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Effect of Short-Term Treatment of SHR With the Novel Calcium Channel Antagonist Mibefradil on Function of Small Arteries
AJH 1997; 10:94 – 100
Effect of Short-Term Treatment of SHR With the Novel Calcium Channel Antagonist Mibefradil on Function of Small Arteries Jin-S. Li and Ernesto L. Schiffrin
Treatment of spontaneously hypertensive rats ( SHR ) and Wistar-Kyoto control rats ( WKY ) for at least 12 weeks with calcium channel antagonists is associated with regression of structural hypertensive changes in the heart and in conduit and small arteries. To establish whether structural or functional changes of small arteries could be corrected with shorter periods of specific antihypertensive treatment, SHR and WKY were treated for 4 weeks with the novel calcium channel blocker mibefradil. Blood pressure rise was significantly reduced by mibefradil treatment in SHR to 165 { 1 mm Hg compared to a systolic blood pressure of 183 { 2 mm Hg in untreated SHR ( P õ.01 ) . Aortic hypertrophy in SHR was slightly reduced by treatment, but small artery hypertrophy in 4 vascular beds ( mesenteric, renal, coronary, and femoral ) was unaffected by administration of mibefradil for 4 weeks. Mibefradil treatment resulted in normalization of endothelium-dependent relaxation in mesenteric
small arteries, with disappearance of acetylcholine-induced contractions, although hypertrophy and remodeling of these small arteries were not significantly affected by treatment. In WKY rats, treatment had no effect on either structure or function of small arteries. These results demonstrate that treatment with the calcium antagonist mibefradil may induce an improvement in altered endothelial function even before regression of cardiovascular hypertrophy and remodeling takes place under treatment, indicating that normalization of abnormal small artery endothelial function in SHR under antihypertensive therapy may be independent of correction of altered small artery structure. q 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997; 10:94 – 100
A
studies have shown that some of these changes may regress toward normal under prolonged antihypertensive treatment.8 – 14 Together with structural abnormalities present in hypertensive blood vessels, functional changes occur.15,16 There is only scant evidence suggesting that antihypertensive treatment is able to correct functional changes as well as structural changes in hypertension, particularly with angiotensin converting enzyme inhibitors.17 – 19 We showed in a previous study that prolonged ( 14 weeks ) antihypertensive treatment of SHR resulted in correction not only of the structural, 14 but also of the functional, changes present in blood vessels, and this was specially prominent for the
rterial structure and function are altered in spontaneously hypertensive rats (SHR).1–7 The changes occurring in these vessels may be those that have been called hypertrophic remodeling 2,3 or eutrophic remodeling.4 – 6 Previous
Received March 18, 1996. Accepted July 9, 1996. From the MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montre´ al, University of Montre´al, Montre´al, Que´bec, Canada. Address correspondence to Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montre´al, 110 Pine Ave. West, Montre´al, Que´ bec H2W 1R7, Canada.
KEY WORDS:
Nitric oxide, endothelial-dependent relaxing factor, endothelial-dependent contracting factor, resistance arteries, antihypertensive therapy, hypertension.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
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acetylcholine-induced endothelium-dependent contractions found in vessels from SHR ( unpublished observations, 1996 ) . In the present investigation we evaluated the shortterm (4 weeks) chronic effects of treatment of SHR and Wistar-Kyoto rats (WKY) with a novel nondihydropyridine calcium channel antagonist, (1S, 2S)-2-[2-[[3-(2benzylimidazolyl)propyl]methylamine]ethyl]-6-fluoro1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl-methoxyacetate dihydrochloride (mibefradil or Ro 40-5967), 20 which has the interest of not only blocking L-type Ca 2/ channels, but also T-type Ca 2/ channels.21 This drug had previously been shown to normalize functional changes after longterm (14 weeks) treatment (authors’ unpublished observations, 1995) in association with correction of structural changes.14 Its effects on functional changes present in hypertensive blood vessels, particularly acetylcholineinduced endothelium-dependent contractions, and on the structure of small resistance-sized arteries of the coronary, renal, mesenteric, and femoral circulations, were investigated.
MATERIALS AND METHODS Animal Experiments Animal experiments were performed following the recommendations of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the Clinical Research Institute. SHR and WKY control rats were bought from Taconic Farms ( Germantown, NY ) , and were received aged 8 weeks. Rats were housed under conditions of constant temperature ( 227C ) and humidity ( 60% ) and exposed to a 12-h dark – light cycle. Systolic blood pressure was taken every 2 weeks by the tail-cuff method, after warming and under slight restraint, and recorded on a model 7 Grass polygraph ( Grass Medical Instruments, Quincy, MA ) fitted with a 7-P8 preamplifier, using a PCPB photoelectric pulse sensor. The average of three pressure readings was recorded. SHR and WKY rats were offered, starting at 10 weeks of age and for 4 weeks, the calcium channel antagonist mibefradil ( kindly provided by Dr J.-P. Clozel, F. Hoffmann-La Roche Ltd., Basel, Switzerland ) at a daily oral dose of 50 mg / kg body weight mixed with powdered chow. Controls were untreated SHR and WKY rats run in parallel to each treated groups and handled in identical fashion. On the day of the experiment, rats were killed by decapitation. The heart and a segment of thoracic aorta 1.5 cm in length were blotted dry and weighed. Preparation of Small Arteries Coronary, renal arcuate, and femoral arteries were obtained as we have described previously.6,14,22 The heart and the kidneys were placed in ice-cold Krebs’ solution. The rat was then placed in the supine position and the skin of the right hind leg was incised. An artery in the popliteal region of about 2 mm in length was dissected. To dissect coronary vessels, the right ventricle was opened to expose coronary arteries
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on the interventricular septum. The interventricular artery was followed to the cardiac apex and then the chordae tendinae and the myocardium were separated and a vessel of 2 mm in length was isolated. For the isolation of renal cortical arteries, the renal capsule was first removed. The kidney was sectioned, and a renal artery was dissected close to the renal cortex and then followed distally. A renal arcuate artery of about 2 mm in length was isolated. Mesenteric small arteries were obtained as previously described.4,23,24 Superior mesenteric arteries were taken from the part of the mesenteric vascular bed that feeds the jejunum 8 to 10 cm distal to the pylorus. A third order branch at 1 mm distance from the intestine and of about 2 mm in length was isolated. The vessels were mounted as ring preparations on an isometric myograph (Living Systems Instrumentation, Burlington, VT). The dissection and mounting were performed in physiological salt solution (PSS) at room temperature. PSS had the following composition (in mmol/L): NaCl, 120; NaHCO3 , 25; KCl, 4.7; KH2PO4 , 1.18; MgSO4 , 1.17; CaCl2 , 2.5; ethylenediaminetetraacetic acid, 0.026; and glucose, 5.5. All solutions were bubbled with 95% O2 and 5% CO2 to give a pH 7.40 to 7.45. Solutions were maintained at 377C.
