Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system (original) (raw)
The 2 α subunits of GC are encoded by different genes. To generate the KO mice, we used Cre/loxP-mediated recombination to delete exon 4 of either the α1 or α2 subunit, respectively (Figure 1). Homozygous α1-KO and α2-KO mice were born at normal Mendelian ratio and did not show apparent alterations or a reduced life expectancy. To confirm the loss of the α1 or α2 subunit, we performed Western blot analysis using subunit-specific antibodies. As can be seen in Figure 2A, the proteins were completely absent in lung and brain of the KO mice. WT-like amounts of the other, nondeleted α subunit were determined in the respective KO animals, indicating that deletion of 1 α subunit is not compensated by upregulation of the other. Interestingly, the loss of the α subunit was accompanied by a decrease in the β subunit content, indicating that the β subunit was not stable without its dimerizing partner. The observed reduction of the β subunit revealed similar expression of α1 and α2 isoforms in brain as well as the dominance of α1-GC in lung.
Gene inactivation of α subunits of NO-sensitive GC. Targeting strategies for α1 (A) or α2 (B) subunit of NO-sensitive GC. Arrowheads, loxP sites; Neo, neomycin resistance; TK, thymidine kinase; DT, diphtheria toxin gene; WT, wild-type loci of the α subunits; filled rectangles, exons, numbered from the first coding exon. B, BamHI; Bl, BglII; EI, EcoRI; EV, EcoRV; N, NheI; S, SpeI; Xb, XbaI, Xh, XhoI. Brackets indicate destroyed restriction sites; newly created sites are in bold. Genotyping of α1 (C) or the α2 (D) mouse line was performed with PCR analysis as described in Methods.
Analysis of GC isoform content in α-KO mice. (A) GC subunit content in brain and lung homogenates analyzed with antibodies against the different subunits (α1, α2, and β1) and quantified with respect to the subunit amount in WT lung (100%; n = 4 of each genotype). (B) Cyclic GMP-forming activity in brain and lung homogenates in the presence of DEA-NO (100 μM) was determined as outlined in detail in Methods (n = 5 of each genotype).
The NO-dependent activity in α1- and α2-KO mice represents the amount of the respective nondeleted GC isoform in a tissue. Thus, we measured NO-stimulated cGMP formation in lung and brain of KO and WT animals (Figure 2B). NO-dependent GC activities in brain amounted to 1.4 ± 0.1 nmol/mg/min in WT and were reduced by about 50% in α1- and α2-deficient animals (0.5 ± 0.1 and 0.9 ± 0.2 nmol/mg/min, respectively). In lung, WT-like activity was measured in α2-KO (4.5 ± 0.7 nmol/mg/min) whereas GC activity in α1-KO was greatly reduced (7% of WT). These findings are in agreement with the Western blot results and confirm α1-GC as the major isoform in lung and similar expression of both isoforms in brain.
In platelets, NO inhibits aggregation via the stimulation of NO-sensitive GC and the subsequent increase in cGMP. Platelets from α2-KO mice showed WT-like behavior; i.e., collagen-induced aggregation was completely inhibited by NO (Figure 3A). In contrast, α1-deficient platelets did not respond to NO at all. Accordingly, in Western blots, only the α1 subunit was detected in WT platelets (Figure 3B) whereas the α2 subunit was undetectable (data not shown), indicating that α2-GC does not exist in this cell type. These results demonstrate that inhibition of platelet aggregation by NO is solely mediated by α1-GC.
Analysis of α-deficient platelets. (A) Aggregation of α-deficient platelets was induced with collagen (4 μg/μl) in the absence or presence of DEA-NO (100 μM). Shown are aggregometric traces. (B) To identify the GC isoform expressed in platelets, Western blot analysis of platelet homogenates from WT, α1-KO, and α2-KO mice was performed.
To assess the physiological impact of the GC isoforms on vascular tone, we studied NO-induced relaxation of aortic rings of α1- and α2-deficient mice. Concentration-response curves for the NO donor S-nitrosoglutathione (GSNO) revealed WT-like behavior of α2-deficient aortic rings (half-maximal effective concentration [EC50] ≅ 0.4 μM), underlining the predominant role of α1-GC in vasorelaxation (Figure 4A). Unexpectedly, α1-deficient rings were completely relaxed by GSNO as well. However, the concentration-response curve for NO was shifted to the right with a 5-fold higher EC50 (2 μM). These findings demonstrate an unpredicted functional role of α2-GC in vascular relaxation. To ensure that the retained NO-induced relaxation in α1-deficient rings was caused by NO-sensitive GC and not by other NO effector molecules, we used the inhibitor of NO-sensitive GC, 1_H_-[1,2,4]oxadiazol[4,3-a]quinoxalin-1-one (ODQ) (11). First, ODQ effects were established in the WT rings to confirm complete inhibition of NO-sensitive GC under the applied conditions. As can be seen in Figure 4B, in the presence of ODQ, NO-induced relaxation of WT rings was fully abolished. The responsiveness of the ODQ-treated rings was confirmed by the subsequent relaxation with ANP. Moreover, ODQ also inhibited the endothelium-dependent relaxation induced by carbachol, demonstrating its inhibitory potential toward endogenously produced NO (Figure 4D). When ODQ was applied to α1-deficient rings, the vascular response to NO was completely abolished, confirming α2-GC as the NO receptor mediating relaxation in α1-KO (Figure 4B). The results are further supported by the inhibition of carbachol-induced relaxation in the α1-deficient rings by ODQ as well (Figure 4D). By measuring NO-stimulated GC activity in α1-deficient aortic tissue (Figure 4C), α2-GC was determined to represent only 6% of the total GC content (α1-KO, 0.24 ± 0.15 nmol/mg/min versus WT, 3.8 ± 0.8 nmol/mg/min). Taken together, the retained relaxation in the α1-deficient rings shows that the loss of the majority of the cGMP-forming NO receptor (94%) does not impair the biological response to but decreases the potency of NO. To study the physiological cGMP response, we induced endothelium-dependent relaxation using carbachol (Figure 4D). Here, a carbachol concentration (30 μM) that evoked an almost complete relaxation of WT and α2-deficient aortic rings (approximately 90%) induced 50% relaxation in the α1-deficient rings. Obviously, the cGMP increase caused by endogenously produced NO is able to induce significant relaxation in the α1-deficient rings; thus, small increases in cGMP exert a profound effect on vascular tone.
