Prolonged exposure to melatonin leads to time-dependent sensitization of adenylate cyclase and down-regulates melatonin receptors in pars tuberalis cells from ovine pituitary.

In photoperiodic mammals, seasonal cycles of growth and reproduction are cued by changes in the duration of the nocturnal profile of secretion of the pineal hormone melatonin. To investigate the likely mode of action of this hormone on target tissues, the effect of prolonged treatment with melatonin on the sensitivity of the adenylate cyclase (AC) system was examined in primary cultures of ovine pars tuberalis (PT) cells. When cells were exposed to melatonin (100 pM or 1 microM) for 16 h, and the hormone was then removed by a series of washes, basal production of cAMP was elevated over that observed in cells not treated with melatonin. Moreover, the rate of accumulation of cAMP after stimulation with forskolin (1 microM) was markedly enhanced in cells previously treated with melatonin compared to that in untreated controls. This sensitization by melatonin of the basal and forskolin-stimulated responses developed gradually and was half-maximal after approximately 8 h of exposure. There was no significant difference between the sensitizing effects of the two melatonin concentrations used. Treatment with melatonin for 24 h reduced the total amount of specific [125I]iodomelatonin binding in PT cell membranes by 30-50%. However, over the same period there was no reduction in the ability of a maximal (1 microM) concentration of melatonin to inhibit forskolin-stimulated cAMP production, indicating the presence of an excess capacity of melatonin receptors in cultured PT cells. Nevertheless, treatment with melatonin for 16 h did result in a 10-fold increase in the IC50 for the inhibition by melatonin of forskolin-stimulated cAMP production. The enhancement of cAMP production after prolonged treatment with melatonin was not masked by the inclusion of isobutylmethylxanthine (1 mM) during the subsequent challenge with forskolin, suggesting that sensitization was not due to a reduction in the activity of cAMP-phosphodiesterase. In control cells, aluminium fluoride caused an inhibition of forskolin-stimulated cAMP production. Prolonged treatment with melatonin abolished the inhibitory effect of aluminium fluoride, suggesting that treatment with melatonin caused a shift in the net balance between the G-protein-mediated stimulatory and inhibitory influences on the AC system. The sensitization of AC was not blocked by the inclusion of cycloheximide (10 micrograms/ml) during prolonged exposure to melatonin, suggesting that de novo protein synthesis is not a requirement for this effect of the hormone. These results constitute the first demonstration of an independent action of melatonin on ovine PT cells that is dependent upon the duration of the endocrine stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroxine 5'-deiodination in brown adipose tissue and pineal gland: implications for thermogenic regulation and role of melatonin.

T4 type II 5'-deiodinase (5'-D II) activity was studied in wild-captured Richardson's ground squirrels. As previously reported for other species, 5'-D II activity was detected in frontal cortex, cerebellum, pineal gland, and brown adipose tissue (BAT); in the median eminence the levels of 5'-D II activity were undetectable with our methodology. When pineal gland, frontal cortex, and cerebellum nyctohemeral profiles were studied, none of them showed variations. Cold exposure for 4 h led to an increase in the enzymatic activity 10-fold above the basal values for BAT, while in the pineal gland the values were doubled; cold exposure failed to change the 5'-D II activity in the frontal cortex. Acute melatonin treatment caused a 7-fold increase in 5'-D II activity in BAT, but did not affect enzyme activity in either the pineal gland or frontal cortex. The data indicate that 5'-D II in Richardson's ground squirrel shows classical localizations. Additionally, two new regulatory factors of 5'-D II are reported, i.e. melatonin for BAT and cold for the pineal gland.

Characterization and measurement of [125I]iodopindolol binding in individual rat pineal glands: existence of a 24-h rhythm in beta-adrenergic receptor density.

A simple procedure has been developed that permits measurement of beta-receptors in membrane preparations from individual rat pineal glands using [125I]iodopindolol ([125I]PIN). [125I]PIN binding to pineal membranes was stereospecific and saturable. Scatchard analysis of saturation isotherms yielded a Kd of 147.3 +/- 54 pM and a Bmax of 11.1 +/- 1.5 fmol/pineal gland. Binding was linear suggesting that [125I]PIN binds to a single population of pineal beta-adrenergic receptors. This procedure was used to evaluate 24-h variations in density of pineal [125I]PIN binding sites in male rats maintained in a 14:10 h light:dark cycle. Binding remained uniformly low during the daytime, increased slightly prior to lights off and peaked after 6 h of darkness decreasing abruptly 2 h later, before lights on. In animals maintained in light at night, the number of binding sites also increased, but did not exhibit the darkness-related decrease. The results demonstrate that beta-adrenergic receptors defined via [125I]PIN binding can be measured in tissue samples equivalent to less than one pineal gland. Moreover, the technique can be used in studies concerning the noradrenergic regulation of pineal function.

