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Enhanced vasoconstrictor responses in eNOS deficient mice
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 8 (2003) 207–213 www.elsevier.com/locate/yniox
Enhanced vasoconstrictor responses in eNOS deficient mice Kathryn Lampinga,b,* and Frank Faracia a
Departments of Internal Medicine and Pharmacology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA b The Department of Veterans Affairs, Medical Services (111), VA Medical Center 601 Highway 6 West, Iowa City, IA 52246, USA Received 5 December 2002; received in revised form 12 May 2003
Abstract Previous studies suggest that vasoconstriction is modulated by nitric oxide (NO). Contractions to ET-1 and/or thromboxane may be enhanced during chronic deficiency in expression or activity of NO synthase (NOS). Multiple isoforms of NOS are expressed within the vessel wall and purely pharmacological approaches cannot define the role of each. We tested the hypothesis that vasoconstriction to endothelin-1 (ET-1) and/or the thromboxane mimetic, U46619, is enhanced under conditions of chronic, selective deficiency in endothelial NOS (eNOS)/)) by examining responses in aorta from eNOS)/) mice compared to wild type (eNOS+/+). ET-1 produced dose-dependent contraction of aorta from eNOS+/+ mice that was increased twofold following acute inhibition of all NOS isoforms with NG -nitro-L -arginine (L -NNA). In eNOS)/) mice, contractions to ET-1 were increased twofold compared to eNOS+/+. L -NNA had no effect. Although contraction of the aorta to thromboxane mimetic U46619 was increased at lower concentrations, maximal contractions to U46619 were not increased following acute inhibition of NOS or in eNOS)/) mice. These studies provide direct evidence that vasoconstriction to ET-1 and thromboxane is augmented in the face of eNOS deficiency, demonstrating that eNOS normally inhibits vascular contractile responses. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Endothelin; Thromboxane; Nitric oxide; Endothelial nitric oxide synthase; Mice
Although substantial research has focused on the ability of the endothelium to produce vasodilator substances, the endothelium also actively produces a variety of vasoconstrictor substances that regulate vascular tone. Under normal conditions the action of these potent endothelial-derived vasoconstrictor substances including endothelin and thromboxane may be tempered by release of endogenous endothelium-derived vasodilator substances. Endothelin and thromboxane A2 (TXA2 ) are the most potent vasoconstrictors identified. The role of these two vasoconstrictors in the regulation of vascular tone has been studied extensively [1,2]. Three isoforms of endothelin have been isolated but the most abundant form is endothelin-1 (ET-1). In several pathological conditions, tissue and/or plasma levels of ET-1 and TXA2 are elevated, potentially contributing to development of atherosclerosis, hypertension, and ischemia [3]. Depending on vascular bed and species vasoconstriction to these agents is modulated by * Corresponding author. Fax: 1-319-339-7040. E-mail address: [email protected] (K. Lamping).
endothelium which has the potential to release vasodilator substances including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor(s) (EDHF) [4,5]. Expression of nitric oxide (NO) and ET-1 is reciprocally regulated possibly through regulation of transcription of preproET-1 and nitric oxide synthase (NOS) genes [6,7]. Thus, with chronic inhibition or absence of nitric oxide ET-1 synthesis and role in regulation of vascular tone may be increased. Although the expression of specific NOS isoforms initially appeared to be somewhat cell specific, recent evidence suggests that multiple isoforms may be present in endothelium and vascular muscle under normal or pathological conditions [4,8–12]. Determination of the role of the different isoforms of NOS in the control of vascular responses has been dependent on the use of pharmacological inhibitors selective for the different NOS isoforms; however, the validity of this approach is dependent upon the specificity and efficacy of the inhibitors. Currently, there are no selective inhibitors of the endothelial isoform (eNOS). In addition, acute
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inhibition of NOS is often incomplete allowing for some continued production of NO to influence vascular function [13,14]. The development of mouse models deficient in the gene for eNOS (eNOS)/)) allows study of the role of eNOS specifically in the regulation of vascular tone. Previous studies using pharmacological approaches to block NOS or cyclooxygenase suggest that NO or cyclooxygenase products inhibit the synthesis and vasoconstrictor effects of ET-1 or TXA2 [2,15–22]. Although vasoconstriction to these agents is increased in disease states, the mechanisms that account for this increase are not clear. Possibilities related to altered endothelial function include decreased production or bioavailability of NO, prostaglandins, and/or EDHF. Altered contractile function of vascular muscle may also contribute. The goal of these studies was to determine whether the vascular effects of ET-1 and/or thromboxane mimetic, U46619, are enhanced under conditions of chronic deficiency of eNOS and whether acute inhibition of eNOS and chronic deficiency of eNOS have similar effects on contractile responses.
