Nitric Oxide Inhibits α2-Adrenoceptor–Mediated Endothelium-Dependent Vasodilation
Abstract—This study was designed to investigate the interaction between the NO/l-arginine pathway and the α2-adrenoceptor–mediated endothelium-dependent vasorelaxation. Reactivity of isolated resistance mesenteric arterial segments from mice lacking the gene for constitutive endothelial NO synthase (eNOS− mice, n=14) and from their wild-type controls (WT mice, n=46) was studied in isometric conditions in the presence of indomethacin (blocker of cyclooxygenase). Oxymetazoline (OXY, 0.01 to 30 μmol/L; a selective α2-adrenoceptor agonist) induced an endothelium-dependent relaxation of eNOS− but not WT arteries preconstricted either with phenylephrine or serotonin. In the presence of Nω-nitro-l-arginine (l-NNA, 100 μmol/L), an inhibitor of NOS, OXY induced an endothelium-dependent relaxation of WT mesenteric arteries. l-NNA had no effect on the relaxation caused by OXY in eNOS− arterial rings. Therefore, the relaxation caused by OXY was independent of NO formation. To demonstrate the inhibitory role of NO on the α2-adrenoceptor–mediated relaxation, subthreshold (0.1 nmol/L) to threshold (1 nmol/L) concentrations of sodium nitroprusside (donor of NO) were added to l-NNA–treated arteries before OXY challenges: in these conditions, the α2-adrenoceptor–mediated relaxation of eNOS− and WT arteries was inhibited. OXY-induced relaxation was restored on readdition of methylene blue (1 μmol/L, inhibitor of guanylate cyclase), suggesting that cGMP may be the mechanism of inhibition of the α2-adrenergic pathway in the presence of NO. Finally, OXY-mediated relaxation was blocked by tetraethylammonium (1 mmol/L) but not glibenclamide (1 μmol/L), suggesting the involvement of an endothelium-derived hyperpolarizing factor that activates Ca2+-activated K+ channels. In conclusion, α2-adrenoceptor activation caused relaxation of isolated murine mesenteric arteries that was functionally blocked by NO through a mechanism that may involve activation of the soluble guanylate cyclase and cGMP formation. The endothelium-dependent α2-adrenoceptor–mediated relaxation is likely to be due to an endothelium-derived hyperpolarizing factor, whose release and/or production is reduced by concurrent NO formation.
The α2-adrenoceptors are widely distributed in the cardiovascular system. Whereas activation of presynaptic α2-adrenoceptors decreases sympathetic activity,1 2 activation of smooth muscle α2-adrenoceptors induces contraction.3 4 α2-Adrenoceptors are also functionally expressed on the vascular endothelium. Their activation induces relaxation of isolated arteries from various vascular beds.5 6 7 Most of the data gathered involving α2-adrenoceptor–induced relaxation have been obtained from studies performed in large conductance vessels.6 8 A few studies, however, have focused on the mechanisms coupling α2-adrenoceptor activation and relaxation of resistance arteries, which are clearly different from those identified in conductance arteries.7 9
α2-Adrenoceptor–mediated relaxation primarily involves endothelium-derived NO in large conductance arteries.10 NOS inhibition antagonizes α2-adrenoceptor–induced relaxation of pig carotid arteries,6 partially blocks relaxation of coronary arteries,6 and has no effect in rabbit cerebral arteries.9 The reason for the variable importance of NO formation in α2-adrenoceptor–mediated relaxation may be related to the difference in intracellular pathways involved in receptor activation. α2-Adrenoceptors classically inhibit the activity of a family of adenylyl cyclases and activate phospholipase C, leading to smooth muscle contraction.11 12 They also inhibit endothelin-1 formation9 and stimulate inward rectifying K+ currents.13 This demonstrates that a wide range of coupling and effector mechanisms can be associated with α2-adrenoceptors.
