Pineal Serotonin Research Articles

Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

Gating of retinal inputs through the suprachiasmatic nucleus: Role of excitatory neurotransmission

The mammalian circadian clock, located in the hypothalamic suprachiasmatic nucleus (SCN) is important in the regulation of many circadian rhythms, including regulation of pineal gland metabolism and melatonin secretion. Transsection of the optic nerves, disrupting the retinohypothalamic pathway, lesion of the SCN, or lesion of the hypothalamic paraventricular nucleus (PVN) abolish the regulation of pineal serotonin N-acetyltransferase activity by light. Therefore, the pathways linking the retina and the pineal gland must be channelled from the retina through the SCN and the PVN. Many lines of evidence indicate that the major neurotransmitter in the retinal afferents is glutamate. The first aim was therefore to study the retinal target neurons by localising glutamate receptors in the rodent SCN. Using in situ hybridisation, we detected NMDA-RI and NMDA-R2C mRNA subunits in the SCN. Using immunocytochemistry, immunoreactivity for the AMPA type receptors GluR 1, GluR 2,3 and GluR 4 was also detected in the SCN. Presentation of a short light pulse during the subjective night [i.e. circadian time (CT) 14 or 19], when light induced phase-shifting of activity-rest cycles can be accomplished, also induces expression of the immediate early-genes c- fos and junB in the rodent SCN. The second aim was to use this cellular correlate of behavioural function to determine the location of potential retinal target neurons in the SCN, and to investigate the hypothesis that glutamatergic neurotransmission mediates the effects of light on the circadian system. Thus, the ability of the NMDA receptor antagonist MK-801 to block light-induced c- fos expression in the SCN was studied. In the rat, this antagonist blocked c - fos mRNA expression in a subpopulation of cells in the ventral SCN at doses of 6, but not 2 mg/kg. In contrast, in the hamster both doses blocked light-induced c- fos expression in the ventral SCN. These data provide support for the hypothesis that glutamate mediates effects of light in the SCN, although it appears that the complexes of NMDA receptor subunits, which are involved in light-induced expression of c fos after light, are relatively insensitive to MK-801. The diversity, heterogeneous distribution, and complexity of glutamate receptor subunits in the SCN suggest that processing of light pulses in the SCN is mediated by several cell types in the SCN. Via an integration process in the clock, the transmission of photic information takes place to other brain structures.

Changes in nocturnal pineal indoleamine metabolism in rats treated with parathion are prevented by β-adrenergic antagonist administration

Parathion, an organophosphorous insecticide, was previously shown to enhance the nighttime rise in pineal N-acetyltransferase (NAT) activity and serum melatonin levels. The purpose of the present study was to test whether parathion acts on the pineal gland by means of a β-adrenergic receptor mechanism. Whereas parathion (total dose 6.5 mg/kg body wt over 6 days) by itself significantly augmented nocturnal pineal NAT activity and serum melatonin levels in otherwise untreated rats, the insecticide was ineffective in reference to this enzyme when it was given in conjunction with the β-adrenergic receptor antagonist propranolol (20 mg/kg body wt, l h before lights off). The augmentation of NAT activity by parathion also caused significant reductions in pineal serotonin (5-HT); again, this response was blocked by propranolol treatment. Neither pineal hydroxyindole- O-methyltransferase (HIOMT) activity nor pineal levels of 5-hydroxytryptophan or hydroxyindole acetic acid (5-HIAA) were significantly changed as a result of either parathion or propranolol treatment. The results are consistent with the idea that parathion influences pineal S-HT metabolism either at the level of the β-adrenergic receptor or via the sympathetic innervation to the pineal gland.

P-Chlorophenylalanine treatment depresses the number of synaptic ribbon profiles in the rat pineal gland, but does not abolish their day-night rhythm

