A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

Abstract

The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (Pi) and H+ ([MgADP], [Pi] and [H+], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k1 [5.6+/-0.1 s-1 for barnacle, n=30; 26.3+/-0.7 s-1 for frog, n=14 (mean+/-SEM)] and k2 [0.6+/-0.1 s-1 in barnacle, n=30; 10.4+/-1.0 s-1 in frog, n=14 (mean+/-SEM)], at 10 degrees C. Decreasing the pH by 0.5 pH units did not significantly affect k1 for barnacle relaxation [5.6+/-0.1 s-1 (mean+/-SEM), n=15] compared to the decrease in k1 of 40% seen in frog. Use of the Ca2+-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa50 value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in koff from the labelled protein [322+/-32 s-1 at pH 7.0 and 381+/-24 s-1 (mean+/-SEM) at pH 6.6]. Increasing [MgADP] by 20 microM (50 microM added ADP) from control values of 50 microM in frog decreased k1 to 12.3+/-0.4 s-1 (mean+/-SEM, n=8), and at 400 microM MgADP, k1=9.6+/-0.1 s-1 (mean+/-SEM, n=12). In barnacle, 500 microM MgADP had a much smaller effect on k1 (4.0+/-0. 9 s-1, mean+/-SEM, n=8). Increasing the free [Pi] from the contaminant level of 0.36 mM to 1.9 mM slowed k1 by approximately 15% in barnacle [4.8+/-0.8 s-1, mean+/-SEM, n=7], compared to a approximately 30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation.

Basal myosin light chain phosphorylation is a determinant of Ca2+ sensitivity of force and activation dependence of the kinetics of myocardial force development

“…MgADP is thought to exert these effects by competing with MgATP for the nucleotide-binding pocket on myosin, thereby slowing cross-bridge detachment (9), relaxation from rigor (22), relaxation from active contraction (19), and overall cycling rate (17). From these results in skeletal muscle, it is tempting to postulate that RLC phosphorylation in cardiac muscle prolongs attached states by influencing MgADP dissociation kinetics.…”

Skeletal muscle contractile performance and ADP accumulation in adenylate kinase-deficient mice

“…Muscle relaxation is ultimately due to the detachment of myosin from actin. Increasing concentrations of ADP prolongs the rate of myosin detachment after full isometric activation (23,30,55). If this applies to our muscle with elevated ADP, there should be a prolonged maintenance of force after the end of stimulation, similar to the delay in early relaxation apparent in AK Ϫ/Ϫ muscle (Fig.…”

“…ADP has been shown to slow the rate of cross-bridge detachment after fully activated isometric contractions in skeletal (23,30,55) and cardiac (52) muscle fibers. This is thought to be the same reason that unloaded shortening velocity is slowed: more cross-bridges populate the strong bound ADP state longer with higher ADP concentrations.…”

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

“…Release of cross-bridge strain accelerates the release of ADP and thereby their detachment from actin according to kinetic studies of nucleotide exchange rates in shortening myofibrils [128]. ADP slows down force relaxation of skeletal and cardiac myofibrils and fibres which proves cross-bridge detachment via ADP release to be an important pathway rate-limiting relaxation kinetics [93,130,137,147]. Fig.…”

“…This problem can be overcome by loading fibres with photo-labile caged compounds that can rapidly release products, e.g. Ca 2+ or Ca 2+ chelators, by light flashes [4,9,30,69,93,96,100,111,149,156]. However, the use of caged compounds has other drawbacks (discussed in [46,117]).…”