Doris Koesling

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.)

SENSITIZING SOLUBLE GUANYLYL CYCLASE TO BECOME A HIGHLY CO-SENSITIVE ENZYME

It took at least a decade to realize that the toxic gas NO is the physiological activator of soluble guanylyl cyclase (sGC), thereby acting as a signaling molecule in the nervous and cardiovascular systems. Despite its rather poor sGC‐activating property, CO has also been implicated as a physiological stimulator of sGC in neurotransmission and vasorelaxation. Here, we establish YC‐1 as a novel NO‐independent sGC activator that potentiates both CO‐ and NO‐induced sGC stimulation. As this potentiating effect is also observed with protoporphyrin IX which activates sGC independently of a gaseous ligand, we conclude that stabilization of the enzymes active configuration is the underlying mechanism of YC‐1′s action. Moreover, the results obtained with YC‐1 reveal that CO is capable of stimulating sGC to a degree similar to NO, and thus provide the molecular basis for CO functioning as a signaling molecule.

Fatal gastrointestinal obstruction and hypertension in mice lacking nitric oxide-sensitive guanylyl cyclase

The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for the β1 subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3–4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor.

Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system.

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (alpha1-GC, alpha2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either alpha1- or alpha2-GC to dissect their biological functions. In platelets, alpha1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, alpha1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, alpha2-GC, representing only 6% of the total GC content in WT, also completely relaxed alpha1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by alpha2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.

Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling

In platelets, the nitric oxide (NO)–induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.

NO activation of guanylyl cyclase

Nitric oxide (NO)‐sensitive guanylyl‐cyclase (GC) is the most important receptor for the signaling molecule NO. Activation of the enzyme is brought about by binding of NO to the prosthetic heme group. By monitoring NO‐binding and catalytic activity simultaneously, we show that NO activates GC only if the reaction products of the enzyme are present. NO‐binding in the absence of the products did not activate the enzyme, but yielded a nonactivated species with the spectral characteristics of the active form. Conversion of the nonactivated into the activated conformation of the enzyme required the simultaneous presence of NO and the reaction products. Furthermore, the products magnesium/cGMP/pyrophosphate promoted the release of the histidine–iron bond during NO‐binding, indicating reciprocal communication of the catalytic and ligand‐binding domains. Based on these observations, we present a model that proposes two NO‐bound states of the enzyme: an active state formed in the presence of the products and a nonactivated state. The model not only covers the data reported here but also consolidates results from previous studies on NO‐binding and dissociation/deactivation of GC.

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal‐transducing enzymes such as protein tyrosine‐kinases and protein tyrosine‐phosphatases. So far, three isoforms of mammalian membrane‐bound guanylyl cyclases (GC‐A, GC‐B, GC‐C), which are stimulated by either natriuretic peptides (GC‐A, GC‐B) or by the enterotoxin of Escherichia coli (GC‐C), have been identified. These proteins belong to the group of receptor‐linked enzymes, with different NH2‐terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane‐bound enzymes, the heme‐containing soluble guanylyl cyclase is stimulated by NO and NO‐containing compounds and consists of two subunits (α1 and β1). Both subunits contain the putative catalytic domain, which is conserved in the membrane‐bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the α1‐ and β1‐subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated β2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue‐specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.—Koesling, D.; Böhme, E.; Schultz, G. Guanylyl cyclases, a growing family of signal‐transducing enzymes. FASEB J. 5: 2785‐2791; 1991.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Molecular cloning and expression of a new α-subunit of soluble guanylyl cyclase Interchangeability of the α-subunits of the enzyme

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the α1‐subunit and was designated α2 as the new subunit is very similar to the α1‐subunit in the middle and C‐terminal part: it is quite diverse in the N‐terminal part. Preceding experiments had shown that coexpression of the α1‐ and β1‐subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221–223]. The finding that the α2‐subunit was able to replace the α1‐ but not the β1‐subunit in expression experiments demonstrates the interchangeability of the α‐subunit isoforms of soluble guanylyl cyclase.

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.