ventitia was removed and each vessel was cut into four rings (3–4 mm in length). Vascular rings were suspended in an organ bath containing 25 ml Krebs solution maintained at 37 °C and aerated with 20% O2 , 5% CO2 , and 75% N2 . The rings were connected to a force transducer to measure isometric tension (contraction). Resting tension was increased stepwise to reach the final tension of 0.75 g and the rings were allowed to equilibrate for at least 60 min. This amount of resting tension was found to be optimal for contraction of murine aorta. Reactivity of aorta was tested with 100 mM KCl before study. Protocols Contractile responses to ET-1 (0.5 nM to 10 lM) and the thromboxane analog [U46619, (9,11-dideoxy-11a,9aepoxy-methanoprostaglandin F2a ), 10–100 nM] were measured in vessels from eNOS (+/+) and eNOS ()/)) mice. For acute inhibition of NOS, vessels were incubated with NG -nitro-L -arginine (L -NNA, 100 lM) for 30 min before measurement of responses to ET-1 or U46619. Drugs
Experimental procedures Animals The animal protocol used in these experiments was reviewed and approved by the University of Iowa Animal Care and Use Committee. Mice for this study (eNOS+/+ n ¼ 20; eNOS)/) n ¼ 16) were derived from breeding of eNOS (+/)) mice with eNOS (+/)) mice to generate eNOS (+/+) and eNOS ()/)) within the same litter. This approach allowed us to use eNOS (+/+) as littermate controls. For these studies, two groups of mice were studied: wild-type control mice (eNOS+/+ littermates) and homozygous eNOS deficient mice (eNOS)/)). These mice were originally generated as a hybrid of 129 X C57BL/6J [23]. Mice used in this study were derived from 7 to 8 generations of backcross breeding to C57BL/6J mice. Mice were fed regular chow and water was available ad libitum. eNOS (+/+) and eNOS ()/)) mice were similar in age and weight (27 1 and 26 1 weeks; 30 1 and 24 1 g), respectively. Mice were genotyped using PCR by the University of Iowa Transgenic Animal Facility. General preparation Mice were anesthetized with pentobarbital (150 mg/ kg, ip) and the aorta was quickly removed. Following removal, aortas were placed in Krebs buffer with the following ionic composition (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2 PO4 , 1.2, NaHCO3 25, and glucose 11 mM. Loose connective tissue on the ad-
U46619 was obtained from Biomol Research Laboratories and dissolved in 95% ethanol. ET-1 was obtained from Phoenix Pharmaceuticals and dissolved in saline with 0.1–1% bovine serum albumin. All other chemicals were obtained from Sigma. All concentrations are final molar concentrations in the organ chamber. Statistical analysis Contractions are presented as grams of tension developed and are presented as means SEM. Responses of vessels from the same mouse were averaged and n represents the number of mice per group. Comparisons were made using a one-way ANOVA with repeated measures followed by Student–Newman–Keuls test to detect individual differences. A value of p < 0:05 was defined as significant.
Results Responses of wild-type eNOS+/+ mice to ET-1 ET-1 produced modest dose-dependent contraction in aorta from wild-type male ðn ¼ 13Þ and female mice ðn ¼ 7Þ. There were no differences in responses to ET-1 in vessels from male compared to female mice (maximal contraction at ET-1 10 nM males 67 11 mg; females 73 16 mg). Therefore, responses of aorta from male and female mice to ET-1 were combined. Following acute inhibition of NOS with L -NNA (100 lM) con-
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Fig. 1. Contractions of aorta from wild type eNOS+/+ mice to ET-1 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100lM). ET-1 produced dose-dependent contractions that were increased by L -NNA. (Values are means SE, *p < 0:05 vs. eNOS+/+, n ¼ 20.)
Fig. 3. Contraction of aorta from eNOS)/) mice to ET-1 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100 lM). Contractions to ET-1 were not affected by L -NNA. (Values are means SE, n ¼ 16.)
tractions to ET-1 were increased and maximal contraction to ET-1 was increased approximately twofold (Fig. 1). Thus, under normal conditions, vasoconstriction to ET-1 was inhibited by NO.
tractions of aorta from wild-type mice in the presence of L -NNA. L -NNA had no effect on contractions to ET-1 of aorta from eNOS)/) mice (Fig. 3). Thus, NO derived from eNOS inhibits contractions of aorta to ET-1.
Responses of eNOS deficient mice to ET-1
Responses of wild-type mice to U46619
In aorta from mice deficient in eNOS, ET-1 produced dose-dependent contractions that were greater than contractions of vessels from wild-type mice (Fig. 2). Similar to the effects of acute inhibition of NOS, chronic deficiency of eNOS increased maximal contractions to ET-1 approximately twofold. Thus, contractions of aorta from eNOS)/) mice to ET-1 were similar to con-
The thromboxane mimetic, U46619, produced dosedependent contractions of aorta that were similar in vessels from male and female mice. Thus, responses to U46619 in vessels from male and female mice were combined. Acute inhibition of NOS with L -NNA increased contraction of aorta to the low concentration of U46619, but did not alter maximal contraction (Fig. 4).
