- Email: [email protected]
Mastoparan inhibits rat renal NAK-ATPase activity
Life Sciences, Vol. 47, pp. 2451-2458 Printed in the U.S.A.
Pergamon Press
MASTOPARAN INHIBITS RAT RENAL NAK-ATPase ACTIVITY Samuel P. Eng, David L Clough, and Chu S Lo Department of Physiology Uniformed Services Universtty of the Health Sciences Bethesda, Maryland 20814-4799 USA (Received in final form October 24, 1990)
Summarv Rat renal NaK-ATPase was inhibited by mastoparan =n a dose dependent fashion. This inhibition reached completion w0than30 seconds. Due to mastoparan's rap=d effects on NaK-ATPase activtty, this inhibition does not appear to involve either a decrease in the rate of synthesis or an increase m their degradation of NaK-ATPase since these processes requ0re a latency period of at least several minutes. In addit0on, the phosphoenzyme intermedtate formed in the presence of mastoparan was greater than that formed m tts absence further mdtcatmg that inhibition of NaKATPase by mastoparan ts not due to a decrease in the number of NaK-ATPase. A posmble mechantsm for the inhibitton ts that mastoparan stabdtzes the phosphoenzyme intermediate and reduces the Vmax of the enzyme by decreasing the rate of turnover of existing enzyme sttes. Neomycin, an inhtbttor of inositol phosphohpid metabohsm, was also found to attenuate the inhibd0on of Na,K-ATPase by mastoparan, suggastmg that the mechamsm of this inhibrtion may revolve degradation of the phosphatidylinositol "pool" Wasp venom contams active amines (1-4), enzymes such as hyaluronidase, phospholipase, and polypephdes such as mastoparan (1-6). Mastoparan is a tetra-decapeptide found in venom from the wasp Vesoa lewi~ii (7,8) This polypeptide has been isolated, characterized, and chemically synthesized (9) and has been demonstrated to be a potent stimulator of phosphohpase A2 activity (10) Some studies indicate that mastoparan also activates phosphatidyhnosttol-specific phospholipase C resulting in the degradation of phosphatidylinositol bisphosphate and the formation of diacylglycerol and mosltol trisphosphate (11) There ts ev=dence that phosphatidyhnositol is requ0red for the activation of the NaK-ATPase =n microsomal membranes prepared from rat kidney (12,13) In vtew of these results, the first objective was to determine the effects of mastoparan on NaK-ATPase activity. We studied the effects of mastoparan on the Vmax of rat kidney Na,K-ATPase and on the Ko (apparent dissociation constant) for Na ÷, K+ and ATP. Mastoparan could inhibit NaK-ATPase by reducing the number of enzyme sites, or by decreasing the rate of turnover of each site. Either of these mechanisms would result in a decrease in the Vmax of the enzyme. The second objective was to determine the effect of mastoparan on the generation of phosphorylated intermediate.
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
2452
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
Alternat=vely, if the mhtbtt0on of NaK-ATPase were medmted through the degradatton of the phosphatidylinositol "pool", then the treatment of microsomal membranes with neomycin, an ammoglycostde ant=biottc that ~s a relat=vely specific inhibitor of inos0tol phospholipid metabohsm (14-19), should attenuate the mhibttton of NaK-ATPase by mastoparan The thtrd objecttve was to determine the effects of neomycin on inh~btt=on of NaK-ATPase by mastoparan Methods Determ=natton of NaK-ATPase. Male, Sprague-Dawley rats (120-180 g body weight) were maintained on rat chow (Agway 1257) ad libitum. Rats were decapitated, and plasma membrane ennched fract=ons were prepared from the kidney cortex and medulla (20). NaK-ATPase actMty (V max) was measured as described previously (21). Approximately 15 I~g of membrane protein was incubated =n 1 ml of a medium which contained I mM EDTA, 20 mM KCI, 3 mM MgCI 2, 100 mM NaCI, 3 0 mM Tns-ATP, and 100 mM Tns-HCI (pH 7 4 at 37°C), or in a med=um of the same composttion but tn the presence of 1 mM ouabain. The m~xture was pre-mcubated for 10 minutes at 37°C m the absence of ATP and the reactaon was started by the addition of Trts-ATP The reactson was terminated after 15 mmutes by the add=tton of 0 2 ml of 30% ice-cold tr0choloacet=c actd Inorgantc phosphate (Pi) was determined by the Ftske and SubbaRow method (22). NaKATPase activity was expressed as the difference between Pi released m the presence and absence of 1 mM ouabam Spec=fic activity was expressed as i~moles ATP hydrolyzed per mg protein per hour Determination of K,. for ATP. Na ÷. and K*: The effect of mastoparan on the Ko's (apparent dissoctatton constant) of the rat kidney NaK-ATPase for ATP, Na*, and K ÷ were determined =n an ATP regenerating system consisting of p-enolpyruvate and pyruvate kinase (23). The pyruvate klnase was dialyzed against 1 litre of distilled water for 12 hr at 4°C pnor to use. To determine the Ko's for Na,K, and ATP the concentratton of each of the three components was varied =ndMdually =nseparate experiments and the concentratton of the remaining two components was held constant at the concentrations given below. Determin~,tion of PhosDhorylated Intermedmte: The phosphorylated intermediate was generated in 400 I~1 of a reaction mixture that contained 100 mM NaCI, 100 mM Tns-HCI, 3 mM MgCI 2, 1 mM EDTA, 1.0 pM [¥-32p]ATP (1.87 mCi/ml), pH 7 4, and 25-40 I~g of the membrane fraction in Eppendorf tubes(20). Tandem tubes containing 20 mM KCI instead of 100 mM NaCI were used for the measurement of non-specJf=c phosphorylation. The reaction was mtt0ated by the add=tton of [y-32P]ATP and was terminated after 15 seconds by the addttion of 0.1 ml 30% trichloroacet0c acid. The mixture was centrifuged in an Eppendorf centrifuge (model 5412) at h=gh speed for 1 minute. The pellet was washed twice with 3 mM ATP in 100 mM Tns-HCI, (pH 6.8). After the final wash, the pellet was dissolved in 200 id of 1N NaOH for 1 hr. The samples were removed and titrated to neutrahty with 1 N HCI and placed =n scintdlatlon vtals contammg 10 ml Aquasol (New England Nuclear) for quantitatlon in a Mark-Ill spectrophotometer (Searle). Protein analvsis: Aliquots of membrane preparat=ons were dagested =n 1N NaOH. concentrations were determined by the method of Lowry et aL (24).
