Characterization of type-II thyroxine 5'-deiodinase activity in rat Harderian gland

Thyroxine 5'-deiodinase activity was studied in male rat Harderian gland homogenates. The reaction rate was proportional to the tissue content in the homogenate and dependent on pH, with an optimum pH of 7.0, and temperature, between 4-37 degrees C. 5'-deiodinase activity was increased by dithiothreitol (DTT) in a dose-dependent manner, and inhibited moderately by propylthiouracil (PTU) and strongly by iopanoic acid (IA). Thyroidectomy enhanced the enzymatic activity (30-fold above the control value) but this increase is totally prevented by the in vivo iopanoic acid treatment. Thyroxine 5'-deiodinase activity was also dependent on T4 concentration (Km = 3.3 nM; Vmax = 10 fmol 125I-released/mg protein/h) and exhibited a nyctohemeral rhythmicity with a maximal activity at 03.00 h (4-fold above basal values) and minimal activity between 12.00-21.00 h.     

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

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 beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-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 beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.     

Ontogeny of type II thyroxine 5'-deiodinase, N-acetyltransferase, and hydroxyindole-O-methyltransferase activities in the rat Harderian gland

The ontogeny and regulation by isoproterenol of type II thyroxine 5'-deiodinase, N-acetyltransferase, and hydroxyindole-O-methyltransferase activities were studied in the rat Harderian gland. Both 5'-deiodinase and N-acetyltransferase activities exhibited maximal values at the first week of age. These activities gradually decreased till the fourth week. However, hydroxyindole-O-methyltransferase activity did not change during the period of time studied. Neither the different killing times (1600 or 0200 h) nor the photoperiod regimens (darkness or light exposure at night) modified the enzyme activities. On the other hand, isoproterenol, a beta-adrenergic agonist, could not to stimulate 5'-deiodinase at first week of life. Nevertheless, while basal 5'-deiodinase activity was diminishing during development, the enzyme was becoming sensitive to the action of isoproterenol. Thus, isoproterenol elicited increases in Harderian gland 5'-deiodinase activity in rats older than two weeks. However, both N-acetyltransferase and hydroxyindole-O-methyltransferase activities were not affected by isoproterenol treatment in either week studied.     

Beta- and alpha-adrenergic receptors are involved in regulating type II thyroxine 5'-deiodinase activity in the rat Harderian gland.

The role of alpha- and beta-adrenergic receptors in regulation of rat Harderian gland type II thyroxine 5'-deiodinase (5'-D) activity was investigated. Our results show that isoproterenol, a beta-adrenergic agonist, and phenylephrine, an alpha-adrenergic agonist, elicited increases in Harderian gland 5'-D activity. The activation was dependent on the time and the dose of the drug. Other adrenergic agonists, i.e., norepinephrine, methoxamine or terbutaline, also clearly increased the enzyme activity. Moreover, administration of propranolol, a beta-adrenergic blocker, or prazosin, an alpha-adrenergic blocker, completely prevented the activation of the enzyme induced by norepinephrine. Results show a clear regulation by adrenergic mechanisms of 5'-D activity in the rat Harderian gland, where alpha- and beta-adrenergic receptors appear to be involved.     

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.     

Evidence that rat pineal thyroxine 5'-deiodinase is primarily stimulated by beta- and not alpha-adrenergic agonists and that its adrenergic-stimulated and spontaneous rhythmic nocturnal rise require RNA and protein synthesis

Pineal thyroxine 5'-deiodinase (5'-D) activity rose greater than 10-fold above the basal level 2-3 hr after 1 mg/kg isoproterenol and returned to near the basal level by 6 hr. The same dose of norepinephrine or phenylephrine was without effect, but phenylephrine modestly potentiated isoproterenol-stimulated 5'-D activity. Either actinomycin D or cycloheximide treatment markedly decreased diurnal isoproterenol stimulation and the spontaneous rhythmic nocturnal rise of pineal 5'-D. The data indicate that pineal 5'-D activity is very similar to pineal serotonin N-acetyl transferase in being primarily stimulated by beta-adrenergic agonists and requiring new RNA and protein synthesis for its activation.     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Seasonal peculiarities of thyroxine influence on the way of serotonin metabolism in the pineal gland of the rat

