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Cilazapril reverses endothelium-dependent vasodilator response to acetylcholine in mesenteric artery from spontaneously hypertensive rats
AJH 1995; 8:928-933
Cilazapril Reverses Endothelium-Dependent Vasodilator Response to Acetylcholine in Mesenteric Artery From Spontaneously Hypertensive Rats Ron-Hua Young, Yu-An Ding, Yen-Mei Lee, and Mao-Hsiung Yen
This study was designed to evaluate the effect of chronic treatment with cilazapril on vascular reactivity of aorta and mesenteric artery from WistarKyoto (WKY) rats and spontaneously hypertensive rats (SHR). Cilazapril (5 mg/kg), an angiotensin converting enzyme inhibitor, was injected intraperitoneally twice a day for 4 weeks. Results demonstrated that acetylcholine (ACh)-induced relaxation in aorta and mesenteric artery from SHR was significantly less than that from WKY, cilazapriltreated WKY, and SHR. The impairment of AChinduced relaxation in SHR was significantly reversed after cilazapril treatment and there were no significant differences among WKY, cilazapriltreated WKY, and SHR. Meanwhile, both N ~nitro-r-arginine (LNNA; 10 -4 mol/L) and methylene blue (MB; 10 -5 mol/L) completely blocked the vasodilator response to ACh in aorta but only partly inhibited in mesenteric artery from WKY,
S
ince F u r c h g o t t and Z a w a d z k i (1980) described the acetylcholine (ACh)-induced end o t h e l i u m - d e p e n d e n t relaxation in rabbit aorta, 1 various substances have been re-
Received May 23, 1994; accepted April 3, 1995. From the Departments of Pharmacology (R-HY, Y-ML, M-HY) and Medicine (Y-AD), Tri-Service General Hospital, National Defense Medical Center, Taipei, Republic of China, Taiwan. This investigation was supported by a grant (NSC 83-0412-B-016-070) from the National Science Council, Taipei, Republic of China, Taiwan. Address correspondence and reprint requests to Dr. MaoHsiung Yen, Department of Pharmacology, National Defense Medical Center, P.O. Box 90048-504, Taipei, Republic of China, Taiwan.
© 1995 by the American Journal of Hypertensi(m, Ltd.
cilazapril-treated WKY, and SHR. These LNNAand MB-resistant vasodilator responses to ACh in mesenteric artery were only slightly inhibited by TEA (10 -3 mol/L) but not by indomethacin (5 x 10 -6 mol/L). These findings suggest that there may be an unidentified endothelium-dependent relaxing factor(s) (EDRF), which exists in the endothelium and may participate in the modulation of blood pressure in SHR. Results further demonstrate that the antihypertensive effect of cilazapril may be partly mediated by the reversing function of endothelium to release EDRF and LNNA-resistant, unidentified relaxing factor(s). Am J Hypertens 1995;8:928-933
KEY WORDS: Cilazapril, vascular reactivity, N wnitro-r-arginine-resistant vasodilation, spontaneously hypertensive rat.
ported to induce endothelium-dependent relaxation in most of the blood vessels. 2 It appears that endothelial cells are a source of endothelium-derived relaxing factors (EDRF) that can profoundly affect vascular tone. 1"3 Recent evidence suggests that nitric oxide (NO) or related nitroso com pounds may account for the vascular response to EDRF. 4 Indeed, in addition to NO, the endothelium most likely releases additional relaxing factors such as endothelium-derived hyperpolarizing factor (EDHF), prostacycline (PGI2), and possibly other substances. 54 Moreover, ma n y reports demonstrated that the vasodilator response to ACh in SHR was significantly less than that in WistarKyoto rats (WKY). Results suggest that the defective
0895-7061/95/$9.50 0895-7061(95)00158-L
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
CILAZAPRtL REVERSES VASCULAR REACTIVITY IN SHR 929
nature of ACh-induced vasodilation in spontaneously hypertensive rats (SHR) may be due to the decrease in synthesis or release of EDRF. This impairment of ACh-induced vasodilation in SHR can be reversed by the long-term treatment of angiotensin converting enzyme inhibitors. 9'1° However, in our previous study, an endothelium-dependent unidentified relaxing factor (other than NO, PGI 2, and EDHF) was demonstrated in mesenteric artery but not in aorta from Sprague-Dawley rats. 11 Further, this study was done to assess the role of this unidentified factor in the modulation of hypertension in SHR. Experiments were undertaken to compare the relaxation induced by ACh in the presence of various inhibitors of nitric oxide, PGI 2, and EDHF in aortic and mesenteric arterial rings from WKY, SHR (as control group), and long-term cilazapril-treated WKY and SHR. In addition to nitric oxide, there may be another unidentified relaxing factor(s) present in mesenteric artery but not in aorta. These factors may be absent in SHR and appear after cilazapril treatment. METHODS