Protein
Statistical analvsis: Data were analyzed by Student's t-test or Duncan's Multiple Range test and analysis of variance when appropriate (25). Values were considered signdicantly ddferent at p < 05. Results EffQcts of mastooaran on Na.K-ATPase acttvdv. Mastoparan (75 i~g/ml) sign=hcantly decreased NaK-ATPase activity compared to the control (without mastoparan) at all tome points (Table I). At th0s dosage a maxtmal inhibition of 20% was achieved in 30 seconds. It also produced a dose dependent decrease in rat kidney NaK-ATPase act=vlty (Table 2) There was no effect of mastoparan on Mg-ATPase actlvd.y (Data not shown).
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2453
TABLE I Inhibition of Rat Renal NaK-ATPase by Mastoparan
Control NaK-ATPase Activity i~mol Pi mg Protein.HR
Time (minutes) 03
539-+ 2.9
P value
427-+ 1.4
<0.5
1.0
5.0
42.8 + 0.6
15 0
444 +- 0 7
41.0-+ 12
<0 5
<0.2
<0.5
Membrane preparations were incubated with mastoparan (75 Fg/ml) for d=fferentamounts of t,me. Control represents untreated membrane preparation. NaK-ATPase activity was measured as described in "Methods'. Values are mean _+ S E.M Number of experiments = 9.
TABLE II Dose Response of Mastoparan on Rat Renal NaK-ATPase Mastoparan (l~g/ml) 0 12.5 25 37.5 50
NaK-ATPase Activity pmoI pi 54_+ 1 mg Protem.HR
P values
48 + 1
< .05
46_ 3
< .05
44_+ 1
< .03
45_+ 2
< .05
75
44_+ 2
< .03
100
41 _+ 1
< .02
Membrane preparations were preincubated at 37°C for 1 minute with varying concentration of mastoparan. Na,K-ATPase was determined as described in "Methods'. Values presented are mean -+ S.E.M. No. of experiments = 10
Effect of mastooaran on the kinetics of activation of NaK-ATPase bv Na +. K +. and ATP: The pattern of activation by sodium of rat kidney Na,K-ATPase treated with mastoparan (75 pg/ml) appears to be essenhally the same as that of the controls (Figure 1) The KD's for Na + obtained from Hill plots (Figure 1, inset) were 15 -+ 0.89 mM for the controls and 16.74 _+ 0 96 mM for the mastoparan treated membranes (n--6, p--.577). The Hill coefficients which are influenced by a combination of the number of binding sites and their degree of interaction were 1.60 _+ 0 09 and 1.37 +_ 0.14 for the controls and mastoparan treated membranes, respectively (n=6, p=.322) (Figure 1, inset). The pattern of activation by K ÷ of rat kidney NaK-ATPase treated with mastoparan and that of the controls is shown in Figure 2. A significant decrease from the control of Ko of 1.03 -+ 0.07 mM to a Ko value of 0.51 _+0.04 mM for the mastoparan treated membranes was observed (n-- 6, p
2454
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
60
50
i40 z o 20
lO
iI
I I I I I I I I J
I
I
i
I
I
20
40
60
80
100
mM[Na+] Figure 1. Activation by Na ÷ of rat renal Na,K-ATPase. Membrane preparations were treated with diluent alone (El, O) or 75 i~g/ml mastoparan (i,O) for 1 minute and activated by varying concentrations of Na* Each point represents the mean _+ S.E.M. (n =6). Inset. Hill Plot of data in Figure 1. Ko (apparent) dissociation constant for Na*) was determined as the antilog of the X intercept. The Hill coefficient was obtained from the slope. Lines were drawn by linear regression.
