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Melanopsin Mediates Light-Dependent Relaxation in Blood Vessels

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Melanopsin Mediates Light-Dependent Relaxation in Blood Vessels Melanopsin mediates light-dependent relaxation in SEE COMMENTARY blood vessels Gautam Sikkaa, G. Patrick Hussmannb, Deepesh Pandeya, Suyi Caoa, Daijiro Horic, Jong Taek Parka, Jochen Steppana, Jae Hyung Kima, Viachaslau Barodkaa, Allen C. Myersd, Lakshmi Santhanama,e, Daniel Nyhana, Marc K. Halushkaf, Raymond C. Koehlera, Solomon H. Snyderf,1, Larissa A. Shimodag, and Dan E. Berkowitza,e,1 aDepartment of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, MD 21287; bDepartment of Neuroscience, Johns Hopkins University, Baltimore, MD 21205; cDepartment of Surgery, Johns Hopkins University, Baltimore, MD 21287; dDepartment of Allergy and Immunology, Johns Hopkins University, Baltimore, MD 21224; eDepartment of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205; fDepartment of Pathology, Johns Hopkins University, Baltimore, MD 21287; and gDivision of Pulmonary Medicine, Johns Hopkins University, Baltimore, MD 21224 Contributed by Solomon H. Snyder, October 24, 2014 (sent for review June 22, 2014) Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been transduction through photosensitive receptors in blood vessels. linked to a number of behavioral responses to light, including These photoreceptors are part of the family of non-image-forming circadian photo-entrainment, light suppression of activity in (NIF) opsins. We now report a signaling cascade mediating nocturnal animals, and alertness in diurnal animals. We report photorelaxation via Opn4, cGMP, and phosphodiesterase 6 a physiological role for Opn4 in regulating blood vessel function, (PDE6) that is regulated by G protein-coupled receptor kinase particularly in the context of photorelaxation. Using PCR, we 2(GRK2). demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demon- Methods −/− strates that vessels from Opn4 mice fail to display photorelaxation, A complete description of methods is provided in SI Methods. which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response Vasoreactivity. Photorelaxation of blood vessels was assessed via force-ten- at ∼430–460 nm. Photorelaxation does not involve endothelial-, nitric sion myography. Aorta was mounted on a myograph using a microscope, oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive bathed in physiological Krebs buffer, and preconstricted. Light was delivered via cold light lamp (40,000–200,000 lux), light diodes [red (620–750 nm), prostanoid signaling but is associated with vascular hyperpolarization, green (495–570 nm) or blue (380–495 nm)], or a monochromator with as shown by intracellular membrane potential measurements. Sig- varying wavelength. naling is both soluble guanylyl cyclase- and phosphodiesterase β 6-dependent but protein kinase G-independent. -Adrenergic re- LaserDopplerFlowmetry.Anesthetized mice were secured in the supine ceptor kinase 1 (βARK 1 or GRK2) mediates desensitization of pho- position. A laser-Doppler flow probe was placed on the proximal ventral torelaxation, which is greatly reduced by GRK2 inhibitors. Blue surface of the tail. The position of the fiber-optic probe was adjusted to light (455 nM) regulates tail artery vasoreactivity ex vivo and tail obtain a stable flux measurement and was secured in place with glue. blood blood flow in vivo, supporting a potential physiological role Relative changes in red blood cell flux were monitored with a Perimed for this signaling system. This endogenous opsin-mediated, light- PeriFlux System 5000 laser-Doppler Flowmeter. activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a signifi- Significance cant pathophysiologic contributor. Non–image-forming opsins such as Opn4 regulate important photorelaxation | opsin | melanopsin | signal transduction | GRK2 physiological functions such as circadian photo-entrainment and affect. The recent discovery that melanopsin (Opn4) func- hotorelaxation, the reversible relaxation of blood vessels to tions outside the central nervous system prompted us to ex- Pcold light, was initially described by Furchgott et al. in 1955 plore a potential role for this receptor in blood vessel regulation. (1). Subsequent studies have attempted to define the signal We hypothesized that Opn4-mediated signaling might explain transduction mechanisms responsible for this phenomenon. the phenomenon of photorelaxation, for which a mechanism The process seems to be cGMP-dependent but endothelial- has remained elusive. We report the presence in blood vessels of independent. The role of nitric oxide (NO) in photorelaxation has Opn4 and demonstrate that it mediates wavelength-specific, been