The present study was designed to investigate the role of NO in the relaxation mediated by α2-adrenoceptor activation in resistance arteries and the possible interactions between NO and the α2-adrenergic pathway. Mesenteric arteries were isolated from mice lacking the gene for eNOS (eNOS− mice) and their WT controls.14 15 This allowed the dissociation of NO action from the α2-adrenoceptor occupation not only pharmacologically (using l-NNA, an inhibitor of NOS)16 but also genetically. The results suggest that NO-dependent activation of guanylate cyclase antagonizes endothelium-dependent α2-adrenoceptor–mediated relaxation.
Materials and Methods
eNOS− mice14 15 and WT mice (male, a combination of C57BL/6 and SV-129 strains, 18 to 23 g) were killed by cervical dislocation. WT mice were littermates from heterozygous matings. The mesenteric bed was dissected and placed in physiological saline of the following composition (mmol/L): NaCl 130, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 14.9, EDTA 0.026, and dextrose 11, aerated with 12% O2/5% CO2/83% N2 (pH 7.4). Segments (2 mm) of the third-order branch of the mesenteric artery were mounted on 17-μm tungsten wires in small vessel myographs as previously described9 17 and maintained in physiological saline containing indomethacin (10 μmol/L, inhibitor of cyclooxygenase) at 37°C. After a 1-hour stabilization period, arterial segments were challenged with a 40 mmol/L KCl physiological saline solution (KCl was substituted for an equivalent concentration of NaCl). Vessels were further stretched until a stable contractile response was reached (usually between 3 and 4 challenges).
Dose-response curves to OXY (0.1 to 100 μmol/L, α2-adrenoceptor agonist) were obtained on tissues preconstricted with PE (10 μmol/L).NOS was then inhibited by incubation of arterial rings with l-NNA (100 μmol/L) for 1 hour before testing.
In a separate series of experiments, OXY-induced relaxation of WT rings in the presence of l-NNA was obtained before and after 30 minutes of preincubation with yohimbine (0.1 μmol/L), a selective α2-adrenoceptor antagonist.
In a separate series of experiments, the endothelium was removed by gentle rubbing of the lumen of the arterial segments with a human hair. Rings were preconstricted with 10 μmol/L PE and challenged with OXY or SNP (0.1 to 30 μmol/L).
Influence of an Exogenous NO Donor
All experiments were performed in the presence of l-NNA (100 μmol/L). Tone was induced by serotonin (10 μmol/L) to eliminate possible interactions between α1- and α2-adrenergic receptor agonists.
Arterial segments of WT mice were constricted with serotonin; when plateauing, SNP (0.1 or 1 nmol/L) was added, and dose-response curves to OXY were obtained. Methylene blue (1 μmol/L) was subsequently added 20 minutes before serotonin stimulation and the addition of SNP. Dose-response curves to OXY were repeated. For each protocol, one arterial segment was run in parallel and similarly challenged but without SNP or methylene blue addition. These experiments were repeated in the presence of 10 μmol/L DMSO, which has been shown to scavenge hydroxyl radicals generated by methylene blue.18 In a series of experiments, ODQ (10 μmol/L) was used as a selective guanylate cyclase inhibitor.
Experiments were reproduced in the presence of deta nonoate (0.1 μmol/L) or SNAP (0.01 μmol/L) as NO donor.
Similar experiments using SNP (1 nmol/L) in the absence of DMSO were repeated in arteries from eNOS− mice.
Effect of K+ Channel Blockers
All experiments were performed in the presence of l-NNA (100 μmol/L). Tone was induced by serotonin (10 μmol/L).
The effects of TEA (1 mmol/l), an inhibitor of KCa channels, and glibenclamide (1 μmol/L), an inhibitor of KATP channels, on OXY-dependent relaxation were investigated in WT arteries. TEA and glibenclamide were added 20 minutes before testing the relaxant effect of OXY. The effect of pinacidil (1 and 10 μmol/L), an activator of KATP channels, was tested before and after the addition of glibenclamide.