Fig. 2. Contraction of aorta from wild type eNOS+/+ mice and eNOS)/) mice to ET-1. ET-1 produced dose-dependent contractions that were increased in aorta from eNOS)/) compared to wild type mice. Maximal contractions were doubled in vessels from eNOS)/) mice compared to eNOS+/+ mice. (Values are means SE, *p < 0:05 vs. eNOS+/+, eNOS+/+ n ¼ 20, and eNOS)/) n ¼ 16.)
Fig. 4. Contraction of aorta from aorta from wild type eNOS+/+ mice to thromboxane mimetic U46619 in the absence and presence of NG nitro-L -arginine (L -NNA, 100 lM). U46619 produced dose-dependent contractions that were increased at the lowest concentration by L -NNA. (Values are means SE, *p < 0:05 vs. eNOS+/+, n ¼ 20.)
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Thus, under normal conditions vasoconstriction to U46619 was modestly inhibited by release of NO. Responses of eNOS deficient mice to U46619 U46619 produced dose-dependent contractions of aorta from eNOS)/) mice. Contractions to U46619 of aorta from eNOS)/) mice were greater than contractions of wild type mice at the lowest concentration of U46619, but were similar at the highest concentration (Fig. 5). Acute inhibition of NOS with L -NNA had no effect on contractions to U46619 in vessels from eNOS)/) mice (Fig. 6). Contractions of aorta to
Fig. 5. Contraction of aorta from wild type eNOS+/+ mice and eNOS)/) mice to U46619. U46619 produced dose-dependent contractions that were increased in aorta from eNOS)/) mice only at the lowest concentration of U46619. (Values are means SE, *p < 0:05 vs. eNOS+/+, eNOS+/+ n ¼ 20, eNOS)/) n ¼ 16.)
Fig. 6. Contraction of aorta from eNOS)/) mice to U46619 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100 lM). Contractions of aorta from eNOS)/) mice to U46619 were not affected by L -NNA. (Values are means SE, n ¼ 16.)
U46619 following acute inhibition of NOS and in vessels from eNOS)/) mice were similar. Thus, NO derived from eNOS plays a modest role in modulating contraction to U46619 in aorta.
Discussion There are several new findings in this study. First, acute inhibition of NOS increased contraction of aorta to ET-1 in wild-type mice. Maximal contractions to ET-1 were approximately doubled. Second, chronic deficiency of eNOS increased contractions to ET-1. Responses of vessels from mice deficient in eNOS to ET1 were similar to responses seen in vessels from wild type mice treated with L -NNA. Thus, with chronic deficiency of eNOS, no additional mechanisms appear to be activated to compensate for the absence of NO and alter vasoconstriction to ET-1. Third, contractions to the thromboxane mimetic U46619 at the lowest concentration were also increased by both acute inhibition of NOS and chronic deficiency of eNOS. Although acute and chronic absence of NOS increased responses to U46619, the effects were relatively more modest than that observed for ET-1 as maximal contractions were not affected. Lastly, there were no gender differences in the vasoconstrictor response to ET-1 or U46619 in aorta from normal mice. The absence of a gender difference in this study is in contrast to our previous study in which vasoconstrictor responses to serotonin and U46619 were greater in carotid artery from male compared to female mice [24]. Role of NO in modulating responses to ET-1 Previous studies from our laboratory [19] and others [25] have demonstrated that responses to ET-1 are dependent on the route of administration with constriction of vessels with abluminal administration and dilation when administered intravascularly. These data suggest that a diffusional barrier or release of endothelial-derived substances plays a major role in responses to ET-1. Subsequent studies have examined the role of endothelium and endothelium-derived vasodilator substances in modulating responses to ET-1. Removal of endothelium or inhibition of NOS and cyclooxygenase all augment contractile responses to ET-1 depending on the vascular bed or species [15–22]. Thus, a variety of substances including NO and cyclooxygenase products may modulate responses to ET-1. Previous studies suggest a reciprocal regulation of the expression of ET-1 and NO [6,7,26]. Several lines of evidence in endothelial cells in vitro and in vivo suggest that in the presence of reduced levels of NO message for and/or protein levels of ET-1 are increased [6,26]. These data would suggest that in pathological
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conditions with reduced production or bioactivity of NO, the role for ET-1 may be increased. In the present study, acute inhibition of NOS or chronic deficiency of eNOS produced similar increases in contractions to ET-1 in mouse aorta. These data suggest that eNOSderived NO may not regulate ET-1 gene expression in all models. Results of the present study provide direct evidence that eNOS-derived NO attenuates contractions to ET-1 in aorta. Although some previous studies suggest that NO can attenuate vasoconstriction to ET-1, the source of NO was not defined. Some lines of evidence suggest that all three isoforms of NOS are normally present in both endothelium and vascular muscle [4,8–12]. The contribution of NO from these three sources in modulating vascular responses is not clear. Although results from the present study do not rule out a contribution of NO from nNOS or iNOS in modulating contractions to ET-1 in aorta, the data suggest that NO from these sources probably has a minimal role in attenuating contractions to