CHARACTERIZATION OF THE PROPERTIES OF CANINE COLONIC SMOOTH-MUSCLE INCULTURE

We have developed and characterized an organ culture system that maint ains the viability of colonic smooth muscles. Morphological, mechanica l, electrical, and molecular properties of cultured canine colonic cir cular muscles were determined. Strips of circular muscle were cultured for up to 6 days. The smooth muscle phenotype was retained during cul ture; muscles contracted to agonists and responded to electrical field stimulation, suggesting that intrinsic nerves also survived in cultur e. Morphological analysis showed identifiable smooth muscle cells, ent eric neurons, and interstitial cells, but some alterations in ultrastr ucture were also observed. Mechanical responses to acetylcholine sugge sted that the muscles developed supersensitivity during the culture pe riod. The resting membrane potentials of cells near the submucosal sur face of the circular muscle layer decreased from -82 mV on day 0 to -5 5 mV on day 3. Similar changes in the resting potential gradient occur when colonic muscles are treated with inhibitors of the Na+-K+-ATPase . Resting potentials of day 3 muscles remained constant in low externa l K+ (0.1 mM), suggesting little contribution of the pump to resting p otential. Northern analysis of RNA from muscles cultured up to 6 days showed that the a2-isoform of the pump decreased. The data suggest tha t organ-cultured strips of smooth muscle may provide a useful tool for evaluating electrical and mechanical events in conjunction with molec ular analysis of functional components.