Joo Han Woo,1,2 Da Yeong Nam,3 Hyun Jong Kim,4 Phan Thi Lam Hong,1,2 Woo Kyung Kim,2,4, and Joo Hyun Nam1,2,
1Department of Physiology, Dongguk University College of Medicine, Gyeongju, (Korea); 2Channelopathy Research Center (CRC), Dongguk University College of Medicine, Goyang (Korea); 3Avixgen, Seoul (Korea); 4Department of Internal Medicine, Graduate School of Medicine, Dongguk University, Goyang (Korea).
Calcium Imaging, Medical Research
Skin photoaging occurs due to chronic exposure to solar ultraviolet radiation (UV), the main factor contributing to extrinsic skin aging. Clinical signs of photoaging include the formation of deep, coarse skin wrinkles and hyperpigmentation. Although melanogenesis and skin wrinkling occur in different skin cells and have different underlying mechanisms, their initiation involves intracellular calcium signaling via calcium ion channels. The ORAI1 channel initiates melanogenesis in melanocytes, and the TRPV1 channel initiates MMP-1 production in keratinocytes in response to UV stimulation. We aimed to develop a drug that may simultaneously inhibit ORAI1 and TRPV1 activity to help prevent photoaging. We synthesized nootkatol, a chemical derivative of valencene. TRPV1 and ORAI1 activities were measured using the whole-cell patch-clamp technique. Intracellular calcium concentration [Ca2+]i was measured using calcium-sensitive fluorescent dye (Fura-2 AM). UV-induced melanin formation and MMP-1 production were quantified in B16F10 melanoma cells and HaCaT cells, respectively. Our results indicate that nootkatol (90 μM) reduced TRPV1 current by 94% ± 2% at –60 mV and ORAI1 current by 97% ± 1% at –120 mV. Intracellular calcium signaling was significantly inhibited by nootkatol in response to ORAI1 activation in human primary melanocytes (51.6% ± 0.98% at 100 μM). Additionally, UV-induced melanin synthesis was reduced by 76.38% ± 5.90% in B16F10 melanoma cells, and UV-induced MMP-1 production was reduced by 59.33% ± 1.49% in HaCaT cells. In conclusion, nootkatol inhibits both TRPV1 and ORAI1 to prevent photoaging, and targeting ion channels may be a promising strategy for preventing photoaging.
Fluorescence measurements were made with an inverted microscope (Nikon Eclipse Ti; Nikon, Osaka, Japan) with a camera (sCMOS pco.edge 4.2; PCO, Kelheim, Germany) and an illuminator (pE-340 fura; CoolLED, Andover, UK). Samples were exposed to excitation wavelengths of 380 nm for 30 msec and 340 nm for 100 msec, and the data were recorded at an emission wavelength of 510 nm.
Product Associated Features
The pE-340fura offers fast controllable illumination for Fura-2 ratiometric calcium imaging.
Korean J Physiol Pharmacol.
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