Ion transport in an immortalized rat submandibular cell line SMG-C6.

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.

Twenty-four hour urinary excretion of 6-hydroxymelatonin sulfate in Down syndrome subjects.

Because of the overexpression of the enzyme superoxide dismutase, individuals with Down syndrome (DS) are believed to suffer from increased oxidative stress as a result of the excessive production of oxygen-based free radicals; their exposure to higher than normal free radical production may account in part for signs of premature aging, early onset of cataracts, and of Alzheimer's disease. Free radicals are normally neutralized by free radical scavengers and other antioxidants. The pineal hormone melatonin is a potent scavenger of both the hydroxyl and peroxyl radicals, both of which are highly toxic, and a stimulator of the antioxidative enzyme glutathione peroxidase. Considering this, we deemed it important to define the day/night rhythm and levels of melatonin production in DS subjects. To do this, we assessed the urinary excretion of the chief melatonin metabolite, 6-hydroxymelatonin sulfate, throughout a 24 hr period in DS subjects; comparisons were made with the metabolite levels in the urine of non-Down siblings and parents of the DS subjects. All 8 non-Down subjects exhibited what was classified as normal urinary excretion of 6-hydroxymelatonin sulfate with the usual low daytime and high night-time levels of the melatonin metabolite. Of 12 DS subjects studied, 10 exhibited the normal day/night rhythm in urinary 6-hydroxymelatonin sulfate levels; 2 subjects were devoid of a rhythm. However, when all the data from each group were averaged, there were no noticeable differences in the absolute levels or 24 hr variations in urinary 6-hydroxymelatonin sulfate excretion between DS and non-Down subjects.

Melatonin and structurally-related, endogenous indoles act as potent electron donors and radical scavengers in vitro.

The radical scavenging properties of melatonin, structurally-related indoles and known antioxidants were investigated in kinetic competition studies using the specific radical trapping reagent 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). In the presence of highly reactive radicals, ABTS is oxidized to the stable thiazoline cation radical, ABTS*(+) which, due to its intense green color, can be measured photometrically at 420 nm absorbance. The indoles melatonin, 5-methoxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptamine as well as the phenolic and thiolic antioxidants ascorbic acid, Trolox, and glutathione inhibited ABTS cation radical formation and catalyzed ABTS radical cation reduction. Melatonin was the most potent radical scavenger and electron donor when compared with the methoxylated indole analogs and the other antioxidants tested. Melatonin, the methoxylated indole analogs and the other antioxidants tested acted as potent electron donors which scavenged initiating and propagating radicals and repaired oxidative damage due to electrophile intermediates.

Melatonin stimulates the activity of the detoxifying enzyme glutathione peroxidase in several tissues of chicks.

The pineal hormone melatonin has been shown to directly scavenge free radicals and to stimulate, in the mammalian brain, at least one enzyme, glutathione peroxidase, which reduces free radical generation. In the present studies, we examined the effect of melatonin on glutathione peroxidase activity in several tissues of an avian species. Melatonin (500 micrograms/kg), when injected into chicks, increased glutathione peroxidase activity within 90 min in every tissue examined. Tissue melatonin levels, measured by radioimmunoassay, also increased following its peripheral administration. Depending on the tissue, the measured increases in melatonin varied from 75% to 1,300% over the control values. The melatonin-induced increases in glutathione peroxidase activity varied with the tissue and were between 22% and 134%. These percentage increases in glutathione peroxidase activity were directly correlated with tissue melatonin content. These results suggest that melatonin induces the activity of the detoxifying enzyme, glutathione peroxidase, in several tissues in the chick. The findings also suggest that melatonin would reduce the generation of highly toxic hydroxyl radicals by metabolizing its precursor, hydrogen peroxide. Because of this ability to stimulate glutathione peroxidase activity, melatonin should be considered as a component of the antioxidative defense system in this avian species.

Red-light-induced suppression of melatonin synthesis is mediated by N-methyl-D-aspartate receptor activation in retinally normal and retinally degenerate rats.

