Background Endothelium-derived substances and the renin-angiotensin system are important regulators of vascular tone. This study was designed to evaluate the effects of age and hypertension on vascular function of rat coronary arteries.
Methods and Results Rings of the left anterior descending coronary artery were isolated from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at 12 (younger) and 72 (older) weeks of age and suspended in myographs (37°C, 95% O2/5% CO2) for isometric tension recording. Systolic blood pressure was higher in SHR than in WKY rats (P<.05) but was unaffected by age in both strains. Active wall tension to KCl 100 mmol/L (mN/mm) was decreased in younger (0.28±0.03, n=9) and older SHR (0.49±0.06, n=13) compared with age-matched WKY rats (0.87±0.05, n=9 and 1.51±0.11, n=11, respectively, P<.05). In both strains, active wall tension to endothelin-1 and serotonin increased with age (n=6 to 10, P<.05) but was decreased in younger and older SHR compared with WKY rats (P<.05). Active wall tension induced by angiotensin I 10−7 mol/L was increased in older SHR (0.19±0.04, n=7) compared with younger SHR (0.04±0.01, n=9) but was similar in younger and older WKY rats (0.10±0.02 versus 0.15±0.03, n=6 to 9) and younger SHR. In younger WKY rats and SHR, pretreatment of coronary arteries with benazeprilat 10−5 mol/L (n=5 for each) almost completely abolished the contractions to angiotensin I 10−7 mol/L. Active wall tension to angiotensin II 10−7 mol/L was comparable in all four groups, but compared with the contraction to KCl 100 mmol/L, the response was already increased in younger SHR (29±3%, n=9) compared with the younger WKY rats (14±3%, n=9, P<.05), but it was unaffected by age in both strains. In vitro treatment of younger WKY rat and SHR coronary arteries with the nonpeptide angiotensin II (AT1) receptor antagonist valsartan 10−5 mol/L (n=3 for each) fully suppressed contractions to angiotensin II 10−7 mol/L. In contrast, endothelium-independent relaxations to the nitrovasodilator sodium nitroprusside, endothelium-dependent relaxations to acetylcholine, and endothelium-dependent contractions to Nω-nitro-l-arginine methyl ester were comparable in all four groups of rats.
Conclusions In summary, in rat coronary arteries, contractile responses to endothelin-1, serotonin, and KCl increase with age but are decreased by hypertension. In contrast, the l-arginine/nitric oxide pathway remains unaffected. The contractions to angiotensin I markedly increased with increasing duration of hypertension in the SHR only. Despite overall reduced contractile responses of SHR coronary arteries, contractions to angiotensin II were maintained. Hence, aging and hypertension affect contractile responses of rat coronary arteries to vasoconstrictor agonists differently.
In the vasculature, age and hypertension are associated with modest thickening of wall elements as well as functional changes of vascular smooth muscle and the endothelium.1 In general, the maximal contractile strength of isolated arteries to stimuli such as catecholamines decreases with age.2 Endothelin also shows a reduced responsiveness with age, at least in mesenteric resistance arteries.3 Controversy exists as to the vascular response to endothelin in hypertension, depending on the animal model of hypertension, the duration of hypertension, the experimental conditions used, and the blood vessels studied.4 In the aorta and isolated mesenteric resistance arteries of spontaneously hypertensive rats (SHR5 ), a marked reduction in the sensitivity of vascular smooth muscle to endothelin has been reported, whereas in renal arteries, the response appears to be augmented.6 In large conduit arteries of SHR, vasoconstrictor responses to serotonin are increased.7 8 9
In the rat aorta, age is associated with increased formation of an endothelium-derived constricting factor (prostaglandin H210 ) as well as a modest decrease in the release of endothelium-derived nitric oxide.11 Conversely, the dilator responses of aging vessels to papaverine or nitrovasodilators are essentially normal.12 Hypertension generally seems to accelerate cardiovascular aging, and in particular, endothelial dysfunction.12 13 In the rat, this process is reversed by antihypertensive treatment.14
Both a circulating and a local vascular renin-angiotensin system have been proposed as important regulators of the cardiovascular system,1 15 although the importance of the latter is controversial.16 17 Indeed, whereas the circulating renin-angiotensin system has been well characterized, less is known about the functional importance of locally produced angiotensin II. Endothelial cells express angiotensin-converting enzyme (ACE), which transforms angiotensin I into angiotensin II.18 In renovascular hypertension, aortic ACE activity increases in the chronic stage of the disease.19 Recently, nonpeptide angiotensin II (AT1) receptor antagonists20 have been developed that have been suggested to represent more effective inhibitors of the renin-angiotensin system than ACE inhibitors (although proof is still lacking and the possibility of an overactivation of other angiotensin II receptor subtypes exists21 ).
