摘要
已知平滑肌收缩机理可简述为:在Ca~(2+)-CaM 存在的条件下,肌球蛋白轻链激酶(MLCK)使肌球蛋白轻链(MLC2)磷酸化,磷酸化MLC2与肌动蛋白(actin)相互作用而产生张力收缩。细胞内Ca~(2+) 降至静息水平及MLC2 脱磷酸化后,平滑肌仍然保持一定的张力,这一现象难以用现有的Ca~(2+) 依赖性调节理论解释,提示在平滑肌收缩过程中还存在着Ca~(2+) 依赖性调节以外途径。本研究以胃、子宫平滑肌及心肌肌球蛋白(myosin)的非Ca~(2+)-CaM 依赖性磷酸化(CIPM)为研究对象,初步揭示了MLCK 使CIPM 的一些特征,期望这些研究有助于进一步揭示平滑肌、心肌张力维持的机理提供新的研究途径。
该部分研究指出: 在Ca~(2+)-CaM 不存在条件下,MLCK 也可使平滑肌myosin 非Ca~(2+) 依赖性磷酸化(CIPM)。与myosin 的Ca~(2+)-CaM 依赖性磷酸化(CDPM)相比,它们的主要区别如下:(1)随孵育温度升高,CIPM 未显示明显变化,而CDPM 随孵育温度升高而明显降低。(2)随孵育时间延长,CIPM 未显示明显变化;CDPM 随孵育时间延长而明显降低。(3) 随着钾离子强度从60 mmol增加至360 mmol,CIPM 未显示明显变化;然而,当钾离子强度超过120 mmol时,CDPM 不再显示双重磷酸化。(4)CDPM 可被ML-9(一种MLCK 抑制剂)显著抑制;而在同样条件下,未观察到ML-9 对CIPM 的抑制效应。(5)CIPM 的Mg2+-ATP 酶的活性比未磷酸化的myosin 活性高,比CDPM 的活性低。这些差异在统计学上均有显著性(***P < 0.001,**P < 0.01 或* P < 0.05)。
The mechanism dealing with smooth muscle contraction can be simply described as that smooth muscle myosin light chains are phosphorylated by smooth muscle myosin light chain kinase (MLCK) in Ca2+-calmodulin (CaM) dependent way (CDPM), and the interaction between phosphorylated myosin and actin induces smooth muscle contraction. Since the rise of intra-cellular Ca2+ and the phsphorylation of myosin is happened in a micro-second range, the phenomena that the tension of smooth muscle may be retained as [Ca2+]i fall down to the basic level and myosin dephosphorylated are difficult to be explained by traditional hypothesis of CDPM. These phenomena suggested that other mechanisms may also be involved in the regulation of smooth muscle contraction. This study is tried to partially reveal the characterization of Ca2+-CaM independent phosphorylation way (CIPM) by MLCK in gizzard, uterus smooth muscle and cardiac muscle and factors involved in the regulation. The author hopes that these results can provide some valuable information to further study the mechanism of CIPM.
Part one The characterization of CIPM by MLCK in gizzard smooth muscle
Part one indicated that smooth muscle myosin may also be phosphorylated by MLCK in the absence of Ca2+-CaM. The major differences between CIPM and CDPM are as follows. (1) With the increase of incubation-temperature, the extent of CIPM by MLCK showed no apparent change. However, the extent of CDPM by MLCK apparently decreased with the increased temperature. (2) With the increase of incubation-time, the extent of CIPM by MLCK showed no apparent changes; in contrast, the extent of CDPM by MLCK was apparently decreased. (3) The increase of ionic
strength from 60 to 360 mmol KCl showed no apparent effects on CIPM. However, when ionic strength was over 120 mmol/L, Ca2+-CaM dependent di-phosphorylation of myosin was not observed. (4) CDPM was significantly inhibited by ML-9 (MLCK inhibitor). By contrast, no inhibitory effects on CIPM by MLCK were observed in the same assay condition. (5) The Mg2+-ATPase activity of CIPM was higher than that of dephosphorylated myosin but lower than that of CDPM (***P <0.001,** P <0.01 or * P <0.05).