At the end of each experiment, rings were maximally contracted (Emax) with 127 mmol/L KCl physiological saline solution.
Measurement of cGMP
Excised WT mesenteric beds (n=3 per group) were cleaned of fat and connective tissues. The vascular bed was fixed to a stainless-steel hook and suspended in an organ bath (10 mL, 37°C). All preparations were incubated without preload. Special attention was paid to make the time sequence equal to that of the mechanical tension studies. After administration of serotonin (10 μmol/L) and SNP (0.01 μmol/L) in the presence or in the absence of methylene blue (1 μmol/L), mesenteric preparations were frozen in liquid nitrogen after rapid blotting. Preparations were stored at −80°C until the time of analysis. Frozen tissues were pulverized with a percussion mortar at the temperature of liquid nitrogen and were resuspended with 2 mL of ice-cold perchloric acid (1N). After acetylation of the samples, cGMP levels were determined by radioimmunoassay.19
The following drugs were used: acetylcholine hydrochloride, DMSO, glibenclamide, HEPES, indomethacin, l-NNA, methylene blue, ODQ, OXY hydrochloride, PE hydrochloride, SNP, and TEA (Sigma Chemical Co). d-NNA and pinacidil were purchased from Research Biochemicals International. SNAP and deta nonoate were purchased from Calbiochem and Cayman Chemicals, respectively. Stock solutions of drugs were dissolved in physiological saline except for indomethacin and pinacidil, which were dissolved in ethanol, and glibenclamide, which was dissolved in DMSO. Deta nonoate was dissolved in 0.01 mol/L NaOH, and subsequent dilutions were performed in saline at pH 7.4. Solutions were prepared daily and kept on ice except for glibenclamide, which was kept at room temperature.
Contractions were expressed as a percentage of Emax produced by 127 mmol/L KCl physiological saline solution. Vasodilations were expressed as a percentage of the preconstricting tone; a maximum vasodilator effect was recorded as 100%.
Data are given as mean±SEM. In all experiments, n equals the number of animals. Statistical differences between the means were determined by ANOVA, followed by a Scheffé F test. When justified by the experimental design, an unpaired Student t test was applied. Statistical significance was reached at P<0.05.
Characterization of eNOS− Mice
A weak depolarizing physiological salt solution containing 40 mmol/L KCl induced greater contractions in eNOS− (70±3% Emax, n=8, P<0.05) compared with WT (27±6% Emax, n=7) segments.
PE (30 μmol/L, a selective α1-adrenoceptor agonist) induced a maximal contractile response in eNOS− arteries (102±6% Emax) but only 61±17% Emax in WT arteries (P<0.05). Similarly, responses obtained with a lower concentration of PE (10 μmol/L) were greater in eNOS− arterial rings (Table 1⇑); the addition of acetylcholine (1 μmol/L) reduced the preconstricting tone by 63±8% in WT arteries and by 15±8% in eNOS− arteries (n=6, P<0.05).
The maximal contractile response induced by 127 mmol/L KCl physiological salt solution was not affected by eNOS gene deletion (Table 1⇑).
A 1-hour incubation period of the tissues with l-NNA (100 μmol/L) raised the basal tone of WT segments (Table 1⇑) but had no effect in eNOS− arteries. In WT segments, PE-induced tone represented 62±13% Emax in the absence and 55±7% Emax in the presence of l-NNA (n=4). In eNOS− segments, this tone represented 67±11% Emax in the absence and 53±12% Emax in the presence of l-NNA (n=4). A 1-hour incubation period of the tissues with d-NNA (100 μmol/L) had no effect on basal tone (data not shown).
OXY (0.1 to 30 μmol/L) induced a significant concentration-dependent relaxation of PE-preconstricted eNOS− but not WT mesenteric arterial segments (Figure 1A⇓ and 1B⇓). After inhibition of NOS with l-NNA, however, OXY induced relaxation of WT segments (Figure 1A⇓). In the presence of l-NNA, OXY-induced relaxation was not different between WT and eNOS− segments. d-NNA did not potentiate relaxation induced by OXY (10 μmol/L) in WT arteries (data not shown).
