The endothelium plays an important role in the circulation by modulating contractile responses of vascular smooth muscle. We designed this study to investigate the alterations of endothelial modulation in hypertension. Rings of femoral arteries were prepared from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), and changes in isometric tension were recorded. In rings with endothelium, norepinephrine (in either the presence or absence of yohimbine) evoked concentration-dependent contractions. Endothelium removal markedly enhanced the contraction; both the maximal response and sensitivity were increased, and these responses were less pronounced in SHR than WKY. In contrast to norepinephrine-induced contractions, the enhancement of prostaglandin F2α- or serotonin-induced contractions after endothelium removal was small and comparable in WKY and SHR; sensitivity was increased, but the maximal response was not. Nω-Nitro-l-arginine methyl ester enhanced the contractions induced by these agonists in arteries with but not without endothelium and thereby abolished the enhancement of the contractions after endothelium removal. Thus, the endothelium plays an inhibitory role against contractions in rat femoral arteries by releasing nitric oxide, but the characteristics of the endothelial inhibition are not identical against various types of contractions. The negative endothelial modulation is more pronounced during α1-adrenoceptor–mediated contractions than during contractions mediated by other receptors. The inhibitory role of the endothelium against α1-adrenoceptor agonist–induced but not serotonin- or prostaglandin F2α–induced contraction is impaired in hypertension.
The endothelium releases relaxing factors (NO, endothelium-derived hyperpolarizing factor, and prostacyclin) and contracting factors (endoperoxides, thromboxane A2, and endothelin) under basal conditions and when stimulated by vasoactive substances or physical stimuli and thereby contributes to the local regulation of vascular tone.1 2 It is generally accepted that endothelial function is altered in hypertension3 4 ; endothelium-dependent relaxations in response to acetylcholine, adenosine, and histamine are depressed in large conduit and small resistance arteries of SHR.5 6 7 8 In most cases, excessive release of endothelium-derived contracting factors can account for the depressed relaxation in SHR4 9 10 11 although reduced release of relaxing factors is also suggested.12 13 14 Besides the direct relaxing or contracting effects of the endothelium, it also can modulate the effects of vasoconstrictor substances through the release of relaxing factors.1 2 6 15 The endothelial modulation must be quite important because vascular smooth muscle is always exposed to many kinds of vasoconstrictor stimuli such as norepinephrine (released from nerve endings) or serotonin (released from platelets) in (patho)physiological states. However, not much is known about the alterations of the endothelium-dependent modulation in hypertension. Thus, we designed the present study to investigate (1) the mechanisms underlying the endothelial modulation of contractions and (2) the alterations of the modulation in hypertension using femoral arteries from normotensive and hypertensive rats.
Male WKY and SHR aged 13 to 16 weeks were obtained from Charles River Japan (Yokohama). The procedures followed were in accordance with our institutional guidelines. The rats were anesthetized with pentobarbital (50 mg/kg IP) and exsanguinated. The femoral arteries were removed and placed into a modified Krebs-Ringer bicarbonate solution of the following composition (mmol/L): NaCl 118.6, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.1, disodium calcium edetate (EDTA) 0.026, and glucose 10.1, pH 7.4 (Krebs' solution). The arteries were cleaned of adhering tissue and cut into rings (1 mm in width) under a dissection microscope. In some preparations, the endothelium was removed by gentle rubbing of the intimal surface with a wooden stick. Endothelium removal was confirmed later by an inability of acetylcholine to relax the arteries.
The rings were suspended horizontally by means of two L-shaped stainless steel holders in the vessel lumen in small organ chambers (5 mL) filled with the Krebs' solution (37°C) aerated with a mixture of 95% O2 and 5% CO2. One of the holders was fixed and the other was connected to a force displacement transducer (UM-203, Kishimoto) for measurement of isometric tension development. The rings were equilibrated under a resting tension of 300 mg for 60 minutes. This tension was found to be optimal for contraction of the preparation as assessed by repeated exposure to a 60 mmol/L KCl solution (obtained by equimolar replacement of NaCl by KCl in the Krebs' solution) under various resting tensions. After the equilibration period, the rings were maximally contracted by the 60 mmol/L KCl solution two times at 45-minute intervals.
