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Interaction of ethacrynic acid and cysteine with renal medullary adenylate cyclase
Life Saisnws Vol . 16, pp . 255-262 Printed in the U .3 .~1 .
Pergamon Press
INTERACTION OF SfHACRYNIC ACID AND CYSTEINB WITH RENAL MBDULLARY ADENYLATE CYCLASE Larry D . Barnes, Yvonne S .F . Hui and Thomas P . Douse Kayo Clinic sad Foundation, Rochester, Minnesota (R~ceived is linal fora Deoenbar 9, 1974) Results of the present study indicate that (1) ethacrynic acid, dihydroethacrynic acid, and the cyateine adduct of ethacrynic acid inhibit plasma membrane-bound aderylate cyclase from canine renal medulla ; (2) reaction with a aulfhydryl group is not essential for inhibition by ethacrynic acid and its derivatives, but may contribute quantitatively to the inhibition ; and (3) cysteine enhances the activity of renal medullary adaaylate cyclasa in the presence of a vasopressin analog or sodium fluoride . Observations support the vier that ethacryaic acid and its cyeteine adduct interfere rich the action of vasopreaain on the distal nephron at the site of renal medutlary aderylate cgclase . Besides the cell defined saluretic effect, probably through its action an the thick ascending limb of Henle~a loop (1), some observations indicate that athacrynic acid may act also on the more distal segments of the nephron and block the antidiuratic action of vaaopreasin in .collecting ducts .
Ethacrynic
acid ras reported to block the hydroosmotic effect of vasopressin but not of exogenona cyclic AMP in the isolated toad bladder (2,3) or in the microparfused collecting ducts (4) .
The cellular site of the ethacrynic acid affect
has not yet been determined ; interference rich the activity of (Na-K) adanoaine triphosphatase (5) and/or adenylata cyclase (6,7) rare caosiderad . Diuretic properties of ethacrynic acid (and also its mechanism of action) rare attributed to its ability to interact rith aulfhydryl groups (8,9) .
On
the other hand, in microperfveed isolated tubule systems the ethacrgnic acid cyeteine adduct ras reported to be even more effective than ethacrynic acid iùelf (1) .
255
Ethacrynic Acid and Renal Adenylate Cyclase
256
Vol . 16, No . 2
The mechanism of interaction of the ethacrynic acid with renal medullary plasma
membrane
adenylate cyclase, especially the role of the aulfhydryl
group, was examined in the present experiments . Methods and Materials Kidneys were removed from dogs
anesthetized with pentobarbital, and the
plasma membrane ,fraction was prepared from the medullary tissue using the method of Fitzpatrick et al . (10) . The adenylate cyclase assay and protein determination were the same as used in our previous study (11) .
No interference of ethacrynic acid with
the ATP-regenerating system, from creative phosphate to maintain the ATP level, or with chromatography was observed under the present conditions .
The etha-
crynic-cysteine adduct (cyateine-ethacrynate) was prepared as outlined elsewhere (1 ) .
Details of this procedure were kindly supplied by Dr . Cragce of
Merck, Sharp and Dohme .
Cysteine-ethacrynate was always prepared fresh
shortly prior to the use in the experiments .
In some experiments a relatively
high concentration of cyateine was included into the incubation mixture . Since cysteine can inactivate vasopresain through reduction of the disulfide bridge, a synthetic analog of the natural vasopreasin,
[1-deamino-l,6-dicarba,-
8-lysine]-vasopresain (DCLVP) in which disulfide moiety was replaced by two methylene groups, was used in the present study.
Such a chemical analog of
vasopressin was shown to be active both in vivo (12) and in vitro (13) ; DCLVP differed from natural [8-arginine]-vasopresain only by the lower affinity towards renal medullary adenylate cyclase .
A concentration (10-6 M) which
stimulated maximally the adenylate cyclase was used in present experiments . Ethacrynic acid and dihydrcethacrynic acid were gifts of Merck, Sharp and Dohme ; DCLVP was kindly donated by Dr . R. Walter of Mt . Sinai School of Medicine, New York . Resul is Ethacrynic acid (10-3M) adenylate cyclase activities :
inhibited the renal medullary plasma membrane basal, stimulated by DCLVP or by sodium
]57
8thacs:ynio 1~cid aad Renal 7läanylate Cyclasa
Vol . 16, No . 2
TABLE I Bffact of the Phenossyacetic Acid Derivatives on Canine Renal Medullarq Ade~late Cyclase in Absence of Cysteine . Specific activities of the enzyme (in pmoles/~,n/mg prôtein ± SBM) wares basal activity - 9 .29 _+ 2 .63 ; atimulated bq 10 -6M DCLVP ~ 36 .96 ± 8 .14 and stimulated by 10-2 M NaF ~ 88 .8 _+ 9 .92 . Significant at +si+G0 .005, paired t-teat .
