potassium. Alternative explanations of the results of others are possible. SIEGEL and GOODWIN (1972) did not rule out activation by traces of potassium in the DPH. Neither SIEGEL and GOODWIN (1972) or FESTOFFand APPEL (1968) described the pH of their DPH preparation, nor indicated whether DPH altered the pH of the assay, or whether DPH was soluble in their assay media. In addition, test and control incubations could have differed in sodium and solubilizer concentrations, as well as in DPH concentrations. The objective of this investigation was to reevaluate the reported effects of DPH on the kinetic parameters of the NaK ATPase isolated from beef and rat brain. Several assay methods and high purity reagents were used. The data indicate that DPH does not alter the kinetic properties of the NaK ATPase in in vitro assays. The data further show that the activation of the NaK ATPase reported by other workers could have been due to a potassium contamination in the DPH used. Thus, the effects of DPH on intracellular
sodium levels cannot be assigned to the NaK ATPase with certainty and may be the result of interaction with other membrane constituents. MATERIALS AND
METHODS
Chemiculs NaATP, Tris, ouabain, glycylglycine, Tris p-nitrophenylphosphate and EGTA (ethylene glycol-bis (P-amino-ethyl ether)N,N’-tetracetic acid) were obtained from Sigma Chemical Co. Diphenylhydantoin (free acid, 990: pure grade) was purchased from Aldrich Chemical Co.. Inc. An additional sample of DPH (free acid) was donated by Parke-Davis. Sodium hydroxide and potassium hydroxide were obtained from Fisher Scientific Co. Imidazole was obtained from Eastman Kodak Co. All other chemicals were obtained from Mallinckrodt. Quartz triple distilled water was used in all experiments. Reagents Tris ATP was prepared by passing NaATP through a Dowex-SOW resin (H+ form) and titrating to pH 7.0 with Tris. Diphenylhydantoin solution was prepared fresh each day by adding NaOH to a final concentration of 2 x lo-” M to a suspension of 1O-3 M DPH. The resulting clear solution had a pH of 10.0. Turbidity measurements at 400 and 600nm showed that DPH did not precipitate from the assay medium. The 10m3 M stock solution of DPH had a potassium concentration of 5.4 x 10mh M as determined by atomic absorption spectroscopy: 1.3 x IO-’ M potassium was present in the DPH and the balance was due to the potassium contamination in the NaOH used to solubilize the DPH. Because of the potassium contamination in the DPH solution, equivalent amounts of NaOH and KOH were added to the control tubes. This amount of NaOH was not sufficient to change the pH of the assay medium. Tissue sources cind preparotiorzs Beef brain microsomes Beef brains, donated by John Roth and Sons (Omaha. Nebraska), were kept on ice until used. The microsomes were isolated according to the procedure of HI’XLIM (1974) and stored at -70-C. This preparation contained both Ca ATPase and Mg ATPase activity, which are insensitive to ouabain. The average NaK ATPase specific activity of the microsomes was 25 prnol of Pi formed/hr per mg of protein. Sodium iodide-treuted beef bruin microsomes Sodium iodide micrbsomes were prepared according to the procedure of NAI\,r 01 ATP
wnl, cn,!mc
1 b
50 rn~ Trls. pH 8-C 50 rn~ Tns. pH X.0
: e I
SO rn~ Tns. pH 8.0 50 X0 50 rn~ Tin. pH X-O 50 IIIMTris. pH 8-O
: I :
50 30 IIIMTrn. Imidazolc, pH X.0 30 IIIMglycylglycine, 30 m*l Imldazole, 30 mM glycylglycine, 30 rn~ tn~ Imldazole, Imidarole, 30 30mM rn~ glycylglycine, glycylglycme, 30 mM Imidazole, 30 mM glycylglycme. 30 rnr.1Imldarole. 30 rn~ glycylplycine. 30 rn~ Imldatolc. 30 rn~ glycylglycmc. 30 rn~ Imida7olr. 30 rn~ gl)cylglycinr, 30 rn~ Inndarolc. 30 IIIMglyc~lglycmc. 30 rn~ Imidarole. 30 mhl glycylglycme. 30 rn~ hmdarole. pH 7 0 30 rn~ Im~darole, pH 7.0
