(Hypertension. 2000;35:231.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Increased Nitrovasodilator Sensitivity in Endothelial Nitric Oxide Synthase Knockout Mice
Role of Soluble Guanylyl Cyclase
From the Institut für Kardiovaskuläre Physiologie (R.P.B., F.-H.S.-W., M.A., A.M., R.B), Klinikum der J.W. Goethe-Universität, and the Institut für Herz- und Kreislaufphysiologie (A.G.), Heinrich-Heine Universität Düsseldorf, Germany.
Correspondence to Ralf P. Brandes, MD, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, 60596 Frankfurt/Main, Germany. E-mail R.Brandes{at}em.uni-frankfurt.de
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Abstract |
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Abstract—Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS-/-) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS-/- mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N
-Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS-/- but increased the sensitivity in WT mice so it was no longer different than that of eNOS-/-. Basal cGMP levels in aortic rings were significantly lower in eNOS-/- mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOS-/- mice than in WT mice. The maximal SNP-induced (10 µmol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS-/- mice than in WT mice. Incubation of aortic segments from WT mice with N-nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 µmol/L). The aortic expressions of the sGC 
1 and ß1 subunits in WT and eNOS-/- mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS-/- mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents.
Key Words: nitric oxide • mice • genes • vasodilator agents
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Introduction |
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Vascular relaxation responses elicited by nitrovasodilator agents and by endothelium-derived nitric oxide (NO) are mainly mediated via the activation of soluble guanylyl cyclase (sGC) and a subsequent increase in intracellular cGMP levels.1 Interactions between endogenous and exogenous NO have been reported to modulate vasodilatory responsiveness.2 3 4 5 Different mechanisms may underlie this phenomenon, such as the downregulation of sGC, sGC desensitization, or inhibition of the cGMP-dependent signal transduction cascade. In the present study, we investigated the mechanism by which endothelium-derived NO affects the nitrovasodilator-induced relaxation in wild-type (WT) mice and in endothelial NO synthase knockout mice (eNOS-/-).
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Methods |
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Animals and Tissue Preparations
WT c57 black b6 mice were purchased from Charles River, and eNOS-/- mice and age-matched control animals were obtained from the Department of Physiology at Heinrich Heine Universität Düsseldorf.6 Mice were housed under conditions that conformed with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication No. 85-23).
Mice were killed by cervical dislocation. The aorta was excised rapidly and freed of surrounding fat and connective tissue. The aortic arch was used for Western blot analysis, whereas the thoracic aorta was cut into rings for cGMP determination and organ chamber studies. For assessment of sGC activity, as many as 3 mouse aortas were pooled.
Organ Chamber Experiments
Aortic rings were mounted on stainless steel wires connected to force transducers and placed in individual organ chambers containing Krebs’ buffer of the following composition (in mmol/L): NaCl 119, KCl 4.7, CaCl2 1.6, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, EDTA 0.026, and glucose 12, gassed with 95% O2/5% CO2, pH 7.4, at 37°C. Diclofenac (10 µmol/L) was present in all experiments to inhibit prostaglandin synthesis. Passive tension was gradually increased to 1g. Each ring was challenged twice with K+-rich Krebs’ buffer. Precontraction was elicited with phenylephrine (0.01 to 1 µmol/L). Phenylephrine concentrations were adjusted to obtain a similar level of precontraction in each ring (
80% of initial KCl-induced contraction). When a stable contraction plateau was obtained, concentration-relaxation curves were performed in response to cumulatively increasing concentrations of various vasodilators in the presence or absence of N-nitro-L-arginine (L-NA; 300 µmol/L, applied 30 minutes before the experiments).7
Determination of Intracellular cGMP
Aortic rings were incubated in HEPES-modified Tyrode’s solution containing the phosphodiesterase inhibitor isobutylmethylxanthine (100 µmol/L). After 27 minutes, rings were stimulated with either solvent or sodium nitroprusside (SNP; 300 nmol/L or 10 µmol/L) for 3 minutes. Thereafter, rings were frozen in liquid nitrogen and homogenized in ice-cold 10% trichloracetic acid. cGMP was extracted with water-saturated diethylether, acetylated, and quantified via radioimmunoassay as described previously.8
Western Blot Analysis
Aortic segments were boiled for 10 minutes in 40 µL agitated Laemmli’s buffer,9 and the supernatants (35 µg protein) were subjected to SDS-PAGE and blotted onto nitrocellulose membranes as described previously.7 Proteins were detected with their respective antibodies linked with the appropriate horseradish peroxidase–coupled secondary antibody (Calbiochem).
