Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKIdependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP 3 RI)-associated protein (IRAG), which decreases hormone-induced IP 3 -dependent Ca 2 þ release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP 3 RI interaction and resulted in hypomorphic IRAG D12/D12 mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol-and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG D12/D12 mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca 2 þ ] i were not decreased by cGMP in aortic smooth muscle cells from IRAG D12/D12 mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG D12/D12 mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP 3 RI.

Frei E, Huster M, Smital P, Schlossmann J, Hofmann F, Wegener JW. Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 297: G834 -G839, 2009. First published July 23, 2009 doi:10.1152/ajpgi.00095.2009.-cGMP-dependent protein kinase I (cGKI) induces relaxation of smooth muscle via several pathways that include inhibition of intracellular Ca 2ϩ signaling and/or involve activation of myosin phosphatase. In the present study, we investigated these mechanisms comparatively in colon and jejunum longitudinal smooth muscle from mice. In simultaneous recordings from colon muscle, 8-bromo-cGMP (8-Br-cGMP) reduced both carbachol-induced tension and carbachol-induced increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i). These effects of 8-Br-cGMP were absent in colon from mice carrying a mutated inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate (IRAG) gene or lacking cGKI. However, in jejunum, 8-Br-cGMP reduced carbachol-induced tension but did not change corresponding [Ca 2ϩ ]i signals. This setting was also observed in jejunum from mice carrying a mutated IRAG gene, whereas no response to 8-Br-cGMP was observed in jejunum from mice lacking cGKI. After inhibition of phosphatase activity by calyculin A, 8-Br-cGMP did not relax jejunum but still relaxed colon muscle. In Western blot analysis, 8-BrcGMP reduced the signal for phosphorylated MYPT-1 in carbacholstimulated jejunum but not in colon. These results suggest that cGMP/cGKI signaling differentially inhibits contraction in the muscles investigated: in jejunum, inhibition is performed without changing [Ca 2ϩ ]i and is dependent on phosphatase activity, whereas in colon, inhibition is mediated by inhibition of [Ca 2ϩ ]i signals.inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate; myosin phosphatase targeting subunit 1; guanosine 3Ј,5Ј-cyclic monophosphate-dependent protein kinase I; colon; jejunum NONADRENERGIC, NONCHOLINERGIC NEURONS of the gut use nitric oxide (NO) as a neurotransmitter to regulate smooth muscle contractility via cGMP/cGMP-dependent protein kinase I (cGKI) signaling (3,13,15,25). Since NO is gaseous, it diffuses directly from the neurons to its receptor, the soluble guanylyl cyclase (sGC) located in smooth muscle cells. Stimulation of sGC increases intracellular levels of cGMP and induces relaxation via activation of cGKI (10). Smooth muscle contraction is controlled by Ca 2ϩ -dependent and Ca 2ϩ -independent signaling pathways (6,15,22,29). It is therefore likely that relaxation by NO/cGMP/cGKI interferes with both Ca 2ϩ -dependent and -independent signaling pathways. Indeed, several mechanisms have been reported by which cGKI mediates relaxation in smooth muscle. Ca 2ϩ -dependent mechanisms involve cGMP/cGKI inhibition of hormone-induced Ca 2ϩ release from intracellular stores that is not observed after deletion of the cGKI gene in mice (18). Further studies have shown that the latter mechanism involves a...

cGMP dependent protein kinase I (cGKI) in intestinal smooth muscle may induce relaxation via several mechanisms that include inhibition of intracellular Ca2+ release by phosphorylation of the inositol‐1,4,5‐phosphat receptor associated G‐kinase substrate (IRAG) and increasing myosin phosphatase activity by phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). In the present study, we investigated whether one or both mechanisms are involved in cGMP‐mediated inhibition of carbachol‐stimulated contraction of colon and jejunum. In simultaneous recordings of tension and intracellular Ca2+ signals, 8‐Br‐cGMP reduced both carbachol‐induced tension and intracellular Ca2+ signals in colon muscle. In contrast, in jejunum muscle, 8‐Br‐cGMP reduced tension but did not change intracellular Ca2+ signals. In colon muscles from mice exhibiting a mutated IRAG (IRAG 12), 8‐Br‐cGMP did not reduce carbachol‐induced tension and Ca2+ signals. Inhibition of phosphatase activity by calyculin A abolished relaxation of carbachol‐induced contraction by 8‐Br‐cGMP in jejunum but not in colon muscle. Western blot analysis showed a larger signal for phosphorylated MYPT1 in carbachol‐stimulated jejunum than in colon muscle. These result suggest that cGMP/cGKI induces relaxation mostly by inhibition of intracellular Ca2+ release in colon and by activation of myosin phosphatase in jejunum.

Inhibition of hormone-induced Ca 2+ release from intracellular stores by cGMP involves a protein complex which consists of at least 3 proteins, i.e. the inositol-1,3,4-phosphat receptor (IP 3 R), the cGMP-dependent protein kinase (cGK), and, as a linker protein between both, the inositol-1,3,4-phosphat receptor associated G-kinase substrate (IRAG [1]). Recently, mutant mice were created which lack the part of the IRAG protein that is supposed to mediate the association between IRAG and the IP 3 R. In these mice (IRAG ∆12 mice), cGMP failed to attenuate both, hormone-induced Ca2+ signals and hormone-induced contractions in vascular preparations [2]. These result confirm the concept that IRAG is an essential mediator of relaxation mediated by cGMP/cGK signalling in vascular smooth muscle.In the present study, we tested this hypothesis in two other smooth muscle types, i.e. from jejunum and colon. In line with the concept above, cGMP failed to relax hormone-induced contractions in colon muscle from IRAG ∆12 mice. In contrast, cGMP clearly relaxed hormoneinduced contractions in jejunum muscle from these mutant mice. Thus, Rho/Rho kinase rather than IRAG seems to be the key player in mediating cGMP/cGK signalling in this particular type of smooth muscle.Next, we questioned whether the mutation of IRAG affects the relaxant effects of cGMP on hormone-independent contractions. For this purpose, muscles were contracted by activation of Ca 2+ influx, either by depolarisation with high K + or by initiating capacitative Ca 2+ entry with thapsigargin. Since IRAG is mainly associated with sarcoplasmatic membrane proteins, we expected that the IRAG mutation would not affect cell membrane Ca 2+ signalling. Surprisingly, the relaxant effects of cGMP on these types of contraction were attenuated in intestinal muscles from IRAG ∆12 mice.The results suggests that (1) the association of IRAG/IP 3 R determines cGMP/cGK-mediated relaxations only in distinct types of smooth muscle, and (2) IRAG participates in cGMP/cGK-mediated relaxation not only during hormone-induced Ca 2+ release but also during hormoneindependent Ca 2+ entry. Thus, cGMP/cGK may target via IRAG, either alone or with the IP 3 R, to, at present, unidentified elements of the Ca 2+ entry machinery in certain types of smooth muscle. Geiselhoringer A, Werner M, Sigl K, Smital P, Worner R, Acheo L, Stieber J, Weinmeister P, Feil R, Feil S, Wegener J, Hofmann F, Schlossmann J: IRAG is essential for relaxation of receptortriggered smooth muscle contraction by cGMP kinase. Embo