Calcium-independent activation of the contractile apparatus in smooth muscle of mouse aorta by protein phosphatase inhibition

Abstract.

Smooth muscle contraction is primarily regulated by reversible phosphorylation of the 20-kDa light chains of myosin (MLC₂₀) involving Ca²⁺-calmodulin-dependent myosin light chain kinase (MLCK) and serine/threonine protein phosphatases (PP).

The aim of this study was to investigate the effects of the protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) inhibitor cantharidin (Cant), its structural analogue endothall (ETA) and microcystin LR (MC) on force of contraction and MLC₂₀-phosphorylation in arterial smooth muscle of mouse aorta. Cant increased force of contraction and MLC₂₀-phosphorylation in intact arterial rings of mouse aorta in the presence of Ca²⁺ whereas ETA and MC were ineffective under the same experimental conditions. In contrast, all compounds induced contraction and led to enhanced MLC₂₀-phosphorylation in nominally Ca²⁺-free solution in fibers of mouse aorta permeabilised (skinned) with Triton X-100.

In addition, Western blot analysis revealed that skinning of mouse aorta did not result in a loss of PP1 and PP2A compared to intact rings. Thus, both PP must be tightly bound to structural proteins, e.g. myosin. The findings indicate a Ca²⁺-independent mechanism of smooth muscle contraction involving inhibition of PP1- and/or PP2A-activities leading to enhanced force and MLC₂₀-phosphorylation of arterial smooth muscle.