1993 Fiscal Year Final Research Report Summary
The molecular biollogical and electrophysiological investigation of the degranulation of human eosinophils
| Project/Area Number |
04670454
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Respiratory organ internal medicine
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| Research Institution | Tohoku University |
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Principal Investigator |
TAMURA Gen Tohoku University School of Medicine Assistant, 医学部, 助手 (70188431)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAUCHI Kouhei Tohoku University School of Medicine Assistant, 医学部, 助手 (20200579)
KAKUTA Yasunori Tohoku University Hospital Assistant, 医学部・附属病院, 助手 (80142933)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Eosinophils / Bronchial Asthma / Degranulation / Calcium Iion / G-Protein |
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| Research Abstract |
The degranulation mechanism of human eosinophils was studied using the patch-clamp technique, northern blot analysis and an image analysis of the intracellular Ca_<2+> concentration ([Ca_<2+>]^1). Purified eosinophils were obtained by an elutriator and the Percoll discontinuous gradient method. Single eosinophils were dialyzed in the whole cell configuration and granule release was observed by measurement of the membrane capacitance. When single eosinophils were dialyzed with a high concentration (10 mumol/L) of Ca_<2+>, the membrane capacitance increased by 30%, indicating slight granule release by high Ca_<2+>. By contrast 10 mumol/L Ca_<2+> and 100 mumol/L GTP^<-gamma->S caused a marked increase in membrane capacitance (+230%), indicating remarkable granule release by Ca_<2+> and GTP^<-gamma->S.Northaern blot analysis revealed the definite expression of a small molecular weight guanine nucleotide binding protein (small G protein) such as smg P21 (rap 1).
Platelet activating factor (PAF)-induced intracelllular Ca_<2+> concentration changes (Ca_<2+>-transients) were then analyzed by using Fura2-acetoxy methyl (Fura2-AM) loaded eosinophils and a computer-driven image analysis system. Basal [Ca_<2+>]^1 of the eosinoophils was 80% nmol/L PAF caused an increase in [Ca_<2+>]^1 in 4 to 6 sec and the response reachd to peak in 8 to 10 sec. The [Ca_<2+>]^1 changes were observed at PAF concentrations of 1 to 100 nmol/L.The maximum response was observed at 10 nmol/L PAF and [Ca_<2+>]^1 increased from 600 to 700 nmol/L.In additon, the accumulation of in inositol (1, 4, 5) trisphosphate (IP^3) was observed. These results indicate that stimulation by PAF increases [Ca_<2+>]^1 in association with an IP^3 accumulation and that the response activates a granule releasing mechanism which is also regulated by G proteins.
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