ET-1. This conclusion is based on the findings that acute but non-selective inhibition of NOS with L -NNA increases contractions to ET-1 similar to selective deletion of eNOS in genetically deficient mice and that NOS inhibition in eNOS)/) mice has no effect on responses to ET-1. In addition to concerns of specificity of pharmacological inhibitors of NOS, studies from Cohen and others suggest that pharmacological inhibitors of NOS do not always completely abolish release of NO from eNOS [13,14]. With acute inhibition of NOS, release of NO could still be detected. Thus, it is difficult to fully define the magnitude of the role of NO from eNOS in modulating vascular reactivity. Surprisingly, in the present study the magnitude of acute inhibition of NOS on contractions to ET-1 is similar to the shift in the response to ET-1 in vessels obtained from eNOS)/) mice. Role of NO in modulating responses to U46619 A role for NO in modulating responses to thromboxane A2 mimetic, U46619, has been variable [27–31]. Although studies suggest that NO can modulate contractile responses to U46619 in some vascular beds often the effects are modest [28,30]. Studies also suggest no role for NO in modulating contractions to U4619 [27,29,31]. In the present study, we chose to test responses to U46619 to serve as a NO-independent vasoconstrictor. However, NO played a modest role in attenuating contractions to U46619 in mouse aorta. Contractions at low concentrations of U46619 were increased with acute inhibition of NOS and in vessels obtained from eNOS)/) mice compared to controls. These findings in aorta are similar to our recent results in carotid artery from eNOS)/) mice [24]. Similar to
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responses with ET-1 the magnitude of the increased response to U46619 following acute inhibition of NOS was similar to the shift in responses of vessels obtained from eNOS)/) mice compared to responses of vessels obtained from eNOS+/+ mice. Thus, acute inhibition of NOS and chronic deficiency of eNOS produce similar effects on responses to two different vasoconstrictors. Compensatory mechanisms in regulation of vascular reactivity Lamping and others [32–37] have previously demonstrated that with chronic absence of eNOS, other vasodilator pathways may increase to compensate for the absence of NO from eNOS. In coronary arteries and the cerebral microcirculation, nNOS appears to upregulate in the face of eNOS deficiency [32,36,37]. In mesenteric arteries from eNOS)/) mice, cyclooxygenase and nNOS upregulate to maintain flow-mediated dilation at normal levels [33,34,38]. However, in aorta and carotid artery from eNOS)/) mice vasodilation to acetylcholine is absent [39,40], indicating no compensatory mechanisms are activated. In the present study, there was no apparent compensatory increase in other vasodilator pathways to maintain normal contractions to ET-1 or U46619. Thus, in contrast to other vascular beds in the mouse, compensatory mechanisms do not appear to upregulate to maintain normal responses of aorta. The role of NO in modulating vascular responses in disease Numerous studies have suggested that reduced NO production or bioactivity and impaired endotheliumdependent vascular responses are a hallmark of vascular disease. Animal models of atherosclerosis, hypertension, diabetes, aging, AlzheimerÕs, and heart failure have all demonstrated reduced NO mediated vascular responses. Perhaps more important than reduced vasodilator responses in these models is the potential for enhanced constrictor responses to a variety of circulating and/or locally produced substances including ET-1, serotonin, and angiotensin II. In atherosclerotic apoE deficient mice, activation of the ET system was demonstrated in addition to decreases in NO release. Levels of ET-1 protein and receptors in atherosclerotic aortic tissue were associated with reduced NO mediated vascular responses [41]. Treatment with ET-1 receptor antagonists reduced lesion size and intimal thickening in addition to restoring NO mediated vascular responses [41]. Although responses to ET-1 were not measured, other studies have demonstrated enhanced contractions to ET-1 in the presence of atherosclerosis [42]. The present study provides the first direct evidence that a decrease in expression of eNOS leads to enhanced vasoconstriction
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to ET-1. These data suggest that altered ET-1 responses may arise as a consequence of dysfunction in the NO system and highlight the intricate balance between ET-1 and NO. In summary, the present study suggests that vascular responses to ET-1 and U46619 in the mouse aorta are attenuated by NO derived from eNOS. The data from gene-targeted mice provide direct evidence that eNOS plays a major role in influencing responses to ET-1 and to a lesser degree responses to thromboxane. Both acute inhibition of NOS and selective deletion of the gene for eNOS enhanced contractile responses of the aorta in mice.
Acknowledgments This work was supported by grants from the National Institutes of Health (HL-39050, NS-24621 and HL-62984, HL-38901) and a grant from the American Heart Association. K.G.L. and F.M.F. are Established Investigators of the American Heart Association. We thank Xiuying Liu for her valuable technical assistance. We also acknowledge Curt Sigmund, Ph.D., and the University of Iowa Transgenic Core for genotyping mice used in these studies.
