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Inhibitory effect of Cu2+ on nitroglycerin-induced vasodilation
European Journal of Pharmacology, 100 (1984) 145-154
145
Elsevier
I N H I B I T O R Y E F F E C T O F C u 2 + ON N I T R O G L Y C E R I N - I N D U C E D V A S O D I L A T I O N TOSHIHARU KAM1TANI Research Laboratories, Fufisawa Pharmaceutical Co. Ltd., Yodogawa-ku, Osaka 532, Japan
Received 19 August 1983, revised MS received 2 December 1983, accepted 17 January 1984
T. KAMITANI, Inhibitory effect of Cu e + on nitroglycerin-induced vasodilation, European J. Pharmacol. 100 (1984) 145-154. The mechanism of nitroglycerin-induced vasodilation was examined in isolated arteries. Nitroglycerin relaxed the large coronary artery preferentially whereas nitroprusside and the so-called non-specific vasodilators showed the same activities on both the large and small coronary arteries. Nitroglycerin-induced vasodilation but not the vasodilation induced by the other agents was antagonized markedly by pretreatment with CuSO4. Other metal ions except Fe 2+ had no antagonizing effect. The Cu2+-induced antagonism was restored by treatment with sulfhydryl reagents. Nitroglycerin formed inorganic nitrite by non-enzymatic reaction with the tissue sulfhydryl groups; the reaction was also inhibited by Cu 2÷. Cu 2÷ suppressed the membrane stabilizing effect of Ca 2+. It seems that nitroglycerin reacts with the sulfhydryl groups on the inner surface of the cell membrane, which may take part in the selectivity of this drug, and subsequently the intermediate(s) formed may activate guanylate cyclase. Nitroglycerin
Vasodilators
Sulfhydryl groups
I. I n t r o d u c t i o n
Nitroglycerin, the most effective drug in the treatment of angina pectoris, reacts with reduced glutathione (GSH) to form oxidized glutathione and inorganic nitrite, a reaction which is catalyzed by the liver enzyme glutathione-dependent organic nitrate reductase (Heppel and Hilmoe, 1950). These authors also observed that in the presence of GSH, a non-enzymatic reaction took place and liberated nitrite. Cupric sulfate inhibited both the enzymecatalyzed and the spontaneous reaction. The notion of a sulfhydryl-containing, specific 'organic nitrate receptor' as the site of action for nitroglycerin and related organic nitro-compounds in vascular smooth muscle was first advanced by Needleman and coworkers (Needleman and Johnson, 1973; Needleman et al., 1973). Based on the findings that nitroglycerin-induced tolerance resulted from the oxidation of critical sulfhydryl groups in the vascular bed and that there was cross-tolerance to all organic nitrates but not to non-nitrate vasodilators, this group proposed a 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Cupric ion
hypothesis: The sulfhydryl groups are oxidized to the disulfide form, the organic nitrate molecule is reduced to the denitrated metabolite, nitrate ion is released and as a consequence of the interaction, relaxation ensues. Recently, considerable evidence has accumulated to support the theory that c G M P is involved 'in the regulation of nitroglycerin-induced smooth muscle relaxation (Schultz et al., 1977; Katsuki and Murad, 1976, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1979; Ignarro and Gruetter, 1980; Kobayashi et al., 1980). When nitroglycerin was given to dogs, an increase in the coronary blood flow was preceded by an increase in the c G M P concentration of the coronary artery (Kobayashi et al., 1980). Sodium nitroprusside, which acts directly on vascular smooth muscle (Kreye et al., 1975), may also induce vasodilation via c G M P elevation (Schultz et al., 1977; Katsuki and Murad, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1982; Ignarro and Gruetter, 1980; Gruetter et al., 1980; Ignarro et al., 1980), although there seems to be a slight dissociation
146
between the elevation of c G M P and the relaxation (Axelsson et al., 1982). There are some differences between the effect of nitroglycerin and of sodium nitroprusside: (1) nitroglycerin fails to activate soluble vascular guanylate cyclase in the absence of an added thiol, whereas sodium nitroprusside does not require the addition of thiols (Ignarro and Gruetter, 1980); (2) nitroglycerin is a weak activator while sodium nitroprusside is a potent activator of this enzyme (Katsuki et al., 1977a) although the vasodilator effects of both drugs are almost the same (Watkins and Davidson, 1980); (3) sodium nitroprusside increased guanylate cyclase activity in most of the preparations whereas the effect of nitroglycerin was tissue specific (Katsuki et al., 1977a); (4) regarding the tolerance and cross-tolerance characteristics of organic nitrate-tolerant vessels, sodium nitroprusside does not have the features of the organic nitrates (Needleman and Johnson, 1975). Since Cu 2+ is known as an inhibitor of glutathione-dependent denitration (Heppel and Hilmoe, 1950) and seems to play an important role in the regulation of guanylate cyclase activity (White et al., 1976; Gerzer et al., 1981), the effect of pretreatment with Cu z+ on nitroglycerin-induced vasodilation was examined in order to clarify the relation between the vasodilating activity of nitroglycerin and denitration. 2. Materials and methods
2.1. Effect on the isolated arteries Under pentobarbital Na anesthesia (35 m g / k g i.p.), the large (2 m m o.d.) and small (0.3 m m i.d.) coronary arteries were removed from mongrel dogs of either sex. The thoracic aorta was removed from male Wistar strain rats aged 7 weeks. Helical strips cut from the large coronary artery, the small coronary artery and the rat thoracic aorta were approximately 15 x 2 ram, 4 x 0.5 m m and 20 x 2 m m respectively. The strips were suspended in organ baths containing Tyrode solution. Each preparation was connected to a strain gauge and the tension was measured isometrically. The bath solution was aerated with a mixture of 95% 02 and 5%
CO 2 and maintained at 370C. Before the experiments were started, the strips were allowed to equilibrate for more than 60 rain in the bath solution. Vasodilator activity was examined under the active tension induced by 35 mM KCI. In the canine vessels this concentration of KCI produced a contraction up to about 65% of the maximum effect, which was reached with 70 mM KC1. In the rat aorta, 35 mM of KC1 induced submaximal contraction. In some experiments 2 mM of BaCI 2 was used instead of 35 mM of KCI. The tension of the strips from the large coronary artery, the small coronary artery and the rat thoracic aorta were adjusted respectively to 1.5 g, 0.12 g and 1.5 g and the test drugs were added cumulatively. At the end of each series of experiments, l0 4 M of papaverine was added to the organ bath. The values presented are relative to the papaverine-induced relaxation.
2.2. Inorganic' nitrite formation from nitroglycerin in the reaction with minced uascular muscles or sulfh ydrvl reagents Mongrel dogs of either sex were anesthetized with pentobarbital Na (35 m g / k g i.p.). The coronary artery and the femoral artery were dissected and minced finely; 200 mg wet weight of the minced vessels was immersed in 1 ml of Tyrode solution and maintained at 37°C. The reaction was started by adding 0.1 ml of nitroglycerin solution (final concentration of nitroglycerin: 10 4 M). The reaction was stopped by adding 1.1 ml of 5% HgCI 2. After centrifuging, the concentration of inorganic nitrite in the supernatant was measured by the colorimetric method of Heppel and Hilmoe (1950). Protein was measured by the biuret procedure. The chemical reaction of nitroglycerin with sulfhydryl reagents was also examined in Tyrode solution. Nitroglycerin (10 -4 M) and sulfhydryl reagents (10 3 M) were dissolved in Tyrode solution kept at 37°C. The concentration of inorganic nitrite formed was measured 30 min after the start of the reaction by the method mentioned above.
2.3. Analysis of tissue sulfllydryl Minced canine vessels (25-50 mg) kept at - 9 7 ° C until used were added to 5 ml of chilled
147 TABLE 1 Vasodilator effect on isolated canine large and small coronary arteries contracted by KCI. The EDso values of nitroglycerin and the 95% confidence limits were calculated by linear regression analysis, n = 5-14. Drug
EDso (M)
Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
EDs0 (S)
Large coronary artery
Small coronary artery
EDs0 (L)
1.7 X 10 -7 (0.5- 4.9X10 -7) 6.7 × 10 - s (0.8- 51.0 x 10 - s ) 9.0 x 10 - 7 (1.7-100.1 x 10 -7 9,5 × 10 -6 (0.9- 62.8 x 10 -6) 7.3 x 10- s (2.1- 21.8x10 - s )
2.3 × 10 6 (0.5-67.5X10 -6) 1.7 x 10 -4 (0.5-14.0 x 10 - 4 ) 4.9 x 10- 7 (1.1-23.7 × 10 -7) 7.7 x 10 -6 (0.8-74.3 x 10 -6) 1.1 x 10- 7 (0.1- 5.4x10 -7)
13.5
1.6X 10-5 (M)
4 .ox 10-9 0
0-
20
20.
40
40"
~ % ~ _ 1 2 5
}*M
1.5
As shown in table 1, nitroglycerin showed far greater selectivity on the large coronary artery than on the small coronary artery. Sodium nitrite had the same tendency but the selectivity was not so pronounced. In contrast, sodium nitroprusside, iproveratril and papaverine had almost the same activities on these two kinds of arteries.
All chemicals except ethacrynic acid were dissolved in distilled water. Ethacrynic acid was dissolved in ethanol.
