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Gen Comp Endocrinol
2014 Sep 01;205:279-86. doi: 10.1016/j.ygcen.2014.04.019.
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Endothelin modulates the circadian expression of non-visual opsins.
Moraes MN
,
Lima LH
,
Ramos BC
,
Poletini Mde O
,
Castrucci AM
.
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The non-visual opsin, melanopsin, expressed in the mammalian retina, is considered a circadian photopigment because it is responsible to entrain the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Pigment dispersion can also be triggered by endothelin-3 (ET-3). Here we show that melanin translocation is greater when a blue light pulse was applied in the presence of ET-3. In addition, we demonstrated that mRNA levels of the melanopsins Opn4x and Opn4m exhibit temporal variation in melanophores under light/dark (LD) cycles or constant darkness, suggesting that this variation is clock-driven. Moreover, under LD cycles the oscillations of both melanopsins show a circadian profile suggesting a role for these opsins in the photoentrainment mechanism. Blue-light pulse decreased Opn4x expression, but had no effect on Opn4m. ET-3 abolishes the circadian rhythm of expression of both opsins; in addition the hormone increases Opn4x expression in a dose-, circadian time- and light-dependent way. ET-3 also increases the expression of its own receptor, in a dose-dependent manner. The variation of melanopsin levels may represent an adaptive mechanism to ensure greater melanophore sensitivity in response to environmental light conditions with ideal magnitude in terms of melanin granule dispersion, and consequently color change.
Fig. 1. Blue light and ET-3 disperse melanin granule in X. laevis melanophores. Photomicrographies of representative cultures (A) kept under DD, (B) stimulated with white light, (C) stimulated with blue-light pulse, (D) treated with 10−8 M ET-3, stimulated with blue-light pulse and (E) treated with 10−8 M ET-3. (F) and (G) graphs (n = 8–15) showing absolute absorbance measured at 530 nm: (F) 1 h after light pulse and/or endothelin treatment (b is different from control, p < 0.05 and c is different from d, p < 0.05, respectively, ANOVA one-way followed by Tukey’s test); (G) ET-3 treatment for 1, 2, and 6 h (b is different from a, p < 0.05, ANOVA two-way followed by Bonferroni’s test).
Fig. 2. Opn4x expression under DD or LD cycle, and treated with ET-3. For Figs. 2 thru 4, cells were kept for 6 days in the following protocols: (A) DD or LD, undisturbed cells; (B) DD or LD and subject to a medium change on the 6th day; (C) treated with 10−9 M ET-3 for 6 h on the 6th day in DD or (D) LD; (E) treated with 10−8 M for 6 h in DD or (F) LD. The treatment (endothelin or medium change) began at 8:00 h and after 6 h a second medium change was performed. Data are presented as the mean (n = 4–12 flasks) ± SEM of Opn4x transcript normalized by 18S RNA transcript and expressed relative to the minimal value in DD. Letter a means statistically different from b, and letter c different from d (p < 0.05, One-way ANOVA followed by Tukey’s test). Asterisk means statistically different from DD conditions or treatment (p < 0.05 Two-way ANOVA followed by Bonferroni’s test).
Fig. 3. Opn4m expression under DD or LD cycle, and treated with ET-3. Data are presented as the mean (n = 4–12 flasks) ± SEM of Opn4m transcript normalized by 18S RNA transcript and expressed relative to the minimal value in DD. Letter a means statistically different from b (p < 0.05, One-way ANOVA followed by Tukey’s test). Asterisk means statistically different from DD conditions or treatment (p < 0.05 Two-way ANOVA followed by Bonferroni’s test).
Fig. 4. ETc receptor expression shows rhythm in response to 10−8 M ET-3. Data are presented as the mean (n = 4–12 flasks) ± SEM of ETc transcript normalized by 18S RNA transcript and expressed relative to the minimal value in DD. Letter a means statistically different from b (p < 0.05, One-way ANOVA followed by Tukey’s test). Asterisk means statistically different from DD conditions or treatment (p < 0.05, Two-way ANOVA followed by Bonferroni’s test).
Fig. 5. Quantitative PCR for (A) Opn4x and (B) Opn4m in melanophores of Xenopus laevis kept in DD and stimulated with blue light (87.85–95.17 μwatts/cm2, 450–475 nm) for 10 min. The cells were stimulated at 8:00 h on the fourth day. Data are presented as the mean (n = 5–9 flasks) ± SEM of gene transcripts normalized by 18S RNA transcript and expressed relative to the smallest control (DD) value. Asterisk means statistically different from the respective control (p < 0.05, Two-way ANOVA followed by Bonferroni’s test).
