Circadian rhythmicity of the activity of hydroxyindole-o-methyl transferase (HIOMT) in the formation of melatonin and 5-methoxytryptophol in the pineal,retina, and harderian gland of the golden hamster

Summary The day and night rhythms in the activity of HIOMT in the formation of melatonin and of 5-methoxytryptophol have been determined in the pineal, retina and Harderian gland of the adult male golden hamster.In all hamsters used there was no detectable HIOMT activity in the deep pineal. In the superficial pineal HIOMT activity, involved in the synthesis of melatonin (Mel), was observed to be high at the end of the dark period and at the middle of the light period. Considering the HIOMT activity involved in the production of 5-methoxytryptophol (5-MTL), an increase in 5-MTL synthesis was observed only during the light period. Comparing the peak of Mel-production with that of 5-MTL it appears that during the light period the pineal produces more 5-MTL than Mel.In the Harderian glands, the circadian course of HIOMT activity involved in the synthesis of Mel seems to run parallel to that of the enzyme implicated in 5-MTL synthesis, both being stimulated at the end of the dark period. The activity of HIOMT in 5-MTL production is, however, always approximately 2 times higher than for Mel synthesis.In the retina the synthesis of Mel and 5-MTL is not significantly higher during the dark period than during the light period. However, the production of 5-MTL is larger than that of Mel.It appears that (1) with the exception of the end of the dark period, the extra-pineal synthesis of Mel and 5-MTL is always higher than that in the pineal; (2) the circadian synthesis of 5-methoxyindoles is different in each organ, and (3) in the pineal the circadian activity of HIOMT involved in 5-MTL formation is different from that of the same enzyme involved in the formation of Mel. The results are discussed.     

Lesions of the paraventricular nucleus area of the hypothalamus disrupt the suprachiasmatic→ spinal cord circuit in the melatonin rhythm generating system

The circadian rhythm in melatonin production in mammals is regulated by a suprachiasmatic (SCN) leads to spinal cord leads to pineal circuit. In the present investigation the possible participation of the paraventricular nucleus of the hypothalamus (PVN) in the SCN leads to spinal cord segment of this circuit was investigated in the rat. Bilateral lesions of the PVN area were produced and one to two weeks later melatonin production was evaluated by measuring the activities of the two pineal enzymes required for the formation of melatonin from serotonin, indoleamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and urinary 6-hydroxymelatonin, the major melatonin metabolite. In some cases pineal melatonin was also measured. Control animals received sham-PVN lesions. Histological examination of the lesions indicated that the PVN were bilaterally destroyed 100% in 12 animals. The nighttime pineal melatonin and urinary 6-hydroxymelatonin values in this group were reduced about 90%, nighttime pineal NAT activity was reduced about 98%, and HIOMT activity about 75%. The urinary 6-hydroxymelatonin values of PVN-lesioned animals and animals with denervated pineal glands were similar. In animals with hypothalamic lesions involving less than 30% of the PVN, nighttime values of NAT, HIOMT, and urinary 6-hydroxymelatonin were normal; in animals with 30 to 95% PVN damage these parameters were altered to a small degree. These studies, together with histochemical observations, indicate the SCN neurons responsible for pineal circadian rhythms project to the PVN area of the hypothalamus.     

Seasonal variations in HIOMT activity during the night in the pineal gland of 21 day old male wistar rats

Summary In the pineal of 21-day old male Wistar rats hydroxyindole-O-methyl-transferase (HIOMT) activities involved in the synthesis of several 5-methoxyindoles were determined during the night in April, June, October and January. A high HIOMT activity for the synthesis of melatonin/5-methoxytryptophol was determined in the months of January and April. In June and October a decrease was observed. The activity maxima coincide with peaks of activity found for the synthesis of 5-methoxytryptophan. Synthesis of 5-methoxytryptamine occurred only in June and October, whereas the synthesis of 5-methoxyindole-3-acetic acid occurred only in January. From these results it may be concluded that January and April are the most active months of those tested for the melatonin/5-methoxytryptophol synthesis in the rat pineal gland. A possible physiological role of the 5-methoxyindoles other than melatonin and 5-methoxytryptophol is discussed.     

Pineal indole metabolism in the mouse

The activity of two pineal enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole-O-methyltransferase, (HIOMT) and the pineal content of serotonin (5-HT) and N-acetylserotonin (NAS) were measured in several strains of mice (Mus domesticus) in order to compare melatonin synthetic pathways among them. Of the strains we examined, C57BL/6J, AKR/J, BALB/c, NZB/BLNJ and wild mice, only wild mice synthesize pineal melatonin. Wild mice had high activity of both SNAT and HIOMT; NZB had SNAT activity but did not have HIOMT; the three other strains had neither SNAT nor HIOMT; the activity of SNAT in wild mice showed a clear daily rhythm but HIOMT activity did not show any significant daily changes. The pineal content of 5-HT in wild and NZB mice was higher during the day than during the night, however in C57BL and AKR, 5-HT levels were higher during the night than during the day. As expected NAS was found in wild and NZB mice.     

