The general goal of this project is to use a single cell approach to study excitation-contraction (E-C) coupling pathways in the vascular smooth muscl cell. The proposed studies are based on the hypothesis that there are at least two different pathways of excitation-contraction coupling in the smoo h muscle cell. A single cell approach will be utilized to allow the accurate measurement of the latency and the time course of the calcium and force signals and to facilitate the use of a skinned preparation. An exceptionall gentle cell isolation method allows attachment of single cells to microtool with relative ease compared to past techniques. The microtools will be attached to a force transducer and a piezoelectric displacement device to allow force and stiffness measurements to be made from individual cells. Th length-tension relationship for the single cell will be determined and compared to the relationship between length and activation-induced stiffnes . Intracellular calcium levels will be measured with fura-2 or controlled by making the cell membrane hyperpermeable with saponin. The [Ca2+]i - force relationship will be studied in intact and chemically skinned cells in the presence of agonists and second messengers which may utilize different E-C coupling pathways. Putative calcium regulatory substances and inhibitors o the putative calcium regulatory mechanisms will be introduced into the skinned cells to test the relative role of these systems in living cells an to compare their effects to the effects of agonists and second messengers. An understanding of the mechanism by which the normal vascular smooth muscl cell contracts is a prerequisite for understanding how to correct inappropriate vascular contraction such as occurs in coronary artery spasm and hypertension.
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