It is largely unknown as to how the day/night rhythm of pineal synaptic ribbon number is regulated. Transcriptional events related to the nocturnal formation of new synaptic ribbons occur early in the morning, when pineal serotonin levels begin to increase. The present study was carried out in order to elucidate as to how altered serotonin levels affect the day/night changes in the number of synaptic ribbon profiles. To this end, male Sprague-Dawley rats received a single dose of p-chlorophenylalanine (pCPA, 300 mg/kg body weight, 72 hours before sacrifice), which depresses tryptophan hydroxylase activity and hence pineal serotonin levels. Control animals received saline injections. Experimental and control animals were killed in the middle of the light period and in the middle of the dark period, respectively. The pineal glands were removed and bisected. One half was used for electron microscopic quantification of synaptic ribbon profile numbers, and the other half for determination of several biochemical parameters (see below). pCPA decreased pineal serotonin levels to c. 30% of the controls and synaptic ribbon profile numbers by c. 30%, both at day and night. The day/night rhythm of the two parameters did not appear to be affected. The rate-limiting enzyme of melatonin synthesis, serotonin N-acetyltransferase, which is only measurable at night, and pineal melatonin levels were not affected at night. The intermediary product of melatonin formation, N-acetylserotonin, was significantly depressed at night. It is concluded that serotonin plays a more important modulatory role on pineal synaptic ribbons than does melatonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased oxidation of pineal serotonin as a possible explanation for reduced melatonin synthesis in the aging Djungarian hamster ( Phodopus sungorus)

Groups of young adult (1.7 months) and old adult (9.7 months) female Djungarian hamsters were exposed to short-day photoperiods (SD; 8L: 16D) or long-day photoperiods (LD; 16L: 8D) for 6 weeks. Pineal and serum melatonin rhythms were dampened in old SD animals when compared with young SD animals. The melatonin precursor serotonin showed diurnal variations with opposite phases to melatonin synthesis. However, pineal serotonin levels were even lower in old SD animals than in young SD animals, contrary to the expected accumulation of the precursor. The synthesis of a metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA), was significantly lower in younger hamsters, under both photoperiods. These results indicate that the age-associated decline in the production of melatonin is a consequence of an increased oxidation of its precursors.

The locus controlling pineal serotonin N-acetyltransferase activity ( Nat-2) is located on mouse chromosome 11

Melatonin is synthesized from serotonin by the enzymes serotonin N-acetyltransferase (SNAT) and hydroxyindole- O-methyltransferase (HIOMT). We have previously reported that C57BL/6 mice do not have SNAT activity because of a mutation in an autosomal gene which is responsible for the absence of normal SNAT activity. In the present study, we have tried to map the loci of Nat-2 (the locus controlling SNAT activity) on chromosomes using a set of the BxH recombinant inbred strains which were derived from an initial cross between C3H/He with SNAT and C57BL/6 without the enzyme. Based on strain distribution patterns (SDPs), a close linkage on chromosome 11 was found between Nat-2, Es-3 (esterase-3), Glk (the locus controlling galactokinase activity) and Myla (myosin alkali light chains expressed in cardiac atrial muscle). The linkage between Nat-2 and Es-3 was confirmed by a conventional linkage test and the recombination frequency between these loci was estimated to be 16.1 ± 3.6% (mean ± S.E.M.).

Development of MEKA (phosducin), Gβ, Gγ and S-antigen in the rat pineal gland and retina

Pinealocytes and retinal photoreceptor cells contain an unusual cytoplasmic complex composed of the Gβγ dimer of GTP-binding regulatory proteins (G-proteins) tightly bound to an acidic 33 kDa phosphoprotein termed MEKA or phosducin; MEKA is a substrate of cyclic AMP-dependent protein kinase. This study characterized the developmental appearance of these and two related proteins, Gγ and S-antigen, in pineal and retinal tissue. MEKA was absent in the pineal gland prior to birth, at a time when it was possible to detect Gβ in pineal cytoplasm, indicating that the appearance of Gβ in the cytoplasm precedes that of MEKA and does not appear to require the presence of MEKA. The absence of MEKA at this time indicates that the cyclic AMP stimulation of pineal serotonin N-acetyltransferase activity is not mediated by MEKA, which has been considered as a possible role of MEKA. After postnatal day 7, pineal MEKA and cytoplasmic Gβ increased in a parallel manner, with peak values occurring at about postnatal day 21. Thereafter, both proteins in the pineal gland decreased in a parallel fashion to 10 and 35% of their peak values, respectively; in contrast, the cytoplasmic protein S-antigen and membrane associated Gβ remained at maximal levels after this time. Whereas both MEKA and Gβ decreased late in development in the pineal gland, these proteins either increased or remained constant in the retina. These tissue-specific patterns were found to differ from those of another cytosolic protein found exclusively in the pineal gland and retina, S-antigen, which remained constant after 21 in the pineal gland but decreased in the retina late in life.

