Smooth Muscle–Specific Deletion of Nitric Oxide–Sensitive Guanylyl Cyclase Is Sufficient to Induce Hypertension in Mice
Background— Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (α and β). Deletion of the β1 subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure.
Methods and Results— To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle–specific knockout mice for the β1 subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks.
Conclusions— We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.
Received July 1, 2009; accepted November 13, 2009.
Arterial hypertension is a condition of chronically elevated blood pressure (BP). Untreated hypertension may lead to damage of end organs such as brain, kidney, or heart and therefore represents a major risk factor for several different diseases, including stroke and myocardial infarction.1 However, the precise mechanisms by which the body controls BP as well as those leading to the pathogenic development of hypertension are still incompletely understood.
Clinical Perspective on p 409
One of the prominent factors for the regulation of vascular tone is nitric oxide (NO). NO is produced in the endothelium by NO synthases (NOS) and acts mainly on NO-sensitive guanylyl cyclase (NO-GC), which is located in medial smooth muscle cells.2 The signal transduction induced by NO is performed intracellularly by the second messenger cyclic guanosine monophosphate (cGMP), which is synthesized by NO-GC. cGMP exerts its effects via cGMP-dependent kinases, channels, or phosphodiesterases.3–5 In vascular smooth muscle, NO and thus cGMP are known to induce relaxation via stimulation of protein kinase G (PKG) with subsequent phosphorylation of several substrate proteins. Global deletion or truncation of some of these substrate proteins in the mouse was shown to cause hypertension (eg, the BK channel,6,7 RGS2,8 or IRAG).9
To gain further insight into the physiological role of NO, we have generated mice deficient in the β1 subunit of NO-GC (GCKO).10 These mice lack NO-GC globally and suffer from an increased systolic BP (SBP), which strongly emphasizes the role of NO and cGMP with regard to the constitutive regulation of vessel tone. However, elevated BP in these mice not only may result from the lack of NO-GC in the vasculature but also may be explained by mechanisms based on the GC deficiency in other organs such as the kidney or the central nervous system.11–14 This notion is corroborated by the fact that GCKO mice, in addition to hypertension, suffer from various phenotypes, including gastrointestinal dysmotility and platelet dysfunction.
We therefore generated a mouse line in which NO-GC can be specifically deleted in smooth muscle cells (smooth muscle NO-GC knockout [SM-GCKO]) to supply more detailed information on the role of NO and cGMP with regard to vessel tone and thus BP regulation. Analysis of these mice provided evidence that the postnatal deletion of NO-GC in smooth muscle cells is sufficient to cause elevation of BP. Our results emphasize the central role of NO as a signaling molecule to constitutively reduce BP via NO-GC in smooth muscle cells and furthermore indicate that elevated BP can be the result of single gene deficiency in smooth muscle cells disturbing vascular relaxation.
All experiments were conducted in accordance with the German legislation on protection of animals and approved by the local animal care committee.
Generation of SM-GCKO Mice
For the generation of smooth muscle–specific KO mice for NO-GC (SM-GCKO), we used our previously produced mouse strain in which exon 10 of the β1 subunit of NO-GC was floxed (β1flox/flox).10 Beta1flox/flox mice backcrossed on C57BL/6 background for >10 generations were crossed with the inducible smooth muscle–specific Cre transgenic mouse line (SMMHC-CreERT2) backcrossed for 7 generations. This mouse line did not show any Cre activity in the absence of tamoxifen, as shown after crossing with the Gt(ROSA)26Sor Cre reporter mouse line.15
Mice strains used were β1WT/WT/SMMHC-CreX/Y+ (=wild-type [WT]) and β1flox/flox/SMMHC-CreX/Y+ (=SM-GCKO). Only for BP measurements were heterozygous mice β1WT/flox/SMMHC-CreX/Y+ also included.
Male mice (WT and SM-GCKO) aged 6 to 8 weeks were injected with tamoxifen (1 mg IP) on 5 consecutive days. The last day of tamoxifen injection was defined as day zero. Mice were euthanized and investigated at the time points indicated in Figure 1.
