| The phosphorylation and dephosphorylation of 20KD myosin light chains(MLC20) by Ca2±/ Calmodulin(CaM) dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) play the key role for the regulation in smooth muscle contraction. However, there are many observations of discrepancy between the level of intracellular Ca2±, MLC20 phosphorylation and smooth muscle contraction. It is still unclear why the tension of smooth muscle is kept longer despite the decline of intracellular Ca2±. The late study showed ACAD, an unsaturated fatty acid which widely exists in organism is capable of enhancing the phosphorylation of MLC20by inhibiting MLCP via many pathways, and then increase the contraction of smooth muscle. Thus, it is suggested ACAD be related to the tension keeping of smooth muscle. Considering ACAD is a multifunctional regulator and the mechanism of it in the tension keeping is not revealed yet, it is necessary to do further study. We mainly investigated the direct effect of ACAD on myosin ATPase activity and the effect on the Ca2±/ CaM independent phosphorylation of myosin(CIPM) by MLCK.1. The directly stimulatory effect of AA on the ATPase activity of myosin.Myosin ATPase activity was measured with malachite green method. Results showed that ACAD exerted a stimulatory effect on the intact myosin excluding effects of other factors. The enhanced extent of ATPase activity on unphosphorylated myosin (Vmax =13.28±1.52folds,n=6) is higher than that on the phosphorylated myosin(Vmax=5.73±1.09folds, n=6). The maximal effect on the unphosphorylated myosin is not changed irrespective of the presence or absence of actin. By contrast, the maximal effect on the phosphorylated myosin greatly reduced in the presence of actin. The effect of ACAD on the myosin fragments were also studied, and the effects on heavy meromyosin(HMM)(Vmax=4.03±0.41folo,n=6) and on myosin subfragmentl (S1) (Vmax =3.77±0.39folds, n=6)were both lower than that on the intact myosin(Vmax=13.28±1.52folds,n = 6).2. The stimulatory effect of ACAD on the CIPM by MLCKThe data from glycerol PAGE and the analysis with Scion Image showed that ACAD had a markedly stimulatory effect on CIPM(P<0.01) but no significant influence on the CDPM. For example, under the condition of 2umol/ml MLCK, the Ca2±-independent phosphorylation extent of MLC20 increased froml8.7%±3.6%(n=6) to 72.9%±7.5% (n=6) with the increase of ACAD from 0uM to 400uM. MLC20 phosphorylation was not detectable with 0.2 umol/ml MLCK, however, myosin was phosphorylated and gradually reached 46.5% phosphorylation with the ACAD concentration from 0 to 400 u M. The coincidence between the increased phosphorylation extent of CIPM by ACAD and the correspondence ATPase activity was proved, too. We also proved that the increase of phosphorylation extent of CIPM is accordant with the enhancement of the ATPase activity.Our results suggested that ACAD influenced the ATPase activiry of myosin through at least two pathways. One is to stimulate directly ATPase activity of myosin in a low concentration (0-100uM), the other is to stimulate indirectly ATPase activity by enhancing CIPM in a relative high concentration (0-400uM). Therefore, it is speculated that ACAD might participate in the regulation of tension keeping in smooth muscle under the condition of low intracellular Ca2±, and the further mechanism of it is worthy to study.
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