So far, little is known about the consequences of age and prolonged hypertension in the coronary circulation. Therefore, this study was designed to evaluate the effects of age and hypertension on vascular smooth muscle and endothelial function and the angiotensin system of rat coronary arteries.
Experiments were performed in younger (12-week-old) and older (72-week-old) normotensive Wistar-Kyoto (WKY) rats and age-matched SHR (Charles River Wiga GmbH). Within the time frame of this study (from an age of 12 to 72 weeks), all 26 WKY rats survived. In contrast, 13 of 26 SHR died. On the day of the experiment (at the age of 12 or 72 weeks), systolic blood pressure was measured in conscious rats by the tail-cuff method (W+W Electronics). Before the tail-cuff blood pressure determination, rats were warmed with an infrared lamp for half an hour. Then the tail cuff and the pressure sensor were secured on each tail, and blood flow was occluded by compression. Systolic blood pressure was recorded at the first detection of blood flow upon gradual release of the occlusion. At least three recordings of the systolic blood pressure were made, and the mean values of these were taken as final readings. Then the rats were anesthetized with pentobarbital 50 mg/kg IP, and the heart was removed and placed into cold (4°C) Krebs-Ringer bicarbonate solution (mmol/L): NaCl 118.6, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.1, edetate calcium disodium 0.026, and glucose 11.1 (control solution).
Left anterior descending coronary arteries were dissected free under a microscope (Wild-Leitz). Rings 1.8 to 2 mm long and 300 μm in ID were mounted in a modified version of the myograph system22 for measurement of isometric force. Organ chambers were filled with 12.5 mL of control solution (37°C, 95% O2/5% CO2). Rings were held in place by two stiff tungsten wires (diameters, 30 and 50 μm) that were carefully passed through the lumen and fastened to clamps attached to a force transducer (Showa Sokki LB-5, Rikadenki) and to a micromanipulator (Narishige) for adjustment of muscle length. The unstretched vessels were allowed to equilibrate for 30 minutes. The optimal passive wall tension of the arterial rings was determined by repeated exposures to potassium chloride (KCl; 100 mmol/L) at increasing levels of passive wall tension. The vessels were then held at the optimal point of passive wall tension at which maximum active wall tension was produced after stimulation with KCl. Passive and active wall tensions were calculated as WT=F/2x, where F stands for the force (in millinewtons) measured by the transducer and x for the longitudinal length (in millimeters) of the vascular preparation. For all subsequent experiments, optimal passive wall tension was 0.54±0.1 mN/mm.