Protocol of Study of Small Arteries After mounting, the vessels were warmed to 377C and allowed to equilibrate in PSS for about 30 min with the vessel internal circumference set to give a wall tension of 0.2 mN / mm. Then media width was measured using a LeitzDiavert inverted light microscope ( Wild Leitz, Heerbrugg, Switzerland ) , at a 3201 magnification at 12 different sites along the wall, which were then averaged. The vessels were then set to L0 , where L0 Å 0.9 L100 and L100 is the internal circumference ( calculated from the distance between the wires ) that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. After this, the vessels were maintained in PSS at 377C for a further 90 min. After the rest period, the vessels were contracted with K-PSS ( PSS in which Na is replaced by K ) , and two stimulations with K-PSS containing 10 mmol / L norepinephrine to verify that they contracted and developed a tension of at least 1.5 mN /mm. Endothelium-dependent relaxation of mesenteric small arteries was evaluated. Vessels were stimulated with a concentration of methoxamine equivalent to an EC80 ( 10 mmol / L ) , and relaxed with a single maximal dose of acetylcholine ( 10 mmol / L ) . Analysis of Data The media cross-sectional area of small arteries ( A ) was obtained from the media thickness ( m) and the circumference of vessels ( L ) , all measured with the vessel relaxed and under no passive stretch ( wall tension of 0.2 mN /mm) , and calculated as A Å Lm / pm2 . Using L0 and the calculated media cross-sectional area, and assuming a constant media volume, the standardized media thickness of blood vessels ( at L0 ) was then calculated. The lumen diameter was obtained as L0 / p.
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TABLE 1. BLOOD PRESSURE AND BODY AND HEART WEIGHT OF RATS TREATED WITH THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Parameter
SHR
SHR / CaCB
Number Systolic BP (mm Hg) BW (g) HW (g) HW/BW (%) Weight of 1.5-cm segment of aorta (mg) Weight of 1.5-cm segment of aorta (mg)/BW (g)
6 183 { 2 342 { 5 1.39 { 0.02 0.405 { 0.008 35.2 { 2.0 10.3 { 0.57
165 334 1.34 0.402 28.2 8.44
6 { 1** {5 { 0.03 { 0.008 { 1.7* { 0.53*
WKY
WKY / CaCB
6 108 { 2** 444 { 4** 1.54 { 0.09 0.347 { 0.020* 39.2 { 1.9 8.83 { 0.42*
6 104 { 2†† 431 { 8†† 1.54 { 0.02† 0.357 { 0.020† 34.7 { 1.4 8.04 { 0.24*
CaCB, calcium channel blocker; BP, blood pressure; BW, body weight; HW, heart weight. * P õ .05; ** P õ .01 v SHR. † P õ .05; †† P õ .01 v SHR / CaCB.
Results are represented as means { SEM. Statistical comparisons were performed by ANOVA followed by the Newman-Keuls post hoc test. Differences were considered statistically significant when P õ.05.
RESULTS Effect of Treatment on Body Weight, Blood Pressure, Heart Weight, and Plasma Renin Activity Body weight of SHR, treated or untreated, was significantly lower than that of age-matched WKY ( Table 1 ) , as we have previously found.14,23 The rise in systolic blood pressure of SHR was blunted by treatment with the calcium channel blocker mibefradil by 18 mm Hg ( Figure 1 and Table 1 ) . Blood pressure of WKY rats was unaffected by treatment. The weight of the heart and aorta segments were significantly elevated in SHR relative to WKY when normalized by body weight. The absolute and the normalized weight of aorta segments but not that of the heart of SHR was significantly re-
duced under treatment with mibefradil, reaching the same value as in WKY for the aorta weight normalized by body weight ( Table 1 ) .