The SMMHC-Cre gene is located on the Y chromosome in this mouse line, and only male offspring carried the SMMHC-Cre gene; thus, in our study, we characterized only male mice. In each experiment, WT littermates were used wherever feasible.
Genotyping of Mice
Genotyping was performed by polymerase chain reaction (PCR) analysis of tail DNA as described previously.10 Amplification of the SMMHC-Cre gene was performed in another multiplex PCR with the primers SMWT1 (TGA CCC CAT CTC TTC ACT CC), SMWT2 (AAC TCC ACG ACC ACC TCA TC), and phCREAS1 (AGT CCC TCA CAT CCT CAG GTT).
Analysis of Murine Tissues
The preparations of brain tissue, platelets, and aorta, as well as Western blot analysis, determination of GC activity, and cGMP radioimmunoassay, were performed as described.15
Mice were killed by inhalation of an overdose of isoflurane, and tissue was snap-frozen with the use of melting isopentane; alternatively, mice were fixed by transcardiac perfusion with 3% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4, and tissue was cryoprotected with the use of 20% sucrose and snap-frozen. Cryosections (10 μm) were cut, air-dried, and incubated overnight with an antibody against the β1 subunit of NO-GC raised in rabbit (1:800 in Antibody Diluent, Invitrogen, Karlsruhe, Germany)10 together with a FITC-labeled mouse anti-α-smooth muscle actin antibody (1:500, clone 1A4, Sigma-Aldrich, Munich, Germany). The rabbit antibody was detected by incubating the sections with an Alexa 555–conjugated antirabbit IgG antibody raised in donkey (Invitrogen; 1:800 in Antibody Diluent) for 1 hour. The sections were mounted in Mowiol and were evaluated with the use of an epifluorescence microscope equipped with appropriate filter sets for Alexa 555 and FITC.
Organ Bath Experiments
Thoracic aortas were cut into rings and mounted in a myograph as described previously.10 If not indicated otherwise, diclofenac (3 μmol/L) and N-nitro-l-arginine methyl ester (L-NAME) (200 μmol/L) were present. Resting tension was set to 5 mN. After equilibration (at least 60 minutes at 37°C), rings were precontracted with phenylephrine (1 μmol/L) or U46619 (10 nmol/L) and relaxed with the following compounds: DEA-NO (Alexis), sildenafil (generous gift from Pfizer, Kent, UK), 8-Br-cGMP (Biolog, Bremen, Germany), or carbachol. Isobutyl methylxanthine (IBMX) (100 μmol/L) was used to induce maximal relaxation. Each experiment was performed in parallel with 2 to 4 aortic rings derived from SM-GCKO and WT mice.
Measurement of BP
SBP was measured in conscious mice by tail-cuff plethysmography (Softron BP-98A, Tokyo, Japan). Measurements of BP in conscious mice were taken on days indicated in Figure 1 and corresponded to n=2 to 8 animals for each genotype and day. Measurement of the L-NAME–induced changes in BP were performed with n=5 mice per genotype.
If not stated otherwise, data are expressed as mean±SEM (n=number of animals). For calculation of statistical tests, SPSS 15.01 (SPSS Inc, Chicago, Ill) was used. For comparison of independent variables, all groups were compared by the Kruskal-Wallis test. If P≤0.05 for the global test, 2 groups were compared by Mann-Whitney U test in a predefined sequence, and comparison was stopped if P>0.05 to prevent α inflation.
For dependent variables, all groups were compared by the Friedman test, and individual groups were compared by the Wilcoxon test. Comparison was stopped if P>0.05 to prevent α inflation. Comparisons of individual groups were only reported if global tests reached significance.
