CD-832, a new dihydropyridine derivative with both nitrate-like and Ca2+ channel antagonist vasodilator activities

European Journal of Pharmacology, 249 (1993) 141-149

141

© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

EJP 53371

CD-832, a new dihydropyridine derivative with both nitrate-like and Ca 2+ channel antagonist vasodilator activities N o r i y u k i M i y a t a *, H i r o k o Y a m a u r a , M a k o t o T a n a k a , K e n z o T a k a h a s h i , K a t s u h a r u T s u c h i d a and Susumu Otomo Research Center, TaishoPharmaceutical Co. Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan Received 14 January 1993, revised MS received 17 August 1993, accepted 20 August 1993

We investigated the effects of CD-832 ((4R)-(-)-2-(nicotinoylamino)ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3nitrophenyl, 3,5-pyridine dicarboxylate), a new dihydropyridine derivative with nitrate ester, on contraction and relaxation responses induced by various vasoactive agents in rabbit aorta. CD-832 potently inhibited the specific binding of [3H](-t-)-PN200110 to rat brain membranes. The IC50 values for [3](+ )-PN200-110 binding were 2.8 nM and 4.9 nM in CD-832 and nifedipine, respectively. CD-832 (10 -8 to 10 -5 M), diltiazem (10 -8 to 10 -5 M) and benidipine (10 -8 to 10 -5 M) inhibited the 64 mM KCl-induced contraction of the aortic strips in a concentration-dependent manner. Neither nitroglycerin (10 -8 to 10 -5 M) nor nicorandil (10 -8 to 10 -5 M) affected the 64 mM KCI-induced contraction in rabbit aorta. CD-832 (10 -8 to 10 -5 M), nitroglycerin (10 -8 to 10 -5 M) and nicorandii (10 -5 M) inhibited the 10 -6 M norepinephrine-induced contraction of the aortic strips. However, diltiazem (10 -5 M) and benidipine (10 -5 M) had no effect on norepinephrine-induced contraction in rabbit aorta. Nitroglycerin (10 -5 M), atrial natriuretic peptide (10 -8 M), nicorandil (10 -5 M) and CD-832 (10 -7 to 10 -5 M ) augmented the isoproterenol-induced relaxation responses of rabbit aorta precontracted with endothelin-1 (1 × 10 - 7 to 2 × 10 -7 M). The effects of nitroglycerin (10 -5 M), nicorandil (10 -5 M) and CD-832 (10 -5 M) on isoproterenol-induced relaxation responses were antagonized by treatment with methylene blue (10 -5 M) and oxyhemoglobin (10 -5 M). The effect of CD-832 (10 -6 M) on isoproterenol-induced relaxation was augmented by treatment with 3 × 10 -5 M zaprinast, a phosphodiesterase V inhibitor. Neither diltiazem nor benidipine affected isoproterenol-induced relaxation responses in rabbit aorta. CD-832 (10 -7 to 10 -5 M) increased the levels of cyclic GMP without affecting the levels of cyclic AMP in rabbit aorta. These results indicate that CD-832 has not only a Ca 2+ channel antagonist-like action but also a nitrate-like action in vascular smooth muscle of the rabbit aorta. CD-832; Ca 2+ channel antagonist; cGMP; cAMP; Aorta vasodilatation; (Rabbit)

1. Introduction CD-832 ((4R)-(-)-2-(nicotinoylamino)ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5pyridine dicarboxylate) has a nitrate ester group in its molecule (fig. 1). CD-832 decreased mean blood pressure and heart rate in the anesthetized dog in a dosedependent manner, and increased coronary blood flow selectively without increasing common carotid blood flow (Takahashi et al., 1992). The coronary selective vasodilator effect of CD-832 was longer-lasting than that of nifedipine, diltiazem or nicorandil. A role of cyclic G M P in smooth muscle relaxation has been proposed (Katsuki et al., 1977a; Lincoln, 1989). Many researchers have reported relaxation responses associated with cyclic G M P in smooth muscle

* Corresponding author.

increased by nitrovasodilators (Grueter et al., 1979; Katsuki et al., 1977b). Subsequent studies with other vasodilators, including atrial natriuretic peptide (Waldman et al., 1984) and agents that cause release of endothelium-derived relaxing factor (Furchgott, 1984; Rapoport and Murad, 1983), have demonstrated a selective increase in cyclic GMP, and in cyclic GMP-dependent protein kinase activation coincident with functional effects of the vasodilators.

NO2 o

@.

H Fig. 1. Chemical structure of CD-832.

