Advances Volume 20 No.

3
In Therapy® May/June 2003

Possible Impact of Nitric Oxide

on the Antihypertensive Effect
of Captopril and Zaprinast
Mohamed A. Ibrahim
Nobunori Satoh
Shiro Ueda
Department of Drug Information and Communication
Graduate School of Pharmaceutical Sciences
Chiba University, Japan

ABSTRACT
The interaction between the renin-angiotensin system and nitric oxide (NO) is
undeniable, but its nature is not fully known. This study investigated the contri-
bution of NO to the acute hypotensive effect of captopril in conscious normoten-
sive rats and the effect on blood pressure of dual administration of captopril and
the phosphodiesterase-5 inhibitor zaprinast. In two separate experiments, rats
were pretreated with the NO inhibitor L-arginine methyl ester (L-NAME) and with
the NO enhancer zaprinast. Pretreatment with L-NAME attenuated and pretreat-
ment with zaprinast potentiated the hypotensive effect of captopril. The hypoten-
sive effect of captoril was not associated with a significant change in the plasma
level of cyclic guanosine monophosphate (cGMP). These findings suggest that NO
contributes to the blood pressure–lowering effect of captopril. The inability of cap-
topril to alter plasma cGMP levels is not consistent with this view, however, and
leads to the conclusion that NO contributes to the acute hypotensive effect of cap-
topril, although the mechanism is not fully understood. Zaprinast potentiates the
hypotensive effect of captopril, and an adjustment in dose should be considered
when this combination is administered.

Keywords: nitric oxide; captopril; zaprinast; blood pressure

INTRODUCTION
The antihypertensive effect of angiotensin-converting enzyme (ACE)
inhibitors has been attributed primarily to the diminished formation of
angiotensin II in both plasma and tissues and to the accumulation of endo-
genous vasodilator kinins and nitric oxide (NO).1,2 Less well established,

©2003 Health Communications Inc. Address reprint requests to

Transmission and reproduction of this material in whole Mohamed A. Ibrahim
or part without prior written approval are prohibited. Department of Drug Information
and Communication
0709 Graduate School of Pharmaceutical
Sciences, Chiba University
1-33 Yayoicho, Inage-ku, Chiba-shi
Chiba 263-8522, Japan
143
however, is the effect of ACE inhibitors on the NO/cyclic guanosine monophosphate
(cGMP) pathway. Some investigators have reported that ACE inhibitors prevent
NO-deficient hypertension and left ventricular hypertrophy without affecting
the NO/cGMP level.3,4 Others noted that enalapril, captopril, and quinapril increased
NO levels by enhancing antioxidant defenses.5,6
Phosphodiesterase-5 (PDE5) inhibitors increase the activity of NO by preventing
degradation of cGMP, an indirect indicator of NO concentrations. The combination
of a PDE5 inhibitor (NO-enhancing agent) and an organic nitrate (NO-releasing
agent) is contraindicated because of a possible protracted decrease in blood pres-
sure.7 In a rat study,8 for example, changes in mean arterial pressure were signifi-
cantly correlated with both plasma and aortic levels of cGMP. Because of the wide-
spread use of PDE5 inhibitors in the treatment of sexual dysfunction, it seemed
worthwhile to investigate the possible role of NO on the blood pressure effect of
ACE inhibitors or PDE5 inhibitors.

MATERIALS AND METHODS

Both experiments used conscious, normotensive, male Sprague-Dowley rats
weighing from 300 to 320 g. Each treatment group consisted of 6 animals. Control
animals received water or dimethylsulfoxide.

Protocol 1 (Role of NO in the Acute Hypotensive Effect of Captopril)

A single dose of captopril 20 mg/kg was administered intraperitoneally. One
hour previously, the animals had received an intraperitoneal injection of 50 mg/kg
of L-arginine methyl ester (L-NAME), an NO synthase inhibitor. Time-matched con-
trol experiments were performed. The tail-cuff method was used to measure mean
arterial blood pressure. The rats were killed by deep anesthesia, and blood samples
were collected.

Protocol 2 (Effect of Captopril in Zaprinast-Pretreated Rats)

This protocol consisted of two separate experiments, both involving zaprinast,
an NO enhancer/PDE5 inhibitor. Two doses of zaprinast were used—12 mg/kg
(subhypotensive) and 24 mg/kg (hypotensive)—both administered intraperitoneal-
ly alone and 1 hour before an intraperitoneal injection of captopril 20 mg/kg.
The chosen doses were based on our preliminary studies and previous reports.8-10

Analysis of cGMP
The concentration of cGMP in a 100-µL aliquot of plasma was determined by
means of an enzyme immunoassay kit (R&D Systems, Inc, Minneapolis, Minn, USA)
and a microplate reader (Titertek Multiscan MCC/340, Dainippon Seiyaku, Japan).