Eight-week-old WKY and SHR, whose stock originated from the Charles River Breeding Laboratories in Japan, were purchased from the Department of Laboratory Animal Science of the National Defense Medical Center, caged individually in clear plastic cages, and kept in an environmentally controlled room maintained at 23°C, relative humidity of 55%, and a light-dark cycle of 12 h/12 h. Animals were randomly divided into four groups: WKY, SHR, and cilazapril-treated WKY and SHR. In treated rats, cilazapril (5 mg/kg, intraperitoneally) was injected twice a day for 4 weeks at 8 weeks of age. The untreated rats were injected with an equal volume of normal saline as control. Systolic blood pressure was measured in conscious rats by the tail-cuff method every week. An average of three readings was analyzed. Values in systolic blood pressure of SHR above 180 mm Hg were used in this experiment. Experimental Animals
Arterial S m o o t h M u s c l e Relaxation After cilazapril treatment, rats weighing 250 to 300 g were killed after
being anesthetized with urethane (1.2 g/kg, intraperitoneally). Their mesenteric arteries and thoracic aorta were excised and placed in cold physiologic salt solution (PSS; 4°C). Excess fat and connective tissue were trimmed from the arteries and they were then cut into rings. Each segment of the vessels was kept to 3 to 4 mm in length and one ring of each vessel was rubbed gently to remove the endothelium. Care was taken to preserve the endothelium of the other ring. The rings were mounted in a tissue bath containing
20 mL PSS bubbled with a mixture of 95% 0 2 and 5% C O 2. The pH value of the PSS solution was 7.2 and the compositions (in mmol/L) were as follows: NaCI, 118; KCI, 4.7; NaHCO3, 25; K H 2 P O 4 , 1.2; MgC12, 1.25; CaC12, 2.5; and glucose, 11. The rings were connected to Grass FTO3C force transducers (Grass Instrument Co., Quincy, MA) and changes in vascular tension were recorded isometrically on a Grass Model 7D polygraph. Before the experiment began, the preparations were progressively stretched and exposed to phenylephrine (PE; 3 x 10 -7 mol/L) at each level of tension until the optimal point of length-tension relationship was reached. The optimal passive t e n s i o n of mesenteric arteries and aorta in cilazapril-treated and untreated WKY and SHR are shown in Table 1. A submaximal contraction was elicited with PE (3 x 10 - 7 and 10 - 6 mol/L) on aortic and mesenteric arterial rings, respectively. Drug was then removed from the bath by several washes with PSS, and the tension was allowed to return to baseline. Tissues were allowed to reequilibrate for 30 min after the drug response to ensure maximum washout of the drug and to minimize the possibility of receptor desensitization. Again, contractions were induced with PE, 3 x 10 _7 mol/L and 10 - 6 mol/L on aortic rings and mesenteric arterial rings, respectively. After the contraction reached a steady state, acetylcholine (ACh; 3 x 10 -8 to 3 x 10 - 6 mol/L) was added to the tissue bath in a cumulative manner. The vessels were then incubated for 15 to 20 rain with one of the inhibitors used: NW-nitro-c-arginine (LNNA; 10 -5 to 10 -3 mol/L), 12 methylene blue (10 6 to 10 -4 mol/L), 13 tetraethylammonium (10 3 mol/L),14 or indomethacin (5 x 10 - 6 mol/L; incubation time 30 min). is The vessels were washed at least three times before incubation with the inhibitors. If the inhibitors by themselves induced an increase in tension or potentiated the contraction evoked by PE, the added concentration of PE was adjusted to match the level of contraction reached in the corresponding control experiment. Chemicals
Phenylephrine, acetylcholine, NW-nitro -
TABLE 1. THE OPTIMAL PASSIVE TENSION OF AORTA AND MESENTERIC ARTERY IN CILAZAPRIL-TREATED A N D UNTREATED WKY AND SHR Optimal Passive Tension (g) WKY
Thoracic aorta Mesenteric artery
Cila, cilazapril.
WKY-Cila SHR (n=6)
SHR-Cila
2.2
2.2
1.8
2.0
1.0
1.0
0.8
1.1
930
YOUNG
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
ET AL
L-arginine, indomethacin, methylene blue, and tetraethylammonium were purchased from Sigma Chemical (St. Louis, MO) and dissolved in distilled water. Nitroglycerin was purchased from the Parke-Davis Company (Morris Plains, NJ) and dissolved in distilled water. Cilazapril was a gift from RocheHoffmann Co., Switzerland, and was also dissolved in distilled water. Statistics All data are expressed as means _+ standard error of the mean (SEM), and compared by a one-factor analysis of variance followed by the Dunnews t D test. A P < .05 was considered of statistical significance.
