Sodium-Potassium-Activated Adenosine Triphosphatase

Transcription

1 Sodium-Potassium-Ativated Adenosine Triphosphatase of Brain Mirosomes: Modifiation of Sodium Inhibition by Diphenylhydantoins GORG J. SIGL and BVRLY B. GOODWIN From the Departments of Neurology and Physiology, Mount Sinai Shool of Mediine of the City University of New York, New York 129 and the Institute for Medial Researh and Studies, New York 11 A B S T R A C T ffets of diphenylhydantoins on (Nae + K+)-ATPase ativity in rat and at brain mirosomes were studied. 5,5-diphenylhydantoin (DPH) in onentrations of 5-2 slg ml' produes an apparent stimulation of the rat brain (Na' + K+) -ativated ATPase of 55-65% in media ontaining 5 mm Na+,.15 mm K+, 3 mm Mg++, and 3 mm ATP. No effets are found on the Mg-ATPase. At onstant K+ levels of.5 mmole/liter and.15 mmole/liter, inreasing the Na+ onentration ativates the enzyme similarly with and without DPH. However, Nat onentrations greater than 5 mmoles/ liter and 1 mmoles/liter, respetively, whih are inhibitory in these low Ke media, produe less inhibition in the presene of DPH. In media ontaining 1 mm Nat, the K+ ativation, on the other hand, is potentiated by DPH. In preparations from at brain qualitatively similar results are obtained. No effet of DPH is seen on the inhibition produed by high K+ in low Na' media. DPH produes an approximately onstant apparent stimulation of 45% in the (Na++ K+) inrements when these ions are varied simultaneously at a fixed ratio of 15 Na+: 1 Ki with at brain extrats. 5- (p-hydroxyphenyl)-5-phenylhydantoin (HPPH) has the same poteny as DPH in reduing the Nat inhibition at high Na: K ratios. The hydantoins appear to at by dereasing the Na+ inhibition that ours at high Na: K ratios. Presented in part at the 23rd Annual Meeting, Amerian Aademy of Neurology, 3 April 1971, New York. Reeived for publiation 21 September 1971 and in revised form 11 November The Journal of Clinial Investigation Volume INTRODUCTION Diphenylhydantoin (DPH)' has been observed to redue the intraellular sodium ion onentration and to inrease Na' onentrations in brains of rats subjeted to eletroshok seizures. It was therefore proposed that the anti-seizure ation of DPH results from stimulation of a metabolially linked proess of ative Nae extrusion (1). There is now onsiderable evidene that ative Na' transport is linked to (Nae + K+)-ativated adenosine triphosphatase (2, 3). Studies on this enzyme have shown that DPH inhibits the enzyme ativity in brain mirosome extrats (4) and in synaptosomal preparations (5, 6) under onditions of low Na: K ratios although it appears to stimulate enzyme ativity in synaptosomes under high Na: K ratios (5). In addition, DPH stimulates K+ transport under ertain onditions in synaptosomes (7). The present study is a further attempt to haraterize the effets of hydantoins on brain mirosomal (Nat + K+) -ATPase. It is found that DPH and its hydroxylated derivative, 5- (p-hydroxyphenyl) -5-phenylhydantoin (HPPH), both at to derease the inhibition of (Na+ + K+)-ATPase produed by high Na: K ratios. MTHODS Materials. Tris-ATP and the sodium salt of 5,5-diphenylhydantoin (DPH) were obtained from Sigma Chemial Co., St. Louis, Mo.; ['y-"p]-atp was obtained from International Chemial and Nulear Corp., Irvine, Calif.; and 5- (p-hydroxyphenyl) -5-phenylhydantoin (HPPH) was a generous gift from Dr. Henn Kutt (8). Diazepam was 1Abbreviations used in this paper: ATPase, adenosine triphosphatase; DPH, diphenylhantoin; DTA, ethylenediaminetetraaetate; HPPH, 5- (p-hydroxyphenyl) -5-phenylhydantoin.