I m n 0 P q r S Dctcrmmahon A B c D E H 1 J K L R S
+ P, 6
pH pH pH pH pH pH pH pH pH pH
6 h 6 6 6 6 7,5 7,5 7.5 75 75 7-5 7.5 75 l-5 7.5 6 6
7.4 7.4 7.4 7-4 74 74 74 74 74 7.4
2 2 2
2 4 4
_
+ + + + + + +
_ _ _ + + + + + + + + + + _
_ _ _ _ _ _
_ _ _
_ _ _ _ _
_ _
_
+ + + + + + + + + + + +
+ + + + + + + + + + + +
_ _ + + _ _ _ + + + + + _
of total ATPare 50 rn~ 7115, pH X.0 50 HIMTrls, pH 8.0 50 rn~ Tns. pH 8 0 50 rn~ Tns, pH 8-O 50 mha Tns. pH 8 0 30 rn~ Imh~olc. 30 IIIMglycyl~lycmc. 30 rn~ Im~darolc. 30 rn~ &c~l~lycmc. 30 “IM Im,datole. 30 rn~ &~ylglycme. 30 rn~ Im~dazole. 30 rn~ &c~l~l~cme. 30 rn~ Imldazole, 30 rn~ glycylglycine. 30 rn~ Imidarole, pH 7.0 30 rn~ Imldazole. pH 7.0
pH PH pH PH pH
6 6 6 6 6 1.5 7.1 71 75 75 6 6
74 7.4 7-4 7.4 7.4
Determination of NaK ATPase = Total ATPasee hydrolysis of ATP). NaK ATPase in absence of DPH calculated as: VS
NaK ATPase in presence of DPH calculated
as:
(Mg ATPase B-b D-d
+ + + + +
_ _
+ Pi associated A-a
A-f
_ + + + + + _
I-i
with protein K-k
I-n
K-p
_ _ _ _
_ _ _
+ Pi due to spontaneous H-h
H-h H-m H-m
R-r
J. D. DEUPRE~
190
amount of Pi formed was determined as described under Mg ATPase assay. The NaK ATPase was assayed above its optimum pH in some cases, since DPH was more soluble at the higher pH. Diphenylhydantoin did not interfere with the measurement of Pi. Poro\sizfm-t/[~perltlr,lr p-nirroph~r7~~lpho.spl7utL7.tr L/.S.\I7)‘. The potassium-dependent p-nitrophenylphosphatasc activity was calculated as the difference in rate of hydrolysis observed in the presence and absence of KCI. The potassium-dependent p-nitrophenylphosphatase activity was determined by pre-incubating the NaI microsomes at 37’C for 10min in 1 ml containing 50 mM Tris, pH 7%: 5 mM MgCl,: with and without 15 mM KCl; with and without DPH. The 10min reaction was initiated by the addition of 6,~mol p-nitrophenylphosphate and terminated by the addition of 1 ml 1.0~ NaOH. The tubes were centrifuged for 10 min at 1000 9 to remove the cloudy material. The centrifugation did not pellet any p-nitrophenol. The amount of p-nitrophenol released was determined by measuring the absorption at 400nm. Ca ATPase assay. The Ca ATPase activity was calculated as the difference in activity obtained in the presence and in the absence of calcium. The test conditions contained DPH solubilized with NaOH. The assay was conducted by pre-incubating the microsomes at 37 C for 10 min in 1 ml containing 67 mM glycylglycine adjusted to pH 7.4 with imidazole, 5 mM MgCl,, 0.125mM EGTA, 10m4~ ouabain, with and without 0.2rn~ CaCl,, with and without DPH. The 5 min reaction was started by the addition of 1.5 /*mol NaATP. The reaction was stopped by the addition. with immediate mixing, of cold 5”,, trichloroacetic acid in chloroform:methanol (1 :l), and the amount of Pi formed was determined as described under Mg ATPase assay. Protein determination. Protein was determined by the method of LOWRY, ROSENBROUGH, FARR and RANDALL (1951) using bovine serum albumin as a standard. Statist ical analysis
Table 2. Effects of DPH on Mg ATPase present somes isolated from beef brain DPH (hll
in micro-
Mvlg ATPar (pmobhr per mgl
Microsomes were assayed using conditions b vs c in Table 1. The Mg ATPase was calculated as the difference between activity in the presence of magnesium and the activity in the absence of magnesium. Values are the mean of 6 determinations i SE.
