Functional and histologic impairments of endothelial cells are causal factors in the genesis and development of atherosclerosis. Hypercholesterolemia, an important risk factor for atherosclerosis (1), interferes with endothelial cell functions (2). Endothelial dysfunctions in the atherosclerotic lesion are postulated to evoke additional histologic changes (3). It is widely recognized that endothelium-dependent vasodilatation and release of nitric oxide (NO) from the endothelium elicited by acetylcholine or other muscarinic agonists are blunted in atherosclerotic arteries and those exposed to hypercholesterolemia in vitro or in vivo, whereas endothelium-independent vasodilatation induced by nitrovasodilators, such as sodium nitroprusside, SIN 1 etc., is unaltered (4-9). Therefore under these pathologic conditions, decreased production or action of endothelial NO and increased degradation of NO by superoxide anions are speculated. However, other investigators have demonstrated that the response to endothelium-derived relaxing factor (EDRF)-releasing substances other than acetylcholine is not impaired in atherosclerotic arteries from various mammals (10), including humans (11,12). If this is the case, mechanisms underlying the depressed action of acetylcholine must be reconsidered.
NO is undoubtedly an important mediator in preventing vascular dysfunction and pathologic change (13), but it may also exert deleterious effects on atherosclerosis (14). In addition, attention is directed to endothelium-derived hyperpolarizing factor (EDHF) that opens K+ channels in smooth muscle cells (15) and is suggested to participate more in lowering peripheral vascular resistance than endothelial NO (16-18). Many compounds, including acetylcholine and substance P, liberate not only endothelial NO but also EDHF from a variety of blood vessels (15), although the nature of EDHF has not been identified. Despite such an important vasodilator property of EDHF, modification by atherosclerosis of responsiveness to this factor has not been investigated.
There are quite a few animal models of atherosclerosis. Among these, heritable, hyperlipidemic (WHHL) rabbits introduced by Watanabe (19) have been reported to show evident, consistent atherosclerotic changes in blood vessels (20,21) and are regarded as a good model of familial hypercholesterolemia. In the present study, endothelium-dependent responses to acetylcholine and substance P and endothelium-independent responses to sodium nitroprusside and 2,2-(hydroxynitrosohydrazino)-bis-ethanamine (NOC18) of isolated carotid arteries from senile Japanese white (JW) and WHHL rabbits (age 20-29 months) were compared. Aims of this study were to determine whether both of the relaxations elicited by endothelium-derived NO and EDHF were impaired by atherosclerosis and whether the responses to acetylcholine and substance P were affected by atherosclerosis in a similar manner, and to clarify functional changes in the arteries with and without atherosclerotic lesions from WHHL rabbits exposed for long period to hyperlipidemia.
MATERIALS AND METHODS
Experiments were carried out with WHHL rabbits (WHHL; n = 18, weighing 3.6-4.9 kg, 20-29 months old) and Japanese white (JW) rabbits (n = 26, weighing 3.4-4.7 kg, 20-29 months old), which were fed cholesterol-free standard lab chow in the Institute for Experimental Animals of the Kobe University School of Medicine. The Animal Care and Use Committee at the Shiga University of Medical Science approved the use of rabbit blood vessels in this study.
Biochemical study of plasma cholesterol
The rabbits were anesthetized with intraperitoneal injections of sodium pentobarbital (50 mg/kg), and blood samples were collected from the aorta for determination of serum cholesterol and triglyceride in some rabbits. Plasma levels of total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol and triglyceride were measured based on the methods of Allian et al. (22) and Spayd et al. (23) using autoanalyzers (Hitachi 7450, Hitachinaka, Japan, and Olympus AU600, Hachioji, Japan).
Carotid arteries from JW and WHHL rabbits were fixed with 2.5% glutaraldehyde (Nacalai Tesque, Kyoto) and embedded in paraffin. Then the sample was cut into 2.5-μm sections and stained with hematoxylin-eosin.