Mesa Drug (lÔ 3M)s
Ns
Baa
s
x
Change ± SBM
DCLVP (10. 6M)
NaF (LÔ ~)
Bthacryaic Acid
4
-93 .3 ± 2 .6*
-97 .7 + 0 .9*
-94 .8 ± 1 .3*
Dihydroethacrynic Acid
5
-47 .8 + 4 .3*
-58 .0 + 4 .6*
-21 .4 ± 7 .8*
Phenoxyacetic Acid
3
+ 5 .6 + 9 .6
+ 8 .2 ± 8 .8
+ 7 .1 ± 7 .7
100
Boscl
0
10's
DCLVPstimulorted 10' 4
10'3
0
10's
10"~
10'3
FIG . 1 Sffecta of Increasing Concentrations of Bthacrynic Acid (B .A . -~-~-) and Dihydroethacrynic Acid (D .B .A. --0--0-) on the Adenylate Cyclaae Activity (x activity) . Le_~t panels basal activity ; right panels activity stimulated by 10 M DCLVP . Each point is the mean of 3-5 independent observations ± SBM .
8thacrynic Acid and Renal Adenylate Cj~clase
258
fluoride .
Vol . 16, No . 2
Dihydrcethacrynic acid also inhibited all three activities in the
similar fashion, only the inhibition vas less pronounced than with the ethacrynic acid .
Phenoxyacetic acid had no effect on the adenylate cyclase
activity (Table I) .
Dihydrcethacrynic acid inhibited adenylate cyclaee in
a similar dose-dependent fashion as ethacrynic acid with the exception that the concentration of this drug required to produce half-maximal inhibition vas about one order higher than that of the ethacrynic acid itself (Figure 1) .
,c
1 Q OI .C
Q .V
û Q
FIG. 2 on Renal [iedullary Adenylate Cyclaae= basal, Effect of Cyatei stimulated by LO~M DCLVP or stimulated by 10 -2M sodium fluoride (NaF) . Numbers in brackets denote number of experiments,
Vol. 16, No . Z
8thacryaic Acid and Renal Adenylats Cyclasa
259
Because of the imetability of the cysteine-ethacrynate, studies with this compound were conducted in the presence of excess (in terms of molar concentration) cysteine to prevent dissociation of the adduct .
Therefore, the
effect of added cysteine on adenylate cyclese activity was examined firnt . M shave in Figure No . 2, inclusion of lÔ ~ cysteine to the incubation mixture had a very small insignificant affect on the basal adenylate cyclaae activity, but greatly increased the ade~rlate cyclase activity stimulated by DCLVP or NaF . TABLE II Sffacts of Cyataine-ßthacrynate and of Dihydroethacrynic Acid on Renal llsdullary Adanylate Cyclase in the Presence of 10-4!! Cysteine . Concentrations of drugs were 10-3M. The activity of adarylata cyclase (in pswles/min/mg protein ~ Sam() were : basal 13 .9 + 2 .3 ; with 10-6 M DCLVP, 65 .0 f 12 .4, and pith 10- K NaF 157 .0 ± 5 .7 . Significant at *PG0 .001 ; **PG0 .025 ; ***PG0 .01 ; paired t-test .
Mean Drugs
Ns
Basels
Cysteineethacrynate
5
Dihydroethacrynic Acid
3
x
Change ± SBti
DCLVP (lô 6)
NaF (lÙ 2)
-51 .12 ± 3 .8*
-56 .3 ± 5 .9*
-34 .0 ± 9 .4**
-35 .04 + 2 .8***
-24 .1 + 2 .4*
+ 5 .7 + 0 :4**
Cystaina-ethacrynate marlaedly inhibited the adenylate cyclasa but the inhibition (in terms of
x
decreased activity) appears to be lass pronounced
than the inhibition by the athacrynic acid itself (Table II) .
