Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase-Activating Peptide

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin- N -acetyltransferase (NAT) activity and N -acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of α-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and β-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant α₁-adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac-Tyr, d -Phe)-GRF 1–29 amide, in 100–1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.     

Day-to-day variation in pineal serotonin N-acetyltransferase activity in stressed and non-stressed male Sprague-Dawley rats

Previous studies involving physical-immobilization stress in laboratory rats have yielded inconsistent results with respect to melatonin synthesis in the pineal gland. As melatonin formation undergoes circadian and infradian rhythms, the aim of the present study was to examine whether stress experiments exhibit day-to-day variation. Toward this end, groups of male Sprague-Dawley rats were stressed by physical immobilization on eight consecutive days, respectively, or left relatively undisturbed, and killed. The pineal gland was rapidly dissected out and serotonin N-acetyltransferase (NAT) activity and melatonin levels were measured. NAT activity was significantly depressed on experimental days 1, 3 and 5, and slightly depressed on day 7. In addition, both in control and experimental animals NAT activity exhibited statistically significant differences between experimental days. Pineal melatonin levels were less variable. On experimental days 3 and 6 immobilization led to a significant increase of pineal melatonin levels. These results show that day-to-day variation is an important factor that influences the outcome of stress experiments and represent another example that NAT activity and pineal melatonin levels do not always show corresponding changes.     

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.     

Changes in pineal indoleamine metabolism in vitamin A deficient rats

Weanling, male rats were fed a vitamin A deficient (VAD) diet from 20 to 77 days of age. The circadian rhythms of the precursors and metabolites of pineal melatonin were measured along with the activity of N-acetyltransferase (NAT). Significant decreases in peak melatonin levels (0100 hours) and in nightime NAT activity (0100 and 0300 hours) were found in the pineals of the VAD rats. In contrast, the contents of serotonin, 5-hydroxytryptophan and 5-hydroxyindole acetic acid were only moderately affected by the deficiency. Daily administration of 25 micrograms melatonin from 20 to 74 days of age markedly reduced NAT activity in control and VAD rats. These data suggest that NAT activity is more sensitive to chronic VAD than any other parameters of melatonin metabolism.     

Pineal melatonin: circadian rhythm and variations during the hibernation cycle in the ground squirrel, Spermophilus lateralis

Variations in pineal melatonin content throughout a 24-hour period and during different phases of the hibernation bout cycle were studied in the golden-mantled ground squirrel (Spermophilus lateralis). In addition to pineal melatonin, the circadian variation in the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were also investigated in summer animals maintained at 22 +/- 2 degrees C, on a light:dark (L:D) schedule of 12:12 hr for 1 month (lights on at 08.00 hr). Pineal glands were collected from six animals in each group at 1200, 1600, 2000, 2400, 0200, 0400, and 0800 hr. Changes in pineal melatonin content during the hibernation bout cycle were investigated in ground squirrels housed at 4 +/- .05 degrees C in relative darkness (1.9-3.4 lux; 10:14 LD). Pineal glands were obtained between 12:00 and 18:00 hr from 30 animals during one of three phases of the cycle (deep hibernation, euthermic interbout, and entrance into hibernation). Pineal melatonin was also measured for comparison in six winter euthermic animals that were housed at 22 +/- 2 degrees C, on a L:D schedule of 10:14 hr. Melatonin was measured in individual pineal glands by radioimmunoassay. The daily melatonin rhythm in S. lateralis was characterized by a marked increase in pineal melatonin during the dark phase, in which peak nighttime values were nearly 20-fold greater than daytime basal levels. The daily rhythm for NAT activity paralleled the changes in melatonin, showing a peak activity at 0200 hr that was 45 times greater than mean daytime values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal modulation of pineal melatonin synthesis in rats and Syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections

Pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the enzyme activities of N-acetyltransferase and hydroxyindole-O-methyltransferase were determined in male albino rats and Syrian hamsters that were injected with insulin twice daily for three days, or injected with streptozotocin to induce diabetes. Neither insulin injections nor streptozotocin diabetes had any effect on pineal melatonin production in rats. In hamsters, diabetes reduced the nocturnal peak of pineal melatonin content by approximately one half, while insulin injections had no effect on pineal melatonin levels; however, insulin injections did cause a slight increase in pineal N-acetyltransferase activity. These findings indicate that the pineal gland of the hamster may be more sensitive to alterations in plasma insulin levels than the same organ in rats.     

