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Reduced endothelium-dependent vasodilation by acetylcholine and bradykinin in isolated nitroglycerin-tolerant blood vessels
Gen. Pharmac. Vol. 25, No. I, pp. 61-67, 1994 Copyright © 1994Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-3623/94 $6.00 + 0.00
Pergamon
Reduced Endothelium-dependent Vasodilation by Acetylcholine and Bradykinin in Isolated Nitroglycerin-tolerant Blood Vessels MASAYUKI FUKAZAWA* and ATSUSHI NAMIKI Third Department of Internal Medicine, Toho University School of Medicine, Ohashi Hospital, 2-17-6 Ohashi, Meguro-ku, Tokyo 153, Japan [Tel. 81-3-3468-1251; Fax 81-3-3468-1269]
(Receh,ed 4 June 1993)
A b s t r a c t - - l . Rings of porcine pulmonary arteries were mounted in tissue organ baths and incubated in
physiological solution. The rings were allowed to equilibrate for > 1 hr under a resting tension of 1.0 g. The presence of endothelium was confirmed by 10 -6 M acetylcholine (ACh)-induced relaxation (60-80%) of 10-6 M norepinephrine (NE) contraction. 2. Relaxation response generated by nitroglycerin (NTG) (10 9-10-5M), ACh (10-9-10-5M), bradykinin (BK) ( I 0 - 1 3 - 1 0 - 6 M ) and nitric oxide (NO) after NE ( 1 0 - 6 M ) contraction was compared before and after 1 hr treatment of NTG (5 × 10 4 M). Then tissues were pretreated with NG-monomethylL-arginine (LNMMA) (I0 4 M) each before and after NTG treatment respectively, and ACh-induced relaxation was compared. 3. After 1 hr treatment with 5 × 10 4 M NTG, the relaxation response of NTG at concentrations > 10 -7 M was attenuated significantly. This indicates that I hr treatment with 5 x 10 -4 M NTG induces NTG tolerance in isolated porcine pulmonary arterial rings. 4. The relaxation response of ACh at concentrations > 10 7 M was attenuated significantly after NTG tolerance induction. 5. Relaxation response of BK at concentrations > I0 -~° M was attenuated significantly after NTG tolerance induction. 6. NTG tolerance had no effect on NO-induced vascular smooth muscle relaxation. 7. The relaxation response ofACh pretreated with LNMMA at concentrations higher than 10 _7 M was attenuated after NTG tolerance. 8. These results demonstrate that ACh releases an endothelium-derived relaxing factor (EDRF) or several EDRFs other than NO which is (are) affected by NTG tolerance.
(cGMP), thereby eliciting vasodilation (Ignarro et al., 1981). N T G tolerance has been recognized clinically, which is developed in dependence on time a n d nitrate concentration. M a n y investigators have reported on the mechanism of N T G tolerance. N e e d l m a n a n d J o h n s o n 111973) and N e e d l m a n et al. (1973) d e m o n strated a reduction in tissue levels of sulfhydryl groups in N T G - t o l e r a n t vessels and proposed that it is the primary reason for N T G tolerance. Moreover, previous studies indicated that a decrease in the b i o t r a n s f o r m a t i o n o f N T G (Brien et al., 1986) as well as a reduction in activation of G C ( W a l d m a n et al., 1986) is responsible for the induction of N T G tolerance. Since F u r c h g o t t a n d Zawadzki (1980) reported that vascular s m o o t h muscle relaxation induced by
INTRODUCTION Nitrovasodilators such as nitroglycerin ( N T G ) are widely used for the treatment of cardiovascular diseases. However, nitrovasodilator-induced tolerance has been recognized and sometimes causes clinical problems. The vasodilating effect of N T G is t h o u g h t to be mediated by the f o r m a t i o n of nitric oxide (NO) in the vascular s m o o t h muscle cells. Organic nitrates are postulated to release nitrite in the presence o f thiols which is then transformed into nitrosothiol and N O with the aid of a n o t h e r thiol. The generated N O activates soluble guanylate cyclase (GC) a n d liberates guanosine 3 ' , 5 ' - m o n o p h o s p h a t e
*To whom all correspondence should be addressed. 61
62
MASAYUKIFUKAZAWAand ATSUSrUNAMiKI
acetylcholine (ACh) was endothelium-dependent, the importance of endothelial cells for vascular reactivity has been recognized (Furchgott et al., 1987). Although it is still unknown whether the endothelium-derived relaxing factor (EDRF) is a single substance, it has been suggested that at least one of the EDRF(s) is NO (Furchgott et al., 1987; Huchinson et al., 1987; Ignarro et al., 1987; Palmer et al., 1987). Therefore, the NO ingredient of the E D R F is especially called endothelium-derived nitric oxide (EDNO). The stimulation of GC and the subsequent cGMP increase in smooth muscle are the common mechanisms promoting NTG- and EDNO-induced vascular relaxation (Rapoport and Murad, 1983; Griffith et at., 1985). Therefore, it may be probable that the effect of EDNO is altered in NTG-tolerant vessels. In this study, we thus investigated the effect of N T G tolerance on the endothelium-dependent relaxation produced by some vasodilators. MATERIALS AND METHODS Tension measurements
