Stephan Steinlechner

Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus Tridecemlineatus)

Pineal melatonin levels were compared in laboratory-raised or wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus) that were either exposed to 10 h of darkness at night or to light which had an irradiance of 400 microW/cm2. In laboratory-born squirrels the period of darkness was associated with a gradual rise in pineal melatonin levels with peak values being reached at 0200 h, 6 h after darkness onset. Thereafter, melatonin levels decreased and were back to low daytime levels by 0800 h, 2 h after light onset. The exposure of laboratory-raised animals to an irradiance of 400 microW/cm2 during the night totally prevented the nocturnal rise in pineal melatonin levels in these animals. In wild-captured ground squirrels the period of darkness at night was associated with a rapid rise in pineal melatonin such that by 2200 h, 2 h after lights out, peak melatonin values were already attained; additionally, melatonin levels remained high throughout the period of darkness but returned to daytime values by 0800 h. Exposure of wild-captured squirrels to a light irradiance of 400 microW/cm2 during the normal dark period was completely incapable of suppressing pineal melatonin levels. The difference in the sensitivity of the pineal gland of laboratory-raised and wild-captured ground squirrels may relate to their previous lighting history.

The Influence of Various Irradiances of Artificial Light, Twilight, and Moonlight on the Suppression of Pineal Melatonin Content in the Syrian Hamster

The purpose of the present studies using artificial light was to determine how the timing and duration of exposure influence the light‐induced suppression of pineal melatonin levels in hamsters. An 8‐min exposure to 0.186 μW/cm2 of cool white fluorescent light caused a continued depression of pineal melatonin even when animals were returned to darkness. In addition, the pineal gland does not appear to change its sensitivity to light throughout the night. A 20‐min exposure to 0.019 μW/cm2 of cool white fluorescent light did not significantly suppress pineal melatonin during any time of the melatonin peak, whereas a 20‐min exposure to 0.186 μW/cm2was capable of always suppressing melatonin. Furthermore, increasing the duration of 0.019‐μW/cm2 exposure to 30, 60, 120, or 180 min does not increase the capacity of this irradiance to depress melatonin.

Influence of light irradiance on hydroxyindole-O-methyltransferase activity, serotonin-N-acetyltranferase activity, and radioimmunoassayable melatonin levels in the pineal gland of the diurnally active Richardson's ground squirrel

When Richardsons ground squirrels were kept under light:dark cycles of 14:10 h there was no nocturnal rise in pineal hydroxyindole-O-methyltransferase (HIOMT) activity. Conversely, the 10 h dark period was associated with large nocturnal rises in both pineal serotonin-N-acetyltransferase (NAT) activity and radioimmunoassayable melatonin levels. The nighttime rises in pineal NAT and melatonin were not suppressed by the exposure of the animals to a light irradiance of 925 mu W/cm2 during the normal dark period. On the other hand, when the light irradiance was increased to 1850 mu W/cm2 the rise in pineal NAT activity was eliminated while the melatonin rise was greatly reduced. When ground squirrels were acutely exposed to a light irradiance of 1850 mu W/cm2 for 30 min beginning at 5.5 h after lights out, pineal NAT activity and melatonin levels were reduced to daytime values within 30 min. The half-time (t 1/2) for each constituent was less than 10 min. Exposure to a light irradiance of either 5 s or 5 min (beginning at 5.5 h into dark period) was equally as effective as 30 min light exposure in inhibiting pineal NAT activity and melatonin levels. When animals were returned to darkness after a 30 min exposure to a light irradiance of 1850 mu W/cm2 at night, both pineal NAT activity and melatonin levels were restored to high nighttime levels within 2 h of their return to darkness. The results indicate that the pineal gland of the wild-captured, diurnal Richardsons ground squirrel is 9000 X less sensitive to light at night than is the pineal gland of the laboratory raised, nocturnal Syrian hamster.

Comparison of the Effects of β‐Adrenergic Agents on Pineal Serotonin N‐Acetyltransferase Activity and Melatonin Content in Two Species of Hamsters

The nighttime rise in pineal melatonin levels can be blocked by administration of the β‐adrenergic receptor antagonist, propranolol, in both Syrian hamsters and rats. Although the administration of β‐adrenergic receptor agonists such as norepinephrine or isoproterenol stimulates pineal melatonin production in the rat, these drugs are without apparent effect on indole production in the Syrian hamster. To determine whether this lack of stimulatory effect in the Syrian hamster is characteristic of this species, a comparison of the effects of norepinephrine and isoproterenol on pineal serotonin N‐acetyltransferase activity and melatonin content was conducted. In contrast to their lack of effect in the Syrian hamster, norepinephrine and isoproterenol stimulated pineal serotonin N‐acetyltransferase activity and melatonin content in the Djungarian hamster. Hourly injection of norepinephrine during a continuation of light into the normal dark period stimulated increases in the activity of serotonin N‐acetyltransferase and melatonin content in the Djungarian hamster but was without effect on these pineal parameters in the Syrian hamster.

