The long-term objective of this project is to understand how potassium(K) decolorization, endothelin (EN), and nifedipine (NIF) regulate coronary artery smooth muscle cell(SMC) free calcium (Ca1). More specifically, how might NIF attenuate K and EN-induced increase in Ca? The overall hypothesis is that EN potentiates K depolarization-induced activation of voltage-gated Ca channels (VGCC), thereby increasing Ca1; NIF is postulated to inhibit these processes by high affinity binding to the VGCC. However, EN also releases Ca from the sarcoplasmic reticulum (SR) and, thus increases Ca without depolarization of SMC. Other than elevated external K, no vasoconstrictor directly causes depolarization of coronary artery SMC, which would activate 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 SMC that are freshly dispersed from bovine conary artery and can be contracted by EN and K. Three measures of SMC Ca regulation will be obtained: 1) whole-cell VGCC currents will be studied with patch-clamp, 2) Ca will be measured with fura-2 microfluorimetry, and 3) contraction will be verified with video recording. Experimental design for achieving specific aims basically involves obtaining these three measures simultaneously during depolarization, and/or exposure to EN, and/or NIF, this allowing precise experimental control to differentiate the actions of these agents on VGCC or the SR.
Specific aims are to determine: 1) NIF inhibition of VGCC. The decrease of VGCC current and Ca caused by NIF is hypothesized to be directly related to the amount of membrane depolarization during voltage- clamp. 2) NIF inhibition of SR Ca release/sequestration. The hypothesis to be tested is the EN-induced Ca release from the SR is inhibited by NIF, primarily due to the inhibition of VGCC needed to refill the SR with Ca. 3) Potentiation of VGCC by EN. EN is hypothesized to shift the voltage- dependence for VGCC activation such that increased Ca influx occurs near the resting membrane potential. 4) NIF inhibition of VGCC that have been potentiated by EN. The significance of this research is the increased understanding of the pivotal role of VGCC in the cellular actions of EN and NIF of the coronary artery.
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