MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

Local corticosterone infusion enhances nocturnal pineal melatonin production in vivo.

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-alpha decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N-acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions.

BACKGROUND AND PURPOSE: We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells. EXPERIMENTAL APPROACH: Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA. KEY RESULTS: RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats. CONCLUSIONS AND IMPLICATIONS: Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro.

BACKGROUND AND PURPOSE: We have previously shown that melatonin inhibits bradykinin-induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin. EXPERIMENTAL APPROACH: RT-PCR assays were performed with rat cultured endothelial cells. The putative effect of melatonin upon arteriolar tone was investigated by intravital microscopy while NO production by endothelial cells in vitro was assayed by fluorimetry, and intracellular Ca(2+) measurements were assayed by confocal microscopy. KEY RESULTS: No expression of the mRNA for the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase, or for the melatonin MT(2) receptor was detected in microvascular endothelial cells. Melatonin fully inhibited L-NAME-sensitive bradykinin-induced vasodilation and also inhibited NO production induced by histamine, carbachol and 2-methylthio ATP, but did not inhibit NO production induced by ATP or alpha, beta-methylene ATP. None of its inhibitory effects was prevented by the melatonin receptor antagonist, luzindole. In nominally Ca(2+)-free solution, melatonin reduced intracellular Ca(2+) mobilization induced by bradykinin (40%) and 2-methylthio ATP (62%) but not Ca(2+) mobilization induced by ATP. CONCLUSIONS AND IMPLICATIONS: We have confirmed that melatonin inhibited NO production both in vivo and in vitro. In addition, the melatonin effect was selective for some G protein-coupled receptors and most probably reflects an inhibition of Ca(2+) mobilization from intracellular stores.

Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

Release of [(3)H]-L-glutamate by stimulation of nicotinic acetylcholine receptors in rat cerebellar slices.

This is a neurochemical study which shows that nicotine acting through alpha7-containing nicotinic acetylcholine receptors promotes the release of [(3)H]-glutamate from rat cerebellar slices. Release evoked by half maximal concentration of nicotine (100 microM) was blocked by alpha-bungarotoxin and in a calcium-free medium, suggesting an effect mediated by an alpha7 receptor. Dihydro-beta-erythroidine and mecamylamine were effective only at very high concentrations, excluding the participation of heteromeric receptors. The effect of nicotine was partially blocked by inhibitors of glutamatergic receptors DL-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating a glutamate-induced glutamate release. Nicotine-evoked response was dependent on activation of tetrodotoxin sensitive sodium channels. Therefore, here we show that glutamate released by stimulation of alpha7-containing nicotinic receptors, located preterminal and/or postsynaptically, evokes a further glutamate release in adult rat cerebellar slices.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 æM melatonin and 0.1 æM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia)

Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 + or - 0.12 and maximal response = 2.14 + or - 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [³H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 + or - 0.06 and Emax = 4.16 + or - 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 æM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [³H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 æM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively

Characterisation of P2Y(1)-like receptor in cultured rat pineal glands.

The rat pineal gland possesses P2 receptors which potentiate the effect of noradrenaline-induced N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) production. In the current study, this receptor was characterised according to agonist selectivity and signal transduction mechanisms. 2-MethylthioATP (2MeSATP), 2-chloroATP (2-ClATP), adenosine 5'-O-2-thiodiphosphate, (ADPbetaS), ATP and ADP, but not UTP, potentiated noradrenaline-induced N'-acetyl-5-HT production in a concentration-dependent manner. 2MeSATP neither induced the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP), nor inhibited its formation when the glands were stimulated by forskolin. The phospholipase C inhibitor 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), but not the inactive analogue, 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione (U73343), blocked the 2MeSATP effect. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-dissulphonic acid (PPADS), which inhibits phospholipase C-coupled P2Y(1) receptors, blocked the 2MeSATP effect. In conclusion, our data strongly suggest that the P2-like receptor that is present in rat pinealocytes and which is responsible for the potentiation of noradrenaline-induced N'-acetyl-5-HT production is a P2Y(1)-like receptor, coupled to a G protein which stimulates phospholipase C.

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca.

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.

Purinergic and noradrenergic cotransmission in the rat pineal gland.

ATP is coreleased with noradrenaline in several noradrenergic synapses, and P2-like receptors were shown to be present in rat pineal glands. A new method of functional investigation was developed to assess the importance of both transmitters (noradrenaline and ATP) in eliciting the synthesis of melatonin and its precursor N'-acetyl-5-hydroxytryptamine (N'-acetyl-5-HT) through transmural electrical field stimulation of cultured pineal glands. Incubation with the beta-adrenoceptor antagonist propranolol (>10(-7) M) blocked almost completely the production of N'-acetyl-5-HT, whilst the P2 receptor antagonists pyridoxalphosphate-6 azophenyl-2',4'-disulfonic acid (PPADS, >3x10(-6) M) and suramin (>10(-6) M) blocked it partially. These findings indicate a physiologically relevant role for the purinergic cotransmission in this system.

Adenosine inhibits the renal plasma-membrane (Ca2+ + Mg2+)-ATPase through a pathway sensitive to cholera toxin and sphingosine.