Time Course of Change in the Systolic Blood Pressure in Conscious WKY, SHR, and Cilazapril-treated WKY and SHR The change in systolic blood pressure increased progressively with time in the SHR group and there was no significant change in WKY. In the cilazaprfl-treated SHR group, the systolic blood pressure was significantly decreased, near to the WKY level, following 1 week of treatment. The blood pressure response in the cilazapril-treated WKY was significantly more reduced than that of untreated WKY after 2 weeks' treatment. Both phenomena were maintained throughout this experiment (Figure 1). Effect of Cilazapril Treatment on ACh-induced Vasodilatation As shown in Figure 2A and B, the vasodilator responses to ACh in aorta and mesenteric arteries were concentration dependent. ACh-induced relaxation in aorta and mesenteric artery from SHR were significantly less than those from WKY and cflazapril-treated WKY and SHR. The impairment of ACh-induced relaxation in SHR was significantly reversed after cilazapril treatment and there was no sig-
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-log[ACh] M FIGURE 2. Effects of increasing concentration of ACh in aorta (A) and mesenteric arteries (B) with intact endothelium. Aorta and mesenteric arteries were precontracted with phenylephrine 3 x 10-7 and 10 -6 tool~L, respectively, and the relaxation induced by ACh is expressed as a percentage of the contraction. Value represents mean +- SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
nificant difference between WKY and cilazapriltreated WKY.
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Age (weeks) FIGURE 1. Time course of change in the systolic blood pressure in conscious WKY, SHR, and cilazapril-treated W K Y and SHR. Value represents mean + SEM, n = 12. *P < .05 cilazapriltreated SHR v SHR. +P < .05 cilazapril-treated W K Y v WKY.
Effects of Nitroglycerin on Aorta and Mesenteric Arteries in WKY, SHR, and Cilazapril-treated WKY and SHR To test whether the function of smooth muscle altered after cilazapril treatment, endothelium-independent vasodilator nitroglycerin (NG) was used in this experiment. Results showed that NG (3 x 10 -s to 3 x 1 0 - 6 moFL) produced concentrationdependent relaxation in aorta and mesenteric arteries in a concentration-dependent manner among WKY, SHR, and cilazapril-treated WKY and SHR groups. However, there was no significant difference among these four groups (Figure 3). Effects of LNNA, MB, Indomethacin, and TEA on the Relaxation Evoked by ACh in Aorta and Mesenteric Artery In aorta, LNNA (10 -6 to 10 -4 mol/L)
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
A
CILAZAPRIL
REVERSES
VASCULAR
REACTIVITY
IN SHR
931
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almost abolished the relaxation induced by ACh, whereas LNNA (10 -s to 10 - 3 mol/L) only partially attenuated vasodilator response to ACh in mesenteric arteries (Figure 4). Similarly, the inhibitor of guanylate cyclase MB (10 -6 to 10 -4 tool/L) totally suppressed the relaxation to ACh in aorta, whereas MB (10 -6 to 10 - 4 moUL) only significantly but not completely reduced the maximal relaxation induced by ACh in mesenteric arteries (Figure 5). Moreover, ACh-induced relaxation in aorta and mesenteric arteries from WKY, and cilazapril-treated WKY and SHR were partially inhibited by TEA (10- 3 mol/L) but not by indomethacin (5 x 10 -6 tool/L) (Figure 6). DISCUSSION The present study demonstrated that endotheliumderived nitric oxide was released after stimulation with ACh in aorta and mesenteric arteries of cilazapril-treated SHR and age-matched WKY. This t-arginine pathway accounts for the endothelium-depen-
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FIGURE 4. Histogram represents the effect of LNNA (10-6 to 10 -4 or 10-3 tool~L) on the relaxation induced by ACh in WKY, SHR, and ciIazapril-treated WKY and SHR. Value represents mean +- SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
dent vasodilator response to ACh in aorta but only partially in mesenteric arteries. As shown in Figure 2, the relaxation induced by ACh in SHR was significantly less than that in WKY or cilazapril-treated WKY and SHR. This result confirms previous reports by demonstrating that endothelium-dependent relaxations induced by ACh were impaired in SHR. 16,17 In addition, Figure 3 showed that the endotheliumindependent relaxing responses to NG in intact rings were not different among WKY, SHR, or cilazapriltreated WKY and SHR. This indicates that the function of smooth muscle does not alter among these groups. Results suggest that the basal release of endothelium-derived nitric oxide must be reduced in hypertension. These findings further support the idea that the increase in arterial pressure occuring in the SHR leads specifically to functional alteration of the luminal surface of the endothelium. LNNA, a specific inhibitor of nitric oxide formation from L-arginine, inhibited the relaxation induced by ACh, demonstrating that nitric oxide is synthesized from L-arginine in the endothelium of rat mesenteric
932
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
Y O U N G ET AL
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FIGURE 5. Histogram represents the effect of MB (10 -6 to
FIGURE 6. Histogram represents the effect of indomethacin (5
10 -4 tool~L) on the relaxation induced by ACh in WKY, SHR, and cilazapril-treated W K Y and SHR. Value represents mean + SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
X 10 -6 moI/L) and TEA (10 -3 tool/L) on the relaxation induced by ACh in WKY, SHR, and cilazapril-treated W K Y and SHR. Values represents mean +- SEM; n = 12. *P < .05 cilazapriltreated SHR v SHR.