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Biology and Chemistry
Nitric Oxide 8 (2003) 207–213 www.elsevier.com/locate/yniox
Enhanced vasoconstrictor responses in eNOS deficient mice Kathryn Lampinga,b,* and Frank Faracia a
Departments of Internal Medicine and Pharmacology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA b The Department of Veterans Affairs, Medical Services (111), VA Medical Center 601 Highway 6 West, Iowa City, IA 52246, USA Received 5 December 2002; received in revised form 12 May 2003
Abstract Previous studies suggest that vasoconstriction is modulated by nitric oxide (NO). Contractions to ET-1 and/or thromboxane may be enhanced during chronic deficiency in expression or activity of NO synthase (NOS). Multiple isoforms of NOS are expressed within the vessel wall and purely pharmacological approaches cannot define the role of each. We tested the hypothesis that vasoconstriction to endothelin-1 (ET-1) and/or the thromboxane mimetic, U46619, is enhanced under conditions of chronic, selective deficiency in endothelial NOS (eNOS)/)) by examining responses in aorta from eNOS)/) mice compared to wild type (eNOS+/+). ET-1 produced dose-dependent contraction of aorta from eNOS+/+ mice that was increased twofold following acute inhibition of all NOS isoforms with NG -nitro-L -arginine (L -NNA). In eNOS)/) mice, contractions to ET-1 were increased twofold compared to eNOS+/+. L -NNA had no effect. Although contraction of the aorta to thromboxane mimetic U46619 was increased at lower concentrations, maximal contractions to U46619 were not increased following acute inhibition of NOS or in eNOS)/) mice. These studies provide direct evidence that vasoconstriction to ET-1 and thromboxane is augmented in the face of eNOS deficiency, demonstrating that eNOS normally inhibits vascular contractile responses. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Endothelin; Thromboxane; Nitric oxide; Endothelial nitric oxide synthase; Mice
Although substantial research has focused on the ability of the endothelium to produce vasodilator substances, the endothelium also actively produces a variety of vasoconstrictor substances that regulate vascular tone. Under normal conditions the action of these potent endothelial-derived vasoconstrictor substances including endothelin and thromboxane may be tempered by release of endogenous endothelium-derived vasodilator substances. Endothelin and thromboxane A2 (TXA2 ) are the most potent vasoconstrictors identified. The role of these two vasoconstrictors in the regulation of vascular tone has been studied extensively [1,2]. Three isoforms of endothelin have been isolated but the most abundant form is endothelin-1 (ET-1). In several pathological conditions, tissue and/or plasma levels of ET-1 and TXA2 are elevated, potentially contributing to development of atherosclerosis, hypertension, and ischemia [3]. Depending on vascular bed and species vasoconstriction to these agents is modulated by * Corresponding author. Fax: 1-319-339-7040. E-mail address: [email protected] (K. Lamping).
endothelium which has the potential to release vasodilator substances including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor(s) (EDHF) [4,5]. Expression of nitric oxide (NO) and ET-1 is reciprocally regulated possibly through regulation of transcription of preproET-1 and nitric oxide synthase (NOS) genes [6,7]. Thus, with chronic inhibition or absence of nitric oxide ET-1 synthesis and role in regulation of vascular tone may be increased. Although the expression of specific NOS isoforms initially appeared to be somewhat cell specific, recent evidence suggests that multiple isoforms may be present in endothelium and vascular muscle under normal or pathological conditions [4,8–12]. Determination of the role of the different isoforms of NOS in the control of vascular responses has been dependent on the use of pharmacological inhibitors selective for the different NOS isoforms; however, the validity of this approach is dependent upon the specificity and efficacy of the inhibitors. Currently, there are no selective inhibitors of the endothelial isoform (eNOS). In addition, acute
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inhibition of NOS is often incomplete allowing for some continued production of NO to influence vascular function [13,14]. The development of mouse models deficient in the gene for eNOS (eNOS)/)) allows study of the role of eNOS specifically in the regulation of vascular tone. Previous studies using pharmacological approaches to block NOS or cyclooxygenase suggest that NO or cyclooxygenase products inhibit the synthesis and vasoconstrictor effects of ET-1 or TXA2 [2,15–22]. Although vasoconstriction to these agents is increased in disease states, the mechanisms that account for this increase are not clear. Possibilities related to altered endothelial function include decreased production or bioavailability of NO, prostaglandins, and/or EDHF. Altered contractile function of vascular muscle may also contribute. The goal of these studies was to determine whether the vascular effects of ET-1 and/or thromboxane mimetic, U46619, are enhanced under conditions of chronic deficiency of eNOS and whether acute inhibition of eNOS and chronic deficiency of eNOS have similar effects on contractile responses.