Possible involvement of neuropeptide Y in the seasonal control of hydroxyindole-O-methyltransferase activity in the pineal gland of the European hamster (Cricetus cricetus)

Hydroxyindole-O-methyltransferase (HIOMT) catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have established (1) that one of these neuropeptides, neuropeptide Y (NPY), stimulates specifically HIOMT activity in rat pinealocytes and (2) that the density of the NPY-immunoreactive (NPY-IR) fibers innervating the pineal gland of the European hamster (Cricetus cricetus) displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryptophol (5-ML) content and of circulating MEL levels in European hamsters when NPYergic innervation is low (end of October) and when it is the highest (mid-December). We report in this study that HIOMT activity is significantly increased by 80% in mid-December compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels (with a fourfold increase occurring in early dawn and decreasing slowly towards the end of the day). These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity.     

Circadian rhythmicity in the methylation of 5-hydroxy-indoles and norepinephrine in the pineal gland of 10 day old rats

Summary In pineals of 10 day old rats 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, N-acetylserotonin/5-hydroxytryptophol and norepinephrine are methylated following a circadian rhythm. During the night HIOMT and COMT activities were measured for the above mentioned substrates, while HIOMT activity for 5-hydroxytryptophan and N-acetylserotonin/5-hydroxytryptophol was also determined during daytime.     

High levels of human chymase expression in the pineal and pituitary glands

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.     

An antiserum against chicken hydroxyindole-O-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors

Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole-O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4-5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.     

Hydroxyindole-O-methyltransferase in rat retinal bipolar cells: persistence following photoreceptor destruction

The presence of hydroxyindole-O-methyltransferase (HIOMT) activity and localization of HIOMT immunoreactivity was examined in albino rat retinas following photoreceptor destruction. Male Sprague-Dawley rats were exposed to high intensity fluorescent light for 4 consecutive days, then placed on a 14:10 h light:dark cycle for two weeks to allow for phagocytic removal of damaged cells from the retina. Histologic examination revealed almost complete destruction and removal of all photoreceptors. The damaged retinas exhibited an increase in HIOMT activity relative to controls, when expressed as activity per mg of protein. HIOMT activity in the pineal glands was not affected. When control and light damaged retinas were examined for HIOMT localization by immunocytochemistry, the control retinas displayed intense HIOMT immunoreactivity in all photoreceptors, and a somewhat lighter labeling in a population of bipolar cells, whereas the light damaged retinas (lacking photoreceptors) showed intense HIOMT immunoreactivity in bipolar cells. These results suggest that the increase in HIOMT activity following photoreceptor destruction is due to increased synthesis of this enzyme in a population of bipolar cells. These HIOMT-immunoreactive bipolar cells may perhaps respond in a compensatory manner to changing levels of melatonin in the retina.     

Alterations in components of the pineal melatonin synthetic pathway by acute insulin stress in the rat and Syrian hamster

Acute insulin stress increased plasma catecholamine levels in both the Syrian hamster and albino rat within 3 h after an intraperitoneal injection of either 5 or 10 units of insulin. In the rat, this stress caused a concurrent increase in pineal serotonin N-acetyltransferase (NAT) activity and melatonin content with no observable change in hydroxyindole-O-methyltransferase (HIOMT) activity. In the hamster, on the other hand, acute insulin stress did not alter pineal NAT activity, but depressed both HIOMT activity and melatonin content up to 3 h after the stress. These results present further evidence that catecholamines do not control hamster pineal melatonin synthesis by the same mechanism as observed in the rat.     

Immunocytochemical demonstration of hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland

The immunocytochemical localization for hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland is described in this paper. Most of the pinealocytes showed immunoreactivities to a HIOMT antiserum and an NSE antiserum, simultaneously. Neither HIOMT-nor NSE-immunoreactivity was observed in a few pinealocytes. On the other hand, dispersed interstitial cells (glial cells) were stained only by an S-100 protein antiserum. No evident HIOMT-immunoreactivity was observed in the retina and intestinal mucosa, where HIOMT has been suggested to occur.     

