Calmodulin antagonists inhibit latch bridges in detergent skinned swine carotid media.
Ca2+-calmodulin-dependent phosphorylation of the 20,000 apparent molecular weight (Mr) myosin light chain (MLC) is widely believed to be the primary regulator of smooth muscle stress development. Stress maintenance, however, has been hypothesized to be the result of a Ca2+-dependent state called latch, defined as stress maintenance by slowly cycling, dephosphorylated latch bridges. This study was designed to examine the role of calmodulin in the Ca2+ dependence of latch. Swine carotid medial fibers were detergent skinned with Triton X-100 and MLC phosphorylation levels were measured by two-dimensional gel electrophoresis. Experiments were performed in which calmodulin antagonists, W-7 or trifluoperazine (TFP), were used to prevent Ca2+-dependent contractions or to relax previously contracted muscle strips. Both protocols resulted in similar decreases in stress with MLC phosphorylation levels correlating to the level of stress under all conditions. In another series of experiments, the skinned fibers were first contracted with Ca2+ and ATP and then exposed to Ca2+ and cytidine 5'-triphosphate (CTP). This resulted in stress maintenance with basal levels of MLC phosphorylation. W-7 relaxed Ca2+ and CTP maintained stress in a concentration-dependent manner. The potency of W-7 for relaxation of the CTP maintained stress was similar to that for inhibition of the ATP developed stress determined in the first set of experiments. The results demonstrate that calmodulin antagonists inhibit stress associated with MLC phosphorylation and also stress independent of MLC phosphorylation. This suggests that a calmodulin-like, Ca2+-binding protein may be important for latch bridge attachment.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002339:Carotid Arteries / Q000378:metabolism / Q000502:physiology*; D003570:Cytidine Triphosphate / Q000494:pharmacology; D003902:Detergents; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects*; D009218:Myosins / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D013449:Sulfonamides / Q000494:pharmacology; D013552:Swine; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D003902:Detergents; D007093:Imidazoles; D013449:Sulfonamides; D014268:Trifluoperazine; C031938:calmidazolium; D003570:Cytidine Triphosphate; C017967:W 7; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium; D002118:Calcium