ventitia was removed and each vessel was cut into four rings (3–4 mm in length). Vascular rings were suspended in an organ bath containing 25 ml Krebs solution maintained at 37 °C and aerated with 20% O2 , 5% CO2 , and 75% N2 . The rings were connected to a force transducer to measure isometric tension (contraction). Resting tension was increased stepwise to reach the final tension of 0.75 g and the rings were allowed to equilibrate for at least 60 min. This amount of resting tension was found to be optimal for contraction of murine aorta. Reactivity of aorta was tested with 100 mM KCl before study. Protocols Contractile responses to ET-1 (0.5 nM to 10 lM) and the thromboxane analog [U46619, (9,11-dideoxy-11a,9aepoxy-methanoprostaglandin F2a ), 10–100 nM] were measured in vessels from eNOS (+/+) and eNOS ()/)) mice. For acute inhibition of NOS, vessels were incubated with NG -nitro-L -arginine (L -NNA, 100 lM) for 30 min before measurement of responses to ET-1 or U46619. Drugs
Experimental procedures Animals The animal protocol used in these experiments was reviewed and approved by the University of Iowa Animal Care and Use Committee. Mice for this study (eNOS+/+ n ¼ 20; eNOS)/) n ¼ 16) were derived from breeding of eNOS (+/)) mice with eNOS (+/)) mice to generate eNOS (+/+) and eNOS ()/)) within the same litter. This approach allowed us to use eNOS (+/+) as littermate controls. For these studies, two groups of mice were studied: wild-type control mice (eNOS+/+ littermates) and homozygous eNOS deficient mice (eNOS)/)). These mice were originally generated as a hybrid of 129 X C57BL/6J [23]. Mice used in this study were derived from 7 to 8 generations of backcross breeding to C57BL/6J mice. Mice were fed regular chow and water was available ad libitum. eNOS (+/+) and eNOS ()/)) mice were similar in age and weight (27 1 and 26 1 weeks; 30 1 and 24 1 g), respectively. Mice were genotyped using PCR by the University of Iowa Transgenic Animal Facility. General preparation Mice were anesthetized with pentobarbital (150 mg/ kg, ip) and the aorta was quickly removed. Following removal, aortas were placed in Krebs buffer with the following ionic composition (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2 PO4 , 1.2, NaHCO3 25, and glucose 11 mM. Loose connective tissue on the ad-
U46619 was obtained from Biomol Research Laboratories and dissolved in 95% ethanol. ET-1 was obtained from Phoenix Pharmaceuticals and dissolved in saline with 0.1–1% bovine serum albumin. All other chemicals were obtained from Sigma. All concentrations are final molar concentrations in the organ chamber. Statistical analysis Contractions are presented as grams of tension developed and are presented as means SEM. Responses of vessels from the same mouse were averaged and n represents the number of mice per group. Comparisons were made using a one-way ANOVA with repeated measures followed by Student–Newman–Keuls test to detect individual differences. A value of p < 0:05 was defined as significant.
Results Responses of wild-type eNOS+/+ mice to ET-1 ET-1 produced modest dose-dependent contraction in aorta from wild-type male ðn ¼ 13Þ and female mice ðn ¼ 7Þ. There were no differences in responses to ET-1 in vessels from male compared to female mice (maximal contraction at ET-1 10 nM males 67 11 mg; females 73 16 mg). Therefore, responses of aorta from male and female mice to ET-1 were combined. Following acute inhibition of NOS with L -NNA (100 lM) con-
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Fig. 1. Contractions of aorta from wild type eNOS+/+ mice to ET-1 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100lM). ET-1 produced dose-dependent contractions that were increased by L -NNA. (Values are means SE, *p < 0:05 vs. eNOS+/+, n ¼ 20.)
Fig. 3. Contraction of aorta from eNOS)/) mice to ET-1 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100 lM). Contractions to ET-1 were not affected by L -NNA. (Values are means SE, n ¼ 16.)
tractions to ET-1 were increased and maximal contraction to ET-1 was increased approximately twofold (Fig. 1). Thus, under normal conditions, vasoconstriction to ET-1 was inhibited by NO.
tractions of aorta from wild-type mice in the presence of L -NNA. L -NNA had no effect on contractions to ET-1 of aorta from eNOS)/) mice (Fig. 3). Thus, NO derived from eNOS inhibits contractions of aorta to ET-1.
Responses of eNOS deficient mice to ET-1
Responses of wild-type mice to U46619
In aorta from mice deficient in eNOS, ET-1 produced dose-dependent contractions that were greater than contractions of vessels from wild-type mice (Fig. 2). Similar to the effects of acute inhibition of NOS, chronic deficiency of eNOS increased maximal contractions to ET-1 approximately twofold. Thus, contractions of aorta from eNOS)/) mice to ET-1 were similar to con-
The thromboxane mimetic, U46619, produced dosedependent contractions of aorta that were similar in vessels from male and female mice. Thus, responses to U46619 in vessels from male and female mice were combined. Acute inhibition of NOS with L -NNA increased contraction of aorta to the low concentration of U46619, but did not alter maximal contraction (Fig. 4).