Concentration-response curves to norepinephrine (10−9 to 10−4 mol/L), phenylephrine (10−8 to 10−4 mol/L), serotonin (10−9 to 10−5 mol/L), or PGF2α (10−9 to 10−4 mol/L) were obtained in rings with or without endothelium. To obtain α-adrenoceptor–mediated responses to norepinephrine and phenylephrine, we performed the experiments using these agonists in the presence of the β-adrenoceptor antagonist timolol (3×10−7 mol/L). In some preparations, the concentration-response curves to norepinephrine were obtained in the presence of the α2-adrenoceptor antagonist yohimbine (3×10−7 mol/L). To study a possible role of NO in the endothelial modulation of the contractions, we obtained the concentration-response curves to norepinephrine, serotonin, and PGF2α in the absence or presence of L-NAME (10−4 mol/L), which inhibits the endogenous production of NO from l-arginine.16 This L-NAME concentration completely prevented endothelium-dependent, endothelium-derived hyperpolarizing factor–independent relaxations to acetylcholine in the femoral arteries. The antagonists or the inhibitor was applied 30 minutes before the start of the experiments and was present throughout the experiments. In other series of experiments, we studied relaxant responses to clonidine or serotonin in arteries with endothelium half-maximally precontracted with PGF2α (WKY, 6×10−6 mol/L; SHR, 3×10−6). When steady contraction was established, clonidine (10−8 to 10−4 mol/L) or serotonin (10−9 to 10−5 mol/L) was added in a cumulative manner.
To avoid possible time-dependent changes in the responsiveness of the endothelium and vascular smooth muscle, we constructed only a single concentration-response curve in each ring preparation.
Drugs and Solutions
The following drugs were used (Sigma Chemical Co, unless otherwise stated): acetylcholine chloride, clonidine hydrochloride, ketanserin tartrate (Research Biochemicals International), l-norepinephrine bitartrate, L-NAME, l-phenylephrine hydrochloride, prazosin hydrochloride (Taito Pfeizer Co), PGF2α, serotonin (5-hydroxytryptamine) creatinine sulfate complex, timolol maleate (Banyu Pharmaceutical Co), and yohimbine hydrochloride.
Calculations and Statistical Analyses
Contractions were expressed as a percentage of the contraction developed by the 60 mmol/L KCl solution. Data are given as mean±SE. The concentration of an agonist causing half-maximal contraction (EC50 value) was calculated for each experiment and expressed as negative log molar (pD2). The shift of concentration-response curves was expressed as concentration shift at pD2 values. In each set of experiments, n equals the number of rats studied. Statistical analysis was performed by Student's unpaired t test and by ANOVA followed by Scheffé's F test. Means were considered to be significantly different at a value of P<.05.
Contraction Induced by Norepinephrine
In WKY arteries with endothelium, norepinephrine (10−9 to 10−4 mol/L) evoked concentration-dependent contractions (pD2: 5.59±0.15; maximal response: 57±4% of 60 mmol/L KCl–induced contraction; n=7) (Fig 1⇓, left). Endothelium removal markedly enhanced the contraction (pD2: 6.52±0.07, P<.0001; maximal response: 124±6%, P<.0001; n=8). The concentration shift and increase in the maximal response were 8.6- and 2.2-fold, respectively. Treatment with L-NAME (10−4 mol/L) enhanced the contraction in arteries with endothelium (pD2: 6.49±0.07, P<.005; maximal response: 119±8%, P<.0001; n=6) (Fig 1⇓, left) but not without endothelium (pD2: 6.54±0.10; maximal response: 117±5%, P=NS; n=6) (not shown). In the presence of L-NAME, the contraction was identical in arteries with and without endothelium.
In SHR arteries with endothelium, norepinephrine also evoked concentration-dependent contractions (pD2: 5.90±0.13; maximal response: 96±5%; n=6) (Fig 1⇑, right). The contraction was greater in SHR than in WKY (pD2: P=NS; maximal response: P<.0001). In SHR, endothelium removal only slightly enhanced the contraction (pD2: 6.62±0.11, P<.005; concentration shift: 5.2-fold; maximal response: 132±7%, P<.005; increase in maximal response: 1.4-fold; n=5). As a result, in arteries without endothelium, the contraction was identical in WKY and SHR. In SHR, L-NAME enhanced the contraction in arteries with endothelium (pD2: 6.65±0.12, P<.005; maximal response: 132±5%, P<.0005; n=7) (Fig 1⇑, right) but not without endothelium (pD2: 6.67±0.12; maximal response: 129±4%, P=NS; n=6) (not shown), as observed in WKY. In the presence of L-NAME, the contraction obtained in arteries with and without endothelium was not different.