Lungs of Yorkshire swine (approx. 6 months old, weighing 100 kg) were obtained at a slaughterhouse. Lungs were dissected within 60min after death, placed in ice-cold physiological solution (NaCI 118, KCI 4.75, CaC12 2.54, KH2PO4 1.19, NaHCO3 25.0, MgSO4 1.2, glucose 11.0 mM) and transported to the laboratory. The pulmonary arteries were carefully cleaned free of adherent and connective tissues, and cut into rings. Rings (outer diameter 4 mm, length 5 mm approx.) were mounted in tissue organ baths containing 10 ml of physiological solution. The bath medium was maintained at 37°C and aerated before and during the experiments with a mixture of 95% 02 and 5% CO 2. Under these conditions, the pH level of 7.4 remained constant. Isometric developed tension was recorded by isometric force-transducers (FD pickup TB-611T, Nihon Kohden, Tokyo, Japan). Before the start of the experiments, the rings were allowed to equilibrate for > 1 hr under a resting tension of 1.0 g. During this period, the physiological solution in the tissue baths was replaced every 15 min, The presence of endothelium was confirmed by 10 -6 M ACh-induced relaxation (60-80%) of 10 - 6 M norepinephrine (NE) contraction. We previously sought the optimal resting tension of pulmonary arterial ring segments of Yorkshire swine, and concluded that 1.0 g was the optimal level (Namiki et al., 1992). Effect o f N T G treatment on the relaxation o f vasodilators
After the tings were precontracted with 1 0 - t M NE, N T G was added cumulatively from 10 9 to
10 -5 M (response before N T G treatment). The tings were then incubated with 5 x 10 -4 M NTG for 1 hr and washed by the physiological solution four times every 15 min. After the rings were precontracted with 1 0 - t M NE, N T G was again added cumulatively from 10 -9 to 10 -5 M (response after NTG treatment). The response before and after N T G treatment was compared in the same tings (Fig. i). A similar comparison was made by the addition of ACh (10-9-10 -5 M), bradykinin (BK) (10-13-10 -6 M), and NO solution (approx. 10-SM) instead of NTG. Furthermore, the tings were pretreated with N Gmonomethyl-L-arginine (LNMMA) (10-4M) for 20 rain before precontraction with NE, and a similar comparison was made by the addition of ACh (10-9-10 -5 M) before and after N T G treatment. In the control study, we performed the same experiments as described above, but incubated the pulmonary arterial tings with 1.1% mannitol (NTG solvent) instead of 5 x 10 -4 M N T G for 1 hr. All tings were pretreated with aspirin (10 -5 M) for 20 min in order to prevent prostaglandin formation, especially prostacyclin (PGI:), the formation of which could be provoked by various chemicals and manipulations (Gryglewski et al., 1986). Chemicals and solutions
The reagents were obtained from the following suppliers and were used without further purification: nitroglycerin aqueous solution (nitroglycerin, 0.5 mg/ml in 5% mannitol solution) from Nippon Kayaku Co. (Tokyo, Japan), dl-norepinephrine from Sankyo Co. (Tokyo, Japan), acetylcholine chloride from Dai-ichi Pharmaceutical Co. (Tokyo, Japan), bradykinin acetate from Sigma Chemical Co. (St Louis, MO, U.S.A.), and L N M M A (acetate salt) from Calbiochem Co. (La Jolla, CA, U.S.A.). All solutions were added directly to the baths, and the concentrations were calculated accordingly. Aqueous solution of NO was prepared in the following manner, essentially same as described by Shikano et al. (1987). NO gas were obtained from Nippon Sanso Co. (Tochigi, Japan) and was bubbled
NTG
I ACh -9 - -5 ~E-6 + ] BK -13--6 [ NO 1 (BEFORE)
t t WW
/[ NTG ACh "9 - - " 5 NE-6 + 1 BK -13~ -6 NTG 5 x 11)~! [ NO I~~IIIIIIIIIIII/A U t lhr t t ~ t (AFTER) W WW WW
Fig. 1. Protocol of tension measurements. NTG - 9 ~ - 5 : NTG 10-9~ 10-SM. ACh - 9 ~ - 5 : ACh 10 - 9 ~ 10 ~M. BK -13 ~ - 6 : BK 10-~3~ 10-6 M. NO, nitric oxide; NE - 6 , norepinephrine; 10-6 M W wash, washed by physiological solution every 15 min. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations.
ACh relaxation in NTG tolerance
63
0 20
i,o
**
**
60 80
"11
100 I
-9
i
I
i
-8
I
i
-7
I
-6
NTG CONCENTRATION
i
I
-5
Log [M]
Fig. 2. NTG dose-response curve before (O) and after (O) 1 hr treatment with 5 x 10-4 M NTG. Percent relaxation was calculated for 10 -6 M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean + SE of eight rings. Asterisks indicate points of significance(**P < 0.01).
for 15 min into a septum-sealed glass flask containing glass-distilled water which had previously been deoxygenated by bubbling with nitrogen for > 1 hr. Aliquots of the NO solution were added into the tissue baths immediately using a gas-tight syringe (Hamilton Co., Reno, NV, U.S.A.). Although actual concentrations of NO are unknown, an estimation of the maximal concentrations is possible. The solubility of NO in H20 at 0°C is 7.34cm3/100ml. Assuming complete saturation with gas, the maximal concentration of NO in solution is approx. 3.3 mM. This concentration was used to calculate the doses of NO administered. We used approx. 10 -5 M NO solution which caused a similar relaxation response to 10 -6 M ACh.
Data analysis Relaxation was expressed as a percentage of the tension induced by NE (10 -6 M). Student's t-test was used for statistical analysis between relaxation before and after 1 hr treatment with NTG. A P value of < 0.05 was considered to be a significant difference. All data are expressed as mean + SE. RESULTS
Relaxation response o f NTG After 1 hr treatment with 5 x 10-4M NTG, the relaxation response of NTG at concentrations > 10-7M was attenuated significantly (Fig. 2). The magnitude of contraction induced by 10 -6 M NE was 1140 _.+87.6 mg before NTG treatment and 1209 _+ 102.1 mg after NTG treatment. There was no statistically significant difference between before and after N T G treatment. This indicates confirmly that
1 hr treatment with 5 x 10 -4 M NTG induces NTG tolerance in isolated porcine pulmonary arterial tings.