Differential regulation of central vasopressin receptors in transgenic rats with low brain angiotensinogen

The consequences of permanent alteration to the brain renin-angiotensin system (RAS) on central vasopressinergic system was studied in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)]. Levels of vasopressin (AVP) and V1a receptor mRNAs were measured by ribonuclease protection assay (RPA) and AVP by radioimmunoassay (RIA). AVP (100 pmol/50 nl) was microinjected into the nucleus tractus solitarii (NTS) of urethane-anesthetized TGR(ASrAOGEN) and Sprague-Dawley (SD) rats and the mean arterial pressure (MAP) and heart rate (HR) baroreflex induced by phenylephrine were evaluated. AVP but not its mRNA levels were significantly lower in the hypothalamus and hypophysis of TGR(ASrAOGEN) rats. Brainstem V1a mRNA levels were significantly higher in TGR(ASrAOGEN) in comparison to SD rats (5.2+/-0.4% vs. 3.3+/-0.2% of beta-actin mRNA, P<0.05). In contrast, the hypothalamic V1a mRNA levels in TGR(ASrAOGEN) were not different from those found in SD rats. AVP microinjections induced a greater decrease in MAP in TGR(ASrAOGEN) in comparison with SD rats (-19.9+/-5.2 vs. -7.5+/-0.7 mm Hg, P<0.01). The significantly higher baroreflex sensitivity observed in TGR compared to that of SD rats was normalized after AVP microinjection. The increased brainstem V1a mRNA levels and sensitivity to AVP in TGR(ASrAOGEN) rats indicates a functional upregulation of AVP receptors in the NTS. The fact that the hypothalamic V1a mRNA levels are not altered indicates that these receptors are differentially regulated in different brain regions. This study demonstrates that a permanent deficit in brain angiotensinogen synthesis can alter the functionality of central vasopressinergic system.     

Electrical stimulation of the hypothalamic nucleus paraventricularis mimics the effects of light on pineal melatonin synthesis

In an attempt to clarify further the role of the hypothalamic paraventricular nuclei (PVN) in the control of pineal function, the effects of 2 min electrical stimulation of these nuclei were investigated in acutely blinded, adult, male Sprague-Dawley rats. Pineal serotonin-N-acetyltransferase (NAT) activity, melatonin content and catecholamine levels were measured by means of radio-enzymatic, radioimmunoassay and high-performance liquid-chromatography methods, respectively. All three pineal parameters underwent significant declines following brief PVN stimulation during the night time. These observations lend credence to the view that the neural pathways transmitting light information to the sympathetic innervation controlling pineal melatonin synthesis.     

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.     

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.     

Swimming-induced suppression of rat pineal melatonin is prevented by pretreatment with calcium channel blockers

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22 degrees C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.     

Altered circadian rhythm reentrainment to light phase shifts in rats with low levels of brain angiotensinogen

In this study, we aimed to investigate the adaptation of blood pressure (BP), heart rate (HR), and locomotor activity (LA) circadian rhythms to light cycle shift in transgenic rats with a deficit in brain angiotensin [TGR(ASrAOGEN)]. BP, HR, and LA were measured by telemetry. After baseline recordings (bLD), the light cycle was inverted by prolonging the light by 12 h and thereafter the dark period by 12 h, resulting in inverted dark-light (DL) or light-dark (LD) cycles. Toward that end, a 24-h dark was maintained for 14 days (free-running conditions). When light cycle was changed from bLD to DL, the acrophases (peak time of curve fitting) of BP, HR, and LA shifted to the new dark period in both SD and TGR(ASrAOGEN) rats. However, the readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. The LA acrophases changed similarly in both strains. When light cycle was changed from DL to LD by prolonging the dark period by 12 h, the reentrainment of BP and LA occurred faster than the previous shift in both strains. The readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. In free-running conditions, the circadian rhythms of the investigated parameters adapted in TGR(ASrAOGEN) and SD rats in a similar manner. These results demonstrate that the brain RAS plays an important role in mediating the effects of light cycle shifts on the circadian variation of BP and HR. The adaptive behavior of cardiovascular circadian rhythms depends on the initial direction of light-dark changes.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