Pharmacological studies on the regulation of N-acetyltransferase activity and melatonin content of the pineal gland of the Syrian hamster

Thus far, all attempts to stimulate melatonin synthesis by β‐adrenergic receptor agonists in the Syrian hamster pineal gland have failed. Neither a wide range of dosages of isoproterenol (0.5 mg/kg to 24 mg/kg), nor prolonged treatment with norepinephrine, the natural neurotransmitter, increased N‐acetyltransferase (NAT) activity or melatonin production. In the present study, the administration of isoproterenol at night was likewise ineffective in advancing or enhancing the normal nightly melatonin peak. Also, we did not find a delayed effect 7 or 8 h after the administration of the drug. Furthermore, we tested the idea of coneurotransmitters such as octopamine or dopamine being possibly necessary for stimulation, but could not find any effect of these substances on melatonin synthesis. In addition, a parasympatholytic agent, atropine, did not increase the responsiveness to sympathomimetic agents. Administration of a phosphodiesterase inhibitor was also ineffective in stimulating NAT activity. On the other hand, isoproterenol did retard the drop in NAT and melatonin after lights‐on at night, indicating that β‐receptors are involved in maintaining elevated melatonin levels.

Tryptophan administration inhibits nocturnal N-acetyltransferase activity and melatonin content in the rat pineal gland. Evidence that serotonin modulates melatonin production via a receptor-mediated mechanism.

In the rat pineal gland, the activity of serotonin N-acetyltransferase (NAT) and the concentration of melatonin are normally high at night; conversely, the concentration of serotonin (5-HT), the precursor of melatonin, is low. Since tryptophan administration increases the concentration of pineal 5-HT at night, we examined its effect of melatonin production. Nighttime tryptophan loading led to substantial increases in pineal 5-hydroxytryptophan, 5-hydroxyindole acetic acid (5-HIAA), and 5-HT but a highly significant reduction in NAT activity in comparison to saline-injected controls. In contrast to other measured indoles, melatonin levels also were significantly diminished by tryptophan loading. Nocturnally high pineal norepinephrine levels were unaltered by tryptophan administration. The idea that high concentrations of 5-HT could lead to substrate inhibition of NAT activity was not supported by kinetic analysis of control NAT levels versus tryptophan-inhibited NAT activity under varied substrate concentrations. Hypotheses to explain these results include the possibility that tryptophan inhibition of melatonin synthesis is mediated by the release of 5-HT from the pinealocyte and its subsequent autocrine action on melatonin production.

Diurnal variation in the serotonin content and turnover in the pineal gland of the syrian hamster

Serotonin and 5-hydroxyindoleacetic acid were measured in the pineal gland of Syrian hamsters by high performance liquid chromatography over a period of 24 h. A distinct 24 h rhythm was detected for both indoles. Turnover studies revealed a higher rate of serotonin synthesis during the day than during the night. We therefore suggest that the serotonin content in the pineal gland of the Syrian hamster is regulated by changes in its synthesis rate, rather than by changes of serotonin catabolism, via N-acetyltransferase activity.

Does maximal serotonin N-acetyltransferase activity necessarily reflect maximal melatonin production in the rat pineal gland?

p-Chlorophenylalanine (PCPA) treatment reduced pineal 5-hydroxytryptamine (5-HT) levels by 68.9% of non-PCPA treated levels in intact rat pineal glands and by 95.9% of non-PCPA treated levels in adrenergically denervated rat pineal glands. Although isoproterenol stimulation resulted in increased activity of serotonin N-acetyltransferase (NAT) activity in all experimental groups, pineal melatonin production was maximal only in non-PCPA treated rats, suggesting that 5-HT concentrations may exert considerable influence on the level of melatonin production in the rat pineal gland, independent of the level of NAT activity.

Simultaneous Determination of N-Acetyltransferase Activity, Hydroxyindole-O-methyl-transferase Activity, and Melatonin Content in the Pineal Gland of the Syrian Hamster

Abstract 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.

Day-night differences in estimated rates of 5-hydroxytryptamine turnover in the rat pineal gland

SummaryRates of 5-hydroxytryptamine synthesis in various brain tissues can be estimated from the linear increase in 5-hydroxytryptophan levels following inhibition of 5-hydroxytryptophan decarboxylation with RO4-4602 or NSD-1015. In addition, NSD-1015 can prevent 5-hydroxytryptamine oxidative-deamination via monoamine oxidase inhibition, leading to linear decreases in a major metabolite of this amine, 5-hydroxyindole acetic acid. In the rat pineal gland we demonstrated similar increases in 5-hydroxytryptophan levels after nocturnal or diurnal injection of RO4-4602 (100 mg · kg−1) or NSD-1015 (200 mg · kg−1). Similar decreases in 5-hydroxyindole acetic acid were also observed after nocturnal or diurnal injection of NSD-1015 or pargyline (an inhibitor of monoamine oxidase) (75 mg · kg−1). 5-Hydroxytryptamine levels increased after nocturnal pargyline injection but remained constant after diurnal pargyline administration. 5-Hydroxytryptamine levels exhibited little change following nocturnal injection of NSD-1015 but decreased linearly after diurnal injection of NSD-1015. We suggest that (1) rat pineal 5-hydroxytryptamine synthesis is increased nocturnally, (2) metabolic utilization, primarily by oxidative-deamination, of 5-hydroxytryptamine is increased diurnally and (3) basal levels of pineal 5-hydroxytryptamine may be stored within adrenergic nerve endings which innervate the pinealocytes responsible for synthesizing this amine, thus “protecting” or otherwise making unavailable this pool of 5-hydroxytryptamine for metabolic utilization.