Part two The characterization of CIPM by MLCK in uterus
The characterization of Ca2+-CaM independent phosphorylation of uterus myosin light chains by MLCK is as following. (1) CIPM by MLCK is much lower than that of CDPM by MLCK. (2) The extent of CIPM is more stable than that of CDPM, and CIPM was less influenced by the changes of incubation-time, incubation-temperature, and increase of the concentration of KCl. (3) Both CIPM and CDPM of uterus were less influenced by achidonic acid (AA) compared to those in gizzard smooth muscle. (4) CIPM by MLCK was less efficient, less energy consumption than that of CDPM by MLCK. (5) It was not observed that CIPM in uterus was affected by oxcytocin. The results suggested that CIPM by MLCK in uterus smooth muscle share similarity to that of CIPM by MLCK in gizzard smooth muscle.
Part three Characterization of cardiac CIPM by MLCK
The characterizations of cardiac CIPM by MLCK and CDPM by MLCK are as follows. (1) The extent of CIPM by MLCK is lower than that of CDPM. CIPM was characterized by less efficient, fewer energy consumption comparing with that of CDPM. (2) CIPM by MLCK in cardiac muscle was more influenced by changes of ionic strength than that of CDPM by MLCK. The extent of CIPM was less influenced by the changes of AA concentration than that of CDPM and this is different from that of CIPM in smooth muscle. (3) Cardiac CIPM and CDPM were more stable than those in smooth muscle. Cardiac CIPM and CDPM by MLCK were less influenced by changes of incubation time, incubation-temperature. CIPM by MLCK in cardiac muscle can be observed only under acid assay condition and these differences are statistically significant (** P < 0.01, or * P < 0.05).
Part four The effects of histamine on CIPM by MLCK in different muscle
The effects of histamine in high, medium and low concentration on CIPM and CDPM in gizzard, uterus sm
引文
1. Trybus KM Regulation of smooth muscle myosin. Cell Motil Cytoskeleton. 1991;18(2):81-5.
2. Kohama K, Ye LH, Hayakawa K, Okagaki T Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin. Trends Pharmacol Sci. 1996 ;Aug;17(8):284-7.
3. Walsh MP Regulation of vascular smooth muscle tone. Can J Physiol Pharmacol 1994; Oct;72 (10):1257.
4. Jiang, H., Stephens, N.L. Calcium and smooth muscle contraction. Mol. Cell Biochem.1994; 135,1-9.
5. Rembold, C.M. Regulation of contraction and relaxation in arterial smooth muscle. Hypertension 1992; 20, 129-137.
6. Walsh, M.P. Calcium-dependent mechanisms of regulation of smooth muscle contraction. Biochem. Cell Biol, 1991; 69, 771-800.
7. Onishi H, Mochizuki N, Morales MF. On the myosin catalysis of ATP hydrolysis. : Biochemistry. 2004; Apr 6;43(13):3757-63.
8. Jiang, H., Stephens, N.L. Calcium and smooth muscle contraction. Mol. Cell Biochem. 1994 ;135,1-9.
9. Sobieszek A. Formation and regulation of myosin light chain kinase and phosphatase complex in smooth muscle: the outlookTsitologiia. 1998;40(6):568-78.
10. Kamm KE, Stull JT. Second messenger effects on the myosin phosphorylation system in smooth muscle. Prog Clin Biol Res. 1989;315:265-78.
11. Hirano K, Derkach DN, Hirano M, Nishimura J, Kanaide H. Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain.Mol Cell Biochem. 2003 Jun;248(1-2):105-14.
12. de Lanerolle P, Paul RJ.Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am J Physiol. 1991 Aug;261(2 Pt 1):L1-14.
13. Lin Y, Ishikawa R, Kohama K. Role of myosin in the stimulatory effect of caldesmon on the interaction between actin, myosin, and ATP. J Biochem (Tokyo ). 1993, 114(2): 279-283.
14. Tchirikov M, Peiper U, Schroder HJ.Contraction kinetics of isolated human myometrium during menstrual cycle and pregnancy. BJOG. 2000 Jan;107(1):62-7.
15. Wachsberger PR, Pepe FA.Purification of uterine myosin and synthetic filament formation. J Mol Biol. 1974 Sep 15;88(2):385-91.
