Andreas Friebe

Regulation of Nitric Oxide-Sensitive Guanylyl Cyclase

&NA; In this review, we outline the current knowledge on the regulation of nitric oxide (NO)‐sensitive guanylyl cyclase (GC). Besides NO, the physiological activator that binds to the prosthetic heme group of the enzyme, two novel classes of GC activators have been identified that may have broad pharmacological implications. YC‐1 and YC‐1‐like substances act as NO sensitizers, whereas the substance BAY 58‐2667 stimulates NO‐sensitive GC NO‐independently and preferentially activates the heme‐free form of the enzyme. Sensitization and desensitization of NO/cGMP signaling have been reported to occur on the level of NO‐sensitive GC; in the present study, an alternative mechanism is introduced explaining the adaptation of the NO‐induced cGMP response by a long‐term activation of the cGMPdegrading phosphodiesterase 5 (PDE5). Finally, regulation of GC expression and a possible modulation of GC activity by other factors are discussed. (Circ Res. 2003;93:96‐105.)

Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule

Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO‐releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM‐3) in vitro and in vivo. CORM‐3 produced a concentration‐dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25–50 μM. Inactive CORM‐3, which does not release CO, did not affect vascular tone. Blockers of ATP‐dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM‐3‐dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM‐3 stimulation. The in vitro and in vivo vasorelaxant activities of CORM‐3 were further enhanced in the presence of YC‐1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM‐3, suggesting that factors produced by the endothelium influence CORM‐3 vascular activities. These results, together with our previous findings on the cardioprotective functions of CORM‐3, indicate that this molecule is an excellent prototype of water‐soluble CO carriers for studying the pharmacological and biological features of CO.

Nitric oxide-sensitive guanylyl cyclase: structure and regulation

By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.

Rapid nitric oxide–induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

Nitric oxide‐sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition

See also Gordge MP. Nitric oxide: a one‐trick pony? This issue, pp 1340–2.

Inhibition of Phosphodiesterase Type 5 by the Activator of Nitric Oxide–Sensitive Guanylyl Cyclase BAY 41-2272

Background—By the formation of cGMP, nitric oxide (NO)–sensitive guanylyl cyclase (GC) acts as the effector for the signaling molecule NO and mediates the relaxation of vascular smooth muscle and the inhibition of platelet aggregation. The compounds YC-1 and BAY 41-2272 are regarded as NO-independent activators and sensitizers of NO-sensitive GC. In vivo effects, for example, lowering blood pressure and prolonging tail-bleeding times, turn the compounds into promising candidates for the therapy of cardiovascular diseases. However, YC-1 has also been shown to inhibit the major cGMP-degrading enzyme phosphodiesterase type 5 (PDE5). The synergistic properties of YC-1 on cGMP formation and degradation lead to an excessive NO-induced cGMP accumulation in cells, explaining the observed physiological effects. We assessed a potential inhibition of PDE5 by the new GC activator BAY 41-2272. Methods and Results—The effects of BAY 41-2272 on NO-sensitive GC and PDE5 activities were tested in vitro. BAY 41-2272 not only sensitized NO-sensitive GC toward activation by NO but also, with comparable potency, inhibited cGMP degradation by PDE5. In intact platelets, BAY 41-2272 greatly potentiated the NO-induced cGMP response that was caused by a synergistic effect of BAY 41-2272 on cGMP formation and degradation. Conclusions—The physiological effects of BAY 41-2272, which are commonly ascribed to the NO-independent activation of NO-sensitive GC, are rather due to the synergism of sensitization of NO-sensitive GC and inhibition of PDE5.

C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons.

Neuronal circuits are shaped during development by the coordinated action of guidance factors and signals that regulate axonal branching. Unlike guidance cues, the molecules and signaling cascades that underlie axonal branching remain to be resolved. Here we show that the secreted molecule C-type natriuretic peptide (CNP) induces a cGMP signaling cascade via its receptor particulate guanylyl cyclase Npr2 which is essential for sensory axon bifurcation at the dorsal root entry zone (DREZ) of the spinal cord. In contrast, another form of sensory axon branching—collateral formation—is not affected by this pathway. We also demonstrate that cGMP signaling via the nitric oxide-stimulated soluble guanylyl cyclase system (NO-GC) is dispensable for sensory axon branching. Functionally, the bifurcation error in CNP mutant mice is maintained at mature stages and results in a reduced input on secondary neurons as detected by patch-clamp recordings.

Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFβ signalling

Objectives We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC. Methods Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCIfl/fl mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor. Results sGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity. Conclusions We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits experimental fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of fibrosis and vascular disease in systemic sclerosis.

Pressure-Overload–Induced Subcellular Relocalization/Oxidation of Soluble Guanylyl Cyclase in the Heart Modulates Enzyme Stimulation

Rationale: Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO–sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload–induced hypertrophy. The latter itself depresses signaling through this pathway by reducing NO generation and enhancing cGMP hydrolysis. Objective: We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms. Methods and Results: C57BL/6 mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, whereas NO- and heme-independent sGC activation by BAY 60–2770 was preserved. Total sGC&agr;1 and &bgr;1 expression were unchanged by TAC; however, sGC&bgr;1 subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2- to 3-fold greater in Cav3-containing lipid raft versus nonlipid raft domains in control and 6-fold greater after TAC. In contrast, BAY 60–2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO- and BAY-stimulated sGC activity in microdomains containing Cav3 for controls but no change within non–Cav3-enriched domains. Conclusions: Pressure overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.