腺嘌呤衍生物在白兔陰莖海綿體平滑肌之藥理活性

在中國傳統中草藥當中，冬蟲夏草在神農百草經被歸類為上品藥，而冬蟲夏草在臨床上具有治療陽痿遺精、改善性功能障礙之效果，其中的Adenosine即為冬蟲夏草的主要成份之一。Adenosine經由化學結構分子修飾後，在Adenine的第四和第八位置進行新鍵結，最後合成出新型的Adenine衍生物Adeno-3。本實驗設計便是探討Adeno-3，研究其對白兔陰莖海綿體平滑肌的藥理作用。在活體實驗中，不同濃度的Adeno-3 (0.2, 0.4, 0.6mg/kg)可使白兔海綿體內壓 ( intracavernous pressure , ICP ) 產生劑量相關性的增加，其促使海綿體內壓增加作用與對照組之磷酸二酯酶第五型抑制劑sildenafil作用效果相近，且其產生的陰莖勃起時間也是與sildenafil的作用時間相近。在離體組織實驗的部份：於phenylephrine誘導收縮的白兔陰莖海綿體組織，分別累積投與從10-9 ∼ 10-4 M不同濃度的 Adeno-3後，對於去除內皮細胞或是保留內皮細胞的海綿體組織，均可產生劑量相關性的海綿體鬆弛作用。但Adeno-3產生之海綿體鬆弛作用會因內皮細胞的去除與前處理60 mM高鉀溶液而降低其鬆弛作用。在經由前處理許多鉀離子通道阻斷劑的部分，Adeno-3產生的海綿體鬆弛作用會受到一氧化氮合成酶(nitric oxide synthase ; NOS) 抑制劑L-NAME (100 µM)，soluble guanylate cyclase （sGC）抑制劑ODQ (1 µM)、鉀離子通道阻斷劑tetraethylammonium chloride (TEA，10 mL)、4-aminopyridine (4-AP，100 µM) 以及 Ca2+-dependent 鉀離子通道阻斷劑charybodotoxin (ChTX，0.1 µM)等試劑的加入而降低其海綿體平滑肌鬆弛作用。其次，以放射性免疫分析方法（RIA）測定來評估cGMP之釋放量，利用［125I］cGMP進行分析，分別投與從10-8 ∼ 10-5 M不同濃度的Adeno-3與sildenafil在白兔海綿體組織初代培養細胞後，可發現Adeno-3與sildenafil呈現劑量相關性增加細胞內cGMP 的釋放量。而在以ODQ (10 µM)前處理後，Adeno-3 (10 µM) 所增加之細胞內cGMP 釋放量則明顯減少。在磷酸二酯酶的活性分析測定方面，從人類血小板之細胞中可分離出PDE5的isozyme，由測試的數據指出Adeno-3 可抑制PDE5約71% 相對於IBMX活性的抑制濃度。在PKG的表現方面，以Western blots的方法來測定白兔海綿體平滑肌之細胞培養，可發現加入不同濃度的Adeno-3 (10-6 ∼ 10-4 M)後，PKG蛋白質量的相對濃度有增加之趨勢，而在前處理ODQ (10 µM)後，則PKG蛋白質量的相對濃度明顯減少。基於以上活體、離體組織實驗，以及進一步進行放射性免疫分析及Western blots方法，可以顯示Adeno-3在白兔海綿體之鬆弛作用方面，可能是部份經由刺激一氧化氮的產生，活化sGC ，開啟鉀離子通道而增加cGMP 之合成，增加PKG的表現，另外也有部分的作用可能是來自於抑制PDE5所產生的影響。

關鍵字

腺嘌呤衍生物；白兔陰莖海綿體；鉀離子通道開啟；蛋白激酶G ；磷酸二酯酶抑制劑

並列摘要

Numerous studies have shown that the Cordyceps sinensis can prevent sexual dysfunction. Adenosine is one of the main compounds in Cordyceps sinensis to improve sexual activities. An Adenine derivative, Adeno-3, was designed to compare with sildenafil on their erectile function. The experiments indicated that Adeno-3 and sildenafil had concentration-dependently elevated the erectile function of rabbit corpus cavernosum smooth muscles. The relaxation of rabbit cavernosa was tested in vitro using an organ bath. In phenylephrine- preconstricted endothelium-intact or denuded rabbit corpus cavernosum, Adeno-3 produced concentration-dependent relaxations. The relaxation was reduced by endothelium removal or by the presence of L-NAME (100 μM), a nitric oxide synthase inhibitor. In addition, the relaxant effect of Adeno-3 was inhibited by pretreatment with a soluble guanylate cyclase (sGC) inhibitors ODQ (1 μM), a K+ channels blocker TEA (10 mM), a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 μM), and a Ca2+- dependent K+ channels blocker charybdotoxin (ChTX, 0.1 μM). Moreover, increased extracellular potassium levels (60 mM K+ solution) resulted in an attenuation of the concentration-dependent relaxant effects of Adeno-3. Adeno-3 and a phosphodiesterase type 5 inhibitor sildenafil (0.1, 1, 10, 100 μM) induced concentration-dependently increases in intracellular cyclic GMP levels in rabbit corpus cavernosum smooth muscle cells. The increase in cyclic GMP contents of Adeno-3 (100 μM) was abolished by pretreatment with a sGC inhibitor ODQ (10 μM) . In phosphodiesterase type 5 inhibitor assay, Adeno-3 (100 μM) inhibited the PDE5 activity about 71% activities caused by IBMX (100 μM) in human platelets. In Western blots experiments, Adeno-3 (1, 10, 100 μM) was found to increase the PKG1α1β expression in rabbit corpus cavernosum smooth muscle cells. And the PKG1α1β expression was decreased by pretreatment with ODQ (10 μM). Both Adeno-3 and sildenafil (0.2, 0.4, 0.6 mg/kg caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose dependent manner. These in vivo activities of ICP for Adeno-3 and sildenafil are consistent with those of in vitro effects of cGMP. It is suggested that the rabbit corpus cavernosum smooth muscle relaxant effects of Adeno-3 might be mediated by the stimulation of NO / sGC / cGMP pathway, the opening of the K+ channels, the increase PKG1α1β expression and PDE5 inhibition effects. Adeno-3 also plays a prominent role in CCSM relaxation-associated increases of ICP and penile erection.

並列關鍵字

adenine derivative ； rabbit corpus cavernosum ； potassium ion channel opening ； PKG expression ； PDE inhibitor

參考文獻

郭義忠碩士畢業論文,高雄醫學大學, 天然藥物研究所,.2002.

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