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 single 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 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting 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 isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.
Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D014661:Vasoconstriction
D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D002118:Calcium