Relaxation response o f ACh in NTG tolerance ACh (10-9-10-5M) relaxed porcine pulmonary arterial rings precontracted with 10-6M NE. The relaxation response of ACh at concentrations > 1 0 - 7 M was attenuated significantly after 1 hr treatment with 5 x 10-4M NTG (Fig. 3).
Relaxation response o f BK in NTG tolerance Furthermore, vasorelaxation of BK was investigated before and after 1 hr treatment with 5 x 10-4 M NTG. BK (10-13-10 -6 M) dose-dependently relaxed tings precontracted with 10-6M NE. Relaxation response of BK at concentrations > 10-1°M was attenuated significantly after NTG tolerance induction. (Fig. 4).
Relaxation response o f NO in NTG tolerance The time-course of relaxation by NO (approx. 10 -5 M) was compared before and after 1 hr treatment with 5 x 10-4 M NTG. NTG tolerance had no effect on NO-induced vascular smooth muscle relaxation (Fig. 5). It has been difficult to obtain dose-response curve of NO, because it is a very unstable substance. Therefore, we compared time course of NO-induced relaxation by single administration of NO before and after NTG tolerance.
Effect o f L N M M A pretreatment LNMMA (10-4 M) caused small contractions, probably due to inhibition of endogenous EDRF. ACh (10-9-10-SM) dose-dependently relaxed rings pretreated with LNMMA for 20 rain and precontracted
64
MASAYUK1 FUKAZAWA a n d ATSUSHI NAMIKI
0
2O
I ..I
F< 40
I
.<
"%,
**
60
**
"%
1: ....... 1 ....... 1 80
100 I
|
I
-9
-8
|
I
.
I
!
I
-7 -6 ACh CONCENTRATION
-5 Log [M]
Fig. 3. ACh dose-response curve before (©) and after (@) 1 hr treatment with 5 x l0 -4 M NTG. Percent relaxation was calculated for 10 -6 M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean _+ SE of eight rings. Asterisks indicate points of significance (*P < 0.05, **P < 0.01).
with 10 -6 M NE. The relaxation response of A C h p r e c o n t r a c t i o n with L N M M A at concentrations higher t h a n 1 0 - 7 M was attenuated after N T G tolerance (Fig. 6). The electrolyte contents a n d pH values of physiological solution in tissue b a t h s were not significantly different between before a n d after 1 hr t r e a t m e n t with 5 x 1 0 - 4 M N T G . There was no statistically significant difference between the dose-response curves by N T G , A C h a n d B K before a n d after 1 hr t r e a t m e n t
~.. . . . . . . . . m • . . . . . . . . . .i
o.,...,
with N T G solvent (1.1% m a n n i t o l solution) (data not shown).
DISCUSSION M a n y nitrovasodilators act t h r o u g h a c o m m o n mechanism, the liberation (generation) of NO. M a n y studies on the m e c h a n i s m o f nitrate tolerance have been reported, but the exact m e c h a n i s m remains u n k n o w n . The general tendency of this p h e n o m e n o n
_
20
"'-.
X
AFTER
Z
40
60 i%
**
80 ......... X
100 I
-13
I
I
I
i
-12
-11
-10
-9
I
I
-8
-7
I
-6
BK CONCENTRATION Fig. 4. BK dose-response curve before (C)) and after (O) 1 hr treatment with 5 x 10-4M NTG. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Percent relaxation was calculated for 10 -6 M NE precontraction. Values are expressed as the mean + SE of eight rings. Asterisks indicate points of significance (**P < 0.01).
ACh relaxation in NTG tolerance
65
%%%%%%%%%%%
20
Z .< x,<
40 AFrER
r ........................ 1
I 1
I 2
60
80
I 0
I 3 min
Fig. 5. Time-course of NO-induced relaxation before (O) and after (Q) I hr treatment with 5 x 1 0 - 4 M NTG. Percent relaxation was calculated for 10-6M NE precontraction. Values are expressed as the mean + SE of 12 rings.
mainly holds true for organic nitrates like N T G or isosorbide dinitrate, while nitrate tolerance is not observed or is observed to a much lesser degree after the application of the prodrug molsidomine, its main metabolite SIN-1 and sodium nitroprusside. N T G and isosorbide dinitrate generate N O in the metabolic activation process, while N O can be generated from SIN-1 or sodium nitroprusside non-enzymatically or without the interaction of any co-factor. Current hypotheses suggest that N T G tolerance may arise from reduced intracelluler metabolic conversion of N T G to NO, diminished G C activation (Waldman et al., 1986) and enhanced c G M P phos-
phodiesterase activity (Axelsson and Andersson, 1983). These metabolic processes need the presence of glutathion and glutathion S-transferase. Therefore, the development of tolerance may be involved in the deficiency of these substances. Many investigators have reported the endothelium-dependent vascular smooth muscle relaxation in N T G tolerance. Prior exposure of rat thoracic aorta to N T G decreased relaxation by A C h slightly in vivo (Molina et al., 1987) and in vitro (Rapoport et al., 1987), and Ljusegren et al. (1988) suggested the existence of a certain degree of cross-tolerance between N T G and A C h in isolated bovine mesenteric artery. However,
0
10
20 3o #. %
40
.......
.......
I
50
I -9
i
I -8
i
I -7
.
ACh CONCENTRATION
I -6
i
i -5
Log[M]
Fig. 6. ACh dose-response curve before (C)) and after (O) l hr treatment with 5 × 10-4 M NTG pretreated with 10-4M LNMMA for 20rain. Percent relaxation was calculated for 10-6M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean + SE of I0 rings. Asterisks indicate points of significance (*P < 0.05, **P < 0.01).
