C. Santana

Inhibition of Pineal Type-II 5′-Deiodinase Does Not Affect the Nocturnal Increase of N-Acetyltransferase Activity and Melatonin Content in Either Euthyroid or Thyroidectomized Rats

The presence of type‐II thyroxine 5′‐deiodinase (5′‐D) activity in rat pineal gland has been previously described. In the present paper, 5′‐D activity, N‐acetyltransferase (NAT) activity, and melatonin content were measured in the same rat pineal. Each of these constituents exhibits a nocturnal increase with peak values at 0100 h for melatonin (1.20 ± 0.12 ng/gland) and at 0300 h for both 5′‐D (39.5 ± 11.9 fmol/gland/h) and NAT (8.38 ± 1.04 nmol/gland/h) activities. In vivo treatment with iopanoic acid (IOP) completely prevented the nocturnal increase in 5′‐D activity (14.1 ± 2.6 fml/gland/h at 0300 h) with no modification in either the NAT activity or melatonin content. Thyroidectomy greatly enhanced the 5′‐D activity during the dark period (102.9 ± 10.2 vs. 31.6 ± 4.2 fmol/gland/h), reaching a peak at 0200 h; thyroidectomy, however, did not affect daytime pineal 5′‐D activity (3.11 ± 0.78 vs. 2.5 + 0.92 fmol/gland/h). Treatment of rats with IOP acid completely inhibited the pineal 5′‐D activity in both control (7.86 ± 0.88 fmol/gland/h) and thyroidectomized animals (2.24 ± 1.10 fmol/gland/h) with no change in the melatonin content of the gland (1.21 ± 0.32 vs. 0.99 ± 0.18 ng/gland).

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

Abstract 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 β-adrenergic activation of the gland.

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

Summary1.Compared to pinealN-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, aβ-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.

Role of postsynaptic α-adrenergic receptors in the β-adrenergic stimulation of melatonin production in the Syrian hamster pineal gland in organ culture

The role played by postsynaptic α‐adrenergic receptors in the stimulation of pineal melatonin production was investigated in the Syrian hamster. The studies were conducted using organ cultured pineal glands collected from both anatomically intact and superior cervical ganglionectomized hamsters. Results obtained indicate that phenylephrine, an a‐adrenergic agonist, by itself has no effect in promoting melatonin production; however, it potentiates the stimulatory effects of isoproterenol, a P‐adrenergic agonist, on pineal melatonin production in nonoperated hamsters. Similar observations were obtained with pineal glands whose presynaptic terminals were removed by prior superior cervical ganglionectomy. However, a longer incubation time was required (4–6 hours vs. 2 hours) with pineal glands taken from ganglionectomized animals. Apparently, β‐adrenergic activation is an absolute requirement to stimulate pineal melatonin production, and an a‐adrenergic receptor mechanism potentiates P‐adrenergic activation. In addition, the findings obtained with denervated pineal glands suggest that the regulation of pineal melatonin production by both α‐ and p‐adrenergic mechanisms is through receptors located on postsynaptic structures.

Effects of Photoperiod or Exogenous Melatonin Administration on the Activity of N-Acetyltransferase and Hydroxyindole-O-Methyltransferase and the Melatonin Content of the Harderian Gland of Two Strains of Female Syrian Hamsters

The activities of N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT) and the melatonin concentration of the Harderian glands of two strains of Syrian hamster females (outbred and inbred LSH/SsLak) exposed to two different photoperiods (14:10 h and 8:16 h) were studied. The Harderian glands of the inbred hamsters showed greater NAT activity than those of the outbred animals. On the other hand, the glands of the outbred hamsters exhibited higher HIOMT activity and melatonin content than those of the inbred LSH/SsLak. Short photoperiod exposure, which produced gonadal regression in the inbred but not in the outbred hamsters, decreased the NAT activity in the inbred animals to the levels of the outbred. HIOMT activity was not affected by the lighting conditions. After the exposure to short days, the melatonin content of the inbred hamster Harderian glands increased to that in the outbred animals. Daily melatonin injections, which caused gonadal regression in the LSH/SsLak but not in the outbred hamsters, did not simulate the effect of the short photoperiod on the Harderian gland NAT activity and melatonin content of the inbred hamsters.

ß-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.

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.

The in vitro activation of cyclic AMP production by either forskolin or isoproterenol in the syrian hamster pineal during the day is not accompanied by an increase in melatonin production

The role of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Forskolin markedly increased cyclic AMP production in pineal glands collected either late in the light period or in the dark period. The effect of forskolin was synergistically enhanced by 3-isobutylmethylxanthine, a phosphodiesterase inhibitor; however, increase in cyclic AMP after isoproterenol was only apparent in the presence of 3-isobutylmethylxanthine. Since beta-adrenergic agonists are able to stimulate melatonin production late in the dark period only, these data suggest that, in the hamster pineal gland, there may be intracellular mechanisms in addition to a cyclic AMP increase required for induction of melatonin production by beta-adrenergic agonists.

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol.

Abstract Herein we show, for the first time, a very marked increase in thyroxine 5′-deiodinase (5′-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5′-D than was isoproterenol, suggesting that, in addition to β-adrenergic receptors, α-adrenergic receptors might be involved in the 5′-D activation. However, phenylephrine, an α-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5′-D activity. The nocturnal increase in pineal 5′-D activity was completely abolished by propranolol, a β-adrenergic receptor blocker, while prazosin, an α-adrenergic receptor blocker, had minimal effect. These results show that the role of α-receptors in promoting the NE-mediated rise in rat pineal 5′-D activity is minor in contrast to the role of β-adrenergic receptors.

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.