We also constructed concentration-response curves to norepinephrine in the presence of 3×10−7 mol/L yohimbine to investigate a possible contribution of α2-adrenoceptors to the inhibition of the contraction by the endothelium (Fig 2⇓). The contraction was enhanced after endothelium removal in WKY (pD2: 5.39±0.04 to 6.28±0.05, concentration shift: 7.8-fold; maximal response: 62±4% to 129±5%, 2.1-fold; both P<.0001; n=5) (Fig 2⇓, left) and SHR (pD2: 5.89±0.08 to 6.50±0.09, 4.1-fold; maximal response: 97±5% to 124±7%, 1.3-fold; both P<.001; n=5) (Fig 2⇓, right). The enhancement observed in the presence of yohimbine was similar to that observed in the absence of the α2-antagonist in both WKY and SHR (Figs 1 and 2⇑⇓).
Contraction Induced by Phenylephrine
Similar enhancement of the contractions by endothelium removal was observed with norepinephrine and the α1-adrenoceptor agonist phenylephrine as a contractile agent (Figs 1 and 3⇑⇓). Endothelium removal augmented the contraction in WKY (pD2: 5.59±0.05 to 6.24±0.07, concentration shift: 4.4-fold; maximal response: 53±6% to 126±2%, 2.4-fold; both P<.0001; n=5) (Fig 3⇓, left) and SHR (pD2: 5.81±0.09 to 6.40±0.10, 3.9-fold; maximal response: 97±5% to 131±7%, 1.3-fold; both P<.005; n=5) (Fig 3⇓, right).
Relaxant Response to Clonidine
The α2-adrenoceptor agonist clonidine (10−8 to 10−4 mol/L) did not relax WKY or SHR arteries with endothelium contracted with PGF2α in the absence or presence of 10−7 mol/L prazosin (data not shown, n=4).
Contraction Induced by Serotonin
In WKY, serotonin (10−9 to 10−5 mol/L) caused concentration-dependent contractions in arteries with endothelium (pD2: 6.61±0.07; maximal response: 124±5%; n=6) (Fig 4⇓, left). In contrast to the contraction induced by norepinephrine (or phenylephrine), serotonin-induced contraction was slightly enhanced after endothelium removal; the pD2 value was increased (7.01±0.03, P<.001; concentration shift: 2.5-fold), but the maximal response was not (132±5%, P=NS, n=6). L-NAME enhanced the contraction in arteries with endothelium (pD2: 6.93±0.09, P<.05; maximal response: 134±4%, P=NS; n=5) (Fig 4⇓, left) but not without endothelium (pD2: 7.01±0.12; maximal response: 133±1%; P=NS; n=5) (not shown). After L-NAME treatment, the contraction was identical in arteries with and without endothelium as observed in norepinephrine-induced contractions.
Endothelium removal had similar effects on serotonin-induced contraction in SHR and WKY; in SHR, the removal increased the pD2 values (6.92±0.07 to 7.31±0.07, P<.005; concentration shift: 2.5-fold; n=6) but not the maximal responses (125±5% to 127±2%, P=NS, n=6) (Fig 4⇑). The pD2 values but not the maximal responses for serotonin were greater in SHR than in WKY in arteries both with (P<.05) and without (P<.005) endothelium. In SHR, L-NAME enhanced the contraction in arteries with endothelium (pD2: 7.24±0.07, P<.05; maximal response: 131±6%, P=NS; n=5) (Fig 4⇑, right) but not without endothelium (pD2: 7.20±0.05; maximal response: 130±2%; P=NS; n=5) (not shown). In the presence of L-NAME, the contraction was identical in arteries with and without endothelium.