66
MASAYUKIFUKAZAWAand ATSUSHINAMIKI
Stewart et al. (1987) mentioned that a moderate degree of NTG tolerance to large coronary artery dilation did not affect the responsiveness to ACh in conscious dogs. The difference in species, vascular beds, duration and concentration of NTG pretreatment was noted in previous reports. In these reports, ACh was used to produce EDRF from endothelial cells. However, substances released by ACh include not only EDNO, but also other factors. To separate the effect of EDNO and other factors from endothelial cells by ACh, we checked vascular response by NO in NTG-tolerant blood vessels. In this study, we examined by using porcine pulmonary arteries by 1 hr treatment with 5 x 10-4 M NTG. In many previous reports, it has been recognized that various vascular beds show NTG tolerance by I hr treatment with 5 x 10-4 M NTG. [SD rat aorta: 2.2 x 10-4M N T G for I hr (Kowaluk and Fung, 1990); SD rat aorta: 4.4 x 10-4M NTG for 1 hr (Rapoport et al., 1987); human coronary artery: 10 -4 M NTG for 1 hr (Waldman et al., 1986)]. In this study, NTG tolerance induction was confirmed by 1 hr treatment with 5 × 10-4M NTG. The present study showed that when porcine pulmonary arterial rings were tolerant to NTG, the endothelium-dependent relaxation by ACh and BK was reduced. Nevertheless, NTG tolerance had no significant effect on relaxation of porcine pulmonary arterial rings by NO. Therefore, NTG tolerance induced by 1 hr treatment with 5 × 10-4 M NTG had no effect on the pathway after NO generation in vascular smooth muscle. When pretreated with aspirin and LNMMA, which inhibited the production of EDNO, ACh-induced relaxation was diminished, but the reduced degree was small. This indicates the existence of ACh-induced relaxation mechanism other than those due to PGI 2 and EDNO in porcine pulmonary arteries. In this study, we observed the reduced relaxation response of ACh after N T G tolerance pretreated with aspirin and LNMMA. Therefore, it is suggested that ACh releases some EDRF(s) other than NO, which is (are) affected by NTG tolerance. The existence of endothelium-derived hyperpolarizing factor (EDHF) has been pointed out in recent years (Chen et al., 1988, 1989a, b) have reported that the endothelium of isolated rat pulmonary arteries releases another factor that contributes to endothelium-dependent relaxation by causing hyperpolarization of the vascular smooth muscle. However, its chemical form and its signal transduction in vascular smooth muscle cells are still unknown. In this study, we used porcine pulmonary arteries. As we mentioned previously, ACh relaxed these arteries through the mechanism other than PGI 2 and
EDNO. It is possible that ACh releases EDHF from endothelial cells and relaxes porcine pulmonary arteries, and that EDHF may be affected by NTG tolerance. EDHF seems to be an endogenous potassium-channel activator, but its specific blocker has not been reported. Further investigations are needed to clarify these points. In many previous reports, it has generally been accepted that NTG-tolerant vessels show parallel reduction in cGMP accumulation and relaxation. However, Kowaluk and Fung (1990) reported dissociation between relaxation-response and cGMP levels during NTG tolerance. We have obtained a similar result. The measurement of cGMP may lead to clarify the mechanism that ACh releases EDRF(s) other than NO, which is (are) affected by NTG tolerance. In conclusion, ACh- and BK-induced endothelium-dependent relaxation was diminished, while NO-induced relaxation was unchanged in isolated NTG-tolerant porcine pulmonary arteries. The relaxation response of ACh pretreated with LNMMA was also attenuated after NTG tolerance. These results show that ACh releases some EDRF(s) other than NO, which is (are) affected by NTG tolerance. Acknowledgements--The authors thank Professor Kiyoshi
Machii and Professor Tetsu Yamaguchi for their valuable comments, and Drs Jo Aikawa, Masao Moroi and Michiro Ishikawa for their helpful discussion.
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Axelsson K. L. and Andersson R. G. G. (1983) Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover. Fur. J. Pharmac. 88, 71-79. Brien J. F., McLaughlin B. E., Breedon T. H., Bennett B. M., Nakatsu K. and Marks G. S. (1986) Biotransformation of glyceryltrinitrate occurs concurrently with relaxation of rabbit aorta. J. Pharmac. exp. Ther. 237, 608~ 14. Chen G. and Suzuki H. (1989a) Direct and indirect actions of acetylcholineand histamine on intrapulmonary artery and vein muscles of the rat. Jap. J. Physiol. 39, 51-65. Chen G. and Suzuki H. (1989b) Some electrical properties of the endothelium-dependenthyperpolarization recorded from rat arterial smooth muscle cells. J. Physiol. 410, 91-106. Chen G., Suzuki H. and Weston A. H. (1988) Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels. Br. J. Pharmac. 95, 1165-1174. Furchgott R. F. and Zawadzki J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376. Furchgott R. F., Khan M. T. and Jothianandan D. (1987) Comparison of endothelium-dependent relaxation and nitric oxide-inducedrelaxation in rabbit aorta. Fedn Proc. 46, 385. Gritiith T. M., Edwards D. H., Lewis M. J. and Henderson A. H. (1985) Evidence that cyclic guanosine monophosphate (cGMP) mediates endothelium-dependent relaxation. Fur. J. Pharmac. 112, 195-202.