142 /3-Adrenoceptor stimulants cause relaxation responses in many types of smooth muscle, and are known to exert their relaxing effects through the elevation of cyclic AMP (Kramer and Hardman, 1980; Hardman, 1984). Isoproterenol-induced relaxation responses of arteries have been demonstrated to be decreased in hypertensive rats (Amer, 1982; Asano et al., 1982; Cohen and Berkowitz, 1976) and diabetic animals (Kamata et al., 1989a; Miyata et al., 1992a). Thus, functional changes in /3-adrenoceptors may play an important role in modulating peripheral vascular resistance in the pathophysiological states of hypertension and diabetes. Recently, it has been reported that the agents known to elevate cyclic GMP augment the isoproterenol-induced relaxation responses in rat and rabbit aorta by enhancing the accumulation of cyclic AMP (Maurice and Haslam, 1990b; Maurice et al., 1991; Miyata et al., 1992b). In the present study, we have investigated the effects of CD-832 on contraction and relaxation responses in rabbit aorta with particular attention to its nitrate-like action. The second purpose of the present study was to examine whether cyclic GMP-elevating agents and CD-832 augment the responsiveness to isoproterenol-induced relaxation in rabbit aorta.

2. Materials and methods

2.1. Binding of [3H]( + )-PN200-110 binding to rat brain membranes The brains of male Wistar rats (250-300 g) were removed and homogenized in 50 mM Tris-HCl buffer (pH 7.4) using a Physcotron homogenizer (Niti-On, Chiba, Japan). The homogenates were centrifuged at 1000 x g for 10 min, and the supernatant fractions were centrifuged at 48000 x g for 10 min. The pellet was re-homogenized and recentrifuged, and the resulting pellet was then suspended in 50 mM Tris-HC1 buffer (pH 7.4). This membrane fraction (1.0 ml) was incubated with 0.5 nM [3H](+)-PN200-110 at 26°C for 80 min in the presence of increasing concentrations of displacing agents. Incubation with 1/zM nifedipine was also included to determine non-specific binding. The reaction was stopped by rapid filtration through glass fiber filters ( G F / B , Whatman). The filters were washed 3 times with ice-cold 50 mM Tris-HCl buffer (pH 7.4). Radioactivity was determined with 10 ml Aquasol-2 in a scintillation spectrometer (Aloka LSC-3500, Tokyo, Japan). IC50 values from competitive inhibition experiments were determined by using the ManquardtLevenberg non-linear curve-fitting procedure of the R S / 1 program (BBN Research System, Cambridge, MA, USA) running on a V A X / V M S system.

2.2. Isolation of the rabbit aorta Male Japanese White rabbits (2.1-4.5 kg) were anesthetized with sodium pentobarbital (40 m g / k g i.v.) and exsanguinated from the carotid artery. A section of thoracic aorta between the aortic arch and the diaphragm was then removed and placed in oxygenated, modified Krebs-Henseleit solution. The solution consisted of (in mM): NaCI 118, KC1 4.7, CaCI 2 1.8, MgSO 4 1.2, NaH2PO 4 1.2, NaHCO 3 25.0, and glucose 11.1. Each aortic strip was cleaned of loosely adhering fat and connective tissue and was cut into eight helical strips 2 mm wide and 15 mm long. In all experiments, the possible influence of endothelium-derived relaxing factor was avoided by denuding each strip from its endothelium by gentle rubbing of the endothelial surface with a disposable cotton applicator. The tissue was placed in a well-oxygenated (95% O z, 5% CO 2) 10-ml bath containing Krebs-Henseleit solution at 37°C with one end connected to a tissue holder and the other to a force-displacement transducer (Nihon Kohden, Tokyo, Japan; TB-612T). The tissue was equilibrated for 60-90 min under a resting tension of 2.0 g. During this time, KHS in the tissue bath was replaced every 20 min. After equilibration, the aortic strip was contracted by treatment with 10 -7 M norepinephrine to ensure stabilization of the smooth muscle. Acetylcholine at a final concentration of 10 -5 M was then added to the bath. Removal of the endothelial cells by rubbing was confirmed by the fact that tonic contraction of the aorta by norepinephrine was not affected by treatment with acetylcholine. Then, 64 mM KC1 o r 10 - 6 M norepinephrine was added to the bath repeatedly at intervals of 20 min until a steady response was obtained (usually 4-6 times). These concentrations of KCI and norepinephrine caused an average 90% of the maximal contraction at each maximal response. We examined the effects of CD-832 (10 -8 to 10 -5 M), benidipine (10 -8 to 10 -5 M), diltiazem (10 -8 to 10 -5 M), nitroglycerin (10 -8 to 10 -5 M) and nicorandil (10 -8 to 10 -5 M) on 64 mM KCl-induced and 10 - 6 M norepinephrine-induced contraction in rabbit aorta. The aortic strips were exposed to these agents for 15 min in Krebs-Henseleit solution, and KC1 or norepinephrine was added to the bath. Because CD-832 and other agents greatly inhibited the norepinephrine-induced and KCl-induced contractions, neither KCI nor norepinephrine was used as the precontractile agent. For isoproterenol-induced relaxation experiments, we used endothelin-1 (1 x 10 - 7 to 2 X 10 - 7 M), as the precontractile agent since the endothelin-l-induced contraction of the rabbit aorta was not affected significantly by CD-832, nitroglycerin or other agents. Endothelin-1 (1 X 10 - 7 to 2 X 10 - 7 M)-induced contractions were an average 100-120% of the maximal contraction induced by 64 mM KC1. When

