Role of SM 22 in the differential regulation of phasic versus 1 tonic smooth muscle

43 Preliminary proteomics studies (actual data is not given here) between tonic vs. phasic smooth 44 muscles identified three distinct protein spots identified to be those of SM22. The latter was 45 found to be distinctly down regulated in the internal anal sphincter (IAS) vs. rectal smooth 46 muscle (RSM) SMC. The major focus of the present studies was to examine the differential 47 molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the 48 IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before 49 and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 siRNA. pFLAG50 SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the 51 RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the 52 SMCs. Further studies revealed significant reverse relationship between the levels of SM22 53 phosphorylation and the amount of SM22-actin binding, in the IAS and RSM SMC. Data 54 showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and 55 reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 56 phosphorylation in these smooth muscles revealed that Y27632 (rho kinase inhibitor) but not Gö 57 6850 (PKC inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We 58 speculate that SM22 plays an important role in the regulation of basal tone via ROCK-induced 59 phosphorylation of SM22. 60 61