ACh relaxation in NTG tolerance Gryglewski R. J., Palmer R. M. J. and Moncada S. (1986) Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320, 454-456. Hutchinson P. J. A., Palmer R. M. J. and Moncada S. (1987) Comparative pharmacology of EDRF and nitric oxide on vascular strips. Eur. J. Pharmac. 141, 445-451. Ignarro L. J., Buga G. M., Wood K. S., Byrns R. E. and Chaudhuri G. (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. natn ,4cad. Sci., U.S.A. 84, 9265-9269. Ignarro L. J., Lippton H., Edwards J. C., Baricos W. H., Hyman A. L., Kadowitz P. J. and Gruetter C. A. (1981) Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates. J. Pharmac. exp. Ther. 218, 739-749. Kowaluk E. A. and Fung H. L. (1990) Dissociation of nitrovasodilator-induced relaxation from cyclic GMP levels during in vitro nitrate tolerance. Eur. J. Pharmac. 176, 91-95. Ljusegren M. E., Ahlner J. and Axelsson K. L. (1988) Studies on vascular smooth muscle tolerance to different cGMP-mediated vasodilators and cross-tolerance to glyceryl trinitrate. Pharmac. Toxicol. 62, 302-307. Molina C. R., Andresen J. W., Rapoport R. M., Waldman S. and Murad F. (1987) Effect of in vivo nitroglycerin therapy on endothelium-dependent and independent vascular relaxation and cyclic GMP accumulation in rat aorta. J. cardiovasc. Pharmac. 10, 371-378. Namiki A., Hirata Y., Ishikawa M., Moroi M., Aikawa J. and Machii K. (1992) Endothelin-l- and endothelin-3-
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induced vasorelaxation via common generation of endothelium-derived nitric oxide. Life Sci. 50, 677q582. Needlman P. and Johnson E. M. Jr (1973) Mechanism of tolerance development to organic nitrates. J. Pharmac. exp. Ther. 184, 709-715. Needlman P., Jakschik B. A. and Johnson E. M. Jr (1973) Sulfhydryl requirement for relaxation of vascular smooth muscle. J. Pharmac Exp. Ther. 187, 324-33 I. Palmer R. M. J., Ferrige A. G. and Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526. Rapoport R. M. and Murad F. (1983) Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP. Circ. Res. 52, 352-357. Rapoport R. M., Waldman S. A., Ginsburg R., Molina C. R. and Murad F. (1987) Effect of glyceryl trinitrate on endothelium-dependent and -independent relaxation and cyclic GMP levels in rat aorta and human coronary artery. J. cardiovasc. Pharmac. 10, 82-89. Shikano K., Ohlstein E. H. and Berkowitz B. A. (1987) Differential selectivity of endothelium-derived relaxing factor and nitric oxide in smooth muscle. Br. J. Pharmac. 92, 483-485. Stewart D. J., Holtz J. and Bassenge E. (1987) Long-term nitroglycerin treatment: effect on direct and endotheliummediated large coronary artery dilatation in conscious dogs. Circulation 75, 847-856. Waldman S. A., Rapoport R. M., Ginsburg R. and Murad F. (1986) Desensitization to nitroglycerin in vascular smooth muscle from rat and human. Biochem. Pharmac. 35, 3525 3531.
Pergamon
Reduced Endothelium-dependent Vasodilation by Acetylcholine and Bradykinin in Isolated Nitroglycerin-tolerant Blood Vessels MASAYUKI FUKAZAWA* and ATSUSHI NAMIKI Third Department of Internal Medicine, Toho University School of Medicine, Ohashi Hospital, 2-17-6 Ohashi, Meguro-ku, Tokyo 153, Japan [Tel. 81-3-3468-1251; Fax 81-3-3468-1269]
(Receh,ed 4 June 1993)
A b s t r a c t - - l . Rings of porcine pulmonary arteries were mounted in tissue organ baths and incubated in
physiological solution. The rings were allowed to equilibrate for > 1 hr under a resting tension of 1.0 g. The presence of endothelium was confirmed by 10 -6 M acetylcholine (ACh)-induced relaxation (60-80%) of 10-6 M norepinephrine (NE) contraction. 2. Relaxation response generated by nitroglycerin (NTG) (10 9-10-5M), ACh (10-9-10-5M), bradykinin (BK) ( I 0 - 1 3 - 1 0 - 6 M ) and nitric oxide (NO) after NE ( 1 0 - 6 M ) contraction was compared before and after 1 hr treatment of NTG (5 × 10 4 M). Then tissues were pretreated with NG-monomethylL-arginine (LNMMA) (I0 4 M) each before and after NTG treatment respectively, and ACh-induced relaxation was compared. 3. After 1 hr treatment with 5 × 10 4 M NTG, the relaxation response of NTG at concentrations > 10 -7 M was attenuated significantly. This indicates that I hr treatment with 5 x 10 -4 M NTG induces NTG tolerance in isolated porcine pulmonary arterial rings. 4. The relaxation response of ACh at concentrations > 10 7 M was attenuated significantly after NTG tolerance induction. 5. Relaxation response of BK at concentrations > I0 -~° M was attenuated significantly after NTG tolerance induction. 6. NTG tolerance had no effect on NO-induced vascular smooth muscle relaxation. 7. The relaxation response ofACh pretreated with LNMMA at concentrations higher than 10 _7 M was attenuated after NTG tolerance. 8. These results demonstrate that ACh releases an endothelium-derived relaxing factor (EDRF) or several EDRFs other than NO which is (are) affected by NTG tolerance.