I0-6
0,8
3.1. Effect on the isolated canine arteries contracted by KCI
2.4. Drugs
6.4x I0-8
0.5
3. Results
glucose-free Tyrode solution, homogenized and centrifuged at 900 x g foc 10 min. Sulfhydryl in the supernatant was measured by the method of Ellman with dithiobisnitrobenzoic acid (1959). Protein was measured by the method of Lowry et al. (1951).
4.0x 10-9
2.5
I
6.4x 10-8 l
I
1.6x I0-5 (M)
I0-6 I
I
I
I
60"
60 500
80-
80" .
~ too" (%)
O:>ntxol
too- (%)
Fig. 1. Effect of CuSO4 on nitroglycerin-induced vasodilation in isolated canine large coronary artery (left panel) and rat thoracic aorta (right panel). Each value represents a percent of the response induced by 10-4 M of papaverine. The values are the mean :h S.E. from 5-21 preparations.
148
3.2. Effect of pretreatment with CuSO 4, other metal ions and sulfhydryl reagents" The effect of pretreatment with CuSO4, other metal ions and sulfhydryl reagents on nitroglycerin-induced vasodilation was examined after washing out the pretreatment drugs.
3.2.1. CuS04 20 min-pretreatment with CuSO 4 in concentrations of 8 × 1 0 - 6 M or higher dose dependently
diminished the vasodilator effect of nitroglycerin in the canine large coronary artery and the rat aorta which were contracted by KC1 (fig. 1). The diminution also appeared when BaCl 2 was used as a vasoconstrictor (table 2). No tachyphylaxis was seen though the effect of nitroglycerin was determined before and after the CuSO 4 treatment in the same preparation in the case of canine large coronary artery (data not shown). When the highest concentration of CuSO 4 was added to the bath, the Tyrode solution became turbid probably because
TABLE 2 Effect of 5 × 10 - 4 M C u S O 4 p r e t r e a t m e n t on EDs0 values of several vasodilators with isolated c a n i n e large c o r o n a r y artery and rat aorta. Dose-response curves to v a s o d i l a t o r s were o b t a i n e d before and after a 20 m i n p r e t r e a t m e n t with C u S O 4 which was w a s h e d out before testing. EDs0 values and the 95% confidence limits were calculated from linear regression analysis. The e x p e r i m e n t was c o n d u c t e d with 5 to 21 preparations. Drug
EDs0 (M) Non-treatment (A)
Ratio P r e t r e a t m e n t with CuSO 4 (B)
(B)/(A)
1.76×10 5 (0.22-14.06 × 10- 5) 1.44× l0 4 (0.25- 8 . 3 8 × I 0 4) 1.10× 10 ~ (0.26- 4 . 6 8 × 1 0 6) 1.46 × 10 s (0.32- 6 . 7 6 × 1 0 - 5 ) 1.28×10 7 (0.28- 5 . 9 3 × 1 0 7)
68.8
3 . 9 8 × 10 ~' ( 0 . 3 2 - 4 8 . 7 0 × 10 - 6 ) 1.90× l0 4 (0.36-10.08 × 1 0 4) 5.59xl0 6 (0.97-32.14 × 10- ~') 1.26×10 s (0.29- 5 . 5 5 × 1 0 5) 2.02 × 10 7 (0.42- 9 . 7 2 × 1 0 7)
96.8
1.71 x 10 -6 (0.22-13.25×10 1.86X 10 4 (0.45- 7 . 7 6 × 1 0 5.53 × 10 - s (1.01-30.35×10 5.42×10 `6 (1.91-15.42 × 10 2.38 × 10 -~ (0.71 8 . 0 0 × 1 0
46.0
Coronary artery contracted by 35 m M KC1 Nitroglycerin NaNO z Nitroprusside Na Papaverine Iproveratril
2.56 × 1 0 - 7 (0.88- 7.43 x 10 - 7 6.01 × 1 0 - 5 (1.07-33.69 × 1 0 - ~ 3.28 × 10 . 7 (0.91-11.87 x 1 0 - v 1.13 × 10 - s (0.32- 3 . 9 8 × 1 0 5 1.17 × 10 7 (0.19- 7 . 0 2 × 1 0 . 7
2.4 3.4 1.3 13
Coronary artery contracted by 2 m M BaCI 2 Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
4.11 × 10 - s (0.44-38.88 × 10 8 2.57×10 5 ( 0 . 5 4 - 1 2 . 3 0 × 10 -5 4.85 x 10 7 (1.00-23.48×10 7 9.67 × 10 6 (2.71-34.59 × 10 6 1 . 6 7 × 10 v (0.40- 7 . 0 5 x 1 0 7
7.4 11.5 1.3 1.2
Rat aorta contracted by 35 m M KCI Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
3 . 7 2 × 10 8 (0.66-20,96 x 10 8 1.36×10 4 (0.35- 5.36 x 1074 1.00 × 10 ~ (0.19- 5 . 3 4 × 10 s 8 . 1 0 x 10 6 (2.91-22.50 × 10 6 1.41 × 10 s (0.31- 6 . 4 9 × 1 0 s
6) 1.4 4) 5.5 s) 0.7 6) 1.7 ~)
149
of the production of CuCO 3 a n d / o r Cu3(PO4) 2. This antagonism was not induced in the case of the other vasodilators, although the effect of N a N O 2 and sodium nitroprusside was more or less inhibited by CuSO4 in the vessels contracted by BaC12 (table 2). This antagonizing effect of CuSO 4 against nitroglycerin could not have been due to SO 2 - because pretreatment with equimolar Na2SO 4 did not cause any change in the nitroglycerin dose-response curve (data not shown). Since KCl-induced contraction was potentiated to some extent after CuSO4 treatment, the effect of nitroglycerin after CuSO 4 treatment was examined under low Ca 2+ conditions in the canine large coronary artery. The Ca 2÷ concentration decreased from 1.8 m M in the normal Tyrode solution to 1.0-1.5 mM. The procedure maintained the tonus of the vessel treated with CuSO4 at the same level as before treatment but did not block the antagonism (EDs0 values; 2.3 x 10 -7 M in normal Tyrode solution and 7.7 × 10-6 M after treatment with CuSO4 under low Ca 2+ conditions). The possibility that nitroglycerin may be unstable in the presence of Cu 2÷ is not likely since nitroglycerin, which was recovered from a mixture of CuSO 4 and nitroglycerin by using Amberlite XAD2 had the same activity as non-treated nitroglycerin in the rat aorta (EDso values; 4.0 × 10 - s and 5.7 × 10 -8 M respectively).
3.2.2. Other metal ions (table 3) Since the antagonizing effect of Cu 2+ against
nitroglycerin also appeared in the rat thoracic aorta (fig. 1), the effect of other metal ions was examined in this preparation. The effect of pretreatment with and without other metal ions was tested in separate preparations removed from the same animal. Fe 2÷ showed the same antagonizing effect as Cu z÷ whereas Ni 2+, Fe 3÷, Co 2+ and Mn 2÷ had no effect at a concentration of 5 × 1 0 - 4 M. Since 5 x 10 - 4 M of Hg 2+ and Cd 2+ inhibited KCl-induced contraction, the effect of these drugs was evaluated at lower concentrations. HgCI 2 8 × 10 -6 M tended to potentiate the vasodilator response of nitroglycerin. On the other hand, 3.2 x 10 _5 M of Cd 2÷ significantly potentiated the nitroglycerin-induced vasodilation in rat aorta. This potentiation was not seen in the canine large coronary artery (data not shown).
3.2.3. Sulfhydryl reagents (fig. 2) 4 mM of reduced glutathione and 1 m M of L-cysteine had no effect on the dose-response curve of nitroglycerin whereas 1 mM of dithiothreitol markedly potentiated the effect of nitroglycerin. All these drugs restored the Cu 2÷-induced antagonism against nitroglycerin.
3.2.4. Ethacrynic acid (fig. 3) The effect of 2.5 × 10-5 M of ethacrynic acid, an alkylating agent of the sulfhydryl groups, was also examined in the rat aorta. A 1 h pretreatment with this drug markedly lessened nitroglycerinand NaNO2-induced vasodilation, whereas the
TABLE 3 Effect of pretreatment with metal ions on nitroglycerin-induced vasodilation in the isolated rat aorta contracted by KCI. EDso values of nitroglycerin and the 95% confidence limits were calculated by linear regression analysis. The experiment was done with 5-8 preparations. Ion
Cone. (M)
ED5o (M) (95% confidence limits)
EDs0 (treatment) EDso (control)
Control Cu 2+ Fe z+ Fe 3+ Ni 2+ Co 2+ M n 2+ Hg 2+ Cd 2+
5 ×10 -4 5 )'(10 - 4 5 >(10 - 4 5 ×10 -4 5 × 1 0 -4 5 × 1 0 -4 8 xl0 -6 3.2 X 1 0 - s
3.72 x 10- s (0.66-20.96 × 1 0 - s) 1 . 7 1 x 1 0 6(0.22-13.25×10-6) 7.81×10-'~(0.82-74.12×10 -7) 1 . 1 6 × 1 0 - 7 ( 0 . 2 0 - 6.66X10 -7) 1 . 1 6 × 1 0 - 7 ( 0 . 1 8 - 7 . 5 8 × 1 0 7) 9.16 x 1 0 - s (1.27-66.14 × 1 0 - 8 ) 1 . 3 2 x 1 0 8(0.18- 9 . 5 1 x 1 0 - s ) 1.97×10 s(0.29-13.46x10 - s ) 8.2 X 10 -9 (l.0 -66.6 × 10 9)
46 21 3 3 2 0.4 0.5 0.2
150 4.0
6.4
1.0
4.0 6.4 1.0 1.6 xl~ 9 xl08 x166 x165
1.6
xl09 xl08 xl06 xl05 i
i
i
i
J
4.o xl09
6.4 xl08
1.0
1.6
xl(~ 6
xlOS(H)
l
50 ~ C u + D T T Cu+GSH
~
Cys
"
r
,
Cu*
SH I00
i0(
lOO
4 mM GSH
i mM
0.25mM
" N~.~t~ICu+DTT lmM ~ ' R C o n t ro 1 ° ~ "~DTT 0.25raM DTT ImM
Dithiothreitol
Cysteine
Fig. 2. Effect of pretreatment with sulfhydryl reagents on nitroglycerin-induced vasodilation and the reversal of CuSO 4 (5 × 10 4 M)-induced antagonism against nitroglycerin in the isolated rat aorta. Each value represents a percent of the response induced by 10 -4 M of papaverine. The values are the mean + S.E. from 3-5 preparations.