2 generously supplied by Rohe Laboratories, Nutley, N. J. Sodium Phenobarbital Injetion, (13 mg/ml in 67.8% propyleneglyol, 1% ethanol, and 1.5% benzyl alohol) was obtained from USV Pharmaeutial Corp., New York. This was diluted 4-fold in the enzyme assay media. DPH and HPPH suspensions in water were made at the time of use. In some experiments where noted, sodium DPH was dissolved in Parke, Davis Solvent for Dilantin (4% propylene glyol, 1% ethanol, adjusted with NaOH to ph 12; Parke, Davis & Co., Detroit, Mih.) this solvent was diluted 175-fold in the enzyme assay media. Diazepam was dissolved in the Parke, Davis Solvent and similarly diluted in the enzyme assay. Mirosomal (Na+ + K+) -ATPase was prepared from rat and at brains in a proedure using NaI (9) and stored in liquid nitrogen. Protein was measured by the method of Lowry, Rosebrough, Farr, and Randall (1). Assay of (Na+ + K+)-ATPase. Brain mirosomes were diluted at the time of assay in.5 M Tris-HCl (ph 7.4) ontaining.1 mm Tris-DTA. Mirosomes, 5-3 jig protein, were inubated at 37C for 3 min in.5 M Tris- HCl (ph 7.4), 3 mm MgCl2, 3 mm Tris-[(y-'P]-ATP (speifi ativity 1' pm/,gmole) plus NaCl and KC1 as indiated, in final volumes of 4,l. The enzyme was preinubated for 15 min at C in the test agent plus the indiated salts before initiation of reation with ATP. Reations were terminated by the addition of 1 /Al of 5% old trihloroaeti aid. Inorgani phosphate was extrated in isobutanol and the radioativity measured as desribed (11). (Nat + K+)-ATPase ativity was obtained by subtrating the hydrolyti rate in the presene of Mg++ from the total rate in Mg++, Na+, and K+. It was asertained that pretreatment of mirosomes with.1 mm ouabain in 3 mm MgCl2 for 3 min at OC abolished 95% of the (Na+ + K+) inrements. RSULTS During preliminary experiments, it was found that DPH stimulates (Na+ + K+)-ATPase ativity of rat or at brain mirosomes in the presene of 15 mm Na and 1 mm K+.' Apparent stimulation of the (Na+ TABL I Stimulation of Rat Brain (Na+ + K+)-A TPase by Hydantoins DPH AMg2+ A(Na++K+)* HPPH &(Na++K+)t AMg2+ Pg ml-, % ontrol pig -ml- % ontrol Other onditions as desribed in Methods. Control values (nmoles mg-' min-'): DPH experiment, AMg2+ = 58, A(Na+ + K+) = 43; HPPH experiment, AMg2+ = 122, A(Na+ +K+) = 119. * 5 mm NaCi plus.15 mm KC1. t 15 mm NaCl plus 1 mm KCl. 'Under the same ation onditions, DPH, 2 /Ag-ml[, had no signifiant effet on (Nat + K+) -ATPase in eletroplax mirosomes from letrophorus eletrius. 5 j :2 (a) I ~ ~... _ o mm NaCI Ė en -! ~~~~~~~I o mm NoCI FIGUR 1 ffets of DPH on rat brain (Na+ + K+)-ATPase under varying Na' onentration (a).5 mm KCl. (b).15 mm KCl. Other onditions as desribed in Methods. o - -, ontrol; *-*, DPH 2 Ag-ml1. + K+) inrements in ATPase ativity is produed by DPH or HPPH in onentrations as low as 3-5 IAg ml (Table I). The largest effets are seen with onentrations of 2-25 ugeml-'. No signifiant effets on the Mge+-ATPase ativity are seen. The ativation of ATPase ativity by sodium ion is shown in Fig. la and b. In separate experiments performed in onstant K+ onentrations of.5 mmole/ liter and.15 mmole/liter, sodium ion is inhibitory in the range of 1 and 2 mmoles/liter, respetively. However, in both ases, the sodium inhibition is redued in the presene of DPH 2 Ag mlv'. On the other hand, the sodium ativation is not signifiantly altered by the DPH. Fig. 2 shows the effet of DPH on potassium ion ativation in 1 mm NaCl. DPH appears to potentiate ativation by the low K+ onentrations where the Na:K ratio is high relative to the ratio whih produes optimal ativation. This effet is negligible when 125 _ zo I_ ' (b) 4 - o.5.8 FIGUR 2 ffet of DPH on rat brain (Na++ K+)-ATPase under varying K+ onentration in 1 mm NaCl. Other onditions as desribed in Methods. -, ontrol; -, DPH 2 Ag*mlt " I1 go A """ _. F Sodium-Potassium-Ativated ATPase of Brain Mirosomes 1165

