Abstract
Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca2+/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [gamma-32P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 microM Ca2+ caused a 30-40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 micrograms/ml) in the presence of calmodulin (10 microM) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca2+ in digitonin-treated cells. In the presence of 1 microM Ca2+, myosin light chain kinase (400 micrograms/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 microM Ca2+. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.
Highlights
At a meeting one year ago Peter Baker overheard one of us (R.W.H.) reminiscing to a colleague how quickly understanding in a new area had developed several years before
Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at t # M .Ca 2§ Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase
In the following study we have investigated the effects of myosin light chain kinase (130-kDa), an important regulator of nonmuscle myosin, on phosphorylation and secretion in digitonin-treated chromaffin cells
Summary
At a meeting one year ago Peter Baker overheard one of us (R.W.H.) reminiscing to a colleague how quickly understanding in a new area had developed several years before. In the following study we have investigated the effects of myosin light chain kinase (130-kDa), an important regulator of nonmuscle myosin, on phosphorylation and secretion in digitonin-treated chromaffin cells. The possible involvement in exocytosis of contractile proteins which respond to Ca 2+ was originally suggested by Douglas and colleagues because of the similar requirement for extracellular Ca z§ of muscle contraction and catecholamine secretion from chromaffin cells [7, 8]. In the following work with digitonin-permeabilized chromaffin cells we deraonstrate that myosin light chain kinase phosphorylates intracellular myosin light chain but does not alter Ca 2+-dependent catecholamine secretion
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