Beta-adrenergic stimulation prior to darkness advances the nocturnal increase of Syrian hamster pineal melatonin synthesis.

Pineal glands of male Syrian hamsters stimulated in vivo with isoproterenol (ISO) for 4 h before the onset of darkness showed a 4-h advance in the timing of the nighttime increases in both N-acetyltransferase activity and melatonin levels. When ISO (1 mg/kg) was administered every 2 h to animals kept in light during the night, a significant increase in melatonin synthesis was observed after 4-6 h. The results suggest that the Syrian hamster pineal gland can respond in vivo to continuous beta-adrenergic stimulation, but a lag period of 4-6 h is required before there is an increase in melatonin synthesis.

The depression in rat pineal melatonin production after saline injection at night may be elicited by corticosterone.

A hind-leg subcutaneous saline injection into rats at night elicits a decrease in N-acetyltransferase (NAT) activity and melatonin content of the pineal gland. The decrement in pineal melatonin production after saline injection is prevented by adrenalectomy. The present studies were undertaken to determine what factor(s) from the adrenal gland cause(s) the drop in pineal melatonin production after saline injection at night. In the first study, groups of intact and adrenal-demedullated male rats were given a saline injection at 23.10 h (3 h, 10 min after lights off) and their pineals were collected 15 or 30 min later. Pineal NAT activity was depressed in both intact and adrenal-demedullated rats at 15 min postinjection as compared to their respective control animals. Pineal melatonin levels exhibited a drop in intact animals at 15 min and in adrenal-demedullated rats at 30 min. In a second study, hypophysectomy was found to prevent the drop in nocturnal pineal NAT activity and melatonin levels normally associated with a hind leg injection of saline. Finally, in a third experiment, groups of hypophysectomized rats were injected i.p. with corticosterone at 23.10 h and killed 10, 25 or 40 min postinjection. Corticosterone injection in hypophysectomized rats produced a response similar to that caused by saline injection in intact animals: NAT activity was depressed at 10 min and melatonin content was lowered at 25 min. These results suggest that the adrenal-mediated depression in melatonin synthesis after saline injection at night in rats may be elicited by an adrenal cortical hormone (corticosterone) and apparently does not involve the release of factors from the adrenal medulla.

Adrenalectomy but not gonadectomy affects rat pineal beta-adrenergic receptor density.

Bilateral adrenalectomy, like chronic pineal denervation by means of superior cervical ganglionectomy, induced an increase in the morning levels of [125I]iodopindolol (IPIN) binding to rat pineal membranes; this change may be related to the reduced input of catecholamines to the pinealocytes as suggested by the decrease in nocturnal pineal melatonin synthesis previously reported in adrenalectomized rats. Castrated rats did not show changes in IPIN binding to pineal membranes when measured either at middark or in chronically superior cervical ganglionectomized rats, suggesting that the regulation of pineal beta-adrenergic receptors is independent from gonadal control.

Darkness-induced changes in noradrenergic input determine the 24 hour variation in beta-adrenergic receptor density in the rat pineal gland: in vivo physiological and pharmacological evidence.

In male rats housed under a 14:10 LD cycle (lights on at 0600 h), pineal beta-adrenergic receptors, assessed as 125Iodopindolol (IPIN) binding to membrane preparations, showed a 24 hour variation characterized by a nocturnal increase that peaked around middark (2300 h-0200 h) and a decrease during the latter half of the dark period. Animals exposed to light for 3 hours into the normal dark period showed a similar increase in IPIN binding that was prevented by a single sc injection (0.5 mg/kg) of isoproterenol (ISO). The decrease in IPIN binding observed after middark was prevented both by moving the animals to light at 0200 h and by propranolol administration (20 mg/kg). Likewise, the reduction in IPIN binding was induced in light exposed animals both by ISO administration (in a dose dependent manner) and by injection of norepinephrine (NE) plus the catecholamine uptake blocker desmethylimipramine (DMI). DMI alone was without effect. Chronic denervation of the pineal gland by superior cervical ganglionectomy (SCGx) increased IPIN binding to levels not higher than those observed at middark. The results suggest that rat pineal beta-adrenergic receptors are regulated in a rhythmic 24 hour pattern. A decrease in density (downregulation) induced by a darkness-associated increase in NE release, occurs late in the night before lights on; recovery from the down regulated state (upregulation) occurs during the light and early dark phase, reaching a maximum density of beta-adrenergic receptors at middark not different from that observed in chronically denervated pineal glands.

