Contractile protein isoforms of single and cultured smooth muscle cells from guinea pig ileum.
Single smooth muscle cells isolated from guinea pig ileum using collagenase and papain produce contractile response to muscarinic agents, while the cultured cells do not. Using fluo-3/AM and a confocal laser scanning fluorescence microscope, it was observed that carbachol, a muscarinic agent, caused an increase in the intracellular Ca2+ of both single and cultured cells. SDS-PAGE and Western Blot analyses revealed the expression of myosin heavy chain isoforms of SM1 (204 kDa) and SM2 (200 kDa) in single smooth muscle cells, and non muscle isoform (196 kDa) of myosin heavy chain only in the cultured cells. With respect to actin isoforms, alpha-actin was predominant in single cells and beta-actin was major in the cultured cells. Two types of tropomyosin monomer, 39 kDa and 41 kDa, were detected in single cells, while the 41 kDa monomer was lost in cultured cells. These differences in contractile protein profiles between single and cultured cells were collaborated with the observation of cells using immunofluorescence microscope with responsible antibodies to isoforms of myosin heavy chain, actin and tropomyosin. These results suggest that the loss of contractility in cultured smooth muscle cells is profoundly related to changes in contractile protein profiles from smooth muscle type to non muscle type.
Iijima
M
M
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Aoki
K
K
Aizawa
H
H
Ohata
H
H
Momose
K
K
D000199:Actins / Q000032:analysis*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D006168:Guinea Pigs; D007082:Ileum / Q000737:chemistry* / Q000166:cytology; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000737:chemistry* / Q000166:cytology; D018995:Myosin Heavy Chains / Q000032:analysis*; D020033:Protein Isoforms; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling / Q000379:methods; D014335:Tropomyosin / Q000032:analysis*
D000199:Actins; D018721:Muscarinic Agonists; D020033:Protein Isoforms; D014335:Tropomyosin; D012967:Sodium Dodecyl Sulfate; D002217:Carbachol; D018995:Myosin Heavy Chains; D002118:Calcium