Relaxant Response to Serotonin
Serotonin (10−9 to 10−5 mol/L) did not relax WKY or SHR arteries with endothelium contracted with PGF2α in the absence or presence of ketanserin (10−6 mol/L; data not shown, n=4).
In WKY, PGF2α (10−9 to 10−4 mol/L) caused concentration-dependent contractions in arteries with endothelium (pD2: 5.20±0.09; maximal response: 121±4%; n=8) (Fig 5⇓, left). In contrast to the contraction induced by norepinephrine, endothelium removal slightly shifted the concentration-response curves to the left (pD2: 5.82±0.07, P<.0001; concentration shift: 4.2-fold) without affecting the maximal response (129±5%, P=NS, n=8). L-NAME enhanced the contraction in arteries with endothelium (pD2: 5.72±0.04, P<.005; maximal response: 124±3%, P=NS; n=6) (Fig 5⇓, left) but not without endothelium (pD2: 5.71±0.08; maximal response: 127±6%, P=NS; n=5) (not shown). After treatment with L-NAME, the contraction was identical in arteries with and without endothelium.
The enhancement of PGF2α-induced contraction after endothelium removal was similar in WKY and SHR (Fig 5⇑). In SHR, endothelium removal increased the pD2 value (5.52±0.11 to 6.29±0.16, P<.005; concentration shift: 5.8-fold) but not the maximal response (120±6% to 125±4%, P=NS, n=6-7), as observed in WKY (Fig 5⇑, right). The pD2 values but not the maximal responses for PGF2α were greater in SHR than in WKY in arteries both with (P<.05) and without (P<.01) endothelium. In SHR, L-NAME enhanced the contraction in arteries with endothelium (pD2: 6.02±0.16, P<.05; maximal response: 129±4%, P=NS; n=6) (Fig 5⇑, right) but not without endothelium (pD2: 6.10±0.14; maximal response: 127±6%, P=NS; n=6) (not shown). In the presence of L-NAME, the contraction was identical in arteries with and without endothelium.
The present results demonstrate that the endothelium plays an inhibitory role against the contractile responses of vascular smooth muscle to norepinephrine, serotonin, or PGF2α by releasing NO in rat femoral arteries. The inhibitory effect against contractions mediated by α1-adrenoceptors is much more pronounced than that against contractions mediated by the other receptors, suggesting that different mechanisms are underlying NO release during contractions induced by different agonists. The endothelial inhibition against α1-agonist–induced but not serotonin- or PGF2α-induced contractions is selectively impaired in hypertension.
The presence of the endothelium reduced the contractions induced by norepinephrine, serotonin, and PGF2α, indicating an inhibitory role of the endothelium against the contractions.1 2 6 15 The negative modulation by the endothelium must be physiologically important because this is observed during the contractions induced by the neurotransmitter norepinephrine and the platelet-derived substance serotonin; platelets release enough serotonin to evoke constriction responses.17 L-NAME enhanced the contractions in arteries with but not without endothelium, and as a result, the inhibitory effect of the endothelium disappeared in the presence of L-NAME. Because L-NAME is a specific and potent inhibitor of the vascular production of NO,16 the endothelium plays an inhibitory role against the contractions by releasing NO. This is of particular importance because NO not only relaxes vascular smooth muscles but also inhibits platelet aggregation.1 During contractions induced by various agonists, the endothelium also plays a protective role in the circulation against the pathogenesis of cardiovascular disorders.
In vivo studies have revealed that NO is continuously released from the endothelium (basal release).18 19 20 However, the basal release alone, if at all, cannot fully explain the endothelial negative modulation of the contractions, because the characteristics of the endothelial inhibition against the contraction induced by norepinephrine were quite different from those against the contraction induced by serotonin or PGF2α. The possibility that NO basally released from the endothelium may differentially affect the contractions to norepinephrine, serotonin, and PGF2α is small because an NO donor, sodium nitroprusside, evoked comparable relaxations in arteries without endothelium contracted with norepinephrine and PGF2α (n=4, data not shown). Furthermore, L-NAME did not evoke contractions in quiescent arteries with endothelium (data not shown, n=7), indicating that basal NO release is not significant at least in this experimental condition. It is possible that NO release is stimulated by the contractions themselves. If this is true, the contraction-induced release of NO must be similar during the norepinephrine-, serotonin-, and PGF2α-induced contractions because the amplitude of the contractions (in arteries without endothelium) induced by these agonists was identical. However, the endothelial inhibition was much more pronounced in norepinephrine-induced than serotonin- or PGF2α-induced contraction; the presence of the endothelium reduced the maximal response to norepinephrine but not to serotonin or PGF2α. This implies that another mechanism rather than contraction-induced NO release may also be involved in the endothelial inhibition, at least against norepinephrine-induced contraction.