Expression and characterization of hydroxyindole O-methyltransferase from a cloned cDNA in Chinese hamster ovary cells

We have used two kinds of expression systems to test whether the cloned cDNA encoding hydroxyindole O-methyltransferase (HIOMT) of the bovine pineal gland was functional or not. First, when mRNA was synthesized in vitro by the SP6 system and injected into Xenopus oocytes, the enzymatic activity was expressed in the oocytes. Second, the cloned cDNA was recombined to a vector under the control of the simian virus 40 early promoter and transfected to Chinese hamster ovary (CHO) cells. The enzymatic activity of the crude supernatant of transfected cells (CHO-HT2) reached to 400 pmol melatonin formed per min per mg of protein, which value was approximately 9% of that of bovine pineal supernatant. The amounts of enzyme protein estimated by immunoblotting were proportional to the enzymatic activity in both CHO and pineal gland. The content of HIOMT protein was 8- to 30-fold larger in pineal gland compared to CHO cells. On the other hand, the content of mRNA encoding the enzyme measured by dot hybridization with [32P]cDNA, was in the same range in both CHO cells and pineal glands. These data suggest that the 11-fold higher enzymatic activity in pineal gland is due to an accumulation of the enzyme protein, not to a high level of the mRNA and also indicate that the cloned cDNA can express an intact hydroxyindole O-methyltransferase enzyme in CHO cells.     

Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians

In most vertebrates and several insects, melatonin (N-acetyl-5-methoxytryptamine) is synthesized enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). In the freshwater planarian Dugesia japonica, which belongs to the most primitive metazoan phylum, activities of NAT and HIOMT, as well as melatonin, were found. The apparent Michaelis constants for substrates of NAT and HIOMT in the planarian were similar to those reported for the mammalian pineal gland and retina. When the planarians were maintained under a 12 h light:12 h dark cycle, the activities of NAT and HIOMT and melatonin levels exhibited a significant diurnal variation, peaking at the mid-dark time. In constant darkness, NAT activity and melatonin levels fluctuated with a circadian (about 24 h) rhythm. These data demonstrate that the planarian synthesizes melatonin through the same pathways as those in most vertebrates and several insects, and that its melatonin synthesis fluctuates in a circadian manner. Thus, it is strongly suggested that the planarian contains a circadian clock controlling melatonin synthesis.     

Parathion (O,O-dimethyl-O-p-nitrophenyl phosphorothioate) induces pineal melatonin synthesis at night

The effects of parathion on male rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels at the end of light period (2000 h) and at night (2300 h and 0100 h) were studied. Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Parathion was administered intragastrically at total doses (over 6 days) of either 6.5 or 13 mg/kg. Control rats received vehicle (corn oil) only. During the study, the rats were exposed to light:dark cycles of 14:10 with light off at 2100 h. Pineal NAT activity was increased at 0100 h following parathion administration at both doses, but HIOMT activity was unaffected. Pineal and serum melatonin levels were increased at night (2300 h and 0100 h) after the 13 mg/kg dose of parathion while the lower dose increased pineal melatonin only at 0100 h. Also, both doses decreased 5-HTP at 2000 h while the lower dose increased it at 2300; 5-HT was significantly decreased at 2300 h and 5-HIAA levels were lower but only significantly so for the 13 mg/kg dose at 2000 h. The results indicate that parathion has significant effects on pineal melatonin synthesis by mechanisms which remain unknown.     

Preliminary investigations on melatonin and 5-methoxytryptophol synthesis in the pineal,retina, and Harderian gland of the mole rat and in the pineal of the mouse “eyeless”

Summary Hydroxyindole-O-methyltransferase (HIOMT) activity for the synthesis of melatonin and 5-methoxytryptophol, both 5-methoxyindoles, was measured in the pineal, the Harderian gland and the retina of the mole rat and in the pineal of the mouse eyeless. In the pineal and the Harderian gland of the mole rat a larger amount of 5-methoxytryptophol than of melatonin is synthesized. 5-Methoxyindole synthesis is extremely high in the Harderian gland, whereas in the retina HIOMT activity is low and variable. In the pineal of the mouse eyeless, a low 5-methoxyindole synthesis showing no circadian rhythm is demonstrated. It is concluded that, besides the generally accepted regulation of the indole metabolism by light, in species with atrophied eyes having Harderian glands (mole rat) and in species without eyes other factors than light might be responsible for the indole metabolism in the pineal gland.     