extreme variation in this essential correction factor, the microsomal enzyme preparation was not used for this study. Sodium iodide microsomes were used for further studies since the Nal treatment removes 902, of the Mg ATPase activity. Eflkcts of DPH on NaK rnicrosomes
ATPase
in beef brain NaI
In the studies reported in this paper ouabain was not used in the NaK ATPase assay since ouabain and DPH may have been acting at the same site on the enzyme. Rather, the NaK ATPase was measured as the difference between activity in the presence and the absence of sodium and potassium. The Mg ATPase activity in the presence of ouabain and a high salt concentration (120mM NaCl + 30m~ KCI) was the same as the activity in the absence of NaCl and KC1 (Table 3). Thus, Mg ATPase activity was not affected by ionic strength. Since the NaK ATPase activity was higher in the presence of the chelator, and heavy metals have been shown to inhibit the enzyme (SPITHT and Ront~~so~. 1973) (see Table 3). EGTA was used in all assays. Diphenylhydantoin solubilizcd with NaOH had no effect on the NaK ATPase when assayed at pH 8.0 in the presence of 50miv1 NaCl and KC1 at concentrations ranging from 0.2 to 1Om~ (Fig. 1). In addition. DPH did not alter NaK ATPasc activity obtained in the presence of 2 or @2m~ KC1 with
The data were analyzed using the Student t-test. A P value greater than 0.05 was considered not significant. RESULTS
Effbcts
ofDPH on My ATPuse
Since one method of determining NaK ATPase was to subtract Mg ATPase activity from the total ATPase, it was necessary to ascertain the effects of DPH on the Mg ATPase in the assay system used. Diphenylhydantoin has been reported to inhibit the Mg ATPase (WILENSKY and LOWDEN, 1972). Diphenylhydantoin at concentrations between lo-* and 10m4 M did not inhibit the Mg ATPase present in microsomal fractions (Table 2). Because of the
i
20 t-t
p,,,,,,,,,, 2
4 6 KCl(mM)
8
IO
Fig. 1, Effects of DPH solubilized with NaOH on the NaK ATPase assayed at high levels of sodium and varying levels of potassium. The NaI microsomes from beef brain were assayed using conditions B-b vs C-c in Table 1.