arteries and released after stimulation with ACh (Figure 4). Our observation confirms the previous studies by using a large number of artery experiments in rats. 18A9 However, in rat mesenteric arteries, the inhibition of the ACh-induced relaxation by LNNA was smaller than that obtained in aorta (Figure 4). This may be related to large stores of r-arginine in the endothelium of mesenteric arteries, making it difficult to completely inhibit the L-arginine pathway with LNNA, or the concomitant release of endotheliumderived relaxing factors other than nitric oxide. There may also be present another type of NO synthase which is not sensitive to LNNA and MB in endothelium. The fact that even a high concentration of LNNA (10 -3 mol/L) did not prevent the relaxations induced by ACh argues strongly against an incomplete inhibition of the endothelial L-arginine pathway. Indeed, particularly in SHR, the maximal inhibition was achieved with 10-4 mol/L of LNNA, with no further effect at higher concentrations of the false substrate. This was consistent with our previous experiment in Sprague-Dawley rats.11 Furthermore, the
inhibitor of soluble guanylate cyclase MB inhibited the relaxation to a comparable extent as did LNNA (Figure 5). Thus, the endothelium of rat mesenteric arteries most likely releases another endotheliumderived relaxing factor(s) distinct from nitric oxide. 6'2° Prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) can be excluded as contributing factors, as indomethacin and TEA did not significantly affect the relaxations induced by ACh (Figure 6). Results suggest that the basal release of the endothelium-derived relaxing factor(s) in addition to nitric oxide, PGI 2, and EDHF may also be impaired in the SHR or that another type of NO synthase which is not sensitive to LNNA and MB may be present in endothelium. In contrast, cilazapril, an inhibitor of angiotensin converting enzyme, markedly reversed the impaired endothelium-dependent relaxation induced by ACh. These results confirm Clozel's report. 9 However, this reversed effect can be fully inhibited either by LNNA or by MB in aorta but only partially inhibited in mesenteric arteries. This LNNA-
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
CILAZAPRIL REVERSES VASCULAR REACTIVITY IN SHR
and MB-resistant vasodilator r e s p o n s e to ACh in mesenteric arteries can not be completely blocked by TEA and indomethacin. Results further suggest that the f u n c t i o n of e n d o t h e l i u m can be r e v e r s e d by cilazapril treatment. These e n d o t h e l i u m - d e p e n d e n t relaxing factors were absent in SHR and present in WKY and in cilazapril-treated WKY and SHR. In conclusion, the e n d o t h e l i u m of mesenteric arteries of rats is capable of forming nitric oxide from L-arginine but the p a t h w a y only accounts in part for the inhibitory role of the e n d o t h e l i u m . In contrast, in hypertension the inhibitory effects of the e n d o t h e l i u m against contractions i n d u c e d by PE are r e d u c e d comp a r e d with a g e - m a t c h e d WKY and cilazapril-treated SHR and WKY. This i m p a i r m e n t of vasodilatation ind u c e d by ACh in SHR was almost completely reversed by cilazapril treatment. These findings suggest that in addition to nitric oxide, an unidentified e n d o t h e l i u m - d e r i v e d relaxing factor(s) m a y also play an i m p o r t a n t role in the maintenance of increased peripheral vascular resistance that occurs in established h y p e r t e n s i o n . Finally, the actual characteristics of this factor(s) n e e d to be investigated further by using bioassay, electrophysiologic, and biochemical approaches. REFERENCES
1. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-376. 2. Peach MJ, Loeb AL, Singer HA, et al: Endotheliumderived vascular relaxing factor. Hypertension 1985;7: I94-100. 3. Moncada S, Palmer RMJ, Higgs EA: Physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109-142. 4. Palmer RMJ, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-526. 5. Komori K, Suzuki H: Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery. Circ Res 1987;61:586-593. 6. Vanhoutte PM: The end of quest? Nature 1987;327: 459-460. 7. Beny JL, Brunet PC: Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially
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mediated vasodilation of pig coronary arteries. Blood Vessels 1988;25:3-11. Feletou M, Vanhoutte PM: Endothelium-dependent hyperpolarization of cannine coronary smooth muscle. J Pharmacol 1988;93:515-524. Clozel JP, Kuhn H, Hefti F: Effects of chronic ACE inhibition and of hydralazine on endothelial function in hypertension rats. J Hypertens 1989;16:532-540. Yen MH, Wu CC: Absence of methylene blue resistant endothelial relaxations of ACh in mesenteric arteries of SHR. Hypertens 1992;10(suppl 4):$54. Wu CC, Chen SJ, Yen MH: Different responses to acetylcholine in the presence of nitric oxide inhibitor in rat aortae and mesenteric arteries. Clin Exper Pharmacol Physiol 1993;20:405--412. Ishii K, Chang B, Kerwin JF, et al: N°'-nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation. Eur J Pharmacol 1990;176:219-233. Martin W, Villani GM, Jothianandan D, et al: Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J Pharmacol Exper Therapeut 1985;232:708-716. Benhan CD, Bolton TB: Patch-clamp studies of slow potential-sensitive potassium channels in longitudinal smooth muscle cells of rabbit jejunum. J Physiol 1983; 340:469-486. Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971;231:232-235. Dohi Y, Thiel MA, Buhler FR, et al: Activation of endothelial L-arginine pathway in resistance arteries. Effect of age and hypertension. Hypertension 1990;16: 170-179. Wu CC, Bohr DF: Role of endothelium in the response to endothelin in hypertension. Hypertension 1990;16: 677-681. Sakuma I, Stuehr D, Gross SS, et al: Identification of arginine as a precursor of endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988;85:8664-8667. Rees DD, Palmer RMJ, Hodson HF: A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 1989;96:418-424. Taylor SG, Weston AH: Endothelium-derived hyperpolarizing factor: a new endogenous inhibitor from the vascular endothelium. Trends Pharmacol Sci 1988; 9:272-274.