Cloning and early expression pattern of two melatonin biosynthesis enzymes in the turbot (Scophthalmus maximus)

Melatonin biosynthesis from serotonin involves the sequential activation of the arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Photoperiod synchronizes a daily rhythm in pineal and retinal melatonin secretion through controlling AANAT activity. Teleost fish possess two Aanat, one expressed in the retina (AANAT1) and the other expressed in the pineal gland (AANAT2). We report here the full-length cloning of Aanat1, Aanat2, SmHiomt and Otx5 (orthodenticle homeobox homolog 5) in the turbot (Scophthalmus maximus, Sm), a flatfish belonging to an evolutionary recent group of Teleost. The temporal expression pattern of the genes investigated is consistent with the idea that OTX5 is needed for photoreceptor specification, and that the pineal gland differentiates before the retina. SmAanat2 expression remained pineal specific during the period of time investigated, whereas SmOtx5 and SmHiomt expressions were seen in both the retina and pineal gland. Our results do not support the existence of a second SmHiomt, as is the case for SmAanat. Neither SmAanat2 nor SmHiomt mRNAs displayed cyclic accumulation in the pineal organ of embryos and larvae maintained under a light-dark cycle from fertilization onward. This is in marked contrast with the situation observed with zebrafish Aanat2, indicating that the molecular mechanisms controlling the development of the pineal melatonin system have been modified during the evolution of Teleost.     

Pineal transplants in oculo: limitations on the ability of collateral sprouts of foreign neurons to establish normal function

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the superior cervical ganglia. This pathway, which carries information concerning environmental lighting to the gland, is important for maintaining a circadian rhythm in the activity of the pineal enzyme serotonin N-acetyltransferase (NAT). Lesioning this pathway blocks the normal nighttime increase in NAT activity. Following transplantation of the pineal gland to the anterior chamber of the eye, the gland becomes reinnervated by collateral sprouts of sympathetic neurons that innervate the iris. In addition, a day-night rhythm in NAT activity is restored to the transplanted glands (Moore, 1975; B?ckstr?m et al., 1976). These findings raise the possibility that the neural regulation of pineal function may be restored to normal by collateral sprouts of foreign neurons. To determine whether this is the case, other aspects of the neural regulation of NAT activity were examined in transplanted and in situ pineal glands. When the dark period was extended into the normal light period, NAT activity decreased to daytime values in both groups, suggesting that, in both cases, darkness is only effective in maintaining high levels of NAT activity at certain times during the day-night cycle. In contrast to their similar responses to extended darkness, the 2 groups of pineal glands responded differently to a brief pulse of light during the dark period. While the light exposure caused a dramatic decrease in nighttime NAT activity in in situ pineal glands, it produced no change in this enzyme activity in transplanted glands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary characterization of bovine pineal prolactin releasing (PPRF) and release-inhibiting factor (PPIF) activity

Summary Bovine pineal glands were subjected to extraction with dilute acetic acid, gel filtration on Sephadex G-25 and subsequent ultrafiltration through Diaflo membranes PM10, UM2 and UM05. Various fractions derived at each step were tested for the presence of substances which stimulate or inhibit prolactin secretionin vitro andin vivo. Both prolactin releasing (PPRF) and release-inhibiting (PPIF) activities were observed. PPRF activity was present in certain fractions derived from Sephadex G-25 and in the PM10 residue (MW<10,000). Whereas both the UM2 residue (MW>1000) and UM05 filtrates (MW<500) was seen to inhibit pituitary prolactin releasein vitro, the UM05 residue (MW>500 and <1000) inhibited prolactin releasein vivo, possibly by stimulating the secretion of the hypothalamic prolactin inhibiting factor. On the basis of its inactivation by trypsin it was concluded that PPIF may be a peptide or contain a peptide moiety indispensible for its biological activity. Experiments are in progress to characterize pineal prolactin-regulating activities and to elucidate further the physiological role of the pineal gland in the regulation of prolactin secretion.Supported by N.I.H. grant # HD-08759 and by the University of Arizona BRS grant 5010-3150-75.     

Molecular and cellular aspects of hydroxyindole-O-methyltransferase expression in the developing chick pineal gland

The pineal gland influences circadian activity and seasonal breeding through the production of an indolic hormone, melatonin. The terminal step of melatonin biosynthesis is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Using an antibody directed against HIOMT, we examined the differentiation of the melatoninergic phenotype in the developing chick pineal gland. HIOMT first appeared 4 days before hatch and rose linearly until the 7th day posthatch. This was correlated with an increased immunoreactivity of the 38 kDa enzyme on Western blots and with an accelerated rate of HIOMT biosynthesis as demonstrated by [35S]methionine labeling. Immunocytochemistry revealed a growing number of HIOMT-positive cells between day 2 before hatch and day 15 posthatch. Until hatching HIOMT was expressed almost exclusively in modified photoreceptors. Parafollicular pinealocytes became HIOMT-positive mostly after hatching. Their different timings of functional differentiation emphasize the existence of two populations of melatonin-producing cells in the chick pineal gland.