Endothelium-Derived Endothelin-1 Reduces Cerebral Artery Sensitivity to Nitric Oxide by a Protein Kinase C–Independent Pathway
Background and Purpose— Nitric oxide (NO) reduces endothelin-1 (ET-1) production and blunts ET-1 dependent vasoconstriction. The direct effects of smooth muscle ETA receptor stimulation on NO-mediated relaxation are unknown. We hypothesized that endothelium-derived ET-1 regulates vascular tone by reducing smooth muscle sensitivity to NO, possibly through activation of protein kinase C (PKC).
Methods— Rings of rabbit middle cerebral artery were mounted on microvessel myographs to measure isometric tension. Dose-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP; an NO donor) were obtained with or without ET-1 receptor blockade. Experiments were performed in the presence of indomethacin (10 μmol/L). Results are expressed as mean±SEM.
Results— In depolarized conditions (40 mmol/L KCl physiological solution), ACh-induced relaxation was entirely NO-dependent, as indicated by its suppression by Nω-nitro-l-arginine (P<0.05). Arterial sensitivity (pD2) to ACh (6.32±0.11, n=6) was increased (P<0.05) to 6.77±0.10 (n=6) by BQ123 (ETA receptor antagonist, 5 μmol/L) but not by BQ788 (ETB receptor antagonist, 5 μmol/L; 6.08±0.22, n=5). Consistent with this finding, blockade of ETA receptors increased (P<0.05) vascular sensitivity to SNP (6.95±0.10, n=8), whereas BQ788 had no influence on arterial sensitivity to SNP (6.17±0.07, n=7) compared with control (6.43±0.13, n=11). In denuded arteries, the sensitivity to SNP (7.10±0.08, n=8) was reduced by exogenous ET-1 (6.51±0.35, n=7, P<0.05). Chelerythrine, a PKC inhibitor, did not alter smooth muscle sensitivity to NO, whereas phorbol 12-myristate 13-acetate, a PKC activator, strongly increased it.
Conclusions— Blockade of ETA but not ETB receptors sensitizes vascular smooth muscle to exogenous and endothelium-derived NO. This suggests that ET-1 regulates smooth muscle sensitivity to NO by a PKC-independent pathway. This represents an alternative pathway by which NO and ET-1 interact to regulate vascular tone.
Endogenous endothelin (ET-1) regulates vascular tone through the activation of specific ETA and ETB receptors.1 ETA receptors are expressed on smooth muscle cells that contract on activation. In contrast, ETB receptors are expressed both on smooth muscle cells to evoke contractions and endothelial cells to induce nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation.2,3 EDHF dilates arteries by increasing membrane potential, which reduces Ca2+-dependent contraction of smooth muscle cells.4 A direct interaction between this factor and ET-1 has not been reported. NO, however, antagonizes ET-1 release from the endothelium.5 In resistance arteries isolated from human and rat brains as well as rabbit mesentery, the tonic release of ET-1 by the endothelium augments myogenic tone6 as well as contractile responses to phenylephrine and serotonin.7,8 These responses are highly increased by NO synthase inhibition.
In addition to modulating vascular reactivity, the endothelium regulates smooth muscle sensitivity to NO.3,9,10 After NO synthase inhibition or endothelial denudation, the potency of NO and its donors to induce relaxation increases, suggesting that NO exerts a feedback down-regulation on smooth muscle sensitivity to NO.11 Moncada and coworkers9 have suggested that removal of NO hypersensitizes the soluble guanylate cyclase to NO, increasing cyclic guanosine monophosphate (cGMP) production and relaxation.
The influence of endothelium-derived ET-1 on smooth muscle sensitivity to NO has not been considered. In this connection, it was reported that blockade of ET-1 receptors improved endothelium-dependent, NO-mediated relaxation in experimental heart failure,12,13 an effect attributed in part to a reduction in free radical generation. Relaxation of isolated cerebral arteries to an NO donor of rats with heart failure was also improved by chronic ETA receptor blockade.3 Endothelium-derived ET-1, therefore, may contribute to the regulation of smooth muscle sensitivity to NO. To explore this possibility, we studied the influence of acute ET-1 receptor blockade on smooth muscle sensitivity to NO of isolated cerebral arteries of the rabbit.