Effect of Treatment on Vascular Structure Small vessels from the coronary, renal ( arcuate arteries ) , mesenteric, and femoral circulations were studied on a myograph under isometric conditions. Treatment of SHR with the calcium channel blocker resulted in little change in the structure of small vessels. Only the media of mesenteric arteries exhibited a slight difference between treated and untreated SHR, at the limit of significance ( P õ .07, t test ) . The expected differences in different small vessels of SHR relative to WKY 6 were found at the level of the lumen diameter ( smaller ) , media width ( larger ) , and the media-to-lumen ratio ( larger ) : particularly in mesenteric small arteries, in which the differences reached statistical significance in both SHR and SHR treated with mibefradil relative to their respective WKY control, to a lesser degree in coronary and femoral arteries, and were not found to be statistically significant in renal cortical small arteries, as depicted in Table 2. Effect of Treatment on Vascular Function Treatment with the calcium channel blocker did not change maximum media stress developed by small arteries ( Table 3 ) . On the other hand, endothelium-dependent responses induced with 10 mmol / L acetylcholine showed presence of contractions in mesenteric arteries from untreated SHR, which could no longer be detected or were significantly reduced in mibefradil-treated SHR ( Figures 2 and 3; Table 3 ) . Relaxation of WKY vessels was complete with acetylcholine and no contractions were detected. DISCUSSION
FIGURE 1. Systolic blood pressure of Wistar-Kyoto ( WKY ) and spontaneously hypertensive rats ( SHR ) treated or not with the calcium channel blocker ( CaCB ) mibefradil for 4 weeks.* P õ .01 SHR / CaCB v WKY; / P õ .05, // P õ .01 SHR / CaCB v SHR. Note that error bars represent SD.
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This study demonstrates that the calcium channel blocker mibefradil, given since age 10 weeks for a period of only 4 weeks, produces a small but significant reduction in blood pressure elevation, but little regression of cardiac hypertrophy or of hypertrophic
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TABLE 2. MORPHOMETRIC PARAMETERS OF SMALL ARTERIES OF RATS TREATED THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Vascular Bed
SHR
Lumen diameter (mm) Coronary 258 Renal 218 Mesenteric 209 Femoral 186 Media width (mm) Coronary 11.5 Renal 12.5 Mesenteric 11.6 Femoral 11.5 Media-to-lumen ratio (%) Coronary 4.5 Renal 5.9 Mesenteric 5.7 Femoral 6.1 Cross-section of media (mm2) Coronary 9102 Renal 9242 Mesenteric 8056 Femoral 7487
SHR / CaCB
WKY
WKY / CaCB
{ { { {
10 13 7 9
254 215 210 181
{ { { {
6 13 9 7
291 246 238 216
{ { { {
16 15 10* 10
293 240 238 232
{ { { {
12† 13 7† 12††
{ { { {
0.2 0.7 0.4 0.4
10.5 12.2 10.5 11.4
{ { { {
0.7 0.8 0.4 0.5
9.8 11.3 9.1 10.2
{ { { {
0.2** 0.6 0.5** 0.4
10.1 10.9 8.6 9.5
{ { { {
1.1 0.4 0.2†† 0.6
{ { { {
0.2 0.4 0.4 0.3
4.3 5.9 5.1 6.2
{ { { {
0.2 0.6 0.3 0.3
3.6 4.9 3.8 4.9
{ { { {
0.4 0.3 0.2** 0.2**
3.5 4.6 3.6 4.1
{ { { {
0.4 0.4 0.1†† 0.5††
{ { { {
334 454 355 301
8762 8985 7528 7056
{ { { {
333 414 214 396
9219 9318 7487 7471
{ { { {
388 303 294 258
9288 8644 7224 7340
{ { { {
485 387 242 359
CaCB Å calcium channel blocker. * P õ .05, ** P õ .01, v SHR. † P õ .05, †† P õ .01, v SHR / CaCB.
and nonhypertrophic remodeling in SHR small vessels, although there is already less aortic hypertrophy. This contrasts with long-term treatment ( 14 weeks ) , which corrects cardiac and small artery hypertrophy.14 However, already after this relatively short interval of treatment ( and even if there is little or no correction of structural changes of small vessels ) , endothelial function, as evaluated by acetylcholine-induced relaxation of precontracted small arteries, is normalized in SHR with disappearance of endothelium-dependent contractions elicited by high doses of acetylcholine. Blood pressure did not continue to rise in SHR when treated with mibefradil in this study, similarly to what was observed in a previous long-term study with this drug at a similar time period, 14 but no dramatic fall in blood pressure was observed. In other studies, other calcium channel antagonists such as nifedipine 25 have induced comparable lowering of
systolic blood pressure elevation in SHR in the short term ( approximately 20 mm Hg ) . We do not know whether vascular structure or endothelial function were improved in SHR after short periods of time of treatment with other such agents, as vessels were only examined after 8 weeks of treatment, and at that time interval only endothelial function was studied and shown to have improved.25,26 After 14 weeks of treatment with mibefradil, the structure of small arteries was corrected to a degree that is similar to that achieved with the angiotensin I-converting enzyme inhibitor cilazapril in the same study.14 Simultaneously, there was disappearance of endothelium-dependent contractions elicited by high concentrations of acetylcholine in SHR small mesenteric arteries under treatment with both mibefradil and cilazapril ( authors’ unpublished observations, 1995 ) . The present study demonstrates that correction of altered endothelial function in SHR precedes correction of struc-
TABLE 3. MAXIMAL MEDIA STRESS DEVELOPED BY SMALL ARTERIES OF RATS TREATED WITH THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Vascular Bed 2
Methoxamine (mN/mm ) Acetylcholine (mN/mm2)
SHR
SHR / CaCB
WKY
WKY / CaCB
150 { 19 135 { 18*
173 { 17 19.5 { 9.5
165 { 14 17.5 { 6.7
197 { 20 15.8 { 5.2
Methoxamine was used at a dose of 10 mmol/L; acetylcholine at a dose of 10 mmol/L was superimposed on the contraction elicited by methoxamine. CaCB, calcium channel blocker. * P õ .01 v other groups.