Cilazapril Reverses Endothelium-Dependent Vasodilator Response to Acetylcholine in Mesenteric Artery From Spontaneously Hypertensive Rats Ron-Hua Young, Yu-An Ding, Yen-Mei Lee, and Mao-Hsiung Yen
This study was designed to evaluate the effect of chronic treatment with cilazapril on vascular reactivity of aorta and mesenteric artery from WistarKyoto (WKY) rats and spontaneously hypertensive rats (SHR). Cilazapril (5 mg/kg), an angiotensin converting enzyme inhibitor, was injected intraperitoneally twice a day for 4 weeks. Results demonstrated that acetylcholine (ACh)-induced relaxation in aorta and mesenteric artery from SHR was significantly less than that from WKY, cilazapriltreated WKY, and SHR. The impairment of AChinduced relaxation in SHR was significantly reversed after cilazapril treatment and there were no significant differences among WKY, cilazapriltreated WKY, and SHR. Meanwhile, both N ~nitro-r-arginine (LNNA; 10 -4 mol/L) and methylene blue (MB; 10 -5 mol/L) completely blocked the vasodilator response to ACh in aorta but only partly inhibited in mesenteric artery from WKY,
S
ince F u r c h g o t t and Z a w a d z k i (1980) described the acetylcholine (ACh)-induced end o t h e l i u m - d e p e n d e n t relaxation in rabbit aorta, 1 various substances have been re-
Received May 23, 1994; accepted April 3, 1995. From the Departments of Pharmacology (R-HY, Y-ML, M-HY) and Medicine (Y-AD), Tri-Service General Hospital, National Defense Medical Center, Taipei, Republic of China, Taiwan. This investigation was supported by a grant (NSC 83-0412-B-016-070) from the National Science Council, Taipei, Republic of China, Taiwan. Address correspondence and reprint requests to Dr. MaoHsiung Yen, Department of Pharmacology, National Defense Medical Center, P.O. Box 90048-504, Taipei, Republic of China, Taiwan.
© 1995 by the American Journal of Hypertensi(m, Ltd.
cilazapril-treated WKY, and SHR. These LNNAand MB-resistant vasodilator responses to ACh in mesenteric artery were only slightly inhibited by TEA (10 -3 mol/L) but not by indomethacin (5 x 10 -6 mol/L). These findings suggest that there may be an unidentified endothelium-dependent relaxing factor(s) (EDRF), which exists in the endothelium and may participate in the modulation of blood pressure in SHR. Results further demonstrate that the antihypertensive effect of cilazapril may be partly mediated by the reversing function of endothelium to release EDRF and LNNA-resistant, unidentified relaxing factor(s). Am J Hypertens 1995;8:928-933
KEY WORDS: Cilazapril, vascular reactivity, N wnitro-r-arginine-resistant vasodilation, spontaneously hypertensive rat.
