P J Gallagher

Vascular smooth muscle contractile elements. Cellular regulation.

For many years the simple view was held that contractile force in smooth muscle was proportional to cytosolic Ca2+ concentrations ([Ca2+],). With the discovery that phosphorylation of myosin light chain by Ca2+/calmodulin-dependent myosin light chain kinase initiated contraction, regulation of the contractile elements developed more complex properties. Molecular and biochemical investigations have identified important domains of myosin light chain kinase: light chain binding sites, catalytic core, pseudosubstrate prototope, and calmodulin- binding domain. New protein phosphatase inhibitors such as okadaic acid and calyculin A should help in the identification of the physiologically important phosphatase and potential modes of regulation. The proposal of an attached, dephosphorylated myosin cross bridge (latch bridge) that can maintain force has evoked considerable controversy about the detailed functions of the myosin phosphorylation system. The latch bridge has been defined by a model based on physiological properties but has not been identified biochemically. Thin-filament proteins have been proposed as secondary sites of regulation of contractile elements, but additional studies are needed to establish physiological roles. Changes in the Ca2+ sensitivity of smooth muscle contractile elements with different modes of cellular stimulation may be related to inactivation of myosin light chain kinase or activation of protein phosphatase activities. Thus, contractile elements in smooth muscle cells are not dependent solely on [Ca2+], but use additional regulatory mechanisms. The immediate challenge is to define their relative importance and to describe molecular-biochemical properties that provide insights into proposed physiological functions.

Regulation of Myosin Light Chain Kinase Activity in Smooth Muscle

Phosphorylation of myosin regulatory light chain by Ca2+/calmodulindependent myosin light chain kinase (MLCK) plays a central role in smooth muscle contractility. The quantitative relation between intracellular Ca2+ concentrations and light chain phosphorylation is not fixed but modulated dynamically by various regulatory processes. Most of the calmodulin in smooth muscle cells is tightly bound to cellular elements even in the absence of Ca2+. Surprisingly, even the most mobile fraction (8% of the total) has a diffusion coefficient in smooth muscle cells sevenfold lower than a freely diffusible, similar size dextran. The possibility is considered that calmodulin available for MLCK activation is limiting. Furthermore, calmodulin availability may be regulated by unidentified processes. The multifunctional Ca2+/ calmodulin-dependent protein kinase II phosphorylates MLCK adjacent to the C-terminus of the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence physiologically increases the Ca z concentration required for light chain phosphorylation. However, in smooth muscle cells the concentration of Ca2+ necessary for MLCK phosphorylation is greater than that required for light chain phosphorylation. Thus MLCK is sensitive to small increases in Ca2+ during the initiation of contraction and subsequently becomes desensitized to Ca2+ after phosphorylation, thereby limiting light chain phosphorylation.