(cGMP), thereby eliciting vasodilation (Ignarro et al., 1981). N T G tolerance has been recognized clinically, which is developed in dependence on time a n d nitrate concentration. M a n y investigators have reported on the mechanism of N T G tolerance. N e e d l m a n a n d J o h n s o n 111973) and N e e d l m a n et al. (1973) d e m o n strated a reduction in tissue levels of sulfhydryl groups in N T G - t o l e r a n t vessels and proposed that it is the primary reason for N T G tolerance. Moreover, previous studies indicated that a decrease in the b i o t r a n s f o r m a t i o n o f N T G (Brien et al., 1986) as well as a reduction in activation of G C ( W a l d m a n et al., 1986) is responsible for the induction of N T G tolerance. Since F u r c h g o t t a n d Zawadzki (1980) reported that vascular s m o o t h muscle relaxation induced by
INTRODUCTION Nitrovasodilators such as nitroglycerin ( N T G ) are widely used for the treatment of cardiovascular diseases. However, nitrovasodilator-induced tolerance has been recognized and sometimes causes clinical problems. The vasodilating effect of N T G is t h o u g h t to be mediated by the f o r m a t i o n of nitric oxide (NO) in the vascular s m o o t h muscle cells. Organic nitrates are postulated to release nitrite in the presence o f thiols which is then transformed into nitrosothiol and N O with the aid of a n o t h e r thiol. The generated N O activates soluble guanylate cyclase (GC) a n d liberates guanosine 3 ' , 5 ' - m o n o p h o s p h a t e
*To whom all correspondence should be addressed. 61
62
MASAYUKIFUKAZAWAand ATSUSrUNAMiKI
acetylcholine (ACh) was endothelium-dependent, the importance of endothelial cells for vascular reactivity has been recognized (Furchgott et al., 1987). Although it is still unknown whether the endothelium-derived relaxing factor (EDRF) is a single substance, it has been suggested that at least one of the EDRF(s) is NO (Furchgott et al., 1987; Huchinson et al., 1987; Ignarro et al., 1987; Palmer et al., 1987). Therefore, the NO ingredient of the E D R F is especially called endothelium-derived nitric oxide (EDNO). The stimulation of GC and the subsequent cGMP increase in smooth muscle are the common mechanisms promoting NTG- and EDNO-induced vascular relaxation (Rapoport and Murad, 1983; Griffith et at., 1985). Therefore, it may be probable that the effect of EDNO is altered in NTG-tolerant vessels. In this study, we thus investigated the effect of N T G tolerance on the endothelium-dependent relaxation produced by some vasodilators. MATERIALS AND METHODS Tension measurements
Lungs of Yorkshire swine (approx. 6 months old, weighing 100 kg) were obtained at a slaughterhouse. Lungs were dissected within 60min after death, placed in ice-cold physiological solution (NaCI 118, KCI 4.75, CaC12 2.54, KH2PO4 1.19, NaHCO3 25.0, MgSO4 1.2, glucose 11.0 mM) and transported to the laboratory. The pulmonary arteries were carefully cleaned free of adherent and connective tissues, and cut into rings. Rings (outer diameter 4 mm, length 5 mm approx.) were mounted in tissue organ baths containing 10 ml of physiological solution. The bath medium was maintained at 37°C and aerated before and during the experiments with a mixture of 95% 02 and 5% CO 2. Under these conditions, the pH level of 7.4 remained constant. Isometric developed tension was recorded by isometric force-transducers (FD pickup TB-611T, Nihon Kohden, Tokyo, Japan). Before the start of the experiments, the rings were allowed to equilibrate for > 1 hr under a resting tension of 1.0 g. During this period, the physiological solution in the tissue baths was replaced every 15 min, The presence of endothelium was confirmed by 10 -6 M ACh-induced relaxation (60-80%) of 10 - 6 M norepinephrine (NE) contraction. We previously sought the optimal resting tension of pulmonary arterial ring segments of Yorkshire swine, and concluded that 1.0 g was the optimal level (Namiki et al., 1992). Effect o f N T G treatment on the relaxation o f vasodilators
After the tings were precontracted with 1 0 - t M NE, N T G was added cumulatively from 10 9 to
10 -5 M (response before N T G treatment). The tings were then incubated with 5 x 10 -4 M NTG for 1 hr and washed by the physiological solution four times every 15 min. After the rings were precontracted with 1 0 - t M NE, N T G was again added cumulatively from 10 -9 to 10 -5 M (response after NTG treatment). The response before and after N T G treatment was compared in the same tings (Fig. i). A similar comparison was made by the addition of ACh (10-9-10 -5 M), bradykinin (BK) (10-13-10 -6 M), and NO solution (approx. 10-SM) instead of NTG. Furthermore, the tings were pretreated with N Gmonomethyl-L-arginine (LNMMA) (10-4M) for 20 rain before precontraction with NE, and a similar comparison was made by the addition of ACh (10-9-10 -5 M) before and after N T G treatment. In the control study, we performed the same experiments as described above, but incubated the pulmonary arterial tings with 1.1% mannitol (NTG solvent) instead of 5 x 10 -4 M N T G for 1 hr. All tings were pretreated with aspirin (10 -5 M) for 20 min in order to prevent prostaglandin formation, especially prostacyclin (PGI:), the formation of which could be provoked by various chemicals and manipulations (Gryglewski et al., 1986). Chemicals and solutions
The reagents were obtained from the following suppliers and were used without further purification: nitroglycerin aqueous solution (nitroglycerin, 0.5 mg/ml in 5% mannitol solution) from Nippon Kayaku Co. (Tokyo, Japan), dl-norepinephrine from Sankyo Co. (Tokyo, Japan), acetylcholine chloride from Dai-ichi Pharmaceutical Co. (Tokyo, Japan), bradykinin acetate from Sigma Chemical Co. (St Louis, MO, U.S.A.), and L N M M A (acetate salt) from Calbiochem Co. (La Jolla, CA, U.S.A.). All solutions were added directly to the baths, and the concentrations were calculated accordingly. Aqueous solution of NO was prepared in the following manner, essentially same as described by Shikano et al. (1987). NO gas were obtained from Nippon Sanso Co. (Tochigi, Japan) and was bubbled
NTG
I ACh -9 - -5 ~E-6 + ] BK -13--6 [ NO 1 (BEFORE)
t t WW
/[ NTG ACh "9 - - " 5 NE-6 + 1 BK -13~ -6 NTG 5 x 11)~! [ NO I~~IIIIIIIIIIII/A U t lhr t t ~ t (AFTER) W WW WW
Fig. 1. Protocol of tension measurements. NTG - 9 ~ - 5 : NTG 10-9~ 10-SM. ACh - 9 ~ - 5 : ACh 10 - 9 ~ 10 ~M. BK -13 ~ - 6 : BK 10-~3~ 10-6 M. NO, nitric oxide; NE - 6 , norepinephrine; 10-6 M W wash, washed by physiological solution every 15 min. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations.