Dihydroatha
crynic acid also inhibited basal aderylate cyclase activity and that stimulated by DCLVP in the presence of cysteine but to a lesser extent in terms of
x
inhibition (PG0 .02) than in an incubation medium pithout the cyateine
(compare Tables I and II) .
Fluoride stimulated ade~.late cyclase pas not
inhibited by dihydroathacrynic acid in the presence of cysteine .
260
Ethaaxynic Acid and Renal Adenylate Cyclase
Vol . 16, No . 2
Discussion The present results show that ethacrynic acid is a potent inhibitor of adenylate cyclase contained in the plasma membranes derived from dog renal medulla.
The finding that the basal activity of the enzyme as well
as hormone
and fluoride-stimulated activities were inhibited to a similar degree suggests that the drug acted on the catalytic subuait of adenylate cyclase rather than on the receptor site for vasopreasin .
The inhibitory effect of ethacrynic
acid on the adenylate cyclase is not dependent on its ability to combine with sulfhydryl groups since dihydrcethacrynic acid, a compound unable to form adducts with aulfhydryl groups inhibits adenylate cyclase in a similar way as ethacrynic acid itself, the inhibition is only quantitatively leas expressed . Dihydrcethacrynic acid exhibits weak but distinct diuretic activity in the dog (3) (Doctor Cragce, personal communication) and inhibits vasopresain effects in the toad urinary bladder (3) .
On the other hand, phenoxyacetic acid, a
parent compound of this class of diuretics, has no noticeable effect on the adenylate cyclase activity . The presence of lÔ ~ cyateine greatly enhanced stimulation of the renal madullary adenylate cyclase by the vasopreasin analog and by sodium fluoride . This suggests that aulfhydryl groups play an important role in the activity of renal medullary adenylate cyclase, especially when stimulated by hormone or sodium fluoride .
Results of experiments studying the effect of cyateine
on the isolated toad bladder were interpreted as that cyateine inhibits the adenylate cyclase (14) .
Our present results would indicate that either major
differences exist in terms of dependence on aulfhydryl groups between mammalien kidney and toad bladder or that the inhibitory effect of cyateine on vasopresain and theophylline actions in toad bladder was not due to a direct interaction with adenylate cyclase in the intact cello . The finding that cyateine-ethacrynate retains its ability to inhibit renal madullary adenylate cyclase further supports the view that the inhibitory effect of ethacrynic acid is not dependent on the interaction with
Vol . 16, No . 2
261
8thacrynic Acid and Raval Adenylata Cyclase
aulfhydryl group of this enayma .
A smaller inhibition pith cysteine-etha-
crynate than pith ethacrynic acid may be due to the stimulatory effect of cyateine present in the incubation mixture phich can maloe the adenylate cyclaae more resistent to the inhibition by ethacrynic acid and its derivative .
This
viep is supported by the fact that inhibition of adenylate cyclase by dihydroethacrynic acid (the compound unable to readily react pith cyateine or other compounds containing sulfhydryl groups) is also leas prominent phen cyateine is present in the incubation mixture .
Since cyeteiae-ethacrynate is unstable and
should be laept in the solution pith exceae cyateine the direct quantitative comparison of the effects of ethacrynic acid and cysteine-ethacrynate caanot be made . The preaant findings support the viep that the inhibitory effect of ethacrynic acid on the hydrooamotic action of vasopresain observed in vivo (2,3,4) is due to the inhibition of plasma membrane-bound renal medullary aderylate cyclaae .
Our results also shop that for this action of ethacrynic
acid the presence of the ~ waaturated system in the molecule is not essential although this feature may contribute quantitatively to the inhibitory effect . Cyateine-ethacrynate, a major metabolite excreted in the urine (15), can have a similar effect on the vasopresain-sensitive segment of the nephron as ethacrynic acid . Açlçnorled~ementa We theak Jan Applequiat for secretarial assistance and Mr . Christopher Wilson for expert technical assistance . This study pas supported by USPHS NIH research graata AM-16105 and 5-SO1-RR-05530-10, by Grant-in-Aid from the American Heart Association pith funds contributed in part by the Minnesota Heart Association and by the Mayo Foundation . Dr . Larry D . Harnea is a recipient of the Mayo Research Fellopship . Dr . Thomas P . Douse is an Established Investigator of the American Heart Association . References 1.