Failure to stimulate melatonin synthesis in the rat pineal gland with an adenosine analog

Recent studies indicate the presence of adenosine binding sites in rat pinealocytes and an effect of their activation on pineal serotonin metabolism. However, controversial data exist, and reports suggest that the role of adenosine in pineal physiology is complex. On this basis, we evaluated the effects of an adenosine analog (N-ethyl-carboxamido-adenosine: NECA) on in vitro and in vivo melatonin production in the rat pineal gland. In the in vitro protocol, pineals were incubated with NECA (0.5 mM, 1 mM, or 2.5 mM) or isoproterenol (ISO: 10 −6 M) for 4 hr. In the in vivo experiments, animals were given NECA (1 mg/kg IP), ISO (0.5 mg/kg IP) or 1 ml saline diluent and sacrificed 2 hr later. The samples were assayed for pineal N-acetyltransferase (NAT) activity and melatonin concentrations. ISO caused the expected marked rise in NAT activity and melatonin levels in both protocols. NECA was ineffective in causing any modification of the parameters measured. We conclude that the adenosine analog NECA may not be involved in the activation of melatonin production. These data contrast with others in which NECA administration resulted in an increase in an increase in melatonin levels. The participation, if any, of the purinergic system in the physiology of the pineal gland is still far from being characterized.

Regulation of pineal serotonin N-acetyltransferase activity.

Regulation of pineal serotonin N-acetyltransferase activity David C. Klein,* Nicolas L. Schaad,* M. A. A. Namboordiri,§ Li Yut and Joan L. Weller* 'Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, U.S.A; $Department of Biology, Georgetown University, Washington, D.C., U.S.A. and tlnstitute for Molecular Virology, University of St. Louis, St.Louis, MO 63 100, U.S.A.

Serotonin content and melatonin production in the pineal gland of the male Djungarian hamster (Phodopus sungorus).

Diurnal variations in serum melatonin levels and pineal concentrations of serotonin (5-HT), N-acetylserotonin (NAS), 5-hydroxyindole acetic acid (5-HIAA), and melatonin were estimated in adult male Djungarian hamsters kept under long-day (LD 16:8) or short-day (LD 8:16) photoperiods. The nocturnal increase in melatonin production was accompanied by a marked drop in pineal serotonin concentrations. Serotonin levels, however, decreased approximately 4 hr before pineal melatonin increased. Correlations of the mean values for pineal serotonin and pineal melatonin indicated significant correlations at both LD 16:8 (r = -0.92, P less than 0.001) and LD 8:16 (r = -0.85, P less than 0.001). The mean levels of pineal serotonin and serum melatonin were correlated as well (LD 16:8, r = -0.91, P less than 0.001; LD 8:16, r = -0.81, P less than 0.01). The levels of pineal serotonin declined at approximately the same time as serum melatonin levels increased. These data suggest that the drop in pineal serotonin is primarily a consequence of melatonin production (as reflected by increasing serum concentrations). Consequently, pineal concentrations of melatonin may not be the best estimate of actual melatonin production, but a measure of how much melatonin is accumulated within the pineal due to high synthesis rates while the release of the hormone from the gland is limited.

Sympathetic denervation and chronic serotonin uptake blockade by fluoxetine do not affect pineal gland 5-hydroxyindole acetic acid: evidence that oxidative deamination of pineal serotonin is a property of the pinealocyte.

This investigation tested the hypothesis that oxidative deamination of 5HT in the pineal gland occurs primarily in cellular compartments other than the pinealocyte (i.e., noradrenergic nerve terminals and glia). Following sympathetic denervation of the pineal gland by bilateral superior cervical sympathectomy, pineal levels of 5HT and its oxidative metabolite, 5HIAA, were measured by HPLC from samples collected at six time points over the 24 h photoperiod. The role of glia in 5HT deamination was further examined by chronic treatment with the 5HT uptake blocker, fluoxetine (10 mg/kg). Sympathectomy abolished the circadian rhythms of both 5HT and 5HIAA, but had no statistically significant effect on the ratio of 5HIAA/5HT compared to sham-operated and intact controls over the 24 h period. Pineal daytime levels of both 5HT and 5HIAA were unaffected by fluoxetine treatment. These findings indicate that the pinealocyte is an important cellular compartment for 5HT oxidative metabolism.

