THIN FILAMENT REGULATION OF FORCE ACTIVATION IS NOT ESSENTIAL IN SINGLE VASCULAR SMOOTH-MUSCLE CELLS

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized s ingle smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 mu M ATP increased force, in-phase stiffness, and quadrature stiffness , while 20 mu M CTP did not increase any of these parameters, suggesti ng that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomeri zation. These results also suggest that for smooth muscle, force activ ation is regulated by myosin light-chain kinase. From rigor, 20 mu M A TP (pCa 9) increased force and quadrature without changing in-phase st iffness. This force increase could be explained if in rigor solution b oth actomyosin (AM) and AM ADP cross bridges exist (2, 32), and ATP-in duced detachment of AM cross bridges is accompanied by AM ADP cross br idges undergoing a force-producing isomerization in combination with c ooperative cross-bridge reattachment (36). Thus results of our experim ents suggest that thin filament-based regulation of force activation i s not essential in smooth muscle, and a population of cross bridges mu st begin in an attached state for force to be produced in the absence of MLC phosphorylation.