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Characteristics of hydroxyindole-O-methyltransferase (HIOMT) activity were examined in pineal gland tissue from 10 muskoxen shot by native hunters in November, 1990. The enzyme preferentially methylated N-acetylserotonin, with other hydroxyindole compounds showing relatively low affinities; activity peaked sharply at pH 8.2. HIOMT was noncompetitively inhibited by its substrate, N-acetylserotonin, and competitively inhibited by its product S-adenosylhomocysteine. The catalytic mechanism appeared to be ordered as described in previous studies: S-adenosylmethionine was the obligatory first substrate, followed by N-acetylserotonin; methyl transfer then occurred and the products, melatonin and S-adenosylhomocysteine, were released sequentially. Interestingly, the inhibition constant (Ki) for N-acetylserotonin was relatively close to the Michaelis-Menten constant (Km), which might allow physiological concentrations of N-acetylserotonin to inhibit HIOMT activity in vivo. This effect could be relevant to the ecology of free-living muskoxen during the dramatic seasonal fluctuations in dietary protein and daily photoperiod associated with their arctic habitat.

Pineal glands were obtained from two young female black rhinoceri that had died as a result of postcapture trauma during a translocation exercise. Hydroxyindole-O-methyltransferase (HIOMT) from these pineal glands showed a peak activity at pH 8.2, although high activity extended over a fairly wide pH range (7.8-8.4). N-acetylserotonin was the best hydroxyindolic substrate for the enzyme, although other hydroxyindoles were methylated, the relative affinities being similar to values previously reported for bovine HIOMT. Kinetic analyses revealed that black rhinoceros HIOMT was subject to substrate inhibition by both substrates at high concentration; this observation is unlikely to have physiological significance. The catalytic mechanism was found to be ordered Bi-Bi, in which S-adenosylmethionine is the obligatory first substrate to bind to the enzyme, such binding allowing for binding of the hydroxyindolic substrate followed by catalysis, products again leaving the catalytic site in a sequential fashion.

An intact pineal gland organ culture technique was developed which utilised radiolabelled tryptophan as the indolic precursor and two-dimensional thin-layer chromatography to separate the various indole metabolites produced. The method was capable of reproducibly separating and quantitating all tryptophan metabolites except 5-methoxytryptophan which co-chromatographed with tryptophan in all the solvent systems evaluated. Noradrenergic stimulation of cultured pineals led to a predictable increase in N-acetyl-serotonin and melatonin production, suggesting that the method was useful for biochemical and pharmacological studies on the pineal gland. Similarly evaluation of the results revealed that a strong linearity existed between N-acetylserotonin and melatonin production and between actual and theoretical methylation as previously reported, again verifying the usefulness of the method developed.

A variety of reports indicate that some kind of interaction may exist between the pineal gland and cations. Of particular interest are the reports that indicate comparatively high levels of copper, manganese, and zinc in the pineal gland and that the pineal gland exhibits a circadian rhythm in calcium, magnesium, potassium, and sodium. There are, unfortunately, no reports suggesting a functional role for these findings. This study investigated circadian rhythms in circulating plasma cations in rats under 12/12 h light-dark cycle and in rats whose pineal function had been suppressed by exposure to constant light for 1 and 7 days. Neither of the treatments affected circulating potassium levels but had some significant effects on sodium concentration at a number of time points without affecting the total amount of sodium circulating in a 24 h period. Calcium, magnesium, and zinc plasma concentrations were little affected by 1 day of constant light, while 7 days of constant light caused a dramatic and highly significant increase in the circulating levels of the three cations. The plasma levels of copper, on the other hand, while also being unaffected by 1 day were significantly depressed by 7 days of constant light. It is apparent, therefore, that the pineal gland may be involved in regulation of circulating levels of the cations measured. The functional significance of these observations is not clear at this stage but warrants further investigation.

The various pineal gland tryptophan metabolites were administered to male rats intraperitoneally (100 micrograms/kg) and rectal temperatures were recorded. Of the compounds tested, hydroxytryptophan, N-acetylserotonin, hydroxytryptophol, and their corresponding methoxyindoles all caused a marked hypothermia, indicating that several indolic products may be involved in thermoregulation. Although the brain penetration of indoles is poor, a central site of action would be most likely, although peripheral actions cannot be excluded. The mechanism of induction of hypothermia may involve peptides, the pituitary-thyroid axis, the adrenal gland, or a combination of these. These results may suggest that the pineal gland integrates environmental cues to act in concert with physiological thermostats in the fine tuning of thermoregulation.

The effect of anticonvulsant drugs on daytime and nighttime N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) levels was investigated. Some of the drugs caused a significant reduction in daytime NAT and HIOMT levels, possibly owing to beta-receptor blockade although other mechanisms cannot be excluded. Sulthiame caused an increase in daytime NAT levels, and this may represent a compensatory mechanism by the pineal gland to allow normal melatonin production. None of the drugs affected nocturnal HIOMT levels, and only phenobarbitone affected NAT levels, apparently causing a shift in the nocturnal peak later in the dark phase.