To study contractions evoked by endothelin-1 10−15 to 10−7 mol/L, serotonin (5-HT) 10−9 to 3×10−5 mol/L, and Nω-nitro-l-arginine methyl ester (L-NAME) 10−7 to 10−4 mol/L, increasing concentrations of the drugs were added in a cumulative fashion to quiescent preparations, while in the case of angiotensin I and angiotensin II, only a single dose was given (10−7 mol/L for both, because of rapid development of tachyphylaxis). The effects of norepinephrine were not studied because of the weak and inconsistent effects of the catecholamine in this preparation (n=5; data not shown). To study the effects of benazeprilat 10−5 mol/L and valsartan 10−5 mol/L on the contractions to angiotensin I 10−7 mol/L and angiotensin II 10−7 mol/L, respectively, vascular rings were pretreated with these drugs for 1 hour. The contractions are given as a percentage of the contraction to KCl 100 mmol/L and/or as an active wall tension in mN/mm. The relaxing effects of acetylcholine 10−9 to 10−4 mol/L and sodium nitroprusside 10−9 to 10−6 mol/L were studied by adding increasing concentrations of the drugs on top of a contraction evoked by serotonin 10−6 mol/L. Concentration-response curves to sodium nitroprusside were constructed after incubation of the vessel with L-NAME 10−4 mol/L and SQ 3074 (thromboxane receptor antagonist) 10−7 mol/L for 30 minutes. The effect of SQ 30741 10−7 mol/L on endothelium-dependent relaxations to acetylcholine or contractions to serotonin were tested by performing concentration-response curves before and after incubation with this drug for 30 minutes.
The following drugs were used (from Sigma Chemical Co unless otherwise stated): pentobarbital (Abbott), endothelin-1 (Nova Biochem), serotonin (5-hydroxytryptamine creatinine sulfate; Serva), acetylcholine hydrochloride, SQ 30741 (Squibb Institute for Medical Research), L-NAME, sodium nitroprusside, angiotensin I, and angiotensin II. Benazeprilat and valsartan were synthesized at the Chemistry Department of CIBA-Geigy. All concentrations of the drugs used in vitro are expressed as final molar concentration in the organ chambers.
Calculations and Statistical Analysis
For statistical analysis, the concentration of an agonist causing half-maximal contraction (EC50) or half-maximal inhibition of a preceding contraction (IC50), the maximal relaxation (percent), the contractions (percent and/or active wall tension), and the area under the concentration-response curve (arbitrary units) were calculated for each experiment. EC50 and IC50 were expressed as negative log molar (pD2 value). Data are given as mean±SEM. In each set of experiments, n is the number of animals studied. Statistical evaluation was done by unpaired Student’s t test or by ANOVA followed by Scheffé’s F test. Means were considered significantly different at P<.05.
Blood Pressure, Heart Weight, and Body Weight
Systolic blood pressure was higher in 12-week-old as well as in 72-week-old SHR compared with age-matched WKY rats (Table 1⇓, P<.005). In both strains of rats, aging for 60 weeks had no significant effect on systolic blood pressure.
Heart Weight, Body Weight, and Heart Weight/Body Weight Ratio
Heart weight was significantly higher in older SHR compared with the age-matched WKY rats and with younger SHR (Table 1⇑, P<.05). Body weight was significantly higher in older WKY rats and older SHR compared with their respective younger counterparts, and body weight was decreased in older SHR compared with age-matched WKY rats (Table 1⇑, P<.05). The heart weight/body weight ratio was significantly greater in 72-week-old SHR compared with their respective younger counterparts and with older WKY rats (Table 1⇑, P<.05).
Vascular Smooth Muscle Function
Contractions to KCl
Active wall tension (mN/mm) induced by KCl 100 mmol/L was reduced in 12-week-old (0.28±0.03, n=9) as well as in 72-week-old (0.49±0.06, n=13) SHR compared with age-matched WKY rats (0.87±0.05, n=9 and 1.51±0.11, n=11, respectively, P<.05). With age, active wall tension to KCl increased only in WKY rats not in SHR (Fig 1⇓; for WKY rats, P<.05).
Contractions to Endothelin-1
Active wall tension (mN/mm) evoked by endothelin-1 10−15 to 10−7 mol/L was decreased in 12-week-old (0.40±0.04, n=9) as well as in 72-week-old SHR (0.81±0.13, n=8) compared with age-matched normotensive WKY rats (1.37±0.09, n=9 and 2.12±0.38, n=6, respectively, P<.05). With age, active wall tension to endothelin-1 increased in both strains of rats (Fig 2⇓; P<.05).
