Circadian regulation of hydroxyindole-O-methyltransferase mRNA levels in rat pineal and retina

Abstract

Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the methylation of acetylserotonin to complete the synthesis of melatonin in the pineal and retina. A complete 1728 nucleotide cDNA encoding rat pineal HIOMT was isolated, characterized, and used to evaluate day/night levels of HIOMT mRNA. As previously reported for HIOMT enzyme activity, HIOMT mRNA levels were also greater in the pineal than in the retina. Northern blot analysis and in situ hybridization were useful for detection of HIOMT mRNA in the pineal but not the retina, whereas the reverse transcriptase-polymerase chain reaction or RNase protection assay revealed transcripts for HIOMT both in the pineal and retina. Investigating HIOMT mRNA levels in rat pineal and retina at 6 time-points throughout a 24 h period revealed higher levels of HIOMT message during darkness. The daily fluctuation in HIOMT mRNA persisted in constant darkness, verifying an endogenous circadian rhythm both in the pineal and retina. In mammalian pineals, sympathetic innervation, synthesizing norepinephrine that activates beta (β) adrenergic receptors, entrain several circadian bodily functions through the synthesis and release of melatonin. A single injection of the β-adrenergic agonist, isoproterenol, induced a dramatic increase of HIOMT mRNA levels in the light-adapted pineal, in vivo. Moreover, a single injection of the β-adrenergic antagonist, propranolol, prevented the nocturnal increase of pineal HIOMT mRNA. Using a combination of methods, it has been shown that the level of HIOMT mRNA fluctuates daily in both the pineal gland and retina. This day/night rhythm can be modulated either by β receptor agonists or antagonists when applied appropriately during the circadian cycle, suggesting that the mRNA changes in HIOMT may be controlled at the transcriptional level.

Introduction

The mammalian pineal gland responds to changes in ambient light by modulating the synthesis and release of melatonin (N-acetyl-5-methoxytryptamine) [42], a hormone that participates in the regulation of circadian and circannual rhythms 17, 19, 35. Serotonin is the precursor from which melatonin is synthesized as it passes through a sequence of reactions that add acetyl- and methyl-groups, utilizing respectively the enzymes arylalkylamine-N-acetyltransferase (NAT, EC 2.3.1.87), and hydroxyindole-O-methyltransferase (S-adenosyl-l-methionine: N-acetylserotonin-O-methyltransferase, HIOMT, EC 2.1.1.4), 1, 41, 43. The synthesis and release of melatonin is elevated during darkness and light exposure of an animal during darkness rapidly inhibits melatonin synthesis and release to the low levels that are found during daylight hours 3, 28, 30. Light evoked visual information from the retina plays upon the circadian oscillator of the suprachiasmatic nucleus in the hypothalamus that provides the input to a neural circuitry sending adrenergic afferents to the pineal [21]. The synaptic release of norepinephrine activates β-adrenergic receptors on the pinealocytes and subsequently an increase in enzyme activity both of NAT and HIOMT 11, 20, 33, 34.

The cDNAs encoding pineal HIOMT have been identified for bovine 13, 14, 18, chicken [36] and human [15]. There is high homology between the HIOMT cDNAs of the different species; however, the transcriptional, translational or posttranslational mechanisms controlling mRNA levels and HIOMT activity are unknown generally for all species. In the chicken pineal, HIOMT mRNA and enzyme expression were increased threefold at midday vs. midnight when animals were exposed to constant light 2, 4. In contrast, rats kept in constant light show a decrease in pineal HIOMT enzyme activity during the daytime 3, 33, 34 and, as demonstrated in this report, a concomitant decrease in HIOMT mRNA levels. The divergent responses between these species may be related partially to the presence in chicken pineal, but not rat, of differentiated photoreceptor cells that respond to light, in vivo.

The rat retina is known to show circadian changes in melatonin synthesis and release that is proposed to effect the daily circadian rhythm of rod outer segment membrane assembly and/or shedding 23, 39. Uncoupled from neuronal input from the suprachiasmatic nucleus, the retina has developed an independent biological clock that is entrained by light directly and the endogenous release of dopamine acts to modulate melatonin and the circadian oscillator 5, 37. HIOMT enzyme activity has been reported in rat retina 8, 26, 27, and an HIOMT mRNA has recently been amplified from both human and bovine retina cDNA 13, 29. The level of HIOMT activity and mRNA are significantly lower in the retina than the pineal.

The level of circulating melatonin in the blood is set primarily by its synthesis and release from the pineal, with the melatonin synthesized in the retina restricted mainly to ocular distribution and function. In order to investigate the level and circadian changes associated with HIOMT mRNA in the pineal and retina, a rat cDNA encoding HIOMT was identified and sequenced. Using cDNA products derived from the rat pineal HIOMT, we utilized several methodologies that demonstrated circadian oscillations of HIOMT mRNA levels in the rat retina and pineal and that show changes in light/dark HIOMT levels. Furthermore, these changes can be mimicked selectively in the rat pineal by the appropriate application in vivo of pharmacological agents that either activate or inhibit β-adrenergic receptors in the pineal, correspondingly increasing or decreasing melatonin synthesis.