Materials and Methods
Our institutional ethical committee approved this study. Experiments were performed in New Zealand white rabbits (Charles River Laboratories, Wilmington, Mass) of either sex (weight 2.8 to 3.0 kg). Rabbits were anesthetized by intravenous injection of pentobarbital (50 mg/kg) and euthanized by exsanguination. The brain was harvested and middle cerebral arteries removed from the cortex. They were placed in ice-cold physiological saline solution (PSS) containing indomethacin (10 μmol/L) and of the following composition (mmol/L): NaCl 130, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 14.9, EDTA 0.026, glucose 10, CaCl2 1.6, and aerated with 12% O2, 5% CO2, 83% N2 (pH 7.4, 37°C). Segments 2 mm in length were mounted on 20-μm tungsten wire in microvessel myographs (IMF, University of Vermont). Vessels were equilibrated for 30 minutes at their optimal tension (150 mg), as previously described.14
Endothelial integrity was determined in each experiment by addition of 1 μmol/L of ACh to rings preconstricted with 40 mmol/L K+. In a series of experiments (n=15), the endothelium was removed mechanically by gentle rubbing with a human hair. The effectiveness of endothelial removal was confirmed by the absence of relaxation to ACh (1 μmol/L) in rings preconstricted with 40 mmol/L KCl PSS.
In the absence or presence of endothelium, parallel series of experiments using 2 arterial segments treated or not for 30 minutes with antagonists or inhibitors were performed. Only 1 dose-response curve to 1 agonist was obtained per arterial segment. Vessels were preconstricted with 40 mmol/L KCl PSS, and dose-response curves to SNP or ACh were obtained. When phorbol 12-myristate 13-acetate (PMA) was used, it was added at the plateau of the contraction induced by 40 mmol/L KCl PSS, prior to the addition of ACh or SNP. In some experiments, vessels were preconstricted with Nω-nitro-l-arginine (L-NNA; 100 μmol/L) and dose-response curves to ACh were obtained; in these conditions, we evaluated the influence of endogenous ET-1 on ACh-mediated relaxation by a K+-sensitive endothelium-derived relaxing factor. The term EDHF used in this study refers therefore to the KCl-sensitive, L-NNA-resistant, and indomethacin-resistant component of endothelium-dependent vasorelaxation.15 Finally, vessels were denuded and preconstricted with 40 mmol/L KCl PSS or ET-1; a dose-response curve to SNP was subsequently constructed.
The concentration of BQ123 (5 μmol/L) reduced ET-1 sensitivity (pD2) of cerebral arteries from 9.31±0.68 to 7.45±0.14 (n=5, P<0.05). BQ788 (5 μmol/L) however, had no influence (data not shown). The inhibitor of PKC (chelerythrine; 1 μmol/L) has been shown to be selective and effective at the concentration used.16,17 PMA was used at a concentration of 0.1 μmol/L.18 Catalase (500 U/mL) and superoxide dismutase (SOD; 250 U/mL) were added 30 minutes before the 40 mmol/L KCl PSS and cumulative addition of SNP.19,20 DMSO (0.014 mol/L), a weak free radical scavenger21,22 used to dissolve ET-1 receptor antagonists did not affect vascular sensitivity to ACh (6.44±0.24, n=6) and SNP (6.31±0.27, n=7) compared with the control sensitivity (6.32±0.11, n=6, and 6.43±0.13, n=11, respectively).
Results are expressed as mean±SEM. In all experiments, n equals the number of rabbits, and only 1 segment from 1 rabbit was used by protocol. Vasorelaxation is expressed as the percent inhibition of the preconstriction. Preconstriction refers to the level of tone induced by any constricting agent (K+, L-NNA or ET-1) before addition of the relaxant agent (Ach, forskolin, or SNP). The half-maximum effective concentration (EC50) of agonist was determined from each individual dose-response curve using a logistic curve-fitting program (Allfit, Dr A. Deléan, Department of Pharmacology, University of Montreal). The pD2 value is the negative log of the EC50 of agonist. Statistical differences between means were determined by ANOVA followed by a Scheffé F test. A probability value of <0.05 was accepted as significant for differences between groups of data.
The following drugs were purchased from Sigma Chemical Co: catalase, dimethyl sulfoxide (DMSO), indomethacin, L-NNA, PMA, SNP, and SOD. ET-1, BQ123, and BQ788 were purchased from American Peptide Company, and chelerythrine chloride was purchased from Cedarlane Laboratories Ltd. All drugs were dissolved in PSS except for indomethacin, which was dissolved in ethanol, and ET-1, BQ123, and BQ788, which were dissolved in PSS and DMSO (2:1, vol:vol). The final concentration of DMSO in the bath was 0.1% (vol:vol) and did not influence the parameters measured.
