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Aims Nitric oxide (NO) and atrial natriuretic peptide (ANP) signalling via cGMP controls smooth muscle tone. One important signalling pathway of cGMP-dependent protein kinase type I (cGKI) is mediated by IRAG (IP3 receptor associated cGKI substrate) which is highly expressed in smooth muscle tissues. To elucidate the role of IRAG for NO- and ANP-mediated smooth muscle tone regulation, cGKI localization, and for its possible function in blood pressure adjustment, we generated IRAG-knockout mice by targeted deletion of exon 3. Methods and results IRAG deletion prevented stable interaction of IP3 receptor type I (IP3RI) with cGKIβ determined by cGMP affinity chromatography. Confocal microscopy in vascular smooth muscle cells (VSMCs) showed that localization of cGKIβ and cGKIα did not change in absence of IRAG. NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic vessels and colon was significantly affected in IRAG-knockout mice. The suppression of cGMP-induced relaxation was not rescued by selective expression of cGKIβ in smooth muscle from cGKIβ-transgenic mice. NO-, ANP-, and cGMP-mediated inhibition of the hormone-induced increase in intracellular calcium concentration measured by Fura2 was suppressed in IRAG-deficient VSMC. Telemetric measurements revealed that IRAG-deficient animals exhibited normal basal tone, but were resistant to blood pressure reduction induced by lipopolysaccharide-treatment. Conclusion These findings indicate that signalling of cGKIβ 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.

Stimulation of cGMP signalling cascade in smooth muscle results in decreased intracellular Ca2+ levels and thus muscle relaxation. Inositoltrisphosphate Receptor 1 (IP3R1) – a Ca2+ release channel located in the membrane of sarcoplasmatic reticulum (SR) – is inhibited by the phosphorylation of IP3R1 associated cGMP kinase dependent substrate (IRAG) via cGMPkinase1β (cGK1β). On the other hand, Ca2+ is pumped back into SR by the Sarco/Endoplasmatic Reticulum Ca2+ ATPase (SERCA), which is inhibited by Phospholamban (PLB). Phosphorylation of PLB by cGK1 suppresses its inhibitory effect on SERCA. To characterize the subcellular distribution of these cGMP signalling proteins upon deletion of IRAG, aortic vascular smooth muscle cells (VSMCs) of IRAG KO mice were immunohistochemically analyzed and compared to VSMCs of littermate wild type animals. Furthermore we investigated the effects of treatment with 8Br-cGMP on the distribution of these proteins in IRAG KO VSMCs versus WT VSMCs.

Results For the functional analysis of cGMP signalling, transgenic murine mutants were used. cGKI signalling pathways include interaction of the cGKIb-isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). NO/cGMP and ANP/cGMP-dependent relaxation of aortic smooth muscle was strongly affected in IRAG-deficient mice. NO/ ANP-dependent inhibition of intracellular calcium release was suppressed in IRAG-deficient vascular smooth muscle cells (VSMC). Furthermore, reduction of store operated calcium entry by cGMP was affected in IRAG-KO VSMC. Basal mean arterial blood pressure, heart rate and activity were not changed in IRAG knockout mice. However, under pathophysiological conditions like sepsis (induced by E. coli lipopolysaccharide application) IRAG knockout mice were resistant to blood pressure reduction.

NO/cGMP signalling via cGMP-dependent kinase I (cGKI) induces a variety of physiological functions comprising relaxation of smooth muscle and inhibition of platelet aggregation. Several signalling pathways of cGKI exist including the interaction of the cGKIβ isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). To get insight into the physiological function of IRAG protein a knockout mutant of the IRAG gene was generated by targeted deletion. Expression of other cGKI substrate proteins in mutant smooth muscle tissues and platelets was not affected upon IRAG deletion. Interestingly, the localization of cGKIβ was unchanged in IRAG-deficient vascular smooth muscle cells. Analysis of smooth muscle contractility suggests that signalling via IRAG is essential for endogenous and exogenous NO/cGMP-dependent contractility of aortic smooth muscle. Furthermore, NO/cGMP-dependent relaxation of murine colon is dependent on this signalling cascade. In platelets, IRAG-deficiency abolishes the NO/cGMP-dependent inhibition of platelet aggregation and granule secretion. These results strongly suggest that IRAG signalling is a predominant physiological signalling pathway of NO/cGMP in smooth muscle and platelets.