ported to induce endothelium-dependent relaxation in most of the blood vessels. 2 It appears that endothelial cells are a source of endothelium-derived relaxing factors (EDRF) that can profoundly affect vascular tone. 1"3 Recent evidence suggests that nitric oxide (NO) or related nitroso com pounds may account for the vascular response to EDRF. 4 Indeed, in addition to NO, the endothelium most likely releases additional relaxing factors such as endothelium-derived hyperpolarizing factor (EDHF), prostacycline (PGI2), and possibly other substances. 54 Moreover, ma n y reports demonstrated that the vasodilator response to ACh in SHR was significantly less than that in WistarKyoto rats (WKY). Results suggest that the defective
0895-7061/95/$9.50 0895-7061(95)00158-L
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
CILAZAPRtL REVERSES VASCULAR REACTIVITY IN SHR 929
nature of ACh-induced vasodilation in spontaneously hypertensive rats (SHR) may be due to the decrease in synthesis or release of EDRF. This impairment of ACh-induced vasodilation in SHR can be reversed by the long-term treatment of angiotensin converting enzyme inhibitors. 9'1° However, in our previous study, an endothelium-dependent unidentified relaxing factor (other than NO, PGI 2, and EDHF) was demonstrated in mesenteric artery but not in aorta from Sprague-Dawley rats. 11 Further, this study was done to assess the role of this unidentified factor in the modulation of hypertension in SHR. Experiments were undertaken to compare the relaxation induced by ACh in the presence of various inhibitors of nitric oxide, PGI 2, and EDHF in aortic and mesenteric arterial rings from WKY, SHR (as control group), and long-term cilazapril-treated WKY and SHR. In addition to nitric oxide, there may be another unidentified relaxing factor(s) present in mesenteric artery but not in aorta. These factors may be absent in SHR and appear after cilazapril treatment. METHODS
Eight-week-old WKY and SHR, whose stock originated from the Charles River Breeding Laboratories in Japan, were purchased from the Department of Laboratory Animal Science of the National Defense Medical Center, caged individually in clear plastic cages, and kept in an environmentally controlled room maintained at 23°C, relative humidity of 55%, and a light-dark cycle of 12 h/12 h. Animals were randomly divided into four groups: WKY, SHR, and cilazapril-treated WKY and SHR. In treated rats, cilazapril (5 mg/kg, intraperitoneally) was injected twice a day for 4 weeks at 8 weeks of age. The untreated rats were injected with an equal volume of normal saline as control. Systolic blood pressure was measured in conscious rats by the tail-cuff method every week. An average of three readings was analyzed. Values in systolic blood pressure of SHR above 180 mm Hg were used in this experiment. Experimental Animals
Arterial S m o o t h M u s c l e Relaxation After cilazapril treatment, rats weighing 250 to 300 g were killed after
being anesthetized with urethane (1.2 g/kg, intraperitoneally). Their mesenteric arteries and thoracic aorta were excised and placed in cold physiologic salt solution (PSS; 4°C). Excess fat and connective tissue were trimmed from the arteries and they were then cut into rings. Each segment of the vessels was kept to 3 to 4 mm in length and one ring of each vessel was rubbed gently to remove the endothelium. Care was taken to preserve the endothelium of the other ring. The rings were mounted in a tissue bath containing
20 mL PSS bubbled with a mixture of 95% 0 2 and 5% C O 2. The pH value of the PSS solution was 7.2 and the compositions (in mmol/L) were as follows: NaCI, 118; KCI, 4.7; NaHCO3, 25; K H 2 P O 4 , 1.2; MgC12, 1.25; CaC12, 2.5; and glucose, 11. The rings were connected to Grass FTO3C force transducers (Grass Instrument Co., Quincy, MA) and changes in vascular tension were recorded isometrically on a Grass Model 7D polygraph. Before the experiment began, the preparations were progressively stretched and exposed to phenylephrine (PE; 3 x 10 -7 mol/L) at each level of tension until the optimal point of length-tension relationship was reached. The optimal passive t e n s i o n of mesenteric arteries and aorta in cilazapril-treated and untreated WKY and SHR are shown in Table 1. A submaximal contraction was elicited with PE (3 x 10 - 7 and 10 - 6 mol/L) on aortic and mesenteric arterial rings, respectively. Drug was then removed from the bath by several washes with PSS, and the tension was allowed to return to baseline. Tissues were allowed to reequilibrate for 30 min after the drug response to ensure maximum washout of the drug and to minimize the possibility of receptor desensitization. Again, contractions were induced with PE, 3 x 10 _7 mol/L and 10 - 6 mol/L on aortic rings and mesenteric arterial rings, respectively. After the contraction reached a steady state, acetylcholine (ACh; 3 x 10 -8 to 3 x 10 - 6 mol/L) was added to the tissue bath in a cumulative manner. The vessels were then incubated for 15 to 20 rain with one of the inhibitors used: NW-nitro-c-arginine (LNNA; 10 -5 to 10 -3 mol/L), 12 methylene blue (10 6 to 10 -4 mol/L), 13 tetraethylammonium (10 3 mol/L),14 or indomethacin (5 x 10 - 6 mol/L; incubation time 30 min). is The vessels were washed at least three times before incubation with the inhibitors. If the inhibitors by themselves induced an increase in tension or potentiated the contraction evoked by PE, the added concentration of PE was adjusted to match the level of contraction reached in the corresponding control experiment. Chemicals
Phenylephrine, acetylcholine, NW-nitro -
TABLE 1. THE OPTIMAL PASSIVE TENSION OF AORTA AND MESENTERIC ARTERY IN CILAZAPRIL-TREATED A N D UNTREATED WKY AND SHR Optimal Passive Tension (g) WKY
Thoracic aorta Mesenteric artery
Cila, cilazapril.
WKY-Cila SHR (n=6)
SHR-Cila
2.2
2.2
1.8
2.0
1.0
1.0
0.8
1.1
930
YOUNG
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
ET AL
L-arginine, indomethacin, methylene blue, and tetraethylammonium were purchased from Sigma Chemical (St. Louis, MO) and dissolved in distilled water. Nitroglycerin was purchased from the Parke-Davis Company (Morris Plains, NJ) and dissolved in distilled water. Cilazapril was a gift from RocheHoffmann Co., Switzerland, and was also dissolved in distilled water. Statistics All data are expressed as means _+ standard error of the mean (SEM), and compared by a one-factor analysis of variance followed by the Dunnews t D test. A P < .05 was considered of statistical significance.