The animals were killed by bleeding from the aorta under deep anesthesia as described. The common carotid arteries were isolated and cut into helical strips of ∼20 mm long, with special care to avoid damage to the endothelium. The reason for the use of arterial strips was to separate the tissues obtained from WHHL rabbits, in which 50-75% of the intimal surface was covered by visible atherosclerotic lesions, from those with no visible atherosclerosis. The strips were vertically fixed between hooks in a muscle bath (20 ml) containing modified Ringer-Locke solution maintained at 37 ± 0.3°C and aerated with a mixture of 95% O2 and 5% CO2. The upper end of the strips was connected to the lever of a force-displacement transducer (Nihon-Kohden Kogyo Co., Tokyo, Japan). The resting tension was adjusted to 2.0 g, optimal for inducing the maximal contraction. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a cotton ball. Endothelial integrity was determined by relaxations induced by 10−6M acetylcholine. Constituents of the bathing media were as follows (mM): NaCl, 120; KCl, 5.4; CaCl2, 2.2; MgCl2, 1.0; NaHCO3, 25.0; and dextrose, 5.6. The pH of the solution was 7.38 to 7.44. Before the start of experiments, all of the strips were allowed to equilibrate in the bathing media for 30 min, during which time the fluid was replaced every 10 min.
Isometric mechanical responses were displayed on an ink-writing oscillograph. The contractile response to 30 mM K+ was first obtained, and the arterial strips were then repeatedly rinsed with fresh media and equilibrated. The arterial strips were partially contracted with phenylephrine (10−7-10−6M) in a range between 30 and 45% of contraction induced by 30 mM K+. Concentration-response curves of acetylcholine, sodium nitroprusside, and NOC18 were obtained by adding the drugs directly to the physiologic buffer in cumulative concentrations. To avoid tachyphylaxis, one of the concentrations of substance P was used, and after repeated rinsing and 30-40 min equilibration, the next concentration was applied. The concentrations used were from 10−10 to 10−8M. In the same strips, a similar experiment was performed to construct the concentration-response curve for substance P. Papaverine (10−4M) was added at the end of each series to obtain the maximal relaxation. The relaxations elicited by test drugs were presented as values relative to those to 10−4M papaverine. All of the strips were treated with 10−6M indomethacin to prevent the formation of prostanoids. Preparations were treated for ≥20 min with blocking agents, except for pertussis toxin (PTX; 100 ng/ml), which was applied for 120 min to the strips. In some experiments, the responses to acetylcholine or substance P were compared in arterial strips with and without the endothelium obtained from the same rabbits.
Statistics and drugs used
The results shown in the text, tables, and figures are expressed as mean values ± SEM. Statistical analyses were made using the Student's paired and unpaired t tests for two groups and the Tukey's test after one-way analysis of variance for three or more groups. The n in the text, tables, and figures denotes the number of strips from different individuals, unless otherwise mentioned.
Drugs used were acetylcholine chloride (Daiichi Co., Tokyo), indomethacin, superoxide dismutase (SOD), L-phenylephrine hydrochloride (Sigma Chemical Co., St. Louis, MO, U.S.A.), sodium nitroprusside (E. Merck, Darmstadt, Germany), NG-nitro-L-arginine (L-NA), substance P, apamin, charybdotoxin (Peptide Institute Inc., Minoh, Japan), pertussis toxin (PTX; Seikagaku Corp., Tokyo), 2,2-(hydroxynitrosohydrazino)bis-ethanamine (NOC18; Dojindo Lab., Kumamoto, Japan), and papaverine hydrochloride (Dainippon Pharmaceutical Co., Osaka, Japan). 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) was a generous gift from Dr. S. Moncada.
Plasma concentrations of total cholesterol, LDL and HDL cholesterol, and triglyceride
Mean values of the total, LDL, and HDL cholesterol and triglyceride in plasma obtained from WHHL (20-29 months) and age-matched JW rabbits are summarized in Table 1. The values, except for the HDL cholesterol level, markedly increased in the WHHL rabbits. In particular, the level of LDL cholesterol in WHHL rabbits is about 18 times that in JW rabbits.