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FIGURE 2. Representative tracings of contraction and relaxation of small mesenteric arteries. Vessels were contracted with an EC80 of methoxamine ( ME, 10 mmol / L ) and then relaxed with 10 mmol / L acetylcholine ( A.Ch.) . CaCB, calcium channel blocker; W, wash.
tural changes, as it is already found when regression of vascular changes of small arteries has not yet occurred, after 4 weeks of mibefradil administration, and despite little difference in systolic blood pressure with untreated SHR, which present endothelial dysfunction. In contrast to small artery structure, which exhibited minor reversal without achieving statistical significance ( P õ .07 ) , the weight of thoracic aorta, a crude index of conduit artery hypertrophy, experienced a significant regression toward the value of WKY rats ( when expressed as relative weight ) . This suggests that conduit artery hypertrophy may depend differently on determinants of growth of vascular wall components ( whether blood pressure, humoral, local growth factors, or intracellular signaling ) than small arteries, and will be favorably affected by treatment of shorter duration. The mechanism for these differences between conduit and small arteries remains to be established. The nature of the endothelial abnormalities present in SHR has been the matter of much study, debate, and controversy. Although initial studies suggested that in experimental hypertension, endothelium-dependent relaxation was blunted, 27 it became apparent that in SHR there was release of vasoconstrictor substances from the endothelium, particularly the socalled endothelium-dependent contracting factor ( EDCF ) .28 This has been identified as a cyclooxygenase product, probably an endoperoxide.29 Typically, EDCF release by high concentrations of acetylcholine is inhibited by indomethacin and other prostaglandin synthase inhibitors.30 We previously showed that the contractions we obtain in our experimental set-up are inhibitable by indomethacin 31 and thus, in this study, the improvement reported may be construed to be a decreased production or contractile effect of EDCF in vessels from rats treated with the calcium channel blocker. This effect of mibefradil could be due to a persistent blockade of voltage-dependent calcium
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channels in the vascular preparations examined. In vitro, mibefradil has been shown to effectively block the contractile action of a thromboxane analogue in intramyocardial arteries of the pig, 32 and therefore could potentially antagonize the actions of EDCF, resulting in more effective acetylcholine-induced relaxation. However, contractile effects of methoxamine were unaffected, suggesting that there was no residual in vitro effect of the drug given in vivo, thus making it unlikely that the contractile action of EDCF was blocked by a persistent in vitro effect of mibefradil. Rather, this suggests that there was decreased production of EDCF in response to acetylcholine as a result of improved endothelial function. It is unlikely as well that this represented increases in the production of nitric oxide, which might antagonize the effects of EDCF, as there is currently evidence that nitric oxide generation is not altered in vessels from SHR, although its degradation may be accelerated.33 Whether an antioxidant effect of the calcium channel antagonist could contribute to reduce nitric oxide degradation was not examined in this study. In conclusion, short-term treatment ( 4 weeks ) of SHR with mibefradil, a novel calcium channel blocker, results in normalization of endothelium-dependent vascular relaxation at the level of small arteries at a time when blood pressure is only slightly reduced, and when regression of vascular hypertrophy in small resistance-sized, which may be found after more prolonged treatment, has not yet occurred. This suggests that improvement of endothelial dysfunction in small arteries of SHR occurs before reversal of structural alterations. Since this functional improvement occurs when blood pressure is only slightly lower in treated SHR in comparison to untreated SHR, this may indi-
FIGURE 3. Bar graph shows contraction following initial relaxation, both induced by 10 mmol / L acetylcholine on methoxamine precontracted small arteries. CaCB, calcium channel blocker.* P õ .01.
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cate that blood pressure per se plays little role in the changes found, but rather that this improvement may be the result of direct beneficial effects of the calcium channel antagonist on endothelial function. These results suggest that treatment of hypertension with these or similar agents may offer benefits through improvement of function of the endothelium of arteries, beyond those provided merely by the reduction of blood pressure.
ACKNOWLEDGMENTS The authors are grateful for the technical help of Mr Andre´ Turgeon and Ms Micheline Vachon. This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, by a grant from the Fondation des Maladies du Coeur du Que´ bec, and by a grant from F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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of hypertension and vascular changes by captopril treatment. Hypertension 1991; 17:141 – 150. 12. Hajdu MA, Heistad DD, Baumbach GL: Effects of antihypertensive therapy on mechanics of cerebral arterioles in rats. Hypertension 1991; 17:308 – 316. 13. Thybo NK, Korsgaard N, Eriksen S, et al: Dose-dependent effects of perindopril on blood pressure and smallartery structure. Hypertension 1994; 23:659 – 666. 14. Li J-S, Schiffrin EL: Effect of calcium channel blockade or angiotensin converting enzyme inhibition on structure of coronary, renal and other small arteries in SHR. J Cardiovasc Pharmacol 1996; 28:68 – 74. 15. Mulvany MJ, Aalkjaer C: Structure and function of small arteries. Physiol Rev 1990; 70:921 – 971. 16. Schiffrin EL: Reactivity of small blood vessels in hypertension. Relationship with structural changes. Hypertension 1992; 19 ( suppl II ) :II-1 – II-9. 17. Clozel M: Mechanism of action of angiotensin converting enzyme inhibitors on endothelial function in hypertension. Hypertension 1991; 18 ( suppl II ) :II-37 – II-42. 