Diphenylhydantoin
I91
and ATPases
Table 3. Effecrs of ionic strength, EGTA and EDTA on ATPase activity TotalATPasc I” p’cscnce of Na + K (umts,mg)
Additions NDX Ouaham EGTA EGTA + ouaham tDTA EDTA + ouahsm
23-7 23 2 ‘I I 21 5 22.9 23.7
40 5 234 444 21.5 42.0 23.9
IfIX: 17 lb 23 7: 90 190:
21-X* Jl.lt Ii).?*
23
I9hi
?09* ?? 3-1
l8.m‘
* Mg ATPase = (Total ATPase in absence of Na + K) - (Pi associated with microsomes + Pi from spontaneous hydrolysis of ATP). ‘r Mg ATPase = (Total ATPase in presence of Na, K and ouabain) - (PI associated with microsomes + Pi from spontaneous hydrolysis of ATP). : NaK ATPase = (Total ATPase in presence of Na + K) - (Total ATPase in absence of Na + K). NaK ATPase = (Total ATPase in presence of Na + K) ~ (Total ATPase in presence of Na. K and ouabam). Microsomes isolated from beef brain were assayed for ATPase activity in the presence of 50m~ Tris, pH 8.0, for 5 min at 37-C, and the Pi produced was determined as described in the Methods. Total ATPase was determined by adding to the incubation mixture 6rn~ MgCI,, 4rn~ Tris ATP with or without l30m~ NaCl and 20m~ KCI. The Pi associated with the enzyme was determined by omitting Mg, Na, K and ATP from the assay medium. The spontaneous hydrolysis of ATP was determined by omitting enzyme from the assay medium. The concentrations of ouabain, EGTA and EDTA were 10m4.~.
PJaCl at concentrations varying between 2.5 and 12Om~ NaCl (Fig. 2). Likewise, lo-’ to 3 x 10m4 M I>PH did not significantly (P > 0.05) alter the NaK ATPase assayed in the presence of 50m~ NaCl and C1.2rn~ KC1 (not shown). In these experiments the NaK ATPase activity in the absence of DPH was compared with the NaK ATPase activity in the presence of DPH (Table 1, B-b compared with C-c). The only difference between test conditions and control c,onditions was the amount of DPH, since the same amount of NaOH and contaminating potassium was added to the control conditions as was added to the test conditions. Changing the pH of the assay medium did not result in activation of the NaK ATPase by DPH. The ?\JaK ATPase activity was not altered by DPH at pH 7.0 or 7.4 when assayed at 50m~ NaCl and
0
20
I 40
I NaCl
I 60
I
I 80
0.2 mM KC1 (l-i compared with J-j, and R-r compared with S-s as listed in Table 1). If DPH was solubilized with KOH, and KOH was added to the control conditions to balance the KOH in the test conditions, there was no apparent stimulation of the NaK ATPase by DPH in assay media containing a high ratio of sodium to potassium (media D-d vs E-e, K-k vs L-l, and K-p vs L-q in Table 1). However, if KOH was not added to the control tubes (A-a vs E-e and H-h vs L-l as in Table 1) a two-fold stimulation of the NaK ATPase by DPH was obtained (Fig. 3). Thus, activation under these conditions would have been due to the potassium and not the DPH.
l - DPH n-t DPH
l -DPH
I
I 100
I 120
(rn~)
I
I
I
I
20
40
60
80
NaCl
l?g. 2. Effects of DPH solubilized with NaOH on the NaK ATPase assayed at low potassium and varying levels of 1. The NaI microsomes from beef brain were assayed using conditions B-b vs C-c in Table 1.
I
I
100
120
(mM)
Fig. 3. Effects of DPH solubilized with KOH on the NaK ATPase assayed at low levels of potassium and varying levels of sodium. The NaI microsomes from beef brain were assayed using conditions A-a vs E-e in Table 1.
192
J. D. DEUPREE
Effects of DPH on NuK ATPase in synaptosomes from rat brain
Interaction of DPH and the NaK ATPase ence of various substrates of the enzyme
To make our conditions as comparable as possible to those reported by FESTOFF and APPEL (1968), synaptosomes were prepared from rat brain using procedures similar to those reported by FESTOFF and APPEL (1968). The assay method was also similar to theirs except the protein concentration in the assay medium was reduced to a level at which the assay was linear for 30min and the modified Fiske-SubbaRow method (CLARK, 1964) was used to determine Pi. The Mg ATPase activity was determined by either omitting sodium and potassium from the assay media or by omitting sodium and potassium and adding ouabain. The NaK ATPase activity was measured as I-i vs J-j, K-k vs L-l, I-n vs J-o, K-p vs L-q, H-h vs K-k, H-h vs L-l, H-m vs L-q, and H-m vs K-p (Table 1). If DPH was solubilized with NaOH, and NaOH was added to control tubes, no activation of the NaK ATPase was seen, and results similar to those in Figures 1 and 2 were obtained. Activation of NaK ATPase, similar to that shown in Figure 3. was obtained only when DPH was solubilized with KOH and KOH was omitted from the control tubes. Thus, apparent stimulation by DPH could be accounted for by an increase in potassium, resulting in a change in the Na:K ratio.