controversial (2–7), with some studies showing that NOS light-dependent vascular relaxation. This photorelaxation signal transduction involves cGMP and phosphodiesterase 6, but not inhibition with L-NAME not only fails to inhibit the response (2) but in some cases enhances and prolongs it (3). Moreover, sev- protein kinase G. Furthermore it is regulated by G protein-cou- eral published reports examining photorelaxation demonstrate pled receptor kinase 2 and involves vascular hyperpolarization. This receptor pathway can be harnessed for wavelength-specific an attenuation of the response with each subsequent light stim- light-based therapy in the treatment of diseases that involve ulation. A number of investigators have proposed that NO de- altered vasoreactivity. pendence results from the photo-release of NO stores from nitrosothiols and that the endothelium and NOS are important Author contributions: G.S., D.P., J.S., V.B., L.S., D.N., M.K.H., R.C.K., S.H.S., L.A.S., and D.E.B. for the repriming of these stores (stores that become depleted designed research; G.S., G.P.H., D.P., S.C., D.H., J.T.P., J.S., J.H.K., A.C.M., L.S., M.K.H., L.A.S., with each photo-stimulation); however, the source of those and D.E.B. performed research; D.P., L.S., L.A.S., and D.E.B. contributed new reagents/ nitrosothiols has not as yet been clearly identified (6). Impor- analytic tools; G.S., G.P.H., S.C., D.H., J.T.P., J.S., J.H.K., V.B., A.C.M., M.K.H., R.C.K., S.H.S., L.A.S., and D.E.B. analyzed data; and G.S., D.N., R.C.K., S.H.S., L.A.S., and D.E.B. wrote tantly, photo-release of NO occurs in the UV-A spectrum at 366 the paper. – nm (4 6), a wavelength at which intravascular nitrosospecies and The authors declare no conflict of interest. nitrite have the potential to release substantial quantities of NO See Commentary on page 17704. (7). However, this wavelength is very different from that at which 1To whom correspondence may be addressed. Email: [email protected] or dberkow1@ others have observed vascular responses. Given the controversy jhmi.edu. surrounding the photorelaxation mechanism, we postulated an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MEDICAL SCIENCES entirely new mechanism: that photorelaxation is mediated by 1073/pnas.1420258111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1420258111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17977–17982 Downloaded by guest on September 27, 2021 RT-PCR. To demonstrate the expression of Opn4, GRK2, PDE5A, and PDE6G, RT-PCR was performed using mRNA isolated from mouse aortas. Adenovirus Encoding shRNA. GRK2 was knocked down in aortic rings with shRNA. Ad-sh-Nontargeted and Ad-shGRK2 encoded viruses were gener- ated using a pAdBLOCK-iT kit (Life Sciences). Western Blot Analysis. Aorta lysates were resolved by 10% (vol/vol) SDS/PAGE buffer, transferred, probed with antibodies, visualized with peroxidase, and enhanced with chemiluminescence system (Pierce). Membrane Potential Measurements. Endothelium-denuded segments of murine thoracic aorta were pinned in place, lumen side down, in a recoding chamber. Intracellular recordings were performed in current clamp (3.0–4.0 kHz sampling rate) mode and recorded on a chart recorder (TA240; Gould Instrument Systems). Results Opsin 4 Expression in Blood Vessels Mediates Photorelaxation. We first demonstrated photorelaxation in mouse aortic rings suspended in an organ chamber in physiological solution and preconstricted with phenylephrine (PE; 1 μM). Exposing the suspended vessels to cold white light at increasing intensities (40,000–2,000,000 lux units) produced a phasic, intensity-dependent vasorelaxation with a maximal response of ∼35% (Fig. 1 A and B). The diverse physiological roles of NIF opsins prompted us to explore their involvement in photorelaxation. Using PCR, we demonstrated +/+ −/− expression of Opn4 in the aorta of Opn4 but not in Opn4 mice (Fig. 1C). Furthermore, the abundance of transcript seems to be higher in aorta than in homogenates of whole brain (Fig. S1A). Expression of Opn4 was further confirmed using a second B Opn4−/− setofPCRprimers(Fig. S1 ). In aortic rings from Fig. 1. Opsin 4 expression in blood vessels and its role in photorelaxation. mice, vasorelaxant responses to light were virtually abolished (A) Summary data and (B) representative trace of ex vivo vasoreactivity + + (Fig. 1D and Fig. S1C), whereas relaxation to the endothelium- demonstrating dose-dependent effects of cold white light on Opn4 / dependent vasodilator acetylcholine was unaltered (Fig. S1D). A mouse aorta compared with no light exposure. Error bars denote SEM, n = 6, small-molecule opsin inhibitor (“opsinamide” with sulfonamide ***P < 0.001. (C) RT-PCR analysis
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