Nocturnal increase of type II thyroxine 5'-deiodinase activity in the Syrian hamster harderian gland is abolished by light exposure and induced by isoproterenol.

The presence of type II 5'-deiodinase activity in the Syrian hamster Harderian gland was investigated. This enzyme exhibited an increase of its activity after animals entered the normal dark phase, with maximal activity occurring at 04.00 hr (8 hr after lights off). The nocturnal increase was prevented by maintaining the animals in light during the night. Isoproterenol subcutaneously injected every 2 hr (1.0 mg/kg body wt) from 20.00 hr through 0.400 hr to animals exposed to light during the normal dark period mimicked the effect of darkness, i.e., with this treatment an increase in 5'-deiodinase activity with maximal peak values at 02.00 hr was observed. The results show that 5'-deiodinase activity in the Syrian hamster Harderian gland exhibits a nyctohemeral profile dependent on beta-adrenergic activation of the gland.

5 alpha-dihydrotestosterone administration converts indolamine metabolism and porphyrin content of the female Syrian hamster Harderian gland to the male type.

The effects of ovariectomy and exogenous androgen administration on the indole and porphyrin metabolism of Syrian hamster Harderian glands were studied. Ovariectomy alone had no effect on any of the parameters analyzed. The administration of either testosterone or 5 alpha-dihydrotestosterone increased the activity of N-acetyltransferase in the Harderian glands. However, androgen treatment failed to change the activity of hydroxyindole-O-methyltransferase. Melatonin content of the glands dropped 20 days after treatment with testosterone and 10 days after the administration of 5 alpha-dihydrotestosterone. The porphyrin content of the Harderian glands was dramatically depressed after the administration of either androgen. It is concluded that the Harderian glands of Syrian hamsters are under an androgenic control involving 5 alpha-dihydrotestosterone.

N-acetyltransferase activity and indole contents of the male Syrian hamster Harderian gland: changes during the light:dark cycle.

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the indole contents of the Harderian glands of male Syrian hamsters were studied throughout a 24-h period. NAT activity exhibited a sharp rise 1 h after lights on, decreasing to basal levels 1 h later. Neither a HIOMT activity nor a melatonin concentration rhythm was detected throughout the 24 h. The 5-hydroxytryptamine (serotonin) concentration was highest during the dark phase reaching a peak at 0300 h; with light onset serotonin levels exhibited a rapid short-term drop. The 5-hydroxytryptophol concentration was highest during the mid- to late photophase; the lowest values to this constituent were measured late in the dark phase and at 1 h after lights on. The 5-hydroxyindole acetic acid concentration of the Harderian glands was rather stable throughout the 24-h period but levels did show a short-lived drop 1 h after light onset. Only a few animals contained detectable amounts of N-acetyl-5-hydroxytryptamine (N-acetylserotonin) in their Harderian glands. In agreement with previous work on the Harderian glands of female Syrian hamsters, the present results in males suggest that light onset is associated with marked changes in Harderian indoleamine metabolism.

Nocturnal increase in the sensitivity of the Syrian hamster pineal gland to isoproterenol is darkness dependent.

The sensitivity of the Syrian hamster pineal gland to stimulation by isoproterenol is greatly increased in the latter half of the daily dark phase. This increased sensitivity requires a period of dark exposure for up to 6.5 hr. Also, if dark-maintained hamsters are exposed to light in the latter half of the night pineal melatonin levels drop precipitously but can be restimulated by isoproterenol administration. As the interval of light exposure continues, however, the pineal sensitivity to isoproterenol decreases.

Stimulatory effect of isoproterenol but not of dibutyryl cyclic AMP on N-acetyltransferase activity and melatonin content of Syrian hamster pineal gland in organ culture.