Pineal gland N-acetyltransferase (NAT) activity and pineal and serum levels of melatonin declined linearly in albino rats acutely exposed to different intensities of red light (600 nm or higher; low, 140 microW/cm2; moderate, 690 microW/cm2; high, 1200 microW/cm2) during the middle of the night. The high intensity red light was as effective as white light (780 microW/cm2) in suppressing NAT activity and pineal and circulating melatonin. Red-light-inhibited nighttime NAT activity and suppressed nocturnal melatonin levels in both retinally degenerate and normal rats. Pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 mg/kg intraperitoneally) completely prevented the red-light-induced inhibition of nighttime melatonin synthesis. Magnesium chloride (300 mg/kg intraperitoneally) reduced the inhibitory effects of low and moderate intensities of red light but was ineffective when high red-light intensity was used. However, both agents failed to antagonize the suppression of nighttime melatonin synthesis elicted by the exposure to white light. Since retinally degenerate and retinally normal animals respond in the same way to both red-light and pharmacological intervention with the NMDA receptor blocker MK-801, the findings indicate that the activation of central hypothalamic NMDA receptors might mediate the photic inhibition of nocturnal melatonin synthesis in the pineal gland elicited by the exposure to red light at night. Red-light-induced suppression of nocturnal melatonin synthesis possibly can be used to investigate the biochemical mechanisms by which light entrains melatonin synthesis and to study the pharmacological and physiological effects of endogenous and synthetic agents that antagonize the NMDA receptor response.

Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals.

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with Nw-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin stimulates brain glutathione peroxidase activity.

Exogenously administered melatonin causes a 2-fold rise in glutathione peroxidase activity within 30 min in the brain of the rat. Furthermore, brain glutathione peroxidase activity is higher at night than during the day and is correlated with high night-time tissue melatonin levels. Glutathione peroxidase is thought to be the principal enzyme eliminating peroxides in the brain. This antioxidative enzyme reduces the formation of hydroxyl radicals formed via iron-catalyzed Fenton-type reactions from hydrogen peroxide by reducing this oxidant to water. Since the hydroxyl radical is the most noxious oxygen radical known, induction of brain glutathione peroxidase might be an important mechanism by which melatonin exerts its potent neuroprotective effects.

Regularly scheduled, day-time, slow-onset 60 Hz electric and magnetic field exposure does not depress serum melatonin concentration in nonhuman primates.

Experiments conducted with laboratory rodents indicate that exposure to 60 Hz electric fields or magnetic fields can suppress nocturnal melatonin concentrations in pineal gland and blood. In three experiments employing three field-exposed and three sham-exposed nonhuman primates, each implanted with an indwelling venous cannula to allow repeated blood sampling, we studied the effects of either 6 kv/m and 50 microT (0.05 G) or 30 kV/m and 100 microT (1.0 G) on serum melatonin patterns. The fields were ramped on and off slowly, so that no transients occurred. Extensive quality control for the melatonin assay, computerized control and monitoring of field intensities, and consistent exposure protocols were used. No changes in nocturnal serum melatonin concentration resulted from 6 weeks of day-time exposure with slow field onset/offset and a highly regular exposure protocol. These results indicate that, under the conditions tested, day-time exposure to 60 Hz electric and magnetic fields in combination does not result in melatonin suppression in primates.

Rapid-onset/offset, variably scheduled 60 Hz electric and magnetic field exposure reduces nocturnal serum melatonin concentration in nonhuman primates.

Experiments with rodents indicate that power-frequency electric field (EF) or magnetic field (MF) exposure can suppress the normal nocturnal increase in melatonin concentration in pineal gland and blood. In a separate set of three experiments conducted with nonhuman primates, we did not observe melatonin suppression as a result of 6 weeks of day-time exposure to combined 60 Hz electric and magnetic fields (E/MF) with regularly scheduled "slow" E/MF onsets/offsets. The study described here used a different exposure paradigm in which two baboons were exposed to E/MF with "rapid" E/MF onsets/offsets accompanied by EF transients not found with slowly ramped E/MF onset/offset; profound reductions in nocturnal serum melatonin concentration were observed in this experiment. If replicated in a more extensive experiment, the observation of melatonin suppression only in the presence of E/MF transients would suggest that very specific exposure parameters determine the effects of 60 Hz E/MF on melatonin.