The pattern of activation by ATP of rat kidney NaK-ATPase treated with mastoparan and that of the controls is shown in Figure 3. The Ko'S for ATP were 0.41 -+ 0.01 mM for the controls and 0.35 -+ 0.01 mM for the mastoparan treated membranes (n--6, P=.059) (Figure 3, inset). The Hill coefficients were 2.20 _+ 0.29 and 2.69 -+ 0.69 for the control and mastoparan treated membranes, respectively (n=6, P=.551) (Figure 3, inset). EffQ~ of mastooaran on the formation of Dhosohorylated intermediate. The amount of phosphorylated intermediate were 88 _+5 and i07 _+4 (pmol/mg protein/15 sec.) in the dduenttreated and mastoparan-treated membrane, respectively (p <.001). E f f ~ of noomvcm on the inhibit=on of NaK-ATPase bv mastooaran: Pre-treatment of the membranes with 2 mM neomycin had no significant effect on NaK-A1 Pase activrty (data not shown). When the membranes were pre-treated with varying concentrations of neomycin and incubated in the presence of mastoparan (75 i~g/ml), NaK-ATPase activity was greater (p < 05) compared to that of membranes treated with mastoparan (75 i~g/ml) alone (Table 3).
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2455
80
70
60
_T_
•~ "~ 50
o 1$ 10 --
z o
0.5
30
20
I~ 10
-1.1 I I I I I -o" -u -o4 .o= o=
I
I
I
04
06
08
t~ll I K÷I
0
10 m M [K+]
20
Figure 2. Activation by K + of rat renal Na, K-ATPase Membrane preparations were treated with diluent alone (n, O) or 75 pg/ml mastoparan (11, O) and activated by varying concentrat=ons of K+. Each point represents the mean -+ S.E.M. (n=8). Inset: Hill Plot of data in Figure 2. Ko (apparent dissociation constant for K*) was determined as the antilog of the x intercept. The Hill coefficient was obtained from the slope. Lines were drawn by linear regression.
D~cusslon In the present study, mastoparan was found to produce a concentration dependent inhib=tJon of Na,K-ATPase activity of membrane fractions prepared from rat kidney, and at 100 i~g/ml, the highest concentration of mastoparan tested, inhibition of the enzyme's Vmax was approx=mately 24%. The mhibrtion does not appear to be due to a decrease in the number of NaK-ATPase s=tes, because the amount of phosphoenzyme intermediate formed in the presence of mastoparan was greater than, rather than less than, that formed in its absence. This is supported by the observation that the inhibition occurred rapidly, reaching completion within 30 seconds or less. It therefore does not appear to be due to a decrease in the rate of synthesis of NaK-ATPase sites or to an increase in the rate of their degradation since these processes would require a latency of several minutes to hours for their effect to become evident. A possible mechanism for the inhibition of NaKoATPase in the presence of less than saturating concentrations of Na+, K+, or
2456
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
50
40
.> ._c ~ 30
<2 o.E ~
20
10
06
-05 -04
-03
-02
-01
0
01
Log [ATP]
0
I 0.2
I I 0.4 0.6 mM ATP
I
I
0.8
1.0
Rgure 3. Activation by ATP of rat renal NaK-ATPase Membrane preparations were treated with diluent alone (El,O) or 75 i~g/ml mastoparan (11,0) and activated by varying concentrations of ATP. Each point represents the mean _+ S.E.M (n=9). Inset: Hill Plot of data in Figure 3. Ko (apparent dissociation constant for ATP) was determined as the antilog of the x intercept from the slope The Hill coefficient was obtained from the slope Lines were drawn by linear regression.
ATP, would be a decrease =nthe enzyme's affinity for one or more of these substrates. However, this does not appear to have occurred in the present experaments, because mastoparan decreased the Ko for K*, and it had no effect on the KD'S for Na ÷ and ATP. Mastoparan also had no effect on the Hill coefficients for the three substrates. Because of the apparent increase in the affinity of the enzyme for K ÷ observed in the presence of mastoparan, inhibition of NaK-ATPase activity by mastoparan occurred almost exclusively at the h~gher range of K + concentrations. The effect of the inhibitor on the Ko therefore appeared to be as important kinetically, as its affect on the Vmax in the lower range of K + concentrations. The mechanism by which mastoparan increased the amount of phosphoenzyme intermediate is obscure. A highly speculative explanation for this effect is that even in the presence of 100 mM Na + and absence of added K ÷, there was a slow but fin=te turn-over of the phosphoenzyme
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2457
Table III Effect of Neomycin on the Activity of Mastoparan Treated Rat Renal NaK-ATPase
Neomycin (mM) Control
0 mM
0.25 mM
0.50 mM
1.0 mM
2 mM
52_+1 6
46-+ 1.4
47 4-+ 1.7
47.6-+1.3
48.5-+1.3
50.5-+1.5
n=10
n=8
n=6
n--6
n=6
n=6
p < .05
p < .05
p < .05
p < .05
NS
Control represents untreated membrane preparation Other membrane preparations were preincubated (15 minutes) with varying concentrations of neomycin then treated w=th mastoparan (75 pg/ml) for 1 minute. NaK-ATpase activity was determined as described in "Methods'. Act=vdy is expressed as pmoles Pi per mg protein per hour. Values represent the mean _+ S.E.M P values given are compared to control.
