Role Of CBmall/cClock In The Monochromatic Light Influencing The Secretion Of Melatonin In Chick Pinealocytes

Avian circadian system is a complex of mutually coupled pacemakers residing in pineal gland,retina and suprachiasmatic nucleus.As one of the biological clock oscillators,the pineal gland is an typical model system for the study of circadian rhythm,because it secretes melatonin into the blood with high level during the subjective night and low level during the subjective day.The circadian oscillator in avian was driven by the core circadian-feedback loop that consist of positive regulators of cellular clock(Bmall,Bmal2 and Clock)and negative regulators of cellular clock(Cry1,Cry2,Per2 and Per3).Previous studies have shown that monochromatic green light could increase the amount of melatonin in chick plasma while maintaining its rhythmic secretion.However,the mechanism underlying the regulation of melatonin secretion by GL remains unclear.In this study,the primary chick pinealocytes were cultured in 12L:12D(LD)and continuous darkness(DD)to investigate the endogenous oscillation and the sensitivity to light stimulation.Then the pinealocytes were cultured under red light(660 nm,RL),green light(560 nm,GL),blue light(480 nm,BL)and white light(400-760 nm,WL)by a light emitting diode(LED)system with a photoperiod of 12L:12D to study the effect of monochromatic light on circadian rhythm of clock genes in chick pinealocytes.This study further investigated the role of cOpnp,cBmall and cClock in the monochromatic green light-induced melatonin secretion in chick pinealocytes using siRNAs interference and overexpression technique.The results showed as follows:1 Endogenous rhythm oscillation and light sensitivity of chick pinealocytesThe expression of clock genes(cBmal1/2,cClock,cCry1/2,and cPer2/3),cAanat and melatonin presented the significant circadian rhythm in chick pinealocytes under DD and LD.Compared with the DD group,LD decreased the mesors of positive clock genes and increased the amplitudes of negative clock genes,as well as the mesors and amplitudes of cAanat and melatonin were reduced.Moreover,LD advanced the acrophases of cClock and cBmal2 but delayed the acrophases of negative clock genes except cCryl.These results indicated that the chick pinealocytes with an independent rhythm oscillator could receive an external optical signal to entrain the circadian rhythm of clock genes.2 Effect of monochromatic light on circadian rhythm of clock genes in chick pinealocytesClock genes:the expression of all genes and melatonin in the chick pinealocytes exposed to various lights presented significant circadian rhythm,except for the loss of cCryl under BL.Compared with WL,GL increased the mesors and amplitudes of positive clock genes,and decreased the mesors of cPer2/3.The amplitudes of cCryl and cPer3,and the mesors of cPer2/3 were notably increased by RL.cAanat and melatonin:cAanat and melatonin under different lights showed circadian rhythm.Compared to WL,GL significantly enhanced the mesors and the amplitudes of cAanat and melatonin,and advanced the acrophases of cAanat and melatonin.While RL and BL had opposite effects.cOpsin:the expression of cOpnp and cpn4-1 mRNA showed remarkable circadian rhythm under different lights.Compared with WL,the mesor and the amplitude of cOpnp under GL were increased as well as acrophase was advanced,but it was opposite to the changes of cOpnp at RL.However,the expression of cOpn4-1 were reduced in all monochromatic lights compared to WL.The above results indicated GL could increase the synthesis and secretion of melatonin,which might be related to the changes that GL increased the expression of the positive clock genes and cOpnp,and reduced the expression of the negative clock genes.3 Role of cOpnp in the GL influencing the clock system of chick pinealocytesThe interference of cOpnp damaged the circadian rhythm of cBmall,cCry2 and cPer3 as well as the cAanat and melatonin.Compared with the control group,si-cOpnp decreased the mesors and the amplitudes of cBmal2 and cClock,and significantly advanced the acrophases of cClcok and cCryl.When cOpnp was overexpressed,the circadian rhythm of cBmal1,cCry2,cAanat and melatonin were disappeared.Compared with the control group,the mesors and the amplitudes of cPer2 and cPer3 were increased,and the acrophase of cCryl was advanced.Those results indicated that cOpnp could regulate the synthesis and secretion of melatonin.4 Role of cBmall in the GL influencing the clock system of chick pinealocytesThe si-cBmall destroyed the circadian rhythm of cAanat and melatonin as well as clock genes except cCry1.Compared with the control group,the expression levels of positive clock genes,cAanat and melatonin were decreased.Overexpression of cBmall disturbed the circadian rhythm of cBmall,cCry1/2,cAanat and melatonin.Overexpression of cBmall significantly increased the levels of all clock genes,cAanat and melatonin.These results indicated that cBmall played a critical role in the regulation of monochromatic green light-induced melatonin synthesis in chick pinealocytes.5 Role of cClock in the GL influencing the clock system of chick pinealocytesThe interference of cClock had no impact the rhythm for cAanat and melatonin as well as cBmall^ cCryl and cPer2,but significantly damped the circadian rhythm of cClock,cBmal2,cCry2 and cPer3.Moreover,si-cClock significantly inhibited the cAanat mRNA and melatonin secretion at subjective night,and reduced the mesors and the amplitudes of cCryl and cPer2.Meanwhile,the phase of cCryl was also changed by the interference of cClock.The overexpression of cClock only abolished the circadian rhythm of cCry1/2,and increased the mesors and amplitudes of cBmal2,cPer2 and cPer3,as well as the acrophases of positive clock genes and melatonin were delayed.Those results suggested that cClock could affect the synthesis of melatonin in chicken pinealocytes under GL,but it did not involved in regulating the circadian rhythm of melatonin.6 Effect of cAMP onclock system in chick pinealocytes under GLThe expression of all clock genes and melatonin were decreased when supplemented PKA antagonist H89 to chick pinealocytes.While the cAMP activator FSK and cAMP analogue Dibutyryl-cAMP supplementation promoted the expression of cAanat and melatonin with increase in the expression of positive clock genes and decrease in expression of negative clock genes.These results showed that the cAMP signaling pathway participated in the maintenance of the circadian rhythm of melatonin and clock genes under green light condition.ConclusionThe chick pinealocytes with an independent oscillator could receive the external different wavelength signal to entrain the clock system oscillators.Monochromatic green light activated cAMP signaling pathway by affecting the expression level of cOpnp,which increased the expression of cBmall,thereby to promote the secretion of melatonin.