Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Abstract

Interleukin-1␤ (IL-1␤) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1␤ on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1␤ (10 ng/ml), contractions elicited by high K ؉ and carbachol were inhibited in a time-dependent manner. IL-1␤ more strongly inhibited the carbachol-induced contractions than high K ؉ with decreasing myosin light chain phosphorylation. In the ␣-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5 -3-O-(thio)triphosphate increased the Ca 2؉ sensitivity of contractile elements, and this G protein-coupled Ca 2؉ sensitization was significantly reduced in the IL-1␤-treated ileum. Among the functional proteins involved in the smooth muscle Ca 2؉ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1␤, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1␤ treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1␤-treated muscles. These results suggest that long term treatment with IL-1␤ decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1␤ to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1␤.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

“…Sato et al (48), however, reported hypocontractility of mouse colonic circular smooth muscle obtained from DSS-treated colon that was mediated by a decrease in CPI-17 expression. Their findings are similar to those previously by others and us in circular smooth muscle exposed to IL-1␤ or obtained from TNBS-treated colon (16,43,52). Shi and co-workers (52) have reported that decrease in circular muscle contraction obtained from DSS-treated rat colon was due to suppression of G␣ q protein, whereas in TNBS-treated rat colon was due to suppression of ␣ 1c 1b subunit of Ca v 1.2 b channels, G␣ q and CPI-17.…”

17.BETA.-Estradiol Induces Gastrointestinal Motility Disorder by Decreasing CPI-17 Phosphorylation Via Changes in Rho-Family G-Protein Rnd Expression in Small Intestine

“…We previously reported that ROCKs and PKC phosphorylate CPI-17 at Thr38 in the rat small intestine [20]. We therefore examined the effects of 17-estradiol on carbachol-induced phosphorylation of CPI-17 in organ-cultured ileal tissues (Fig.…”

“…Organ culture was performed as described previously [19,20]. Ileal tissues were isolated from the distal ileal region of Wistar rats (age, 7 weeks; male) and the circular ileal smooth muscle layer was carefully separated from the mucosal layer.…”

“…Western blot analysis: Western blot analysis was performed as described previously [19,20]. Muscle strips were homogenized in homogenizing buffer to extract Rnd2, Rnd3, -smooth muscle actin and heat shock protein 27 (HSP27).…”

“…Moreover, the organ culture method makes it possible to dissociate the influence of systemic cellular and humoral immune responses, thus allowing the direct effects of drugs on cell function to be investigated at constant drug concentrations. It has been reported that organ tissue cultures of intestinal cells without fetal calf serum exhibit well-maintained morphology and cellular functions, including muscle contractility, for several days [8,12,19,20].…”