vasodilation induced by sodium nitroprusside was decreased only slightly. In contrast, the effect of iproveratril and papaverine was not disturbed at all.
brain microsomes, we examined whether the antagonizing effect of Cu 2+ on the nitroglycerininduced vasodilation in the canine large coronary artery was due to inhibition of Na+-K + ATPase. However, the dose-response curves of nitroglycerin with or without 1 0 - 7 M of ouabain did not differ. The EDs0 values were 6.9 × 10 _7 M and 2.0 × 10 7 M respectively. Though Na+-K + ATPase was also inhibited by a 1 h treatment with Na ÷ free Tyrode
3. 3. Effect of Na +-K + A TPase inhibition Since Ting-Beall et al. (1973) reported that Cu 2+ strongly inhibited Na+-K ÷ ATPase of chicken 4.0x 6o4X 10-9 10-8
1.6x 10-6 10-5
1.6x 10-6 10-5 °
I
50-
~ .2
t
Control
i00-
N i t r o g l ~ (%) 1.6x 10-6 10-5
i ~
50-
2.5x 10-4
i
I
.k.
Papaverinel i00- (%) ~
i00-
)
0
50 -
2.5x 10-4
10-3 0
I
1.6x 10-8 I
0-9 <
2.5x 4.0x 10-7 10-6 (M) I *
E.A.
50- ~
~
%
~
Nitroprusside i00- (%)
l',lat~2 (%l
4.0x
6.4x
0 -9
10 -8
I
I
1.6x 10 -6
10 -5 (M)
i
E.A. _[prover I o at n _~%~.._ i "A _
io0- (%) ril
Fig. 3. Effect of pretreatment with 2.5 × 10 -5 M of ethacrynic acid on vasodilation of the isolated rat aorta. Each value represents a percent of the response induced by 10 -4 M of papaverine. The values are the mean + S.E. from 5 preparations.
151 solution in which N a + was replaced by equimolar Li +, the effect of nitroglycerin remained unchanged ( E D s 0 : 4 . 8 × 10 -7 M in n o r m a l Tyrode solution, 5.0 × 10 -7 M in N a + free Tyrode solution).
TABLE 4 Effect of CuSO4 on inorganic nitrite liberation from nitroglycerin in response to the addition of minced canine femoral and coronary arteries. Each value represents the mean 5: S.E. of 3 experiments, a p < 0.05, b p < 0.001. Conc. of CuSO4 (M)
3.4. Effect of Cu 2 + on Ca 2 +-induced vasodilation
Liberated NOf (nmol/g tissue per 30 rain)
Femoral artery It is k n o w n that excess a m o u n t s of Ca 2+ show a m e m b r a n e - s t a b i l i z i n g effect a n d cause vasodilation. To ascertain the site of action of Cu 2+, the effect of CuSO 4 p r e t r e a t m e n t o n the vasodilation i n d u c e d by CaCI 2 was examined i n ' t h e rat aorta contracted by KCI. As shown in fig. 4, more than 6.8 m M of CaC12 caused dose-dependent relaxation. P r e t r e a t m e n t with 5 × 10 -4 M of CuSO 4 shifted the dose-response curve to the right a n d the m a x i m a l relaxation b y CaC12 was suppressed.
3.5. Inorganic nitrite liberation from nitroglycerin in minced vascular smooth muscles Nitroglycerin metabolizing enzyme, which has already been shown to be in the liver (Heppel and Hilmoe, 1950), was extracted according to the m e t h o d of Heppel and Hilmoe (1950). However, we could not detect any enzyme activity in the
20
0
t~rmal
1.8..........---~
jL
6.,8-',.~1.,8
, ~1,.8
, 41;8,61.8
Control 3.2X10 -s
39.8 ___2.8 31.35:3.3 5.0 × 10 - 4 24.2 _ 2.2 a Control (heat 100°C, 5 rain) 119.65:6.8b 5.0 × 10-4 (heat 100°C, 5 min) 48.75:3.1
Coronary artery Control 3.2x10 -5 5 . 0 × 10 - 4
64.2 + 4.6 44.35:2.5 " 28.4+_7.7 a
c a n i n e coronary artery a n d the rabbit aorta. Since the enzyme might have been inactivated in the course of the extraction procedure, it was tested for in minced vessels. As shown in table 4, m i n c e d c a n i n e coronary arteries and femoral arteries liberated inorganic nitrite slowly. Nitrite formation was inhibited by the same c o n c e n t r a t i o n of C u S O 4 as antagonized the nitroglycerin-induced vasodilation. However, contrary to our expectations, the formation of inorganic nitrite in the vessels was potentiated after heat t r e a t m e n t (100°C, 5 min), which suggests that this response was not catalyzed by the enzyme.
(.~
3.6. Chemical reaction of nitroglycerin with sulfhydryl reagents at physiological p H
-40-
Control I
~
-60 -
(%7
Fig. 4. Effect of C u S O 4 o n high Ca2+-induced vasodilation in isolated canine large coronary artery. In this experiment 10 -4 M of papaverine was added to the bath at the beginning of the experiment because high Ca2+ markedly inhibited the response to papaverine. Each value represents a percent of the response induced by papaverine. The values are the mean 5: S.E. from 5-7 preparations.
Nitroglycerin 0.1 m M was reacted with 1 m M of sulfhydryl reagents in Tyrode solution. A d d i n g reduced glutathione, dithiothreitol a n d cysteine to nitroglycerin formed 2.00 + 0.12, 2.05 + 0.18 a n d 5.08 + 0.14 ~ m o l n i t r i t e / m l per 30 m i n respectively, whereas oxidized glutathione a n d histidine did not cause such a reaction.
3. 7. Analysis of tissue sulfhydryl Sulfhydryl in the 900 × g s u p e r n a t a n t from the large and small coronary arteries was 55.7 and 44.4 ~ m o l / g p r o t e i n respectively.
