InsP3R-associated cGMP Kinase Substrate (IRAG) Is Essential for Nitric Oxide-induced Inhibition of Calcium Signaling in Human Colonic Smooth Muscle

Abstract

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.

Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle

“…None of these effects of cGMP were observed in colon muscle from IRAG ⌬12/⌬12 mice. Similarly, cGMP did not reduce bradykinin-induced Ca 2ϩ transients after inactivation of IRAG in cultured human colon cells (4). These results confirm the view that cGMP/cGKI-induced inhibition of contraction is mediated by IRAG-controlled reduction of intracellular Ca 2ϩ signals in colon smooth muscle, most probably via interaction with the IP 3 R and, consequently, inhibition of IP 3 -induced Ca 2ϩ release from intracellular Ca 2ϩ stores (8).…”

Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

“…The finding that 8-Br-cGMP, and cyclic nucleotide signaling in general, desensitizes or reduces maximal smooth muscle contractile force is not new, as the effects of cyclic nucleotides on [Ca 2ϩ ] i (25,26,(47)(48)(49), RhoA activation (14,15,19,30,32,50), and MYPT1/CPI-17 phosphorylation (20,21,23,24,31,32,37) have been well documented. Mechanistically, a cGKI-mediated decrease in [Ca 2ϩ ] i is unlikely to be dominant in our studies of the cerebral vasculature, as contraction mediated by TXA 2 R activation is predominantly through Ca 2ϩ -independent pathways (9 -11).…”

Cyclic GMP-dependent Protein Kinase Iα Inhibits Thrombin Receptor-mediated Calcium Mobilization in Vascular Smooth Muscle Cells

“…Ca 2ϩ efflux from IP 3 -sensitive intracellular stores also is inhibited by PKGI␤-mediated phosphorylation of the IP 3 receptor-associated cyclic GMP kinase substrate (IRAG) in a complex of sarcoplasmic reticulum membrane proteins, including PKGI␤, IRAG, and the IP 3 receptor (18 -21). The presence of IRAG is essential in the cyclic GMP-dependent inhibition of Ca 2ϩ signaling in colonic SMCs, suggesting that PKGI␤ is a principal regulator of intracellular Ca 2ϩ levels in these cells (19). However, in VSMCs derived from PKGI knock-out mice blood vessels, the transfection of PKGI␣, but not PKGI␤, decreases noradrenaline-induced Ca 2ϩ mobilization (9).…”