3 TABL I I ffets of Hydantoins on Rat Brain (Na+ + K+)-A TPase at High Na+:K+ Ratio* Addition Mg++ Mg+++Na+ +K+ A (Na+ +K+) % Control.1 mmoie/liter pmoles mg11min1 (SMt) Control.96 (.3).23 (.3).134 DPH.92 (.2).357 (.12) HPPH.94 (.3).366 (.7) Phenobarbital.95 (.1).239 (.14) Diazepam.95 (.2).23 (.11) mm FIGuR 3 Ativation of rat brain ATPase by ATP plus MgCl2. Tris-ATP and MgCls were varied in equimolar proportions. Other onditions as desribed in Methods. A -- A, no univalent ations added; solid line, 8 mm NaCl plus 5 mm KC1 added;, ontrol; *, DPH 22 /Ag' m[1. maximum rates are obtained at the optimal Na: K ratio. The absene of any signifiant DPH effet in the presene of 8 mm Nae plus 5 mm K+ is onfirmed in Fig. 3. Thus, the prinipal effet of DPH is not on the maximum veloity but rather on the inhibition seen at high Na: K ratios. This may be manifested as an inrease in the observed veloity whih is a resultant of both the stimulatory and inhibitory ationi effets. The DPH effet is most reproduible under onditions of 15 mm Nae and 1 mm K+, taking into aount the amount of enzyme ativity required for reliable measurements under these onditions. HPPH has the same effet as DPH in reduing the sodium ion inhibition at high Na: K ratios (Fig. 4). No effet is seen on the ativation phase of the Na+ urve. Table II ompares the effets of DPH and HPPH with phenobarbital and diazepam in the same experiment under idential onditions and shows that both HPPH and DPH produe an apparent stimulation '.3 'l,. 2 ;n,u.1 L 5 16 mm NoCI FIGUR 4 ffet of HPPH on rat brain (Na+ + K+)-ATPase under varying Na+ onentration in.5 mm KCL. Assay media ontained 1:175 dilution of Parke, Davis Solvent. Other onditions as desribed in Methods. -, ontrol; * ---, HPPH 25, g-ml-. Other onditions as desribed in Methods. DPH and diazepam were dissolved in Parke, Davis Solvent (see Methods). The final dilution of this solvent in the assay media was 175-fold. The same amount was added to ontrol tubes and those with HPPH. It was asertained that this amount of solvent did not alter the enzyme ativity or DPH effet. The sodium salt of phenobarbital was used as desribed in Methods. * 15 mm NaCi plus 1 mm KC1. N = 3. under these onditions of 13% and 98%, respetively. Phenobarbital and diazepam have no signifiant effet under these onditions. Mirosome preparations from at brain yield results qualitatively similar to those obtained with rat brain. In the presene of 15 mm Nae and 1 mm K+, DPH stimulates the (Na + K')-ATPase by 44% and does not affet the Mg'-ATPase ativity. The inhibition produed by pretreatment with ouabain is not altered by DPH under these onditions (Table III). Ouabain inhibition was also unaffeted when mirosomes were treated first with DPH in an experiment otherwise similar to that of Table III. Fig. 5 shows that in.5 mm K+, NaCl ativation is not altered by DPH, whereas the inhibition in high Nat is redued. Fig. 6 shows TABL III ffets of DPH plus Ouabain on Cat Brain (Na+ + K+)-A TPase Ativity Additions AMge+ &(Na+ + K+)* % of ontrol Ouabain (.5 mm) DPH (2ug-mlh1) Ouabain plus DPH 5.9 Mirosomes were pretreated in.5 mm ouabain, 25 mm Tris-HCl (ph 7.4), and 1.5 mm MgCl2 at C for 3 min. The ouabain-treated mirosomes were then preinubated an additional 15 min at C with and without DPH and finally assayed for ATPase ativity for 3 min at 37C as desribed in Methods. Ouabain onentrations in the pretreated samples were onstant throughout the assay. Controls were preinubated for idential periods. Control values (nmolesmg-l-min-l): AMg2+ = 1.2, A(Na+ +K+) = 2.3. * 15 mm NaCl plus 1 mm KCl G. J. Siegel and B. B. Goodwin