152 4. Discussion
Faro and McGregor (1968) and Winbury et al. (1969) proposed that nitroglycerin induced the preferential relaxation of the large coronary artery and subsequently facilitated redistribution of the blood to the ischemic region in patients with angina pectoris. This finding was also confirmed with isolated coronary arteries (Schnaar and Sparks, 1972; Kamitani et al., 1977). This characteristic of nitroglycerin was reconfirmed in the present study (table 1). NaNO 2 showed the same selectivity although the selectivity was less than that of nitroglycerin. In contrast, sodium nitroprusside and the so-called non-specific vasodilators, iproveratril and papaverine, had almost the same effect on the two kinds of arteries. Needleman et al. (1973) proposed that vasodilation was produced via interaction with the sulfhydryl groups of the vessels because ethacrynic acid, a sulfhydryl-alkylating agent, markedly decreased the tissue sulfhydryl groups and decreased vascular sensitivity to all the vasodilators tested. However, we did not obtain the same effect (fig. 3). The effect of nitroglycerin and NaNO 2 was markedly antagonized by ethacrynic acid whereas the antagonizing effect against sodium nitroprusside was only slight and the effect of iproveratril and papaverine was not disturbed at all, although the relaxation appeared more slowly than in the non-treated vessels. It is likely that the non-specific vasodilators do not essentially require the sulfhydryl groups for vasodilation. Needleman et al. (1969) showed that organic nitrate esters, which show potent vasodilator activity, seemed to be rapidly denitrated in the presence of reduced glutathione; this might suggest that intermediate(s) or inorganic nitrite released from nitroglycerin triggered the vasodilation. Afterward, these workers showed that nitroglycerin receptors were present in certain critical sulfhydryl groups by using nitroglycerin-tolerant vessels (Needleman and Johnson, 1973; Needleman et al., 1973). To study why nitroglycerin causes different responses on the large and small coronary arteries and whether there were differences in the requirement of the tissue sulfhydryl groups, we examined
the effect of cupric sulfate which is known as an inhibitor of nitroglycerin metabolizing enzyme (Heppel and Hilmoe, 1950) on nitroglycerin-induced vasodilation. As was expected. CuSO4 antagonized the effect of nitroglycerin (fig. 1) and the antagonism seemed to result from a disturbance of the reaction between nitroglycerin and the tissue sulfhydryl groups. The reasons are as follows: (1) inhibition of the nitroglycerin-induced vasodilation by cupric sulfate disappeared after treatment with sulfhydryl reagents (fig. 2); (2) dithiothreitol, a potent sulfhydryl reagent, potentiated the vasodilator response to nitroglycerin (fig. 2); (3) nitroglycerin liberated inorganic nitrite in the minced vessels and the nitrite liberation was inhibited by the same concentrations of CuSO 4 as antagonized the nitroglycerin-induced vasodilation although the denitration did not seem to be enzymatically catalyzed (table 4); (4) nitroglycerin also liberated inorganic nitrite even in the chemical reaction with sulfllydryl reagents at physiological pH. Since the effect of NaNO 2 was hardly disturbed by treatment with CuSO 4 (table 2) whereas it was markedly inhibited by ethacrynic acid (fig. 3) there seems to be a difference between the critical sulfhydryl groups for nitroglycerin- and N O [ - i n d u c e d vasodilation. This may suggest that nitroglycerininduced vasodilation is not essentially due to nitrite ion formation but to intermediate(s) synthesized in the course of nitroglycerin degradation, as was suggested in the review by Litchfield (organic nitrite or nitrated glutathione) (1973) and by Ignarro and Gruetter (S-nitrosothiols) (1980). CuSO 4 may disturb such intermediate formations. It has already been reported that Cu 2 + penetrated the cell membrane (Neumann and Silverberg, 1966). Although CuSO 4 may be an inhibitor of Na+-K + ATPase (Ting-Beall et al., 1973), the effect did not seem to take part in the antagonism against nitroglycerin because ouabain or Na + free conditions did not prevent the antagonism. It is unlikely that Cu 2+ had any effect on the contractile proteins because the ion had no effect on glycerinated rabbit aorta contracted by ATP (data not shown). Since the mitochondrial population in the small coronary artery was more numerous than in the large coronary artery, al-
153
though nitroglycerin-induced vasodilation was more marked in the large coronary artery (Kamitani et al., 1977), it is unlikely that mitochondria play an important role in the Cu 2÷induced antagonism against nitroglycerin-induced vasodilation. Since CuSO4 diminished the membrane stabilizing activity of Ca 2 + (fig. 4) the possible site of action of Cu 2+ seems to be on the cell membrane though the possibility remains that the effect of CuSO4 on the membrane stabilizing activity of Ca z+ and its effect on nitroglycerin-induced relaxation may be separate events. CdCI 2, which is also an inhibitor of sulfhydryl groups and does not seem to penetrate the cell membrane of the vessels (Toda, 1976), induced a significant potentiation of nitroglycerin-induced vasodilation in the rat aorta (table 3), which suggests that the outer surface of the cell membrane does not seem to be a major site in nitroglycerin vasodilation. The Cd2÷-in duced potentiation might result from the blocking of nitroglycerin degradation on the outer surface of the cell membrane. On the basis of these findings, the sulfhydryl groups on the inner surface of the cell membrane seem to be most probable site for Cu2÷-induced antagonism against nitroglycerin. The sulfhydryl groups, which are able to bind Cu 2 + on the inner surface of the cell membrane, may have a role in organic nitrate tolerance, because NaNO 2 and sodium nitroprusside are not cross-tolerant in nitroglycerin-tolerant vessels (Needleman and Johnson, 1973, 1975; Needleman et al., 1973). Recent evidence supports the theory that cGMP is involved in the regulation of vascular relaxation induced by nitroglycerin, N a N O 2 and sodium nitroprusside (Schultz et al., 1977; Katsuki and Murad, 1976, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1979, 1982; Ignarro and Gruetter, 1980; Kobayashi et al., 1980; Gruetter et al., 1980; Ignarro et al., 1980). There seem to be some differences among these drugs: (1) nitroglycerin and N a N O 2 failed to activate soluble vascular guanylate cyclase in the absence of an added thiol whereas sodium nitroprusside did not require the thiols (Ignarro and Gruetter, 1980); (2) nitroglycerin specifically required cysteine while N a N O 2 required one of several thiols or ascorbate to activate soluble guanylate cyclase from bovine coronary arteries (Ignarro and Gruetter, 1980); (3)
sodium nitroprusside increased guanylate cyclase activity in most of the preparations, whereas nitroglycerin was tissue specific (Katsuki et al., 1977a); (4) nitroglycerin is a weak activator while sodium nitroprusside is a potent activator of this enzyme (Katsuki et al., 1977a), although the vasodilator effects of both drugs are almost the same (Watkins and Davidson, 1980). Several reports described Cu 2÷ (White et al., 1976; Gerzer et al., 1981) and sulfhydryl groups (Brhme et al., 1978; Braughler, 1983) as important constituents of guanylate cyclase. However, since Cu 2÷ could not inhibit N a N O 2- or nitroprussideinduced vasodilation, the inhibitory effect of Cu 2+ on nitroglycerin-induced vasodilation does not seem to be due to its direct effect on guanylate cyclase. Taking account of these findings we concluded that: Nitroglycerin reacts non-enzymatically with the sulfhydryl groups, which are thought to be on the inner surface of the cell membrane and prone to inhibition by Cu 2÷ and ethacrynic acid, and subsequently forms an active intermediate(s). This intermediate(s) may activate guanylate cyclase in the same way as sodium nitroprusside to induce an increase in cGMP and subsequent vasodilation. The selectivity of nitroglycerin on the large coronary artery (table 1), which was not the case for nitroprusside, may be derived from the difference in the concentrations of the sulfhydryl groups on the inner surface of the cell membrane but not from the effect on guanylate cyclase. This proposal gains support from the finding that sulfhydryl concentrations in the large coronary artery were higher than in the small coronary artery. There seem to be two kinds of sulfhydryl groups required for nitrate- and nitrite-induced vasodilation. One is preferentially acceptable to Cu z+ and the other to ethacrynic acid. The site of action of N a N O 2 seems to be on the latter, because the vascular effect of this drug was inhibited by pretreatment with ethacrynic acid but not with Cu 2+.
References Axelsson, K.L., R.G.G. Andersson and J.E.S. Wikberg, 1982, Vascular smooth muscle relaxation by nitro compounds:
154 reduced relaxation and c G M P elevation in tolerant vessels and reversal of tolerance by dithiothreitol, Acta Pharmacol. Toxicol. 50, 350. Axelsson, K . L , J.E.S. Wikberg and R.G.G. Andersson, 1979, Relationship between nitroglycerin, cyclic G M P and relaxation of vascular smooth muscle, Life Sci. 24, 1779. Brhme, E., H. Graf and G. Schultz, 1978, Effects of sodium nitroprusside and other smooth muscle relaxants on cyclic G M P formation in smooth muscle and platelets, Adv. Cycl. Nucl. Res. 9, 131. Braughler, J.M., 1983, Soluble guanylate cyclase activation by nitric oxide and its reversal, Biochem. Pharmacol. 32, 811. EIlman, G . L , 1959, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82, 70. Fam, W.M. and M. McGregor, 1968, Effect of nitroglycerin and dipyridamole on regional coronary resistance, Circ. Res. 22, 649. Gerzer, R., E. BOhme, F. H o f m a n n and G. Schultz, 1981, Soluble guanylate cyclase purified from bovine lung contains heine and copper, FEBS Lett. 132, 71. Gruetter, D.Y., C.A. Gruetter, B.K. Barry, W.H. Baricos, A.L. Hyman, P.J. Kadowitz and L.J. Ignarro, 1980, Activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and nitrosoguanidine - Inhibition by calcium, lanthanum and other cations, enhancement by thiols, Biochem. Pharmacol. 29, 2943. Heppel, L.A. and R.J. Hilmoe, 1950, Metabolism of inorganic nitrite and nitrate esters. II. The enzymatic reduction of nitroglycerin and erythritol tetranitrate by glutathione, J. Biol. Chem. 183, 129. Ignarro, L.J., B.K. Barry, D.Y. Gruetter, J.C. Edwards, E.H. Ohlstein, C.A. Gruetter and W.H. Baricos, 1980, Guanylate cyclase activation by nitroprusside and nitrosoguanidine is related to formation of S-nitrosothiol intermediates, Biochem. Biophys. Res. C o m m u n . 94, 93. Ignarro, L.J. and C.A. Gruetter, 1980, Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite, Biochim. Biophys. Acta 631, 221. Kamitani, T., K. Nakano, J. Mori, S. Katsuki and F. Honda, 1977, Local specificity in responses of canine coronary vessels to oxygen deficiency and antianginal drugs, Arch. Int. Pharmacodyn. Ther. 225, 257. Katsuki, S., W. Arnold, C. Mittal and F. Murad, 1977a, Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine, J. Cycl. Nucl. Res. 3, 23. Katsuki, S., W.P. Arnold and F. Murad, 1977b, Effects of sodium nitroprusside, nitroglycerin and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues, J. Cych Nucl. Res. 3, 239. Katsuki, S. and F. Murad, 1976, Cyclic G M P and cyclic A M P levels during contraction and relaxation of bovine tracheal smooth muscle, Pharmacologist 18, 220. Katsuki, S. and F. Murad, 1977, Regulation of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophos-