M . Burg and N . Green, Kidney Int . 4, 301-309 (1973)
2.
F .R. Cobb and T .J . McManus, Abat . of 3rd Int . Cong . Neph ., Washington, D .C ., p . 172 (1966)
262
8thacrynic Acid and Renal Adenylate Cyclase
Vol . 16, No . 2
3.
P,J . Bentley, J . Endocrin . 43, 347-357 (1969)
4.
M, Abramov, J . Clin . Invest . 53, 796-804 (1974)
5.
J .E, Baer and R.H, Baer, Drugs Affecting Kidney Function and Metabolism , in "Progress in Biochemical Pharmacology ", edited by Edwarda, Karger-Basel, PP " 59-93 (1972)
6.
K,M. Jakobs, K. Schultz and G. Schultz, Nawvn- Schmiedebergs Arch . Pharmacol . 273, 248-266 (1972)
7.
H, Sbel, Naunvn-Schmiedeberg'a Arch . Pharmacol . 281, 301-314 (1974)
8.
R. Romorn and E.J, Cafruny, J. Pharmacol . Ex t . Thera . 148, 367-372 (1965 )
9. 10 .
D,A. Koechel nnd E. CafrvAy, J. Med. Chem. 16, 1147-1152 (1973) D.F . Fitzpatrick, C . R. Davenport, L . Forte and E.J . Landon, J. Biol . Chem. 244, 3561-3569 (1969)
il .
T .P, Dousa and L .D . Barnes, J. Clin . Invest . 54, 252-262 (1974)
12 .
V, Pliaka, J. Rudinger, T .P . Dousa aad J.H . Cort, Am. J . Phvaiol . 215, 916-920 (1968)
13 .
T .P . Doua a, 0. Hechtar,
LL . Schvartz and R. Walter, Proc . Natl . Acad .
Sçi . U.S .A. 68, 1693-1697 (1971) 14 .
J .S . Handler and J . Orloff, Am . J. Physiol . 206, 505-509 (1964)
15 .
K.H . Beyer, J.E . Baer, J.K. Michaela on and H .F . Rusao, J . Pharm. Expt . Thera . 147, 1-22 (1965)
Pergamon Press
INTERACTION OF SfHACRYNIC ACID AND CYSTEINB WITH RENAL MBDULLARY ADENYLATE CYCLASE Larry D . Barnes, Yvonne S .F . Hui and Thomas P . Douse Kayo Clinic sad Foundation, Rochester, Minnesota (R~ceived is linal fora Deoenbar 9, 1974) Results of the present study indicate that (1) ethacrynic acid, dihydroethacrynic acid, and the cyateine adduct of ethacrynic acid inhibit plasma membrane-bound aderylate cyclase from canine renal medulla ; (2) reaction with a aulfhydryl group is not essential for inhibition by ethacrynic acid and its derivatives, but may contribute quantitatively to the inhibition ; and (3) cysteine enhances the activity of renal medullary adaaylate cyclasa in the presence of a vasopressin analog or sodium fluoride . Observations support the vier that ethacryaic acid and its cyeteine adduct interfere rich the action of vasopreaain on the distal nephron at the site of renal medutlary aderylate cgclase . Besides the cell defined saluretic effect, probably through its action an the thick ascending limb of Henle~a loop (1), some observations indicate that athacrynic acid may act also on the more distal segments of the nephron and block the antidiuratic action of vaaopreasin in .collecting ducts .
Ethacrynic
acid ras reported to block the hydroosmotic effect of vasopressin but not of exogenona cyclic AMP in the isolated toad bladder (2,3) or in the microparfused collecting ducts (4) .
The cellular site of the ethacrynic acid affect
has not yet been determined ; interference rich the activity of (Na-K) adanoaine triphosphatase (5) and/or adenylata cyclase (6,7) rare caosiderad . Diuretic properties of ethacrynic acid (and also its mechanism of action) rare attributed to its ability to interact rith aulfhydryl groups (8,9) .
On
the other hand, in microperfveed isolated tubule systems the ethacrgnic acid cyeteine adduct ras reported to be even more effective than ethacrynic acid iùelf (1) .
