Although B2-adrenergic agonists are widely used for the acute treatment for asthma, these therapeutic agents are not ideal. They are not always effective in severe asthma, must be redosed frequently, are often inadequate as sole therapy and are associated with increased mortality. B2 agonists are thought to reduce symptoms of asthma by directly relaxing airway smooth muscle via the Gs/cAMP- PKA pathway but the events downstream from PKA are not known. Although PKA-independent mechanisms have been suggested, the signaling intermediates in a PKA-independent pathway have never been identified. Exciting preliminary data demonstrate B-adrenergic agonist-mediated relaxation of intact airway smooth muscle and actin deploymerization of primary cultures of human airway smooth muscle cells via a novel PKA-independent signaling pathway involving Src tyrosine kinases as well as by PKA-dependent inhibition of RhoA. RhoA is a monomeric G proteins that plays a central role in the dynamic regulation of the actin cytoskeleton in airway smooth muscle cells and in maintaining contraction in intact smooth muscle. We now seek to identify the intermediates in the PKA-independent pathway and to determine whether the PKA-dependent and/or PKA-independent signaling involve inhibition of RhoA. We hypothesize that B-adrenergic agonists induce airway smooth muscle relaxation and actin depolymerization by a novel PKA-independent pathway that involves Gs activation of Lck tyrosine kinase with subsequent activation of P190 RhoGAP leading to RhoA inhibition as well as by PKA-dependent inhibition of RhoA and dephosphorylation of cofilin. We propose: to confirm preliminary data demonstrating that B-adrenergic agonists inhibit contraction of intact airway smooth muscle by PKA-independent as well as by PKA-dependent mechanisms and to identify intermediates involved (Aim 1); to identify the Src tyrosine kinase involved in the PKA-independent pathway and explore the upstream (Gs protein) and downstream (P190 RhoGAP) intermediates (Aim 2); and to determine if B-adrenergic agonists induce actin depolymerization by PKA-induced phosphorylation and inhibition of RhoA;
(Aim 3). Identification of intermediates of a signaling pathway that relaxes airway smooth muscle in a PKA-independent manner would provide new selective targets for asthma drug therapy and would be a significant advance in the field. Identifying novel pathways in airway smooth muscle that mediate relaxation will lead to new therapeutic intervention strategies that may be employed to impair the ability of airway smooth muscle to contract and cause acute airway narrowing in asthma.
