2001 Fiscal Year Final Research Report Summary
A Study of Electroporation of Cardiac Myocytes by Simultaneous Recording of Membrane current and Cellular Fluorescence
| Project/Area Number |
12670046
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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 physiology
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| Research Institution | JUNTENDO UNIVERSITY |
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Principal Investigator |
OCHI Rikuo School of Medicine, Juntendo University, Professor, 医学部, 教授 (10049025)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Electroporation / Plasma membrane / hyperpolarization / inward currents / ethidium bromide / lysophosphatidylcholine / Cardiac myocyte |
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| Research Abstract |
Strong electrical pulses produce membrane rupture that is associated with multiple pores (electroporation). Ion permeation through these pores can influence various cellular activities. In cardiac myocytes irregular inward currents (I_M) elicited by hyperpolarization has been assumed to represent electroporation. We examined it by simultaneously recording membrane currents and fluorescence of ethidium bromide (EB) that is membrane impermeable, binds with DNA and increases cellular fluorescence. Whole cell patch clamp technique and fluorescence microscope equipped with CCD camera were applied to rabbit ventricular myocytes supervising by K^+ -free solution with 5μM glibenclamide and 10 μg/ml EB. 40 s-negative square pulses from holding potential of -20 mV to potentials between -80 and -180 mV increased I_M with increase in hyperpolarization mainly by its conductance increase and it was associated with cellular EB fluorescence increase. The conductance produced by -80 and -1 6 0 mV steps were, 9.0±1.8 (n=14) and 66.2±25.4 pS/pF (n=7), and in these cells EB fluorescence increase was 0.062±0.005 and 0.321±0.075 in relative unit in the nuclei and 0.042±0.004 and 0.069±0.005 in the cytoplasm. EB fluorescence increased approximately in parallel with time integral of I_M, but nuclear EB fluorescence could rise also after I_M. LPC (10μM) enhanced I_M conductance to 103.0±21.8 pS/pF (n=5) and nuclear EB fluorescence to 0.34±0.12 in 5 min at -80 mV. Thus, hyperpolarization transiently ruptures membrane to produce pores to allow ethidium^+ (and Ca^<2+>) permeation and even more extensive membrane poration is produced by LPC at normal resting potential in cardiac myocytes.
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