The NaK ATPase appears to take on a number of different conformation states depending on the cations bound to it (BANCRJE~.WOXG and SEN, 1972). It is possible that DPH binds to only one conformation state of the enzyme, or that DPH takes a prolonged time to react with the NaK ATPase. To test this possibility, the NaK ATPase present in NaI microsomes (9 mg protein/ml) isolated from beef brain was incubated at 4’C in the presence of 10m4 M DPH containing 2 x 1O-4 M NaOH, or in 2 x 10m4~ NaOH without the DPH. The following additions were made as indicated: 50m1~1 NaCl, 0.15 mM KCl, 3.3 mM Tris ATP, 4 mM MgClz. After 24 hr the NaI microsomes were diluted and assayed using conditions B-b vs C-c in Table 1. Diphenylhydantoin did not have any significant effect on the NaK ATPase on prolonged incubation with various cations (not shown).
Effects of DPH on NaK ATPase in Nal microsomes from beef brain under conditions qf saturating sodium and potassium Diphenylhydantoin at concentrations between 10d8 and 2 x 10m4 M did not have any significant effect on the NaK ATPase in the presence of saturating levels of sodium and potassium (not shown). This was demonstrated by assaying NaK ATPase present in NaI microsomes prepared from beef brain in the presence of 120m~ NaCl, 20m~ KC1 and assay conditions B-b vs C-c in Table 1. Sodium hydroxide was added to both test and control conditions. Eflect of DPH on the Michaelis constant for ATP $v the NaK ATPase in Nal microsomes from beef bruin Most experiments testing for an effect of DPH on NaK ATPase have been conducted at saturating levels of ATP and would not indicate whether DPH was altering the binding of ATP to the enzyme. The effects of DPH on the ATP binding to the Na ATPase were estimated by determining the Michaelis constant (K,) for ATP in both the presence and absence of DPH. A K, of 0.24 mM for ATP for the NaK ATPase in NaI microsomes isolated from beef brain was obtained using the method of Lineweaver and Burk. This agrees with the K, values previously reported in the literature (KLINE, HEXUM, DAHL and HOKIN, 1971). The K, for ATP was not affected by 10m4h.1 DPH solubilized with 10e4 M NaOH (not shown). Assay conditions B-b vs C-c were used. A MgCI, to ATP ratio of 2 was maintained at all levels of ATP.
in the pres-
Ef,Cect ok’ DPH on the potussiunz-cieperztlent p-nitrophen!,/pho.sphatLIsr
It is possible that DPH only alters one of the steps in the NaK ATPase reaction. Since a potassium-dependent p-nitrophenylphosphatase reaction is thought to reflect the last step in the NaK-dependent ATP hydrolysis (ASKARI and KOYAL, 1968), the effects of DPH on this reaction were measured. Diphenylhydantoin in concentrations of lo-’ to 10m4M had no effect on the potassium-dependent p-nitrophenylphosphatase present in beef brain NaI microsomes assayed as described in the Methods (not shown). In addition, 10e4M DPH did not affect the potassium-dependent p-nitrophenylphosphatase at K, levels of potassium (not shown). Eflect of DPH and calcium on NaK ATPase microsomes from beef brain
in Nal
Since it has been postulated that DPH displaces calcium from the membrane (PINCUS and LEE, 1973; PINCUS et al., 1970), it is conceivable that DPH could activate the NaK ATPase by removing calcium from the enzyme. To test this hypothesis, the NaK ATPase was incubated with varying concentrations of calcium in the presence and absence of DPH. Increasing concentrations of calcium inhibited the NaK ATPase as has been reported by others (SKOU, 1962), but DPH did not reverse the inhibition by calcium (Table 4).’ EfSrct of DPH on Ca ATPase in microsomes from beef brain To determine if DPH was inhibiting the active transport of calcium, the effect of DPH on the Ca ATPase was studied. The Ca ATPase present in beef brain microsomes was not significantly (P > 0.05) inhibited by DPH when measured as described in the Methods (not shown).