Effect of BQ123 on Endothelium-Dependent and Endothelium-Independent Relaxation
Incubation of intact vessels with BQ123 (ETA receptor antagonist; 5 μmol/L) increased cerebral artery sensitivity to ACh (Figure 1 and Table 1) and SNP (Figure 2 and Table 2). Inhibition of ETB receptors with BQ788 (5 μmol/L), however, had no effect on vascular sensitivity to NO compared with control. Furthermore, in arteries precontracted with L-NNA (100 μmol/L, in the presence of indomethacin), ACh-induced relaxation was not affected by BQ123 (Table 1 and Figure 3) but was blocked by further addition of high external K+ (40 mmol/L KCl). cAMP-dependent, endothelium-independent relaxation induced by forskolin (1 nmol/L to 30 μmol/L) of arteries precontracted with 40 mmol/L KCl (pD2=7.69±0.76, n=5) was not affected by BQ123 (pD2=7.44±0.96, n=5).
Effect of Endothelial Denudation on Relaxation Induced by SNP
Endothelial denudation facilitated the relaxation induced by SNP (Figure 4), as demonstrated by the increase in vascular sensitivity to SNP (Table 2). In these conditions, BQ123 (5 μmol/L) did not affect vascular sensitivity to SNP (Table 2). In denuded arteries preconstricted with ET-1 (428±45 mg, n=7), however, the sensitivity to SNP was reduced to 6.51±0.35 (P<0.05) compared with the sensitivity to SNP (7.10±0.08, n=7) of denuded arteries precontracted with 40 mmol/L KCl (445±97 mg).
Influences of Free Radical Scavengers, Chelerythrine, and PMA on Cerebral Artery Reactivity to NO
Combined addition of catalase and superoxide dismutase did not influence vascular sensitivity to SNP (Table 2). Chelerythrine (1 μmol/L), a PKC inhibitor, did not influence vascular reactivity to endogenous and exogenous NO released by ACh and SNP, respectively (Tables 1 and 2⇑). Accordingly, sensitivity to SNP (6.47±0.51, n=6) of intact arteries precontracted with ET-1 (358±44 mg) was not increased by pretreatment with chelerythrine (6.56±0.26, n=5), which did not influence the level of precontraction induced by ET-1 (435±30 mg).
The level of precontraction induced by 40 mmol/L KCl PSS (290±51, n=11) was further increased by addition of PMA, the PKC activator (422±47 mg, n=10; P<0.05). In these conditions, vascular sensitivity to ACh was increased (8.21±0.56, n=5; P<0.05) compared with the sensitivity to ACh in the absence of PMA (6.32±0.11, n=6). Combined addition of PMA and BQ123 did not further increase ACh sensitivity (8.06±0.44). Similarly, the sensitivity to SNP was increased by PMA (7.67±0.42, n=5; P<0.05) compared with the vascular sensitivity to SNP in the absence of PMA (6.43±0.13, n=11). As for ACh-induced relaxation, BQ123 did not further increase the sensitivity to SNP in the presence of PMA (7.35±0.92, n=5).
Effect of L-NNA on the Relaxation Induced by SNP
BQ123 and BQ788 did not affect L-NNA– (Table 1) or 40 mmol/L KCl PSS–induced contraction (Tables 1 and 2⇑). Similarly, chelerythrine and catalase combined with superoxide dismutase did not influence the precontraction induced by 40 mmol/L K+ (Table 2). Endothelial denudation, PMA, and L-NNA, however, increased the level of precontraction induced by 40 mmol/L KCl PSS. The sensitivity to NO was sensitive to BQ123, PMA, and L-NNA. In the absence of endothelium, precontraction levels induced by ET-1 (428±45 mg, n=7) and 40 mmol/L KCl (445±97 mg, n=7) were increased compared with that of intact arteries (Tables 1 and 2⇑); nonetheless, vascular sensitivity of SNP-induced relaxation was reduced by ET-1. The level of the preconstriction induced by 40 mmol/L KCl PSS, ET-1, PMA, or L-NNA was therefore not predictive of the vascular sensitivity to NO.