In the presence of l-NNA, yohimbine (0.1 μmol/L), a selective α2-adrenoceptor antagonist, competitively shifted the dose-response to OXY in WT arterial segments preconstricted with serotonin (10 μmol/L) (Figure 2⇓).
Influence of an Exogenous NO Donor
We considered the possibility that NO antagonized the α2-adrenoceptor–dependent relaxation through a guanylate cyclase–dependent mechanism. Segments were constricted with serotonin (10 μmol/L). l-NNA (100 μmol/L) was present in all experiments.
In WT arteries, SNP (0.1 and 1 nmol/L) did not significantly affect serotonin-induced tone (Table 2⇓). Similarly, pretreatment with methylene blue (1 μmol/L) had no effect on serotonin-induced tone; addition of 1 nmol/L SNP had no vasoactive effect (Table 2⇓).
In eNOS− mesenteric arteries, serotonin-induced tone was not significantly altered by 1 nmol/L SNP (Table 2⇑). Methylene blue, either alone or in combination with 1 nmol/L SNP, had no significant effect on preconstricting tone (Table 2⇑).
OXY induced the relaxation of serotonin-preconstricted arteries (Figure 3⇓). In the presence of SNP (1 nmol/L), the relaxation mediated by OXY was significantly diminished in WT and eNOS− arteries, but methylene blue reversed SNP-mediated inhibition (Figure 3⇓). Similar data were obtained in WT segments in the presence of a lower concentration of SNP (0.1 nmol/L) (Figure 3A⇓).
In the presence of methylene blue alone, the relaxation mediated by OXY was not different in WT compared with eNOS− mesenteric arteries (Figure 4A⇓). To exclude the possibility of a nonspecific effect of methylene blue,18 20 21 22 23 dose-response curves to OXY were obtained in WT arterial segments in the presence of methylene blue (1 μmol/L) before and after the addition of DMSO (10 μmol/L), a hydroxyl radical scavenger.18 As shown in Figure 4B⇓, DMSO had no effect on α2-adrenoceptor–mediated responses.
SNP was light-exposed for 7 days. After this inactivation, SNP (1 nmol/L) did not inhibit the OXY-induced relaxation of serotonin-preconstricted WT arteries (n=2) (data not shown).
OXY (10 μmol/L) induced a potent relaxation of l-NNA–treated PE (10 μmol/L)–preconstricted rings of WT mesenteric arteries (Table 3⇓). SNAP (10 nmol/L) significantly attenuated the dilatory response to OXY, which was recovered by methylene blue (Table 3⇓). Similarly, deta nonoate (0.1 μmol/L) decreased OXY-induced relaxation; this inhibitory effect was blocked by methylene blue (Table 3⇓).
The relaxation mediated by OXY (10 μmol/L) was not affected by ODQ alone (10 μmol/L), a selective guanylate cyclase inhibitor,22 in l-NNA–treated vessels (79±6% versus 76±6% relaxation). In the presence of SNP (1 nmol/L), ODQ restored OXY-induced relaxation of l-NNA–treated segments (86±5% relaxation, P<0.05) compared with control without ODQ (23±10% relaxation).
SNP (0.01 μmol/L) increased cGMP content in the mesenteric bed from 0.2±0.2 to 0.74±0.1 pmol/mg tissue (P<0.05). The increase in cGMP induced by SNP was prevented by prior addition of methylene blue (1 μmol/L) in the organ chamber; in the presence of methylene blue, levels of cGMP were below the detection limit of the assay in the presence or in the absence of 0.01 μmol/L SNP.
Effects of K+ Channel Blockers
In the presence of TEA (1 mmol/L), the relaxation mediated by OXY was abolished in WT mice (Figure 5⇓), whereas glibenclamide (1 μmol/L) had no effect. Relaxations induced by pinacidil were decreased from 13±6% to 0±0% at 1 μmol/L and from 42±5% to 21±6% at 10 μmol/L in the presence of glibenclamide (n=5, P<0.05).