Diphenylhydantoin Table 4. Effects of calcium and DPH on NaK ATPase present in Nal microsomes isolated from beef brain
-
Cdlctum
Wathout
(NI
li~rni,i hr
0
-
10 6 IO 5 ,i, 4 IO ’
NaK DPH per mg,
ATPew
WithDPH (pmal hr per mg)
54x5 k 035 5665 f 1.65 56.70i 0 20 .lj55 f 095
1335 i I35
5665 - 0.65 5X.25 + 0.65 575 + O-20 46.1? 0.50 13.15+ 005
P
“S “S “.S “_S. n.5.
‘The NaI microsomes were assayed using conditions B-b vs C-c in Table 1 with 120 mM NaCl and 20m~ KCI. T,e values are expressed as the mean of 2 experiments rln in duplicate f SD.
DISCUSSION
In most of these experiments the ouabain-inhibited assay used by FESTOFFand APPEL (1968) was not used bN:cause of possible competition in binding between olabain and DPH. Rather, we tried to reaffirm the results of other investigators using an assay system that was more generally applicable to studying the ell’ects of drugs. In addition, the NaK ATPase was assayed at pH 8.0, which was above its optimum pH, to further assure that DPH was in solution during the enzyme assay. No effects of DPH on kinetic parameters were seen under these conditions or at pH 7 0 and 7.4. Under conditions which should closely approach those used by SIEGEL and GWDWIN (1972) there was no stimulation or inhibition of the NaK ATPase incubated with DPH solubilized with either NaOH or KOH provided the corresponding cation was also added to the control tubes. As seen in Figure 3, activation of the NaK ATPase occurred only when the trace amount of potassium added along with the DPH was not balanced by adding potassium to the control tube. A 2 x 10e5 M potassium contamination would account for their apparent two-fold stimulation (see Fig. 2 in SIEGEL and GOODWIN, 1972). This strongly suggests that the stimulation of NaK ATPase ty DPH at high Na:K ratio was due to a potassium c,ontamination in the DPH, thereby causing an imbalance in potassium levels in the test and control conditions. In addition, the DPH used in the results reported here was determined to be 99’;:, pure (infrared spectrometery by Aldrich Chemical Co.). Pure DPH (free acid) donated by Parke-Davis and Company and solubilized with NaOH, did not activate the NaK ATPase at a high Na:K ratio. These results indicated that lack of DPH activation was not due lo a single lot of DPH. Using a synaptosomal preparation similar to that reported by FESTOFF and APPEL (1968), stimulation of the NaK ATPase by DPH at a high ratio of Na:K was not observed unless DPH solubilized with KOH was used and a potassium imbalance existed between test conditions and control conditions. The lack of etfect of DPH on NaK ATPase was noted when Mg ATPase was estimated, using either ouabain inhibi-
and ATPases
193
tion or omission of sodium and potassium from the medium. The activation reported by FESTOFF and APPEL (1968) could be explained by a 3 x 10e5 M potassium contamination in DPH. Their solutions reportedly contained less than lo-‘M sodium and potassium, but no description was given of the procedure used to solubilize DPH. A 10e3 M DPH solution requires 10 -3 M NaOH or KOH for solubilization, and the DPH must have contained some sodium or potassium. In addition, FESTOFFand APPEL (1968) used an enzyme preparation which contained a high concentration of Mg ATPase. Extreme variability among assays was observed when DPH was added to the Mg ATPase (Table 2). Although FESTOFF and APPEL (1968) did not report the effects of DPH on the Mg ATPase, that variability could account for the stimulation of the NaK ATPase observed when very low activities of enzyme were measured at high ratios of sodium to potassium. LEWIN and BLECK (1971) injected DPH into rats, sacrificed the rats, isolated the microsomal fractions and assayed the NaK ATPase. In the process of isolating the microsomal fraction, the DPH should have been substantially diluted, and since DPH was not added to the assay medium, it becomes questionable as to whether the reported activation was directly related to DPH. The results reported here, as well as by SIEGEL and GWDWIN (1972), SPAIN and CHIDSEY (1971), GIBSON and HARRIS (1969), SPERELAKISand HENN (1970), indicated that DPH did not affect the maximal velocity of. the NaK ATPase since the drug did not significantly alter the rate of reaction at saturating sodium, potassium, and MgATP levels. FESTOFF and APPEL (1968) reported a less than 10% inhibition of the NaK ATPase at 50 mM sodium and 1Om~ potassium, which may have been within experimental error of the assay. However, RAWSON and PINCUS (1968) reported a 20% inhibition (statistical significance was not given) of the enzyme under these conditions. WILENSKYand LOWDEN (1972) reported an 18% activation of the NaK ATPase at 10m4M DPH and a 36’% inhibition at 2 x 10e4 M DPH (statistical significance was not given) and a statistically significant inhibition of the Mg ATPase with 1O-4 M DPH. These variations could have been due to the high concentration of Mg ATPase in comparison with the NaK ATPase in the microsomal preparation, thus obscuring the effects of DPH on the NaK ATPase. Since DPH did not alter the K, for ATP, this would indicate that the anticonvulsant did not interfere with the binding of ATP to the NaK ATPase. Nor did DPH appear to slowly activate the NaK ATPase, since the NaK ATPase was not activated following a 24 hr incubation of the NaK ATPase with DPH. The presence of specific ligands (Mg, ATP, Na and K) was not important since DPH did not activate the NaK ATPase when they were present. Diphenylhydantoin did not activate the third step in the NaK ATPase reaction since it had no effect
194
J. D. DEUPREE
on the potassium-dependent p-nitrophenylphosphatase reaction. LEWIN, WALKER and BLECK (1973) reported that potassium-dependent p-nitrophenylphosphatase activity was the same in microsomes isolated from the brains of rats receiving DPH as compared with the enzyme from rats not treated with DPH. In addition, DPH did not affect the K, for potassium for this reaction, indicating that DPH did not compete with potassium in the reaction. There is some indication that DPH may have been blocking nerve conduction in a manner similar to that of local anaesthetics (PINCUS and LEE, 1973). It has been postulated that local anaesthetics and DPH displaced calcium from the membrane (HASBANI, PINCUS and LEE, 1974) and that the NaK ATPase may have been slightly inhibited in riro by calcium (TOBIN, AKERA, BASKIN and BRODY. 1973). It is not likely that DPH activated the NaK ATPase by displacing calcium from the enzyme since DPH did not reverse the inhibition of the NaK ATPase by calcium. In addition. FESTOFF and APPEL (1968) reported activation of the NaK ATPase under conditions where calcium would have been chelated by EGTA. Although DPH may have blocked calcium uptake into the nerve (HASBANI~‘1al., 1974; PINCUS and LEE, 1973), the anticonvulsant probably did not affect the active transport of calcium. since DPH did not inhibit the Ca ATPase. If a drug exerts a direct effect on an enzyme, then some parameter of enzyme activity (either the K, of one or more of the substrates or the V,,,, for the enzyme) should be altered by the drug under in vitro conditions. The results reported here show that DPH did not alter the velocity of the enzyme at saturating and non-saturating levels of sodium or potassium; did not affect the K,, for ATP; did not slowly affect the enzyme; did not affect the enzyme in the presence of specific ligands; did not affect the partial reaction of the NaK ATPase, as measured by p-nitrophenylphosphatase activity: and did not relieve the calcium inhibition of the NaK ATPase. In addition, the reported activation of NaK ATPase at a high ratio of Na:K by DPH appeared to be due to potassium contamination of the DPH. Therefore, I conclude that DPH does not affect the NaK ATPase from rat or beef brain, and the pharmacological action of DPH must be due to the interaction at sites other than the NaK ATPase. Ackno~lrd~rment.~~K~~~~ SORIA provided excellent techn~cal assistance and KII 761SIILI\~ONS performed the atomic absorption spectrum analysis.
