Receptor binding properties of di (1,N6-ethenoadenosine) 5', 5'''-P1, P4-tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum.

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes.

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.

Human diadenosine triphosphate hydrolase: preliminary characterisation and comparison with the Fhit protein, a human tumour suppressor.

Human platelets diadenosine triphosphatase was characterised and compared with the Fhit protein, a human tumour suppressor with diadenosine triphosphatase activity. Both enzymes exhibit similar Km, are similarly activated by Mg2+, Ca2+ and Mn2+, and inhibited by Zn2+ and suramin. However, they are differentially inhibited by Fhit antibodies and exhibit differences in gel-filtration behaviour.

Fluorimetric detection of enzymatic activity associated with the human tumor suppressor Fhit protein.

The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP. We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme. Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme.

Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin.

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). Ki values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5 x 10(-6) M and 3 x 10(-7) M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.

Reciprocal effects of chronic diazepam and melatonin on brain melatonin and benzodiazepine binding sites.

Recent reports indicate that benzodiazepines can suppress melatonin levels and that melatonin can increase brain benzodiazepine binding. We have studied the possibility of reciprocal effects of chronic diazepam and melatonin on brain melatonin and benzodiazepine binding sites. Daily injections (3 weeks) of diazepam markedly reduced 125I-melatonin binding site density in the medulla-pons but not cortex of male rats, whereas benzodiazepine binding was not significantly affected. Melatonin, administered via the drinking water, significantly enhanced benzodiazepine (3H-RO 15-1788) binding in the medulla-pons and slightly reduced it in the cortex, but did not affect 125I-melatonin binding. Diazepam and melatonin combination reversed the suppression by diazepam of 125I-melatonin binding in the medulla-pons and the suppression by melatonin of benzodiazepine (3H-RO 15-1788 and 3H-flunitrazepam) binding in the cerebral cortex. These results indicate benzodiazepine-mediated suppression of brain melatonin binding sites that can be abrogated by melatonin administration.

Age-related changes in 2-[(125)I]-iodomelatonin binding sites in the brain of sea breams (Sparus aurata, L.).

The pineal organ of fish, through its 24h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g., reproduction, growth). The target sites for melatonin are poorly known in fish, especially marine species. A radioligand study was undertaken using the gilthead sea bream (Sparus aurata) maintained under natural temperature and photoperiod (at 28°N latitude). This species exhibits the property of changing sex during growth. Brains of one year-old males were collected at 16:00h and brains of three year-old females at 03:00, 10:00, 16:00 and 23:00h. Membrane homogenate receptor assays were run using 2-[(125)I]iodomelatonin as a ligand. Binding sites were detected in brains of young and old fish. In the younger, the exhibited a Bmax between 3.52 and 4.29 fmol mg protein(-1) and a KD between 358-380 pmol l(-1). In the older fish, the KD varied according to a daily pattern: values were three times higher at 03:00 and 10:00h (500-600 pmol l(-1)) than at 16:00 and 23:00h (150-300 pmol l(-1)). The number of sites also were higher at 03:00 and 10:00h (180-200 fmol mg protein(-1)) than at 16:00 and 23:00h (95-110 fmol mg protein(-1)). Melatonin and iodomelatonin displaced 2-[(125)I]iodomelatonin binding in a dose dependent manner, the second being more potent than the first. Binding was also inhibited by GTP. The results provide the first evidence for the presence of membrane melatonin binding sites in the brain of an exclusively marine fish. They suggest that their number and affinity varies during growth and throughout a light/dark cycle. Future experiments will aim to precise the anatomical location and role of these binding sites.

The modulatory effect of melatonin on the dopamine-glutamate interaction in the anterior hypothalamus during ageing.

We investigated the effects of melatonin on the dopamine-glutamate interaction in the anterior hypothalamus of young, middle-aged and aged rats. In young rats, under the effects of amphetamine, melatonin produced an inhibition of dopamine release and a significant increase in glutamate and aspartate release. In middle-aged and aged rats, the inhibitory effects of melatonin on amphetamine-evoked dopamine release were maintained, but no effects on glutamate or aspartate release were found. These results suggest that, during ageing, the modulatory effect of melatonin on dopamine release in rat anterior hypothalamus is preserved whereas the dopamine-glutamate interaction is disrupted with age.

Melatonin antagonizes alpha-melanocyte-stimulating hormone enhancement of melanogenesis in mouse melanoma cells by blocking the hormone-induced accumulation of the c locus tyrosinase.

Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24-48 h treatment and at concentrations of melatonin higher than the physiologic levels of the circulating hormone. Although these effects on the melanogenic activities were modest, incubation of melanocytes with melatonin prior to the addition of the melanotropin mediated a dramatic inhibition of alpha-melanocyte-stimulating-hormone-(alpha-MSH)-induced melanogenesis. This inhibitory effect was evident at melatonin concentrations as low as 10 nM. Inhibition was nearly total at 0.1 mM melatonin, even at high concentrations of alpha-MSH (1 microM). The inhibitory effect of melatonin on alpha-MSH stimulation of melanogenesis was investigated. Melatonin appeared to act at least at two stages. Pharmacological concentrations of melatonin diminished the number of alpha-MSH receptors to about 75% of the control values without an apparent effect on receptor affinity, as determined by receptor-binding studies using 125I-[N-Leu4-D-Phe7]alpha-MSH as a probe. Physiological concentrations of melatonin also appeared to interfere with the intracellular events coupling increased cAMP levels and induction of the c locus tyrosinase, since it strongly inhibited the theophylline-mediated stimulation of melanogenesis. The inhibition of tyrosinase stimulation was higher in the microsomal than in the melanosomal fractions of cells which were treated with melatonin, then exposed to either alpha-MSH (1 microM) or theophylline (1 mM), suggesting that one of the main effects of melatonin might be inhibition of the induction of tyrosinase de novo synthesis.

Dopamine-glutamic acid interaction in the anterior hypothalamus: modulatory effect of melatonin.

The aim of the present study was to investigate the role of melatonin as neuromodulator. For that, the effects of melatonin on the extracellular concentrations of excitatory amino acids, glutamic and aspartic acids, were investigated in the anterior hypothalamus and parieto-temporal cortex of the conscious rat using an intracerebral perfusion system. Melatonin at doses of 250 nM and 1 microM produced no effects on extracellular glutamate and aspartate concentrations in these two areas of the brain. Since amphetamine releases dopamine we perfused melatonin into the anterior hypothalamus and parieto-temporal cortex after a previous injection of amphetamine. Interestingly, we found a release of glutamic acid (p < 0.01) and aspartic acid (p < 0.01) produced by melatonin only when the increase (157%) of the extracellular dopamine concentration evoked by amphetamine was inhibited by melatonin in the anterior hypothalamus. The possibility is discussed of melatonin exerting its effects when the dopaminergic system is activated.

Changes in forebrain Na,K-ATPase activity and serum hormone levels during sexual behavior in male rats.

We have previously shown that Na,K-ATPase activity (the enzymatic machinery for the sodium pump) in brain areas such as the medial basal hypothalamus (MBH) and the preoptic-suprachiasmatic region (POSC) can be changed by experimental manipulations of gonadal function. We now report enzyme levels in brain regions as related to hormonal changes occurring during sexual behavior. Male rats were exposed to receptive females and decapitated immediately after displaying one of the following behavioral events: the start of copulatory activity, first ejaculation, and the beginning of a second copulatory series. A group of noncopulating animals were used as control. The variables measured included serum levels of LH, PRL and testosterone and Na,K-ATPase activity in MBH, POSC and parietal cerebral cortex (CC). A steady increase in enzyme activity in the POSC, but not the MBH or CC, was found in copulating animals. Serum LH levels changed in a similar fashion. A sharp increase in serum PRL levels, seemingly related to ejaculation, was also observed. These data are consistent with our previous findings on monoaminergic neurotransmission in brain regions related to male sexual behavior.

Influence of melatonin on the testicular regression induced by subcutaneous testosterone pellets in male rats kept in long or short photoperiod.

Daily afternoon injections of 25 micrograms melatonin for 12 weeks had no effect on testicular weights of male rats kept in long photoperiod (14L:10D); similarly, exposure of rats to short photoperiod (2L:22D) had no effect on gonadal weight. However, rats maintained in a long or short photoperiod and implanted every 2 weeks with a 15 mm Silastic pellet containing testosterone showed a significant reduction in testicular weight; this effect was more pronounced in rats exposed to a short photoperiod. Melatonin injections in testosterone-treated rats in a long photoperiod exacerbated the inhibitory effects of testosterone alone. Subcutaneous 2-weekly implants of a beeswax pellet containing 1 mg melatonin reversed the effects of the melatonin injections on relative testicular weights but not those due to short photoperiod exposure. Testosterone implants significantly reduced pituitary LH values in long and short photoperiod-exposed animals, more particularly in those exposed to short photoperiod. Melatonin injections alone or in combination with melatonin pellets did not further exaggerate the depression in pituitary LH due to testosterone alone in long photoperiod-exposed animals; similarly melatonin pellets did not reverse the depression in pituitary LH observed. No significant differences in plasma prolactin concentrations or in thyroxine concentrations or free thyroxine index were observed after any combination of treatments. We therefore suggest that the effects observed with short photoperiod may be due to melatonin.

