The pineal hormone melatonin is involved in photic regulations of various kinds, including adaptation to light intensity, daily changes of light and darkness, and seasonal changes of photoperiod lengths. The melatonin effects are mediated by the specific high-affinity receptors localized on plasma membrane and coupled to GTP-binding protein. Two different G proteins coupled to the melatonin receptors have been described, one sensitive to pertussis toxin and the other sensitive to cholera toxin. On the basis of the molecular structure, three subtypes of the melatonin receptors have been described: Mel1A, Mel1B, and Mel1C. The first two subtypes are found in mammals and may be distinguished pharmacologically using selective antagonists. Melatonin receptor regulates several second messengers: cAMP, cGMP, diacylglycerol, inositol trisphosphate, arachidonic acid, and intracellular Ca2+ concentration ([Ca2+]i). In many cases, its effect is inhibitory and requires previous activation of the cell by a stimulatory agent. Melatonin inhibits cAMP accumulation in most of the cells examined, but the indole effects on other messengers have been often observed only in one type of the cells or tissue, until now. Melatonin also regulates the transcription factors, namely, phosphorylation of cAMP-responsive element binding protein and expression of c-Fos. Molecular mechanisms of the melatonin effects are not clear but may involve at least two parallel transduction pathways, one inhibiting adenylyl cyclase and the other regulating phospholipide metabolism and [Ca2+]i.

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The pineal gland has proven to be an excellent model for the study of adrenergic control systems. Noradrenaline, released from sympathetic nerve terminals in the pineal gland, regulates a large nocturnal increase in melatonin synthesis by stimulating the activity of arylalkylamine N-acetyltransferase (NAT, EC 2.3.1.87) 30-70-fold. An essential step in both the induction and maintenance of high NAT activity is an increase in intracellular cyclic AMP. Noradrenaline acts via beta-adrenoceptors to increase pineal cyclic AMP by activating adenylate cyclase, and the activation of pineal alpha 1-adrenoceptors potentiates beta-adrenergic stimulation not only of NAT but of both cyclic AMP and cyclic GMP. Here we describe investigations designed to test whether alpha 1-adrenergic potentiation of beta-adrenergic stimulation of pineal cyclic AMP involves protein kinase C. Our results suggest that kinase activation is involved and the data provide the first demonstration of a synergistic interaction between Ca2+-phospholipid-dependent protein kinase (protein kinase C) and neurotransmitter-dependent stimulation of cyclic AMP.

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.

In the Djungarian hamster Phodopus sungorus, the daily temporal pattern of synthesis and release of pineal hormone melatonin, mainly the length of the period of elevated melatonin levels, may be involved in transferring the information on day length to the neuroendocrine-gonadal axis. The present study investigated the time course of adjustment of the rhythm in melatonin production and concentration to the change from long to short photoperiods. Adult female Djungarian hamsters, maintained on a regime of 16 h of light and 8 h of darkness per day (LD 16:8) were transferred to the LD regime 8:16 and the daily rhythms in the pineal melatonin concentration and in the pineal N-acetyltransferase activity, as an indicator of melatonin formation, were studied at various intervals following the transfer. Under LD 16:8, the nocturnal melatonin concentration was elevated for 4.8 h. After 3 days on LD 8:16, no extension of the period of high melatonin levels occurred. 2, 4 and 6 weeks after the transfer to LD 8:16, the period of elevated melatonin levels lasted for 8.1, 9.3 and 11.5 h, respectively. Extension of the melatonin pattern proceeded first predominantly into the morning hours. Only after this extension was completed, a considerable extension into the evening hours began. Extension of the N-acetyltransferase rhythm on short photoperiods proceeded in the same way as that of the melatonin rhythm. The data show that while a change in the photoperiod might be seen by hamsters within 2 weeks after the transfer to LD 8:16, the full shortening of the photoperiod might be recognized only within 6 weeks or later. It is suggested that the gradual extension of the melatonin rhythm might play a role in the time course of adjustment of the reproductive system of Djungarian hamsters to short photoperiods.

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The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.

Circadian rhythms, endogenously generated in suprachiasmatic nuclei (SCN), seem to be under the direct influence of melatonin. Therefore, the effect of iontophoretically applied melatonin on electrical activity of SCN neurons was investigated in vitro. Usually, melatonin had an inhibitory effect. In the 3-h periods before (2.00-5.00 p.m.) or after (5.00-8.00 p.m.) the light-dark transition the percentage of SCN neurons sensitive to melatonin was very high (80% and 100%, respectively). However, efficacy of melatonin was low in the periods preceeding (20%) and following (33%) this 6-h time interval.