The role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle: a flash photolysis study.
The effect of [MgADP] on relaxation from isometric tension, initiated by reducing free [Ca2+] through photolysis of the caged photolabile Ca2+ chelator diazo-2, was determined at 20 degrees C in alpha-toxin permeabilized tonic (rabbit femoral artery, Rf) and phasic (rabbit bladder, Rb) smooth muscle. In Rf, the shape of the relaxation curve was clearly biphasic, consisting of a slow "plateau" phase followed by a monotonic exponential decline with rate constant k. The duration of the plateau (d = 44 +/- 4 s, mean +/- SEM, n = 28) was well correlated (R = 0.92) with the total t1/2 of relaxation that was 66 +/- 3 s (n = 28) in the presence of 20 mM creatine phosphate (CP), and was prolonged in the absence of CP (t1/2 = 83 +/- 3 s, n = 7); addition of 100 microM MgADP further slowed relaxation (t1/2 = 132 +/- 7 s, n = 14). In Rb, a plateau was not detectable and t1/2 (= 15 +/- 2 s, n = 6) was not affected by 100 microM MgADP. In Rf the Q10 between 20 degrees C and 30 degrees C was 4.3 +/- 0.4 for d-1 and 2.8 +/- 0.3 for k (n = 8; p = 0.006). The regulatory myosin light chain (MLC20) in Rf was dephosphorylated at 0.07 +/- 0.02 s-1, from 42 +/- 3% before to 20 +/- 2% after photolysis of diazo-2, reaching basal values at a time when force had fallen by only 40%. We conclude that, in the presence of ATP, as during rigor, the affinity of dephosphorylated cross-bridges for MgADP is significantly higher in tonic than in phasic smooth muscle and contributes to the maintenance of force at low levels of phosphorylation. The MgADP dependence of the post-dephosphorylation phase of relaxation is consistent with its being rate-limited by the slow off-rate of ADP from cross-bridges that were dephosphorylated while in force-generating ADP-bound (AM*D) cross-bridge states. The fourfold faster off-rate of ADP from AM*D in the phasic, Rb, compared to tonic, Rf, smooth muscle is a major determinant of the different kinetics of relaxation in the two types of smooth muscle.
Khromov
A
A
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908, USA.
Somlyo
A V
AV
Trentham
D R
DR
Zimmermann
B
B
Somlyo
A P
AP
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003979:Diazonium Compounds; D004533:Egtazic Acid / Q000494:pharmacology; D004548:Elasticity; D005263:Femoral Artery / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008433:Mathematics; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D010642:Phenoxyacetates; D010766:Phosphorylation; D010782:Photolysis; D011817:Rabbits; D010738:Type C Phospholipases / Q000494:pharmacology; D001743:Urinary Bladder / Q000187:drug effects / Q000502:physiology
D002614:Chelating Agents; D003979:Diazonium Compounds; D018994:Myosin Light Chains; D010642:Phenoxyacetates; C063685:diazo-2; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D010738:Type C Phospholipases; D002118:Calcium