Generation of SM-GCKO Mice
For the generation of smooth muscle–specific KO mice for NO-GC (SM-GCKO), we used our previously produced mouse strain in which exon 10 of the β1 subunit of NO-GC was floxed (β1flox/flox).10 β1flox/flox mice were crossed with a mouse line expressing a fusion protein of Cre recombinase with a modified estrogen receptor binding domain (CreERT2) under the control of the smooth muscle–specific smooth muscle myosin heavy chain promoter (SMMHC).16 In these mice, Cre-mediated recombination was observed to occur exclusively in smooth muscle cells. Smooth muscle–specific deletion of the β1 subunit and thus of NO-GC10 was performed as shown in the time scheme in Figure 1: Male mice (6 to 8 weeks old) were treated with tamoxifen (1 mg IP) on 5 consecutive days. The last day of tamoxifen injection was defined as day 0; mice were analyzed on days 5, 10, 14, 20, 32, 53, and 72. As controls, we used WT and SM-GCKO mice not receiving tamoxifen (WTpre and SM-GCKOpre animals) as well as tamoxifen-injected WT animals. All control animals carried the Cre transgene (ie, all WT and SM-GCKOpre animals).
Immunohistochemical Analysis of SM-GCKO Tissues
The successful smooth muscle–specific deletion of NO-GC was shown by immunohistochemistry (Figure 2). Smooth muscle in large and small vessels (aorta, femoral artery and vein, myocardial arterioles) was identified with the use of an antibody specific for smooth muscle actin (green staining) in WT and SM-GCKO animals (tamoxifen >50 days). The expression of NO-GC was detected with the use of an antibody specific for the β1 subunit.10 With this antibody, strong NO-GC signals (red staining) were obtained in WT smooth muscle, whereas no NO-GC–specific signal was detected in smooth muscle from SM-GCKO>50 days animals. NO-GC expression in brain (WTpre versus SM-GCKOpre versus WT>50 days versus SM-GCKO>50 days, P=0.898, Kruskal-Wallis test) and platelets (α1, α2, and β1 subunits) was not affected by the smooth muscle–specific KO (Figure 3A and 3B; Figures I and II in the online-only Data Supplement), as shown by Western blotting analysis.
Deletion of NO-GC and NO-Stimulated cGMP Production Over Time
Next we studied the deletion of NO-GC in aortic smooth muscle over time. Homogenates were prepared from the aorta of mice euthanized according to the time scheme in Figure 1. Aortic protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting with the use of antibodies against the α1 and β1 subunits of NO-GC. Extracellular signal–regulated kinase (p42/p44 Erk) was used as loading control. The representative blot in Figure 4A clearly demonstrates the time-dependent loss of NO-GC in SM-GCKO aorta; the quantitative analysis is shown in Figure 4B. NO-GC was reduced >75% 5 days after the last tamoxifen injection (P=0.01, Mann-Whitney U test) and was further reduced with time (SM-GCKO 5 days after tamoxifen injection versus SM-GCKO >50 days after tamoxifen injection, P=0.016, Mann-Whitney U test). Semiquantitative PCR for the β1 subunit showed an ≈90% reduction in mRNA expression 5 days after tamoxifen injection, which parallels the reduction in protein (Figure V in the online-only Data Supplement). In contrast, NO-GC expression in WT aorta was not affected by the tamoxifen treatment. Figure 4C shows that concomitant to the reduction in NO-GC expression, DEA-NO–stimulated cGMP production declined with similar kinetics (SM-GCKOpre versus SM-GCKO 5 days after last tamoxifen injection, P=0.001, Mann-Whitney U test).
