These findings indicate that signalling of cGKIbeta via IRAG is an essential functional part for regulation of smooth muscle tone and of intracellular calcium by NO (exogenously applicated or endogenously synthesized) and by ANP. IRAG signalling does not modulate basal tone but might be important for blood pressure regulation under pathophysiological conditions.

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg·kg −1 ·d −1 ) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.PKGI | PDE | cardiac failure | hypertension | NO/cyclic GMP system

a b s t r a c tWe analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Ia and Ib and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositoltrisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI-and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.

Schlossmann J, Desch M. IRAG and novel PKG targeting in the cardiovascular system. Am J Physiol Heart Circ Physiol 301: H672-H682, 2011. First published June 10, 2011; doi:10.1152/ajpheart.00198.2011.-Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1␤ isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1␤ and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system. inositol trisphosphate receptor-associated guanosine 3=,5=-cyclic monophosphatekinase substrate; nitric oxide; guanosine 3=,5=-cyclic monophosphate-dependent protein kinase THIS ARTICLE is part of a collection on Hypertension and Novel Modulators of Vascular Tone. Other articles appearing in this collection, as well as a full archive of all collections, can be found online at http://ajpheart.physiology.org/.Nitric oxide (NO) leads to cGMP synthesis and thereby activates cGMP-dependent protein kinase (PKG), mediating various cardiovascular functions. The identification of PKG substrates including the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG) has lead to a new view of PKG1 isozyme PKG1␤ and PKG1␣ function. Intracellular targeting and function of these isozymes are modulated by different substrates. The recognition of these substrates is mediated by the NH 2 -terminal leucine/isoleucine zipper (LZ) domains of PKG1. In this review the current view of the molecular interaction and function of IRAG compared with other substrates regulated by PKG are described, mainly focusing on the cardiovascular system. Furthermore, the subtle pathways for cGMP/PKG signaling are discussed. Function of NO/cGMP/PKG Signaling CascadeNO/cGMP signaling regulates several tasks in the cardiovascular sy...

Signalling of cGK (cGMP-dependent protein kinases) are mediated through phosphorylation of specific substrates. Several substrates of cGKI and cGKII were identified meanwhile. Some cGKI substrates are specifically regulated by the cGKIalpha or the cGKIbeta isozyme. In various cells and tissues, different cGK substrates exist that are essential for the regulation of diverse functions comprising tissue contractility, cell motility, cell contact, cellular secretion, cell proliferation, and cell differentiation. On the molecular level, cGKI substrates fulfill various cellular functions regulating e.g. the intracellular calcium and potassium concentration, the calcium sensitivity, and the organisation of the intracellular cytoskeleton. cGKII substrates are involved e.g. in chloride transport, sodium/proton transport and transcriptional regulation. The understanding of cGK signalling and function depends strongly on the identification of further specific substrates. In the last years, diverse approaches ranging from biochemistry to genetic deletion lead to the identification and establishment of several substrates, which raised new insights in the molecular mechanisms of cGK functions and elucidated new cellular cGK functions. However, the analysis of the dynamic signalling of cGK in tissues and cells will be necessary to discover new signalling pathways and substrates.