intermediate, and that mastoparan increased the steady state amount of phosphoenzyme intermediate by slowing its breakdown (the rate limiting step). Stabilization ofthe phosphoenzyme intermediate could help explain the decrease in Vmax produced by mastoparan since thin would decrease the rate of NaK-ATPaseturnover. Neomycin, wh0ch is an inhibitor of inositol phosphohp=d metabolism (14-19), was found to attenuate the inhibition of NaK-ATPase activity by mastoparan. We did not study the effect of neomycin on the mastoparan induced increase in the NaK-ATPase phosphorylated intermediate. However, we observed that neomycin attenuates mastoparan's inhibit=on of Na*-dependent net AIB uptake and completely suppressed mastoparan increases in AIB efflux (26). Since renal NaK.ATPase activity appears to require the presence of phosphatidylinositol (12, 13), the mechanism of the inhibition of NaK-ATPase by mastoparan may involve degradation of the phosphatidylinositol "pool" (27) Acknowledoment This work was supported by USUHS Grant CO7623 and American Heart Association, Maryland Affiliated, Inc. Reference 1. 2 3. 4.
5. 6.
7. 8 9. 10.
M. SCHATHER and E. THAIN, Brit. J. Pharmacology, 9:352 (1954). R.G. GELLER, H. YOSHIDA, M.A. BEAVAN, A. HORAKOVA, F.L. ATKINS, H. YAMABE, and J.J PISANO, Toxicon 14:27-33 (1976). H. YOSHIDA, R.G GELLER, and J.J. PISANO, Biochemistry, 15:61-64 (1976). E. HABERMANN, Science 117:314-322 (1972). R.A.SHIPOLINI, European J. Biochemistry 20: 459- (1971). E. HABERMANN, Biochemistry 329: 1- (1957). Y. HIRAI, Y. UENO, T. YASUHARA, H. YOSHIDA, and T. NAKAJIMA, Chem Pharm. Bull 27:1945-1946 (1979). Y. HIRAI, Y. UENO, T. YASUHARA, H. YOSHIDA, and T. NAKAJIMA, Biomedical Res. 1: 185-197 (1980). Y. HIRAI, T. YASUHARA, H. YOSHIDA, T. NAKAJIMA, M. FUJINO, and C. KITADA, Chem. Pharm. Bull. 27:1942-1944 (1979). A. ARGIOLOS, and J. PISANO, J. BIOL. CHEM. 258:13697-13702 (1983).
2458
11. 12. 13. 14. 15. 16. 17 18. 19 20. 21. 22. 23. 24. 25. 26. 27.
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
Y. OKANO, H. TAKAGI, T. TOHMATSU, S. NAKASHIMA, Y. KURODA, IL SAITO, and Y. NOZAWA, FEBS Letters 188 (2): 363-366, (1985). J. MANDERSLOOT, B. ROELOFSEN, and J. DE GIER, Biochem. Biophys. Acta. 508: 478485 (1978). B. ROELOFSEN and M. VAN LINDE-SIBENIUS, Biochem. Biophys. Acta., 647: 302-306, (1981). V. LANG, G. PRYHITKA, and J. BUCKLEY, Can. J. Biochem. 55:1007-1012 (1977). S. LODHI, N. WEINER, and J. SCHACHT, Biochem. Biophys. Acta. 557:1-8 (1979). J.J. LIPSKY, J. Pharmacol. Exp. Ther. 220: 287-292, (1982). P. MARCHE, S. KOUTOUZOV, and A. GIRARD, J. Pharmacol. Exp. Ther. 227:415-420 (1983). J. SCHACHT, J. Lipid Res. 19:1063-1067 (1978). D. SCHWERTZ, J. KREISBERG, and M. VENKATACHALAM, J. Pharmacol. Exp. Ther. 251. 48-55 (1984). C S. Lo and T.N. Lo, Endocnnology 109.1473.1478 (1981) C.S. LO, T. AUGUST, U.l. LIBERMAN, and I. EDELMAN, J. Biol. Chem. 251 (24): 7826-7833 (1976). C. FISKE and Y. SUBBAROW, J. Biol. Chem. 66: 375-400, (1925). A. SCHWARTZ, J. ALLEN, and S. HARIGAYA, J. Pharmacol. EXp. Ther. 168:31-41 (1989). O.H. LOWRY, N.J. ROSEBOROUGH, A.L. FARR, and R.J. RANDALL, J Biol. Chem. 195: 265-275 (1951). G.W. SNEDECOR and W.G. COCHRAN in Statistical Analvsis. 7th edition, Iowa State Press (1980). S.P. ENG, D.L. CLOUGH, and C.S. LO Life Sci. 46:865-873 (1990). S.P. ENG and C.S. Lo, Life Sci 46:273-279 (1990).