Vasorelaxing properties of α-deficient aortic rings. Functional responses of aortic rings were determined as described in Methods. (A) Cumulative concentration-response curves of GSNO-induced relaxation of α1- and α2-deficient rings (n = 3 in each group). (B) GSNO-induced relaxation of WT and α1-deficient rings in the presence of the inhibitor of NO-sensitive GC, ODQ. Shown are the original recordings using 10 μM GSNO and 20 μM ODQ. ANP (10 nM) was added to confirm integrity of the rings. (C) NO-stimulated GC activities (100 μM DEA-NO) determined in aortic homogenates (n = 5 per genotype). (D) Carbachol-induced relaxation of WT and α-deficient aortic rings in the absence or presence of ODQ (n = 3–8 per genotype). Experiments used 30 μM carbachol and 20 μM ODQ.
In an attempt to judge the cGMP increases in aorta, we determined the cGMP content in aortic rings with and without exogenous NO as well as in the presence of the GC inhibitor ODQ (Figure 5). In WT and α2-deficient aortas, steady state levels of cGMP (~12 pmol/mg) were comparable and were significantly reduced by ODQ (~6 pmol/mg), demonstrating the impact of endogenously produced NO. The addition of NO yielded comparable cGMP increases in WT and α2-deficient rings. In contrast, cGMP levels in the α1-deficient aortic rings did not differ in the absence or presence of ODQ (~6 pmol/mg) and did not significantly increase upon addition of NO. The results show that the cGMP formed by the residual α2-GC is not detectable and strongly suggest that local cGMP increases are responsible for the induction of the biological response.
Cyclic GMP levels determined in intact aortic rings of WT, α1 -, and α2 -deficient mice. Cyclic GMP content was determined in rings incubated without any addition or in the presence of ODQ (20 μM, 15 min) or GSNO (100 μM, 5 min) (n = 3–6 per genotype). *P < 0.05, differences considered significant.
To detect a putative increased responsiveness toward cGMP in the rings with the low GC content, we induced relaxation with the stimulator of the membrane-bound GC-A, ANP, and the direct activator of the cGKI, 8–(p-chlorophenylthio)–cGMP (8-pCPT-cGMP) (Figure 6). An increased sensitivity toward both substances was observed in the α1-deficient rings, indicating an as yet unknown regulatory potential within the cGMP signaling cascade. We then asked whether the observed increase in ANP sensitivity was supported by a reduction of the cGMP-degrading PDEs. We evaluated the different PDE isoforms present in aortas (PDE1, PDE3, and PDE5) by measuring PDE activities under various experimental conditions (different substrate concentrations, presence of milrinone, sildenafil, EGTA, Ca2+/calmodulin; Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI27657DS1). However, we did not find any significant reduction of cGMP-degrading activities in the α1-deficient aortas.
Increased vascular reactivity of the α1 -deficient aortic rings. (A) Cumulative concentration-response curves of ANP-induced relaxation of α1- and α2-deficient aortic rings (n = 3 per genotype). (B) Cumulative concentration-response curves of 8-pCPT-cGMP–induced relaxation in the α1-deficient aortic rings (n = 4 per genotype). *P < 0.05, differences considered significant.
The importance of NO/cGMP signaling for the maintenance of blood pressure is well established. To monitor the impact of the GC isoforms, systolic blood pressure was assessed by tail-cuff measurements. The systolic values obtained in the α2-KO animals did not differ significantly from those in WT, as shown in Figure 7A, whereas blood pressure in α1-deficient mice was moderately elevated (mean 111 ± 2 mmHg versus 104 ± 2 mmHg). These results indicate that α2-GC was not sufficient to completely substitute for the loss of the major α1-GC. To confirm the functional role of α2-GC in the α1-KOs, mice were treated with the NO synthase inhibitor l-NAME (Figure 6B). l-NAME treatment led to an increase in blood pressure in the α1-KO mice (21%), demonstrating that NO-induced cGMP synthesis, although being greatly reduced in these mice, retains its importance for regulation of smooth muscle tone in vivo.
Systolic blood pressure in α-deficient mice. (A) Systolic blood pressure as measured in conscious mice by tail-cuff plethysmography as described in Methods. Means (indicated by horizontal lines) of 111 and 104 mmHg were determined for the α1-KO mice and their WT siblings, respectively; for the α2-KO mice and the respective WT littermates, means of 111 and 110 mmHg were determined. The 95% confidence interval of the difference between α1-KO and WT mice is 1–15 mmHg. (B) Increase of blood pressure induced by l-NAME treatment of α1-KO mice (n = 5 per genotype). *P < 0.05, differences considered significant.