Abstract
The pre-myosin light chain (MLC20) phosphorylation components of the lag phase (td) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2+ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin (CaM) shortened the td (470 ms at 0 added CaM) that followed Ca2+ release, but its effect (td = approximately 200 ms) saturated at 40 microM. Photolysis of caged ATP following preequilibration with identical [Ca4CaM] shortened td to 41 ms. The rate of phosphorylation was very fast (3.5 s-1 at 22 degrees C in the presence of 5 microM exogenous CaM) following photolysis of caged ATP, and, following Ca2+ release, phosphorylation was accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA was followed by a td of 194 ms at 5 microM CaM and a rate of MLC20 phosphorylation intermediate between these parameters following photolysis of, respectively, NP-EGTA and caged ATP. In the presence of the normal, total endogenous CaM content (37 +/- 4 microM) of protal vein smooth muscles td was 565 ms. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (> 2.5 microM) to shorten the td. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 microM and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC20 phosphorylation and force development (t1/2 and td) initiated by Ca2+ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td of an additional component, possibly a prephosphorylation activation/isomerization of the Ca4CaM myosin light chain kinase complex (Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 microM CaM) following simultaneous photolysis of NP-EGTA and caged ATP suggests that preincubation with ATP (prior to photolysis of NP-EGTA) may inhibit the formation of a preactive Ca2CaM myosin light chain kinase complex.
Highlights
The pre-myosin light chain (MLC20) phosphorylation components of the lag phase of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2؉ from 4-(2-nitrophenyl)-EGTA (NP-EGTA)
In order to compare the CaM dependence of the delay with the steady state response to Ca2ϩ, we first determined the effects of exogenous CaM on steady state tension in strips permeabilized with -escin (Kobayashi et al, 1989; Iizuka et al, 1994), by cumulatively increasing [Ca2ϩ] in the presence of variable [CaM]
Increasing, at pCa 4.5, the exogenous CaM concentration from 10 nM to 40 M did not change the steady state level of tension, indicating that, exogenous CaM increased the sensitivity for Ca2ϩ (Fig. 2), at this high free [Ca2ϩ] it had no effect on maximum isometric force
Summary
The pre-myosin light chain (MLC20) phosphorylation components of the lag phase (td) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2؉ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). The rate of phosphorylation was very fast (3.5 s؊1 at 22 °C in the presence of 5 M exogenous CaM) following photolysis of caged ATP, and, following Ca2؉. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (>2.5 M) to shorten the td. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td of an additional component, possibly a prephosphorylation activation/isomerization of the Ca4CaM myosin light chain kinase complex
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