Pergamon Press
MASTOPARAN INHIBITS RAT RENAL NAK-ATPase ACTIVITY Samuel P. Eng, David L Clough, and Chu S Lo Department of Physiology Uniformed Services Universtty of the Health Sciences Bethesda, Maryland 20814-4799 USA (Received in final form October 24, 1990)
Summarv Rat renal NaK-ATPase was inhibited by mastoparan =n a dose dependent fashion. This inhibition reached completion w0than30 seconds. Due to mastoparan's rap=d effects on NaK-ATPase activtty, this inhibition does not appear to involve either a decrease in the rate of synthesis or an increase m their degradation of NaK-ATPase since these processes requ0re a latency period of at least several minutes. In addit0on, the phosphoenzyme intermedtate formed in the presence of mastoparan was greater than that formed m tts absence further mdtcatmg that inhibition of NaKATPase by mastoparan ts not due to a decrease in the number of NaK-ATPase. A posmble mechantsm for the inhibitton ts that mastoparan stabdtzes the phosphoenzyme intermediate and reduces the Vmax of the enzyme by decreasing the rate of turnover of existing enzyme sttes. Neomycin, an inhtbttor of inositol phosphohpid metabohsm, was also found to attenuate the inhibd0on of Na,K-ATPase by mastoparan, suggastmg that the mechamsm of this inhibrtion may revolve degradation of the phosphatidylinositol "pool" Wasp venom contams active amines (1-4), enzymes such as hyaluronidase, phospholipase, and polypephdes such as mastoparan (1-6). Mastoparan is a tetra-decapeptide found in venom from the wasp Vesoa lewi~ii (7,8) This polypeptide has been isolated, characterized, and chemically synthesized (9) and has been demonstrated to be a potent stimulator of phosphohpase A2 activity (10) Some studies indicate that mastoparan also activates phosphatidyhnosttol-specific phospholipase C resulting in the degradation of phosphatidylinositol bisphosphate and the formation of diacylglycerol and mosltol trisphosphate (11) There ts ev=dence that phosphatidyhnositol is requ0red for the activation of the NaK-ATPase =n microsomal membranes prepared from rat kidney (12,13) In vtew of these results, the first objective was to determine the effects of mastoparan on NaK-ATPase activity. We studied the effects of mastoparan on the Vmax of rat kidney Na,K-ATPase and on the Ko (apparent dissociation constant) for Na ÷, K+ and ATP. Mastoparan could inhibit NaK-ATPase by reducing the number of enzyme sites, or by decreasing the rate of turnover of each site. Either of these mechanisms would result in a decrease in the Vmax of the enzyme. The second objective was to determine the effect of mastoparan on the generation of phosphorylated intermediate.
0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
2452
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
Alternat=vely, if the mhtbtt0on of NaK-ATPase were medmted through the degradatton of the phosphatidylinositol "pool", then the treatment of microsomal membranes with neomycin, an ammoglycostde ant=biottc that ~s a relat=vely specific inhibitor of inos0tol phospholipid metabohsm (14-19), should attenuate the mhibttton of NaK-ATPase by mastoparan The thtrd objecttve was to determine the effects of neomycin on inh~btt=on of NaK-ATPase by mastoparan Methods Determ=natton of NaK-ATPase. Male, Sprague-Dawley rats (120-180 g body weight) were maintained on rat chow (Agway 1257) ad libitum. Rats were decapitated, and plasma membrane ennched fract=ons were prepared from the kidney cortex and medulla (20). NaK-ATPase actMty (V max) was measured as described previously (21). Approximately 15 I~g of membrane protein was incubated =n 1 ml of a medium which contained I mM EDTA, 20 mM KCI, 3 mM MgCI 2, 100 mM NaCI, 3 0 mM Tns-ATP, and 100 mM Tns-HCI (pH 7 4 at 37°C), or in a med=um of the same composttion but tn the presence of 1 mM ouabain. The m~xture was pre-mcubated for 10 minutes at 37°C m the absence of ATP and the reactaon was started by the addition of Trts-ATP The reactson was terminated after 15 mmutes by the add=tton of 0 2 ml of 30% ice-cold tr0choloacet=c actd Inorgantc phosphate (Pi) was determined by the Ftske and SubbaRow method (22). NaKATPase activity was expressed as the difference between Pi released m the presence and absence of 1 mM ouabam Spec=fic activity was expressed as i~moles ATP hydrolyzed per mg protein per hour Determination of K,. for ATP. Na ÷. and K*: The effect of mastoparan on the Ko's (apparent dissoctatton constant) of the rat kidney NaK-ATPase for ATP, Na*, and K ÷ were determined =n an ATP regenerating system consisting of p-enolpyruvate and pyruvate kinase (23). The pyruvate klnase was dialyzed against 1 litre of distilled water for 12 hr at 4°C pnor to use. To determine the Ko's for Na,K, and ATP the concentratton of each of the three components was varied =ndMdually =nseparate experiments and the concentratton of the remaining two components was held constant at the concentrations given below. Determin~,tion of PhosDhorylated Intermedmte: The phosphorylated intermediate was generated in 400 I~1 of a reaction mixture that contained 100 mM NaCI, 100 mM Tns-HCI, 3 mM MgCI 2, 1 mM EDTA, 1.0 pM [¥-32p]ATP (1.87 mCi/ml), pH 7 4, and 25-40 I~g of the membrane fraction in Eppendorf tubes(20). Tandem tubes containing 20 mM KCI instead of 100 mM NaCI were used for the measurement of non-specJf=c phosphorylation. The reaction was mtt0ated by the add=tton of [y-32P]ATP and was terminated after 15 seconds by the addttion of 0.1 ml 30% trichloroacet0c acid. The mixture was centrifuged in an Eppendorf centrifuge (model 5412) at h=gh speed for 1 minute. The pellet was washed twice with 3 mM ATP in 100 mM Tns-HCI, (pH 6.8). After the final wash, the pellet was dissolved in 200 id of 1N NaOH for 1 hr. The samples were removed and titrated to neutrahty with 1 N HCI and placed =n scintdlatlon vtals contammg 10 ml Aquasol (New England Nuclear) for quantitatlon in a Mark-Ill spectrophotometer (Searle). Protein analvsis: Aliquots of membrane preparat=ons were dagested =n 1N NaOH. concentrations were determined by the method of Lowry et aL (24).