It is likely that norepinephrine, but not serotonin or PGF2α, directly stimulates endothelial NO synthesis via specific receptors in the femoral artery of rats. Norepinephrine induces vasodilation mediated by activation of endothelial β-adrenoceptors in rat aorta.21 However, the present experiments were performed in the presence of the β-adrenoceptor antagonist timolol, suggesting that β-adrenoceptor–mediated NO release can be excluded. Furthermore, in the absence of the β-adrenoceptor antagonist (and in the presence of the α1-adrenoceptor antagonist prazosin [10−6 mol/L]), norepinephrine (10−8 to 10−4 mol/L) evoked comparable relaxations in arteries with and without endothelium precontracted with PGF2α (n=4, data not shown). The activation of endothelial α2-adrenoceptors by norepinephrine also induces NO release in some vessels.22 23 24 In the present study, however, the presence of the α2-adrenoceptor antagonist yohimbine did not modify the endothelial inhibition against the contraction induced by norepinephrine. Furthermore, the α2-adrenoceptor agonist clonidine did not evoke relaxations in arteries with endothelium precontracted with PGF2α. These results suggest that endothelial α2-adrenoceptors are not involved in the endothelial inhibition against norepinephrine-induced contraction. Thus, we speculate that norepinephrine stimulates NO release through an activation of endothelial α1-adrenoceptors. Indeed, similar endothelial inhibition was observed against the contractions induced by norepinephrine and the α1-adrenoceptor agonist phenylephrine. The participation of endothelial α1-adrenoceptors in the depression of norepinephrine-induced contraction has also been suggested in the rat aorta.25
The fact that the inhibitory effect of the endothelium was similar against PGF2α- and serotonin-induced contractions suggests that serotonin does not stimulate NO release via endothelial serotonergic receptors in the rat femoral artery. Indeed, serotonin did not relax femoral arteries with endothelium contracted with PGF2α.
In SHR, the contraction induced by norepinephrine in arteries with endothelium was greater than in WKY. Since the contraction in arteries without endothelium was identical in WKY and SHR, the response of the hypertensive vascular smooth muscle to norepinephrine is not altered. These results suggest that the contraction (in arteries with endothelium) is augmented in SHR because of a reduced inhibitory effect of the hypertensive endothelium against norepinephrine-induced contraction. The reduced inhibition may be related to a reduced production and/or release of NO in the hypertensive endothelium as endothelium-independent relaxation induced by sodium nitroprusside was not different in WKY and SHR (data not shown, n=6). NO plays a vascular protective role in the circulation. Thus, it is possible that the impairment of endothelial function may be related to the increased incidence of cardiovascular disorders in hypertension. In contrast, the endothelial inhibition against serotonin- or PGF2α-induced contractions was not impaired in SHR even though NO mediates the inhibition. This again implies that the mechanism underlying NO release during the contraction induced by norepinephrine is different from that during the contractions induced by serotonin or PGF2α. High blood pressure selectively impairs the mechanism that is activated during the contraction induced by α1-adrenoceptor agonists.
In conclusion, the endothelium plays an inhibitory role against contractions in rat femoral arteries. NO released from the endothelium is responsible for the inhibition, but the characteristics of the endothelial inhibition were not similar against various types of contractions. The negative endothelial modulation is more pronounced during contractions mediated by α1-adrenoceptors than during contractions mediated by other receptors. Hypertension impairs the inhibitory role of the endothelium (NO production and/or release) against α1-adrenoceptor agonist–induced but not serotonin- or PGF2α-induced contraction.
Selected Abbreviations and Acronyms
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|SHR||=||spontaneously hypertensive rat(s)|
- Received March 4, 1996.
- Revision received April 1, 1996.
- Revision received May 13, 1996.
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