255
Ethacrynic Acid and Renal Adenylate Cyclase
256
Vol . 16, No . 2
The mechanism of interaction of the ethacrynic acid with renal medullary plasma
membrane
adenylate cyclase, especially the role of the aulfhydryl
group, was examined in the present experiments . Methods and Materials Kidneys were removed from dogs
anesthetized with pentobarbital, and the
plasma membrane ,fraction was prepared from the medullary tissue using the method of Fitzpatrick et al . (10) . The adenylate cyclase assay and protein determination were the same as used in our previous study (11) .
No interference of ethacrynic acid with
the ATP-regenerating system, from creative phosphate to maintain the ATP level, or with chromatography was observed under the present conditions .
The etha-
crynic-cysteine adduct (cyateine-ethacrynate) was prepared as outlined elsewhere (1 ) .
Details of this procedure were kindly supplied by Dr . Cragce of
Merck, Sharp and Dohme .
Cysteine-ethacrynate was always prepared fresh
shortly prior to the use in the experiments .
In some experiments a relatively
high concentration of cyateine was included into the incubation mixture . Since cysteine can inactivate vasopresain through reduction of the disulfide bridge, a synthetic analog of the natural vasopreasin,
[1-deamino-l,6-dicarba,-
8-lysine]-vasopresain (DCLVP) in which disulfide moiety was replaced by two methylene groups, was used in the present study.
Such a chemical analog of
vasopressin was shown to be active both in vivo (12) and in vitro (13) ; DCLVP differed from natural [8-arginine]-vasopresain only by the lower affinity towards renal medullary adenylate cyclase .
A concentration (10-6 M) which
stimulated maximally the adenylate cyclase was used in present experiments . Ethacrynic acid and dihydrcethacrynic acid were gifts of Merck, Sharp and Dohme ; DCLVP was kindly donated by Dr . R. Walter of Mt . Sinai School of Medicine, New York . Resul is Ethacrynic acid (10-3M) adenylate cyclase activities :
inhibited the renal medullary plasma membrane basal, stimulated by DCLVP or by sodium
]57
8thacs:ynio 1~cid aad Renal 7läanylate Cyclasa
Vol . 16, No . 2
TABLE I Bffact of the Phenossyacetic Acid Derivatives on Canine Renal Medullarq Ade~late Cyclase in Absence of Cysteine . Specific activities of the enzyme (in pmoles/~,n/mg prôtein ± SBM) wares basal activity - 9 .29 _+ 2 .63 ; atimulated bq 10 -6M DCLVP ~ 36 .96 ± 8 .14 and stimulated by 10-2 M NaF ~ 88 .8 _+ 9 .92 . Significant at +si+G0 .005, paired t-teat .
Mesa Drug (lÔ 3M)s
Ns
Baa
s
x
Change ± SBM
DCLVP (10. 6M)
NaF (LÔ ~)
Bthacryaic Acid
4
-93 .3 ± 2 .6*
-97 .7 + 0 .9*
-94 .8 ± 1 .3*
Dihydroethacrynic Acid
5
-47 .8 + 4 .3*
-58 .0 + 4 .6*
-21 .4 ± 7 .8*
Phenoxyacetic Acid
3
+ 5 .6 + 9 .6
+ 8 .2 ± 8 .8
+ 7 .1 ± 7 .7
100
Boscl
0
10's
DCLVPstimulorted 10' 4
10'3
0
10's
10"~
10'3
FIG . 1 Sffecta of Increasing Concentrations of Bthacrynic Acid (B .A . -~-~-) and Dihydroethacrynic Acid (D .B .A. --0--0-) on the Adenylate Cyclaae Activity (x activity) . Le_~t panels basal activity ; right panels activity stimulated by 10 M DCLVP . Each point is the mean of 3-5 independent observations ± SBM .
8thacrynic Acid and Renal Adenylate Cj~clase
258
fluoride .
Vol . 16, No . 2
Dihydrcethacrynic acid also inhibited all three activities in the
similar fashion, only the inhibition vas less pronounced than with the ethacrynic acid .
Phenoxyacetic acid had no effect on the adenylate cyclase
activity (Table I) .
Dihydrcethacrynic acid inhibited adenylate cyclaee in
a similar dose-dependent fashion as ethacrynic acid with the exception that the concentration of this drug required to produce half-maximal inhibition vas about one order higher than that of the ethacrynic acid itself (Figure 1) .
