1999 Fiscal Year Final Research Report Summary
Elucidation of intracellular mechanism for desensitization of ET_A endotheline receptor by using isolated single cardiomyocytes.
| Project/Area Number |
10670103
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | National Institute of Health Sciences |
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Principal Investigator |
ONO Kageyoshi National Institute of Health Sciences, Senior Research Scientist, 生薬部, 主任研究官 (40177259)
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| Project Period (FY) |
1998 – 1999
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| Keywords | endothelin / desensitization / RGS / electrophysiology / potassium channel / acetylcholine / G-proteion signaling |
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| Research Abstract |
It is well known that endothelin receptors are susceptible to desensitization. In negative chronotropic response in the heart mediated by ET_AR endothelin receptor (ET_AR), I have discovered that there exists a clear species difference in the desensitization of ET_AR, by utilizing which intracellular mechanism for the desensitization of ET_AR was investigated. Intracellular environment of single isolated cardiomyocytes was modified through the patch-clamp pipette. By using isolated sinoatrial node cells precise electrophysiological mechanism for the negative chronotropic effect of ET-1 is also studied intensively.
All the ET_ARs so far studied has a phosphorylation site in its 3rd intracellular loop, targeted by protein kinase C (PK-C). It turned out in this study that all the nine amino acid residues, which are possible targets for phosphorylation, in the 2nd through the 4th intracellular loops are conserved in ET_AR of guinea pig, compared with all other species studied, indicating th
… More
at the lack of desensitization of ET_AR in guinea pig heart is not caused by the lack of any phosphorylation cite in guinea pig.
Pretreatment of rat heart with 300 nM staurosporine completely blocked the development of desensitization of ET_AR.Intracellular perfusion of 10 mM EGTA, selective inhibitors of PK-C or PK-C inhibiting peptides in rat isolated cardiomyocytes also blocked desensitization as measured by repeatability of activating I_<K(ACh)> channel current by ET-1. These findings indicate that phosphorylation of possibly ET_AR itself by PK-C is necessary for the development of desensitization of ET_AR.In contrast in guinea pig heart where ET_AR does not desensitize, application of PMA under the presence of okadaic acid plus isoproterenol successfully produced desensitization of ET_AR, indicating ET_AR desensitization requires, in addition to PK-C, an yet unknown factor recruited by β-adrenoceptor stimulation. Possible involvement of G-protein coupled receptor kinase, GRK was then investigated. Since GRKs has a RGS (regulator of G-protein signaling) domain, effect of intracellularly applied RGSs on the speed of activation and spontaneous decay of I_<k(ACh)> current upon application of ACh or ET-1 and the speed of recovery of the current upon washing-off was studied. The results showed that several RGS inhibits the development of desensitization of mAChR or ET_AR by accelerating the re-setting of Gi protein-I_<K(ACh)> channel cycle. Less
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