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標題: | 小分子G蛋白在血管平滑肌收縮角色之探討 Small G protein rho in vascular smooth muscle contraction |
作者: | 黃肅惠 |
出版年 : | 1994 |
學位: | 碩士 |
摘要: | 鈣離子為平滑肌收縮的主要活化因數。過去的實驗顯示GTP結合蛋白(GTP-binding protein)的活化可以提高平滑肌收縮時收縮器(contractile apparatus)對鈣離子的敏感度。本實驗以electroporation的方法將小分子G蛋白rho負載到尾動脈平滑肌束細胞中,以探討其於血管平滑肌收縮時所扮演之角色。若將GTPγS(一種不被細胞水解的GTP類似物)負載到細胞內,可增強血管肌束對24mM KCl使胞膜去極化(depolarization)所引起的收縮,並於0.1mM時達最佳效果;但是對較高濃度的KCl及phenylephrine(α1 adrenergic agonist)引起的收縮,則無顯著效應。以GDPβS(一種不被細胞水解的GDP類似物)取代GTPγS時,則36mM及51mM KCl所引起的收縮,隨GDPβS濃度的增加而受到抑制。另一方面,自大腸桿菌純化出來的rho蛋白接上GTPγS(呈活化態)或與肉毒桿菌Clostridium botulinum分離出來之C3酵素進行核醣基化(ribosylation;呈非活化態)後,以electorporation負載到平滑肌束時,並不影響phenylephrine所引起的收縮反應;而C3酵素單獨負載時,不但抑制phenylephrine所引起的收縮,亦同時降低細胞內鈣離子濃度的增加,但是並不影響胞內鈣離子濃度和等長收縮的關係。相對地,KCl所引起血管肌束的收縮作用,皆不受這些蛋白分子負載的影響。由這些結果顯示:1)GTPγS可促進24mM KCl引起的收縮,此促進的機制似與rho蛋白的活化無關;2)rho蛋白參與在phenylephrine所引起的血管收縮訊息傳遞途徑中,對胞膜去極化刺激引起之收縮則無影響;3)rho蛋白似乎不影響phenylephrine刺激的平滑肌收縮之鈣離子-張力的關係。 Elevation of intracellular Ca2+ concentration ([Ca2+]i) is the major activator of smooth muscle contraction. It has been reported that Ca2+ sensitivity of the contractile apparatus could be regulated by the activation of GTP-binding protein(s) (G-proteins). In order to clarify the possible function of a small G protein, rho, in smooth muscle contraction, purified rho was introduced into intact rat tail artery helical strips by electroporation. Gunanosine 5'-0-(3-thiotriphosphate) (GTPγS), a non-hydrolysable analogue of GTP, potentiated 24mM KCl-induced contractions, but had no effect on isometric force activated by 36mM KCl, 51 mM KCl or different concentrations of an α1-adrenergic agonist, phenylephrine. Guanosine 5'-0-(2-thiodiphosphate) (GDPβS), a non-hydrolysable analogue of GDP that competitively inhibits the binding of guanine nucleotides to G proteins, inhibited the contraction induced by 36 and 51mM KCl. The introduction of the GTPγS-bound active form or the ADP-ribosylated inactive form of rho into muscle strips did not affect the contraction induced by phenylephrine. On the other hand, the introduction of C3 exoenzyme from Clostridium botulinum, known to ADP-ribosylate rho and thus impairing its function, significantly inhibited phenylephrine - activated contraction. Furthermore, the inhibitory effect of C3 exoenzyme was diminished by the co-introduction of GTPγS-bound form of rho (rho-GTPγS). Simultaneous measurements of force and aequorin light signals showed that C3 exoenzyme caused concomitant decreases in [Ca2+]i and force activated by phenylephrine, but did not alter the relationships between [Ca2+]i and force. Potassium-activated contractions were not affected by the introduction of rho-GTPγS, ADP-ribosylated rho or C3 exoenzyme. These results suggest that 1) the potentiation effect of GTPγS on 24mM KCl-induced contractions does not appear to be mediated by rho; 2) rho is likely to be involved in the signaling pathways activated during phenylephrine-induced contraction, but not in those coupled to contractions activated by potassium depolarization; and 3) rho does not appear to affect the relationships between [Ca2+]i and force in phenylephrine-induced contraction. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76061 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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