50-Hz SINUSOIDAL MAGNETIC FIELD EFFECT ON IN VITRO PINEAL N-ACETYLTRANSFERASE ACTIVITY

Some studies have shown a decrease in pineal N-acetyltransferase (NAT) activity and/or blood melatonin concentration in rodents exposed to extremely low-frequency (ELF) and low magnetic flux density electromagnetic fields. The mechanism/s involved in such effects are not known. It has been hypothesized that the magnetic fields (MF) could act on the pineal gland directly and/or indirectly through the retina. The aim of this work was to study whether MFs could modify NAT activity through a direct effect on the gland. Pineal glands obtained from rats sacrificed in the middle of the dark period were exposed during a 1-h incubation to 10-, 100-, or 1,000-μT, 50-Hz, sinusoidal MFs. The results showed that the glands exposed to the highest magnetic flux density responded with a significant decrease in NAT activity. The data obtained from these experiments support the idea that the pineal gland can be directly affected by ELF electromagnetic fields.     

Indole metabolism in the pineal organ of the pigeon with special reference to melatonin-synthesizing cells

By means of radioimmunoassay a clear-cut peak of melatonin concentration was found in the pineal organ of the pigeon at the middle of the scotophase (Coisin et al. 1982a). The aim of the present study was to identify the cell type responsible for the nocturnal indole metabolism, including melatonin synthesis, in the pineal of this avian species. After a short-term incubation or organ culture in the presence of [3H]-indolic precursors, [3H]-5-hydroxytryptophan or [3H]-5-hydroxytryptamine, the relative amounts of deaminated and acetylated products occurring in the pineal organ were measured by the use of thin layer chromatography and liquid-scintillation counting. It was possible to modify the relative amounts of deaminated and acetylated indoles by the application of some inhibitors of monoamine oxidase and cyclic nucleotide phosphodiesterase. Irrespective of the experimental conditions, high-resolution autoradiography combined with the above-mentioned radiochemical experiments showed that the cells of the receptor line (modified photoreceptor cells) are responsible for indole storage and metabolism, and very probably also for melatonin biosynthesis. The other cell types of the pineal parenchyma did not display significant labeling.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

σ Receptor Modulation of Noradrenergic-Stimulated Pineal Melatonin Biosynthesis in Rats

Abstract: Because σ receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the σ-receptor ligands 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine potentiated the enhancement of N -acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)- N -allylnormetazocine enhanced significantly the physiological increases in both pineal N -acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific σ-receptor antagonist. These findings suggest that, in rats, the activation of pineal σ-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it pontentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.     

Elevated daytime rat pineal and serum melatonin levels induced by isoproterenol are depressed by swimming

Isoproterenol (1 mg/kg) was subcutaneously injected into adult male rats during the day to stimulate pineal N-acetyltransferase (NAT) activity and pineal and serum melatonin levels. Two hours after isoproterenol administration when levels of each of these variables had increased significantly, the experimental animals swam for 10 min in 22 degrees C water. At 15 min after swimming onset, pineal and serum melatonin levels were highly significantly depressed compared to those in control animals that did not swim. The high NAT level was not influenced by swimming. In a second study, isoproterenol injected rats swam for either 1, 3, 6 or 10 min and were sampled 15 min after the onset of swimming. The reduction in the elevated pineal melatonin in these animals was correlated with the length of the swim, i.e., as the duration of swim increased the percent reduction in pineal melatonin also increased. Neither pineal NAT nor hydroxyindole-O-methyltransferase (HIOMT) activities were influenced by swimming. The results suggest that elevated pineal and serum melatonin induced by isoproterenol can be depressed with no effect on the activity of the enzymes which convert serotonin to melatonin.