,c
1 Q OI .C
Q .V
û Q
FIG. 2 on Renal [iedullary Adenylate Cyclaae= basal, Effect of Cyatei stimulated by LO~M DCLVP or stimulated by 10 -2M sodium fluoride (NaF) . Numbers in brackets denote number of experiments,
Vol. 16, No . Z
8thacryaic Acid and Renal Adenylats Cyclasa
259
Because of the imetability of the cysteine-ethacrynate, studies with this compound were conducted in the presence of excess (in terms of molar concentration) cysteine to prevent dissociation of the adduct .
Therefore, the
effect of added cysteine on adenylate cyclese activity was examined firnt . M shave in Figure No . 2, inclusion of lÔ ~ cysteine to the incubation mixture had a very small insignificant affect on the basal adenylate cyclaae activity, but greatly increased the ade~rlate cyclase activity stimulated by DCLVP or NaF . TABLE II Sffacts of Cyataine-ßthacrynate and of Dihydroethacrynic Acid on Renal llsdullary Adanylate Cyclase in the Presence of 10-4!! Cysteine . Concentrations of drugs were 10-3M. The activity of adarylata cyclase (in pswles/min/mg protein ~ Sam() were : basal 13 .9 + 2 .3 ; with 10-6 M DCLVP, 65 .0 f 12 .4, and pith 10- K NaF 157 .0 ± 5 .7 . Significant at *PG0 .001 ; **PG0 .025 ; ***PG0 .01 ; paired t-test .
Mean Drugs
Ns
Basels
Cysteineethacrynate
5
Dihydroethacrynic Acid
3
x
Change ± SBti
DCLVP (lô 6)
NaF (lÙ 2)
-51 .12 ± 3 .8*
-56 .3 ± 5 .9*
-34 .0 ± 9 .4**
-35 .04 + 2 .8***
-24 .1 + 2 .4*
+ 5 .7 + 0 :4**
Cystaina-ethacrynate marlaedly inhibited the adenylate cyclasa but the inhibition (in terms of
x
decreased activity) appears to be lass pronounced
than the inhibition by the athacrynic acid itself (Table II) .
Dihydroatha
crynic acid also inhibited basal aderylate cyclase activity and that stimulated by DCLVP in the presence of cysteine but to a lesser extent in terms of
x
inhibition (PG0 .02) than in an incubation medium pithout the cyateine
(compare Tables I and II) .
Fluoride stimulated ade~.late cyclase pas not
inhibited by dihydroathacrynic acid in the presence of cysteine .
260
Ethaaxynic Acid and Renal Adenylate Cyclase
Vol . 16, No . 2
Discussion The present results show that ethacrynic acid is a potent inhibitor of adenylate cyclase contained in the plasma membranes derived from dog renal medulla.
The finding that the basal activity of the enzyme as well
as hormone
and fluoride-stimulated activities were inhibited to a similar degree suggests that the drug acted on the catalytic subuait of adenylate cyclase rather than on the receptor site for vasopreasin .
The inhibitory effect of ethacrynic
acid on the adenylate cyclase is not dependent on its ability to combine with sulfhydryl groups since dihydrcethacrynic acid, a compound unable to form adducts with aulfhydryl groups inhibits adenylate cyclase in a similar way as ethacrynic acid itself, the inhibition is only quantitatively leas expressed . Dihydrcethacrynic acid exhibits weak but distinct diuretic activity in the dog (3) (Doctor Cragce, personal communication) and inhibits vasopresain effects in the toad urinary bladder (3) .
On the other hand, phenoxyacetic acid, a
parent compound of this class of diuretics, has no noticeable effect on the adenylate cyclase activity . The presence of lÔ ~ cyateine greatly enhanced stimulation of the renal madullary adenylate cyclase by the vasopreasin analog and by sodium fluoride . This suggests that aulfhydryl groups play an important role in the activity of renal medullary adenylate cyclase, especially when stimulated by hormone or sodium fluoride .
Results of experiments studying the effect of cyateine
on the isolated toad bladder were interpreted as that cyateine inhibits the adenylate cyclase (14) .
