FLASH-PHOTOLYSIS STUDIES OF RELAXATION AND CROSS-BRIDGE DETACHMENT - HIGHER SENSITIVITY OF TONIC THAN PHASIC SMOOTH-MUSCLE TO MGADP

The effects of MgADP and inorganic phosphate (P-i) on cross-bridge detachment were determined in tonic (rabbit femoral artery) and phasic (rabbit bladder and guinea pig portal vein) smooth muscles permeabilized with staphylococcal alpha-toxin. Relaxation from rigor was induced by photolysis of ATP (1.2-1.5 mM) from caged ATP. The initial one second of relaxation from rigor was resolved into two exponential components: a rapid component with normalized amplitudes, A(f), of 8, 15 and 26% and rate constants, k(f) (in s(-1)) of 26, 36 and 30 in rabbit femoral artery, guinea pig portal vein, and rabbit bladder; the respective rate constants of the second, slower component, k(s), were 0.07, 0.2 and 0.1. Removal of residual endogenous ADP with apyrase treatment increased the amplitude A(f) and accelerated k(s); addition of MgADP reduced A(f). The combination or. these effects (increases in A(f) and k(s)) decreased the t(1/2) of relaxation from control values by factors of 2.6 (femoral artery), 6.7 (portal vein) and 10 (bladder). P-i (30 mM) further increased the amplitudes A(f). The affinity of MgADP for myosin cross-bridges, estimated as the reduction of the relative amplitude of the rapid component, A(f), was significantly higher in tonic than in phasic smooth muscle: the K-D of MgADP was 1.1+/-0.3 mu M in rabbit femoral artery- and 4.9+/-1.0 mu M in rabbit bladder. The higher affinity of tonic smooth muscle myosin for MgADP correlated with its relatively high LC(17b) isoform content (58+/-4.2%) in contrast to the lower affinity of the phasic, bladder detrusor smooth muscle that contained only the LC(17a) isoform. The t(1/2) of relaxation from rigor was markedly slowed down by MgADP (10-200 mu M) in femoral artery, but not in bladder smooth muscle. We suggest that: 1. The high affinity of myosin for MgADP and the consequent maintenance of a strongly bound, AM.ADP state by dephosphorylated cross-bridges, as well as cooperative reattachment of unphosphorylated cross-bridges, contribute to the maintenance of 'latch'. The acceleration of relaxation by P-i is consistent with the reversal of step(s), including cooperative attachment of nonphosphorylated cross-bridges, that precede entry into strongly bound, force-generating states. The higher affinity for myosin and the greater slowing of relaxation by MgADP in tonic, than phasic, smooth muscle suggest that MgADP may have a greater role in force maintenance at low levels of MLC(20) phosphorylation in the tonic muscles. 2. The absence or low expression of the LC(17b) isoform and/or the presence of a seven amino acid insert near the catalytic site of the myosin heavy chain in phasic smooth muscle may be responsible for their lower affinity for MgADP and for their resultant faster kinetics. 3. The marked slowing by MgADP of the late phases of relaxation from rigor in femoral artery suggests that in tonic smooth muscle the nucleotide affects not only rigor bridges, but also additional state(s) of cooperatively cycling cross-bridges. The lack of effect of MgADP on the later phases of relaxation from rigor in bladder smooth muscle suggests that in this, phasic, smooth muscle MgADP affects primarily cross-bridges in the rigor (AM) state.