Michael Russwurm

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.

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.

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.

Major occurrence of the new α2β1 isoform of NO-sensitive guanylyl cyclase in brain

Abstract NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (α 1 β 1 ), another heterodimer (α 2 β 1 ) has recently been identified to be associated with PSD-95 in brain. Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the α 2 β 1 heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the α 2 β 1 and α 1 β 1 isoforms were comparable; in all other tissues, the α 1 β 1 heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain. In sum, the major occurrence of the α 2 β 1 heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies.

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.

Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.

Crystal structure of the guanylyl cyclase Cya2

Cyclic GMP (cGMP) is an important second messenger in eukaryotes. It is formed by guanylyl cyclases (GCs), members of the nucleotidyl cyclases class III, which also comprises adenylyl cyclases (ACs) from most organisms. To date, no structures of eukaryotic GCs are available, and all bacterial class III proteins were found to be ACs. Here we describe the biochemical and structural characterization of the class III cyclase Cya2 from cyanobacterium Synechocystis PCC6803. Cya2 shows high specificity for GTP versus ATP, revealing it to be the first bacterial GC, and sequence similarity searches indicate that GCs are also present in other bacteria. The crystal structure of Cya2 provides first structural insights into the universal GC family. Structure and mutagenesis studies show that a conserved glutamate, assisted by an interacting lysine, dominates substrate selection by forming hydrogen bonds to the substrate base. We find, however, that a second residue involved in substrate selection has an unexpected sterical role in GCs, different from its hydrogen bonding function in the related ACs. The structure identifies a tyrosine that lines the guanine binding pocket as additional residue contributing to substrate specificity. Furthermore, we find that substrate specificity stems from faster turnover of GTP, rather than different affinities for GTP and ATP, implying that the specificity-determining interactions are established after the binding step.

A phosphodiesterase 2A isoform localized to mitochondria regulates respiration

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.

Isoforms of NO-sensitive guanylyl cyclase

By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (α1β1, α2β1) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the α2β1 isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the α2 subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the α2β1 isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.