Relative contractions to endothelin-1 10−15 to 10−7 mol/L (as a percentage of the contraction to KCl 100 mmol/L) were modestly decreased in older SHR compared with the older WKY rats. The area under the concentration-response curve was significantly reduced in older SHR compared with older WKY rats (Table 2⇓; P<.05).
Contractions to Serotonin (5-Hydroxytryptamine)
Active wall tension (mN/mm) induced by serotonin 10−9 to 3×10−5 mol/L was decreased in younger (0.30±0.04, n=10) and older (0.59±0.09, n=10) SHR compared with the age-matched WKY rats (1.12±0.06, n=9 and 2.00±0.16, n=8, respectively, P<.05). With age, active wall tension to serotonin increased in WKY rats as well as in SHR rats (Fig 3⇓; P<.05).
Relative contractions to serotonin 10−9 to 3×10−5 mol/L (as a percentage of the contraction to KCl 100 mmol/L) were significantly decreased in younger SHR compared with the younger WKY rats (Table 2⇑; log shift at EC50, 6.3-fold; P<.05) but were unaffected by age in both strains.
The thromboxane receptor antagonist SQ 30741 10−7 mol/L did not affect contractions to serotonin 10−9 to 3×10−5 mol/L in all four groups of animals (data not shown).
Relaxations to Sodium Nitroprusside
The nitrovasodilator sodium nitroprusside 10−9 to 10−6 mol/L induced potent relaxations (percentage of the contraction to 5-HT 10−6 mol/L) in coronary arteries obtained from 12-week-old and 72-week-old SHR and age-matched WKY rats. These endothelium-independent relaxations, however, did not differ statistically from each other in all four groups of rats (Table 3⇓).
Vascular Angiotensin System
Contractions to Angiotensin I
Active wall tension (mN/mm) induced by angiotensin I 10−7 mol/L was increased in older SHR (0.19±0.04, n=7) compared with younger SHR (0.04±0.01, n=9) but was similar in younger and older WKY rats (0.10±0.02 versus 0.15±0.03, n=6 to 9) and younger SHR (Fig 4⇓, top).
The relative responses to angiotensin I 10−7 mol/L (as a percentage of the contraction to KCl 100 mmol/L) were similar in younger and older WKY rats (12±3% versus 10±2%, n=6 to 9) and younger SHR (16±2%, n=8) but were increased in older SHR (36±6%, n=7, P<.05; Fig 4⇑, bottom).
The response to angiotensin I 10−7 mol/L was almost completely blocked by pretreatment of the coronary arteries with the ACE inhibitor benazeprilat 10−5 mol/L for 1 hour both in younger WKY rats (2.2±1.8%, n=5, P<.001 versus control) and younger SHR (1.9±1.2%, n=5, P<.001 versus control).
Contractions to Angiotensin II
Active wall tension (mN/mm) evoked by angiotensin II 10−7 mol/L was comparable in all four groups of rats (WKY: younger, 0.12±0.02; older, 0.12±0.02; SHR: younger, 0.09±0.01; older, 0.13±0.03; n=6 to 9, P=NS; Fig 5⇓, top).
The relative responses to angiotensin II 10−7 mol/L (as a percentage of the contraction to KCl 100 mmol/L) were significantly increased in 12-week-old (29±3%, n=8) as well as in 72-week-old SHR (34±7%, n=7) compared with their age-matched normotensive counterparts (14±3% and 8±1%, respectively, n=6 to 9, P<.05) but were unaffected by age in both strains (Fig 5⇑, bottom).
In 12-week-old WKY rats and SHR, the contractions to angiotensin II 10−7 mol/L were fully suppressed by pretreatment of the coronary rings with valsartan 10−5 mol/L, a nonpeptide angiotensin II (AT1) receptor antagonist (n=3, P<.001 versus control).
Endothelium-Dependent Contractions to L-NAME
L-NAME 10−7 to 10−4 mol/L induced marked contractions (as a percentage of the contraction to KCl 100 mmol/L) in preparations with but not in those without endothelium (data not shown). These endothelium-dependent contractions, however, were comparable in all four groups of rats (WKY: younger, 61±24%; older, 77±23%; SHR: younger, 64±29%; older, 60±15%; n=6 to 9).