143 some blocking agents were added after the development of a steady contraction induced by 1 x 10 -7 to 2 x 10 -7 M endothelin-1, direct inhibitory effects by blockers were observed. However, these agents added after the development of the contraction had inhibitory effects greater than those of agents added 15 min before the strips were precontracted by endothelin-1. Accordingly, to examine the effects of CD-832 (10 -8 to 10 -5 M), nitroglycerin (10 -8 to 10 -5 M), atrial natriuretic peptide (10 -1° to 10 -8 M), nicorandil (10 -7 to 10 -5 M), benidipine (10 -5 M), diltiazem (10 -s M), methylene blue (10 -5 M), zaprinast (3 x 10 -s M) and oxyhemoglobin (10 -5 M), the aortic strips were treated with these agents for 15 min and were then precontracted with 1 x 10 -7 to 2 x 10 -7 M endothelin-1. The precontractile force induced by these concentrations of endothelin-1 did not differ significantly among the treatment groups studied (table 2). When the endothelin-l-induced contraction reached a plateau, isoproterenol (10 -8 to 10 -5 M) was added cumulatively to the bath. Because endothelin-l-induced contraction was desensitized, and the second application of endothelin-1 failed to contract the aortic strips, we examined only one concentration-response curve in each aortic strip.

2.3. Measurement of cyclic nucleotides Basal or drug-induced changes in levels of cyclic G M P and cyclic AMP were measured in a separate series of experiments. Endothelium-denuded rabbit aortic strips were allowed to equilibrate in tubes that contained Krebs-Henseleit solution, gassed with 95% O 2 and 5% CO 2, at 37°C for 60 min. During this time the Krebs-Henseleit solution in the tube was replaced every 20 min. After equilibration, the aortic strips were treated with CD-832 (10 -7 to 10 -5 M) for 1, 5, and 15 min. The tissues were then frozen in liquid N 2, homogenized in 1 ml of 6% trichloroacetic acid, and centrifuged at 3000 X g for 10 rain. The supernatants were extracted 3 times in three volumes of water-saturated ether, and were stored at - 80 °C for 2 days until being assayed for cyclic G M P or cyclic AMP. Following succinylation, the levels of cyclic G M P or cyclic AMP were determined using radioimmunoassay kits as previously described by Kamata et al. (1989b) and Miyata et al. (1992b).

endothelin-1. The significance of differences between mean values was determined by analysis of variance. If analysis of variance demonstrated a significant difference between means, Dunnet's test was used to determine which treatment group was significantly different from the mean control value. The level of significance was P < 0.05.

2.5. Drugs Norepinephrine hydrochloride, methylene blue, diltiazem and hemoglobin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetylcholine chloride was purchased from Tokyo Kasei (Tokyo, Japan). Endothelin-1 was purchased from the Peptide Institute (Osaka, Japan). Nitroglycerin was purchased from Nippon Kayaku Co. (Tokyo, Japan). CD-832, benidipine, nicorandil and cromakalim were synthesized in the Taisho Research Center. [3H](+)-PN200110 was purchased from D u P o n t / N e w England Nuclear (Wilmington, DE, USA). Radioimmunoassay kits for cyclic G M P and cyclic AMP were purchased from Yamasa Shouyu Co. (Chiba, Japan). CD-832, benidipine, diltiazem and cromakalim were dissolved in 100% dimethyl sulfoxide. Zaprinast was dissolved in 10% triethanolamine. The final concentration of dimethyl sulfoxide and triethanolamine in the tissue bath, < 0.1% and < 0.03%, respectively, did not affect the tissue contractile or relaxation response. The other drugs were dissolved in distilled water. Oxyhemoglobin was prepared by methods previously described by Martin et al. (1985).