Assessment of GC Activity in Aortic Protein Extracts
After preparation of the aorta, samples (at least 1 mouse aorta per data point) were incubated in Krebs’ buffer (37°C in 5% CO2) in the presence or absence of L-NA (300 µmol/L) and SNP (10 µmol/L). After 2 hours, the tissue was shock frozen and homogenized in liquid nitrogen, dissolved in 200 µL ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 7.0, 0.25 mol/L sucrose, 200 µmol/L EDTA, 10 mmol/L dithiothreitol, 2 mmol/L benzamidine, and 10 µg/mL leupeptin), and cleared through centrifugation (13 000g for 30 minutes). GC activity was assessed in the supernatant on the basis of the formation of [32P]cGMP as described previously.10 Briefly, aortic extracts (5 µg protein) were incubated at 37°C for 10 minutes in a Tris-HCl–buffered solution (30 mmol/L, pH 7.4, 100 µL) containing 50 µmol/L [-32P]GTP (0.2 µCi), 100 µmol/L unlabeled cGMP, 3 mmol/L MgCl2, 100 µg/mL bovine 
-globulin, 5 mmol/L creatine phosphate,100 µg/mL creatine phosphokinase (1 U), 3 mmol/L glutathione, 0.5 mmol/L isobutylmethylxanthine, and 0.5 mmol/L DTPA in the presence or absence of 100 µmol/L SNP. Reactions were stopped by the addition of 0.4 mL zinc acetate (120 mmol/L) and 0.5 mL sodium carbonate (120 mmol/L). After centrifugation (10 000g for 10 minutes) 0.95 mL supernatant was loaded onto acid alumina, and [32P]cGMP was isolated and determined as described previously.10
Materials
8-para-Chlorophenylthio-cGMP (8p-cpt-cGMP) was obtained from Biolog. The rabbit sGC ß1 subunit antibody was kindly provided by Dr Peter Yuen (Memphis, Tenn).11 The mouse monoclonal eNOS antibody was from Transduction Laboratories. The polyclonal 1-sGC antibody (SA-6735) was obtained through immunization of a rabbit with a keyhole limpet hemocyanin–conjugated peptide directed against a consensus sequence of human and rat 1-sGC [amino acids 634 to 648: H2N-CKD VED GNA NFL GKA S-CONH2] at Eurogentec. [-32P]GTP was purchased from NEN Life Science. All other chemicals were purchased from Sigma and were dissolved in water.
Statistical Analysis
All values are given as mean±SEM. Relaxations are expressed as percent deviation from the precontraction levels. The maximal relaxant effects (Emax) and the concentration of half-maximal relaxation (EC50) were calculated from individual concentration-response curves. The number of experiments refers to the number of animals. Statistical significance was tested with 2-way ANOVA for repeated measures, followed by Newman-Keuls test. Statistical significance was accepted at the 0.05 level of probability. Densitometry of blots was carried out with the PC version of NIH Image (Scion Corp).
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Results |
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Organ Chamber Studies
The endothelium-dependent vasodilator acetylcholine (10-9 to 10-5 mol/L) relaxed aortas from WT mice but not from eNOS-/- mice (Emax 86±4% versus 2±5%, P<0.0001). SNP (10-11 to 10-5 mol/L) induced complete relaxation of aortic rings from WT mice and eNOS-/- mice. The sensitivity to SNP was significantly greater in eNOS-/- mice than in WT mice. In rings from WT mice, the NOS inhibitor L-NA shifted the concentration-response curve to SNP to the left, whereas relaxations in eNOS-/- mice were unaffected. SNP-induced relaxations in aortic rings from WT mice and eNOS-/- mice in the presence of L-NA did not differ (Figure 1 and Table). Relaxations in response to the G-kinase I-activator 8p-cpt-cGMP were similar in aortas from WT mice and eNOS-/- mice. L-NA had no effect on 8p-cpt-cGMP–induced relaxation in either strain (Figure 2 and Table). Relaxations in response to the KATP channel opener cromakalim (10-9 to 10-5 mol/L) were slightly greater in preparations from WT mice than in preparations from eNOS-/- mice (n=14, P<0.02), but Emax and EC50 values were not significantly different (Figure 3 and Table).