ACh relaxation in NTG tolerance
63
0 20
i,o
**
**
60 80
"11
100 I
-9
i
I
i
-8
I
i
-7
I
-6
NTG CONCENTRATION
i
I
-5
Log [M]
Fig. 2. NTG dose-response curve before (O) and after (O) 1 hr treatment with 5 x 10-4 M NTG. Percent relaxation was calculated for 10 -6 M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean + SE of eight rings. Asterisks indicate points of significance(**P < 0.01).
for 15 min into a septum-sealed glass flask containing glass-distilled water which had previously been deoxygenated by bubbling with nitrogen for > 1 hr. Aliquots of the NO solution were added into the tissue baths immediately using a gas-tight syringe (Hamilton Co., Reno, NV, U.S.A.). Although actual concentrations of NO are unknown, an estimation of the maximal concentrations is possible. The solubility of NO in H20 at 0°C is 7.34cm3/100ml. Assuming complete saturation with gas, the maximal concentration of NO in solution is approx. 3.3 mM. This concentration was used to calculate the doses of NO administered. We used approx. 10 -5 M NO solution which caused a similar relaxation response to 10 -6 M ACh.
Data analysis Relaxation was expressed as a percentage of the tension induced by NE (10 -6 M). Student's t-test was used for statistical analysis between relaxation before and after 1 hr treatment with NTG. A P value of < 0.05 was considered to be a significant difference. All data are expressed as mean + SE. RESULTS
Relaxation response o f NTG After 1 hr treatment with 5 x 10-4M NTG, the relaxation response of NTG at concentrations > 10-7M was attenuated significantly (Fig. 2). The magnitude of contraction induced by 10 -6 M NE was 1140 _.+87.6 mg before NTG treatment and 1209 _+ 102.1 mg after NTG treatment. There was no statistically significant difference between before and after N T G treatment. This indicates confirmly that
1 hr treatment with 5 x 10 -4 M NTG induces NTG tolerance in isolated porcine pulmonary arterial tings.
Relaxation response o f ACh in NTG tolerance ACh (10-9-10-5M) relaxed porcine pulmonary arterial rings precontracted with 10-6M NE. The relaxation response of ACh at concentrations > 1 0 - 7 M was attenuated significantly after 1 hr treatment with 5 x 10-4M NTG (Fig. 3).
Relaxation response o f BK in NTG tolerance Furthermore, vasorelaxation of BK was investigated before and after 1 hr treatment with 5 x 10-4 M NTG. BK (10-13-10 -6 M) dose-dependently relaxed tings precontracted with 10-6M NE. Relaxation response of BK at concentrations > 10-1°M was attenuated significantly after NTG tolerance induction. (Fig. 4).
Relaxation response o f NO in NTG tolerance The time-course of relaxation by NO (approx. 10 -5 M) was compared before and after 1 hr treatment with 5 x 10-4 M NTG. NTG tolerance had no effect on NO-induced vascular smooth muscle relaxation (Fig. 5). It has been difficult to obtain dose-response curve of NO, because it is a very unstable substance. Therefore, we compared time course of NO-induced relaxation by single administration of NO before and after NTG tolerance.
Effect o f L N M M A pretreatment LNMMA (10-4 M) caused small contractions, probably due to inhibition of endogenous EDRF. ACh (10-9-10-SM) dose-dependently relaxed rings pretreated with LNMMA for 20 rain and precontracted
64
MASAYUK1 FUKAZAWA a n d ATSUSHI NAMIKI
0
2O
I ..I
F< 40
I
.<
"%,
**
60
**
"%
1: ....... 1 ....... 1 80
100 I
|
I
-9
-8
|
I
.
I
!
I
-7 -6 ACh CONCENTRATION
-5 Log [M]
Fig. 3. ACh dose-response curve before (©) and after (@) 1 hr treatment with 5 x l0 -4 M NTG. Percent relaxation was calculated for 10 -6 M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean _+ SE of eight rings. Asterisks indicate points of significance (*P < 0.05, **P < 0.01).
with 10 -6 M NE. The relaxation response of A C h p r e c o n t r a c t i o n with L N M M A at concentrations higher t h a n 1 0 - 7 M was attenuated after N T G tolerance (Fig. 6). The electrolyte contents a n d pH values of physiological solution in tissue b a t h s were not significantly different between before a n d after 1 hr t r e a t m e n t with 5 x 1 0 - 4 M N T G . There was no statistically significant difference between the dose-response curves by N T G , A C h a n d B K before a n d after 1 hr t r e a t m e n t
~.. . . . . . . . . m • . . . . . . . . . .i
o.,...,
with N T G solvent (1.1% m a n n i t o l solution) (data not shown).
DISCUSSION M a n y nitrovasodilators act t h r o u g h a c o m m o n mechanism, the liberation (generation) of NO. M a n y studies on the m e c h a n i s m o f nitrate tolerance have been reported, but the exact m e c h a n i s m remains u n k n o w n . The general tendency of this p h e n o m e n o n
_
20
"'-.
X
AFTER
Z
40
60 i%
**
80 ......... X
100 I
-13
I
I
I
i
-12
-11
-10
-9
I
I
-8
-7
I
-6
BK CONCENTRATION Fig. 4. BK dose-response curve before (C)) and after (O) 1 hr treatment with 5 x 10-4M NTG. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Percent relaxation was calculated for 10 -6 M NE precontraction. Values are expressed as the mean + SE of eight rings. Asterisks indicate points of significance (**P < 0.01).
