| Smooth muscle of the gastrointestinal tract propels food through the GI tract to allow for digestion and absorption of nutrients. Similar to skeletal muscle, the contraction of visceral SMCs is caused by the sliding of actin and myosin filaments activated by a rise in intracellular Ca2+. The biggest source of Ca2+ influx is through voltage dependent calcium channels (VDCC), and the VDCC of interest in GI visceral SMCs is CaV1.2. TS1 and TS2 mice have gain of function and knockdown mutations in CaV1.2 channels, respectively, as determined by function21. Taking advantage of these two mutations, visceral SMCs were isolated for up to 3 weeks in order to observe the effects on Ca2+ handling. Wild type SMCs responded to ATP in Ca2+ containing buffer and ATP in Ca2+ free buffer, but didn't respond readily to ATP in Ca2+ free buffer containing EGTA suggesting that the Ca2+ necessary for contraction comes from the external cellular environment. Wild type cells also responded to Cch and Substance P in Ca2+ containing buffer. TS2 cells responded to ATP in Ca2+ containing buffer and Cch in Ca2+ containing buffer. To assess the state of the cultured SMCs after one, two, and three weeks in culture, fluorescence imaging was performed. The fluorescence assessed the presence of smoothelin, a marker of smooth muscle cells, CaV1.2 antibody, a marker for CaV1.2 channels, and Hoescht die, a marker of the nucleus. It was found that smoothelin is present in smooth muscle cells up to three weeks in culture, suggesting that the cells did not dedifferentiate. CaV1.2 was seen to have higher expression in the nucleus, suggesting that the channels are co-localized to the nucleus. Since the fluorescence marker used for the CaV1.2 channel marks the C-terminal end of the protein, it is likely that this segment of the channel protein is cleaved and co-localized to the nucleus such that it inhibits the CaV1.2 channel gene 39. In summary, visceral SMCs were successfully cultured for up to three weeks although Ca2+ handling was likely altered due to the cleavage of the C-terminal end and inactivation of the CaV1.2 channel. |