A review of the evidence supporting melatonin's role as an antioxidant.

This survey summarizes the findings, accumulated within the last 2 years, concerning melatonin's role in defending against toxic free radicals. Free radicals are chemical constituents that have an unpaired electron in their outer orbital and, because of this feature, are highly reactive. Inspired oxygen, which sustains life, also is harmful because up to 5% of the oxygen (O2) taken in is converted to oxygen-free radicals. The addition of a single electron to O2 produces the superoxide anion radical (O2-.); O2-. is catalytic-reduced by superoxide dismutase, to hydrogen peroxide (H2O2). Although H2O2 is not itself a free radical, it can be toxic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical (.OH). The .OH is the most toxic of the oxygen-based radicals and it wreaks havoc within cells, particularly with macromolecules. In recent in vitro studies, melatonin was shown to be a very efficient neutralizer of the .OH; indeed, in the system used to test its free radical scavenging ability it was found to be significantly more effective than the well known antioxidant, glutathione (GSH), in doing so. Likewise, melatonin has been shown to stimulate glutathione peroxidase (GSH-Px) activity in neural tissue; GSH-PX metabolizes reduced glutathione to its oxidized form and in doing so it converts H2O2 to H2O, thereby reducing generation of the .OH by eliminating its precursor. More recent studies have shown that melatonin is also a more efficient scavenger of the peroxyl radical than is vitamin E. The peroxyl radical is generated during lipid peroxidation and propagates the chain reaction that leads to massive lipid destruction in cell membranes. In vivo studies have demonstrated that melatonin is remarkably potent in protecting against free radical damage induced by a variety of means. Thus, DNA damage resulting from either the exposure of animals to the chemical carcinogen safrole or to ionizing radiation is markedly reduced when melatonin is co-administered. Likewise, the induction of cataracts, generally accepted as being a consequence of free radical attack on lenticular macromolecules, in newborn rats injected with a GSH-depleting drug are prevented when the animals are given daily melatonin injections. Also, paraquat-induced lipid peroxidation in the lungs of rats is overcome when they also receive melatonin during the exposure period. Paraquat is a highly toxic herbicide that inflicts at least part of its damage by generating free radicals.(ABSTRACT TRUNCATED AT 400 WORDS)

The pineal melatonin rhythm and its regulation by light in a subterranean rodent, the valley pocket gopher (Thomomys bottae).

The daytime and nightime levels of pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin were measured in adult male and female valley pocket gophers, Thomomys bottae. This species was chosen for study because it is a subterranean rodent that inhabits burrows whose openings to the surface are closed. Therefore, under field conditions it is estimated that the pocket gopher spends roughly 99% of its time in absolute darkness in underground burrows. When wild captured pocket gophers were maintained under a light:dark cycle (light intensity during the day of roughly 140 microW/cm2), nightime levels of pineal NAt activity and melatonin content were higher than values measured during the day; on the other hand, HIOMT activity in the pineal gland was similar in the day and at night. When pocket gophers were exposed to an extended light period (220 microW/cm2) 4 hr into the night, the rise in melatonin synthesis normally associated with darkness onset was not inhibited. Also, when gophers were acutely exposed to a light intensity of 400 microW/cm2 for 1 hr beginning 4 hr after darkness onset, neither high nocturnal levels of pineal NAT nor pineal melatonin contents were reduced. Finally, when pocket gophers were exposed to a 600 microW/cm2 light intensity at either 4 hr or 8 hr into the dark period, pineal melatonin synthesis remained elevated at a level comparable to that measured in dark-exposed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole.

