| Co-Investigator(Kenkyū-buntansha) |
MURAKI Katsuhiko Nagoya City University, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学研究科, 助教授 (20254310)
OHYA Susumu Nagoya City University, Graduate School of Pharmaceutical Sciences, Assistant Professor, 大学院・薬学研究科, 助手 (70275147)
OHWADA Tomohiko The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (20177025)
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| Budget Amount *help |
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 2004: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2002: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
Although the increase in intracellular Ca^<2+> concentration ([Ca^<2+>]i) is commonly observed in responses to various types of stimuli, the excess increase in [Ca^<2+>]i, or in other words, overload of cells with calcium is one of the key steps and very popular in the process of accumulation in cellular damages under pathophysiological settings. To minimize calcium overload, cells have various systems to extrude Ca^<2+> to and/or prevent Ca^<2+> entry from outside. The Ca^<2+> entry is usually due to opening of two separate types of Ca^<2+> entry channels; voltage-dependent Ca^<2+> channels (VDCCs) and non selective cation channels. Ion channels, whose activities are directly modulated by [Ca^<2+>]i, strongly contributes to the regulation of Ca^<2+> entry via the changes in membrane potential. Large conductance Ca^<2+> activated K^+ (BK) channels are ubiquitously expressed in excitable cells except cardiac myocytes and also expressed in some non-excitable cells. The activation of BK c
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hannel induces membrane hyperpolarization, reduces VDCC activity and minimizes Ca^<2+> overload in excitable cells. We surveyed low molecular natural products mainly from plants to find out new prototype of BK channel opener, since this type of agents may reduce the hyper contractility of smooth muscle tissues or Ca^<2+> over load in neurons under pathophysiological conditions. Among over 60 natural products and their synthesized derivatives, we found pimaric acid (PiMA)and related compounds as potent openers of BK channel. Effects of PiMA and other compounds on BK channels were examined using HEK293 cells, in which either the a-subunit of BK channel (HEKBKα) or both α and 01 (HEKBKαβ1) subunits was heterologously expressed. Effects of these compounds (10μM) on the membrane potential of HEKBKαβ1 were monitored by use of DiBAC_4(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BKαβ1 opening under whole cell voltage-clamp showed that these six compounds activated BKαβ1 in a very similar concentration range (1-10 μM), in contrast abietic acid, sclareol and methyl pimarate had no effect. PIMA did not affect the charybdotoxin-induced block of macroscopic BKaβ1 current. Single channel recordings of BKαβ1 in inside-out patches showed that 10 μM PiMA did not change channel conductance, but significantly increased its open probability due to increase in sensitivity to Ca^<2+> and voltage. Since co-expression of β1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKα subunit. PiMA was selective to BK over cloned small and intermediate Ca^<2+> activated K^+ channels. It can be concluded that PiMA (>1μM) increases Ca^<2+> and voltage-sensitivity of BKα when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BKα activation. Moreover, we found that BK channel-like K^+ channels, which may be expressed in mitochondria of cardiac myocytes, are also activated PiMA. The protective effects of PiMA to reduced cell injury in ischemic conditions were also detected in rat cardiac myocytes. Taken together, we found a useful compound, PiMA, as a prototype of BK channel opener and obtained basic information about the activity-structure relationships for BK channel opener. Less
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