This proposal seeks to define the cellular and subcellular processes that regulate calcium influx in human airway smooth muscle (ASM) cells. In airway smooth muscle, agents that are effective at blocking voltage-dependent calcium channels (VDCC), do not inhibit agonist-induced force production in muscle strips, and do not effectively antagonize bronchospasm in vivo. The focus of this proposal will be the identification and characterization of sustained, receptor-activated calcium influx mechanisms in cultured human ASM cells that retain important functional responses to autacoids implicated in asthma and other airway diseases. Measurements of cytosolic calcium using the fluorescent calcium indicator fura-2 will be combined with conventional microelectrode voltage-clamp and patch clamp techniques to examine specific mechanisms of calcium entry and determine the effect of this influx on cytosolic calcium concentration. The hypothesis that sustained calcium influx following receptor-binding occurs via an influx pathway distinct from voltage-activated calcium channels, will be tested by a comparison of the permeability characteristics, pharmacologic sensitivity, and voltage-dependence of sustained, receptor-activated calcium influx and voltage-dependent calcium channels. If receptor-activated calcium influx occurs via VDCC, the cation permeability, antagonist potency, and voltage-dependence of the influx should be predictable from direct measurements of VDCC. A second goal will be to determine the extent to which agonists activate VDCC, and the degree to which activation of these channels contributes to a sustained rise in cytosolic calcium. A direct search for receptor-activated calcium channels distinct from VDCC, which have biophysical properties that match macroscopic agonist-triggered calcium influx, will also be conducted. Finally, the molecular mechanisms by which receptor-binding couples to calcium influx will be determined.
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