16. Cohen c,lowey s,kucera J. Structural studies on uterine myosin. J Biol Chem. 1961 May;236:PC23-4.
17. Arner A, Lofgren M, Morano I. Smooth, slow and smart muscle motors J Muscle Res Cell Motil. 2003;24(2-3):165-73.
18. Tetaert D, Moreau O, Han KK, Hildebrand HF, Biserte G. Isolation and characterization of "L17" light chain of myosin from bovine uterine smooth muscle Biochimie. 1977;59(3):337-9.
19. Haeberle JR, Hott JW, Hathaway DR. Regulation of isometric force and isotonic shortening velocity by phosphorylation of the 20,000 dalton myosin light chain of rat uterine smooth muscle. Pflugers Arch. 1985 Feb;403(2):215-9.
20. Nguyen T,Hayes E,Louis A,etal Maximal actomyosin ATPase activity and invitro myosin motility are unaltered in buman mitral regurgitation heart failure [J].J Biol Chem, 1978, 253:8659.
21. Shiverick KT, Thomas LL, Alpert NR Purification of cardiac myosin. Application to hypertrophied myocardium.Biochim Biophys Acta. 1975 May 30;393(1):124-33.
22. Nguyen TT, Hayes E, Mulieri LA, Leavitt BJ, ter Keurs HE, Alpert NR, Warshaw DM. Maximal actomyosin ATPase activity and in vitro myosin motility are unaltered in human mitral regurgitation heart failure. Circ Res. 1996 Aug;79(2):222-6.
23. Lin,Y. ,Ishikawa, Okagaki T, Ye LH , Kohama, K. Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon. Cell Motil Cytoskeleton.;29(3):250-8, 1994.
24. Gallagher PJ, Herring BP, Griffin SA, Stull JT.Molecular characterization of a mammalian smooth muscle myosin light chain kinase. J Biol Chem. 1991 Dec 15;266(35):23936-44.
25. Pato MD, Lye SJ, Kerc E. Purification and characterization of pregnant sheep uterus myosin light chain kinase. Arch Biochem Biophys. 1991 May 15; 287(1): 24-32.
26. Pato MD, Kerc E, Lye SJ. Phosphorylation and partial sequence of pregnant sheep uterus myosin light chain kinase. Mol Cell Biochem. 1995 Aug-Sep; 149-150:59-69.
27. Okagaki T, Higashi-Fujime S, Ishikawa R, Takano-Ohmuro H, Kohama K. In vitro movement of actin filaments on gizzard smooth muscle myosin: Requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon. J Biochem (Tokyo). 1991, 109(6): 858-866.
28. Lin Y. and Kohama K. N-terminal myosin-binding fragment of talin Biochem. Biophys. Res. Comm. 249,656-659, 1998.
29. Tansey MG, Luby-Phelps K, Kamm KE, et al. Ca(2+)-dependent phosphorylation of myosin light chain kinase decreases the Ca(2+) sensitivity of light chain phosphorylation within smooth muscle cells. J Biol Chem. 1994 Apr 1;269(13):9912-20.
30. Ikebe, M., and D. J. Hartshorne. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J. Biol. Chem. 1985.260: 10027-10031.
31. Jaworowski A, Arner A.Temperature sensitivity of force and shortening velocity in maximally activated skinned smooth muscle. J Muscle Res Cell Motil. 1998 Apr;19(3):247-55.
32. Sunano S, Miyazaki E. A comparison of the effects of temperature and metabolic inhibition on the contraction of smooth muscle. Jpn J Physiol. 1981;31(4):445-56.
33. Sobieszek A. Regulation of myosin light chain kinase: kinetic mechanism, autophosphorylation, and cooperative activation by Ca2+ and calmodulin. Can J Physiol Pharmacol. 1994 Nov;72(11):1368-76.
34. Filenko AM, Sobieszek A. Effect of preincubation on smooth muscle myosin light chain kinase activity Ukr Biokhim Zh. 1998 May-Jun;70(3):39-43.
35. Jiang MJ, Morgan KG. Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. Pflugers Arch. 1989 Apr; 413(6):637-43.
36. Andrews MAW, Maughan DW, Nosek TM, et al. Ion-specific and general ionic effects on contraction of skinned fast-twitch skeletal muscle from the rabbit. J Gen Physiol 1991 98:1105–112.
37. Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 7(72):248-254, 1976.