Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle

Abstract

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-P(i)) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2+-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 μm in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+<1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+=0.5 μM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab). MLC-P(i), quantitated by an immunoblotting assay, was increased from basal values of 0.13±0.03 mol P(i)/mol 20-kDa myosin light chain (n=4) to 0.62±0.01 (n=9) in contracting solution. MLC-P(i), however, was lowered to only 0.36±0.01 (n=7) after 120 minutes in the presence of anti-MLCK Fab in contrast to the complete inhibition of force. Moreover, anti-MLCK Fab also relaxed a contraction induced by ATPγS without any apparent decrease in MLC-P(i). These data suggest that antibodies inhibiting specific contractile protein function can be used as functional probes in the skinned fiber model system. In addition to inhibiting MLCK catalytic activity, anti-MLCK Fab may relax smooth muscle by a mechanism involving a structural role for MLCK.