Time-Dependent Loss of NO-Induced Smooth Muscle Relaxation
Next we monitored the functional effects of time-dependent NO-GC loss in organ bath experiments. NO-dependent aortic relaxation did not differ between WTpre mice and tamoxifen-injected animals as well as heterozygous animals (untreated animals versus animals 20 days after tamoxifen injection versus 50 days after tamoxifen injection, P>0.3 for every concentration, Kruskal-Wallis test; Figure 5A; original tracings can be found in Figure III in the online-only Data Supplement), indicating that the tamoxifen treatment by itself did not influence NO signaling. The relaxing response of aorta from SM-GCKOpre animals was similar to that seen in WT or heterozygous animals (pre and tamoxifen-injected). However, in contrast to WT and heterozygous animals, aortas from tamoxifen-injected SM-GCKO mice showed a time-dependent decrease in NO responsiveness; surprisingly, not until >50 days after tamoxifen treatment did we see a total loss of NO-mediated relaxation (Figure 5B; comparison for all time points, P<0.05 from 10 to 0.001 μmol/L DEA-NO, Kruskal-Wallis test). As a consequence, we considered SM-GCKO >50 days as complete SM-GCKO mice, and the majority of the experiments were performed with the use of mice >50 days after tamoxifen treatment (Figures 2, 3, 6, 7, and 8⇑⇓⇓⇓). In conclusion, time-dependent deletion of NO-GC in aortic smooth muscle cells induces a gradual reduction of NO sensitivity that depended on the time after tamoxifen injection and finally causes a complete loss of NO responsiveness.
Time-Dependent Loss of Endothelium-Derived Relaxing Factor–Induced Relaxation
Because deletion of NO-GC in aortic smooth muscle is paralleled by a reduced relaxation from pharmacological NO, we next studied the response toward endogenously produced NO. Carbachol was applied at a maximally effective concentration (30 μmol/L) to aortic rings precontracted with U46619 (10 nmol/L) in the absence of L-NAME and diclofenac. In aortic rings from WTpre and WT>50 days animals, carbachol led to an almost 70% relaxation (Figure 6A and 6B). Although SM-GCKOpre rings were relaxed by carbachol similar to WT and WT>50 days rings (P=0.804, Kruskal-Wallis test), this relaxation was totally abolished in rings from SM-GCKO>50 days animals (P=0.016, SM-GCKOpre versus SM-GCKO>50 days animals, Mann-Whitney U test; Figure 6A and 6B).
Figure 6C compares the time-dependent reduction in GC expression to the time-dependent loss of NO-induced relaxation (pharmacological NO and endothelium-derived NO). The relaxation induced by a maximally effective carbachol concentration equaled that elicited by 0.1 to 1 μmol/L DEA-NO; thus, DEA-NO concentrations >1 μmol/L may be supraphysiological, at least compared with the carbachol-induced NO production. Surprisingly, GC reduction clearly preceded the reduction of NO responsiveness: Reduction of GC protein by almost 75% led to only a slight loss of NO response, as seen on day 5, indicating that activation of a minor fraction of GC molecules in the smooth muscle cell is sufficient to elicit a full NO response.
Relaxing Effect of Phosphodiesterase 5 Inhibition
Sildenafil is a specific inhibitor of phosphodiesterase 5, which is the main cGMP-degrading enzyme in smooth muscle.5 To test the effect of sildenafil, we administered increasing concentrations to aortic rings precontracted with U46619 (10 nmol/L; original tracing in Figure 7A and quantitative analysis in Figure 7B) in the absence of L-NAME and diclofenac. Aortas from WT>50 days animals relaxed on addition of sildenafil. Similar responses were seen in WTpre and SM-GCKOpre aortas (not shown). In contrast, phosphodiesterase 5 inhibition by sildenafil did not induce relaxation in aorta from SM-GCKO>50 days mice; only at very high concentrations (10 μmol/L) was relaxation observed, which is probably due to nonspecific inhibition of a phosphodiesterase other than phosphodiesterase 5 (comparison SM-GCKO>50 days versus WT>50 days for all time points, P<0.05 from 10 000 to 1 nmol/L, Mann-Whitney U test).
The intactness of the NO/GC/PKG cascade downstream from GC was investigated with the use of the membrane-permeable activator of PKG, 8-Br-cGMP (Figure 7C and 7D). This cGMP analogue led to similar relaxation of precontracted aortic smooth muscle from WTpre, SM-GCKOpre, WT>50 days, and SM-GCKO>50 days animals (P=0.392, Kruskal-Wallis test), demonstrating undisturbed signal transduction by PKG and beyond. Administration of a NO donor subsequent to 8-Br-cGMP administration did not lead to additional relaxation in the SM-GCKO>50 days aorta (Figure 7C).