The purpose of this study was to compare the response of Syrian hamster pineal glands in organ culture either to isoproterenol, a beta-adrenergic agonist, or to dibutyryl cyclic AMP. When pineal glands were collected at night, hamsters were exposed to light for 30 min to depress pineal N-acetyltransferase (NAT) activity and melatonin values to low levels. Pineal glands were removed and placed in organ culture containing either isoproterenol or dibutyryl cyclic AMP and subsequent changes in NAT activity and melatonin levels were measured. At night, isoproterenol (10(-7) or 10(-6) M) induced an increase in the NAT activity and melatonin levels in both pineals and culture media. However, dibutyryl cyclic AMP was either ineffective or minimally effective in stimulating these parameters at either different incubation times (2, 4, and 6 h) or drug concentrations (0.1, 0.5, and 1.0 mM). Conversely, when rat pineal glands were incubated with either isoproterenol (10(-7)) or dibutyryl cyclic AMP (0.5 mM) dramatic rises in NAT activity and melatonin levels were observed. In another experiment, hamster pineal glands were collected from animals killed either late in the light period (19.00 h) or in the latter half of the dark period. Isoproterenol promoted NAT activity and melatonin production only in glands collected in the latter half of the dark phase.

Androgenic control of N-acetyltransferase activity in the harderian glands of the Syrian hamster is mediated by 5 alpha-dihydrotestosterone.

N-acetyltransferase (NAT) activity in the Harderian glands of intact and gonadectomized male and female Syrian hamsters was evaluated. The exogenous administration of 5 alpha-dihydrotestosterone (DHT) to castrated males and intact females produced an increase in NAT values, which reached the values present in the glands of intact males. The administration of a 5 alpha-reductase inhibitor to intact males led to a decrease in NAT activity, suggesting that testosterone is converted in DHT within the glands. It is concluded that NAT activity in the Syrian hamster Harderian glands is under androgenic control, the active steroid being DHT.

Forskolin, an activator of adenylate cyclase activity, promotes large increases in N-acetyl transferase activity and melatonin production in the Syrian hamster pineal gland only during the late dark period.

The exposure of organ cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in serotonin N-acetyltransferase activity and melatonin content in a dose-related manner (1-100 microM) when glands were collected in the second half of the dark period. However, addition of forskolin to glands collected anytime during the light period or at the beginning of the dark period failed or only modestly stimulated either pineal N-acetyltransferase activity or melatonin levels. Similar results were obtained with isoproterenol. The results suggest that intrapinealocyte regulatory mechanisms may determine the nocturnal rise in the Syrian hamster pineal gland.

N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin levels in the Harderian glands of the female Syrian hamster: changes during the light:dark cycle and the effect of 6-parachlorophenylalanine administration.

The activities of NAT and HIOMT and the melatonin content of the Harderian glands of female Syrian hamsters were studied. When hamsters were kept under a light:dark cycle of 14:10 (lights on at 06.00 h), NAT activity exhibited a sharp, short term rise at one hour after lights on. Simultaneously, the activity of HIOMT, which forms melatonin, exhibited a rapid decline. Melatonin levels, like HIOMT activity, also showed a precipitous drop at one hour after light onset. After the respective changes, both NAT and HIOMT activity reverted back to night time levels. Melatonin levels remained depressed for several hours but by 1400 h (8 hours after lights on), nighttime melatonin values were re-established. Treatment of female hamsters with PCPA, a trytophan hydroxylase inhibitor, led to depressed levels of Harderian melatonin without affecting the activities of either NAT or HIOMT.

Differential responses of rat pineal thyroxine type II 5'-deiodinase and N-acetyltransferase activities to either light exposure, isoproterenol, phenylephrine, or propranolol.

1. Compared to pineal N-acetyl transferase (NAT) activity, which exhibited a dramatic drop following acute light exposure at night, nocturnal rat pineal thyroxine type II 5'-deiodinase (5'-D) activity was minimally influenced by the same light exposure. The injection of cycloheximide, a potent inhibitor of protein synthesis, although it did curtail the rise in NAT activity for at least 2 hr, did not elicit decreases in the activities of either 5'-D or NAT enzymes. Propranolol, a beta-adrenergic blocker, either delayed the continued nocturnal rise in 5'-D activity when injected at 0000 hr or slightly enhanced the fall in 5'-D activity when injected at 0200 hr. These results suggest that interruption of the synthesis of proteins is responsible for the slow deterioration of 5'-D activity induced by either light or propranolol. 2. The slight fall in 5'-D activity induced by light at night was prevented by isoproterenol; phenylephrine, however, did not prevent the fall and the effect of isoproterenol + phenylephrine was similar to that obtained with isoproterenol alone. On the other hand, the light-inhibited NAT activity recovered after the injection of isoproterenol; phenylephrine did not elicit any effect, but the injection of both isoproterenol and phenylephrine simultaneously caused a greater NAT response than that induced by isoproterenol alone. 3. When injected during the day, phenylephrine had no effect on either pineal 5'-D or NAT activities; however, the injection of either isoproterenol alone or isoproterenol + phenylephrine elicited 5-fold and 10-fold increases in nocturnal, light-suppressed 5'-D and NAT activities, respectively. During the day, phenylephrine did not potentiate the effects of isoproterenol on NAT activity as it did at night. When the effects of isoproterenol on the 5'-D activity were compared to rats exposed to light during the day and at night, the activity of 5'-D reached a higher level at night than during the day.

