臺灣博碩士論文加值系統

本篇論文主要是探討大鼠血管平滑肌細胞上 BK接受器的藥理特性及其訊
號傳遞的交互調節作用。首先以[3H]-BK結合分析試驗、IPs累積以及鈣離
子移動作用來確立細胞上接受器的亞型, 結果得知細胞上表現的為BK-B2
接受器亞型, 接受器的最大密度 (Bmax) 為47.3*4.4 fmol/mg protein,
解離常數 (KD) 為1.7*0.2 nM。BK 以及Kallidin 可以濃度相關的方式引
起 IP3 及細胞內鈣離子濃度上升, 而這個情形會受到B2 選擇性拮抗劑
Hoe 140及 [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK 的抑制, 由pKB值的計
算可與接受器結合測試得到一致的結果:細胞上表現的是B2 接受器亞型。
此?以百日咳毒素前處理細胞並不影響BK 誘發的反應, 顯示接受器偶合
的是百日咳毒素不敏感的G蛋白-Gq蛋白。而在細胞外鈣對BK 所誘發反應
的影響實驗中可知, 細胞內所偶合的PLC 為鈣離子依賴型, BK 所導致的
鈣離子濃度上升包含了內質網的釋放及外鈣內流二種機轉, 而細胞外鈣內
流是透過包含L型通道在內的非選擇性陽離子通道達成。利用PMA 處理30
分鐘活化PKC 可以抑制BK 所引起的IPs生成及鈣離子反應, 而長期24小時
處理使PKC 受下移調節 (down regulation) 則二者反應都有反轉且增強
之現象, 以西方墨點轉漬法分析可知參與調控作用的可能為PKC-alpha,
betaI, betaII, delta, epsilon, zeta 等六種, 其中除了PKC-zeta 外,
其餘五種均與BK 引發之反應有逆相關的調節情形, PKC 作用的位置初步
推測在接受器之後。除了PKC 的調節外, 以forskolin 等藥物提升 cAMP
的濃度活化PKA, 發現PKA 與PKC 類似, 短期處理可以抑制BK 所誘發的
IPs累積及鈣離子生成, 長期24小時則有反轉的情形, PKA 的作用點初步
推測可能是G蛋白或PLC-beta。

The pharmacological properties of Bradykinin (BK) receptors in
rat vascular smooth muscle cells (RVSMCs) were characterized by
a radioligand [3H]-BK binding assay, inositol phosphates (IPs)
accumulation, and Ca2+ mobilization. Analysis of binding
isotherms yielded KD of 1.7*0.2 nM and Bmax of 47.3*4.4 fmol/mg
protein. The specific binding of [3H]-BK to RVSMCs was inhibited
by the B2 receptor selective antagonists (Hoe 140 and [D-Arg0,
Hyp3, Thi5,8, D-Phe7]-BK) and agonists (BK and Kallidin) with a
best fit by a one-binding-site model. BK and Kallidin stimulated
IPs accumulation and Ca2+ mobilization in a concentration-
dependent manner. The BK-induced these responses were inhibited
by B2 receptor selective antagonists Hoe 140 and [D-Arg0, Hyp3,
Thi5,8, D-Phe7]-BK. Incubation of RVSMCs in the absence of
external Ca2+ caused a decrease in IPs accumulation and Ca2+
mobilization in response to BK. Ca2+ influx was required for the
BK-induced responses. The Ca2+ was influxed via non-selective
cation channel including L-type Ca2+ channel. Furthermore,
pretreatment of cells with pertussis toxin did not alter BK-
induced responses. In RVSMCs, B2 receptor seems coupled to a
pertussis toxin-insensitive G protein, might be Gq protein.
Short term pretreatment of RVSMCs with PMA for 30 min attenuated
the BK-induced IPs accumulation and Ca2+ mobilization. But long
term pretreatment for 24 hrs, the inhibitory effect was
reversed. According to western blotting detection, the BK-
induced responses were inversely correlated with the
translocation of PKC-a, bI, bII, d and e from cytosol to
membrane. The inhibitory effect was located at post-receptor
level. In addition, the cAMP elevating agents including
forskolin and L-858051 also inhibited BK-induced responses when
short term pretreatment for 30 min. The effect was mediated by
PKA activation and the target site might be at G protein or PLC-
b.