Recently, we characterized blue light–mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and G_αs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic β₂-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with G_αs, which increases cAMP levels, activating PKA and modulated by GRK2.

最近，我们表征了蓝光介导的气道平滑肌 (ASM) 松弛（光松弛），并暗示了视蛋白 3（OPN3）（一种非典型视蛋白）的参与。在本研究中，我们表征了光松弛的细胞信号机制。我们使用来自 OPN3 缺失小鼠的气管证实了 OPN3 在蓝光光松弛中的功能作用（最大松弛 52 ± 13% 与野生型小鼠 90 ± 4.3%，P  < 0.05）。然后，我们证明了共定位OPN3和G _α小号使用共IP和原代人细胞ASM邻近连接测定法，将其用暗对照（23±3.6对相比通过用蓝色光处理在小鼠气管增加cAMP的进一步支持14 ± 2.6 pmol cAMP/环，P < 0.05)。通过使用 Rp-cAMPS 抑制光松弛显示下游 PKA（蛋白激酶 A）参与（P  < 0.0001）。此外，我们观察到小鼠气管中慢性 β ₂激动剂暴露引起的脱敏的收敛机制，并将这一发现与原代人 ASM 细胞中 OPN3 和 GRK2（G 蛋白受体激酶）的共定位相关联。最后，与未转染的细胞相比，人 ASM 细胞中 OPN1LW（同一视蛋白家族中的一种红光光感受器）的过表达模型显示红光暴露后细胞内 cAMP 水平增加（48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg蛋白质，磷 < 0.01)，表明对组织渗透性更强的光波长存在保守的光松弛机制。总之，这些结果表明 ASM 中的蓝光光松弛是由 OPN3 受体与 G _{α s}相互作用介导的，G _{α s}会增加 cAMP 水平，激活 PKA 并受 GRK2 调节。