ACh relaxation in NTG tolerance
65
%%%%%%%%%%%
20
Z .< x,<
40 AFrER
r ........................ 1
I 1
I 2
60
80
I 0
I 3 min
Fig. 5. Time-course of NO-induced relaxation before (O) and after (Q) I hr treatment with 5 x 1 0 - 4 M NTG. Percent relaxation was calculated for 10-6M NE precontraction. Values are expressed as the mean + SE of 12 rings.
mainly holds true for organic nitrates like N T G or isosorbide dinitrate, while nitrate tolerance is not observed or is observed to a much lesser degree after the application of the prodrug molsidomine, its main metabolite SIN-1 and sodium nitroprusside. N T G and isosorbide dinitrate generate N O in the metabolic activation process, while N O can be generated from SIN-1 or sodium nitroprusside non-enzymatically or without the interaction of any co-factor. Current hypotheses suggest that N T G tolerance may arise from reduced intracelluler metabolic conversion of N T G to NO, diminished G C activation (Waldman et al., 1986) and enhanced c G M P phos-
phodiesterase activity (Axelsson and Andersson, 1983). These metabolic processes need the presence of glutathion and glutathion S-transferase. Therefore, the development of tolerance may be involved in the deficiency of these substances. Many investigators have reported the endothelium-dependent vascular smooth muscle relaxation in N T G tolerance. Prior exposure of rat thoracic aorta to N T G decreased relaxation by A C h slightly in vivo (Molina et al., 1987) and in vitro (Rapoport et al., 1987), and Ljusegren et al. (1988) suggested the existence of a certain degree of cross-tolerance between N T G and A C h in isolated bovine mesenteric artery. However,
0
10
20 3o #. %
40
.......
.......
I
50
I -9
i
I -8
i
I -7
.
ACh CONCENTRATION
I -6
i
i -5
Log[M]
Fig. 6. ACh dose-response curve before (C)) and after (O) l hr treatment with 5 × 10-4 M NTG pretreated with 10-4M LNMMA for 20rain. Percent relaxation was calculated for 10-6M NE precontraction. Concentrations of agents are expressed as log molar concentrations and represent final bath concentrations. Values are expressed as the mean + SE of I0 rings. Asterisks indicate points of significance (*P < 0.05, **P < 0.01).
66
MASAYUKIFUKAZAWAand ATSUSHINAMIKI
Stewart et al. (1987) mentioned that a moderate degree of NTG tolerance to large coronary artery dilation did not affect the responsiveness to ACh in conscious dogs. The difference in species, vascular beds, duration and concentration of NTG pretreatment was noted in previous reports. In these reports, ACh was used to produce EDRF from endothelial cells. However, substances released by ACh include not only EDNO, but also other factors. To separate the effect of EDNO and other factors from endothelial cells by ACh, we checked vascular response by NO in NTG-tolerant blood vessels. In this study, we examined by using porcine pulmonary arteries by 1 hr treatment with 5 x 10-4 M NTG. In many previous reports, it has been recognized that various vascular beds show NTG tolerance by I hr treatment with 5 x 10-4 M NTG. [SD rat aorta: 2.2 x 10-4M N T G for I hr (Kowaluk and Fung, 1990); SD rat aorta: 4.4 x 10-4M NTG for 1 hr (Rapoport et al., 1987); human coronary artery: 10 -4 M NTG for 1 hr (Waldman et al., 1986)]. In this study, NTG tolerance induction was confirmed by 1 hr treatment with 5 × 10-4M NTG. The present study showed that when porcine pulmonary arterial rings were tolerant to NTG, the endothelium-dependent relaxation by ACh and BK was reduced. Nevertheless, NTG tolerance had no significant effect on relaxation of porcine pulmonary arterial rings by NO. Therefore, NTG tolerance induced by 1 hr treatment with 5 × 10-4 M NTG had no effect on the pathway after NO generation in vascular smooth muscle. When pretreated with aspirin and LNMMA, which inhibited the production of EDNO, ACh-induced relaxation was diminished, but the reduced degree was small. This indicates the existence of ACh-induced relaxation mechanism other than those due to PGI 2 and EDNO in porcine pulmonary arteries. In this study, we observed the reduced relaxation response of ACh after N T G tolerance pretreated with aspirin and LNMMA. Therefore, it is suggested that ACh releases some EDRF(s) other than NO, which is (are) affected by NTG tolerance. The existence of endothelium-derived hyperpolarizing factor (EDHF) has been pointed out in recent years (Chen et al., 1988, 1989a, b) have reported that the endothelium of isolated rat pulmonary arteries releases another factor that contributes to endothelium-dependent relaxation by causing hyperpolarization of the vascular smooth muscle. However, its chemical form and its signal transduction in vascular smooth muscle cells are still unknown. In this study, we used porcine pulmonary arteries. As we mentioned previously, ACh relaxed these arteries through the mechanism other than PGI 2 and
EDNO. It is possible that ACh releases EDHF from endothelial cells and relaxes porcine pulmonary arteries, and that EDHF may be affected by NTG tolerance. EDHF seems to be an endogenous potassium-channel activator, but its specific blocker has not been reported. Further investigations are needed to clarify these points. In many previous reports, it has generally been accepted that NTG-tolerant vessels show parallel reduction in cGMP accumulation and relaxation. However, Kowaluk and Fung (1990) reported dissociation between relaxation-response and cGMP levels during NTG tolerance. We have obtained a similar result. The measurement of cGMP may lead to clarify the mechanism that ACh releases EDRF(s) other than NO, which is (are) affected by NTG tolerance. In conclusion, ACh- and BK-induced endothelium-dependent relaxation was diminished, while NO-induced relaxation was unchanged in isolated NTG-tolerant porcine pulmonary arteries. The relaxation response of ACh pretreated with LNMMA was also attenuated after NTG tolerance. These results show that ACh releases some EDRF(s) other than NO, which is (are) affected by NTG tolerance. Acknowledgements--The authors thank Professor Kiyoshi
Machii and Professor Tetsu Yamaguchi for their valuable comments, and Drs Jo Aikawa, Masao Moroi and Michiro Ishikawa for their helpful discussion.
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