3. Results

3.1. Inhibition of [3H]( + )-PN200-110 binding by CD832 CD-832 inhibited the specific binding of [3H](+)PN200-110 to rat brain membranes in a concentrationdependent manner (fig. 2). The inhibitory potency of CD-832 was similar to that of nifedipine. The ICs0 values for [3H](+)-PN200-]10 binding were 2.8 nM and 4.9 nM in CD-832 and nifedipine, respectively.

3.2. Effects of CD-832 and other drugs on KCl-induced and norepinephrine-induced contractions

2.4. Statistical analysis All data are reported as the means + S.E.M. The inhibitory effect of each drug is expressed as the percentage inhibition of a steady contractile force induced by 64 mM KCI or 10 -6 M norepinephrine. The relaxation in response to each agent is expressed as the percentage decrease of contractile force induced by

In aortic strips without endothelium, the 64 mM KCl-induced and 10 -6 M norepinephrine-induced contractions were 2.17 + 0.16 g (n = 25) and 2.65 + 0.11 g (n = 35), respectively. CD-832 (10 -8 to 10 -5 M) inhibited the 64 mM KCl-induced contraction of the aortic strips without endothelium in a concentration-dependent manner (fig.

144 100

TABLE 1 Mean inhibitory concentration (ICs0) for benidipine, CD-832 and diltiazem on 64 mM KCl-induced contraction in rabbit aorta.

o

IC50 value Benidipine CD-832 Diltiazem

"6

2.1 _+0.7× 10 -7 M (n = 5) 8.95:3.5 x 10 -7 M (n = 5) 1.0_+0.2x 10 -6 M (n = 5)

n = number of animals.

duced contraction only at a high concentration, 10 -5 M (fig. 3B). However, neither benidipine (10 -8 to 10 -5 M) nor diltiazem (10 -8 to 10 -5 M) affected the 10 -6 M norepinephrine-induced contraction in rabbit aorta (fig. 3B).

-LOG M

Fig. 2. Inhibitory effects of CD-832 (e) and nifedipine (111) on the specific binding of [3H](+ )-PN200-110 to rat brain membranes. Rat brain membranes were incubated with increasing concentrations of displacing agents in the presence of 0.5 nM [3H](+)-PN200-110. The value was obtained from three separate experiments, each done in duplicate.

3A). Both diltiazem (Sato et al., 1971) and benidipine (Karasawa et al., 1988) also inhibited the KCl-induced contraction of the aortic strips in a concentration-dependent manner (fig. 3A). Table 1 shows the mean inhibitory concentration values (IC50) for diltiazem, benidipine and CD-832. However, neither nitroglycerin (10 -8 to 10 -5 M) nor nicorandil (10 -8 to 10 -5 M) affected 64 mM KCl-induced contraction in rabbit aorta (fig. 3A). In contrast, both nitroglycerin (10 -8 to 10 -5 M) and CD-832 (10 -8 to 10 -5 M) inhibited the 10 -6 M norepinephrine-induced contraction of the rabbit aortic strips in a concentration-dependent manner (fig. 3B). Nicorandil inhibited the 10 -6 M norepinephrine-in-

3.3. Effects of CD-832 and other drugs on endothelin-1induced contraction in rabbit aorta Table 2 shows 1 x 10 -7 to 2 x 10 -7 M endothelin1-induced contractile force in aortic strips in the absence or in the presence of the various drugs listed. Thus, treatment with these agents had no significant effects on the endothelin-l-induced contraction responses in any of the aortic strips studied ( P > 0.05, table 2). Furthermore, we confirmed that the endothelin-l-induced contractile tone was maintained at a constant level for at least 2 h.

3.4. Effects of various agents on isoproterenol-induced relaxation response of the aorta precontracted with endothelin-1 Isoproterenol (10 -8 to 10 -5 M) slightly relaxed, in a concentration-dependent manner, the rabbit aortic strips precontracted with endothelin-1 (fig. 4).

TABLE 2 Effects of CD-832 and other agents on endothelin-l-induced contraction of the rabbit aorta. Endothelin-1 (1 x 10 -7 to 2 x 10 -7 M) Control NTG

ANP

CD

Nic

10 -8 M 10- 7 M 10-6 M 10 -5 M 10-10 M 10 -9 M 10 -8 M 10 - 8 M 10 -7 M 10 -6 M 10-5 M 10 -7 M 10 - 6 M 10 -5 M

1.88 + 0.35 1.82+0.20 1.70 -I-0.21 1.45 + 0.15 1.90 + 0.19

g (n g (n g (n g (n g (n

= = = = =

5) 5) 5) 5) 5)

1.86 + 0.18 g (n = 5)