18. Takase H, Moreau P, Ku¨ ng CF, et al: Antihypertensive therapy prevents endothelial dysfunction in chronic nitric oxide deficiency — effect of verapamil and trandolapril. Hypertension 1996; 27:25 – 31. 19. Schiffrin EL, Deng LY: Comparison of effects of angiotensin I-converting enzyme inhibition and beta blockade on function of small arteries from hypertensive patients. Hypertension 1995; 25 ( Part 2 ) :699 – 703. 20. Osterreider W, Holck M: In vitro pharmacologic profile of Ro 40-5967, a novel Ca 2/ channel blocker with potent vasodilator but weak inotropic action. J Cardiovasc Pharmacol 1989; 13:754 – 759. 21. Mishra SK, Hermsmeyer K: Selective inhibition of Ttype Ca 2/ channels by Ro 40-5967. Circ Res 1994; 75:144 – 148. 22. Li J-S, Schiffrin EL: Chronic endothelin receptor antagonist treatment of young spontaneously hypertensive rats. J Hypertens 1995; 13:647 – 652. 23. Li JS, Larivie` re R, Schiffrin EL: Effect of a nonselective endothelin antagonist on vascular remodeling in DOCA-salt hypertensive rats. Hypertension 1994; 24: 183 – 188. 24. Deng LY, Schiffrin EL: Effects of endothelin on small arteries of DOCA-salt hypertensive rats. Am J Physiol 1992; 262:H1782 – H1787. 25. Tschudi MR, Criscione L, Novosel D, et al: Antihypertensive therapy augments endothelium-dependent relaxations in coronary arteries of spontaneously hypertensive rats. Circulation 1994; 2212 – 2218. 26. Dohi Y, Criscione L, Pfeiffer K, Lu¨ scher TF: Angiotensin blockade or calcium antagonists improve endothelial dysfunction in hypertension: studies in perfused mesenteric resistance arteries. J Cardiovasc Pharmacol 1994; 24:372 – 379. 27. Lockette W, Otsuka Y, Carretero O: The loss of endothelium-dependent vascular relaxation in hypertension. Hypertension 1986; 8 ( suppl II ) :II-61 – II-66. 28. Lu¨ scher TF, Vanhoutte PM: Endothelium-dependent contractions to acetylcholine in the aorta of the sponta-
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Effect of Short-Term Treatment of SHR With the Novel Calcium Channel Antagonist Mibefradil on Function of Small Arteries Jin-S. Li and Ernesto L. Schiffrin
Treatment of spontaneously hypertensive rats ( SHR ) and Wistar-Kyoto control rats ( WKY ) for at least 12 weeks with calcium channel antagonists is associated with regression of structural hypertensive changes in the heart and in conduit and small arteries. To establish whether structural or functional changes of small arteries could be corrected with shorter periods of specific antihypertensive treatment, SHR and WKY were treated for 4 weeks with the novel calcium channel blocker mibefradil. Blood pressure rise was significantly reduced by mibefradil treatment in SHR to 165 { 1 mm Hg compared to a systolic blood pressure of 183 { 2 mm Hg in untreated SHR ( P õ.01 ) . Aortic hypertrophy in SHR was slightly reduced by treatment, but small artery hypertrophy in 4 vascular beds ( mesenteric, renal, coronary, and femoral ) was unaffected by administration of mibefradil for 4 weeks. Mibefradil treatment resulted in normalization of endothelium-dependent relaxation in mesenteric
small arteries, with disappearance of acetylcholine-induced contractions, although hypertrophy and remodeling of these small arteries were not significantly affected by treatment. In WKY rats, treatment had no effect on either structure or function of small arteries. These results demonstrate that treatment with the calcium antagonist mibefradil may induce an improvement in altered endothelial function even before regression of cardiovascular hypertrophy and remodeling takes place under treatment, indicating that normalization of abnormal small artery endothelial function in SHR under antihypertensive therapy may be independent of correction of altered small artery structure. q 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997; 10:94 – 100
A
studies have shown that some of these changes may regress toward normal under prolonged antihypertensive treatment.8 – 14 Together with structural abnormalities present in hypertensive blood vessels, functional changes occur.15,16 There is only scant evidence suggesting that antihypertensive treatment is able to correct functional changes as well as structural changes in hypertension, particularly with angiotensin converting enzyme inhibitors.17 – 19 We showed in a previous study that prolonged ( 14 weeks ) antihypertensive treatment of SHR resulted in correction not only of the structural, 14 but also of the functional, changes present in blood vessels, and this was specially prominent for the
rterial structure and function are altered in spontaneously hypertensive rats (SHR).1–7 The changes occurring in these vessels may be those that have been called hypertrophic remodeling 2,3 or eutrophic remodeling.4 – 6 Previous
Received March 18, 1996. Accepted July 9, 1996. From the MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montre´ al, University of Montre´al, Montre´al, Que´bec, Canada. Address correspondence to Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montre´al, 110 Pine Ave. West, Montre´al, Que´ bec H2W 1R7, Canada.
KEY WORDS:
Nitric oxide, endothelial-dependent relaxing factor, endothelial-dependent contracting factor, resistance arteries, antihypertensive therapy, hypertension.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
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acetylcholine-induced endothelium-dependent contractions found in vessels from SHR ( unpublished observations, 1996 ) . In the present investigation we evaluated the shortterm (4 weeks) chronic effects of treatment of SHR and Wistar-Kyoto rats (WKY) with a novel nondihydropyridine calcium channel antagonist, (1S, 2S)-2-[2-[[3-(2benzylimidazolyl)propyl]methylamine]ethyl]-6-fluoro1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl-methoxyacetate dihydrochloride (mibefradil or Ro 40-5967), 20 which has the interest of not only blocking L-type Ca 2/ channels, but also T-type Ca 2/ channels.21 This drug had previously been shown to normalize functional changes after longterm (14 weeks) treatment (authors’ unpublished observations, 1995) in association with correction of structural changes.14 Its effects on functional changes present in hypertensive blood vessels, particularly acetylcholineinduced endothelium-dependent contractions, and on the structure of small resistance-sized arteries of the coronary, renal, mesenteric, and femoral circulations, were investigated.