Protein
Statistical analvsis: Data were analyzed by Student's t-test or Duncan's Multiple Range test and analysis of variance when appropriate (25). Values were considered signdicantly ddferent at p < 05. Results EffQcts of mastooaran on Na.K-ATPase acttvdv. Mastoparan (75 i~g/ml) sign=hcantly decreased NaK-ATPase activity compared to the control (without mastoparan) at all tome points (Table I). At th0s dosage a maxtmal inhibition of 20% was achieved in 30 seconds. It also produced a dose dependent decrease in rat kidney NaK-ATPase act=vlty (Table 2) There was no effect of mastoparan on Mg-ATPase actlvd.y (Data not shown).
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2453
TABLE I Inhibition of Rat Renal NaK-ATPase by Mastoparan
Control NaK-ATPase Activity i~mol Pi mg Protein.HR
Time (minutes) 03
539-+ 2.9
P value
427-+ 1.4
<0.5
1.0
5.0
42.8 + 0.6
15 0
444 +- 0 7
41.0-+ 12
<0 5
<0.2
<0.5
Membrane preparations were incubated with mastoparan (75 Fg/ml) for d=fferentamounts of t,me. Control represents untreated membrane preparation. NaK-ATPase activity was measured as described in "Methods'. Values are mean _+ S E.M Number of experiments = 9.
TABLE II Dose Response of Mastoparan on Rat Renal NaK-ATPase Mastoparan (l~g/ml) 0 12.5 25 37.5 50
NaK-ATPase Activity pmoI pi 54_+ 1 mg Protem.HR
P values
48 + 1
< .05
46_ 3
< .05
44_+ 1
< .03
45_+ 2
< .05
75
44_+ 2
< .03
100
41 _+ 1
< .02
Membrane preparations were preincubated at 37°C for 1 minute with varying concentration of mastoparan. Na,K-ATPase was determined as described in "Methods'. Values presented are mean -+ S.E.M. No. of experiments = 10
Effect of mastooaran on the kinetics of activation of NaK-ATPase bv Na +. K +. and ATP: The pattern of activation by sodium of rat kidney Na,K-ATPase treated with mastoparan (75 pg/ml) appears to be essenhally the same as that of the controls (Figure 1) The KD's for Na + obtained from Hill plots (Figure 1, inset) were 15 -+ 0.89 mM for the controls and 16.74 _+ 0 96 mM for the mastoparan treated membranes (n--6, p--.577). The Hill coefficients which are influenced by a combination of the number of binding sites and their degree of interaction were 1.60 _+ 0 09 and 1.37 +_ 0.14 for the controls and mastoparan treated membranes, respectively (n=6, p=.322) (Figure 1, inset). The pattern of activation by K ÷ of rat kidney NaK-ATPase treated with mastoparan and that of the controls is shown in Figure 2. A significant decrease from the control of Ko of 1.03 -+ 0.07 mM to a Ko value of 0.51 _+0.04 mM for the mastoparan treated membranes was observed (n-- 6, p
2454
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
60
50
i40 z o 20
lO
iI
I I I I I I I I J
I
I
i
I
I
20
40
60
80
100
mM[Na+] Figure 1. Activation by Na ÷ of rat renal Na,K-ATPase. Membrane preparations were treated with diluent alone (El, O) or 75 i~g/ml mastoparan (i,O) for 1 minute and activated by varying concentrations of Na* Each point represents the mean _+ S.E.M. (n =6). Inset. Hill Plot of data in Figure 1. Ko (apparent) dissociation constant for Na*) was determined as the antilog of the X intercept. The Hill coefficient was obtained from the slope. Lines were drawn by linear regression.