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Western Blot Analysis
eNOS protein was detected in aortic tissue from WT mice but not from eNOS-/- mice. The expression of the sGC subunits 1 and ß1 was not significantly different in aortas from WT mice and eNOS-/- mice (P=0.8; Figure 4).
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cGMP Measurements
Basal cGMP levels were 50 times greater in aortic rings from WT mice than in rings from eNOS-/- mice (10.28±2.07 versus 0.20±0.06 pmol/mg cGMP; n=9). L-NA markedly reduced the cGMP levels in aortic rings from WT mice but had no effect in rings from eNOS-/- mice (0.47±0.12 versus 0.20±0.06 pmol/mg cGMP; n=9, P=NS; data not shown). SNP induced a concentration-dependent increase in cGMP levels in aortas from both strains. cGMP accumulation in response to SNP (300 nmol/L) was significantly greater in rings from eNOS-/- mice than in rings from WT mice. The maximal SNP-induced (10 µmol/L) increase in cGMP was comparable in both strains (Figure 5).
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sGC Activity
The SNP-stimulated (100 µmol/L) sGC activity in aortic homogenates was significantly greater in samples from eNOS-/- mice than in samples from WT mice (n=6). Incubation of intact aortas with L-NA (300 µmol/L) for 2 hours significantly increased the SNP-stimulated sGC activity (n=6; Figure 6A). This increase was prevented when SNP (10 µmol/L) was present during incubation with L-NA (Figure 6B).
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Discussion |
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In the present study, we demonstrate that aortic rings from mice lacking eNOS exhibit an increased sensitivity to nitrovasodilator agents but not to downstream targets of the cGMP pathway (G-kinase) and cGMP-unrelated vasodilator mechanisms, such as opening of KATP channels by cromakalim. A comparable effect could be observed after the application of an NOS inhibitor to aortas from WT mice. Although sGC protein expression was identical in aortas of WT and eNOS-/- mice, the latter exhibited an increased sensitivity of sGC to SNP in both intact aortas and aortic homogenates. Inhibition of NOS in aortas from WT mice increased the SNP-stimulated sGC activity in protein extracts, and this effect was prevented by concomitant incubation with SNP.
Numerous studies have demonstrated that the acute inhibition of NOS increases the sensitivity of arterial segments to NO donors,2 3 4 5 whereas the exposure to higher doses of exogenous NO has the opposite effect.12 Moreover, it has been reported that increasing the vascular generation of NO by inducing the inducible NOS13 or by overexpressing eNOS14 impairs relaxation to nitrovasodilator agents, whereas in eNOS-/- mice, the sensitivity to this group of vasodilators is increased.15 16
NO may alter the sGC-cGMP transduction cascade in several ways, affecting the expression as well as the activity of sGC and targets farther downstream. For example, it has been suggested that excessive amounts of NO decrease sGC expression,17 18 19 whereas in heart failure,20 a situation associated with increased scavenging of endothelial NO by superoxide anions, and in pregnancy,21 sGC expression was enhanced. Excessive amounts of NO have also been demonstrated to impair sGC activity,22 23 whereas the acute inhibition of endothelial NO formation may induce a supersensitivity of sGC to nitrovasodilator agents.3
In the present study, knockout of the eNOS gene had no effect on the expression of the sGC 1 and ß1 subunit proteins, which suggests that the amount of NO generated by eNOS is not sufficient to alter sGC expression and in this way affect nitrovasodilator sensitivity. eNOS-/- mice do not appear to have a higher sensitivity to relaxants in general, because contraction did not differ between the 2 strains (data not shown) and relaxations to the KATP channel opener cromakalim were even slightly greater in aortas from WT mice than in those from eNOS-/- mice.