To prove that endogenously produced cGMP is generally capable of relaxing smooth muscle even in the absence of NO-GC in SM-GCKO aortas, we administered atrial natriuretic peptide to stimulate cGMP production from the membrane-bound guanylyl cyclase GC-A (Figure 7E). There was no difference in atrial natriuretic peptide–induced relaxation of phenylephrine-precontracted aorta between WTpre, SM-GCKOpre, WT>50 days, and SM-GCKO>50 days animals (P=0.882, Kruskal-Wallis test). Thus, cGMP is in fact capable of relaxing aortic smooth muscle even in SM-GCKO animals. In addition, the unchanged response toward atrial natriuretic peptide indicates that no quantitative compensation takes place on the level of GC-A in SM-GCKO mice.
Time-Dependent Development of Hypertension
Next we investigated whether the gradual loss of NO-induced aortic relaxation in SM-GCKO mice relates to a change in BP. WT and heterozygous mice did not show an alteration in SBP after tamoxifen injection (Figure 8A). Thus, tamoxifen has no long-term influence on SBP. However, in SM-GCKO mice, tamoxifen injection led to an increase in SBP. A first elevation of SBP, compared with WT or SM-GCKOpre animals, was detected 10 days after the last tamoxifen injection. Maximal SBP levels (30 mm Hg above that seen in SM-GCKOpre mice) were reached after ≈30 days (differences between WT and SM-GCKO mice were statistically significant at days 27, 36, and 46 [P<0.01, Mann-Whitney U test] and were not tested for the other days because n <4 observations per time point were made). Hypertension in SM-GCKO mice is long-lasting because even 150 days after tamoxifen treatment, SBP remained elevated (30 mm Hg; data not shown). These data clearly show that the gradual reduction of NO-GC in aortic smooth muscle results in steady elevation of SBP with no compensation taking place.
Finally, we tested whether blocking NOS activity in vivo still had an effect on the elevated SBP in SM-GCKO mice. Expression of endothelial NOS was similar in WT and SM-GCKO mice (Figure VI in the online-only Data Supplement). L-NAME (50 mg/mL) was administered to WT>50 days and SM-GCKO>50 days mice via drinking water (Figure 8B). After 1 day, a pronounced increase in SBP in WT animals (20 mm Hg) was detected (day 0 versus day 1, P=0.043, Wilcoxon test). BP stayed elevated for at least 5 days with continuous L-NAME supply. In contrast, no change in SBP was detected in SM-GCKO>50 days mice on L-NAME exposure (day 0 versus day 1 versus day 3 versus day 5, P=0.178, Friedman test), demonstrating that endogenous NO only affects BP in the presence of smooth muscle NO-GC.
Hypertension is one of the most prevailing risk factors in the Western world and is responsible for a variety of cardiovascular diseases such as coronary heart disease and heart failure. Many different mechanisms are held responsible for the development of elevated BP.12,17 The NO/cGMP signaling cascade is known to regulate BP. However, to date, it is not clear whether this regulation takes place primarily via direct action of NO on vascular smooth muscle tone or via effects on the kidney or in the central nervous system.
To explore the importance of this cascade relative to the regulation of BP, several genetic mouse models have been generated that, interestingly, all show increased BP. These include KO mice for endothelial NOS,18–20 NO-GC10,21,22 PKG,23 BK channel,6 and the knockin model of a leucine zipper mutant of PKG.24 In addition, the pharmacological inhibition of NOS with the use of L-NAME or similar inhibitors is known to cause increases in BP. Although these models clearly show the involvement of the NO/cGMP signaling cascade in the regulation of BP, they fail to identify the cell type in which abrogation of this signaling pathway leads to elevated BP.