Pineal serotonin is resistant to depletion by serotonergic neurotoxins in rats

Administration of 3,4-methylenedioxymethamphetamine (MDMA) or para-chloroamphetamine (pCA) to adult rats is neurotoxic to serotonin (5HT) nerve terminals and cell bodies. MDMA (10 mg/kg) reduces 5HT levels in the frontal cortex, medial basal hypothalamus, and striatum acutely and 17 days after a series of multiple injections. The acute reductions occur within 1-2 hr after injection of doses greater than 3 mg/kg. A single injection of pCA reduces 5HT levels in the above mentioned brain regions as well as in the brain stem. However, none of these treatments are able to alter 5HT levels in the pineal gland. It appears that the pineal does not contain the 5HT reuptake system that is thought to be necessary for the neurotoxicity of MDMA and pCA.

Attenuated nocturnal rise in pineal and serum melatonin in a genetically cardiomyopathic Syrian hamster with a deficient calcium pump

Day and nighttime melatonin production in the pineal gland was compared in normal and cardiomyopathic (polydystrophic) adult male Syrian hamsters. These strains of hamsters were selected for comparison because the cardiomyopathetic hamster displays a deficient transmembrane Ca(2+)-pump in a number of tissues, and intracellular CA2+ concentrations ([Ca2+]i) play a central role in the nocturnal increase in pineal melatonin synthesis. Daytime levels of all constituents measured, i.e., pineal N-acetyltransferase (NAT) activity, pineal and serum melatonin levels, and pineal 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindole acetic acid (5-HIAA) contents, were comparable in control and dystrophic hamsters. In contrast, the nighttime rises in pineal NAT activity and pineal and serum melatonin levels were significantly attenuated in the dystrophic hamsters. By comparison, the pineal contents of 5-HTP, serotonin, and 5-HIAA were essentially the same in both groups of hamsters with both pineal serotonin and 5-HIAA values exhibiting the usual nighttime drop. It is presumed that the alterations in nocturnal melatonin production in the pineal gland of the cardiomyopathic hamster may relate to a generalized deficiency in the Ca(2+)-pump in pinealocyte plasma membranes, which leads to unusually high [Ca2+]i, causing a depression of NAT activity; this leads to the commensurate decline in pineal and serum melatonin levels. Harderian gland NAT activity and melatonin levels were essentially similar in the two groups of animals, although NAT activity was slightly depressed in the dystrophic hamsters killed during the day. The reduced amounts of intrascapular brown fat in the cardiomyopathic hamster is speculated to be a result of the diminished amount of melatonin produced in these animals.

EFFECT OF STRESS ON SEROTONIN, NOREPINEPHRINE, EPINEPHRINE AND CORTICOSTERONE CONTENTS IN THE SOFT‐SHELLED TURTLE

1. Adult soft-shelled turtles were exposed to hyperosmotic and dehydration stresses. 2. Acute treatment for 0.5, 1 or 2 h with sodium chloride (3.6%, single intramuscular injection, 2 mL volume) caused depletion of pineal serotonin contents followed by elevation of norepinephrine and epinephrine levels. In addition, it depleted corticosterone and norepinephrine from the adrenal gland. 3. The serotonin level also decreased with a concomitant increase of 5-hydroxyindoleacetic acid but without any discernible change in catecholamine content after chronic treatment with sodium chloride (3.6%, 0.5 mL daily for 7 days). 4. Dehydration for 7 days brought about depletion of serotonin and epinephrine levels and elevation of norepinephrine level. 5. The findings suggest that hyperosmotic stress has a definite influence on pineal-paraphyseal serotonin, 5-hydroxyindoleacetic acid, norepinephrine and epinephrine concentrations, and adrenal corticosterone and norepinephrine contents in Lissemys turtles. Dehydration stress also modulates pineal-paraphyseal serotonin, norepinephrine and epinephrine concentrations.

Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0–15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30–180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.

Lindane may enhance nocturnal pineal N-acetyltransferase activity via β-adrenergic receptors

Lindane, a chlorinated hydrocarbon pesticide, was previously shown to enhance the nighttime rise in pineal N-acetyltransferase (NAT) activity and melatonin as well as serum melatonin levels. The purpose of the present study was to test whether lindane acts on the pineal gland by means of a beta-adrenergic receptor mechanism. Whereas lindane (total dose 17.8 mg/kg b.wt. over 6 days) by itself significantly augmented the nocturnal levels of pineal NAT activity in otherwise untreated rats, the pesticide was ineffective in reference to this enzyme when it was given in conjunction with the beta-adrenergic receptor antagonist propranolol (20 mg/kg b.wt., one hour before lights off). The augmentation of NAT activity by lindane also caused significant reductions in pineal serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA); again, both these responses were blocked by propranolol treatment. Neither pineal 5-hydroxytryptophan nor pineal or serum melatonin levels were significantly changed as a result of either lindane or propranolol treatment. The results are consistent with the idea that lindane influences pineal 5-HT metabolism either at the level of the beta-adrenergic receptor or via the sympathetic innervation to the pineal gland.

