PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Inhibition of avian and mammalian hydroxy-indole-o-methyl-transferase (HIOMT) with low molecular weight fractions of mammalian pineal glands

Partially purified fractions from sheep pineal gland extracts, which show a fluorescence like pteridines, were shown to suppress the enzyme hydroxy-indole-o-methyltransferase (HIOMT) isolated from avian pineals and retinas. The results described demonstrate that the mechanism of inhibition of HIOMT activity is independent of the species and organ. A blocking effect was found in experiments whether retinal or pineal HIOMT were used and the effect was found as well with bovine HIOMT. It is shown that low molecular weight molecules show inhibitory activity and accompany the high molecular weight enzyme HIOMT.     

Distribution of hydroxyindole-O-methyltransferase mRNA in the rat brain: an in situ hybridisation study

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes. Received: 26 June 1997 / Accepted: 15 September 1997     

Regulation of hydroxyindole-O-methyltransferase gene expression in Japanese quail (Coturnix coturnix japonica).

Hydroxyindole-O-methyltrasferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.     

Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase activity, and melatonin content in the pineal gland of the Syrian hamster

The activities of serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin content were measured in Syrian hamster pineal glands at 2-hr intervals over a period of 24 hr. NAT and HIOMT are the two enzymes which catalyze the formation of melatonin from serotonin. The use of micromethods for determination of the enzyme activities allowed concurrent measurement of NAT and melatonin or HIOMT and melatonin in the same gland. HIOMT activity showed no significant diurnal rhythm whereas NAT activity and melatonin content exhibited distinct peak values late in the dark phase as described previously. Despite an apparent parallelism between the NAT activity rhythm and melatonin content, no correlation exists between these parameters in single pineal glands.     

Regulation of Rat Pineal Hydroxyindole-O-Methyltransferase in Neonatal and Adult Rats

The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole-O-methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine-treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the K_m of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.     

The organochlorine insecticide 1,2,3,4,5,6-hexachlorocyclohexane (lindane) but not 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) augments the nocturnal increase in pineal N-acetyltransferase activity and pineal and serum melatonin levels

The effect of organochlorine insecticides lindane (1,2,3,4,5,6-hexachlorocyclohexane) and DDT (1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane) were studied in terms of their effects on the rat pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity and pineal and serum melatonin levels during the day (2000h) and at night (2300 and 0100h). Additionally, pineal levels of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were estimated. Nocturnal NAT activity was increased after lindane administration; likewise, lindane augmented pineal and serum melatonin levels at 2300h. Conversely, DDT was without a statistically significant effect on either NAT activity or on pineal or serum melatonin levels. Neither lindane nor DDT significantly influenced pineal HIOMT values either during the day or at night. Likewise, neither insecticide consistently influenced pineal levels of either 5-HTP, 5-HT or 5-HIAA. The results indicate that the organochlorine insecticide, lindane, modifies pineal melatonin synthesis in vivo.     

Day-night differences in the response of the pineal gland to swimming stress

The effect of swimming stress on pineal N-acetyltransferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content was studied during the day and night in adult male rats. At night, elevated pineal activity was suppressed by light exposure before the animals swam. During the day, swimming for 2 hr did not stimulate NAT activity unless the animals were pretreated with desmethylimipramine (DMI), a norepinephrine uptake blocker. Pineal melatonin content after daytime swimming exhibited a weak rise, unless DMI was injected, in which case melatonin levels showed a highly significant increase. Swimming at night caused a greater (compared to daytime levels) increase in NAT activity in both noninjected and DMI-injected rats. Melatonin levels at night were highly significantly stimulated (compared to daytime values) even without pretreatment of the rats with DMI. The greater response of the rat pineal to swimming stress at night may relate either to an increase in the number of beta-adrenergic receptors in the pinealocyte membrane at night or to a reduced capacity of the sympathetic neurons in the pineal to take up excess circulating catecholamines. Pineal HIOMT activity was not influenced by swimming (with or without DMI) either during the day or at night.     

Gonadal steroid regulation of pineal melatonin synthesis.

The regulation of gonadal function by the vertebrate pineal, primarily through the biological actions of melatonin has received much attention from investigators during the past decade (1–5). Melatonin is synthesized from serotonin through the activities of the pineal enzymes N-acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT). NAT converts serotonin to N-acetyl serotonin by coupling of the acetyl function from Acetyl Coenzyme A to serotonin. HIOMT transfers the methyl group from S-adenosylmethionine to N-acetyl serotonin, to form melatonin.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

Long-term effects of constant light or darkness on chicken pineal hydroxyindole-O-methyltransferase expression: biochemical and cellular aspects

1. Chickens kept in constant light, as opposed to constant darkness, display a twofold increase in the activity of pineal hydroxyindole-O-methyltransferase (HIOMT), the last acting enzyme in the melatonin pathway. 2. Using an immunological approach, we presently show that this regulation of HIOMT activity reflects changes in the concentration of a single molecular form of the enzyme protein (a 38 kDa polypeptide). Immunohistofluorescence indicates that these concentration changes concurrently affect modified photoreceptors and pinealocyte-like cells in the chicken pineal organ. 3. Together, the present data support the hypothesis that environmental lighting might regulate the expression of the HIOMT gene.     

Regulation of pineal enzymes by photoperiod, gonadal hormones and melatonin in Coturnix quail.

The photoperiodic and hormonal regulation of melatonin-synthesizing enzymes was determined in pineals of Coturnix quail. N-Acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT) were twofold higher in pineals of female and male Coturnix quail during exposure to darkness (16L:8D). Castration decreased pineal HIOMT activity in both female and male Coturnix, while selective gonadal steroids restored activity. NAT was not affected by castration or gonadal steroids. Implantation of melatonin into female Coturnix decreased both HIOMT and NAT activities. These results suggest that NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of the external perceptive environment and the internal hormonal milieu, with both enzymic activities modulated by the feedback inhibitory influence of endogenous melatonin.     

Photoperiodic and gonadal steroid regulation of pineal hydroxyindole-O-methyl transferase and N-acetyl transferase in Coturnix quail.

The activities of the melatonin-synthesizing enzymes were determined in pineals of Coturnix quail in response to photoperiodicity and gonadal hormones. Both hydroxyindole-O-methyl transferase (HIOMT) and N-acetyl transferase (NAT) were two-fold higher during exposure to darkness in female and male Coturnix maintained in a gonad-stimulating photoperiod (16L:8D). Castration decreased HIOMT activity in both female and male Coturnix. Administration of diethylstilbestrol, estradiol benzoate and progesterone into castrated females, and testosterone propionate and androstenedione into castrated males, restored HIOMT activity similar to that of intact controls. NAT was not affected by castration or gonadal steroids. These results suggest that the activity of pineal NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of photoperiodic and gonadal steroid regulation.