Statistical Analysis
Results were expressed as means ± SD and analyzed by an unpaired Student’s t test.
A value of P<.05 was considered statistically significant.

M. A. Ibrahim et al
Effect of NO on Antihypertensive Drugs
144
RESULTS
Effect on Mean Arterial Pressure (Fig 1)
Pretreatment with L-NAME significantly attenuated the hypotensive effect of
captopril. Pretreatment with zaprinast 12 mg/kg did not affect blood pressure but
potentiated the hypotensive effect of captopril. Zaprinast 24 mg/kg alone caused
a significant decrease in blood pressure. When administered 1 hour before captopril,
this dose significantly reduced blood pressure compared with either drug alone.

Fig 1. Effects on mean arterial pressure.

Control
Captopril, 20 mg/kg
L-NAME
120 Captopril + L-NAME
Zaprinast 12 mg
Zaprinast 12 mg + captopril
Zaprinast 24 mg
100 *,†
Zaprinast 24 mg + captopril
Mean Arterial Pressure, mm Hg

* *
*,‡
80
*,§

60

40

20

*
P<.05 vs control rats.
†
P<.05 vs captopril-treated rats.
‡
P<.05 vs either captopril- or zaprinast (12 mg/kg)-treated rats.
§
P<.05 vs either captopril- or zaprinast (24 mg/kg)-treated rats.

Effect on Plasma cGMP (Fig 2)

Compared with control rats, animals that received captopril did not demonstrate
significant changes in plasma cGMP level. L-NAME caused a significant decrease in
cGMP; however, in L-NAME–pretreated rats, administration of captopril did not
significantly alter plasma cGMP compared with control animals. Zaprinast 12 mg/
kg significantly increased plasma cGMP, which rose further with 24 mg/kg com-
pared with control and 12 mg/kg. Plasma cGMP remained significantly unaffected
by administration of captopril in zaprinast-pretreated rats compared with zaprinast-
treated rats.

Advances In Therapy®
Volume 20 No. 3, May/June 2003
145
 Fig 2. Effects on plasma cGMP.

60
Control
Captopril, 20 mg/kg
L-NAME
50 Captopril + L-NAME †
cGMP Concentration, pmol/mL

Zaprinast 12 mg
Zaprinast 12 mg + captopril †,||
40 Zaprinast 24 mg
Zaprinast 24 mg + captopril
†
†,§
30

20

10 †
† ,‡

* P=NS vs control rats.

† P<.05 vs control rats.
‡P=NS vs L-NAME–treated rats.
§P=NS vs zaprinast (12 mg/kg)-treated rats.
||
P=NS vs. zaprinast (24 mg/kg)-treated rats.

DISCUSSION
The ability of L-NAME to significantly attenuate the hypotensive effect of capto-
pril suggests that ACE inhibitors act by releasing NO or enhancing its cardiovascu-
lar effect, or both. The significant potentiation of captopril’s hypotensive effect by
zaprinast supports this view. Not consistent with this premise, however, is the
inability of captopril to significantly change plasma cGMP levels. In agreement with
our results, one study11 suggested that vascular NO contributed to basal blood pres-
sure in conscious spontaneously hypertensive and normotensive rats, a conclusion
based on measurements of blood pressure, not NO levels. In a similar acute study,10
the angiotensin II antagonist GR 138950 did not increase urine and plasma cGMP
levels, although it is possible that any rise in cGMP level was restricted to the vas-
culature, failed to reach the plasma, and thus was not detected. This explanation
seems unlikely, however, because captopril did not significantly affect cGMP con-
centration or NO synthase activity in the left ventricle, aorta, kidney, and brain of
normal rats.4,12
An alternative mechanism to explain our findings would involve the equilibrium
existing between NO and angiotensin II. Consequently, when either ACE inhibitors
or an angiotensin antagonist eliminates the activity of angiotensin II, the actions of

M. A. Ibrahim et al
Effect of NO on Antihypertensive Drugs
146
NO are overexpressed, even in the presence of normal levels of these substances.13,14
In other studies,5,6 however, enalapril, captopril, and quinapril increased NO levels
by enhancing antioxidant defenses and bradykinin. The possible explanation for
such contradictory results may be that cGMP levels could not be restored in acute or
relatively short-term experiments. Consistent with this hypothesis are reports that
treatment with the ACE inhibitor trandolapril or captopril for 4 to 6 weeks prevent-
ed the increase in systolic blood pressure in L-NAME–induced hypertension yet did
not improve NO synthase activity or cGMP levels.4,10,12 In light of these findings,
pretreatment with zaprinast potentiated the hypotensive effect of captopril, thereby
supporting the involvement of the NO pathway in this ACE inhibitor’s acute hypo-
tensive effect.

