KMUP-1和KMUP-3在大鼠氣管平滑肌細胞對抗SIPHGOSINE-1-PHOSPHATE(S1P)和U46619誘發有關RHO KINASE和PROTEIN KINASE C的蛋白質表現

KMUP-1 and KMUP-3 inhibit Siphgosine-1- phosphate (S1P) and U46619-induced Rho Kinase and Protein Kinase C in rat tracheal smooth muscle cells.

陳玫勳(Mei-Hsun Chen)

指導教授 ： 陳英俊

高雄醫學大學/醫學院/藥理學科研究所/碩士(2006年)

摘要

在氣管平滑肌收縮路徑中，G 蛋白調解的鈣離子敏感性牽涉了Rho kinase（ROK）與Protein Kinase C（PKC）兩條路徑。Sphingosine-1-phosphate（S1P）與U46619兩種刺激劑皆可活化ROK 與PKC的表現。而S1P屬於神經鞘酯類（sphingolipid）代謝物，U46619是thromboxane A2 衍生物，兩者皆會引起氣管平滑肌收縮。本實驗室已經證明KMUP-1與KMUP-3在不同濃度（1-100 μM）可使收縮的氣管產生放鬆，本研究則深入探討其涉入的機轉。而由結果顯示，除了S1P所誘導PKCα較無明顯抑制趨勢，KMUP-1與KMUP-3（0.1-100 μM）皆可明顯抑制由S1P（2 μM）或U46619（1 μM）誘發的ROKα及PKCα表現，且呈現劑量相關性。
除此之外，本研究中以KMUP-1、KMUP-3、Y27632（Rho kinase抑制劑）、Dexamethasone、Terbutaline、Chelerythrine chloride（Protein kinase C抑制劑）、8-bromo-cGMP與8-bromo-cAMP（10 μM），來比較對於PKCα及ROKα抑制能力。數據顯示除了S1P所誘導PKCα較無明顯抑制趨勢（數據未提供），而相較於Dexamethasone與Terbutaline，KMUP-1與KMUP-3皆可發揮較佳的抑制效果，且KMUP-1的抑制效果亦與Y27632、Chelerythrine chloride相近。
另外，細胞內鈣離子濃度實驗中亦發現，由S1P刺激後增加的細胞內鈣離子濃度皆會被KMUP-1所抑制，但KMUP-3對於細胞內鈣離子濃度較無明顯抑制趨勢。
由於KMUP-1和KMUP-3可以藉由抑制氣管平滑肌細胞的ROK、 PKC 蛋白質表現及降低細胞內鈣離子濃度，而達到抑制由鈣離子依賴性及鈣離子敏感性所導致的收縮現象。透過各項實驗結果，針對KMUP-1與KMUP-3的效能，可提供臨床上另一種治療氣喘的新方法。

關鍵字

大鼠氣管平滑肌細胞

並列摘要

G protein-mediated Ca2+ sensitization in contraction of tracheal smooth muscle（TSM）is involved in Rho kinase （ROK）and Protein Kinase C（PKC）activation pathways. The expression of ROK and PKC are activated by Sphingosine-1-phosphate （S1P）and U46619. S1P, a bioactive sphingolipid metabolite, and U46619, a thromboxane A2 analogue, both can induce TSM contractions. Our laboratory has demonstrated that KMUP-1 and KMUP-3（1-100 μM）relax the contraction of rat TSM. The results indicated that the expression of ROK and PKCα induced by S1P（2 μM）or U46619（1 μM）in cultured TSM cells were dose-dependently inhibited by KMUP-1 and KMUP-3（0.1 - 100 μM）, analyzed by western blotting, except the expression of PKCα induced by S1P（2 μM）.
After further investigation for the discrepancy between KMUP-1, KMUP-3, Y27632, Chelerythrine chloride, Dexamethasone, Terbutaline, 8-bromo-cGMP and 8-bromo-cAMP（10 μM）, KMUP-1 and KMUP-3 possess similar inhibition activities on the expression of ROKα and PKCα proteins as Y27632 and Chelerythrine chloride. Relative to 10μM Dexamethasone and 10μM Terbutaline, both of 10μM KMUP-1 and 10μM KMUP-3 have batter inhibitions on the expression of ROKα and PKCα protein。
In addition, the intracellular concentration of Ca2+ induced by S1P was significantly decreased by KMUP-1. However, KMUP-3 has slight inhibition on the intracellular concentration of Ca2+。
From the results, in this study, it is proposed that KMUP-1 could inhibit the contraction of airway smooth muscle by mediating the intracellular concentration of Ca2+。Those new medicines, KMUP-1 and KMUP-3, developed by our laboratory, could possess well capabilities to inhibit the contraction induced by mechanisms of Ca2+ dependent and Ca2+ sensitization due to dose-dependently inhibitions of PKCα, ROKα and intracellular concentration of Ca2+. In conclusion, KMUP-1 and KMUP-3 may indicate the batter clinical application in the treatment of asthma.

並列關鍵字

rat tracheal smooth muscle cells ； S1P ； U46619 ； Rho Kinase

參考文獻

1.Allen IC, Hartney JM, Coffman TM, Penn RB, Wess J, Koller BH (2006). Thromboxane A2 induces airway constriction through an M3 muscarinic acetylcholine receptor-dependent mechanism .Am J Physiol Lung Cell Mol Physiol. Mar; 290(3): L526-33.

2.Ammit AJ, Hastie AT, Edsall LC, Hoffman RK, Amrani Y, Krymskaya VP, Kane SA, Peters SP, Penn RB, Spiegel S, Panettieri RA Jr (2001). Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J. May; 15(7): 1212-4.

3.Amrani Y, Panettieri RA (2003). Airway smooth muscle: contraction and beyond. Int J Biochem Cell Biol. Mar; 35(3): 272-6. Review

4.Barnes PJ (1998). Pharmacology of airway smooth muscle. Am J Respir Crit Care Med. Nov; 158(5 Pt 3): S123-32. Review

5.Billington CK, Penn RB (2003). Signaling and regulation of G protein-coupled receptors in airway smooth muscle. Respir Res. 4:2. Epub 2003 Mar 14. Review.