The present study was designed to investigate the involvement of NO in α2-adrenoceptor–mediated endothelium-dependent vasorelaxation of mesenteric resistance arteries in conditions in which cyclooxygenase activity was inhibited. The results suggests that NO, by activating the soluble guanylate cyclase, inhibits a TEA-sensitive endothelium-dependent α2-adrenergic pathway.
Characterization of eNOS− Mice
The physiological characteristics of mice lacking the gene for eNOS (eNOS− mice) have been previously reported.14 15 Compared with WT mice, their arterial pressure is slightly higher or unchanged. They also lack endothelium-dependent relaxation of the aorta in response to acetylcholine. In small arteries of the mesenteric bed, relaxation to acetylcholine was reduced by 75% in eNOS− mice, but not abolished. Similar results were obtained for chronic NO-deficient hypertension.24 Other endothelium-derived factors, such as EDHF, have been shown to be involved in the regulation of small artery tone and are likely to contribute to the vasorelaxant properties of acetylcholine.25 26 27 28 We also observed an increased smooth muscle sensitivity to contractile agents. The maximal contraction elicited by 127 mmol/L KCl physiological salt solution was not different between the 2 groups of arteries, but responses to PE, serotonin, and an intermediate depolarizing solution were significantly greater in eNOS− arteries. Such a change in reactivity may be related to the increased wall thickness. In this connection, a recent study24 showed that chronic NOS inhibition increased media thickness in rat mesenteric and cerebral arteries without a change in lumen diameter. These structural changes had no effect on maximal constriction and either increased or did not affect sensitivity to norepinephrine, serotonin, and endothelin-1, depending on the vascular bed studied. Thus, eNOS gene deletion may increase smooth muscle sensitivity to contractile agents independent of structural changes.
Endothelium-dependent relaxation induced by α2-adrenoceptor agonists has been described in several species and vascular beds.3 4 5 6 7 9 29 Although α2-adrenoceptor occupancy triggers the release of NO from large vessel endothelium,10 inhibitors of NOS do not fully antagonize relaxations mediated by α2-adrenoceptor agonists.6 Furthermore, in rat cerebral arteries, NO appears to be involved but does not mediate the dilator response of α2-adrenoceptor agonists.7 The present study suggests that in mice, NO is not responsible for the endothelium-dependent relaxation mediated by OXY, a selective α2-adrenoceptor agonist,9 29 30 but antagonizes α2-adrenoceptor agonist–mediated relaxation, since (1) l-NNA unmasked OXY-induced relaxation in WT mice but was not required in eNOS− mice (Figure 1⇑) and (2) the addition of a subthreshold concentration of SNP, a NO donor, blunted the relaxation in eNOS− and WT mice (Figure 3⇑). At the concentration of SNP used in the present study, it has been proven difficult to detect an increase in cGMP production in rat aortic strips.31 We used 10 nmol/L SNP to demonstrate that, indeed, this NO donor stimulated the guanylate cyclase, inducing a 3-fold increase in endogenous cGMP content. In the presence of methylene blue (1 μmol/L), both basal and SNP-stimulated cGMP formation were decreased below the range of the assay, suggesting that at the concentration used, methylene blue efficiently blocked the soluble guanylate cyclase, as proposed by others.23 31 32 33 34 By use of a similar approach, it has been shown that a stable nonhydrolyzable analogue of cGMP decreased the effect of EDHF in rat mesenteric arteries after inhibition of NOS; this suggests the existence of a guanylate cyclase–dependent regulation of EDHF production and/or activity.35 Similarly, Miyamoto et al36 showed that NO inhibited bradykinin receptor, but not epinephrine receptor, coupling to G proteins. Thus, in our hands, the inhibitory effect of NO on α2-adrenoceptor agonist–induced relaxation most likely involves the activation of guanylate cyclases, since inhibition of this enzyme restored the vasorelaxant properties of OXY (Figure 3⇑).