Time Course of Change in the Systolic Blood Pressure in Conscious WKY, SHR, and Cilazapril-treated WKY and SHR The change in systolic blood pressure increased progressively with time in the SHR group and there was no significant change in WKY. In the cilazaprfl-treated SHR group, the systolic blood pressure was significantly decreased, near to the WKY level, following 1 week of treatment. The blood pressure response in the cilazapril-treated WKY was significantly more reduced than that of untreated WKY after 2 weeks' treatment. Both phenomena were maintained throughout this experiment (Figure 1). Effect of Cilazapril Treatment on ACh-induced Vasodilatation As shown in Figure 2A and B, the vasodilator responses to ACh in aorta and mesenteric arteries were concentration dependent. ACh-induced relaxation in aorta and mesenteric artery from SHR were significantly less than those from WKY and cflazapril-treated WKY and SHR. The impairment of ACh-induced relaxation in SHR was significantly reversed after cilazapril treatment and there was no sig-
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-log[ACh] M FIGURE 2. Effects of increasing concentration of ACh in aorta (A) and mesenteric arteries (B) with intact endothelium. Aorta and mesenteric arteries were precontracted with phenylephrine 3 x 10-7 and 10 -6 tool~L, respectively, and the relaxation induced by ACh is expressed as a percentage of the contraction. Value represents mean +- SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
nificant difference between WKY and cilazapriltreated WKY.
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Age (weeks) FIGURE 1. Time course of change in the systolic blood pressure in conscious WKY, SHR, and cilazapril-treated W K Y and SHR. Value represents mean + SEM, n = 12. *P < .05 cilazapriltreated SHR v SHR. +P < .05 cilazapril-treated W K Y v WKY.
Effects of Nitroglycerin on Aorta and Mesenteric Arteries in WKY, SHR, and Cilazapril-treated WKY and SHR To test whether the function of smooth muscle altered after cilazapril treatment, endothelium-independent vasodilator nitroglycerin (NG) was used in this experiment. Results showed that NG (3 x 10 -s to 3 x 1 0 - 6 moFL) produced concentrationdependent relaxation in aorta and mesenteric arteries in a concentration-dependent manner among WKY, SHR, and cilazapril-treated WKY and SHR groups. However, there was no significant difference among these four groups (Figure 3). Effects of LNNA, MB, Indomethacin, and TEA on the Relaxation Evoked by ACh in Aorta and Mesenteric Artery In aorta, LNNA (10 -6 to 10 -4 mol/L)
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
A
CILAZAPRIL
REVERSES
VASCULAR
REACTIVITY
IN SHR
931
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-log[NG] M FIGURE 3. Line graphs showing the effects of nitroglycerin (NG) in aorta (A) and mesenteric arteries (B) with endothelium in WKY, SHR, and cilazapril-treated WKY and SHR. Value represents mean +- SEM; n = 12.
almost abolished the relaxation induced by ACh, whereas LNNA (10 -s to 10 - 3 mol/L) only partially attenuated vasodilator response to ACh in mesenteric arteries (Figure 4). Similarly, the inhibitor of guanylate cyclase MB (10 -6 to 10 -4 tool/L) totally suppressed the relaxation to ACh in aorta, whereas MB (10 -6 to 10 - 4 moUL) only significantly but not completely reduced the maximal relaxation induced by ACh in mesenteric arteries (Figure 5). Moreover, ACh-induced relaxation in aorta and mesenteric arteries from WKY, and cilazapril-treated WKY and SHR were partially inhibited by TEA (10- 3 mol/L) but not by indomethacin (5 x 10 -6 tool/L) (Figure 6). DISCUSSION The present study demonstrated that endotheliumderived nitric oxide was released after stimulation with ACh in aorta and mesenteric arteries of cilazapril-treated SHR and age-matched WKY. This t-arginine pathway accounts for the endothelium-depen-
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ACh LNNA 10 -5 M + A C h LNNA 10 .4 M + A C h L N N A I0 3 M + A C h
FIGURE 4. Histogram represents the effect of LNNA (10-6 to 10 -4 or 10-3 tool~L) on the relaxation induced by ACh in WKY, SHR, and ciIazapril-treated WKY and SHR. Value represents mean +- SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
dent vasodilator response to ACh in aorta but only partially in mesenteric arteries. As shown in Figure 2, the relaxation induced by ACh in SHR was significantly less than that in WKY or cilazapril-treated WKY and SHR. This result confirms previous reports by demonstrating that endothelium-dependent relaxations induced by ACh were impaired in SHR. 16,17 In addition, Figure 3 showed that the endotheliumindependent relaxing responses to NG in intact rings were not different among WKY, SHR, or cilazapriltreated WKY and SHR. This indicates that the function of smooth muscle does not alter among these groups. Results suggest that the basal release of endothelium-derived nitric oxide must be reduced in hypertension. These findings further support the idea that the increase in arterial pressure occuring in the SHR leads specifically to functional alteration of the luminal surface of the endothelium. LNNA, a specific inhibitor of nitric oxide formation from L-arginine, inhibited the relaxation induced by ACh, demonstrating that nitric oxide is synthesized from L-arginine in the endothelium of rat mesenteric
932
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
Y O U N G ET AL
A Aorta
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i
~
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IND+ACh
T
I
TEA+ACh
FIGURE 5. Histogram represents the effect of MB (10 -6 to
FIGURE 6. Histogram represents the effect of indomethacin (5
10 -4 tool~L) on the relaxation induced by ACh in WKY, SHR, and cilazapril-treated W K Y and SHR. Value represents mean + SEM; n = 12. *P < .05 cilazapril-treated SHR v SHR.