In intact Wistar pubertal male rats held on LD 8:16 (winter) and 16:8 (summer) pineal melatonin (M) production and other pineal indoles content serotonin (S), 5-methoxytriptamine (5-MT), N-acetylserotonin (N-aS), 5-hydroxyindoleacetic acid (5-HIAA) and 5-methoxyindoleacetic acid (5-MIAA) were investigated in basic conditions and after 10-days T4 administration. The results suggest, that intact control in winter comparing with summer demonstrates high M, N-aS, 5-HIAA and 5-MIAA level, but low 5-MT concentrations, S level was not changed. T4 administration in winter produced a pronounced augmentation of all parameters (excluding serotonin), in summer M production not only increased, but appears to be dependent on the active metabolism of his precursors (S, 5-MT, N-aS). The experimental data favour more profound comprehension to the mechanisms of pineal activity regulation.     

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.     

Comparison of isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate stimulation of thyroxine 5'-deiodinase activity in cultured pineal glands from euthyroid and hypothyroid rats

Thyroxine 5'-deiodinase was increased by isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate in a dose- and time-related manner in cultured rat pineal gland. Basal and stimulated activity was higher in glands from hypothyroid than from euthyroid animals. Our data suggest direct beta-adrenergic stimulation of intracellular cyclic AMP may be involved in the regulation of pineal thyroxine 5'-deiodinase activity.     

Rhythmic control of endocannabinoids in the rat pineal gland

Endocannabinoids modulate neuroendocrine networks by directly targeting cannabinoid receptors. The time-hormone melatonin synchronizes these networks with external light condition and guarantees time-sensitive and ecologically well-adapted behaviors. Here, the endocannabinoid arachidonoyl ethanolamide (AEA) showed rhythmic changes in rat pineal glands with higher levels during the light-period and reduced amounts at the onset of darkness. Norepinephrine, the essential stimulus for nocturnal melatonin biosynthesis, acutely down-regulated AEA and other endocannabinoids in cultured pineal glands. These temporal dynamics suggest that AEA exerts time-dependent autocrine and/or paracrine functions within the pineal. Moreover, endocananbinoids may be released from the pineal into the CSF or blood stream.     

Iodothyronine 5'-deiodinating activity in the pineal gland.

1. The presence of an iodothyronine 5'-deiodinating activity has been described in the pineal gland of various rodents, and it has been identified as a type II 5'-deiodinase isoenzyme since it is relatively insensitive to inhibition by propylthiouracil and its activity increases during hypothyroidism. 2. 5'-Deiodinase activity in the rat pineal gland follows a nyctohemeral profile, exhibiting basal values during the day and maximal values at night. The nocturnal increase is dependent on the noradrenergic input from the superior cervical ganglia, and both in vivo and in vitro studies show that beta-adrenergic receptors are primarily involved in the activation of the enzyme. 3. Day-night differences in rat pineal 5'-deiodinase activity are found beginning at 2 weeks of age, with rhythms increasing in amplitude until maximal differences are reached in adult animals. During the maturation of the rhythm, changes in regulation of enzyme activation are observed. Thus, during the first 2-3 weeks of age, alpha-adrenergic receptors appear to be as important as beta-adrenergic receptors in regulating the deiodinating activity of the pineal. However, in adults, no role of alpha-adrenergic receptors has been described. 4. Although regulation of 5'-deiodinase activity in the pineal gland is well established, few data are available concerning the physiological significance of the enzyme in the gland. Of the studies that have been performed, those attempting to demonstrate a relationship between pineal 5'-deiodinase activity and other pineal rhythms, e.g. those of melatonin production and N-acetyltransferase activity, indicates that the latter rhythms do not rely on the cyclic production of T3. The alternate possibility that the 5'D rhythm depends on the cyclic production of melatonin remains to be examined.     

Tentative immunohistochemical demonstration of melatonin in the rat pineal gland

Summary In the present study an attempt was made to demonstrate melatonin in the rat pineal gland by means of immunohistochemistry. The anti-body used was raised against 5-methoxy-N-acetyltryptophan which is chemically similar to melatonin. Specific fluorescence was demonstrable only in pineals from rats killed during the night, when melatonin formation is high. It was restricted to parenchymal cells lying in a marginal zone of the organ. These results are discussed in relation to a subdivision of the pineal parenchyma into cortical and medullary areas.Supported by a grant of the Deutsche Forschungsgemeinschaft (VO 135/4) within the Schwerpunktprogramm Neuroendokrinologie     

Diurnal variation of dopamine content in the rat pineal gland

Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl-³H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.