Fig. 2. Contraction of aorta from wild type eNOS+/+ mice and eNOS)/) mice to ET-1. ET-1 produced dose-dependent contractions that were increased in aorta from eNOS)/) compared to wild type mice. Maximal contractions were doubled in vessels from eNOS)/) mice compared to eNOS+/+ mice. (Values are means SE, *p < 0:05 vs. eNOS+/+, eNOS+/+ n ¼ 20, and eNOS)/) n ¼ 16.)
Fig. 4. Contraction of aorta from aorta from wild type eNOS+/+ mice to thromboxane mimetic U46619 in the absence and presence of NG nitro-L -arginine (L -NNA, 100 lM). U46619 produced dose-dependent contractions that were increased at the lowest concentration by L -NNA. (Values are means SE, *p < 0:05 vs. eNOS+/+, n ¼ 20.)
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Thus, under normal conditions vasoconstriction to U46619 was modestly inhibited by release of NO. Responses of eNOS deficient mice to U46619 U46619 produced dose-dependent contractions of aorta from eNOS)/) mice. Contractions to U46619 of aorta from eNOS)/) mice were greater than contractions of wild type mice at the lowest concentration of U46619, but were similar at the highest concentration (Fig. 5). Acute inhibition of NOS with L -NNA had no effect on contractions to U46619 in vessels from eNOS)/) mice (Fig. 6). Contractions of aorta to
Fig. 5. Contraction of aorta from wild type eNOS+/+ mice and eNOS)/) mice to U46619. U46619 produced dose-dependent contractions that were increased in aorta from eNOS)/) mice only at the lowest concentration of U46619. (Values are means SE, *p < 0:05 vs. eNOS+/+, eNOS+/+ n ¼ 20, eNOS)/) n ¼ 16.)
Fig. 6. Contraction of aorta from eNOS)/) mice to U46619 in the absence and presence of NG -nitro-L -arginine (L -NNA, 100 lM). Contractions of aorta from eNOS)/) mice to U46619 were not affected by L -NNA. (Values are means SE, n ¼ 16.)
U46619 following acute inhibition of NOS and in vessels from eNOS)/) mice were similar. Thus, NO derived from eNOS plays a modest role in modulating contraction to U46619 in aorta.
Discussion There are several new findings in this study. First, acute inhibition of NOS increased contraction of aorta to ET-1 in wild-type mice. Maximal contractions to ET-1 were approximately doubled. Second, chronic deficiency of eNOS increased contractions to ET-1. Responses of vessels from mice deficient in eNOS to ET1 were similar to responses seen in vessels from wild type mice treated with L -NNA. Thus, with chronic deficiency of eNOS, no additional mechanisms appear to be activated to compensate for the absence of NO and alter vasoconstriction to ET-1. Third, contractions to the thromboxane mimetic U46619 at the lowest concentration were also increased by both acute inhibition of NOS and chronic deficiency of eNOS. Although acute and chronic absence of NOS increased responses to U46619, the effects were relatively more modest than that observed for ET-1 as maximal contractions were not affected. Lastly, there were no gender differences in the vasoconstrictor response to ET-1 or U46619 in aorta from normal mice. The absence of a gender difference in this study is in contrast to our previous study in which vasoconstrictor responses to serotonin and U46619 were greater in carotid artery from male compared to female mice [24]. Role of NO in modulating responses to ET-1 Previous studies from our laboratory [19] and others [25] have demonstrated that responses to ET-1 are dependent on the route of administration with constriction of vessels with abluminal administration and dilation when administered intravascularly. These data suggest that a diffusional barrier or release of endothelial-derived substances plays a major role in responses to ET-1. Subsequent studies have examined the role of endothelium and endothelium-derived vasodilator substances in modulating responses to ET-1. Removal of endothelium or inhibition of NOS and cyclooxygenase all augment contractile responses to ET-1 depending on the vascular bed or species [15–22]. Thus, a variety of substances including NO and cyclooxygenase products may modulate responses to ET-1. Previous studies suggest a reciprocal regulation of the expression of ET-1 and NO [6,7,26]. Several lines of evidence in endothelial cells in vitro and in vivo suggest that in the presence of reduced levels of NO message for and/or protein levels of ET-1 are increased [6,26]. These data would suggest that in pathological
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conditions with reduced production or bioactivity of NO, the role for ET-1 may be increased. In the present study, acute inhibition of NOS or chronic deficiency of eNOS produced similar increases in contractions to ET-1 in mouse aorta. These data suggest that eNOSderived NO may not regulate ET-1 gene expression in all models. Results of the present study provide direct evidence that eNOS-derived NO attenuates contractions to ET-1 in aorta. Although some previous studies suggest that NO can attenuate vasoconstriction to ET-1, the source of NO was not defined. Some lines of evidence suggest that all three isoforms of NOS are normally present in both endothelium and vascular muscle [4,8–12]. The contribution of NO from these three sources in modulating vascular responses is not clear. Although results from the present study do not rule out a contribution of NO from nNOS or iNOS in modulating contractions to ET-1 in aorta, the data suggest that NO from these sources probably has a minimal role in attenuating contractions to ET-1. This conclusion is