Smooth muscle tone is predominately regulated by the rise and fall of free intracellular cytosolic calcium concentration (1). Thus, modulation of intracellular calcium handling is one important mechanism by which inhibitory neurotransmitters induce smooth muscle relaxation (2). In the human colon, nitric oxide has been shown to be the principal inhibitory transmitter in nerve-muscle interaction (3). NO 1 -dependent relaxation of gastrointestinal smooth muscle has been demonstrated throughout the gut of various species (4 -9) and has been established as a crucial event contributing to the maintenance of normal gut function (10, 11).The molecular target of NO in smooth muscle is the soluble guanylate cyclase, which is strongly activated by NO (12, 13). Increased levels of cGMP lead to smooth muscle relaxation predominately via activation of cGMP-dependent protein kinase (cGK) (14, 15). The crucial role of cGK for gastrointestinal smooth muscle function became apparent in cGKI-deficient mice showing a selective lack of NO-dependent smooth muscle relaxation associated with severe gastrointestinal dysfunction and marked hypertrophy of gastrointestinal smooth muscle (16). The mechanisms of cGKI-dependent smooth muscle cell relaxation, however, have not been fully understood (for a review, see Ref. 14).In a recent study (see Ref. 26), a new protein has been identified as molecular target of cGKI in smooth muscle microsomal membranes and has been termed IRAG (InsP 3 R-associated cGMP kinase substrate). IRAG precipitated together with cGKI␤ and InsP 3 receptor type I in a complex which was found to be localized at the endoplasmic reticulum membrane. In transfected COS cells, the coexpression of IRAG with cGKI was essential for the cGMP-dependent inhibition of InsP 3 -dependent calcium release. This inhibition has further been demonstrated to depend on the cGKI-mediated phosphorylation of IRAG at Ser 696 . No interaction between cGKI and the InsP 3 receptor was observed when IRAG was absent (17).To date, only little is known about IRAG expression and its function in gastrointestinal tissues. Furthermore, evidence for a functional role of IRAG in smooth muscle tissue is lacking. We show here that IRAG is expressed in human colon and that the suppression of IRAG protein expression in human colonic smooth muscle cells is sufficient to abolish the inhibitory effect of sodium nitroprusside and 8-pCPT-cGMP on bradykinin-induced calcium release in these cells. EXPERIMENTAL PROCEDURESTissue Preparation-Tissues from human colon and rectum were obtained from surgical resections for colorectal malignant disease. The tissues were macroscopically and microscopically free of tumor.

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Nitric oxide (NO) induces relaxation of colonic smooth muscle cells predominantly by cGMP/cGMP-dependent protein kinase I (cGKI)-induced phosphorylation of the inositol 1,4,5-trisphosphate receptor (IP(3)R)-associated cGMP kinase substrate (IRAG), to block store-dependent calcium signaling. In the present study we analyzed the structure and function of the human IRAG/MRVI1 gene. We describe four unique first exon variants transcribed from individual promoters in diverse human tissues. Tissue-specific alternative splicing with exon skipping and alternative splice donor and acceptor site usage further increases diversity of IRAG mRNA variants that encode for NH(2)- and COOH-terminally truncated proteins. At the functional level, COOH-terminally truncated IRAG variants lacking both the cGKI phosphorylation and the IP(3)RI interaction site counteract cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. Since COOH-terminally truncated IRAG mRNA isoforms are widely expressed in human tissues, our results point to an important role of IRAG variants as negative modulators of nitric oxide/cGKI-dependent signaling. The complexity of alternative splicing of the IRAG gene impressively demonstrates how posttranscriptional processing generates functionally distinct proteins from a single gene.

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