Effects of short-term cold exposure on pineal biosynthetic function in rats.

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.

Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Spermophilus richardsonii).

Melatonin synthesis in the pineal gland, which is primarily regulated by the environmental lighting regime, can also be influenced by other factors that elicit modifications in sympathetic tone. The objectives of this study were to determine if forced swimming alters the normal pattern of melatonin production in the pineal gland of the Richardson's ground squirrel (Spermophilus richardsonii). In early June, the squirrels were forced to swim for 10 min during the photophase or during the scotophase. In mid-July squirrels swam only during the scotophase. Animals were sacrificed 15, 30, or 60 min after the onset of swimming. Activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were assessed by radioenzyme assay, and pineal melatonin content was measured by radioimmunoassay. Daytime swimming elicited no major changes in enzyme activity or pineal melatonin. In June, swimming at night prevented the normal rises in NAT activity and pineal melatonin seen in nonswimming controls. In contrast, the pineals of squirrels that were tested 6 weeks later in mid-July did not appear to be as sensitive to nighttime swimming, as there were only minor differences in both NAT activity and melatonin content compared to controls. These results demonstrate that forced nighttime swimming, unlike several other aversive stimuli, can evoke changes in the normal pattern of pineal melatonin production in this species. Furthermore, the pineal's response to such stimuli may not be stable over the course of the active season.

mRNA transcription determines the lag period for the induction of pineal melatonin synthesis in the Syrian hamster pineal gland.

The nocturnal pattern of Syrian hamster pineal melatonin synthesis is characterized by a 6-8 h lag period, followed by a late-night, short-duration peak in both N-acetyltransferase (NAT) activity and melatonin content. Administration of cycloheximide (20 mg/kg body weight) given either at the time of lights out or 4 h into the dark phase to Syrian hamsters blocked the nocturnal increase in both pineal NAT activity and melatonin content. Actinomycin D (5 mg/kg body weight) prevented the nocturnal increase in both constituents only when it was administered at darkness onset, being significantly less effective when injected after 4 h of dark exposure. Reinduction of melatonin production by isoproterenol (2 mg/kg body weight) administration to acutely light-exposed animals during late darkness was prevented by cycloheximide, but not by actinomycin D administration. The results suggest that whereas Syrian hamster pineal melatonin production requires protein synthesis both early and late in the dark phase, the transcription of a putative NAT-related mRNA, which occurs only during the early night, seems to determine the lag period in melatonin synthesis and pineal responsiveness to beta-adrenergic receptor agonist stimulation.

Adrenalectomy prevents changes in rat pineal melatonin content and N-acetyltransferase activity induced by acute insulin stress.

The activity of N-acetyltransferase (NAT) and the content of melatonin (MEL) in the rat pineal have been shown to be sensitive to several types of stressors. This study was designed to assess the role of the adrenals in mediating the effect of one such stressor, insulin-induced hypoglycemia, on pineal synthetic activity. Intact and bilaterally adrenalectomized (ADX) adult male rats were kept under light:dark cycles of 14:10 (lights on 0600 h) and injected intraperitoneally with 10 IU insulin at 1300 h, and groups (n = 8) were killed 2, 3, or 4 h postinjection. Plasma catecholamines were assayed by means of high performance liquid chromatography and radioimmunoassay was used to assess pineal NAT activity and MEL content. All injected groups were rendered hypoglycemic by insulin administration. Compared to uninjected controls, plasma epinephrine in hypoglycemic intact rats rose after 2 h, whereas epinephrine did not change in hypoglycemic ADX animals. The increase in epinephrine in intact animals was correlated with a rise in NAT activity at 2 h. Moreover, pineal MEL content at 2, 3, and 4 h was significantly greater than control values. In contrast, no changes in pineal biosynthetic function were found in ADX rats. This differential response by intact and ADX rats suggests that an adrenal product (possibly epinephrine) is responsible for mediating the stimulatory effects of acute insulin-induced hypoglycemic stress on the rat pineal.