phate levels and contractility in bovine tracheal smooth muscle, Mol. Pharmacol. 13, 330. Kobayashi, A., Y. Suzuki, T. Kamikawa, H. Hayashi and N. Yamazaki, 1980, The effects of nitroglycerin on cyclic nucleotides in the coronary artery in vivo, Life Sci. 27, 1679. Kreye, V.A.W., G.D. Baron, J.B. Li~th and H. Schmidt-Gayk, 1975, Mode of action of sodium nitroprusside on vascular smooth muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 288, 381. Litchfield, M.H., 1973, Recent views on the mechanisms of nitrate ester metabolism, Drug Metab. Rev. 2, 239. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. Needleman, P., D.L Blehm and K.S. Rotskoff, 1969, Relationship between glutathione-dependent denitration and the vasodilator effectiveness of organic nitrates, J. Pharmacol. Exp. Ther. 165, 286. Needleman, P., B. Jakschik and E.M. Johnson, Jr., 1973, Sulfhydryl requirement for relaxation of vascular smooth muscle, J. Pharmacol. Exp. Ther. 187, 324. Needleman, P. and E.M. Johnson Jr., 1973, Mechanism of tolerance development to organic nitrates, J. Pharmacol. Exp. Ther. 184, 709. Needleman, P. and E.M. Johnson Jr., 1975, Sulfhydryl reactivity of organic nitrates: Tolerance to vasodilation., 2rid International Symposium. Odense 1075, 208, Karger, Basel, 1976. N e u m a n n , P.Z. and M. Silverberg, 1966, Active copper transport in m a m m a l i a n tissues a possible role in Wilson's disease, Nature 210, 414. Schnaar, R.L. and H.V. Sparks, 1972, Response of large and small coronary arteries to nitroglycerin, N a N O 2, and adenosine, Am. J. Physiol. 223, 223. Schultz, K.D., K. Schultz and G. Schultz, 1977, Sodium nitroprusside and other smooth muscle-relaxants increase cyclic G M P levels in rat ductus deferens, Nature 265, 750. Ting-Beall, H.P., D.A. Clark, C.H. Suelter and W.W. Wells, 1973, Studies on the interaction of chick brain microsomal (Na + + K + )-ATPase with copper, Biochim. Biophys. Acta 291, 229. Toda, N., 1976, Mechanism of inhibitory action of Cd 2+ on muscle contractility as related to Ca 2+, Jap. J. Pharmacol. 26, Suppl. 5p. Watkins, R.W. and I.W.F. Davidson, 1980, Comparative effects of nitroprusside and nitroglycerin: actions on phasic and tonic components of arterial smooth muscle contraction, European J. Pharmacol. 62, 191. White, A.A., K.M. Crawford, C.S. Patt and P.J. Lad, 1976, Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide, J. Biol. Chem. 251, 7304. Winbury, M.M., B.B. Howe and M.A. Hefner, 1969, Effect of nitrates and other coronary dilators on large and small coronary vessels: an hypothesis for the mechanism of action of nitrates, J. Pharmacol. Exp. Ther. 168, 70.
145
Elsevier
I N H I B I T O R Y E F F E C T O F C u 2 + ON N I T R O G L Y C E R I N - I N D U C E D V A S O D I L A T I O N TOSHIHARU KAM1TANI Research Laboratories, Fufisawa Pharmaceutical Co. Ltd., Yodogawa-ku, Osaka 532, Japan
Received 19 August 1983, revised MS received 2 December 1983, accepted 17 January 1984
T. KAMITANI, Inhibitory effect of Cu e + on nitroglycerin-induced vasodilation, European J. Pharmacol. 100 (1984) 145-154. The mechanism of nitroglycerin-induced vasodilation was examined in isolated arteries. Nitroglycerin relaxed the large coronary artery preferentially whereas nitroprusside and the so-called non-specific vasodilators showed the same activities on both the large and small coronary arteries. Nitroglycerin-induced vasodilation but not the vasodilation induced by the other agents was antagonized markedly by pretreatment with CuSO4. Other metal ions except Fe 2+ had no antagonizing effect. The Cu2+-induced antagonism was restored by treatment with sulfhydryl reagents. Nitroglycerin formed inorganic nitrite by non-enzymatic reaction with the tissue sulfhydryl groups; the reaction was also inhibited by Cu 2÷. Cu 2÷ suppressed the membrane stabilizing effect of Ca 2+. It seems that nitroglycerin reacts with the sulfhydryl groups on the inner surface of the cell membrane, which may take part in the selectivity of this drug, and subsequently the intermediate(s) formed may activate guanylate cyclase. Nitroglycerin
Vasodilators
Sulfhydryl groups
I. I n t r o d u c t i o n
Nitroglycerin, the most effective drug in the treatment of angina pectoris, reacts with reduced glutathione (GSH) to form oxidized glutathione and inorganic nitrite, a reaction which is catalyzed by the liver enzyme glutathione-dependent organic nitrate reductase (Heppel and Hilmoe, 1950). These authors also observed that in the presence of GSH, a non-enzymatic reaction took place and liberated nitrite. Cupric sulfate inhibited both the enzymecatalyzed and the spontaneous reaction. The notion of a sulfhydryl-containing, specific 'organic nitrate receptor' as the site of action for nitroglycerin and related organic nitro-compounds in vascular smooth muscle was first advanced by Needleman and coworkers (Needleman and Johnson, 1973; Needleman et al., 1973). Based on the findings that nitroglycerin-induced tolerance resulted from the oxidation of critical sulfhydryl groups in the vascular bed and that there was cross-tolerance to all organic nitrates but not to non-nitrate vasodilators, this group proposed a 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Cupric ion
hypothesis: The sulfhydryl groups are oxidized to the disulfide form, the organic nitrate molecule is reduced to the denitrated metabolite, nitrate ion is released and as a consequence of the interaction, relaxation ensues. Recently, considerable evidence has accumulated to support the theory that c G M P is involved 'in the regulation of nitroglycerin-induced smooth muscle relaxation (Schultz et al., 1977; Katsuki and Murad, 1976, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1979; Ignarro and Gruetter, 1980; Kobayashi et al., 1980). When nitroglycerin was given to dogs, an increase in the coronary blood flow was preceded by an increase in the c G M P concentration of the coronary artery (Kobayashi et al., 1980). Sodium nitroprusside, which acts directly on vascular smooth muscle (Kreye et al., 1975), may also induce vasodilation via c G M P elevation (Schultz et al., 1977; Katsuki and Murad, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1982; Ignarro and Gruetter, 1980; Gruetter et al., 1980; Ignarro et al., 1980), although there seems to be a slight dissociation
146
between the elevation of c G M P and the relaxation (Axelsson et al., 1982). There are some differences between the effect of nitroglycerin and of sodium nitroprusside: (1) nitroglycerin fails to activate soluble vascular guanylate cyclase in the absence of an added thiol, whereas sodium nitroprusside does not require the addition of thiols (Ignarro and Gruetter, 1980); (2) nitroglycerin is a weak activator while sodium nitroprusside is a potent activator of this enzyme (Katsuki et al., 1977a) although the vasodilator effects of both drugs are almost the same (Watkins and Davidson, 1980); (3) sodium nitroprusside increased guanylate cyclase activity in most of the preparations whereas the effect of nitroglycerin was tissue specific (Katsuki et al., 1977a); (4) regarding the tolerance and cross-tolerance characteristics of organic nitrate-tolerant vessels, sodium nitroprusside does not have the features of the organic nitrates (Needleman and Johnson, 1975). Since Cu 2+ is known as an inhibitor of glutathione-dependent denitration (Heppel and Hilmoe, 1950) and seems to play an important role in the regulation of guanylate cyclase activity (White et al., 1976; Gerzer et al., 1981), the effect of pretreatment with Cu z+ on nitroglycerin-induced vasodilation was examined in order to clarify the relation between the vasodilating activity of nitroglycerin and denitration. 2. Materials and methods
2.1. Effect on the isolated arteries Under pentobarbital Na anesthesia (35 m g / k g i.p.), the large (2 m m o.d.) and small (0.3 m m i.d.) coronary arteries were removed from mongrel dogs of either sex. The thoracic aorta was removed from male Wistar strain rats aged 7 weeks. Helical strips cut from the large coronary artery, the small coronary artery and the rat thoracic aorta were approximately 15 x 2 ram, 4 x 0.5 m m and 20 x 2 m m respectively. The strips were suspended in organ baths containing Tyrode solution. Each preparation was connected to a strain gauge and the tension was measured isometrically. The bath solution was aerated with a mixture of 95% 02 and 5%
CO 2 and maintained at 370C. Before the experiments were started, the strips were allowed to equilibrate for more than 60 rain in the bath solution. Vasodilator activity was examined under the active tension induced by 35 mM KCI. In the canine vessels this concentration of KCI produced a contraction up to about 65% of the maximum effect, which was reached with 70 mM KC1. In the rat aorta, 35 mM of KC1 induced submaximal contraction. In some experiments 2 mM of BaCI 2 was used instead of 35 mM of KCI. The tension of the strips from the large coronary artery, the small coronary artery and the rat thoracic aorta were adjusted respectively to 1.5 g, 0.12 g and 1.5 g and the test drugs were added cumulatively. At the end of each series of experiments, l0 4 M of papaverine was added to the organ bath. The values presented are relative to the papaverine-induced relaxation.
