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 pollen-sensitized dogs. This alteration is manifest physiologically in smooth muscle tissue from sensitized animals as it demonstrates faster shortening velocity and increased shortening capacity. One of the mechanisms underlying the defect is increased myosin light chain kinase activity; it is not known whether modulation of myosin phosphatase activity contributes to enhanced myosin light chain phosphorylation in sensitized canine smooth muscle. We describe a myosin phosphatase assay that we have used to compare the enzyme's activity in crude tracheal smooth muscle tissue homogenates from control and sensitized airway smooth muscle. Twenty kilodalton myosin light chain phosphorylation was initiated with Mg2+-ATP, and maximum levels were reached within 40 s; peak phosphorylation levels were stable for at least 3 min. The relative stoichiometry of 20 kD myosin light chain phosphorylation was estimated by chemiluminescent immunoblot assay. Smooth muscle phosphatase activity was estimated by the rate of decline in peak light chain phosphorylation, while myosin light chain kinase was inhibited indirectly with trifluoperazine, with EGTA, or directly by a synthetic peptide inhibitor. Okadaic acid, an inhibitor of phosphatase activity, curbed the decline in light chain phosphorylation seen after myosin light chain kinase inhibition, indicating that the light chain dephosphorylation observed was the result of smooth muscle phosphatase activity. Addition of okadaic acid to the samples led to a 30 to 40% increase in the peak myosin light chain phosphorylation attained for all samples. This indicates that similar populations of phosphatases were present in the homogenates of both control and sensitized tissues. Peak light chain phosphorylation levels were 20% higher in tracheal homogenates from sensitized animals; however, no difference in phosphatase activity was measured between control and sensitized samples. These results indicate that in airway smooth muscle from hyperresponsive, ragweed pollen-sensitized dogs, increased maximum velocity of shortening, consequent to the increased myosin light chain phosphorylation previously reported by us, is not contributed to by any change in myosin light chain phosphatase activity but is mainly the result of the increased myosin light chain kinase activity previously reported by us.