Proinflammatory action of sphingolipids in primary cultured rat intestinal smooth muscle cells

Abstract

Postoperative ileus or paralysis of the bowel is a transient but life-threatening impairment of bowel motility that occurs after surgery. Several pathogenic mechanisms responsible for postoperative dysmotility have been proposed including the involvement of autonomic nervous system, inhibitory humoral agents, anesthetic agents and local factors such as inflammation. Studies on inflammation progressively revealed a key role of sphingolipids in inflammatory responses. We have, therefore, investigated the involvement of sphingolipids in postoperative inflammation using a standardized surgical rodent model of intestinal manipulation. <br /> Our data demonstrate for the first time a direct correlation between the bioactive sphingolipids, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), and postoperative intestinal inflammation. Sphingolipid analysis by mass spectrometry of both intestinal <i>muscularis</i> and <i>mucosa</i> after manipulation revealed a time dependent increase of S1P and C1P only in the <i>muscularis</i> layer when compared to control animals. We therefore examined the potential role of these two bioactive sphingolipids in postoperative inflammation using primary cultured rat intestinal smooth muscle (RISM) cells. Our studies indicate that S1P induces an appreciable proinflammatory response in these cells by elevating the cyclooxygenase-2 (COX-2) protein expression and formation of prostaglandin E₂ (PGE₂), the most abundant COX-2 product. S1P-induced COX-2 expression was strongly inhibited by pertussis-toxin (PTX), indicating the involvement of G_i/o protein coupled S1P receptors. Studies with pharmacological inhibitors indicate that S1P-induced COX-2 expression is mediated by both, extracellular-signal regulated kinase (ERK) and p38 mitogenic-activated protein kinase (p38 MAPK). In addition, an increased secretion of interleukin-6 (IL-6), after S1P administration was observed. We have also shown that S1P is involved in tumor necrosis factor-α (TNF-α) induced IL-6 secretion. Indeed, an increased sphingosine kinase (SK) activity was observed upon stimulation of RISM cells with TNF-α. C1P also enhanced PGE2 synthesis but without any effect on COX-2 expression. However, C1P stimulated arachidonic acid release, which is indicative for the activation of phospholipase A2 (PLA2). Moreover, a direct correlation between mechanical stress and activation of S1P and C1P formation was established in RISM cells. Collectively, our results demonstrate that intestinal smooth muscle cells represent a major target for both, C1P and S1P activity. Thus, the elevated content of the two bioactive sphingolipids in this tissue could at least in part explain postoperative intestinal dysmotility. In addition, in the present study the role of sphingolipids in lipopolysaccharide (LPS)-induced inflammatory reactions was addressed using a well-established sepsis model. We found an increase in the content of S1P and C1P after LPS administration indicating for the first time a direct involvement of these two bioactive sphingolipids in LPS signalling in an animal model. Moreover, studies on RISM cells reveal that SK mediates LPS-induced IL-6 secretion with a possible activation of ceramide kinase (CK). <br /> The findings that the two bioactive sphingolipids specifically increase in the muscle layer after intestinal manipulation and upon LPS administration, as well as the fact that they induce and mediate pro-inflammatory responses in RISM cells, make them interesting targets for development of novel anti-inflammatory drugs. Our data raise the possibility to overcome postoperative- or sepsis-induced ileus by a targeted disruption of the sphingolipid-mediated inflammatory pathway.