We initially reported that NO has an inhibitory effect on relaxations induced by the G-kinase I activator 8-bromo-cGMP and that 8-bromo-cGMP inhibits NO-induced vasodilation by shifting the concentration-responses curve, probably as the result of the fact that the 2 vasodilators activate the same downstream target (G-kinase I).24 In the present study, endogenously formed NO had no effect on relaxations induced by 8p-cpt-cGMP, which was apparent by the lack of effect of L-NA on 8p-cpt-cGMP relaxations and the similarity of the concentration-response curves to this substance in aortas from WT and eNOS-/- mice. It is not known whether species differences or different properties of the 2 G-kinase I activators (8p-cpt-cGMP lacks the inhibitory effect on phosphodiesterases and the cAMP-like activity of 8-bromo-cGMP25 ) are responsible for this marked difference, but certainly alterations of the cGMP effector pathway are excluded as a cause for the increased nitrovasodilator sensitivity in eNOS-/-.
Because neither sGC expression nor the pathways downstream of sGC appear to be altered in eNOS-/- mice, an increased activity or sensitivity of sGC in eNOS-/- mice is likely to be the underlying mechanism. Indeed, SNP-stimulated sGC activity was significantly higher in eNOS-/- mice than in WT mice, and the accumulation of cGMP in aortic rings from eNOS-/- mice stimulated with SNP (300 nmol/L) was also higher than that in aortas from WT mice. This effect could be simulated in aortas from WT mice, because the inclusion of L-NA in the organ chamber experiments not only increased the relaxant sensitivity to SNP to a similar level as in eNOS-/- mice but also increased the SNP-stimulated sGC activity in aortic homogenates. SNP (10 µmol/L) prevented this effect of L-NA on the SNP-stimulated sGC activity and masked the differences in cGMP accumulation in intact aortic segments from both strains. It has been reported that the S-nitroso-N-acetyl-DL-penicillamine (another NO donor)-stimulated sGC activities in lungs from eNOS-/- and WT mice are identical.15 One possible explanation for this conflicting result is that the amount of NO produced from lung endothelial cells is likely to be very different from that from aortic endothelial cells because of the markedly different hemodynamic forces exerted on the cells. The fact that basal cGMP levels were 50-fold higher in WT mice than in eNOS-/- mice emphasizes that basal NO release from endothelial cells is substantial in the aorta of WT mice. It is perhaps important to note that lung homogenates usually contain blood contaminants that may interfere with the determination of SNP-stimulated sGC activity due to the scavenging of NO by the hemoglobin.
It was previously reported that acute inhibition of endothelial NO release results in a supersensitivity of sGC to NO in rat aorta, but the underlying molecular mechanism at the enzyme level has not been clarified.3 In another study, oxidation of the heme iron of sGC in rat aortic smooth muscle cells was suggested as a possible mechanism to explain the loss of NO responsiveness after short-term treatment with higher doses of exogenous NO.23 This hypothesis is consistent with our present observation that a relatively short incubation of mouse aorta with SNP prevented the L-NA–induced increase in SNP-stimulated sGC activity in aortic protein extracts. Although the oxidation of sGC heme iron (from ferrous to ferric) by the sGC inhibitors ODQ26 or NS202827 renders sGC insensitive to activation by NO, oxidation of the heme iron of sGC by NO has not yet been demonstrated. Therefore, it remains to be shown whether endothelium-derived NO desensitizes vascular sGC by partially oxidizing its heme iron and whether the absence of the continuous basal oxidation of sGC by NO account for the higher nitrovasodilator sensitivity of eNOS-/- mice. If this mechanism is operative in endothelium-intact aorta of WT mice (and other species), it is expected to be readily reversible, because exposure of the intact aorta to L-NA for 120 minutes was sufficient to resensitize sGC to NO. The presence of an endogenous mechanism that resensitizes oxidized sGC to NO is supported by the observation that NS2028-induced inhibition of nitrovasodilator-induced relaxation is reversible, although this compound oxidizes the heme moiety of sGC.27 Recently, indirect evidence for such a mechanism was provided in endothelium-denuded bovine coronary arteries, in which a flavin-dependent enzymatic process appears to sensitize oxidized sGC to NO.28
In summary, we demonstrated that the aorta of eNOS-/- mice exhibits a higher sensitivity to SNP than the aorta of WT mice. This effect can be attributed to an enhanced sensitivity, but not expression, of sGC and is due to the lack of endothelium-derived NO in these animals.