4 1 7o5 C mm NaCI FIGUR 5 ffet of DPH on at brain (Nae + K+)-ATPase under varying Na+ onentration in.5 mm KC1. Other onditions as desribed in Methods. - -, ontrol; *-*, DPH 2 jg 'ml-. that in 15 mm NaCl, the K+ ativation is further inreased by DPH where the Na: K ratio is large, as in the ase of rat brain. No signifiant DPH effet was found at optimal ation ratios. In a separate experiment performed with 15 mm NaCl as in Fig. 6, but with higher KCl onentrations, the maximal observed rates were obtained with 12.5 mm KCl: 31.2 and 34.9 nmoles-mg'-mini' for ontrol and DPH treated samples, respetively. These rates did not vary signifiantly at KCl levels from 12.5 to 1 mmoles/liter. Their averages over this range were, in nmoles mg' min': 3.3 (SM.37) for ontrol and 31.5 (SM 1.25) for DPH samples (n = 4). The effets of high KC1 onentrations in low [NaCl] are shown in Fig. 7. In 1 mmoles/liter of NaCl inhibition by K+ ours in onentrations of 1 mmoles/liter or higher and this ation of K+ is not appreiably altered by DPH. Hene, the inhibition due to high K: Na ratios is not affeted by DPH, in ontrast to inhibition 5 - ar- I.,~~~~~~~ / o 1 1 ~~~~~~~~~~ 7 3, 2 1 n vn FIGuR 7 Inhibition of at brain (Na + K+)-ATPase by exess K+ in 1 mm NaCl. Other onditions as desribed in Methods., ontrol; *, DPH 2 /Ag ml1. by high Na:K ratios. This observation is shown also when Nae is varied; i.e., no effet of DPH is seen on the ativation by low Na+ onentrations where the K: Na ratio is high (Figs. 1, 4, and 5). When the sodium and potassium ion onentrations are varied simultaneously to maintain a onstant ratio of Na: K = 15, DPH produes an average apparent stimulation of about 45% in the (Nat + Ke) inrements throughout the total ation onentration range (Fig. 8). Thus, the DPH effet is independent of total ioni strength and of the onentration of either ation alone. DISCUSSION There is evidene that the (Na' + K+) -ativated ATP hydrolysis proeeds through a multi-stage reation mehanism in whih Nae ativates enzyme phosphorylation and K+ ativates enzyme dephosphorylation (3, 11). It is well known that exesses of either Nae or K+ exert inhibition of a mutually ompetitive nature (12, 13), but there is disagreement on the basis of 4 I I I I I ' 4 3 CP C~ ~~~m C - 2 C. I 1 1-I' I I ill FIGUR 6 ffet of DPH on at brain (Nae + K+)-ATPase under varying K+ onentration in 15 mm~ NaCl. Other onditions as desribed in Methods. -, ontrol; *-@, DPH 21A -gml' loo 5 34 mm NaCI hia I oq FIGuR 8 ffet of DPH on at brain (Na + K+)-ATPase. NaCl and KCl were varied simultaneously in the pro-.portion of 15: 1. Other onditions as desribed in Methods. -, ontrol; -, DPH 2 /Ag ml1. Sodium-Potassium-Ativated ATPase of Brain Mirosomes 1167

5 kineti data as to whether the respetive binding sites are neessarily separate or idential. In the present study it is observed that DPH dereases the inhibition due to high Na: K ratios but has no effet on inhibition due to relative exess of K+. The redution of Na' inhibition may be explained by assuming that DPH antagonizes the Na' effet relative to K+ at a site whih when oupied by Na' is inhibitory. This site might be either idential with or interating with a K+ site, for example, the site involved in dephosphorylation. Conversely, if DPH were also to antagonize Na' ativation of enzyme phosphorylation, then one would expet to observe an apparent inhibition by DPH at low Na: K ratios. This was not observed in the present study. In addition, it has been reported that DPH has no effet on myoardial (Na' + K+)- ATPase ativity in the presene of 1 mm Na+ plus 1 mm K+ (14). The fat that suh inhibition by DPH was, however, reported at low Na: K ratios in other types of brain enzyme preparations (4-) might indiate that under ertain onditions more of the ation binding sites beome suseptible to the