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and PINCUS, J. H. (1968). The effect of diphenylhydantoin on sodium. potassium, magnesiumactivated adenosine triphosphatase in microsomal fractions of rat and guinea pig brain and on whole homogenatcs of human brain. Biochcm. Pharmuc. 17: 573~ 579.
Diphenylhydantoin SIEGEL,G. J. and
GOODWIN, B. B. (1972). Sodium-potassium-activated adenosine triphosphatase of brain microsomes: modification of sodium inhibition by diphenylhydantoin. .r. clin. Invest. 51: 1165-1169. SKOU, J. C. (1957). The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biodim. biophys. Acta 23: 394401. SKOU, J. C. (1962). Preparation from mammalian brain and kidney of the enzyme system involved in active transport of Na+ and K+. Biochim. biophys. Acta 58: 314325. SKOU, J. C. (1965). Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol. Rec. 45: 596617. SPAIN, R. C. and CHIDSEY,C. A. (1971). Myocardial Na/K adenosine triphosphatase activity during reversal of ouabain toxicity with diphenylhydantoin. J. Pharmac. exp. Ther. 179: 594598. SPECHT,S. C. and ROBINSON, J. D. (1973). Stimulation of the (Naf + K+)-dependent adenosine triphosphatase by amino acids and phosphatidylserine: chelation of trace metal inhibitors, Archs Biochem. Biophgs. 154: 314323. ~PWELAKIS, N. and HENN, F. A. (1970). Effect of diphenylhydantoin on membrane potentials and Na-K-ATPase of cultured chick heart cells. Am. .I. Phrsiol. 218: 12241227.
and ATPases
195
SWANSON, P. D. and CRANE, P. 0. (1972). Diphenylhydantoin and movements of radioactive sodium into electrically stimulated cerebral slices. Biochem. Pharmac. 21: 2899-2905. TOBIN, T., AKERA, T., BASKIN, S. I. and BRODY, T. M. (1973). Calcium ion and sodium- and potassium-dependent adenosine triphosphatase: its mechanism of inhibition and identification of the Et-P intermediate. Molec. Pharmac. 9: 336-349. UESUGI, S., DULAK, N. C., DIXON, J. F., HEXUM, T. D., DAHL, J. L., PERDUE, J. F. and HOKIN, L. E. (1971). Studies on the characterization of the sodium-potassium transport adenosine triphosphatase_VI. Large scale partial purification and properties of a lubrol-solubilized bovine brain enzyme. J. biol. Chem. 246: 531-543. WILENSKY. A. J. and LOWDEN, J. A. (1972). The inhibitory effect of diphenylhydantoin on microsomal ATPases. Life Sci. 11: 319-327. WOODBURY, D. M. (1955). Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats. J. Pharmuc. exp. Ther. 115: 74-95. WOODBURY, D. M. and KEMP, J. W. (1971). Pharmacology and mechanism of action of diphenylhydantoin. Pspchiar. Neural. Neurochir. 74: 91-115.