1.83+0.21 1.45_+0.18 1.21 _+0.17 1.375:0.32 1.32+0.21 1.72 5:0.11 1.51 _+0.22 1.645:0.09 1.57_+0.18

g (n g(n g (n g (n g (n g (n g (n g (n g (n

= = = = = = = = =

5) 5) 6) 5) 5) 5) 5) 5) 5)

NTG + MB + Oxy Nic + MB +Oxy CD + MB +Oxy CD + Zap Ben Dil Cro

10 -5 M 10- 5 M 10- 5 M 10 -5 M 10-5 M 10 -5 M 10 -5 M 10- 5 M 10 -5 M 10 -6 M 3 x 10-5 M 10 -5 M 10 -5 M 10 -5 M

2.72 -+0.62 g (n = 4) 1.99 + 0.33 g (n = 4) 1.71 -I- 0.21 g (n = 5)

2.23_+0.02 g (n = 5) 1.63 _+0.27 g (n = 4) 1.73_+0.34 g (n = 4) 1.10 _+0.08 g (n 1.20_+ 0.19 g (n 1.51 _+0.18 g (n 0.98_+0.15 g (n

= = = =

5) 4) 6) 4)

Values are expressed as means5:S.E.M, n = number of animals. NTG, nitroglycerin; ANP, atrial natriuretic peptide; CD, CD-832; Nic, nicorandil; MB, methylene blue; Oxy, oxyhemoglobin; Zap, zaprinast; Ben, benidipine; Dil, diltiazem; Cro, cromakalim. Endothelin-l-induced contractions were expressed as tension development in grams.

145

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Z O I--

=_- 50 1Z

~ "

"

-='

Bo

• (5)

(5)

Z

o I-=_-so "I-

(5) [5) ~

1

5

1 100

-LOG M

-LOG M

Fig. 3. Concentration-response curves for inhibitory effects of CD-832 ([]), benidipine ( • ) , diltiazem (A), nitroglycerin ( • ) and nicorandil (e) on 64 mM KCl-induced (A) and 10 -6 M norepinephrine-induced (B) contractions in rabbit aorta. All data are reported as the means + S.E.M. The inhibitory effect of each drug was expressed as the percentage inhibition of a steady contractile force induced by 64 mM KC1 or 10 -6 M norepinephrine. Numbers in parentheses are the number of animals.

(10 -8 M) (fig. 4). Both CD-832 ( > 10 -7 M) and nicorandil (10 -5 M) also augmented the isoproterenol-induced relaxation responses of the aortic strips (fig. 4).

The isoproterenol-induced relaxation responses of the aortic strips were augmented by pretreatment with nitroglycerin (10 -5 M) and atrial natriuretic peptide

A

g

o

I z

Z

o

_o

~5o ._1 LU IX

"*'~

'

T

ae

ANP

NTG

"* ~ , ~ s ~

10(3

100

-LOG (ISO) M

-LOG (ISO) M

C

D

Z

o

*"" ~

IX a~

(5)

~e

Nicorandil

CD-832 100

10(; -LOG (ISO) M

-LOG (ISO) M

Fig. 4. (A) Effects of nitroglycerin (10 -8 M ( • ) , 10 -7 M (zx), 10 -6 M ( • ) , and 10 -5 M ([])) on isoproterenol (control (e))-induced relaxation responses of endothelium-denuded rabbit aorta. (B) Effects of atrial natriuretic peptide (10 - l ° M (©), 10 -9 M ( A ) , and 10 -8 M (zx)) on isoproterenol (control (o))-induced relaxation responses of endothelium-denuded rabbit aorta. (C) Effects of CD-832 ( 1 0 - s M ( • ) , 1 0 - 7 M (zx), 10 -6 M ( l I), and 10 -5 M ([])) on isoproterenol (control (e))-induced relaxation responses of endothelium-denuded rabbit aorta. (D) Effects of nicorandil (10 -7 M ( 0 ) , 10 -6 M ( • ) , and 10 -5 M ( A ) ) on isoproterenol (control (e))-induced relaxation responses of endothelium-denuded rabbit aorta. Aortic strips were exposed to nitroglycerin, atrial natriuretic peptide, CD-832 and nicorandil for 15 min, and 1 × 10 -7 to 2 × 10 -7 M endotbelin-1 was then added to the bath. The results are expressed as the percentage decrease of contractile force induced by endothelin-I and the means + S.E.M. Numbers in parentheses are the number of animals. * P < 0.05, * * P < 0.01, significantly different as compared with control.

