Mechanisms and Modulation of Opsin 3 Mediated Airway Smooth Muscle Relaxation

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Project Summary/ Abstract Light-mediated relaxation of smooth muscle has been recently demonstrated. Non-visual opsins expressed on smooth muscle have been implicated as the key mediator of relaxation via light within the visible spectrum. However the mechanism of relaxation has yet to be fully understood. Our group has demonstrated that this phenomenon is also present in airway smooth muscle with light-mediated relaxation modulated by opsin receptors coupled to G protein signaling pathways. In preliminary studies, we demonstrate that airway smooth muscle photorelaxation is wavelength- and intensity-dependent and that light induces an interaction between the opsin 3 receptor and Gαs demonstrated by co-immunoprecipitation. This was associated with an increase in intracellular cyclic AMP, a known key second messenger of airway smooth muscle relaxation. We propose to identify the opsin receptors subtype(s), the G protein(s) and the cellular signaling pathways (e.g. cAMP, cGMP, and cytoskeletal regulatory proteins (RhoA, CPI-17 and myosin light chain-20) that modulate airway smooth muscle photorelaxation. We will also explore the mechanism of activation by modulating the light wavelength sensitivity of airway smooth muscle. Normally opsin receptors bind 11-cis-retinal (a chromophore and co- activator with photons of light) to form a Schiff base. The 11-cis-retinal allows for photoisomerization that changes the conformation of the opsin receptor, which induces downstream cellular signaling. Derivatives of retinal have been shown to shift the sensitivity of opsins to longer wavelengths. In our proposal we demonstrate that the use of 11-cis 2,3 didehydroretinal shifts airway smooth muscle sensitivity to longer wavelengths. We propose to evaluate additional retinal derivatives in cellular, in vivo and ex vivo models to identify chromophores that can shift the wavelength sensitivity of endogenous opsins to respond to longer wavelengths of light that may penetrate the body. In the eye, the sensitivity to different wavelengths of light is not only a function of the specific ligand but also a function of the subtype of opsin receptor expressed. Transfection of variants of OPN1 will allow evaluation of specific ligands at varying wavelengths of light in airway smooth muscle that activate cellular signaling congruent with relaxation. We will explore the direct activation of opsin receptors by carotene metabolites in the absence of light. We will demonstrate that the endogenous metabolite of carotenes, β-ionone, is a phytochemical that can directly activate opsin 3 in the absence of light. We will show that the same pro- relaxant cellular signaling occurs in airway smooth muscle when the opsin 3 receptor is directly activated by - ionine as compared to activation by chromophores/light. These proposed studies will allow for a better understanding of the activation and signaling of a previously uncharacterized receptor expressed in airway smooth muscle that modulates pro-relaxant signaling pathways.