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)-ATPase activity. Inhibition occurs up to 0.1 microM and then gradually disappears as the adenosine concentration increases to 100 microM, an effect mimicked by the adenosine analog N6-cyclohexyladenosine, which preferentially binds to A1-type receptors. In contrast, the A2 receptor agonist 5', N-ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocks the inhibitory effect of 0.1 microM adenosine and stimulates (Ca2+ + Mg2+)-ATPase activity in the presence of 1 mM adenosine, a concentration high enough to occupy the low-affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 microM cholera toxin or 1 microM sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5'-[gamma-thio]triphosphate, inositol 1,4, 5-trisphosphate or the diacylglycerol analog 12-O-tetradecanoylphorbol 13-acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G-proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)-ATPase resident in basolateral membranes of kidney proximal tubules.

Melatonin modulation of presynaptic nicotinic acetylcholine receptors located on short noradrenergic neurons of the rat vas deferens: a pharmacological characterization

Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP) = cytisine > nicotine > carbachol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response during the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllycaconitine inhibited the nicotine-induced response only at 21:00 h. Since = 7 nicotinic acetylcholine receptors (nAChRs) have low affinity for nicotine in binding assays, we suggest that a mixed population composed of alpha 3 Beta 4 - plus alpha7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h) is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity binding site, probably an alpha nAChR subtype

Circadian rhythm in experimental granulomatous inflammation is modulated by melatonin.

Biological rhythms are detected in a variety of physiological and pathological conditions in man and animals, such as rheumatoid arthritis and asthma. Here we describe a circadian rhythm in experimental infectious and non-infectious granuloma. After 30 days of BCG (Bacillus Calmette-Guerin) or nystatin inoculation in the left hind foot of C57B1/6 mice, there is an oscillation with a period of approximately 24 hr in the variation of paw thickness, indicating a circadian rhythm. The acrophase occurred during the light phase, between 9:00 and 13:00 hr, while the nadir occurred in the dark phase, between 21:00 and 01:00 hr. The vascular permeability around the granulomatous lesions was higher at 12:00 hr than at 24:00 hr. This is in agreement with the observation that the thickness of a paw with granulomatous lesion is larger during the light phase. This rhythmic variation was eliminated by either pinealectomy or superior cervical ganglionectomy, which greatly reduce melatonin levels in the blood. Nocturnal replacement of melatonin in pinealectomized mice led to the re-establishment of the circadian rhythm. Thus, the rhythm of the granulomatous lesion is due to the rhythmic melatonin release by the pineal gland. This approach opens new questions regarding the modulation of chronic inflammation in inflammatory diseases that present rhythmic symptoms throughout the day.

Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens.

Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on the sympathetic nerve terminals of the rat vas deferens. The pD2 values for nicotine were significantly higher at night (4.20 +/- 0.01) than in the afternoon (3.80 +/- 0.07). Exogenous melatonin shifted the concentration-response curves for nicotine to the left, mimicking the effect of darkness. Melatonin modifies both the displacement and the saturation curves of [3H](-)nicotine binding. In membranes from animals killed at 15:00 h, the binding of [3H](-)nicotine (5-6 nM) was first potentiated and then inhibited by sequential concentrations of (-)nicotine. Higher concentrations of [3H](-)nicotine (50-60 nM) were displaced by all concentrations of nonlabeled ligand. However, when membranes from tissues exposed to melatonin (exogenous or endogenous) were tested, the lower [3H](-)nicotine concentration was displaced progressively by increasing concentrations of nonlabeled ligand. Equilibrium binding studies show a single class of high-affinity nicotinic binding sites with an apparent Kd value of 16 nM and an average maximal number of binding sites of 66 fmol mg-1 protein when animals were killed at the afternoon. Melatonin, although it did not change the properties of high-affinity binding sites, induced the appearance of a second population of lower apparent affinity (Kd = 36.7 nM; Bmax = 185.4 fmol/mg). Melatonin does not modify the functional response and the displacement of [3H](-)nicotine by dimethylphenylpiperazinium. The data suggest that nicotinic neuronal acetylcholine receptors stimulated by dimethylphenylpiperazinium do not change between the light and dark phases. Rather, the higher sensitivity to nicotine in prostatic portions incubated with exogenous melatonin, and in organs from animals killed at night, after the rise of endogenous melatonin, is probably due to the appearance of low-affinity neuronal nicotinic ACh binding sites.

Presence of P2-purinoceptors in the rat pineal gland.

1. The effects of noradrenaline, ATP, adenylyl-imidodiphosphate (AMP-PNP), adenosine, alpha,beta-methylene-ATP and the P2-purinoceptor antagonist, suramin on N'-acetyl-5-hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2. Noradrenaline (3 nM-1 microM) increased N'-acetyl-5-hydroxytryptamine production as measured both in the gland and the culture medium. 3. In noradrenaline (10 nM)-stimulated pineal glands, ATP (0.03 nM-1 mM) or AMP-PNP (0.1 microM-1 mM) increased N'-acetyl-5-hydroxytryptamine production in a concentration-dependent manner. 4. Alpha,beta-Methylene-ATP at the concentration of 0.1 mM, but not 3 microM, attenuated the enhancement by ATP (0.1 mM) of noradrenaline (10 nM)-induced N'-acetyl-5-hydroxytryptamine production. 5. Suramin (0.1 mM) blocked the potentiating effect of ATP (0.1 mM), but not the potentiating effect of adenosine (0.1 mM) in glands incubated with noradrenaline (10 nM). 6. These findings suggest that the rat pineal gland possesses P2-purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N'-acetyl-5-hydroxytryptamine production.