2.2. Inorganic' nitrite formation from nitroglycerin in the reaction with minced uascular muscles or sulfh ydrvl reagents Mongrel dogs of either sex were anesthetized with pentobarbital Na (35 m g / k g i.p.). The coronary artery and the femoral artery were dissected and minced finely; 200 mg wet weight of the minced vessels was immersed in 1 ml of Tyrode solution and maintained at 37°C. The reaction was started by adding 0.1 ml of nitroglycerin solution (final concentration of nitroglycerin: 10 4 M). The reaction was stopped by adding 1.1 ml of 5% HgCI 2. After centrifuging, the concentration of inorganic nitrite in the supernatant was measured by the colorimetric method of Heppel and Hilmoe (1950). Protein was measured by the biuret procedure. The chemical reaction of nitroglycerin with sulfhydryl reagents was also examined in Tyrode solution. Nitroglycerin (10 -4 M) and sulfhydryl reagents (10 3 M) were dissolved in Tyrode solution kept at 37°C. The concentration of inorganic nitrite formed was measured 30 min after the start of the reaction by the method mentioned above.
2.3. Analysis of tissue sulfllydryl Minced canine vessels (25-50 mg) kept at - 9 7 ° C until used were added to 5 ml of chilled
147 TABLE 1 Vasodilator effect on isolated canine large and small coronary arteries contracted by KCI. The EDso values of nitroglycerin and the 95% confidence limits were calculated by linear regression analysis, n = 5-14. Drug
EDso (M)
Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
EDs0 (S)
Large coronary artery
Small coronary artery
EDs0 (L)
1.7 X 10 -7 (0.5- 4.9X10 -7) 6.7 × 10 - s (0.8- 51.0 x 10 - s ) 9.0 x 10 - 7 (1.7-100.1 x 10 -7 9,5 × 10 -6 (0.9- 62.8 x 10 -6) 7.3 x 10- s (2.1- 21.8x10 - s )
2.3 × 10 6 (0.5-67.5X10 -6) 1.7 x 10 -4 (0.5-14.0 x 10 - 4 ) 4.9 x 10- 7 (1.1-23.7 × 10 -7) 7.7 x 10 -6 (0.8-74.3 x 10 -6) 1.1 x 10- 7 (0.1- 5.4x10 -7)
13.5
1.6X 10-5 (M)
4 .ox 10-9 0
0-
20
20.
40
40"
~ % ~ _ 1 2 5
}*M
1.5
As shown in table 1, nitroglycerin showed far greater selectivity on the large coronary artery than on the small coronary artery. Sodium nitrite had the same tendency but the selectivity was not so pronounced. In contrast, sodium nitroprusside, iproveratril and papaverine had almost the same activities on these two kinds of arteries.
All chemicals except ethacrynic acid were dissolved in distilled water. Ethacrynic acid was dissolved in ethanol.
I0-6
0,8
3.1. Effect on the isolated canine arteries contracted by KCI
2.4. Drugs
6.4x I0-8
0.5
3. Results
glucose-free Tyrode solution, homogenized and centrifuged at 900 x g foc 10 min. Sulfhydryl in the supernatant was measured by the method of Ellman with dithiobisnitrobenzoic acid (1959). Protein was measured by the method of Lowry et al. (1951).
4.0x 10-9
2.5
I
6.4x 10-8 l
I
1.6x I0-5 (M)
I0-6 I
I
I
I
60"
60 500
80-
80" .
~ too" (%)
O:>ntxol
too- (%)
Fig. 1. Effect of CuSO4 on nitroglycerin-induced vasodilation in isolated canine large coronary artery (left panel) and rat thoracic aorta (right panel). Each value represents a percent of the response induced by 10-4 M of papaverine. The values are the mean :h S.E. from 5-21 preparations.
148
3.2. Effect of pretreatment with CuSO 4, other metal ions and sulfhydryl reagents" The effect of pretreatment with CuSO4, other metal ions and sulfhydryl reagents on nitroglycerin-induced vasodilation was examined after washing out the pretreatment drugs.
3.2.1. CuS04 20 min-pretreatment with CuSO 4 in concentrations of 8 × 1 0 - 6 M or higher dose dependently
diminished the vasodilator effect of nitroglycerin in the canine large coronary artery and the rat aorta which were contracted by KC1 (fig. 1). The diminution also appeared when BaCl 2 was used as a vasoconstrictor (table 2). No tachyphylaxis was seen though the effect of nitroglycerin was determined before and after the CuSO 4 treatment in the same preparation in the case of canine large coronary artery (data not shown). When the highest concentration of CuSO 4 was added to the bath, the Tyrode solution became turbid probably because
TABLE 2 Effect of 5 × 10 - 4 M C u S O 4 p r e t r e a t m e n t on EDs0 values of several vasodilators with isolated c a n i n e large c o r o n a r y artery and rat aorta. Dose-response curves to v a s o d i l a t o r s were o b t a i n e d before and after a 20 m i n p r e t r e a t m e n t with C u S O 4 which was w a s h e d out before testing. EDs0 values and the 95% confidence limits were calculated from linear regression analysis. The e x p e r i m e n t was c o n d u c t e d with 5 to 21 preparations. Drug
EDs0 (M) Non-treatment (A)
Ratio P r e t r e a t m e n t with CuSO 4 (B)
(B)/(A)
1.76×10 5 (0.22-14.06 × 10- 5) 1.44× l0 4 (0.25- 8 . 3 8 × I 0 4) 1.10× 10 ~ (0.26- 4 . 6 8 × 1 0 6) 1.46 × 10 s (0.32- 6 . 7 6 × 1 0 - 5 ) 1.28×10 7 (0.28- 5 . 9 3 × 1 0 7)
68.8
3 . 9 8 × 10 ~' ( 0 . 3 2 - 4 8 . 7 0 × 10 - 6 ) 1.90× l0 4 (0.36-10.08 × 1 0 4) 5.59xl0 6 (0.97-32.14 × 10- ~') 1.26×10 s (0.29- 5 . 5 5 × 1 0 5) 2.02 × 10 7 (0.42- 9 . 7 2 × 1 0 7)
96.8
1.71 x 10 -6 (0.22-13.25×10 1.86X 10 4 (0.45- 7 . 7 6 × 1 0 5.53 × 10 - s (1.01-30.35×10 5.42×10 `6 (1.91-15.42 × 10 2.38 × 10 -~ (0.71 8 . 0 0 × 1 0
46.0
Coronary artery contracted by 35 m M KC1 Nitroglycerin NaNO z Nitroprusside Na Papaverine Iproveratril
2.56 × 1 0 - 7 (0.88- 7.43 x 10 - 7 6.01 × 1 0 - 5 (1.07-33.69 × 1 0 - ~ 3.28 × 10 . 7 (0.91-11.87 x 1 0 - v 1.13 × 10 - s (0.32- 3 . 9 8 × 1 0 5 1.17 × 10 7 (0.19- 7 . 0 2 × 1 0 . 7
2.4 3.4 1.3 13
Coronary artery contracted by 2 m M BaCI 2 Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
4.11 × 10 - s (0.44-38.88 × 10 8 2.57×10 5 ( 0 . 5 4 - 1 2 . 3 0 × 10 -5 4.85 x 10 7 (1.00-23.48×10 7 9.67 × 10 6 (2.71-34.59 × 10 6 1 . 6 7 × 10 v (0.40- 7 . 0 5 x 1 0 7
7.4 11.5 1.3 1.2
Rat aorta contracted by 35 m M KCI Nitroglycerin NaNO 2 Nitroprusside Na Papaverine Iproveratril
3 . 7 2 × 10 8 (0.66-20,96 x 10 8 1.36×10 4 (0.35- 5.36 x 1074 1.00 × 10 ~ (0.19- 5 . 3 4 × 10 s 8 . 1 0 x 10 6 (2.91-22.50 × 10 6 1.41 × 10 s (0.31- 6 . 4 9 × 1 0 s
6) 1.4 4) 5.5 s) 0.7 6) 1.7 ~)
149
of the production of CuCO 3 a n d / o r Cu3(PO4) 2. This antagonism was not induced in the case of the other vasodilators, although the effect of N a N O 2 and sodium nitroprusside was more or less inhibited by CuSO4 in the vessels contracted by BaC12 (table 2). This antagonizing effect of CuSO 4 against nitroglycerin could not have been due to SO 2 - because pretreatment with equimolar Na2SO 4 did not cause any change in the nitroglycerin dose-response curve (data not shown). Since KCl-induced contraction was potentiated to some extent after CuSO4 treatment, the effect of nitroglycerin after CuSO 4 treatment was examined under low Ca 2+ conditions in the canine large coronary artery. The Ca 2÷ concentration decreased from 1.8 m M in the normal Tyrode solution to 1.0-1.5 mM. The procedure maintained the tonus of the vessel treated with CuSO4 at the same level as before treatment but did not block the antagonism (EDs0 values; 2.3 x 10 -7 M in normal Tyrode solution and 7.7 × 10-6 M after treatment with CuSO4 under low Ca 2+ conditions). The possibility that nitroglycerin may be unstable in the presence of Cu 2÷ is not likely since nitroglycerin, which was recovered from a mixture of CuSO 4 and nitroglycerin by using Amberlite XAD2 had the same activity as non-treated nitroglycerin in the rat aorta (EDso values; 4.0 × 10 - s and 5.7 × 10 -8 M respectively).
