Influence of Staurosporine on Cholinergic Agonist- and K+ -Induced Catecholamine Secretion in The Perfused Rat Adrenal Gland : Staurosporine이 흰쥐 관류부신에서 콜린성 효능약 및 칼륨에 의한 카테콜아민 분비작용에 미치는 영향

국문 초록 (Abstract)

본 연구에서는 흰쥐 적출관류 부신을 이용하여 Streptomyces staurosporeus에서 분리된 alkaloid이며 protein kinases의 강력한 억제제인 staurosporine이 ACh, DMPP, McN-A-343, 고농도 K^(+), Bay-K-8644 및 cyclopiazonic acid에 의한 catecholamine (CA) 분비작용에 미치는 영향을 연구하여 다음과 같은 실험결과를 얻었다.
Staurosporine (30-300 nM)을 부신정맥내로 20 분간 주입하였을때, ACh (5.32 mM), 막탈분극을 일으키는 고농도의 K^(+) (56 mM), 니코틴 수용체 효능약인 DMPP (100 uM), M_(1)-무스카린 수용체 효능약인 McN-A-343 (100 uM), 칼슘채널 개방약인 Bay-K-8644 (10 uM) 및 세포내 칼슘 유리약물인 cyclopiazonic acid (10 uM)에 의한 CA 분비작용은 비교적 용량의존적으로 억제되었다. 또한 protein kinases억제제로 알려진 tamoxifen (2 uM) 존재하에서도 ACh, 고농도의 K^(+), DMPP, McN-A-343, Bay-K-8644 및 cyclopiazonic acid에 의한 CA 분비작용은 억제되었다.
그러나, staurosporine (100 nM) 과 protein kinases의 selecive activator로 알려진 phorbol-12,13-dibutyrate (100 nM)를 20분간 동시에 전처치시 ACh, 고농도의 K^(+), DMPP, McN-A-343, Bay-K-8644 및 cyclopiazonic acid 의 CA 분비효과에 대한 staurosporine 단독처치시의 억제효과가 상당히 감약되었다.
이상과 같은 실험결과 staurosporine은 콜린성 니코틴 및 무스카린 수용체 흥분작용과 막탈분극에 의한 CA 분비효과에 현저한 억제작용을 나타내며, 이는 protein kinases의 활성화를 억제함으로서 흰쥐의 부신 수질 chromaffin 세포내로 세포외칼슘 유입 및 세포질내 칼슘 유리에 대한 직접적인 억제작용에 기인되며, 또한 staurosporine-sensitive protein kinases가 흰쥐 부신수질에서 CA 분비작용을 조절하는데 modulator 로써 역할을 하는 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

It has been known that staurosporine is an alkaloid with antifungal activity isolated from Streptomyces staurosporeus (Omura et al. 1977) and a more potent inhibitor of protein kinases (Tamoki et al., 1986). In the present study, it was attempted to examine the effect of staurosporine on secretion of catecholamines (CA) evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat atlrenal gland. The perfusion of staurosporine (30-300 nM) into an adrenal vein for 20 min produced relatively dose-dependent inhibition in CA secretion evoked by ACh (5.32 mM), high K^(+)(56 mM), DMPP (100 uM for 2 min), McN-14-343 (100 uM for 2 min), cyclopiazonic acid (10 uM for 4 min) and Bay-K-8644 (10 uM for 4 min). Also, in the presence of tamoxifen (2 uM), which is also known to be a protein kinase inhibitor, CA secretory responses evoked by ACh, high K^(+) , DMPP, McK-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with staurosporine (100 nM) under the presence of phobol-12,13-dibutyrate (100 nM). a specific activator of protein kinases (for 20 min), the inhibitory effect of staurosporine on CA secretory responses evoked by ACh, high K^(+), DMPP, McK-A-343, Ray- K-8644 and cyclopiazonic acid was considerably recovered to a considerable extent of the control release as compared to those in the presence of staurosporine only.
These results denionstmte that staurosporine used in the present study causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and niuscarinic) receptors as well as by membrane depolarization, indicating strongly that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells through preventing activation of protein kinases. Furthermore, these findings suggest strongly that these staurosporine-sensitive protein kinases also play a modulatory role in regulating the rat adrenomedullary CA secretion.

목차 (Table of Contents)

• CONTENTS
• 국문초록 = 1
• Ⅰ. INTRODUCTION = 3
• Ⅱ. MATERIALS AND METHODS = 5
• Esperimental Procedure = 5
• Perfusion of Adrenal Gland = 5
• Drug Administration = 7
• Collection of Perfusate = 7
• Measurement of Catecholamines = 8
• Statistical Analysis = 8
• Drugs and Their Sources = 8
• Ⅲ. RESULTS = 10
• Effect of 30 nM staurosporine on CA secretion evoked by ACh, excess K^(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid from the perfused rat adrenal glands = 10
• Effect of 100 nM staurosporine on CA secretion evoked by ACh, excess K^(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid from the perfused rat adrenal glands = 16
• Effect of 300 nM staurosporine on CA secretion evoked by ACh, excess K^(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid from the perfused rat adrenal glands = 21
• Effect of 2 uM tamoxifen on CA secretion evoked by ACh, excess K^(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid from the perfused rat adrenal glands = 26
• The effects of staurosporine plus phorbol-12,13-dibutyrate on CA release evoked y ACh, excess K^(+), DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid from the perfused rat adrenal glands = 32
• Ⅳ. DISCUSSION = 38
• Ⅴ. SUMMARY = 44
• REFERENCES = 45