Swimming depresses nighttime melatonin content without changing N-acetyltransferase activity in the rat pineal gland.

Recently, it was shown that a 1.5-ml subcutaneous saline injection depressed N-acetyltransferase (NAT) activity and melatonin content in the rat pineal gland at night. The present studies were undertaken to determine if another perturbation, swimming, could duplicate this response. Rats swam at 23.10 h (lights out at 20.00 h) for 10 min and were killed 15 and 30 min after the unset of swimming. Pineal NAT activity was found to be unaffected while melatonin content was depressed dramatically. Hydroxyindole-O-methyltransferase (HIOMT) activity as well as the content of serotonin (5HT), 5-hydroxytryptophan (5HTP) and 5-hydroxyindoleacetic acid (5HIAA) were not changed by this treatment. In a second study, pineal melatonin again was depressed without a concomitant drop in NAT activity. Mean serum melatonin at 15 min after onset of swimming was increased although the rise was not statistically significant. In the final study, it was found that NAT activity was slightly increased in intact rats and unchanged in adrenalectomized rats at 7 min after swimming onset. At 15 min both intact and adrenalectomized animals had NAT activity values similar to those of controls. Pineal melatonin content in intact and adrenalectomized rats plummeted to 50% of control values at 7 min and fell further to 25% at 15 min. While the rate of melatonin synthesis was not directly measured, lack of change in the activities of the enzymes involved in melatonin synthesis and the contents of two melatonin precursors suggests that swimming depresses pineal melatonin content by enhancing melatonin efflux from the gland.

Elevated ambient temperature retards the atrophic response of the neuroendocrine-reproductive axis of male Syrian hamsters to either daily afternoon melatonin injections or to short photoperiod exposure.

Adult male Syrian (golden) hamsters, maintained under either 22 +/- 2 or 32 +/- 2 degrees C, were treated with 8 or 11 weeks of exposure to either long photoperiod (14:10), short photoperiod (8:16), or to long photoperiod with a daily afternoon melatonin injection. By 8 weeks, the animals kept at 22 degrees C and treated with daily afternoon melatonin injection exhibited a dramatic reduction in testicular and accessory sex organ weight, but the animals kept at 32 degrees C and treated in the same way exhibited only slight decreases in testicular and accessory organ weights. Short photoperiod caused a slight decrease in testicular and accessory organ weights of hamster kept at 22 degrees C, while it had no significant effects on reproductive organ weights of the animals maintained under 32 degrees C. By 11 weeks, the daily afternoon melatonin injection elicited further reduction in testicular and accessory organ weights of the animals maintained under both 22 and 32 degrees C. However, the reduction in animals kept at 32 degrees C was not as great as that in animals kept at 22 degrees C. Although short photoperiod caused an obvious decline in reproductive organ weights of the animals at 22 degrees C, only a slight decrease was seen in hamsters at 32 degrees C. As with reproductive organ weights, testosterone levels were depressed more rapidly and completely in animals maintained at 22 degrees C. These results indicate that elevated ambient temperature changes the rate at which the gonads of hamsters regress in response to daily afternoon melatonin injections or short photoperiod.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine or isoproterenol stimulation of pineal N-acetyltransferase activity and melatonin content in the Syrian hamster is restricted to the second half of the daily dark phase.