MATERIALS AND METHODS Animal Experiments Animal experiments were performed following the recommendations of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the Clinical Research Institute. SHR and WKY control rats were bought from Taconic Farms ( Germantown, NY ) , and were received aged 8 weeks. Rats were housed under conditions of constant temperature ( 227C ) and humidity ( 60% ) and exposed to a 12-h dark – light cycle. Systolic blood pressure was taken every 2 weeks by the tail-cuff method, after warming and under slight restraint, and recorded on a model 7 Grass polygraph ( Grass Medical Instruments, Quincy, MA ) fitted with a 7-P8 preamplifier, using a PCPB photoelectric pulse sensor. The average of three pressure readings was recorded. SHR and WKY rats were offered, starting at 10 weeks of age and for 4 weeks, the calcium channel antagonist mibefradil ( kindly provided by Dr J.-P. Clozel, F. Hoffmann-La Roche Ltd., Basel, Switzerland ) at a daily oral dose of 50 mg / kg body weight mixed with powdered chow. Controls were untreated SHR and WKY rats run in parallel to each treated groups and handled in identical fashion. On the day of the experiment, rats were killed by decapitation. The heart and a segment of thoracic aorta 1.5 cm in length were blotted dry and weighed. Preparation of Small Arteries Coronary, renal arcuate, and femoral arteries were obtained as we have described previously.6,14,22 The heart and the kidneys were placed in ice-cold Krebs’ solution. The rat was then placed in the supine position and the skin of the right hind leg was incised. An artery in the popliteal region of about 2 mm in length was dissected. To dissect coronary vessels, the right ventricle was opened to expose coronary arteries
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on the interventricular septum. The interventricular artery was followed to the cardiac apex and then the chordae tendinae and the myocardium were separated and a vessel of 2 mm in length was isolated. For the isolation of renal cortical arteries, the renal capsule was first removed. The kidney was sectioned, and a renal artery was dissected close to the renal cortex and then followed distally. A renal arcuate artery of about 2 mm in length was isolated. Mesenteric small arteries were obtained as previously described.4,23,24 Superior mesenteric arteries were taken from the part of the mesenteric vascular bed that feeds the jejunum 8 to 10 cm distal to the pylorus. A third order branch at 1 mm distance from the intestine and of about 2 mm in length was isolated. The vessels were mounted as ring preparations on an isometric myograph (Living Systems Instrumentation, Burlington, VT). The dissection and mounting were performed in physiological salt solution (PSS) at room temperature. PSS had the following composition (in mmol/L): NaCl, 120; NaHCO3 , 25; KCl, 4.7; KH2PO4 , 1.18; MgSO4 , 1.17; CaCl2 , 2.5; ethylenediaminetetraacetic acid, 0.026; and glucose, 5.5. All solutions were bubbled with 95% O2 and 5% CO2 to give a pH 7.40 to 7.45. Solutions were maintained at 377C.
Protocol of Study of Small Arteries After mounting, the vessels were warmed to 377C and allowed to equilibrate in PSS for about 30 min with the vessel internal circumference set to give a wall tension of 0.2 mN / mm. Then media width was measured using a LeitzDiavert inverted light microscope ( Wild Leitz, Heerbrugg, Switzerland ) , at a 3201 magnification at 12 different sites along the wall, which were then averaged. The vessels were then set to L0 , where L0 Å 0.9 L100 and L100 is the internal circumference ( calculated from the distance between the wires ) that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg. After this, the vessels were maintained in PSS at 377C for a further 90 min. After the rest period, the vessels were contracted with K-PSS ( PSS in which Na is replaced by K ) , and two stimulations with K-PSS containing 10 mmol / L norepinephrine to verify that they contracted and developed a tension of at least 1.5 mN /mm. Endothelium-dependent relaxation of mesenteric small arteries was evaluated. Vessels were stimulated with a concentration of methoxamine equivalent to an EC80 ( 10 mmol / L ) , and relaxed with a single maximal dose of acetylcholine ( 10 mmol / L ) . Analysis of Data The media cross-sectional area of small arteries ( A ) was obtained from the media thickness ( m) and the circumference of vessels ( L ) , all measured with the vessel relaxed and under no passive stretch ( wall tension of 0.2 mN /mm) , and calculated as A Å Lm / pm2 . Using L0 and the calculated media cross-sectional area, and assuming a constant media volume, the standardized media thickness of blood vessels ( at L0 ) was then calculated. The lumen diameter was obtained as L0 / p.
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TABLE 1. BLOOD PRESSURE AND BODY AND HEART WEIGHT OF RATS TREATED WITH THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Parameter
SHR
SHR / CaCB
Number Systolic BP (mm Hg) BW (g) HW (g) HW/BW (%) Weight of 1.5-cm segment of aorta (mg) Weight of 1.5-cm segment of aorta (mg)/BW (g)
6 183 { 2 342 { 5 1.39 { 0.02 0.405 { 0.008 35.2 { 2.0 10.3 { 0.57
165 334 1.34 0.402 28.2 8.44
6 { 1** {5 { 0.03 { 0.008 { 1.7* { 0.53*
WKY
WKY / CaCB
6 108 { 2** 444 { 4** 1.54 { 0.09 0.347 { 0.020* 39.2 { 1.9 8.83 { 0.42*
6 104 { 2†† 431 { 8†† 1.54 { 0.02† 0.357 { 0.020† 34.7 { 1.4 8.04 { 0.24*
CaCB, calcium channel blocker; BP, blood pressure; BW, body weight; HW, heart weight. * P õ .05; ** P õ .01 v SHR. † P õ .05; †† P õ .01 v SHR / CaCB.
