MAPK Activation and Cell Viability after H2O2 Stimulation in Cultured Feline Ileal Smooth Muscle Cells

Recent data have shown the importance of oxidative stresses in the pathogenesis of inflammatory bowel disease, crohn's disease and ulcerative colitis. H2O2, reactive oxygen species (ROS) donor, has been reported to act as a signaling molecule involved in a variety of cellular functions such as apoptosis and proliferation. In the present study, we investigated viability of cultured ileal smooth muscle cells (ISMC) after stimulation with H2O2. Trypan blue method revealed that the cell viability of ISMC treated with 1 mM H2O2 was not different from that of controls at up to 2 h time point, while treatment of ISMC with 1 mM H2O2 for 48 h finally induced significant decrease in the cell viability. Therefore, we evaluated whether H2O2 was capable of ERKs activation in ISMC for the short-term exposure and examined whether tyrosine kinase was involved in the process of ERK activation by H2O2 in ISMC. We also investigated the effects of H2O2 on activation of SAPK/JNK and p38 MAP kinase in ISMC. Thus, ISMC were cultured and exposed to H2O2, and western blot analysis was performed with phospho- specific MAP kinase antibodies. Robust activation of ERK occurred within 30 min of 1 mM H2O2 treatment. H2O2-induced ERK activation was attenuated by a tyrosine kinase inhibitor, genistein, indicating that tyrosine kinase was probably involved in the ERK activation by H2O2. H2O2 was a moderate activator of SAPK/JNK, while p38 MAP kinase was not activated by H2O2. We suggest that ERK activation induced by short-term H2O2 treatment plays a critical role in cellular protection in the early stage of response to oxidative stress. The present study suggests the necessity of identification of MAPK signaling pathways affected by ROS, since it could ultimately elucidate cellular consequences involved in initiation and perpetuation of intestinal tissue damage in the diseases such as crohn's disease and ulcerative colitis, resulted from excessive ROS.

Cooperation of Gbeta and Galphaq Protein in Contractile Response of Cat Lower Esophageal Sphincter (LES)

We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gbeta protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1, but not PLC-beta2, PLC-beta4, PLC-gamma2, PLC-delta1, and PLC-delta2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-beta1 or PLC-beta3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-gamma1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-beta1 or PLC-beta3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-beta1 and PLC-beta3 antibody completely abolished the contraction, suggesting that PLC-beta1 and PLC-beta3 have a synergism to inhibit the contraction in LES. PLC-beta1, -beta3 or -gamma1 antibody did not reduce the contraction of LES cells in response to DAG (10 (-6) M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC beta3, but not of PLC beta1 was additive (Fig. 6). In contrast, when Gbeta antibody was incubated, the inhibitory effect of the incubation of PLC beta1, but not of PLC beta3 was additive. This data suggest that Gq/11 or Gbeta may activate cooperatively different PLC isozyme, PLCbeta1 or PLCbeta3 respectively.