Seven experiments were performed to investigate the sensitivity of the hamster pineal gland to exogenously administered norepinephrine (NE). In these studies NE (1 mg/kg) administration was preceded (10 min earlier) by the injection of the catecholamine uptake inhibitor desmethylimipramine (DMI; 5 mg/kg). When DMI and NE were given at night, the hamsters were exposed to light to depress pineal N-acetyltransferase activity and melatonin values to low levels; the drugs were then given 20 (DMI) and 30 (NE) min later, and the subsequent changes in pineal N-acetyltransferase and melatonin were monitored. The combination of DMI and NE administration anytime during the normal light period or during the first 4 h of the normal dark period failed to stimulate either pineal N-acetyltransferase activity or melatonin levels. Conversely, DMI followed by NE (injected either intraperitoneally or subcutaneously) in the second half of the dark phase typically stimulated pineal melatonin production. Likewise, the NE agonist isoproterenol promoted pineal melatonin production only in the latter half of the dark phase. If hamsters were exposed to continual light at night or if they were superior cervical ganglionectomized, a procedure which sympathetically denervates the pineal gland, the stimulatory effect of NE on melatonin production was significantly suppressed. Thus, the hamster pineal gland is sensitive to NE only during the latter half of the normal dark period and both darkness and an intact sympathetic innervation to the pineal gland are required for the gland to develop maximal sensitivity to the catecholamine. Also, the hamster pineal seems not to exhibit a supersensitivity response to NE following a period of reduced exposure to the catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Depression in rat pineal N-acetyltransferase activity and melatonin content produced by a hind leg saline injection is time and darkness dependent.

It has recently been shown that a 1.5-ml subcutaneous saline injection into the dorsal aspect of the hind limb induces a dramatic and rapid fall in N-acetyltransferase activity and melatonin content of the rat pineal gland at night. Since many studies have shown the opposite response to stress during the day, the first experiment was undertaken to test whether the timing of the saline injection at night influences the response of the pineal gland. In the present studies, rats were kept under light:dark (LD) cycles of 14:10 with lights out daily at 2000 h. Groups of rats were then given a saline injection at one of the following times: 2315, 0015, 0115, 0215, or 0315. Early in the dark phase (2315 and 0015) the saline injection depressed both the N-acetyltransferase (NAT) activity and the melatonin content of the pineal. As the animals were treated later in the dark period, the response became more blunted and, finally, disappeared. In the second experiment, animals that were kept in light during the usual dark period showed no pineal response when subjected to a hind leg injection of saline at either 2315 or 0315. Additionally, no response was seen in the two pineal parameters when rats had darkness onset delayed by 4 h (to 2400) and were then treated with saline at 0410. The results of these studies indicate that the pineal response to saline injection is time dependent. Also, if the nighttime rise in melatonin is suppressed by light exposure, a saline injection has no further inhibitory effect on pineal NAT activity or melatonin levels.

Influence of delta-sleep inducing peptide on melatonin synthesis in the rat pineal gland.

The influence of delta-sleep inducing peptide (DSIP) on rat pineal N-acetyltransferase (NAT) activity and melatonin levels was examined. Young adult rats received an injection of either saline or DSIP (either 15, 30 or 60 nmol/kg) at 20:00 h, immediately before lights out. DSIP at a dose of 15 nmol/kg significantly retarded the nighttime rise of pineal NAT activity and melatonin levels at 3 h into the dark phase. At the other two nocturnal time points (01:00 and 03:00 h) and the following morning (08:00 h) pineal NAT activity and melatonin levels were similar in all groups. The results confirmed a short-lived, dose-specific effect of DSIP on serotonin metabolism in the rat pineal gland.

Effects of monoamine neurotoxins injected in different brain areas on gonadotropin and androgen secretion in the male.

In order to investigate the relative contribution of the serotoninergic and catecholaminergic innervation of different brain structures to the control of gonadotropin secretion in the male, adult rats were given bilateral injections of the selective neurotoxins, 5,7-dihydroxytryptamine and 6-hydroxydopamine, into the mediobasal hypothalamus, the medial preoptic area, the amygdala, and the medial forebrain bundle. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone, testosterone, pituitary follicle-stimulating hormone, and LH content were measured 15 days later. The medial preoptic area 5,7-dihydroxytryptamine injections were followed by 2- to 3-fold increases of serum LH and testosterone levels and pituitary LH concentration. The 6-hydroxydopamine injections in the medial forebrain bundle resulted in a 60% decrease of pituitary LH content, associated with serum levels of LH and testosterone which were lowered, although not significantly. None of the treatments seemed to influence either serum or pituitary concentrations of follicle-stimulating hormone. These results suggest a hitherto undocumented major function of the serotoninergic innervation of the medial preoptic area in the control of LH secretion in the male and confirm previous reports indicating that some of the catecholaminergic fibers carried by the medial forebrain bundle can be modulatory for LH secretion.