CONCLUSIONS
NO contributes to the acute hypotensive effect of captopril in conscious nor-
motensive rats, but the mechanism of this effect is not fully established. The PDE5
inhibitor zaprinast potentiates the hypotensive effect of captopril. Consequently, an
adjustment in dose should be considered when these drugs are administered as
combination therapy.

ACKNOWLEDGMENT
We thank Dr. Shin-ichi Yamagata and all laboratory colleagues for help with
the technical notes.

REFERENCES
1. Carretero OA, Miyazaki S, Scicli AG. Role of kinins in the acute antihypertensive effect of
the converting enzyme inhibitor, captopril. Hypertension. 1981;3:18-22.
2. Whittle BJR, Lopez-Belmonte J, Rees DD. Modulation of the vasodepressor actions of acetyl-
choline, bradykinin, substance P and endothelin in the rat by a specific inhibitor of nitric oxide
formation. Br J Pharmacol. 1989;98:646-652.
3. Bernatova I, Pechanova O, Simko F. Effect of captopril in L-NAME-induced hypertension
on the rat myocardium, aorta, brain and kidney. Exp Physiol. 1999;84:1095-1105.
4. Pechanova O, Bernatova I. Effect of captopril on cyclic nucleotide concentrations during
long-term NO synthase inhibition. Physiol Res. 2000;49:55-63.
5. De Cavanagh EMV, Inserra F, Ferder L, Fraga CG. Enalapril and captopril enhance glutathione-
dependent antioxidant defenses in mouse tissues. Am J Physiol Regul Integr Comp Physiol. 2000;
278:R572-R577.
6. Bachetti T, Comini L, Pasini E, Cargnoni A, Salvatore C, Ferrari R. ACE-inhibition with quinapril
modulates the nitric oxide pathway in normotensive rats. J Mol Cell Cardiol. 2001;33:395-403.
7. Ishikura I, Beppu S, Hamada T, Khandheria BK, Seward JB, Nehra A. Effect of sildenafil citrate
(Viagra) combined with nitrate on the heart. Circulation. 2000;102:2516-2521.
8. Dundore RL, Habeeb PG, Pratt PF, Becker LT, Clas DM, Buchholz RA. Differential hemodynamic
responses to selective inhibitors of cyclic nucleotide phosphodiesterases in conscious rats.
J Cardiovasc Pharmacol. 1992;19:937-944.

Advances In Therapy®
Volume 20 No. 3, May/June 2003
147
 9. Chang KC, Peng YI, Tsai YF, Tseng YZ, Chen HI. Hypotensive effects of captopril on physical
properties of the arterial system in young and adult rats. Biogerontology. 2001;2:45-54.
10. Anderson IK, Drew GM. Investigation of the inhibitory effect of N-nitro-L-arginine methyl
ester on the antihypertensive effect of the angiotensin AT1 receptor antagonist, GR 138950.
Br J Pharmacol. 1997;122:1385-1394.
11. Minami N, Imai Y, Hashimoto J, Abe K. Contribution of vascular nitric oxide to basal blood
pressure in conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats.
Clin Sci. 1995;89:177-182.
12. Bernatova I, Pechanova O, Simko F. Captopril prevents NO-deficient hypertension and left
ventricular hypertrophy without affecting nitric oxide synthase activity in rats. Physiol Res.
1996;45:311-316.
13. De Nicola L, Blantz RC, Gabbai FB. Nitric oxide and angiotensin II: glomerular and tubular
interaction in the rat. J Clin Invest. 1992;89:1248-1256.
14. Conrad KP, Whittemore SL. NG-monomethyl-L-arginine and nitroarginine potentiate pressor
responsiveness of vasoconstrictors in conscious rats. Am J Physiol. 1992;262:R1137-R1144.

M. A. Ibrahim et al
Effect of NO on Antihypertensive Drugs
148