It has been reported that the effects of methylene blue may be caused by its ability to generate hydroxyl radicals and inhibit eNOS in addition to its ability to inhibit guanylate cyclase.18 20 21 22 23 First, an effect of methylene blue on eNOS can be ruled out in eNOS− mice, since this enzyme is not expressed. Second, DMSO, a potent antioxidant, did not alter the effect of methylene blue on vascular reactivity in the present study, contrary to what has been previously observed by others.18 20 23 Thus, the effects of methylene blue at the concentration used in the present study appear to be related to the inhibition of the soluble guanylate cyclase. These results are further strengthened by the effect of ODQ, a selective guanylate cyclase inhibitor,22 which mimics the effect of methylene blue. Therefore, NO did not directly inhibit α2-adrenoceptors or their intracellular coupling pathway. Rather, a cGMP-dependent kinase phosphorylation is likely to be involved. α2-Adrenoceptors are targets for phosphorylation,37 inducing short-term desensitization.38 This may represent a mechanism of action for guanylate cyclase–mediated inhibition of the α2-adrenoceptor pathway. Alternatively, cGMP, via activation of a kinase, may modulate α2-adrenoceptor/G protein coupling, as recently demonstrated for bradykinin receptors in cultured endothelial cells.36 Interestingly, since the pharmacological inhibition of NO formation in WT animals also revealed the α2-adrenoceptor–dependent relaxing pathway, it is likely that the genetic manipulation did not induce the de novo expression of a compensatory mechanism.
The pathway activated during α2-adrenoceptor occupation is consistent with the involvement of an agent stimulating a TEA-sensitive smooth muscle cell pathway. TEA is a well-characterized inhibitor of KCa channels.39 40 41 Such a TEA-sensitive pathway has been shown to be a key regulator of the mesenteric and brain circulations.26 27 28 Our data are consistent with previous reports showing that in some vascular preparations, including the mesenteric bed, receptor-mediated endothelium-dependent smooth muscle cell relaxation could be blocked by inhibitors of KCa channels but not KATP channels in the absence of NO production.40 41 42 43 44 From our data, however, we cannot identify the nature of the factor involved.
Recent studies have demonstrated an interaction between NO and EDHF (for review, see References 35 and 3935 39 ), a possible candidate for the α2-adrenoceptor–dependent pathway. In most vascular preparations, EDHF does not play a major role in the basal regulation of tone, and it is NO-cGMP that is predominantly involved. Some disease states may interfere with NO by either limiting its production or, when oxygen-derived free radicals are increased, limiting NO access to or activation of guanylate cyclase. Such alterations in NO-dependent relaxation have been reported in hypercholesterolemia,45 diabetes mellitus,46 and hypertension.47 Thus, according to the present data, a NO-independent mechanism, spared by the disease, may take on greater importance.
In conclusion, suppression of NO formation, either genetically or pharmacologically, reveals an endothelium-dependent α2-adrenoceptor–mediated relaxation. The mediator of this mechanism is likely to be an agent that activates smooth muscle KCa channels. This demonstrates that compensatory pathways can be physiologically expressed and are likely to be activated when NO-dependent responses are depressed.
Selected Abbreviations and Acronyms
|d-NNA, l-NNA||=||Nω-nitro-d-arginine and -l-arginine|
|EDHF||=||endothelium-derived hyperpolarizing factor|
|KATP channel||=||ATP-sensitive K+ channel|
|KCa channel||=||Ca2+-activated K+ channel|
This study was supported by Fonds de la Recherche de l’Institut de Cardiologie de Montréal, the Heart and Stroke Foundation of Québec, and the Totman Medical Research Fund. The authors are grateful to Dr Johanne Tremblay for cGMP measurements.
- Received May 21, 1997.
- Accepted April 1, 1998.
- © 1998 American Heart Association, Inc.
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