Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. A laser-flash photolysis study.

Abstract

A B STR ACT The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig portal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an -45 ~ angle. In the absence of Ca 2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some crossbridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM P~, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca 2+, was estimated to be 0.1-2.5 x 105 M-~s -~, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca "+, force development occurred at a rate (0.4 s -~) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP~,S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca 2+, consistent Address reprint requests to Dr. Avril V. Somlyo,

siRNA-mediated knockdown ofh-caldesmon in vascular smooth muscle

“…Caldesmon has been proposed to play a role in maintaining the high level of force during the latch state (31); however, it has not been fully determined as to whether or not this is one of its physiological roles with regards to contraction. Another area to be investigated with regards to a possible role for caldesmon is the theory of cooperativity, which suggests that cooperative activation of nonphosphorylated cross bridges occurs via phosphorylated cross bridges (36,39,39). It has been proposed that caldesmon may regulate the cooperative activation of cross bridges, but there has been little evidence supporting this potential role for caldesmon in vivo.…”

Agonist-induced Ca2+ Sensitization in Smooth Muscle

“…Tissue Preparation and Force Measurements-Mouse or rabbit portal veins were dissected, denuded of endothelium, cut into small strips (150 -250 m wide, 2-3 mm long), and mounted on a bubble plate or on a wire myograph system for force measurements (20) or in a muscle trough for photolysis experiments (21). The magnitude of contraction with 154 mM K ϩ was measured prior to stimulation with different agonists (20).…”

“…Apparatus and Photolysis Techniques-The UV laser for laser flash photolysis, a computer-controlled muscle trough system for solution exchange, the force transducer, and the data collection system have been described in detail previously (21,22). Protocols for Ca 2ϩ measurements, loading of the AM ester of Fluo3 into intact portal vein, pretreatment with cyclopiazonic acid, and introduction of caged compounds and their photolysis are described in the supplemental Experimental Procedures.…”

Smooth muscle myosin: regulation and properties

“…Positive strain on the cross-bridges may also contribute to the slowing of the cycle. We have suggested that, given the high ratio of actin to myosin filaments in smooth muscle (Ashton et al 1975), it is possible that following the initial attachment of cross-bridges, at least some of the ATPase breakdown occurs through the slower associated state hydrolysis pathway (White et al 1997) and/ or cooperative cycling of non-phosphorylated myosin (Himpens et al 1988;Somlyo et al 1988;Vyas et al 1992;Butler & Siegman 1998).…”

“…This high force-low phosphorylation state (catch-like state, also known as latch) occurs in smooth muscle after a decline in Ca 2þ concentration that initiates dephosphorylation of RLC (Dillion et al 1981). The high affinity of smooth muscle myosin for ADP (Vyas et al 1992;Fuglsang et al 1993;Nishiye et al 1993;Butler & Siegman 1998;Cremo & Geeves 1998;Gollub et al 1999), cycling of dephosphorylated cross-bridges (Vyas et al 1992;Butler & Siegman 1998;Somlyo et al 1988) and the effects of strain and RLC thiophosphorylation on product release (Khromov et al 2004) contribute to this catch-like state and will be one focus of this review along with a survey of some of the structure-linked changes evident in smooth muscle myosin.…”