The pattern of activation by ATP of rat kidney NaK-ATPase treated with mastoparan and that of the controls is shown in Figure 3. The Ko'S for ATP were 0.41 -+ 0.01 mM for the controls and 0.35 -+ 0.01 mM for the mastoparan treated membranes (n--6, P=.059) (Figure 3, inset). The Hill coefficients were 2.20 _+ 0.29 and 2.69 -+ 0.69 for the control and mastoparan treated membranes, respectively (n=6, P=.551) (Figure 3, inset). EffQ~ of mastooaran on the formation of Dhosohorylated intermediate. The amount of phosphorylated intermediate were 88 _+5 and i07 _+4 (pmol/mg protein/15 sec.) in the dduenttreated and mastoparan-treated membrane, respectively (p <.001). E f f ~ of noomvcm on the inhibit=on of NaK-ATPase bv mastooaran: Pre-treatment of the membranes with 2 mM neomycin had no significant effect on NaK-A1 Pase activrty (data not shown). When the membranes were pre-treated with varying concentrations of neomycin and incubated in the presence of mastoparan (75 i~g/ml), NaK-ATPase activity was greater (p < 05) compared to that of membranes treated with mastoparan (75 i~g/ml) alone (Table 3).
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2455
80
70
60
_T_
•~ "~ 50
o 1$ 10 --
z o
0.5
30
20
I~ 10
-1.1 I I I I I -o" -u -o4 .o= o=
I
I
I
04
06
08
t~ll I K÷I
0
10 m M [K+]
20
Figure 2. Activation by K + of rat renal Na, K-ATPase Membrane preparations were treated with diluent alone (n, O) or 75 pg/ml mastoparan (11, O) and activated by varying concentrat=ons of K+. Each point represents the mean -+ S.E.M. (n=8). Inset: Hill Plot of data in Figure 2. Ko (apparent dissociation constant for K*) was determined as the antilog of the x intercept. The Hill coefficient was obtained from the slope. Lines were drawn by linear regression.
D~cusslon In the present study, mastoparan was found to produce a concentration dependent inhib=tJon of Na,K-ATPase activity of membrane fractions prepared from rat kidney, and at 100 i~g/ml, the highest concentration of mastoparan tested, inhibition of the enzyme's Vmax was approx=mately 24%. The mhibrtion does not appear to be due to a decrease in the number of NaK-ATPase s=tes, because the amount of phosphoenzyme intermediate formed in the presence of mastoparan was greater than, rather than less than, that formed in its absence. This is supported by the observation that the inhibition occurred rapidly, reaching completion within 30 seconds or less. It therefore does not appear to be due to a decrease in the rate of synthesis of NaK-ATPase sites or to an increase in the rate of their degradation since these processes would require a latency of several minutes to hours for their effect to become evident. A possible mechanism for the inhibition of NaKoATPase in the presence of less than saturating concentrations of Na+, K+, or
2456
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
50
40
.> ._c ~ 30
<2 o.E ~
20
10
06
-05 -04
-03
-02
-01
0
01
Log [ATP]
0
I 0.2
I I 0.4 0.6 mM ATP
I
I
0.8
1.0
Rgure 3. Activation by ATP of rat renal NaK-ATPase Membrane preparations were treated with diluent alone (El,O) or 75 i~g/ml mastoparan (11,0) and activated by varying concentrations of ATP. Each point represents the mean _+ S.E.M (n=9). Inset: Hill Plot of data in Figure 3. Ko (apparent dissociation constant for ATP) was determined as the antilog of the x intercept from the slope The Hill coefficient was obtained from the slope Lines were drawn by linear regression.
ATP, would be a decrease =nthe enzyme's affinity for one or more of these substrates. However, this does not appear to have occurred in the present experaments, because mastoparan decreased the Ko for K*, and it had no effect on the KD'S for Na ÷ and ATP. Mastoparan also had no effect on the Hill coefficients for the three substrates. Because of the apparent increase in the affinity of the enzyme for K ÷ observed in the presence of mastoparan, inhibition of NaK-ATPase activity by mastoparan occurred almost exclusively at the h~gher range of K + concentrations. The effect of the inhibitor on the Ko therefore appeared to be as important kinetically, as its affect on the Vmax in the lower range of K + concentrations. The mechanism by which mastoparan increased the amount of phosphoenzyme intermediate is obscure. A highly speculative explanation for this effect is that even in the presence of 100 mM Na + and absence of added K ÷, there was a slow but fin=te turn-over of the phosphoenzyme
Vol. 47, No. 26, 1990
Mastoparan on Renal NaK-ATPase
2457
Table III Effect of Neomycin on the Activity of Mastoparan Treated Rat Renal NaK-ATPase
Neomycin (mM) Control
0 mM
0.25 mM
0.50 mM
1.0 mM
2 mM
52_+1 6
46-+ 1.4
47 4-+ 1.7
47.6-+1.3
48.5-+1.3
50.5-+1.5
n=10
n=8
n=6
n--6
n=6
n=6
p < .05
p < .05
p < .05
p < .05
NS
Control represents untreated membrane preparation Other membrane preparations were preincubated (15 minutes) with varying concentrations of neomycin then treated w=th mastoparan (75 pg/ml) for 1 minute. NaK-ATpase activity was determined as described in "Methods'. Act=vdy is expressed as pmoles Pi per mg protein per hour. Values represent the mean _+ S.E.M P values given are compared to control.