We have previously shown that mice lacking NO-GC reveal a prominent increase in SBP concomitant with a totally abolished NO responsiveness of vascular and gastrointestinal smooth muscle. Our data indicate a dysregulation of smooth muscle as primary cause for the BP elevation. However, global deletion of NO-GC does not allow assessment of the physiological importance of the enzyme in a single individual cell type. We therefore asked next whether the lack of NO-GC in smooth muscle cells alone would be sufficient to explain the BP increase seen in total KO animals or whether NO-GC in other cell types contributes to the regulation of BP. Indeed, smooth muscle–specific KO mice for NO-GC (SM-GCKO) showed an increase in SBP identical to that seen in general GCKO mice (+30 mm Hg). Thus, lack of NO effect on smooth muscle cells is a simple explanation for the hypertension in our mice.
In our inducible model, tamoxifen induced a time-dependent loss of NO-induced aortic smooth muscle relaxation. Aortic NO responsiveness was inversely related to BP, as shown in Figure IV in the online-only Data Supplement. SBP became noticeably elevated around day 10 and increased further until reaching maximal values after ≈30 days. Aortic NO response was significantly diminished at day 10 and was almost abolished 30 to 50 days after tamoxifen. Interestingly, GC expression and cGMP production were reduced comparatively earlier than NO responsiveness in aortic rings after tamoxifen injection (Figure 6C). For example, 5 days after the last tamoxifen dose, GC expression as well as NO-induced cGMP synthesis was reduced by almost 75%, with only minor reduction in the NO responsiveness of the aortic rings. This “gap” indicates that smooth muscle cells can suffer from a loss of 80% to 90% of NO-GC without showing hypertension and a major reduction in the NO response. This indicates a relative “overexpression” of NO-GC in smooth muscle: Expression of high amounts of NO-GC increases the sensitivity of the cells toward NO released by the endothelium; consequently, reduction in NO-GC expression initially does not impair physiological regulation except for the fact that higher NO concentrations are needed to induce relaxation. These data corroborate the concept of NO-GC as spare receptor published previously by our group.18
In comparison to other proteins, the reduction of GC expression after induction is surprisingly slow. From the Western blot data in Figure 4B, a half-life of NO-GC of ≈3 to 4 days can be calculated, indicating a slow turnover of NO-GC on the protein and/or on the mRNA level. The slow decrease in NO-GC and NO-mediated effects is not based on a slow action of the Cre recombinase because the use of exactly this SMMHC-Cre mouse was shown to result in a fast smooth muscle–specific elimination of the G proteins Gαq/Gα11 and a decrease in mean arterial BP by ≈10 to 15 mm Hg within 3 days.16 The slow onset of hypertension and reduced aortic NO responsiveness in our mice indicates a comparatively long half-life of NO-GC. Consequently, short-term regulation of NO-GC expression (and thus NO-GC–mediated cellular effects) is unlikely to occur in vivo, at least in smooth muscle cells.
Injection with tamoxifen led to a decrease in the β1 as well as the α1 subunit (Figure 4A). The reduction of the dimerizing partner α1 appears surprising. However, earlier in vitro studies in our laboratory indicated that single subunits are not stable and are rapidly degraded in the absence of the respective dimerizing subunit. Furthermore, we recently showed that deletion of either the α1 or α2 subunit in the mouse led to the reduction of the amount of β1 subunit similar to that of the respective deleted α subunit.18 Although the genes of the α1 and β1 subunits are localized in line on chromosome 3 in the mouse, the α2 subunit is found on chromosome 9, which makes posttranscriptional regulation of subunit expression likely.