Endothelium-Dependent Relaxations to Acetylcholine
Acetylcholine 10−9 to 10−4 mol/L evoked strong but comparable endothelium-dependent relaxations in all four groups of rats (Table 3⇑), which were not affected by the thromboxane receptor antagonist SQ 30741 10−7 mol/L (data not shown).
The present study demonstrates that in isolated rat coronary arteries, contractile responses to endothelin-1, serotonin, and KCl increase with age but are decreased in hypertension. The response to angiotensin I is normal in younger SHR but increases with increasing duration of hypertension. Despite reduced contractile responses of SHR coronary arteries, absolute contractions to angiotensin II are normal and the relative contraction is augmented already at a younger age, most likely because of upregulation of angiotensin II receptors. In contrast, endothelium-independent relaxations to the nitrovasodilator sodium nitroprusside and the endothelial l-arginine/nitric oxide pathway remain unaffected.
The fact that active wall tension to receptor-operated agonists such as endothelin-1 and serotonin as well as receptor-independent agonists such as KCl increased with age in both normotensive and hypertensive rats suggests that intracellular excitation-contraction mechanisms and/or the contractile machinery become more efficient with age. Changes in the release of norepinephrine from adrenergic nerve endings are unlikely to be involved, because α-receptor activation does not evoke significant contractions in rat coronary arteries (similar to other species23 ). Alterations in receptor characteristics such as affinity and receptor number can be excluded, since the relative contractions (as a percentage of the contraction to KCl), which give an indication of the sensitivity to an agonist, were unaffected by age. These results are in contrast with the findings obtained in blood vessels from different vascular beds, such as the rabbit and rat aorta, in which the vasoconstrictor responses to serotonin, norepinephrine, and KCl decrease with age.24 25 This indicates that in the rat, in contrast to other vascular beds, coronary flow may be endangered by augmented contractions with increasing age, particularly in the presence of high local concentrations of endothelin-1 and serotonin. This concept is supported by the fact that in isolated coronary resistance arteries of normotensive WKY rats, serotonin-induced vasoconstriction also is augmented with increasing age.26 With endothelin, the concentration-response curve became biphasic in older WKY rats. It is likely that, as in other preparations,27 the first phase is mediated by ETB receptors and the second phase by ETA receptors.
In contrast to age, hypertension reduced contractile responses evoked by endothelin-1, serotonin, and KCl. The relative contractions (as a percentage of the contraction to KCl) to endothelin-1 and serotonin, on the other hand, were only modestly decreased with hypertension. This would indicate that receptor affinity and/or receptor number are only modestly altered in hypertension; hence, the main cause of the reduced vasoconstrictor responses must be impaired excitation-contraction coupling of the contractile machinery. A contribution of the endothelium to the reduced responsiveness of vascular smooth muscle function can be excluded, since the basal release of endothelium-derived nitric oxide was normal in WKY rats as well as SHR even up to an age of 72 weeks (see below).
The renin-angiotensin system is an important regulatory mechanism of the vasculature.1 18 Recently, a circulating as well as a vascular renin-angiotensin system has been proposed,15 17 and several angiotensin II receptors have been characterized and cloned.28 Endothelial cells play a crucial role in the activation of the biologically inactive angiotensin I into the powerful vasoconstrictor angiotensin II, since ACE is located on the endothelial cell membrane.18 The relative as well as absolute contractions to angiotensin I were increased in older compared with young SHR but were comparable in younger and older WKY rats. This indirectly suggests that the activity of ACE may increase with increasing duration of hypertension in the coronary circulation of the SHR. Indeed, in rat coronary arteries, the response to angiotensin I was almost completely blocked by ACE inhibitors such as benazeprilat.21 Hence, the contractile response to angiotensin I closely reflects ACE activity. An increased ACE activity would increase local vascular angiotensin II levels in chronic stages of hypertension. Similar results were found in the aorta,19 lung, and mesenteric artery29 of 16-week-old but not 8-week-old two-kidney, one-clip hypertensive rats.
