| Project/Area Number |
13470365
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | Asahikawa Medical College (2002)
Nagoya University (2001) |
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Principal Investigator |
TAKAI Akira ASAHIKAWA MEDICAL COLLEGE, DEPT OF PHYSIOLOGY, PROFESSOR, 医学部, 教授 (50126869)
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| Co-Investigator(Kenkyū-buntansha) |
UEMURA Daisuke NAGOYA UNIV, GRAD SCH OF SCIENCE, DEPT OF BIOORGANIC CHEMISTRY, PROFESSOR, 大学院・理学研究科, 教授 (00022731)
ISOBE Minoru NAGOYA UNIV, GRAD SCH OF BIO-AGRICULTURAL SCIENCES, DEPT OF BIOORGANIC CHEMISTRY, PROFESSOR, 大学院・生命農学研究科, 教授 (00023466)
YOSHIDA Akitoshi ASAHIKAWA MEDICAL COLLEGE, DEPT OF PHYSIOLOGY, PROFESSOR, 医学部, 教授 (70125417)
NARUSE Kenji NAGOYA UNIV, GRAD SCH OF MEDICINE, DEPT OF MEDICAL BIOPHYSICS, ASSOC. PROFESSOR, 大学院・医学系研究科, 助教授 (40252233)
三宅 養三 名古屋大学, 大学院・医学研究科, 教授 (30166136)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2002: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Keywords | Visual accommodation / Intraocular pressure / Ciliary muscle / Aqueous humor outflow / Whole-cell voltage clamp / trp channel / Molecular biology / Recombinant DNA |
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| Research Abstract |
In the ciliary muscle, the tonic contraction requires a sustained influx of Ca^<2+> through the cell membrane. Very little has hitherto been known about the route(s) of Ca^<2+> influx in this tissue that lacks voltage-gated Ca^<2+> channels. To identify ion channels as the Ca^<2+> entry pathway we investigated effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. We have also examined the expression of the trp channel gene in this smooth muscle tissue by RT-PCR using several sense and anti-sense constructs for human and murine trp's spanning 100-130 amino acid segments which almost entirely cover the putative pore forming region of each trp. The major results obtained are summarized as follows:
a. Experiments were carried out at 30℃ using pipettes filled with K^+-free solution containing 100 mM Cs aspartate, 5 mM-BAPTA ([Ca^<2+>]_i=70 nM) and 200 μM-GTP (pH 7.0). CCh evoked an inward current showing polarity reversal at holding potential
… More
near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCC_L, and NSCC_S) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li^+, Na^+, Cs^+, Mg^<2+>, Ca^<2+>, Sr^<2+> and Ba^<2+>, estimated by total ion replacement procedures, were 0.9 : 1.0 : 1.5 : 0.2 : 0.3 : 0.4 : 0.5 for NSCC_L and 1.0 : 1.0 : 1.8 : 2.5 : 2.6 : 3.2 : 5.0 for NSCC_S.
b. Both NSCC_L and NSCC_S were dose-dependently inhibited by 1-100 μM of La^<3+>, Gd^<3+> and SKF96365, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the phasic component.
c. Experiments with in-situ membrane patches using pipettes filled with PSS identified a carbachol-activated channel with very similar γ(31±1 pS) and τ(10±1 ms; n=8).
d. Replacement of GTP in the pipette solution with GTPγS gradually caused a spontaneous opening of the channel in the absence of CCh. The response to CCh was irreversibly inhibited (rather than augmented) by bath application of 1 μM-thapsigargin.
e. The results of RT-PCR indicated that the smooth muscle of the bovine ciliary body expresses a relatively high levels of trp's very similar to human trp3 and trp6, which are thought to construct non-selective cation channels controlled by G-protein-linked pathways. Less
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