Abstract
Smooth muscle cell cultures are used frequently to investigate the cellular mechanisms of contraction. We tested the hypothesis that cell culture alters the expression of select cell-signaling proteins of excitation-contraction coupling in colon smooth muscle cells without altering the contractile phenotype. We used muscularis externa (ME) tissues, freshly dispersed cells (FC), primary cell cultures (PC), and resuspensions of cell cultures (RC). Colon smooth muscle cells retained their phenotype in all states. We investigated expression of 10 cell-signaling proteins of excitation-contraction coupling in all four types of tissue. Expression of all these proteins did not differ between ME and FC (P > 0.05). However, expression of the α(1C)-subunit of Ca(v)1.2b, myosin light chain kinase, myosin phosphatase target subunit 1, and 17-kDa C kinase-potentiated protein phosphatase-1 inhibitor (CPI-17) decreased in PC and RC vs. ME and FC (all P < 0.05). Expression of Gα(i3), serine/threonine protein phosphatase-1 β-catalytic subunit, and Rho kinase 1 increased in PC and RC vs. ME and FC (all P < 0.05). Cell culture and resuspension downregulated expression of α-actin and calponin, but not myosin heavy chain. The net effect of these molecular changes was suppression of cell reactivity to ACh in RC vs. FC. Overexpression of CPI-17 in PC partially reversed the suppression of contractility in resuspended cells. Methylation-specific PCR showed increased methylation of the Cpi-17 gene promoter in PC vs. ME (P < 0.05). We concluded that smooth muscle cells retain their contractile phenotype in culture. However, reactivity to ACh declines because of altered expression of specific cell-signaling proteins involved in excitation-contraction coupling. DNA methylation of the Cpi-17 promoter may contribute to its gene suppression.