Pineal glands were incubated in the presence of [3H] methoxytryptophan with and without methoxamine, epinephrine, and norepinephrine. The beta-adrenoceptor-stimulated pineal glands were capable of converting methoxytryptophan to methoxytryptamine, melatonin, methoxyindole acetic acid, and methoxytryptophol, albeit in small quantities. Only methoxyindole acetic acid was detectable after incubation of unstimulated and alpha-adrenergic-agonist-treated pineal glands. These results support the proposal that melatonin can be formed from methoxytryptophan although this is a minor synthetic pathway, and the classic pathway from serotonin via N-acetylserotonin should be considered to be responsible for the majority of pineal melatonin production.

The effect of inhibitory cations on hydroxyindole-O-methyltransferase (HIOMT) was investigated in order to elucidate the mechanisms involved. Copper inhibition of HIOMT can be classified as partial, classic noncompetitive inhibition with respect to S-adenosyl methionine and as partial, mixed noncompetitive inhibition with respect to N-acetylserotonin. Binding of copper to the enzyme impaired but did not prevent catalysis, and the difference in inhibitory pattern observed with both substrates may be due to the obligatory order of substrate binding to HIOMT. Calcium and magnesium were uncompetitive inhibitors of HIOMT with respect to both substrates and binding of these cations to the enzyme prevents catalysis. The cations presumably bind to HIOMT in the vicinity of the catalytic site and in some way prevent or impair the catalytic process, possibly by interfering with substrate binding to the enzyme, interfering with methyl transfer or product release after catalysis, or a combination of these effects. The different inhibitory patterns observed may result from differences in molecular size or outer electron shell valence of the cations.

N-Acetyltransferase (NAT) activity at night and following isoproterenol treatment in intact animals was higher in rats pretreated with magnesium than in controls. This effect was probably due to enhancement of agonist binding and/or adenylate cyclase activation caused by magnesium ions. A similar effect was observed in vitro, where magnesium increased norepinephrine-stimulated NAT activity in organ-cultured pineal glands, suggesting that magnesium acts primarily on the pineal gland as opposed to some other peripheral or central site. This enhancement of NAT activity by magnesium would presumably lead to increased melatonin production; and as melatonin has been shown to decrease serum magnesium levels, a negative feedback mechanism may exist.

The effect of pteridines and sex hormones on hydroxyindole-O-methyltransferase (HIOMT) catalysis was examined. Equivalent quantities of partially purified bovine HIOMT was incubated in the presence and absence of the various test substances and the results fitted to the Michaelis-Menten equation. The pteridines (triampterene and xanthopterin) had no noticeable effect on HIOMT catalysis, while all the sex hormones examined were found to be competitive inhibitors of the enzyme with respect to the hydroxy-substrate. Testosterone concentrations causing noticeable inhibition were above physiological while estrone, estradiol, and progesterone all caused inhibition within the physiological range; estrone was the most potent and progesterone the least potent inhibitor. The possible significance of these findings are discussed.

The effect of different incubation temperatures on the activity of trout hydroxyindole-O-methyltransferase (HIOMT) was determined in order to study the potential effects of changes in environmental temperature on production of melatonin. In the study, N-acetylserotonin (NAS) was found to inhibit trout pineal HIOMT, inhibitory concentration decreasing with a decrease in incubation temperature. An increase in incubation temperature was accompanied by an increase in maximal velocity (Vm) and a decrease in affinity of the substrate NAS for the enzyme, while the affinity of the inhibitor NAS remained more or less constant with increase in temperature up to 30 degrees C. It was interesting to note that at subinhibitory concentrations of NAS, possibly concentrations within the normal physiological range, there was a balance between increased velocity and decreased affinity for substrate, with increase in temperature resulting in a constant rate of melatonin production. On the basis of these results it is suggested that substrate inhibition might be involved in modulation of melatonin production and that trout pineal HIOMT can maintain a constant rate of melatonin production over a wide range of temperatures by a combination of compensatory changes in enzyme kinetic parameters.

Administration of methoxyindoles to rats in both the morning and the afternoon caused significant changes in circulating plasma cation levels. All the methoxyindoles except melatonin were without effect at doses below 100 micrograms/kg; the observed changes caused by doses of 100 micrograms/kg can, therefore, probably be best described as pharmacological rather than having physiological significance. Melatonin at all doses tested (25, 50, and 100 micrograms/kg) led to a decrease in plasma magnesium levels 3 h after administration in the morning, and this finding is discussed in the light of a possible feedback mechanism between melatonin and circulating magnesium. Melatonin administration in the afternoon was without effect on magnesium levels. Zinc levels were also decreased significantly 3 h after a morning dose of melatonin (50 and 100 micrograms/kg), and the possible functional significance of this observation is discussed. Most of the observed effects can be considered nonspecific and pharmacological, and further work is needed to fully elucidate the involvement, if any, of the pineal gland in regulation of cations.

The effect of various cations on activity of hydroxyindole-O-methyltransferase (HIOMT) and N-acetyltransferase (NAT) from pineal gland was studied. Copper inhibited both enzymes while calcium and magnesium inhibited HIOMT; the mechanisms of these inhibitions are unknown at this stage. Calcium, magnesium, potassium, and sodium initially stimulated NAT activity, and as the cation concentration increased inhibitory activity was observed. These differential effects on NAT activity may in some way be involved in pineal regulation of cation levels.