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Acknowledgments |
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This work was supported by a Young Investigator’s Grant from the Klinikum der J.W. Goethe-Universität to Dr Brandes and by the SFB 553, projects B1 and C3.
Received September 14, 1999; first decision October 12, 1999; accepted October 19, 1999.
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 L. M. Bevers, E. E. van Faassen, T. D. Vuong, Z. Ni, P. Boer, H. A. Koomans, B. Braam, N. D. Vaziri, and J. A. Joles Low albumin levels increase endothelial NO production and decrease vascular NO sensitivity Nephrol. Dial. Transplant., December 1, 2006; 21(12): 3443 - 3449. [Abstract] [Full Text] [PDF]
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C. L. Oltman, L. L. Richou, E. P. Davidson, L. J. Coppey, D. D. Lund, and M. A. Yorek Progression of coronary and mesenteric vascular dysfunction in Zucker obese and Zucker diabetic fatty rats Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1780 - H1787. [Abstract] [Full Text] [PDF]
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R. Dumitrascu, N. Weissmann, H. A. Ghofrani, E. Dony, K. Beuerlein, H. Schmidt, J.-P. Stasch, M. J. Gnoth, W. Seeger, F. Grimminger, et al. Activation of Soluble Guanylate Cyclase Reverses Experimental Pulmonary Hypertension and Vascular Remodeling Circulation, January 17, 2006; 113(2): 286 - 295. [Abstract] [Full Text] [PDF]
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S. Kloss, R. Srivastava, and A. Mulsch Down-Regulation of Soluble Guanylyl Cyclase Expression by Cyclic AMP Is Mediated by mRNA-Stabilizing Protein HuR Mol. Pharmacol., June 1, 2004; 65(6): 1440 - 1451. [Abstract] [Full Text] [PDF]
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C. G. Kevil, A. W. Orr, W. Langston, K. Mickett, J. Murphy-Ullrich, R. P. Patel, D. F. Kucik, and D. C. Bullard Intercellular Adhesion Molecule-1 (ICAM-1) Regulates Endothelial Cell Motility through a Nitric Oxide-dependent Pathway J. Biol. Chem., April 30, 2004; 279(18): 19230 - 19238. [Abstract] [Full Text] [PDF]
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K. Sabrane, S. Gambaryan, R. P. Brandes, R. Holtwick, M. Voss, and M. Kuhn Increased Sensitivity to Endothelial Nitric Oxide (NO) Contributes to Arterial Normotension in Mice with Vascular Smooth Muscle-selective Deletion of the Atrial Natriuretic Peptide (ANP) Receptor J. Biol. Chem., May 9, 2003; 278(20): 17963 - 17968. [Abstract] [Full Text] [PDF]
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P. A. Ortiz and J. L. Garvin Cardiovascular and renal control in NOS-deficient mouse models Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2003; 284(3): R628 - R638. [Abstract] [Full Text] [PDF]
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A. Godecke, M. Ziegler, Z. Ding, and J. Schrader Endothelial dysfunction of coronary resistance vessels in apoE-/- mice involves NO but not prostacyclin-dependent mechanisms Cardiovasc Res, January 1, 2002; 53(1): 253 - 262. [Abstract] [Full Text] [PDF]
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K. Schmidt, P. Andrew, A. Schrammel, K. Groschner, V. Schmitz, G. Kojda, and B. Mayer Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2053 - H2061. [Abstract] [Full Text] [PDF]
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J. M. STULAK, A. LERMAN, M. R. PORCEL, J. A. CACCITOLO, J. C. ROMERO, H. V. SCHAFF, C. NAPOLI, and L. O. LERMAN Renal Vascular Function in Hypercholesterolemia Is Preserved by Chronic Antioxidant Supplementation J. Am. Soc. Nephrol., September 1, 2001; 12(9): 1882 - 1891. [Abstract] [Full Text] [PDF]
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D. Li, V. E. Laubach, and R. A. Johns Upregulation of lung soluble guanylate cyclase during chronic hypoxia is prevented by deletion of eNOS Am J Physiol Lung Cell Mol Physiol, August 1, 2001; 281(2): L369 - L376. [Abstract] [Full Text] [PDF]
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M. B. Hussain, R. J. MacAllister, and A. J. Hobbs Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1151 - H1159. [Abstract] [Full Text] [PDF]
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