Histologic changes in the carotid artery
Figure 1 demonstrates macroscopic views of the left and right carotid arteries from a WHHL rabbit. The arteries were longitudinally cut to visualize histologic changes in the intimal surface. Atherosclerotic lesions of the white-colored and thickened intima are quite evident in the regions indicated by arrows, and in some other regions, no or only slight changes are observed in the same arteries from WHHL rabbits. Conversely, there was no atherosclerosis in the arteries from JW rabbits. Microscopic pictures in the region of atherosclerosis of the artery from a WHHL rabbit and in the JW rabbit artery are compared in Fig. 2. The fibrous plaque, including smooth muscle cells, collagen and elastin fibers, and white blood cells, occupies most of the lumen of the artery. Regions of the artery without atherosclerosis from WHHL rabbits (n = 3) did not show any histologic change (data not shown), contrasting to the artery with atherosclerosis shown in Fig. 2B.
Subsequent functional studies were carried out with helical strips of the carotid artery from JW rabbits and those with the thickened intima that covered more than half of the intimal surface and with no visible change from WHHL rabbits.
Responses to acetylcholine of arterial strips
The addition of acetylcholine (10−8-10−5M) produced a concentration-related relaxation in carotid arterial strips from JW and WHHL rabbits treated with 10−6M indomethacin and partially contracted with phenylephrine. The response was totally abolished by removal of the endothelium from the strips of 10 JW and 10 WHHL rabbits and also by treatment with 10−7M atropine (n = 10 and 6, respectively). Concentration-response curves of acetylcholine in the JW rabbit arteries and those with and without atherosclerotic lesions from WHHL rabbits are compared in Fig. 3, left. The responses of JW and WHHL nonatherosclerotic arteries did not differ, whereas those of the atherosclerotic arterial strips were significantly reduced. Treatment with L-NA at 10−4M, which is sufficient to inhibit NO formation (24), moderately attenuated the acetylcholine-induced relaxation. The L-NA-resistant relaxation induced by 10−5M acetylcholine was significantly less in the WHHL rabbit arteries than in those from JW rabbits (Fig. 3, right). However, mean values of the L-NA-resistant relaxations relative to the total relaxation did not differ in WHHL (28% and 72%) and JW (40% and 90%) rabbit arteries. The relative values of the L-NA-sensitive relaxations also did not differ in two groups (44% and 72% vs. 50% and 90%, respectively). Relaxations to sodium nitroprus-side (10−10-10−6M) and NOC18 (10−8-10−5M) were compared in the arteries treated with indomethacin (10−6M) and L-NA (10−4M) from JW and WHHL rabbits. Both mean values of the maximal relaxation and the median effective concentration (EC50) in the arteries from JW rabbits and the WHHL rabbit arteries with and without atherosclerosis did not significantly differ (Table 2).
In endothelium-nondenuded JW and atherosclerotic WHHL rabbit arterial strips, the relaxation induced by acetylcholine was significantly attenuated by L-NA (10−4M), and the additional treatment with charybdotoxin (10−7M) almost abolished the remaining response (Fig. 4). Typical recordings of the response are illustrated in Fig. 5. In six of nine strips from JW rabbits treated with L-NA and charybdotoxin, slight relaxations by acetylcholine were abolished by apamin (10−6M). NOC18 (10−8-10−5M), an NO donor, relaxed the carotid arteries treated with indomethacin (10−6M) and L-NA (10−4M) from JW rabbits in a concentration-dependent manner. The relaxations were not affected by combined treatment with charybdotoxin (10−7M) plus apamin (10−6M), but were abolished by treatment with 1H[1,2,4]oxadiazole[4,3-a]-quinoxalin-1-one (ODQ, 10−6M), a guanylate cyclase inhibitor. The modifications of the NOC18 (10−6M)-induced relaxations by charybdotoxin plus apamin and ODQ are summarized in Table 3.