146

A

Z

z

_o LU

W

15)

Nicorandil

100

lOO - L O G (ISO) M

- L O G (ISO) M

Fig. 5. (A) Effects of methylene blue (10 -5 M ( l ) ) and oxyhemoglobin (10 -5 M (rn)) on potentiating effects of 10 5 M nitroglycerin (10 -5 M (0)) in isoproterenol (control (o))-induced relaxation responses of rabbit aorta. (B) Effects of methylene blue (10 -5 M ( • ) ) and oxyhemoglobin (10 -5 M (rn)) on potentiating effects of nicorandil (10 -5 M (0)) in isoproterenol (control (e))-induced relaxation responses of rabbit aorta. Aortic strips were exposed to these agents for 15 min, and 2 x 10 - 7 M endothelin-1 was then added to the bath. The results are expressed as the p e r c e n t a g e d e c r e a s e of contractile force induced by endothelin-1 and the m e a n s + S.E.M. N u m b e r s in parentheses are the n u m b e r of animals. * P < 0.05, * * P < 0.01, significantly different as compared with 10 5 M nitroglycerin and nicorandil.

3.5. Effects of methylene blue and oxyhemoglobin on potentiating effects of nitroglycerin and nicorandil The effects of 10 -5 M nitroglycerin and 10 -5 M nicorandil on isoproterenol-induced relaxation responses were antagonized by treatment with 10 -5 M methylene blue and 10 -5 M oxyhemoglobin, respectively (fig. 5). However, neither methylene blue nor oxyhemoglobin itself had any effect on isoproterenol-induced relaxation responses in rabbit aorta (data not shown).

3.6. Effects of methylene blue, oxyhemoglobin and zaprinast on CD-832-induced effects The effects of 10 -5 M CD-832 on isoproterenol-induced relaxation responses were also antagonized by

A o

treatment with 10 -5 M methylene blue and 10 -5 M oxyhemoglobin, respectively (fig. 6). The effect of 10 - 6 M CD-832 o:a isoproterenol-induced relaxation responses tended to be augmented by treatment with 3 x 10 -5 M zaprinast (fig. 6). We confirmed that the vehicle (0.03% triethanolamine) did not alter the effect of CD-832 on isoproterenol-induced relaxation responses.

3. 7. Effects of diltiazem, benidipine and cromakalim on isoproterenol-induced relaxation responses In contrast to the effects of nitro compounds, diltiazem (10 -5 M), benidipine (10 -5 M) and cromakalim (10 -5 M) had no effect on the isoproterenol-induced relaxation response of the rabbit aortic strips precontracted with endothelin-1 (fig. 7).

B o

Z

Z

_o

o

xSO

_

LIJ OC

CD-832 tO0

cil

100

-LOG (ISO) M

-LOG (ISO) M

Fig. 6 (A) Effects of methylene blue (10 -5 M ( i ) ) and oxyhemoglobin (10 -5 M (13)) on potentiating effects of CD-832 (10 -5 M (©)) on isoproterenol (control (o))-induced relaxation responses of rabbit aorta. (B) Effects of zaprinast (3 × 10 -5 M ( • ) ) on potentiating effects of CD-832 (10 - 6 M ( • ) ) in isoproterenol (control (o))-induced relaxation responses of rabbit aorta. Aortic strips were exposed to these agents for 15 min, and then 2 X 10-7 M endothelin-1 was added to the bath. T h e results are expressed as the percentage of decreased tension of contractile force induced by endothelin-1; the m e a n s + S . E . M , are given. N u m b e r s in parentheses are the n u m b e r of animals. * P < 0.05, ** P < 0.01, significantly different as compared with 10 -5 M or 10 - 6 M CD-832, respectively.

147 0

•~

(6)

(4) (5) (4)

Ia.I er #

100 - L O G (ISO) M

Fig. 7. Effects of benidipine (10 -5 M (zx)), diltiazem (10 - 5 M (©)) and cromakalim (10 - s M ( A ) ) on isoproterenol (e)-induced relaxation responses of e n d o t h e l i u m - d e n u d e d rabbit aorta. Aortic strips were exposed to these agents for 15 min, and 10 -7 M endothelin-1 was then added to the bath. T h e results are expressed as the percentage decrease of contractile force induced by endothelin-1 and the m e a n s ± S . E . M , are given. N u m b e r s in parentheses are the n u m b e r of animals.

3.8. Effects of CD-832 on levels of cyclic nucleotides The basal levels of cyclic G M P in rabbit aorta were 69.5 + 7.7 f m o l / m g wet tissue. Table 3 shows the time course of cyclic G M P elevation which was stimulated by 10 -5 M CD-832. In aortic strips, the accumulation of cyclic G M P in response to 10 -5 M CD-832 was found to reach maximal concentrations 1 min after exposure to CD-832. CD-832 (10 -7 to 10 -5 M) increased the levels of cyclic G M P in a concentration-dependent manner (table 3). The basal levels of cyclic AMP in rabbit aorta were 991.0 + 120.0 f m o l / m g wet tissue. However, CD-832 (10 -5 M) did not affect the levels of cyclic AMP in rabbit aorta (table 3).

