The Study Of Bidirectional Regulation Of The Combination Of Stimulatory And Inhibitory Agents On Smooth Muscle Myosin And Smooth Muscle Contractility

It is generally accepted that the interaction between actin and phosphorylated myosin by myosin light chain kinase (MLCK) in a Ca2+-CaM dependent manner is the principal mechanism in regulation of smooth muscle contraction. Moreover, in living system, we may notice that bidirectional regulation is also a common mechanism in modulating smooth muscle contractility. And in our previous study, we demonstrated that bidirectional regulation was observed via a combination of the stimulatory agent emodin and the inhibitory agent quercetin, which enhanced myosin activities for partially phosphorylated myosin and inhibited those activities for fully phosphorylated myosin. To determine whether the previously observed phenomenon is unique or represents a rule in regulating smooth muscle contraction, we have selected several stimulatory and inhibitory agents, using them concurrently in pairs, i.e., one stimulatory agent with one inhibitory agent. The results of this study are described as follows.Part one The characteristics of stimulatory and inhibitory agents on smooth muscle myosin and smooth muscle contractilityWe have chosen Mg2+-ATPase activity and phosphorylation of myosin light chains as molecular level indexes and isolated gastro-intestinal smooth muscle strips to determine the effects of each agent on the activities of smooth muscle myosin and on smooth muscle contractility, respectively. The results indicate that (a) berberine and synephrine exert inhibitory effects, while hesperidin produces stimulatory effects on myosin Mg2+-ATPase activities in the presence of Ca2+/calmodulin and in a dose-dependent manner; (b) berberine and synephrine exert inhibitory effects, while hesperidin produces stimulatory effects on myosin phosphorylation in the presence of Ca2+/calmodulin and in a dose-dependent manner; (c) hesperidin increased while berberine, synephrine, capsaicin and baicalin inhibited the muscle contraction of isolated rat intestine and gastric strips in a dose-dependent manner.Part two Counterbalance on smooth muscle myosin function and smooth muscle contractility by a combination of stimulatory and inhibitory agentsThe methods of this study were as described in Part one. In this study, we provided evidence of bidirectional regulation using one stimulatory and one inhibitory agent concurrently, i.e., hesperidin with berberine, hesperidin with synephrine, hesperidin with quercetin, emodin with berberine, and emodin with synephrine. The common characteristics of these combinations are showed as that inhibitory effects are observed when myosin is fully phosphorylated and gastric smooth muscle stripe is present in a relatively high contractile state, and that stimulatory effects are observed when myosin is partially phosphorylated and gastric smooth muscle stripe is in a relatively low contractile state. These results indicate that counterbalance is a common feature of bidirectional regulation using stimulatory and inhibitory agents concurrently. The significances of the counterbalance resulting from bidirectional regulation are as follows. (a) The smooth muscle is not over-contracted or over-relaxed if the combination is applied, offering a possible way to modify smooth muscle malfunction. (b) The phenomena of bidirectional regulation resulted from concurrent use of one stimulatory and one inhibitory agent in regulating myosin activity and contractility of smooth muscle suggest a possible rule in regulating the contractility of smooth muscle. (c) This counterbalance might be useful to minimize the side effects of agents used alone. (d) The results might provide some valuable information for drug clinical applicaiton. This study provides an alternative viewpoint for the further investigation of mechanisms of regulation of smooth muscle contraction and for the further exploration of new medicines for diseases of smooth muscle.