Airway hyperreactivity to pharmacologic bronchoconstricting agents appears to be a basic element of asthma. Relief of bronchospasm through the administration of beta-adrenergic agonists is a basic component in the therapeutic control of the disease. While the precise underlying mechanisms of either the disease or the treatment are not well understood, it is known that both contraction and relaxation of airway smooth muscle (ASM) are closely associated with cytosolic calcium levels. The influx of calcium from extracellular sources is important in sustained contraction of ASM, and both voltage-independent and voltage- dependent influx pathways have been identified. Stimulation of ASM by contractile agonists results in the hydrolysis of phosphoinositol-bis- phosphate to inositoltrisphosphate and diacylglycerol. These putative second messengers mediate increases in cytosolic calcium levels and the initiation of contraction, and they have been associated with activation of extracellular calcium influx in many tissues, including smooth muscle. On the other hand, stimulation of this tissue by beta-adrenergic agonists is associated with decreased cytosolic calcium and decreased tension mediated by cAMP through cAMP-dependent kinase (A-kinase). Recent studies and preliminary data demonstrate that voltage-dependent calcium channels (VDCC) are activated by contractile agonists and inhibited by beta-adrenergic agonists. Calcium channel modulation occurs independent of agonist induced membrane potential changes, and is mediated through molecular pathways activated by agonist stimulation. It is hypothesized that contractile and relaxant substances regulate VDCC activity. The goal of this proposal is to determine the specific mechanisms underlying receptor-activated modulation of VDCC in ASM. The roles of phosphoinositol metabolic pathways and cAMP mediated pathways in agonist induced calcium channel modulation will be examined using on-cell voltage clamp techniques and concurrent intracellular microinjection of various pathway components. The results of these experiments should clarify the mechanisms underlying receptor-coupled VDCC regulation in ASM. A more thorough understanding of the regulation of calcium channels in ASM will likely provide a rational basis for their therapeutic modulation in airway disease.