NO and ET-1 produce opposite responses to establish an important balance between these 2 systems in the control of vascular tone. It has been demonstrated5 that NO can counteract the production and the vasoconstrictor effect of ET-1. In this study, we tested the hypothesis that endothelium-derived ET-1 could regulate smooth muscle sensitivity to NO. Our data demonstrate that ET-1, by a PKC-independent pathway, directly decreases smooth muscle sensitivity to SNP, an NO donor. Indeed, vascular sensitivity to SNP was similar in denuded vessels precontracted with ET-1 and intact arteries precontracted by high K+. In these latter conditions, vascular sensitivity to SNP was lower when compared with that of intact arteries precontracted by high K+ in the presence of BQ123 and that of denuded arteries precontracted by high K+. This mechanism also regulates vascular sensitivity of endothelium-derived NO.
Control experiments10,11 have confirmed that inhibition of NO synthase with L-NNA and endothelial denudation augmented vascular sensitivity to SNP-induced relaxation. It has been demonstrated in vivo9 that the absence of NO in the vasculature leads to a specific supersensitivity to nitrovasodilators related to an upregulation of the enzymatic activity of the soluble guanylate cyclase. Our observation that vascular sensitivity to SNP- and ACh-induced relaxation are increased in the presence of ETA-receptor blockade (Tables 1 and 2⇑ and Figures 2 and 3⇑) suggested to us that endothelium-derived NO was not the only factor involved in the regulation of smooth muscle sensitivity to NO.
The regulatory effects of ETA-receptor blockade on the relaxation mediated by SNP and ACh are specific to NO. BQ123 did not influence the K+-sensitive, EDHF-like relaxation induced by ACh in the presence of L-NNA and indomethacin (Figure 4) nor the forskolin-mediated, cAMP-dependent relaxation.
The mechanism by which ET-1 reduces smooth muscle sensitivity to NO remains unknown. The effects of ET-1 are mediated by activation of smooth muscle ETA receptors but not vascular, either endothelial or muscular ETB receptors. Acutely, ET-1-dependent activation of two known pathways may contribute to this phenomenon. First, ET-1 has been shown to activate NAD(P)H-dependent superoxide production in phagocytes.23 This oxidase represents the most important source of superoxide in endothelial and smooth muscle cells.24–26 It has been demonstrated26 that free radicals interfere with NO-mediated dilatation. As shown in Table 2, however, the free radical scavengers SOD combined with catalase had no effect on vascular sensitivity to endogenous and exogenous NO. Other investigators12,24 have obtained similar results with SOD from aorta of rats and in control conditions. Münzel and coworkers,27 however, have observed that in rabbit aortic segments, pretreatment with liposomal SOD slightly enhanced vascular sensitivity to nitroglycerine and ACh. Other studies have demonstrated the implication of free radicals in the reduction of NO-mediated activation of the soluble guanylate cyclase and subsequent cGMP formation in vascular smooth muscle cells.22,28 Thus, it seems that ET-1–dependent free radical generation has limited effects in physiological conditions.
Second, ET-1 could decrease vascular sensitivity to NO by activating the PKC pathway. Chelerythrine, a PKC inhibitor, did not influence smooth muscle sensitivity to endogenous and exogenous NO (Tables 1 and 2⇑). Furthermore, chelerythrine did not increase smooth muscle sensitivity to NO of vessels precontracted by ET-1. In contrast, activation of the PKC pathway with PMA strongly increased NO sensitivity. Altogether, these data suggest that ET-1–dependent PKC activation is not involved in the reduction of smooth muscle sensitivity to NO. In fact, PKC activation increased cerebral artery sensitivity to NO, an effect opposite to that of ET-1.
As an alternative possibility, ET-1 and NO could counteract each other by physiological antagonism, one factor being constrictor and the other dilator. This possibility can be ruled out when using an NO donor such as SNP in denuded vessels. For a similar level of precontraction, vascular sensitivity to SNP was reduced by ET-1. In all experimental conditions, the level of precontraction was not predictive of the efficacy of NO. There is, therefore, a direct interaction between the cGMP relaxant pathway activated by NO and the intracellular pathway activated by ETA receptors. It should be pointed out that the effects of ET-1 on smooth muscle sensitivity to NO were independent of the endothelium. As a consequence, the improvement of ACh-induced NO-dependent relaxation by blockade of ETA receptors was not due to an acute improvement of the endothelial function but rather to a better efficacy of the smooth muscle relaxant pathway.
In conclusion, our results show that blockade of ETA but not ETB receptors sensitizes vascular smooth muscle to exogenous NO and endothelium-derived NO by a PKC-independent pathway. This represents an additional pathway of cross-reactivity between NO and ET-1 in vascular tissues.
- Received February 27, 2001.
- Revision received June 11, 2001.
- Accepted June 13, 2001.
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