DPH ation. Further information on the mehanism of the DPH effet may be obtained from diret studies of the phosphorylation and ATP-ADP transphosphorylation reations (11). An important question is whether the observed DPH effet on (Na+ + K+)-ATPase an be related to the drug's anti-seizure ativity. Woodbury observed a diret orrelation between the ratio of extraellular to intraellular brain sodium ion onentration and the eletroshok seizure threshold in rats. Based on the observations that DPH inreased the rate of sodium flux in normal rat brains and dereased the intraellular brain sodium onentrations in normal rats and in rats subjeted to eletroshok seizures, he proposed that the anti-seizure ativity results from stimulation of ative sodium extrusion (1). Festoff and Appel (5) found that DPH appears to stimulate (Nat + K+)-ATPase ativity in synaptosomes from rat erebrum when the Na: K ratio is in the range 5: 1. The experiments reported here with NaI-extrated brain mirosomes onfirm the apparent stimulatory effet of DPH at high Na: K ratios and show that this effet results from a derease in Na inhibition. No qualitative differene between rat and at brain preparations regarding this effet was noted under the onditions tested. Although the optimal ondition for the effet in this study is a Na: K ratio of 15: 1, this value is quite broad and differenes in this ratio may be due to differenes in membrane preparations used, extration proedures, or relative extents of Na+ inhibition. Of greater signifiane is the fat that the DPH effet depends on 1168 G. J. Siegel and B. B. Goodwin speifi ioni proportions in the range whih an regulate the enzyme ativity. Regulation of (Na + K+) -ATPase is ompliated; it depends on a balane among both ativation and inhibition effets by both sodium and potassium. It has been found in studies of red ells that ativation of the sodium pump and (Nae + K+)-ATPase is due to intraellular Na+ and extraellular K+ while, on the other hand, extraellular Na+ is inhibitory (15, 16). It is plausible that in vivo DPH might produe an inreased rate of transport under a given set of onditions through a redution in the relative inhibition due to extraellular Na+ similar to its effet on (Na' + K+) - ATPase in vitro. However, as desribed above, other studies have indiated an inhibitory effet of DPH at low Na: K ratios whih suggests that DPH under some onditions may also derease Na+ ativation. Therefore, the net result of the DPH ation will depend on its relative effets on the two Na+ ations. It is diffiult to draw omparisons on a moleular basis between ations on (Na+ + K+) -ATPase in vitro and anti-seizure ativity beause other fators suh as absorption, metabolism, exretion, and permeability might limit a drug's poteny in vivo. HPPH in very high doses was reported ineffetive against eletroshok-indued seizures in rats (17) yet it exhibits the same poteny as DPH in its effets on (Na + K+)- ATPase. HPPH is the main exretory produt of DPH (18) and is found onjugated with gluuroni aid in the urine (19). The liver is an important site for the formation of HPPH (8). If the ation on (Na+ + K+)- ATPase is ritial for the anti-seizure ativity of the hydantoins, then it might be supposed that the nervous system ation of HPPH is limited by a more rapid rate of exretion or dereased permeation into brain in the onjugated form rather than by hydroxylation per se. Alternatively, it remains possible that the hydantoin ation as measured on (Na+ + K+)-ATPase in vitro is itself not suffiient to aount for the antiseizure ativity of the hydantoins. Studies of HPPH metabolism and onentration within the brain may help answer these questions. The observation that DPH stimulates the (Na+ + K+)-ATPase, although onsistent with Woodbury's hypothesis, is not onfirmatory, as disussed by several authors (5, 2). In addition, reent studies with lobster nerves have shown that DPH is able to lower the intraneuronal Na+ onentration without affeting K+ in the presene of ouabain or yanide inhibition (21). Thus, DPH has multiple ations that may modify Na+ transloation and it is not known whih of these, if not a ombination, is important physiologially in the ontrol of seizures. Further physiologi studies omparing HPPH and DPH effets on seizures and on