based on the findings that acute but non-selective inhibition of NOS with L -NNA increases contractions to ET-1 similar to selective deletion of eNOS in genetically deficient mice and that NOS inhibition in eNOS)/) mice has no effect on responses to ET-1. In addition to concerns of specificity of pharmacological inhibitors of NOS, studies from Cohen and others suggest that pharmacological inhibitors of NOS do not always completely abolish release of NO from eNOS [13,14]. With acute inhibition of NOS, release of NO could still be detected. Thus, it is difficult to fully define the magnitude of the role of NO from eNOS in modulating vascular reactivity. Surprisingly, in the present study the magnitude of acute inhibition of NOS on contractions to ET-1 is similar to the shift in the response to ET-1 in vessels obtained from eNOS)/) mice. Role of NO in modulating responses to U46619 A role for NO in modulating responses to thromboxane A2 mimetic, U46619, has been variable [27–31]. Although studies suggest that NO can modulate contractile responses to U46619 in some vascular beds often the effects are modest [28,30]. Studies also suggest no role for NO in modulating contractions to U4619 [27,29,31]. In the present study, we chose to test responses to U46619 to serve as a NO-independent vasoconstrictor. However, NO played a modest role in attenuating contractions to U46619 in mouse aorta. Contractions at low concentrations of U46619 were increased with acute inhibition of NOS and in vessels obtained from eNOS)/) mice compared to controls. These findings in aorta are similar to our recent results in carotid artery from eNOS)/) mice [24]. Similar to
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responses with ET-1 the magnitude of the increased response to U46619 following acute inhibition of NOS was similar to the shift in responses of vessels obtained from eNOS)/) mice compared to responses of vessels obtained from eNOS+/+ mice. Thus, acute inhibition of NOS and chronic deficiency of eNOS produce similar effects on responses to two different vasoconstrictors. Compensatory mechanisms in regulation of vascular reactivity Lamping and others [32–37] have previously demonstrated that with chronic absence of eNOS, other vasodilator pathways may increase to compensate for the absence of NO from eNOS. In coronary arteries and the cerebral microcirculation, nNOS appears to upregulate in the face of eNOS deficiency [32,36,37]. In mesenteric arteries from eNOS)/) mice, cyclooxygenase and nNOS upregulate to maintain flow-mediated dilation at normal levels [33,34,38]. However, in aorta and carotid artery from eNOS)/) mice vasodilation to acetylcholine is absent [39,40], indicating no compensatory mechanisms are activated. In the present study, there was no apparent compensatory increase in other vasodilator pathways to maintain normal contractions to ET-1 or U46619. Thus, in contrast to other vascular beds in the mouse, compensatory mechanisms do not appear to upregulate to maintain normal responses of aorta. The role of NO in modulating vascular responses in disease Numerous studies have suggested that reduced NO production or bioactivity and impaired endotheliumdependent vascular responses are a hallmark of vascular disease. Animal models of atherosclerosis, hypertension, diabetes, aging, AlzheimerÕs, and heart failure have all demonstrated reduced NO mediated vascular responses. Perhaps more important than reduced vasodilator responses in these models is the potential for enhanced constrictor responses to a variety of circulating and/or locally produced substances including ET-1, serotonin, and angiotensin II. In atherosclerotic apoE deficient mice, activation of the ET system was demonstrated in addition to decreases in NO release. Levels of ET-1 protein and receptors in atherosclerotic aortic tissue were associated with reduced NO mediated vascular responses [41]. Treatment with ET-1 receptor antagonists reduced lesion size and intimal thickening in addition to restoring NO mediated vascular responses [41]. Although responses to ET-1 were not measured, other studies have demonstrated enhanced contractions to ET-1 in the presence of atherosclerosis [42]. The present study provides the first direct evidence that a decrease in expression of eNOS leads to enhanced vasoconstriction
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to ET-1. These data suggest that altered ET-1 responses may arise as a consequence of dysfunction in the NO system and highlight the intricate balance between ET-1 and NO. In summary, the present study suggests that vascular responses to ET-1 and U46619 in the mouse aorta are attenuated by NO derived from eNOS. The data from gene-targeted mice provide direct evidence that eNOS plays a major role in influencing responses to ET-1 and to a lesser degree responses to thromboxane. Both acute inhibition of NOS and selective deletion of the gene for eNOS enhanced contractile responses of the aorta in mice.
Acknowledgments This work was supported by grants from the National Institutes of Health (HL-39050, NS-24621 and HL-62984, HL-38901) and a grant from the American Heart Association. K.G.L. and F.M.F. are Established Investigators of the American Heart Association. We thank Xiuying Liu for her valuable technical assistance. We also acknowledge Curt Sigmund, Ph.D., and the University of Iowa Transgenic Core for genotyping mice used in these studies.
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