The long-term goal of this research program is to determine the cellular and molecular ion transport mechanisms underlying coronary artery contraction. Most vasoactive agents regulate membrane ion channels and intracellular Ca release in vascular endothelial and smooth muscle cells. The immediate goal of this research program is the project described here. The purpose of this project is to understand how potassium (K) depolarization, endothelin (ET), and nifedipine (NIF) regulate myoplasmic free Ca (Ca/im) in coronary artery smooth muscle. NIF is postulated to inhibit these processes by high affinity binding to the VGCC. ET also releases Ca from the sarcoplasmic reticulum (SR) and may activate ligand- gated channels (LGC), thus increasing Ca/im with minimal depolarization of the cell and negligible activation of VGCC. Therefore, additional mechanisms, including interactions between VGCC and the SR, should be determined for the action of NIF. Methods involve the use of single cells that are freshly dispersed from bovine and porcine coronary artery. Three measures of Ca/im regulation are: 1) whole-cell VGCC and LGC currents studied with patch-clamp, 2) Ca/im measured with fura-2 microfluorometry, and 3) contraction monitored by video.
Specific aims are to determine: 1) NIF inhibition of VGCC. The decrease of VGCC current and Ca/im caused by NIF is hypothesized to be related directly to the amount of membrane depolarization during voltage-clamp. 2) NIF inhibition of SR Ca release/sequestration. Any direct effects of NIF on Ca release channels will be determined in lipid bilayers. 3) Potentiation of VGCC or LGC by ET. ET is hypothesized to shift the voltage-dependence for VGCC activation and activate LGC such that increased Ca influx occurs near the resting membrane potential. 4) NIF inhibition of VGCC or LGC that have been potentiated by ET. The environment at this institution is very conducive to achieving these specific aims. Physical facilities meet research needs and scientific interactions with other cardiovascular and membrane physiologists are frequent, because these specialties comprise 80% of the research activities in the department. The institution's development plans are to support basic cardiovascular research if extramural funding is available; otherwise, teaching and service activities would take priority. This RCDA would enhance my individual scientific development by protecting valuable time for intense research and by acquisition of new laboratory skills in the molecular biology of ion channels. These activities would foster synthesis, refinement, and important growth of knowledge in ion transport mechanisms in coronary artery smooth muscle.
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