Hepatic DNA adduct formation induced by the chemical carcinogen, safrole, was suppressed by both endogenous pineal melatonin release and by the exogenous administration of melatonin to rats. DNA damage after administration of of melatonin to rats. DNA damage after administration of 100 mg/kg safrole (i.p.) was measured by the P1 enhanced 32P-postlabeling analysis method. The RAL (relative adduct labeling) x 10(7) of carcinogen modified DNA in the liver of untreated controls and in safrole treated animals killed during the day, at night, after pinealectomy and pinealectomy plus melatonin injection (0.15 mg/kg x 4 or a total of 0.6 mg/kg) was 0, 12.6 +/- 0.75, 10.9 +/- 0.72, 13.6 +/- 1.12 and 5.7 +/- 0.53 respectively. For the same groups of animals, circulating melatonin levels at the termination of the study were 31 +/- 3, 29 +/- 2, 276 +/- 31, 24 +/- 1 and 13,950 +/- 1016 pg/ml serum respectively. The higher the melatonin concentration in the serum the lower was DNA adduct formation in the rat liver. Thus, high nocturnal levels of melatonin were protective against safrole-induced DNA damage. These findings indicate that the functional pineal gland plays an important role in oncostatic actions of carcinogens such as safrole. At physiological levels, melatonin seemed to prevent especially the formation of what was referred to as the N1 DNA adduct. Melatonin's ability to suppress DNA adduct formation may relate to its inhibitory effect on a mixed function oxidase, cytochrome p-450, and on the recently identified hydroxyl radical scavenging capacity of the indole. The oncostatic action of melatonin is also suggested by its nuclear accumulation and DNA stabilization characteristics. At pharmacological levels melatonin is extremely potent in preventing DNA modification induced by the chemical carcinogen, safrole.

Nuclear localization of melatonin in different mammalian tissues: immunocytochemical and radioimmunoassay evidence.

Melatonin was detected by an improved immunocytochemical technique in the cell nuclei of most tissues studied including several brain areas, pineal gland, Harderian gland, gut, liver, kidney, and spleen from rodents and primates. Cryostat sections from tissues fixed in Bouin's fluid, formalin, or acetone/ethanol were used. The nuclear staining appeared primarily associated with the chromatin. The nucleoli did not exhibit a positive reaction. The melatonin antiserum was used in the range of 1:500 to 1:5,000. Incubation of the antibody with an excess of melatonin resulted in the complete blockade of nuclear staining. Pretreatment of the sections with proteinase K (200-1,000 ng/ml) prevented the positive immunoreaction. In a second aspect of the study, we estimated the concentration of melatonin by means of radioimmunoassay in the nuclear fraction of several tissues including cerebral cortex, liver, and gut. The subcutaneous injection of melatonin (500 micrograms/kg) to rats resulted, after 30 min, in a rapid increase in the nuclear concentration of immunoreactive melatonin which varied in a tissue-dependent manner. However, samples collected 3 h after the injection showed that melatonin levels had decreased to control values. Pinealectomy in rats resulted in a clear reduction in the nuclear content of melatonin in the cerebral cortex and liver but not in the gut. The results of these studies suggest that melatonin may interact with nuclear proteins and that the indole may have an important function at the nuclear level in a variety of mammalian tissues.

The pineal hormone melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in vivo.

Melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in a dose-dependent manner. Total DNA-adduct formation after in vivo administration of 300 mg/kg safrole measured by 32P-postlabeling analysis of carcinogen-modified DNA in rat liver was 36,751 +/- 2290 counts/min/10 micrograms DNA. Coadministration of 300 mg/kg safrole with either 0.2 mg/kg (low dose) or 0.4 mg/kg (high dose) melatonin reduced DNA-adduct formation induced by safrole to 22,182 +/- 987 counts/min/10 micrograms DNA and 462 +/- 283 counts/min/10 micrograms DNA, respectively. Circulating melatonin concentrations at the termination of the study in safrole, low melatonin and high melatonin groups were 50 +/- 8, 3140 +/- 430 and 10,040 +/- 2610 pg/ml serum, respectively. The results suggest that melatonin protects against safrole associated DNA damage.