3.2.2. Other metal ions (table 3) Since the antagonizing effect of Cu 2+ against
nitroglycerin also appeared in the rat thoracic aorta (fig. 1), the effect of other metal ions was examined in this preparation. The effect of pretreatment with and without other metal ions was tested in separate preparations removed from the same animal. Fe 2÷ showed the same antagonizing effect as Cu z÷ whereas Ni 2+, Fe 3÷, Co 2+ and Mn 2÷ had no effect at a concentration of 5 × 1 0 - 4 M. Since 5 x 10 - 4 M of Hg 2+ and Cd 2+ inhibited KCl-induced contraction, the effect of these drugs was evaluated at lower concentrations. HgCI 2 8 × 10 -6 M tended to potentiate the vasodilator response of nitroglycerin. On the other hand, 3.2 x 10 _5 M of Cd 2÷ significantly potentiated the nitroglycerin-induced vasodilation in rat aorta. This potentiation was not seen in the canine large coronary artery (data not shown).
3.2.3. Sulfhydryl reagents (fig. 2) 4 mM of reduced glutathione and 1 m M of L-cysteine had no effect on the dose-response curve of nitroglycerin whereas 1 mM of dithiothreitol markedly potentiated the effect of nitroglycerin. All these drugs restored the Cu 2÷-induced antagonism against nitroglycerin.
3.2.4. Ethacrynic acid (fig. 3) The effect of 2.5 × 10-5 M of ethacrynic acid, an alkylating agent of the sulfhydryl groups, was also examined in the rat aorta. A 1 h pretreatment with this drug markedly lessened nitroglycerinand NaNO2-induced vasodilation, whereas the
TABLE 3 Effect of pretreatment with metal ions on nitroglycerin-induced vasodilation in the isolated rat aorta contracted by KCI. EDso values of nitroglycerin and the 95% confidence limits were calculated by linear regression analysis. The experiment was done with 5-8 preparations. Ion
Cone. (M)
ED5o (M) (95% confidence limits)
EDs0 (treatment) EDso (control)
Control Cu 2+ Fe z+ Fe 3+ Ni 2+ Co 2+ M n 2+ Hg 2+ Cd 2+
5 ×10 -4 5 )'(10 - 4 5 >(10 - 4 5 ×10 -4 5 × 1 0 -4 5 × 1 0 -4 8 xl0 -6 3.2 X 1 0 - s
3.72 x 10- s (0.66-20.96 × 1 0 - s) 1 . 7 1 x 1 0 6(0.22-13.25×10-6) 7.81×10-'~(0.82-74.12×10 -7) 1 . 1 6 × 1 0 - 7 ( 0 . 2 0 - 6.66X10 -7) 1 . 1 6 × 1 0 - 7 ( 0 . 1 8 - 7 . 5 8 × 1 0 7) 9.16 x 1 0 - s (1.27-66.14 × 1 0 - 8 ) 1 . 3 2 x 1 0 8(0.18- 9 . 5 1 x 1 0 - s ) 1.97×10 s(0.29-13.46x10 - s ) 8.2 X 10 -9 (l.0 -66.6 × 10 9)
46 21 3 3 2 0.4 0.5 0.2
150 4.0
6.4
1.0
4.0 6.4 1.0 1.6 xl~ 9 xl08 x166 x165
1.6
xl09 xl08 xl06 xl05 i
i
i
i
J
4.o xl09
6.4 xl08
1.0
1.6
xl(~ 6
xlOS(H)
l
50 ~ C u + D T T Cu+GSH
~
Cys
"
r
,
Cu*
SH I00
i0(
lOO
4 mM GSH
i mM
0.25mM
" N~.~t~ICu+DTT lmM ~ ' R C o n t ro 1 ° ~ "~DTT 0.25raM DTT ImM
Dithiothreitol
Cysteine
Fig. 2. Effect of pretreatment with sulfhydryl reagents on nitroglycerin-induced vasodilation and the reversal of CuSO 4 (5 × 10 4 M)-induced antagonism against nitroglycerin in the isolated rat aorta. Each value represents a percent of the response induced by 10 -4 M of papaverine. The values are the mean + S.E. from 3-5 preparations.
vasodilation induced by sodium nitroprusside was decreased only slightly. In contrast, the effect of iproveratril and papaverine was not disturbed at all.
brain microsomes, we examined whether the antagonizing effect of Cu 2+ on the nitroglycerininduced vasodilation in the canine large coronary artery was due to inhibition of Na+-K + ATPase. However, the dose-response curves of nitroglycerin with or without 1 0 - 7 M of ouabain did not differ. The EDs0 values were 6.9 × 10 _7 M and 2.0 × 10 7 M respectively. Though Na+-K + ATPase was also inhibited by a 1 h treatment with Na ÷ free Tyrode
3. 3. Effect of Na +-K + A TPase inhibition Since Ting-Beall et al. (1973) reported that Cu 2+ strongly inhibited Na+-K ÷ ATPase of chicken 4.0x 6o4X 10-9 10-8
1.6x 10-6 10-5
1.6x 10-6 10-5 °
I
50-
~ .2
t
Control
i00-
N i t r o g l ~ (%) 1.6x 10-6 10-5
i ~
50-
2.5x 10-4
i
I
.k.
Papaverinel i00- (%) ~
i00-
)
0
50 -
2.5x 10-4
10-3 0
I
1.6x 10-8 I
0-9 <
2.5x 4.0x 10-7 10-6 (M) I *
E.A.
50- ~
~
%
~
Nitroprusside i00- (%)
l',lat~2 (%l
4.0x
6.4x
0 -9
10 -8
I
I
1.6x 10 -6
10 -5 (M)
i
E.A. _[prover I o at n _~%~.._ i "A _
io0- (%) ril
Fig. 3. Effect of pretreatment with 2.5 × 10 -5 M of ethacrynic acid on vasodilation of the isolated rat aorta. Each value represents a percent of the response induced by 10 -4 M of papaverine. The values are the mean + S.E. from 5 preparations.
151 solution in which N a + was replaced by equimolar Li +, the effect of nitroglycerin remained unchanged ( E D s 0 : 4 . 8 × 10 -7 M in n o r m a l Tyrode solution, 5.0 × 10 -7 M in N a + free Tyrode solution).
TABLE 4 Effect of CuSO4 on inorganic nitrite liberation from nitroglycerin in response to the addition of minced canine femoral and coronary arteries. Each value represents the mean 5: S.E. of 3 experiments, a p < 0.05, b p < 0.001. Conc. of CuSO4 (M)
3.4. Effect of Cu 2 + on Ca 2 +-induced vasodilation
Liberated NOf (nmol/g tissue per 30 rain)
Femoral artery It is k n o w n that excess a m o u n t s of Ca 2+ show a m e m b r a n e - s t a b i l i z i n g effect a n d cause vasodilation. To ascertain the site of action of Cu 2+, the effect of CuSO 4 p r e t r e a t m e n t o n the vasodilation i n d u c e d by CaCI 2 was examined i n ' t h e rat aorta contracted by KCI. As shown in fig. 4, more than 6.8 m M of CaC12 caused dose-dependent relaxation. P r e t r e a t m e n t with 5 × 10 -4 M of CuSO 4 shifted the dose-response curve to the right a n d the m a x i m a l relaxation b y CaC12 was suppressed.
3.5. Inorganic nitrite liberation from nitroglycerin in minced vascular smooth muscles Nitroglycerin metabolizing enzyme, which has already been shown to be in the liver (Heppel and Hilmoe, 1950), was extracted according to the m e t h o d of Heppel and Hilmoe (1950). However, we could not detect any enzyme activity in the
20
0
t~rmal
1.8..........---~
jL
6.,8-',.~1.,8
, ~1,.8
, 41;8,61.8
Control 3.2X10 -s
39.8 ___2.8 31.35:3.3 5.0 × 10 - 4 24.2 _ 2.2 a Control (heat 100°C, 5 rain) 119.65:6.8b 5.0 × 10-4 (heat 100°C, 5 min) 48.75:3.1
Coronary artery Control 3.2x10 -5 5 . 0 × 10 - 4
64.2 + 4.6 44.35:2.5 " 28.4+_7.7 a
c a n i n e coronary artery a n d the rabbit aorta. Since the enzyme might have been inactivated in the course of the extraction procedure, it was tested for in minced vessels. As shown in table 4, m i n c e d c a n i n e coronary arteries and femoral arteries liberated inorganic nitrite slowly. Nitrite formation was inhibited by the same c o n c e n t r a t i o n of C u S O 4 as antagonized the nitroglycerin-induced vasodilation. However, contrary to our expectations, the formation of inorganic nitrite in the vessels was potentiated after heat t r e a t m e n t (100°C, 5 min), which suggests that this response was not catalyzed by the enzyme.
