Phase II study of irinotecan and etoposide in patients with metastatic non-small-cell lung cancer

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.     

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

Group interviewing for the purpose of selection

Relation between retinal melatonin and corneal mitotic rhythms in the Japanese quail was investigated in experiments manipulating the ocular physiology by treatments with formoguanamine hydrochloride (FG) and eye-lid suture. In experiment 1, we investigated the effects of FG, which is known to induce photoreceptor degeneration, on retinal melatonin and corneal mitotic rhythms. FG-treatment completely abolished the retinal melatonin rhythms in both LD 12:12 and constant darkness (DD), but the corneal mitotic rhythm was maintained with high mitotic rate in darkness under a LD cycle and subjective night under DD. The result suggests that 1) the photoreceptor cells in the retina are the site for melatonin production and/or for the oscillator which drives the circadian rhythm in retinal melatonin, and 2) melatonin is not involved in generation of the corneal mitotic rhythm. In experiment 2, we investigated the effects of eye-lid suture, which is known to induce eye enlargement and bulgy cornea, on the retinal melatonin and corneal mitotic rhythms. Eye-lid suture abolished the corneal mitotic rhythm in both LD and DD, with a high mitotic rate being maintained throughout 24 hr. But retinal melatonin maintained its rhythm with high levels in darkness under a LD cycle and in subjective night under DD. The result suggests that 1) bulgy cornea in the sutured eye was induced by the increase in mitotic rate in the light period, and 2) disappearance of the corneal mitotic rhythm does not have a relation to retinal melatonin. These results suggest that retinal melatonin is not involved in generation of the corneal mitotic rhythm and that there are two circadian clock systems in the eye.     

Circadian clock functions localized in xenopus retinal photoreceptors

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.     

Circadian rhythms in cultured mammalian retina

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.     

The tau mutation affects temperature compensation of hamster retinal circadian oscillators

Neural retinas of the golden hamster (Mesocricetus auratus) express circadian rhythms of melatonin synthesis when cultured in constant darkness. Retinas from wild-type hamsters synthesize melatonin with a period close to 24 h, while retinas obtained from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 h, synthesize melatonin with a period close to 20 h. The retinal circadian oscillators of both wild-type and tau mutant hamsters are temperature compensated; however, temperature compensation is adversely affected by the mutation.     

Unusual responses to light and darkness of ocular melatonin in European sea bass

Regulation by light and darkness of melatonin rhythms in the plasma and eye of the European sea bass (Dicentrarchus labrax) was studied. During light-dark cycles, plasma and ocular melatonin exhibited day-night changes with higher levels at mid-dark and at mid-light, respectively. Circulating melatonin levels were low in constant light but high in constant darkness (DD); ocular melatonin levels showed the reverse pattern. Plasma melatonin exhibited circadian rhythm for 1 cycle but the rhythm was no longer apparent on day 2. There was no circadian rhythm in ocular melatonin. Acute light exposure in DD decreased plasma melatonin but increased ocular melatonin. These results suggest that circulating melatonin may be used as a signal for darkness but ocular melatonin is used as a signal for the light phase.     

Photic and circadian regulations of melatonin rhythms in fishes

Photic and circadian regulations of melatonin rhythms in the pineal organ and the retina of several teleosts were studied to investigate the regulatory mechanisms of melatonin rhythms in fishes. In the eyecup preparations of the goldfish, Carassius auratus, both time of day and lighting conditions affected melatonin production, with high melatonin production observed only in the dark-treated group incubated during the 'subjective' night. Thus, in the goldfish retina, local photoreceptors and an ocular circadian clock seem to regulate melatonin production, as in the zebrafish retina and in the pineal organ of a number of teleosts, including the goldfish. However, this circadian regulation of melatonin rhythms is not universal among fishes. Although the superfused pineal organ of the masu salmon Oncorhynchus masou secreted melatonin in a rhythmic fashion under light-dark (LD) cycles, the rhythm disappeared under constant darkness (DD), as in the rainbow trout, with a large amount of melatonin released both during the subjective day and the subjective night. These results suggest that all salmonids lack circadian regulation of melatonin rhythms. Furthermore, when ocular melatonin rhythms were compared in two cyprinids, the ugui Tribolodon hakonensis and the oikawa Zacco platypus occupying different ecological niches, ocular melatonin contents exhibited daily variations, with higher values during the dark phase of LD cycles in both species. The rhythmic changes persisted in the ugui under DD, with higher levels at subjective midnight than at subjective midday; however, ocular melatonin levels in the oikawa were consistently high under DD. Thus, the circadian regulation of melatonin rhythms in fishes is influenced not only by phylogeny, but also by the ecological niches of the animals. These results suggest that the physiological functions of melatonin in the circadian and photoperiodic systems differ among fishes.     

Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D4-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D4-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparation prelabeled with [3H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D20-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D3/D4 DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D4-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retina maintained in "eye-cup" preparations. It is suggested that D4-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

What makes a mother? Interviews with women involved in egg donation and surrogacy

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.     

Effects of near-ultraviolet light on the nocturnal serotonin N-acetyltransferase activity of rat pineal gland

Effects of near-ultraviolet (UV-A; 325-390 nm, peak at 365 nm) light on the activity of the pineal serotonin N-acetyltransferase (NAT; a penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in rats. Acute exposure of dark-adapted animals to UV-A radiation produced a marked suppression of NAT activity of the pineal gland, the effect being dependent on exposure time. The decrease in the night-time NAT activity evoked by a 1-min pulse of UV-A light (as well as by a 15-s pulse of broad-band visible light) gradually deepened during the first 40 min of treatment of animals with constant darkness, then the enzyme activity began to rise reaching control values by 3 h. Treatment of rats with a protein synthesis inhibitor, cycloheximide, attenuated this night-driven reactivation of the pineal NAT activity. The presented results provide evidence that UV-A light is a powerful signal capable of controlling melatonin biosynthesis in rat pineal gland.     

Subclavian venogram as a guide to lead implantation

Melatonin synthesis in retinal photoreceptors is stimulated at night by a circadian oscillator and suppressed acutely by light. To identify photoreceptor mechanisms involved in the acute suppression of melatonin synthesis, an action spectrum was measured for dark-adapted Xenopus laevis eyecups at night. Intensity-response curves at six wavelengths from 400 to 650 nm were parallel, suggesting that a single photopigment predominates in melatonin suppression. Half-saturating intensities at 400, 440, 480, and 533 nm were not significantly different from one another, at 1-2 x 10(8) quanta cm(-2) s(-1). Significantly higher intensities of 580- and 650-nm light were required for melatonin suppression. These results indicate a predominant role for the principal green-absorbing rods in acute regulation of retinal melatonin synthesis in response to light, and argue against an important role for the red-absorbing cones. Higher than expected sensitivity at short wavelengths suggests that photoreceptors sensitive to blue and/or violet light may also contribute to melatonin suppression.     

Differences in tryptophan binding to hepatic nuclei of NZBWF1 and Swiss mice: insight into mechanism of tryptophan''s effects

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.     

The butyrylcholinesterase gene is neither independently nor synergistically associated with late-onset AD in clinic- and community-based populations

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.