Results are represented as means { SEM. Statistical comparisons were performed by ANOVA followed by the Newman-Keuls post hoc test. Differences were considered statistically significant when P õ.05.
RESULTS Effect of Treatment on Body Weight, Blood Pressure, Heart Weight, and Plasma Renin Activity Body weight of SHR, treated or untreated, was significantly lower than that of age-matched WKY ( Table 1 ) , as we have previously found.14,23 The rise in systolic blood pressure of SHR was blunted by treatment with the calcium channel blocker mibefradil by 18 mm Hg ( Figure 1 and Table 1 ) . Blood pressure of WKY rats was unaffected by treatment. The weight of the heart and aorta segments were significantly elevated in SHR relative to WKY when normalized by body weight. The absolute and the normalized weight of aorta segments but not that of the heart of SHR was significantly re-
duced under treatment with mibefradil, reaching the same value as in WKY for the aorta weight normalized by body weight ( Table 1 ) .
Effect of Treatment on Vascular Structure Small vessels from the coronary, renal ( arcuate arteries ) , mesenteric, and femoral circulations were studied on a myograph under isometric conditions. Treatment of SHR with the calcium channel blocker resulted in little change in the structure of small vessels. Only the media of mesenteric arteries exhibited a slight difference between treated and untreated SHR, at the limit of significance ( P õ .07, t test ) . The expected differences in different small vessels of SHR relative to WKY 6 were found at the level of the lumen diameter ( smaller ) , media width ( larger ) , and the media-to-lumen ratio ( larger ) : particularly in mesenteric small arteries, in which the differences reached statistical significance in both SHR and SHR treated with mibefradil relative to their respective WKY control, to a lesser degree in coronary and femoral arteries, and were not found to be statistically significant in renal cortical small arteries, as depicted in Table 2. Effect of Treatment on Vascular Function Treatment with the calcium channel blocker did not change maximum media stress developed by small arteries ( Table 3 ) . On the other hand, endothelium-dependent responses induced with 10 mmol / L acetylcholine showed presence of contractions in mesenteric arteries from untreated SHR, which could no longer be detected or were significantly reduced in mibefradil-treated SHR ( Figures 2 and 3; Table 3 ) . Relaxation of WKY vessels was complete with acetylcholine and no contractions were detected. DISCUSSION
FIGURE 1. Systolic blood pressure of Wistar-Kyoto ( WKY ) and spontaneously hypertensive rats ( SHR ) treated or not with the calcium channel blocker ( CaCB ) mibefradil for 4 weeks.* P õ .01 SHR / CaCB v WKY; / P õ .05, // P õ .01 SHR / CaCB v SHR. Note that error bars represent SD.
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This study demonstrates that the calcium channel blocker mibefradil, given since age 10 weeks for a period of only 4 weeks, produces a small but significant reduction in blood pressure elevation, but little regression of cardiac hypertrophy or of hypertrophic
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TABLE 2. MORPHOMETRIC PARAMETERS OF SMALL ARTERIES OF RATS TREATED THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Vascular Bed
SHR
Lumen diameter (mm) Coronary 258 Renal 218 Mesenteric 209 Femoral 186 Media width (mm) Coronary 11.5 Renal 12.5 Mesenteric 11.6 Femoral 11.5 Media-to-lumen ratio (%) Coronary 4.5 Renal 5.9 Mesenteric 5.7 Femoral 6.1 Cross-section of media (mm2) Coronary 9102 Renal 9242 Mesenteric 8056 Femoral 7487
SHR / CaCB
WKY
WKY / CaCB
{ { { {
10 13 7 9
254 215 210 181
{ { { {
6 13 9 7
291 246 238 216
{ { { {
16 15 10* 10
293 240 238 232
{ { { {
12† 13 7† 12††
{ { { {
0.2 0.7 0.4 0.4
10.5 12.2 10.5 11.4
{ { { {
0.7 0.8 0.4 0.5
9.8 11.3 9.1 10.2
{ { { {
0.2** 0.6 0.5** 0.4
10.1 10.9 8.6 9.5
{ { { {
1.1 0.4 0.2†† 0.6
{ { { {
0.2 0.4 0.4 0.3
4.3 5.9 5.1 6.2
{ { { {
0.2 0.6 0.3 0.3
3.6 4.9 3.8 4.9
{ { { {
0.4 0.3 0.2** 0.2**
3.5 4.6 3.6 4.1
{ { { {
0.4 0.4 0.1†† 0.5††
{ { { {
334 454 355 301
8762 8985 7528 7056
{ { { {
333 414 214 396
9219 9318 7487 7471
{ { { {
388 303 294 258
9288 8644 7224 7340
{ { { {
485 387 242 359
CaCB Å calcium channel blocker. * P õ .05, ** P õ .01, v SHR. † P õ .05, †† P õ .01, v SHR / CaCB.
