MYOSIN LIGHT-CHAIN PHOSPHATASE-ACTIVITY IN RAGWEED POLLEN-SENSITIZED CANINE TRACHEAL SMOOTH-MUSCLE

We have reported that myosin light chain phosphorylation is increased in contracting airway smooth muscle from hyperresponsive. ragweed poll en-sensitized dogs. This alteration is manifest physiologically in smo oth muscle tissue from sensitized animals as it demonstrates faster sh ortening velocity and increased shortening capacity. One of the mechan isms underlying the defect is increased myosin light chain kinase acti vity; it is not known whether modulation of myosin phosphatase activit y contributes to enhanced myosin light chain phosphorylation in sensit ized canine smooth muscle. We describe a myosin phosphatase assay that we have used to compare the enzyme's activity in crude tracheal smoot h muscle tissue homogenates from control and sensitized airway smooth muscle. Twenty kilodalton myosin light chain phosphorylation was initi ated with Mg2+-ATP, and maximum levels were reached within 40 s; peak phosphorylation levels were stable for at least 3 min. The relative st oichiometry of 20 kD myosin light chain phosphorylation was estimated by chemiluminescent immunoblot assay. Smooth muscle phosphatase activi ty was estimated by the rate of decline in peak light chain phosphoryl ation, while myosin light chain kinase was inhibited indirectly with t rifluoperazine, with EGTA, or directly by a synthetic peptide inhibito r. Okadaic acid, an inhibitor of phosphatase activity, curbed the decl ine in light chain phosphorylation seen after myosin light chain kinas e inhibition, indicating that the light chain dephosphorylation observ ed was the result of smooth muscle phosphatase activity. Addition of o kadaic acid to the samples led to a 30 to 40% increase in the peak myo sin light chain phosphorylation attained for all samples. This indicat es that similar populations of phosphatases were present in the homoge nates of both control and sensitized tissues. Peak light chain phospho rylation levels were 20% higher in tracheal homogenates from sensitize d animals; however, no difference in phosphatase activity was measured between control and sensitized samples. These results indicate that i n airway smooth muscle from hyperresponsive, ragweed pollen-sensitized dogs, increased maximum velocity of shortening, consequent to the inc reased myosin light chain phosphorylation previously reported by us, i s not contributed to by any change in myosin light chain phosphatase a ctivity but is mainly the result of the increased myosin light chain k inase activity previously reported by us.