Despite a reduced contractile machinery of SHR coronary arteries, the absolute increase in tension to angiotensin II was maintained in both younger and older SHR, and the relative contractile responses (as a percentage of the contraction to KCl) were already increased in younger SHR compared with the younger WKY rats but were unaffected by age in both strains. Since a reduced basal release of endothelium-derived nitric oxide can be excluded as a contributing factor (see below), this augmented response could be due to upregulation of angiotensin II receptors. However, in the absence of concentration-response curves to angiotensin II (which are impossible to perform because of the rapid occurrence of tachyphylaxis) or of angiotensin receptor binding data, this interpretation remains speculative. The angiotensin II receptor involved in rat coronary arteries is of the AT1 subtype in nature.28 Indeed, the nonpeptide AT1 receptor antagonist valsartan20 fully suppressed the response to angiotensin II. The additive effect of an increased response to angiotensin I (which may reflect an increased ACE activity) and maintained contractions to angiotensin II (despite otherwise reduced contractile responses) in chronic stages of hypertension may increase coronary vascular tone and contribute to proliferative changes of the vascular wall in the rat coronary circulation.
Endothelial cells are a source of relaxing factors that can profoundly affect vascular tone.1 A physiologically important endothelium-derived relaxing factor is nitric oxide, which is formed from the amino acid l-arginine.1 30 The endothelium releases nitric oxide under basal conditions and after stimulation with acetylcholine.1 30 31 32 The endothelial l-arginine pathway can be inhibited by analogues of the amino acid, such as L-NAME.33 In this study, the contractions evoked by L-NAME were used as an indirect measurement to judge basal nitric oxide production. Contractions to L-NAME were entirely endothelium dependent in this preparation. Since neither the basal nor acetylcholine-induced stimulation of nitric oxide was affected by age or hypertension, the ability of the coronary endothelium to modulate vascular tone seems to be maintained in WKY rats as well as SHR even up to the age of 72 weeks. Thus, the endothelium of rat coronary arteries, in contrast to peripheral12 34 35 36 and cerebral arteries,9 must be protected from functional alterations induced by age and hypertension. It remains possible that selective defects with receptor-operated agonists other than acetylcholine occur; in our experience, however, acetylcholine is the only reliable endothelium-dependent vasodilator in this preparation.37 Endothelium-independent relaxations to the nitrovasodilator sodium nitroprusside did not differ with age or hypertension: this demonstrates that the vascular responsiveness to endothelium-derived nitric oxide remained unaffected as well. The fact that the response to sodium nitroprusside was tested in preparations with endothelium38 is unlikely to confound these results, since endothelial function remained unchanged in all groups. In contrast to the rat aorta34 and renal artery,9 SQ 30741, a thromboxane-receptor antagonist, did not affect endothelium-dependent relaxations to acetylcholine. Hence, acetylcholine does not stimulate the formation of a cyclooxygenase-dependent endothelium-derived contracting factor such as prostaglandin H2 or thromboxane A2 in epicardial coronary arteries of the SHR rat.
In conclusion, in rat coronary arteries, age increases contractile responses, whereas hypertension is associated with a decreased contractility to endothelin-1, serotonin, and KCl but not to the angiotensins. The specific increase of the responsiveness to angiotensins may be important for the alterations of the coronary circulation in hypertension.
This work was supported by the Swiss National Research Foundation (grant 32-32541.91), the Karl Maier Foundation (Liechtenstein), the Sandoz Foundation for Gerontological Research, and the Sandoz Research Foundation. The authors thank Zhihong Yang, MD, for discussion and Leoluca Criscione, PhD, for kindly supplying benazeprilat and valsartan.
- Received July 26, 1994.
- Revision received November 21, 1994.
- Accepted November 26, 1994.
- Copyright © 1995 by American Heart Association
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