4. Discussion In the present study, we found that CD-832, a dihydropyridine derivative with a nitrate ester moiety,

has both Ca 2+ channel antagonist-like and nitrate-like activities. CD-832 potently inhibited the specific binding of [3H](+)-PN200-110 binding to rat brain membranes. The inhibitory potency of CD-832 was similar to that of nifedipine from the IC50 values. From these results, it is indicated that CD-832 has a high affinity for the Ca 2 + channel in rat brain membranes. CD-832 (10 -8 to 10 -5 M) inhibited the 64 mM KCl-induced contraction in rabbit aortic strips in a concentration-dependent manner. Benidipine, a dihydropyridine derivative (Karasawa et al., 1988), and diltiazem, a benzothiazepine derivative (Sato et al., 1971), also inhibited the 64 mM KCl-induced contraction in a concentration-dependent manner. CD-832 had a potency equal to that of diltiazem and one quarter that of benidipine. In contrast to the effects of Ca 2+ channel antagonists, neither nitroglycerin nor nicorandil affected the KCl-induced contraction of rabbit aortic strips. These results indicate that the agents known to elevate cyclic GMP have no effect on 64 mM KCl-induced contraction in rabbit aorta. On the other hand, nitroglycerin, nicorandil, and CD-832 inhibited the 10 -6 M norepinephrine-induced contraction in a concentration-dependent manner. The inhibitory effects of CD-832 on norepinephrine-induced contraction in rabbit aorta were of an extent similar to the nitroglycerin effect. However, neither benidipine nor diltiazem had any effect on norepinephrine-induced contraction in rabbit aorta. These results indicate that the effects of CD-832 on norepinephrine-induced contraction responses in rabbit aorta were not due to the Ca 2+ channel antagonist-like action. In the present study, Ca 2÷ channel antagonists had no effect on endothelin-l-induced contractile force in rabbit aorta. This observation is in agreement with the findings of Ohlstein et al. (1990). Recently, it has been reported that the agents known to elevate cyclic G M P augment the isoproterenol-induced relaxation responses in rat and rabbit aorta by enhancing the accumulation of cyclic AMP (Maurice and Haslam, 1990b;

TABLE 3 CD-832-induced production of cyclic G M P and cyclic A M P in rabbit aorta without endothelium. Agents None CD-832 CD-832

Cyclic G M P ( f i n o l / m g tissue)

Cyclic A M P ( f m o l / m g tissue)

10 - 7 M 10 - 6 M

(15 min) (15 min)

69.5± 7.7 80.4± 4.8 130.6± 10.5 a

(n = 5) (n = 5) (n = 5)

10 - s M

(1 min) (5 min) (15 min)

250.7+43.0 b 230.9 ± 32.8 b 184.7+28.9 a

(n = 5) (n = 5) (n = 5)

991.0+ 120.0 (n = 5)

925.0± 120.0 (n = 5)

Values are m e a n s ± S . E . M , a Statistically significant ( P < 0.05) difference from value without CD-832; b statistically significant ( P < 0.01) difference from value without CD-832. n = n u m b e r of animals. Aortic strips were exposed to CD-832 for 1 min, 5 min and 15 min.