Our present results would indicate that either major
differences exist in terms of dependence on aulfhydryl groups between mammalien kidney and toad bladder or that the inhibitory effect of cyateine on vasopresain and theophylline actions in toad bladder was not due to a direct interaction with adenylate cyclase in the intact cello . The finding that cyateine-ethacrynate retains its ability to inhibit renal madullary adenylate cyclase further supports the view that the inhibitory effect of ethacrynic acid is not dependent on the interaction with
Vol . 16, No . 2
261
8thacrynic Acid and Raval Adenylata Cyclase
aulfhydryl group of this enayma .
A smaller inhibition pith cysteine-etha-
crynate than pith ethacrynic acid may be due to the stimulatory effect of cyateine present in the incubation mixture phich can maloe the adenylate cyclaae more resistent to the inhibition by ethacrynic acid and its derivative .
This
viep is supported by the fact that inhibition of adenylate cyclase by dihydroethacrynic acid (the compound unable to readily react pith cyateine or other compounds containing sulfhydryl groups) is also leas prominent phen cyateine is present in the incubation mixture .
Since cyeteiae-ethacrynate is unstable and
should be laept in the solution pith exceae cyateine the direct quantitative comparison of the effects of ethacrynic acid and cysteine-ethacrynate caanot be made . The preaant findings support the viep that the inhibitory effect of ethacrynic acid on the hydrooamotic action of vasopresain observed in vivo (2,3,4) is due to the inhibition of plasma membrane-bound renal medullary aderylate cyclaae .
Our results also shop that for this action of ethacrynic
acid the presence of the ~ waaturated system in the molecule is not essential although this feature may contribute quantitatively to the inhibitory effect . Cyateine-ethacrynate, a major metabolite excreted in the urine (15), can have a similar effect on the vasopresain-sensitive segment of the nephron as ethacrynic acid . Açlçnorled~ementa We theak Jan Applequiat for secretarial assistance and Mr . Christopher Wilson for expert technical assistance . This study pas supported by USPHS NIH research graata AM-16105 and 5-SO1-RR-05530-10, by Grant-in-Aid from the American Heart Association pith funds contributed in part by the Minnesota Heart Association and by the Mayo Foundation . Dr . Larry D . Harnea is a recipient of the Mayo Research Fellopship . Dr . Thomas P . Douse is an Established Investigator of the American Heart Association . References 1.
M . Burg and N . Green, Kidney Int . 4, 301-309 (1973)
2.
F .R. Cobb and T .J . McManus, Abat . of 3rd Int . Cong . Neph ., Washington, D .C ., p . 172 (1966)
262
8thacrynic Acid and Renal Adenylate Cyclase
Vol . 16, No . 2
3.
P,J . Bentley, J . Endocrin . 43, 347-357 (1969)
4.
M, Abramov, J . Clin . Invest . 53, 796-804 (1974)
5.
J .E, Baer and R.H, Baer, Drugs Affecting Kidney Function and Metabolism , in "Progress in Biochemical Pharmacology ", edited by Edwarda, Karger-Basel, PP " 59-93 (1972)
6.
K,M. Jakobs, K. Schultz and G. Schultz, Nawvn- Schmiedebergs Arch . Pharmacol . 273, 248-266 (1972)
7.
H, Sbel, Naunvn-Schmiedeberg'a Arch . Pharmacol . 281, 301-314 (1974)
8.
R. Romorn and E.J, Cafruny, J. Pharmacol . Ex t . Thera . 148, 367-372 (1965 )
9. 10 .
D,A. Koechel nnd E. CafrvAy, J. Med. Chem. 16, 1147-1152 (1973) D.F . Fitzpatrick, C . R. Davenport, L . Forte and E.J . Landon, J. Biol . Chem. 244, 3561-3569 (1969)
il .
T .P, Dousa and L .D . Barnes, J. Clin . Invest . 54, 252-262 (1974)
12 .
V, Pliaka, J. Rudinger, T .P . Dousa aad J.H . Cort, Am. J . Phvaiol . 215, 916-920 (1968)
13 .
T .P . Doua a, 0. Hechtar,
LL . Schvartz and R. Walter, Proc . Natl . Acad .
Sçi . U.S .A. 68, 1693-1697 (1971) 14 .
J .S . Handler and J . Orloff, Am . J. Physiol . 206, 505-509 (1964)
15 .
K.H . Beyer, J.E . Baer, J.K. Michaela on and H .F . Rusao, J . Pharm. Expt . Thera . 147, 1-22 (1965)