and nonhypertrophic remodeling in SHR small vessels, although there is already less aortic hypertrophy. This contrasts with long-term treatment ( 14 weeks ) , which corrects cardiac and small artery hypertrophy.14 However, already after this relatively short interval of treatment ( and even if there is little or no correction of structural changes of small vessels ) , endothelial function, as evaluated by acetylcholine-induced relaxation of precontracted small arteries, is normalized in SHR with disappearance of endothelium-dependent contractions elicited by high doses of acetylcholine. Blood pressure did not continue to rise in SHR when treated with mibefradil in this study, similarly to what was observed in a previous long-term study with this drug at a similar time period, 14 but no dramatic fall in blood pressure was observed. In other studies, other calcium channel antagonists such as nifedipine 25 have induced comparable lowering of
systolic blood pressure elevation in SHR in the short term ( approximately 20 mm Hg ) . We do not know whether vascular structure or endothelial function were improved in SHR after short periods of time of treatment with other such agents, as vessels were only examined after 8 weeks of treatment, and at that time interval only endothelial function was studied and shown to have improved.25,26 After 14 weeks of treatment with mibefradil, the structure of small arteries was corrected to a degree that is similar to that achieved with the angiotensin I-converting enzyme inhibitor cilazapril in the same study.14 Simultaneously, there was disappearance of endothelium-dependent contractions elicited by high concentrations of acetylcholine in SHR small mesenteric arteries under treatment with both mibefradil and cilazapril ( authors’ unpublished observations, 1995 ) . The present study demonstrates that correction of altered endothelial function in SHR precedes correction of struc-
TABLE 3. MAXIMAL MEDIA STRESS DEVELOPED BY SMALL ARTERIES OF RATS TREATED WITH THE CALCIUM CHANNEL BLOCKER MIBEFRADIL FOR 4 WEEKS Vascular Bed 2
Methoxamine (mN/mm ) Acetylcholine (mN/mm2)
SHR
SHR / CaCB
WKY
WKY / CaCB
150 { 19 135 { 18*
173 { 17 19.5 { 9.5
165 { 14 17.5 { 6.7
197 { 20 15.8 { 5.2
Methoxamine was used at a dose of 10 mmol/L; acetylcholine at a dose of 10 mmol/L was superimposed on the contraction elicited by methoxamine. CaCB, calcium channel blocker. * P õ .01 v other groups.
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FIGURE 2. Representative tracings of contraction and relaxation of small mesenteric arteries. Vessels were contracted with an EC80 of methoxamine ( ME, 10 mmol / L ) and then relaxed with 10 mmol / L acetylcholine ( A.Ch.) . CaCB, calcium channel blocker; W, wash.
tural changes, as it is already found when regression of vascular changes of small arteries has not yet occurred, after 4 weeks of mibefradil administration, and despite little difference in systolic blood pressure with untreated SHR, which present endothelial dysfunction. In contrast to small artery structure, which exhibited minor reversal without achieving statistical significance ( P õ .07 ) , the weight of thoracic aorta, a crude index of conduit artery hypertrophy, experienced a significant regression toward the value of WKY rats ( when expressed as relative weight ) . This suggests that conduit artery hypertrophy may depend differently on determinants of growth of vascular wall components ( whether blood pressure, humoral, local growth factors, or intracellular signaling ) than small arteries, and will be favorably affected by treatment of shorter duration. The mechanism for these differences between conduit and small arteries remains to be established. The nature of the endothelial abnormalities present in SHR has been the matter of much study, debate, and controversy. Although initial studies suggested that in experimental hypertension, endothelium-dependent relaxation was blunted, 27 it became apparent that in SHR there was release of vasoconstrictor substances from the endothelium, particularly the socalled endothelium-dependent contracting factor ( EDCF ) .28 This has been identified as a cyclooxygenase product, probably an endoperoxide.29 Typically, EDCF release by high concentrations of acetylcholine is inhibited by indomethacin and other prostaglandin synthase inhibitors.30 We previously showed that the contractions we obtain in our experimental set-up are inhibitable by indomethacin 31 and thus, in this study, the improvement reported may be construed to be a decreased production or contractile effect of EDCF in vessels from rats treated with the calcium channel blocker. This effect of mibefradil could be due to a persistent blockade of voltage-dependent calcium
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channels in the vascular preparations examined. In vitro, mibefradil has been shown to effectively block the contractile action of a thromboxane analogue in intramyocardial arteries of the pig, 32 and therefore could potentially antagonize the actions of EDCF, resulting in more effective acetylcholine-induced relaxation. However, contractile effects of methoxamine were unaffected, suggesting that there was no residual in vitro effect of the drug given in vivo, thus making it unlikely that the contractile action of EDCF was blocked by a persistent in vitro effect of mibefradil. Rather, this suggests that there was decreased production of EDCF in response to acetylcholine as a result of improved endothelial function. It is unlikely as well that this represented increases in the production of nitric oxide, which might antagonize the effects of EDCF, as there is currently evidence that nitric oxide generation is not altered in vessels from SHR, although its degradation may be accelerated.33 Whether an antioxidant effect of the calcium channel antagonist could contribute to reduce nitric oxide degradation was not examined in this study. In conclusion, short-term treatment ( 4 weeks ) of SHR with mibefradil, a novel calcium channel blocker, results in normalization of endothelium-dependent vascular relaxation at the level of small arteries at a time when blood pressure is only slightly reduced, and when regression of vascular hypertrophy in small resistance-sized, which may be found after more prolonged treatment, has not yet occurred. This suggests that improvement of endothelial dysfunction in small arteries of SHR occurs before reversal of structural alterations. Since this functional improvement occurs when blood pressure is only slightly lower in treated SHR in comparison to untreated SHR, this may indi-
FIGURE 3. Bar graph shows contraction following initial relaxation, both induced by 10 mmol / L acetylcholine on methoxamine precontracted small arteries. CaCB, calcium channel blocker.* P õ .01.
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cate that blood pressure per se plays little role in the changes found, but rather that this improvement may be the result of direct beneficial effects of the calcium channel antagonist on endothelial function. These results suggest that treatment of hypertension with these or similar agents may offer benefits through improvement of function of the endothelium of arteries, beyond those provided merely by the reduction of blood pressure.
ACKNOWLEDGMENTS The authors are grateful for the technical help of Mr Andre´ Turgeon and Ms Micheline Vachon. This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, by a grant from the Fondation des Maladies du Coeur du Que´ bec, and by a grant from F. Hoffmann-La Roche Ltd., Basel, Switzerland.
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