148

Maurice et al., 1991; Miyata et al., 1992b). We have investigated whether or not nitroglycerin, atrial natriuretic peptide, nicorandil and CD-832 augment the responsiveness to isoproterenol-induced relaxation in rabbit aorta. As shown in fig. 4, both nitroglycerin, an activator of soluble guanylate cyclase, and atrial natriuretic peptide, an activator of particulate guanylate cyclase (Waldman et al., 1984), augmented the isoproterenolinduced relaxation responses of the rabbit aortic strips. Nicorandil, a nitrate ester with a potassium channel opening action (Taira, 1987, 1989; Miyata et al., 1990), also augmented the isoproterenol-induced relaxation responses of the aortic strips precontracted with endothelin-1. It appears that various cyclic GMP increasing agents can augment isoproterenol-induced relaxation responses of the rabbit aorta. On the other hand, CD-832 also augmented the isoproterenol-induced relaxation responses of the aortic strips in a concentration-dependent manner. The extent of the potentiating effect of CD-832 on isoproterenol-induced relaxation response was similar to that of nitroglycerin. Generally, cyclic GMP is synthesized in vascular smooth muscle tissue through soluble guanylate cyclase and particulate guanylate cyclase. The former is stimulated by endothelium-derived relaxing factor and nitro compounds (Rapoport and Murad, 1983), and the latter is stimulated by atrial natriuretic peptide (Waldman et al., 1984; Winquist et al., 1984). Soluble guanylate cyclase, but not particulate guanylate cyclase, is inhibited by methylene blue (Martin et al., 1985). In the present study, the potentiating effects of nitroglycerin and nicorandil on isoproterenol-induced relaxation responses were significantly antagonized by treatment with methylene blue and oxyhemoglobin, which react chemically with nitric oxide (Martin et al., 1985). The effects of nitroglycerin, and nicorandil on isoproterenol-induced relaxation may be due to increased levels of cyclic GMP. Since cromakalim, a K ÷ channel opener (Weir and Weston, 1986) had no effect on isoproterenol-induced relaxation responses of the rabbit aorta (fig. 7), the potentiating effects of nicorandil may not be due to the activating action of K ÷ channels. The effects of CD-832 on isoproterenol-induced relaxation responses were significantly inhibited by treatment with methylene blue and oxyhemoglobin, while they tended to be augmented by treatment with zaprinast, a selective inhibitor of cyclic GMP phosphodiesterase (Souness et al., 1989). Zaprinast (3 × 10 -5 M) was shown previously not to augment the isoproterenol-induced relaxation response of the rabbit aorta precontracted with endothelin-1 (Miyata et al., 1992b). On the other hand, benidipine, diltiazem and cromakalim had no effect on isoproterenol-induced relaxation responses of the rabbit aorta precontracted with endothelin-1 in the pre-

sent study. Furthermore, high concentrations of CD-832 did not affect the levels of cyclic AMP in rabbit aorta without endothelium. Therefore, the present results suggest strongly that the effects of CD-832 on isoproterenol-induced relaxation responses of rabbit aorta precontracted with endothelin-1 may be due to the nitrate-like action, but not to the Ca 2÷ channel antagonist-like action, potassium channel opening action, or the direct inhibitory effect of cyclic AMP phosphodiesterase. Because the rabbit aortic strips were denuded of their endothelial cells using a cotton applicator in all experiments, CD-832 was not likely to have released endothelium-derived nitric oxide. Recently, it has been reported that vascular smooth muscle contains the cyclic GMP-inhibitable cyclic AMP phosphodiesterase (Kauffman et al., 1987; Silver et al., 1988). The molecular basis of the synergistic inhibition of platelet aggregation by nitrovasodilators and activators of adenylate cyclase has been reported (Maurice and Haslam, 1990a). The basis was shown to be the ability of cyclic GMP to enhance the accumulation of cyclic AMP in platelets by inhibiting a specific cyclic GMP-inhibitable cyclic AMP phosphodiesterase (Maurice and Haslam, 1990a). Recently, it has also been reported that the nitro compound augments the isoproterenol-induced responses in rat aorta by enhancing the accumulation of cyclic AMP (Maurice et al., 1990b; Maurice and Haslam, 1991). From the above-mentioned reports and the present data, it is suggested that increased cyclic GMP induced by CD-832 might enhance the isoproterenol-induced relaxation response in vascular smooth muscle, through enhancing the accumulation of cyclic AMP by inhibiting a cyclic GMP-inhibitable cyclic AMP phosphodiesterase. The present results were in agreement with other findings (Maurice and Haslam, 1990b; Maurice et al., 1991; Miyata et al., 1992b). In conclusion, it is indicated that CD-832 has not only a Ca 2+ channel antagonist-like action but also a nitrate-like action in rabbit aorta, possibly mediated through the increased levels of cyclic GMP. References Amer, M.S., 1982, Cyclic adenosine monophosphate and hypertension in rats, Science 179, 807. Asano, M., K. Aoki and T. Matsuda, 1982, Reduced beta- adrenoceptor interaction of norepinephrine enhances contraction in the femoral artery from spontaneously hypertensive rats, J. Pharmacol. Exp. Ther. 223, 207. Cohen, M.L. and B.A. Berkowitz, 1976, Decreased vascular relaxation in hypertension, J. Pharmacol. Exp. Ther. 196, 396. Furchgott, R.F., 1984, The role of endothelium in the response of vascular smooth muscle to drugs, Annu. Rev. Pharmacol. Toxicol. 24, 175. Grueter, C.A., B. Barry, D. McNamara, D. Grueter, P. Kadowitz and L. Ignarro, 1979, Relaxation of bovine coronary artery and activa-

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