Responses to substance P of arterial strips
Concentration-related relaxations were elicited by the addition of substance P (10−10-10−8M) in the arterial strips from JW rabbits and those (atherosclerotic and nonsclerotic) from WHHL rabbits (Fig. 6). Although the maximal responses did not differ, the responses to the peptide at 10−10-10−9M in WHHL rabbit arteries were greater than in those from JW rabbits. Endothelium denudation abolished the response to 3 × 10−9M substance P in JW (n = 7) and WHHL rabbit arteries (n = 5). Figure 7 summarizes the effect of L-NA (10−4M) and charybdotoxin (10−7M) on the response to 10−9M substance P. L-NA partially inhibited, and additional treatment with charybdotoxin almost abolished the response in both of the JW and WHHL rabbit arteries. In six of nine strips from JW rabbits exposed to L-NA and charybdotoxin, apamin (10−6M) abolished the remaining relaxation. The inhibitory effect of L-NA was significantly greater in the WHHL than JW rabbit arteries (71.6 ± 7.6% vs. 52.7 ± 3.8% inhibition; p < 0.05, unpaired t test).
Effects of PTX and SOD
To determine whether the endothelium-dependent relaxations were mediated by G protein sensitive to PTX, effects of this toxin (100 ng/ml for 120 min) on the responses to acetylcholine and substance P were evaluated in the arteries from JW rabbits. The data are summarized in Table 4. No inhibition was obtained in the relaxations induced by these agonists, but the response after long exposure to the control bathing media or those containing PTX were rather greater than that seen before the exposure.
The addition of SOD (200 U/ml) to phenylephrine-contracted strips with the endothelium elicited relaxations of 11.9 ± 2.9 (n = 13), 7.9 ± 1.8% (n = 11), and 9.7 ± 2.1% (n = 6) in the JW, WHHL-nonatherosclerotic and WHHL-atherosclerotic rabbit arteries, respectively, the difference being statistically insignificant.
In WHHL rabbits of 20-29 months old, plasma total cholesterol, LDL cholesterol, and triglyceride concentrations were evidently higher than those in age-matched JW rabbits. Carotid arteries isolated from the WHHL rabbits showed patchy distribution of marked thickening of the intima and atherosclerotic histologic changes. Advanced atherosclerosis of the aorta and focal coronary atherosclerosis are observed in 15-month-old WHHL rabbits (20). Inheritability of coronary atherosclerosis in our WHHL rabbits has also been reported (25).
In the WHHL arterial strips in which the inner surface was largely covered by the thickened intima, endothelium-dependent relaxations induced by acetylcholine were attenuated compared with those in JW and WHHL nonatherosclerotic arteries. No difference was seen in the responses to acetylcholine of JW and WHHL rabbit arteries without atherosclerosis. Therefore it appears that the inhibition of acetylcholine actions is associated with atherosclerotic lesions rather than prolonged exposure to hypercholesterolemic plasma in vivo. Similar findings that depression of acetylcholine-induced relaxation is strictly dependent on the presence of fatty streaks and is not related to the plasma LDL levels have been reported (26,27). Because of the significant attenuation by L-NA, the relaxation would be mediated partly by NO derived from the endothelium. Relaxations induced by sodium nitroprusside and NOC18, NO donors, did not differ in the control and atherosclerotic arteries, suggesting that the sensitivity to NO of the smooth muscle was not impaired by atherosclerosis. Therefore it is unlikely that NO synthase is induced in the atherosclerotic lesions (14), because excessive amounts of NO may desensitize vascular smooth muscle cells to the vasodilator effects of NO. Similar findings on muscarinic agonists and nitroprusside were also obtained in the WHHL aorta (6) and subcutaneous arteries of atherosclerotic humans (12). Superoxide generation is postulated to increase in atherosclerotic lesions (28-30). However, in the present study, the SOD-induced relaxation in WHHL rabbit arteries was comparable to that in JW rabbit arteries. Our preliminary study demonstrated that the relaxant response to SOD was correlated directly to the amount of generated superoxide (31) that was measured by a lucigenin method (32). Increased degradation of NO by extracellular superoxide from endothelium and smooth muscle would be excluded in WHHL rabbit carotid arteries with atherosclerosis and those exposed for long periods to hypercholesterolemia in vivo.