6 membrane properties in vitro will be pertinent to this issue. ACKNOWLDGMNTS This work was supported partially by Grant MH from the National Institute of Mental Health and Grant GB 8754 from The National Siene Foundation. RFRNCS 1. Woodbury, D. M ffet of diphenylhydantoin on eletrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremi and postital rats. J. Pharmaol. xp. Therap. 115: Skou, J. C Further investigations on a Mg2 + Na+-ativated adenosinetriphosphatase, possibly related to the ative, linked transport of Na+ and K+ aross the nerve membrane. Biohim. Biophys. Ata. 42: Albers, R. W Biohemial aspets of ative transport. Ann. Rev. Biohem. 36: Rawson, M. D., and J. H. Pinus The effet of diphenylhydantoin on sodium, potassium, magnesiumativated adenosine triphosphatase in mirosomal frations of rat and guinea pig brain and on whole homogenates of human brain. Biohem. Pharmaol. 17: Festoff, B. W., and S. H. Appel ffet of diphenylhydantoin on synaptosome sodium-potassium- ATPase. J. Clin. Invest. 47: Formby, B The in tivo and in vitro effet of diphenylhydantoin and phenobarbitone on K+-ativated phospholydrolase and (Na+, K+)-ativated ATPase in partiulate membrane frations from rat brain. J. Pharm. Pharmaol. 22: sueta, A. V., and. L. Reilly The effets of diphenylhydantoin on potassium transport within synapti terminals of the epileptogeni foi. Neurology. 21: Kutt, H., and K. Verebely Metabolism of diphenylhydantoin by rat liver mirosomes. I. Charateristis of the reation. Biohem. Pharmaol. 19: Albers, R. W., G. J. Koval, and G. J. Siegel Studies on the interation of ouabain and other ardioative steroids with sodium-potassium-ativated adenosine triphosphatase. Mol. Pharmaol. 4: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Siegel, G. J., and R. W. Albers Sodium-potassium-ativated adenosine triphosphatase of letrophorus eletri organ. IV. Modifiation of responses to sodium and potassium by arsenite plus 2,3-dimeraptopropanol. J. Biol. Chem. 242: Ahmed, K., J. D. Judah, and P. G. Sholefield Interation of Na+ and K+ with a ation-dependent ATPase system from rat brain. Biohim. Biophys. Ata. 12: Robinson, J. D Interations between monovalent ations and the (Na+ + K+) -dependent adenosine triphosphatase. Arh. Biohem. Biophys. 139: Gibson, K., and P. Harris Dephenylhydantoin and human myoardial mirosomal (Nat,K+)-ATPase. Biohem. Biophys. Res. Commun. 35: Garrahan, P. J., and I. M. Glynn The sensitivity of the sodium pump to external sodium. J. Physiol. 192: Priestland, R. N., and R. Whittam The influene of external sodium ions on the sodium pump in erythroytes. Biohem. J. 19: Nitz, R.., W. Persh, and A. Shmidt Zur hemie und antikonvulsiven Wirkung neuer Hydantoinderivate. Arzneimittel-Forshung. 5: Butler, T. C The metaboli onversion of 5,5- diphenylhydantoin to 5-(p-hydroxyphenyl)-5-phenylhydantoin. J. Pharmaol. xp. Therap. 119: Maynert,. W The metaboli fate of diphenylhydantoin in the dog, rat, and man. J. Pharmaol. xp. Therap. 13: Crane, P., and P. D. Swanson Diphenylhydantoin and the ations and phosphates of eletrially stimulated brain slies. Neurology. 2: Pinus, J. H., I. Grove, B. B. Marino, and G.. Glaser Studies on the mehanism of ation of diphenylhydantoin. Arh. Neurol. 22: 566. Sodium-Potassium-Ativated ATPase of Brain Mirosomes 1169