An N-acetylserotonin/melatonin radioimmunoassay (NAS/Mel RIA) and a Mel RIA were developed to measure NAS and Mel contents in tissues of rats and chickens. Anti-NAS and anti-Mel sera were produced by immunization of rabbits with NAS-M-bovine serum albumin (BSA) and Mel-M-BSA, respectively. Anti-NAS serum used in the NAS/Mel RIA reacts equally well with NAS and Mel, while anti-Mel serum used in the Mel RIA reacts specifically with Mel. The NAS and Mel levels in the pineal and Harderian gland, the retina and the brain of rats and chickens and Mel levels in chicken serum were determined using these RIAs. Levels of NAS and Mel in the rat pineal demonstrated diurnal rhythms with high levels during the dark period and low levels during the light period. The indole levels determined in this study correlate well with those obtained by other methods.
The pineal gland plays an important role in seasonal adaptation including variation in energy requirement. Animals exhibiting seasonal changes in their energy expenditure would be benefited if their cardiac and pulmonary systems respond to the pineal photoperiodic signal, melatonin, appropriately. Thus, we would like to hypothesize that melatonin receptors are present in the heart and lung. Using a specific labeled melatonin agonist, 2-[125I]iodomelatonin, binding sites were demonstrated in the lung and heart of birds and other animals. In the chicken lung, there were high affinity (equilibrium dissociation constant, Kd = 9.11 ± 0.73 pmol/l) and low density (maximum number of binding sites, Bmax =1.29 ± 0.16 fmol/mg protein) 2-[125I]iodomelatonin binding sites that were highly specific to melatonin. Similar binding with lower density was demonstrated in the quail and frog lungs. In the duck heart, specific 2-[125I]iodomelatonin binding sites with a Kd of 30.5 ± 3.5 pmol/l and a Bmax of 0.46 ± 0.1 fmol/mg protein (n = 4) were demonstrated. Competitive studies suggested that these binding sites were specific to melatonin. Thus, saturable and reversible 2-[125I]iodomelatonin binding was present in the lung and heart membrane preparations of birds and possibly other animals. The picomolar affinity, femtomolar density and highly specific pharmacological profile of these binding sites suggest that they can be classified as ML-1 melatonin receptors. The 2-[I25I]iodomelatonin binding sites described in the lung and heart as well as those binding sites demonstrated in other peripheral tissues suggest the ubiquitous direct action of melatonin on peripheral tissues.
Several studies have shown sex hormone effects on pineal function. In order to clarify the role of adrenergic mechanisms in these effects, we investigated the pineal response to adrenergic stimulation and pineal β-adrenergic receptors following castration and/or sex hormone treatment in 2-month-old male and female Sprague-Dawley rats. The urinary 6-sulphatoxymelatonin (aMT6s) response to isoproterenol (ISO) was compared in castrated and sham-operated animals. Ovariectomy caused an increase and orchiectomy a decrease in ISO-induced urinary aMT6s excretion. The melatonin response to ISO was examined in pineal glands and serum samples obtained from castrated, sex-hormone-treated castrated and sham-operated rats. Consistently, ovariectomy increased pineal and serum melatonin responses to ISO, while orchiectomy decreased the responses; oestradiol and testosterone treatments, respectively, reversed these effects. 3H-dihydroalprenolol binding was measured in single pineal glands from castrated, sex-hormone-treated castrated and sham-operated rats. Ovariectomy increased the density of β-adrenoceptors, whereas orchiectomy decreased the density; oestradiol and testosterone replacement, respectively, blocked these effects. No significant effect on receptor Kd values was found. These data suggest that sex hormones regulate pineal melatonin production by modifying β-adrenergic mechanisms.
In order to characterize the effects of septal lesionsupon adrenal stress responses, plasma corticosterone levels were examined 2 days after surgery in normal and sham-operated rats and in rats with septal lesions 0, 5, 15, 30, and 60 min after either 5 sec of handling or 3 min of novel environment at both the crest and trough of the adrenal cycle. Baseline levels and diurnal variations were comparable in lesioned and non-lesioned groups. Non-lesioned rats reacted to stress with an elevation of corticosterone which peaked and leveled off 15 min after the end of stress. Their response was greater following 3 min of novel environment than after 5 sec of handling. 60 min after stress, corticosterone levels had begun to return to baseline levels at trough but not at crest of the adrenal cycle. Rats with a septal lesion had an overall stress-response pattern similar to that observed in non-lesioned animals, but the response latency of septally-lesioned rats was much shorter than that of non-lesioned animals, and their corticosterone levels at 0, 5, and 15 min after stress were greater than those of non-lesioned rats. The mean magnitude of overresponse in rats with septal lesions was greater at trough than at the crest of the cycle. It is concluded that baseline adrenal function is not altered by destruction of the septal nuclei but that septally-lesioned rats have a reduced threshold for adrenal activation which is demonstrated by elevated corticosterone levels during the first 15 min following stimulation and that this increased sensitivity to stimulation is greater at trough than at the crest of the adrenal cycle.
The existence of melatonin receptors in adrenal glands is suggested by effects on rodent adrenal function which follow pinealectomy or in vivo and in vitro melatonin treatment. In 1992, Persengiev and coworkers reported specific binding of 2-[125I]iodomelatonin to rat adrenal tissue. In order to study adrenal binding further we have done binding studies on adrenal membranes from ducks sacrificed at midlight. We observed binding of 2-[125I]iodomelatonin which was specific, rapid, saturable, stable, reversible and of high affinity. Scatchard plots were linear and Hill coefficients were close to unity supporting the existence of a single class of 2-[125I]iodomelatonin binding sites. On Scatchard analysis the Kd was in the physiological range (27.4 pmol/l) together with a Bmax of 3.38 fmol/mg protein. Sites were highly specific to melatonin and its two synthetic analogs, 2-iodomelatonin and 6-chloromelatonin in pharmacological studies. In autoradiographic studies on the chicken adrenal gland, one class of 2-[125I]iodomelatonin binding site was demonstrated with a Kd of 58.8 pmol/l and Bmax of 182 fmol/g tissue. Kinetic and pharmacological studies indicated that these sites are saturable, reversible, and of high specificity and affinity. These findings in the chicken are similar to those of the duck adrenal data obtained by radioreceptor assay. In preliminary studies, no significant difference was found between 2-[125I]iodomelatonin binding to the adrenal glands collected at midlight and middark. These high-affinity binding sites are similar to those reported in a wide variety of tissues and are consistent with the hypothesis of direct action of melatonin on the adrenal gland. Further investigations on factors regulating the adrenal 2-[125I]iodomelatonin binding sites as well as on the transduction mechanisms of the melatonin-receptor complex in the adrenal gland are essential in order to establish the physiological functions of these sites.
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