Relaxations to substance P, dependent on the endothelium and mediated partly by NO, were not reduced but rather were potentiated in the WHHL rabbit arteries with and without atherosclerosis. The literature has demonstrated that EDRF-mediated responses to substance P, histamine, and Ca2+ ionophore A23187 are not impaired by atherosclerosis in human coronary and subcutaneous arteries (11,12). Therefore NO production from L-arginine by eNOS would not be reduced in atherosclerotic lesions. In addition, the thickened intima does not seem to interfere with the access of NO released from the endothelium to smooth muscle. The eNOS protein and mRNA have been reported to increase in the WHHL rabbit aorta (21). If this is true in the carotid artery from WHHL rabbits in old age, the potentiated response to substance P is considered to be due to increased production of endothelial NO. Lewis et al. (12) have suggested that reduced reactivity to acetylcholine with preserved responses to substance P of isolated subcutaneous arteries from patients with hypercholesterolemia may be of predictive value for disease progression. However, this is unlikely the case in WHHL rabbits, because the attenuated response to acetylcholine is observed only in atherosclerotic arteries, but not in nonatherosclerotic arteries of old WHHL rabbits (age 20-29 months). The presence of an altered degradation pathway for substance P in the arteries of WHHL rabbits may be excluded because peptides (substance P and bradykinin)-induced, endothelium-dependent relaxations were not different between those in atherosclerotic and nonatherosclerotic human coronary artery (11). Similar distinct changes in the response to different agonists have also been observed in the aortae from younger WHHL rabbits (11-14 months) (6) and rabbit carotid arteries with neointima formation (10).
According to Shimokawa et al. (33), atherosclerosis impairs the response of porcine coronary arteries only to some EDRF-releasing substances that transmit information of NO synthesis via PTX-sensitive G protein, such as in the case of thrombin, UK 14,304, an α2-adrenoceptor agonist, and serotonin. In the present study, although only the acetylcholine-induced relaxation was blunted by atherosclerosis, responses to acetylcholine and substance P of the JW rabbit arteries treated with PTX under almost same conditions as those used by these authors were not inhibited. Similar findings with these agonists were also obtained in pulmonary arteries isolated from rabbits pretreated with PTX (34). Therefore PTX-sensitive G protein would not be involved in the endothelium-dependent responses to acetylcholine and substance P of rabbit arteries.
L-NA-resistant, endothelium-dependent relaxations induced by acetylcholine and substance P in rabbit carotid arteries were almost abolished by charybdotoxin, as reported in rat mesenteric (18), rabbit carotid (35), and rat coronary arteries (36). The relaxing substance(s) other than NO that activates charybdotoxin-sensitive Ca2+-dependent K+ channels is expected to be involved in these responses. In the rabbit carotid artery, L-NA (10−4M) may not be sufficient to eliminate NO, suggesting the existence of the L-NA-resistant NO-formation pathway (37). The authors concluded that solely NO is an EDRF and also an EDHF (37). However, the present study clearly shows that L-NA-resistant acetylcholine-induced relaxation was abolished by combined treatment with charybdotoxin plus apamin, whereas the relaxations caused by NOC18, an NO donor, were not affected by the K+ channel inhibitors. The NOC18-induced relaxations were abolished by ODQ, a guanylate cyclase inhibitor. Therefore L-NA-resistant relaxations caused by acetylcholine are not solely mediated by NO but by Ca2+-dependent K+ channel opening substance(s), as suggested (35). Atherosclerosis does not seem to alter the endothelial function that produces charybdotoxin and apamin-sensitive K+ channel opening substance(s), because magnitudes of the substance P-induced relaxation in the JW and WHHL rabbit arteries treated with high concentrations of L-NA did not differ.
It is concluded that impaired responses to acetylcholine of arteries with atherosclerotic lesions would not be due to suppression of functional processes that acetylcholine and substance P share to synthesize NO in the endothelium. Dysfunction of muscarinic receptors may be involved in the impaired acetylcholine response. As far as the arteries used are concerned, relaxations induced by opening of charybdotoxin-sensitive Ca2+-activated K+ channels are not modified by atherosclerosis.
Acknowledgment: We thank Kaoru Toyosawa (Department of Toxicology Research laboratories, Dainippon Pharmaceutical Co. Ltd.) for wonderful technical assistance for histologic study. This work was supported in part by the Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Culture and Sports, Japan.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
Atherosclerosis; Endothelium-dependent relaxation; Carotid artery; Nitric oxide; Heritable hyperlipidemic rabbit