intermediate, and that mastoparan increased the steady state amount of phosphoenzyme intermediate by slowing its breakdown (the rate limiting step). Stabilization ofthe phosphoenzyme intermediate could help explain the decrease in Vmax produced by mastoparan since thin would decrease the rate of NaK-ATPaseturnover. Neomycin, wh0ch is an inhibitor of inositol phosphohp=d metabolism (14-19), was found to attenuate the inhibition of NaK-ATPase activity by mastoparan. We did not study the effect of neomycin on the mastoparan induced increase in the NaK-ATPase phosphorylated intermediate. However, we observed that neomycin attenuates mastoparan's inhibit=on of Na*-dependent net AIB uptake and completely suppressed mastoparan increases in AIB efflux (26). Since renal NaK.ATPase activity appears to require the presence of phosphatidylinositol (12, 13), the mechanism of the inhibition of NaK-ATPase by mastoparan may involve degradation of the phosphatidylinositol "pool" (27) Acknowledoment This work was supported by USUHS Grant CO7623 and American Heart Association, Maryland Affiliated, Inc. Reference 1. 2 3. 4.
5. 6.
7. 8 9. 10.
M. SCHATHER and E. THAIN, Brit. J. Pharmacology, 9:352 (1954). R.G. GELLER, H. YOSHIDA, M.A. BEAVAN, A. HORAKOVA, F.L. ATKINS, H. YAMABE, and J.J PISANO, Toxicon 14:27-33 (1976). H. YOSHIDA, R.G GELLER, and J.J. PISANO, Biochemistry, 15:61-64 (1976). E. HABERMANN, Science 117:314-322 (1972). R.A.SHIPOLINI, European J. Biochemistry 20: 459- (1971). E. HABERMANN, Biochemistry 329: 1- (1957). Y. HIRAI, Y. UENO, T. YASUHARA, H. YOSHIDA, and T. NAKAJIMA, Chem Pharm. Bull 27:1945-1946 (1979). Y. HIRAI, Y. UENO, T. YASUHARA, H. YOSHIDA, and T. NAKAJIMA, Biomedical Res. 1: 185-197 (1980). Y. HIRAI, T. YASUHARA, H. YOSHIDA, T. NAKAJIMA, M. FUJINO, and C. KITADA, Chem. Pharm. Bull. 27:1942-1944 (1979). A. ARGIOLOS, and J. PISANO, J. BIOL. CHEM. 258:13697-13702 (1983).
2458
11. 12. 13. 14. 15. 16. 17 18. 19 20. 21. 22. 23. 24. 25. 26. 27.
Mastoparan on Renal NaK-ATPase
Vol. 47, No. 26, 1990
Y. OKANO, H. TAKAGI, T. TOHMATSU, S. NAKASHIMA, Y. KURODA, IL SAITO, and Y. NOZAWA, FEBS Letters 188 (2): 363-366, (1985). J. MANDERSLOOT, B. ROELOFSEN, and J. DE GIER, Biochem. Biophys. Acta. 508: 478485 (1978). B. ROELOFSEN and M. VAN LINDE-SIBENIUS, Biochem. Biophys. Acta., 647: 302-306, (1981). V. LANG, G. PRYHITKA, and J. BUCKLEY, Can. J. Biochem. 55:1007-1012 (1977). S. LODHI, N. WEINER, and J. SCHACHT, Biochem. Biophys. Acta. 557:1-8 (1979). J.J. LIPSKY, J. Pharmacol. Exp. Ther. 220: 287-292, (1982). P. MARCHE, S. KOUTOUZOV, and A. GIRARD, J. Pharmacol. Exp. Ther. 227:415-420 (1983). J. SCHACHT, J. Lipid Res. 19:1063-1067 (1978). D. SCHWERTZ, J. KREISBERG, and M. VENKATACHALAM, J. Pharmacol. Exp. Ther. 251. 48-55 (1984). C S. Lo and T.N. Lo, Endocnnology 109.1473.1478 (1981) C.S. LO, T. AUGUST, U.l. LIBERMAN, and I. EDELMAN, J. Biol. Chem. 251 (24): 7826-7833 (1976). C. FISKE and Y. SUBBAROW, J. Biol. Chem. 66: 375-400, (1925). A. SCHWARTZ, J. ALLEN, and S. HARIGAYA, J. Pharmacol. EXp. Ther. 168:31-41 (1989). O.H. LOWRY, N.J. ROSEBOROUGH, A.L. FARR, and R.J. RANDALL, J Biol. Chem. 195: 265-275 (1951). G.W. SNEDECOR and W.G. COCHRAN in Statistical Analvsis. 7th edition, Iowa State Press (1980). S.P. ENG, D.L. CLOUGH, and C.S. LO Life Sci. 46:865-873 (1990). S.P. ENG and C.S. Lo, Life Sci 46:273-279 (1990).