Food restriction retards aging of the pineal gland

The effects of chronic (40%) food restriction from 6 weeks of age were studied in aging male Fisher 344 rats. When compared with 3-month-old, ad libitum fed rats, pineal N-acetyltransferase (NAT) activity had declined to less than 30% and pineal and serum levels of melatonin to 40% after 28 months when feeding had been ad libitum. Food restriction significantly retarded this development (P less than 0.05) giving NAT and melatonin levels which were twice as high as in the ad libitum fed group. Nighttime levels of pineal serotonin (5-HT) were similar in food-restricted and ad libitum fed old rats but were nearly twice as high (P less than 0.05) as in young rats. There was also a tendency for increased production of 5-hydroxyindoleacetic acid (5-HIAA) in the pineal gland with higher levels of 5-HT. It is concluded that aging in the rat (Fisher 344) is accompanied by a reduction of pineal NAT activity, thereby reducing the production of melatonin and causing a buildup of 5-HT in the pineal gland. It is furthermore proposed that food restriction, which markedly increases the life span and reduces age-related physiological deterioration and diseases in many animals, may mediate some of its effects through a sustained pineal activity in old age.

Effect of insulin on serotonin, 5-hydroxyindole-acetic acid, norepinephrine, epinephrine and corticosterone contents in chick

Pineal serotonin (5-HT), 5-hydroxyindole-acetic acid (5-HIAA), norepinephrine (NE), epinephrine (E) and adrenal corticosterone, NE and E contents were measured spectrofluorometrically 5, 15, 30 and 60 min after insulin treatment to induce hypoglycemic stress. In the pineal gland, hypoglycemic stress caused dramatic decrease of 5-HT, resulted in initial decrease followed by an increase of 5-HIAA and brought about changes of NE and E. Corticosterone content in the adrenal gland increased significantly following treatment with insulin. Adrenal NE and E contents plummetted to control value followed by quick resynthesis. The findings indicate that insulin-induced hypoglycemia greatly modulates the activities of pineal and adrenal glands.

Opposite Effects of EGTA and Neutral Surfactants on the Loss of Chicken Pineal Serotonin N‐Acetyltransferase Activity

The effects of some general purpose drugs on the deactivation and activity measurement of the chicken pineal gland enzyme serotonin N-acetyl transferase (EC 2.3.1.5.) were studied. The drugs used were EGTA and two neutral surfactants, Nonidet P40 and Triton X-100. Enzyme activity showed significant variations ranging from 2.8 +/- 1.3 nmol/gland/h when Nonidet P40 was added to the homogenate buffer, to 31.8 +/- 1.7 nmol/gland/h when EGTA was present. This striking variation seemed to be caused by the ability of these compounds to modify the rate of NAT deactivation acting either as accelerating agents, as in the case of the detergents or as braking agent, as in the case of EGTA.

Calcium influx through voltage-sensitive calcium channels regulates in vivo serotonin N-acetyltransferase (NAT) activity in hen retina and pineal gland

The involvement of transmembrane transport of Ca 2+ in regulation of retinal and pineal serotonin N-acetyltransferase (NAT) activity was studied in vivo in hens. Intramuscular administration to hens of organic antagonists of voltage-sensitive calcium channels (VSCC), nimodipine, nifedipine and verapamil, resulted in a marked inhibition of the nocturnal increase of NAT activity in retina and pineal gland. In both tissues the inhibitory effect of nimodipine on the night-time rise of NAT activity was abolished by Bay K 8644, a VSCC agonist. Bay K 8644 administered to hens at the end of the light phase of the light-dark cycle significantly enhanced the dark-dependent increase in retinal and pineal NAT activity. It is suggested that the nocturnal increase of NAT activity in hen retina and pineal gland is similarly regulated in vivo by Ca 2+ influx through the VSCC.