To test whether endogenous NO still participates in the regulation of BP in the absence of NO-GC, we used L-NAME to inhibit NOS. In WT animals, SBP increased from basal levels of 103±1 to 120±2 mm Hg within 1 day on L-NAME administration. In contrast, L-NAME did not influence SBP in SM-GCKO animals (before L-NAME, 133±1 mm Hg; 1 day L-NAME, 133±2 mm Hg). Two important conclusions can be drawn from this experiment. First, endogenous NO appears not to act via other BP-modulating effectors apart from NO-GC in vivo. Second, NO-GC in tissues other than smooth muscle does not directly govern SBP because the hypertension seen in SM-GCKO mice is not augmented by NOS inhibition. This conclusion is corroborated by the fact that GCKO mice (in which NO-GC is knocked out globally) reveal an increase in SBP identical to that seen in SM-GCKO animals. Interestingly, SBP in WT fed with L-NAME did not reach the levels seen in animals lacking NO-GC. This discrepancy may be explained by several factors. First, although L-NAME is known to be an effective inhibitor of NOS, it may not completely inhibit NO-mediated signaling, leaving some residual NO to be produced. As spare receptor, NO-GC is known to be extremely sensitive toward NO; thus, even very low NO concentrations may be perceived and signaled further toward the respecting cGMP effectors. Second, other NO sources such as nitrites/nitrates may still act on NO-GC under NOS-inhibiting conditions.25,26 Third, blockade of NOS with the use of L-NAME reduces the NO-stimulated cGMP synthesis in smooth muscle cells. However, NO-GC is known to catalyze a basal cGMP turnover. This basal cGMP production can be expected to still occur in the L-NAME–treated mice, whereas in the SM-GCKO mice, in which NO-GC is globally deleted, cGMP production from this enzyme is fully abolished. Conceivably, nonstimulated cGMP production still negatively affects vessel tone in L-NAME–treated WT mice; however, the physiological importance of the nonstimulated cGMP synthesis of the enzyme is still not clear to date.
The inherent problem of a constitutive KO mouse is the lack of the respective protein during the animals’ ontogeny. During the development of the embryo, compensatory mechanisms may veil or counteract the impact of the deletion. The use of an inducible system allows deleting NO-GC at stages of finished development. Long-term compensatory mechanisms are unlikely to occur, and these animals very likely reveal the unperturbed phenotype of the KO. Because the BP phenotype was identical in both general (GCKO) and inducible (SM-GCKO) mouse models, we conclude that deletion of NO-GC in smooth muscle cells is the common and sole mechanism for the development of hypertension.
The excellent technical help of Ulla Krabbe and Friedrich Eichhorst is acknowledged.
Sources of Funding
The work was supported by Deutsche Forschungsgemeinschaft (FR 1725), Deutsche Forschungsgemeinschaft Excellence Cluster ‘Inflammation at Interfaces,’ Forschungsförderung der Ruhr-Universität Bochum (FoRUM; F617-2008), and Medizinische Fakultät der Universität zu Lübeck (A31-2007).
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Hypertension is one of the most prevalent risk factors in the Western world and contributes to a variety of cardiovascular diseases, including coronary heart disease or heart failure. Many different mechanisms have been suggested to contribute to the development of elevated blood pressure. One model to explain the development of hypertension is based on intrinsic changes in the kidney leading to altered sodium handling, volume expansion, and elevated vascular resistance. However, increased vascular resistance may also be the consequence of changes in vessel tone and morphology. Only a few studies have investigated the possibility that hypertension may result solely from changes in the vascular system. Vessel tone and thus blood pressure are known to be regulated by the nitric oxide (NO)/cGMP signaling cascade, in part through shear-mediated production of NO in the endothelium and subsequent activation of NO-sensitive guanylyl cyclase (NO-GC) to induce vessel relaxation. We have previously shown that mice lacking NO-GC are hypertensive; however, global deletion of NO-GC in mice does not allow for the identification of the cell and/or tissue type responsible for the resultant elevated blood pressure. In this study, we developed a mouse model in which NO-GC was specifically deleted in smooth muscle cells of adult mice using a tamoxifen-inducible system. Here, we show that these animals gradually develop hypertension within 3 to 4 weeks that is paralleled by a loss of NO sensitivity of smooth muscle cells. In summary, our data provide evidence that deletion of NO-GC solely in vascular smooth muscle is sufficient to cause hypertension.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.890962/DC1.