X 10 -6 moI/L) and TEA (10 -3 tool/L) on the relaxation induced by ACh in WKY, SHR, and cilazapril-treated W K Y and SHR. Values represents mean +- SEM; n = 12. *P < .05 cilazapriltreated SHR v SHR.
arteries and released after stimulation with ACh (Figure 4). Our observation confirms the previous studies by using a large number of artery experiments in rats. 18A9 However, in rat mesenteric arteries, the inhibition of the ACh-induced relaxation by LNNA was smaller than that obtained in aorta (Figure 4). This may be related to large stores of r-arginine in the endothelium of mesenteric arteries, making it difficult to completely inhibit the L-arginine pathway with LNNA, or the concomitant release of endotheliumderived relaxing factors other than nitric oxide. There may also be present another type of NO synthase which is not sensitive to LNNA and MB in endothelium. The fact that even a high concentration of LNNA (10 -3 mol/L) did not prevent the relaxations induced by ACh argues strongly against an incomplete inhibition of the endothelial L-arginine pathway. Indeed, particularly in SHR, the maximal inhibition was achieved with 10-4 mol/L of LNNA, with no further effect at higher concentrations of the false substrate. This was consistent with our previous experiment in Sprague-Dawley rats.11 Furthermore, the
inhibitor of soluble guanylate cyclase MB inhibited the relaxation to a comparable extent as did LNNA (Figure 5). Thus, the endothelium of rat mesenteric arteries most likely releases another endotheliumderived relaxing factor(s) distinct from nitric oxide. 6'2° Prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) can be excluded as contributing factors, as indomethacin and TEA did not significantly affect the relaxations induced by ACh (Figure 6). Results suggest that the basal release of the endothelium-derived relaxing factor(s) in addition to nitric oxide, PGI 2, and EDHF may also be impaired in the SHR or that another type of NO synthase which is not sensitive to LNNA and MB may be present in endothelium. In contrast, cilazapril, an inhibitor of angiotensin converting enzyme, markedly reversed the impaired endothelium-dependent relaxation induced by ACh. These results confirm Clozel's report. 9 However, this reversed effect can be fully inhibited either by LNNA or by MB in aorta but only partially inhibited in mesenteric arteries. This LNNA-
AJH-SEPTEMBER 1995-VOL. 8, NO. 9
CILAZAPRIL REVERSES VASCULAR REACTIVITY IN SHR
and MB-resistant vasodilator r e s p o n s e to ACh in mesenteric arteries can not be completely blocked by TEA and indomethacin. Results further suggest that the f u n c t i o n of e n d o t h e l i u m can be r e v e r s e d by cilazapril treatment. These e n d o t h e l i u m - d e p e n d e n t relaxing factors were absent in SHR and present in WKY and in cilazapril-treated WKY and SHR. In conclusion, the e n d o t h e l i u m of mesenteric arteries of rats is capable of forming nitric oxide from L-arginine but the p a t h w a y only accounts in part for the inhibitory role of the e n d o t h e l i u m . In contrast, in hypertension the inhibitory effects of the e n d o t h e l i u m against contractions i n d u c e d by PE are r e d u c e d comp a r e d with a g e - m a t c h e d WKY and cilazapril-treated SHR and WKY. This i m p a i r m e n t of vasodilatation ind u c e d by ACh in SHR was almost completely reversed by cilazapril treatment. These findings suggest that in addition to nitric oxide, an unidentified e n d o t h e l i u m - d e r i v e d relaxing factor(s) m a y also play an i m p o r t a n t role in the maintenance of increased peripheral vascular resistance that occurs in established h y p e r t e n s i o n . Finally, the actual characteristics of this factor(s) n e e d to be investigated further by using bioassay, electrophysiologic, and biochemical approaches. REFERENCES
1. Furchgott RF, Zawadzki JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-376. 2. Peach MJ, Loeb AL, Singer HA, et al: Endotheliumderived vascular relaxing factor. Hypertension 1985;7: I94-100. 3. Moncada S, Palmer RMJ, Higgs EA: Physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109-142. 4. Palmer RMJ, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-526. 5. Komori K, Suzuki H: Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery. Circ Res 1987;61:586-593. 6. Vanhoutte PM: The end of quest? Nature 1987;327: 459-460. 7. Beny JL, Brunet PC: Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially
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