UBC Theses and Dissertations
Changes in protein phosphorylation during changes in vascular smooth muscle tone Marshall, Caroline Louise
It is now generally accepted that changes in protein phosphorylation play an important role in mediating smooth muscle tone. An attempt was made to determine whether nitrogen oxide containing vasodilators such as sodium nitroprusside and nitroglycerin exert their relaxant effect via a phosphorylation reaction. Isoelectric focusing was initially used as a method of detecting phosphorylation. However, due to the smaller percentage of protein in smooth muscle with a relative increase in other cell constituents, crude smooth muscle homogenates were deemed to be too complex for analysis by this technique alone. Phosphorylation changes are commonly studied by incubating the muscle in labelled inorganic phosphate, thus labelling the ATP pools. Proteins are separated by SDS-polyacrylamide gel electrophoresis. Following staining and drying, gels are exposed to X-ray film and phosphorylation levels determined. By comparing aortic strips relaxed in Ca⁺⁺-free, 5 mM EGTA Krebs with muscle strips contracted in K⁺ (124 mM) Krebs, a significant difference between the phosphorylation levels of myosin light chain was quantitated. By reproducing this well documented phenomena, we demonstrated that we had established a working methodology in the laboratory. In view of the controversy in the literature concerning sustained levels of myosin light chain phosphorylation during sustained K⁺-induced contractions, a K⁺-time course study was performed. Levels of myosin light chain phosphorylation increased significantly within the first 2 min of contraction and were maintained for 12 min, though a non-significant decrease was observed after 2 min. Tension peaked at 4 min and thereafter remained constant. Finally, the effect of nitroglycerin (10⁻M) on K⁺-induced contractions was briefly examined. Nitroglycerin caused a 70% relaxation within 2 min and significantly decreased myosin light chain phosphorylation levels. There also appeared to be an increase in phosphorylation of a 160kD protein with nitroglycerin treatment, which due to technical difficulties could not be quantitated.