Maureen E. Troiani

Norepinephrine or Isoproterenol Stimulation of Pineal N-Acetyltransferase Activity and Melatonin Content in the Syrian Hamster Is Restricted to the Second Half of the Daily Dark Phase

Seven experiments were performed to investigate the sensitivity of the hamster pineal gland to exogenously administered norepinephrine (NE). In these studies NE (1 mg/kg) administration was preceded (10 min earlier) by the injection of the catecholamine uptake inhibitor desmethylimipramine (DMI; 5 mg/kg). When DMI and NE were given at night, the hamsters were exposed to light to depress pineal N-acetyltransferase activity and melatonin values to low levels; the drugs were then given 20 (DMI) and 30 (NE) min later, and the subsequent changes in pineal N-acetyltransferase and melatonin were monitored. The combination of DMI and NE administration anytime during the normal light period or during the first 4 h of the normal dark period failed to stimulate either pineal N-acetyltransferase activity or melatonin levels. Conversely, DMI followed by NE (injected either intraperitoneally or subcutaneously) in the second half of the dark phase typically stimulated pineal melatonin production. Likewise, the NE agonist isoproterenol promoted pineal melatonin production only in the latter half of the dark phase. If hamsters were exposed to continual light at night or if they were superior cervical ganglionectomized, a procedure which sympathetically denervates the pineal gland, the stimulatory effect of NE on melatonin production was significantly suppressed. Thus, the hamster pineal gland is sensitive to NE only during the latter half of the normal dark period and both darkness and an intact sympathetic innervation to the pineal gland are required for the gland to develop maximal sensitivity to the catecholamine. Also, the hamster pineal seems not to exhibit a supersensitivity response to NE following a period of reduced exposure to the catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Swimming Depresses Nighttime Melatonin Content without Changing N-Acetyltransferase Activity in the Rat Pineal Gland

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.

Neither the pituitary gland nor the sympathetic nervous system is responsible for eliciting the large drop in elevated rat pineal melatonin levels due to swimming

Since the pineal gland is an end organ of the sympathetic nervous system, stress might increase the synthesis of its hormone, melatonin. The stress of a 10 min swim, which elicits a marked rise in circulating catecholamines, causes a dramatic depression of high pineal melatonin levels at night within 15 min after swimming onset. N-acetyltransferase (NAT) activity is unaffected by the treatment at 15 or 30 min after swimming onset. Within 90 min after initiation of a 15 min swim, high nighttime pineal melatonin levels are restored while NAT values remain elevated. The swimming-induced reduction in high pineal melatonin levels is not influenced by either hypophysectomy, superior cervical ganglionectomy, prazosin (α1-adrenergic receptor blocker) pretreatment, yohimbine (α2-adrenergic receptor blocker) pretreatment, or reserpine (amine depletor) pretreatment. These results indicate that neither hormones secreted from the pituitary gland nor catecholamines secreted from the sympathetic nerves are involved in eliciting the dramatic reduction in elevated pineal melatonin levels in the rat.

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

The activity of N‐acetyltransferase (NAT) 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 bilaterally adrenalectomized (ADX) adult male rats were kept under light:dark cycles of 14:10 (lights on 0600 h) injected intraperitoneally with 10 IU insulin at 1300 h, groups (n = 8) were killed 2, 3, or 4 h postinjection. Plasma catecholamines were assayed by means of high performance liquid chromatography radioimmunoassay was used to assess pineal NAT activity 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, 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 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.

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.

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.

Harderian Gland N-Acetyltransferase Activity in the Male Syrian Hamster: Effects of Gonadectomy, Short Photoperiod Exposure, or Subcutaneous Melatonin Implants

The activities of NAT and HIOMT and the melatonin concentration in the Harderian glands of intact, gonadectomized, and gonadally-regressed male Syrian hamsters were studied. To produce gonadal regression, hamsters were exposed to either artificial or naturally short photoperiods. NAT activity of castrated and gonadally-regressed hamsters was always less in comparison to that of animals with intact gonads. Castrated hamsters exposed to long days showed higher NAT activity than that of castrated animals exposed to short photoperiods indicating that light may have some influence on Harderian NAT independent of the gonadal status. Also, gonadally-regressed hamsters exposed to long photoperiods exhibited higher NAT activity in comparison to gonadally-regressed animals exposed to short days. The HIOMT activity and melatonin content of Harderian glands in all these groups of male Syrian hamster were very low.

Elevated ambient temperature retards the atrophic response of the neuroendocrine-reproductive axis of male Syrian hamsters to either daily afternoon melatonin injections or to short photoperiod exposure.

Adult male Syrian (golden) hamsters, maintained under either 22 +/- 2 or 32 +/- 2 degrees C, were treated with 8 or 11 weeks of exposure to either long photoperiod (14:10), short photoperiod (8:16), or to long photoperiod with a daily afternoon melatonin injection. By 8 weeks, the animals kept at 22 degrees C and treated with daily afternoon melatonin injection exhibited a dramatic reduction in testicular and accessory sex organ weight, but the animals kept at 32 degrees C and treated in the same way exhibited only slight decreases in testicular and accessory organ weights. Short photoperiod caused a slight decrease in testicular and accessory organ weights of hamster kept at 22 degrees C, while it had no significant effects on reproductive organ weights of the animals maintained under 32 degrees C. By 11 weeks, the daily afternoon melatonin injection elicited further reduction in testicular and accessory organ weights of the animals maintained under both 22 and 32 degrees C. However, the reduction in animals kept at 32 degrees C was not as great as that in animals kept at 22 degrees C. Although short photoperiod caused an obvious decline in reproductive organ weights of the animals at 22 degrees C, only a slight decrease was seen in hamsters at 32 degrees C. As with reproductive organ weights, testosterone levels were depressed more rapidly and completely in animals maintained at 22 degrees C. These results indicate that elevated ambient temperature changes the rate at which the gonads of hamsters regress in response to daily afternoon melatonin injections or short photoperiod.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Short‐Day Photoperiods and of N‐(2,4‐Dinitrophenyl)‐5‐Methoxytryptamine, a Putative Melatonin Antagonist, on Melatonin Synthesis in the Harderian Gland of the Syrian Hamster, Mesocricetus auratus

The Harderian glands of Syrian hamsters contain melatonin and the enzymes N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT) which synthesize melatonin from serotonin. Because the Harderian glands share this metabolic pathway with the pineal gland, we examined the effects of short‐day photoperiods, which stimulate pineal‐mediated gonadal regression, and N‐(2,4‐dinitrophenyl)‐5‐methoxytryptamine (ML‐23), which has been described as a melatonin antagonist, on melatonin synthesis in the Harderian glands of the hamster.