(.~
3.6. Chemical reaction of nitroglycerin with sulfhydryl reagents at physiological p H
-40-
Control I
~
-60 -
(%7
Fig. 4. Effect of C u S O 4 o n high Ca2+-induced vasodilation in isolated canine large coronary artery. In this experiment 10 -4 M of papaverine was added to the bath at the beginning of the experiment because high Ca2+ markedly inhibited the response to papaverine. Each value represents a percent of the response induced by papaverine. The values are the mean 5: S.E. from 5-7 preparations.
Nitroglycerin 0.1 m M was reacted with 1 m M of sulfhydryl reagents in Tyrode solution. A d d i n g reduced glutathione, dithiothreitol a n d cysteine to nitroglycerin formed 2.00 + 0.12, 2.05 + 0.18 a n d 5.08 + 0.14 ~ m o l n i t r i t e / m l per 30 m i n respectively, whereas oxidized glutathione a n d histidine did not cause such a reaction.
3. 7. Analysis of tissue sulfhydryl Sulfhydryl in the 900 × g s u p e r n a t a n t from the large and small coronary arteries was 55.7 and 44.4 ~ m o l / g p r o t e i n respectively.
152 4. Discussion
Faro and McGregor (1968) and Winbury et al. (1969) proposed that nitroglycerin induced the preferential relaxation of the large coronary artery and subsequently facilitated redistribution of the blood to the ischemic region in patients with angina pectoris. This finding was also confirmed with isolated coronary arteries (Schnaar and Sparks, 1972; Kamitani et al., 1977). This characteristic of nitroglycerin was reconfirmed in the present study (table 1). NaNO 2 showed the same selectivity although the selectivity was less than that of nitroglycerin. In contrast, sodium nitroprusside and the so-called non-specific vasodilators, iproveratril and papaverine, had almost the same effect on the two kinds of arteries. Needleman et al. (1973) proposed that vasodilation was produced via interaction with the sulfhydryl groups of the vessels because ethacrynic acid, a sulfhydryl-alkylating agent, markedly decreased the tissue sulfhydryl groups and decreased vascular sensitivity to all the vasodilators tested. However, we did not obtain the same effect (fig. 3). The effect of nitroglycerin and NaNO 2 was markedly antagonized by ethacrynic acid whereas the antagonizing effect against sodium nitroprusside was only slight and the effect of iproveratril and papaverine was not disturbed at all, although the relaxation appeared more slowly than in the non-treated vessels. It is likely that the non-specific vasodilators do not essentially require the sulfhydryl groups for vasodilation. Needleman et al. (1969) showed that organic nitrate esters, which show potent vasodilator activity, seemed to be rapidly denitrated in the presence of reduced glutathione; this might suggest that intermediate(s) or inorganic nitrite released from nitroglycerin triggered the vasodilation. Afterward, these workers showed that nitroglycerin receptors were present in certain critical sulfhydryl groups by using nitroglycerin-tolerant vessels (Needleman and Johnson, 1973; Needleman et al., 1973). To study why nitroglycerin causes different responses on the large and small coronary arteries and whether there were differences in the requirement of the tissue sulfhydryl groups, we examined
the effect of cupric sulfate which is known as an inhibitor of nitroglycerin metabolizing enzyme (Heppel and Hilmoe, 1950) on nitroglycerin-induced vasodilation. As was expected. CuSO4 antagonized the effect of nitroglycerin (fig. 1) and the antagonism seemed to result from a disturbance of the reaction between nitroglycerin and the tissue sulfhydryl groups. The reasons are as follows: (1) inhibition of the nitroglycerin-induced vasodilation by cupric sulfate disappeared after treatment with sulfhydryl reagents (fig. 2); (2) dithiothreitol, a potent sulfhydryl reagent, potentiated the vasodilator response to nitroglycerin (fig. 2); (3) nitroglycerin liberated inorganic nitrite in the minced vessels and the nitrite liberation was inhibited by the same concentrations of CuSO 4 as antagonized the nitroglycerin-induced vasodilation although the denitration did not seem to be enzymatically catalyzed (table 4); (4) nitroglycerin also liberated inorganic nitrite even in the chemical reaction with sulfllydryl reagents at physiological pH. Since the effect of NaNO 2 was hardly disturbed by treatment with CuSO 4 (table 2) whereas it was markedly inhibited by ethacrynic acid (fig. 3) there seems to be a difference between the critical sulfhydryl groups for nitroglycerin- and N O [ - i n d u c e d vasodilation. This may suggest that nitroglycerininduced vasodilation is not essentially due to nitrite ion formation but to intermediate(s) synthesized in the course of nitroglycerin degradation, as was suggested in the review by Litchfield (organic nitrite or nitrated glutathione) (1973) and by Ignarro and Gruetter (S-nitrosothiols) (1980). CuSO 4 may disturb such intermediate formations. It has already been reported that Cu 2 + penetrated the cell membrane (Neumann and Silverberg, 1966). Although CuSO 4 may be an inhibitor of Na+-K + ATPase (Ting-Beall et al., 1973), the effect did not seem to take part in the antagonism against nitroglycerin because ouabain or Na + free conditions did not prevent the antagonism. It is unlikely that Cu 2+ had any effect on the contractile proteins because the ion had no effect on glycerinated rabbit aorta contracted by ATP (data not shown). Since the mitochondrial population in the small coronary artery was more numerous than in the large coronary artery, al-
153
though nitroglycerin-induced vasodilation was more marked in the large coronary artery (Kamitani et al., 1977), it is unlikely that mitochondria play an important role in the Cu 2÷induced antagonism against nitroglycerin-induced vasodilation. Since CuSO4 diminished the membrane stabilizing activity of Ca 2 + (fig. 4) the possible site of action of Cu 2+ seems to be on the cell membrane though the possibility remains that the effect of CuSO4 on the membrane stabilizing activity of Ca z+ and its effect on nitroglycerin-induced relaxation may be separate events. CdCI 2, which is also an inhibitor of sulfhydryl groups and does not seem to penetrate the cell membrane of the vessels (Toda, 1976), induced a significant potentiation of nitroglycerin-induced vasodilation in the rat aorta (table 3), which suggests that the outer surface of the cell membrane does not seem to be a major site in nitroglycerin vasodilation. The Cd2÷-in duced potentiation might result from the blocking of nitroglycerin degradation on the outer surface of the cell membrane. On the basis of these findings, the sulfhydryl groups on the inner surface of the cell membrane seem to be most probable site for Cu2÷-induced antagonism against nitroglycerin. The sulfhydryl groups, which are able to bind Cu 2 + on the inner surface of the cell membrane, may have a role in organic nitrate tolerance, because NaNO 2 and sodium nitroprusside are not cross-tolerant in nitroglycerin-tolerant vessels (Needleman and Johnson, 1973, 1975; Needleman et al., 1973). Recent evidence supports the theory that cGMP is involved in the regulation of vascular relaxation induced by nitroglycerin, N a N O 2 and sodium nitroprusside (Schultz et al., 1977; Katsuki and Murad, 1976, 1977; Katsuki et al., 1977a,b; Axelsson et al., 1979, 1982; Ignarro and Gruetter, 1980; Kobayashi et al., 1980; Gruetter et al., 1980; Ignarro et al., 1980). There seem to be some differences among these drugs: (1) nitroglycerin and N a N O 2 failed to activate soluble vascular guanylate cyclase in the absence of an added thiol whereas sodium nitroprusside did not require the thiols (Ignarro and Gruetter, 1980); (2) nitroglycerin specifically required cysteine while N a N O 2 required one of several thiols or ascorbate to activate soluble guanylate cyclase from bovine coronary arteries (Ignarro and Gruetter, 1980); (3)
sodium nitroprusside increased guanylate cyclase activity in most of the preparations, whereas nitroglycerin was tissue specific (Katsuki et al., 1977a); (4) nitroglycerin is a weak activator while sodium nitroprusside is a potent activator of this enzyme (Katsuki et al., 1977a), although the vasodilator effects of both drugs are almost the same (Watkins and Davidson, 1980). Several reports described Cu 2÷ (White et al., 1976; Gerzer et al., 1981) and sulfhydryl groups (Brhme et al., 1978; Braughler, 1983) as important constituents of guanylate cyclase. However, since Cu 2÷ could not inhibit N a N O 2- or nitroprussideinduced vasodilation, the inhibitory effect of Cu 2+ on nitroglycerin-induced vasodilation does not seem to be due to its direct effect on guanylate cyclase. Taking account of these findings we concluded that: Nitroglycerin reacts non-enzymatically with the sulfhydryl groups, which are thought to be on the inner surface of the cell membrane and prone to inhibition by Cu 2÷ and ethacrynic acid, and subsequently forms an active intermediate(s). This intermediate(s) may activate guanylate cyclase in the same way as sodium nitroprusside to induce an increase in cGMP and subsequent vasodilation. The selectivity of nitroglycerin on the large coronary artery (table 1), which was not the case for nitroprusside, may be derived from the difference in the concentrations of the sulfhydryl groups on the inner surface of the cell membrane but not from the effect on guanylate cyclase. This proposal gains support from the finding that sulfhydryl concentrations in the large coronary artery were higher than in the small coronary artery. There seem to be two kinds of sulfhydryl groups required for nitrate- and nitrite-induced vasodilation. One is preferentially acceptable to Cu z+ and the other to ethacrynic acid. The site of action of N a N O 2 seems to be on the latter, because the vascular effect of this drug was inhibited by pretreatment with ethacrynic acid but not with Cu 2+.
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