Modulation of sympathetic neurotransmission by melatonin.

Melatonin is of considerable interest for its regulatory influence on a variety of physiological processes including biological rhythms and neuroendocrine functions. We showed that melatonin potentiates sympathetic neurotransmission in the prostatic portion of the rat vas deferens, by increasing contractions in response to noradrenaline and ATP released by acetylcholine stimulation of presynaptic nicotinic receptors. Melatonin in vitro (100 pg/ml; for 4 h) increased the maximal acetylcholine-induced contraction only when the hypogastric ganglion was present, and this effect was blocked by cycloheximide (100 micrograms/ml). Melatonin also modulated the sympathetic trophic influence on smooth muscle, since it reduced [35S]methionine incorporation into the vas deferens in the hypogastric ganglion-vas deferens preparation. Thus, it is suggested that the regulation of protein synthesis might be one of the mechanisms whereby melatonin modulates endogenous rhythms and synchronizes them to the environmental light cycle.

Age-related changes in norepinephrine release and its modulation by presynaptic alpha-2 adrenoceptors in the rat vas deferens.

Our objective was to extend the study of age-related changes in the modulation of norepinephrine release by presynaptic alpha-2 adrenoceptors. The experimental model utilized was the epididymal and prostatic portion of the rat vas deferens, because the two portions differ in age-related changes and pattern of sympathetic neurotransmission. The functional influence of presynaptic alpha-2 adrenoceptors was evaluated by quantifying the potentiating effect of yohimbine on the contraction induced by transmural stimulation. Yohimbine-induced potentiation was much more pronounced in the epididymal portion of young rats and was reduced by aging only in this portion. Potassium-induced release of 3H-norepinephrine was significantly higher in the epididymal than in the prostatic portion, and an age-related reduction in 3H-norepinephrine release occurred only in the epididymal portion. However, aging did not change the inhibitory effect of clonidine on the contraction induced by transmural stimulation (0.2 Hz or single pulse) in either portion. Thus the age-related decline in alpha-2 feedback regulation is probably not directly related to changes in prejunctional alpha-2 adrenoceptor activity but may be associated with age-related changes in the amount of norepinephrine that stimulates this receptor. However, the lower overflow of neurotransmitter in the epididymal portion did not translate into age-related changes in the contractile responses to transmural stimulation (2-20 Hz). In the prostatic portion, aging increased the functional neurotransmission, partially because of a reduction in neuronal norepinephrine uptake. The results suggest that age-related changes are specific for each portion and may be associated with the pattern of neurotransmission of the tissue.

Age-related changes in melatonin modulation of sympathetic neurotransmission.

An age-related reduction in the maximal acetylcholine (ACh)-induced contraction in the prostatic portion of rat vas deferens during the day was observed. This contraction is due to the release of norepinephrine and adenosine triphosphate from sympathetic nerve terminals. Male Wistar rats (4 and 24 months-old) were housed on a light/dark cycle (12:00 h/12:00 h, lights on at 6:00). The diurnal variation of ACh-induced contraction was determined on animals sacrificed every 3 h during the day. Aging reduced the maximal contraction and shortened the length of the nocturnal response. In both young and old rats, the response to ACh increased at 21:00, but it remained elevated until 12:00 in young rats and until 24:00 in old rats. Both nocturnal administration of melatonin for 2 consecutive days (9 mg/kg/day, s.c.) and melatonin incubation (100 pg/ml, 4 h) of the vas deferens from old rats sacrificed at 15:00 h significantly potentiated the ACh-induced contraction. However, this potentiation was smaller than that observed in young rats. The results presented here show the loss of maximal contractile response to ACh with age and a qualitative change in the rhythm characteristics of this phenomenon. Moreover, the age-related decrease in the ACh-induced contraction in the prostatic portion of the rat vas deferens is dependent not only on a reduction in the plasma nocturnal increase of melatonin concentration but also on a lower responsiveness of short sympathetic neurons to melatonin.

Presynaptic modulation by melatonin of the nicotine-induced calcium-dependent release of norepinephrine from rat vas deferens.

This study demonstrates that melatonin potentiates the nicotine-induced calcium-dependent release of [3H]-norepinephrine from the rat vas deferens. Slices of the prostatic portion of the rat vas deferens were labelled in vitro with [3H]-norepinephrine and superfused with physiological solution. Nicotine (1 mM, 4 min) induced a calcium-dependent release of norepinephrine during the first (N1 = 1.43 +/- 0.16%) and the second (N2 = 1.11 +/- 0.2%, n = 22) nicotine stimulations. The ratio N2/N1 between two consecutive periods of nicotine stimulations was: 0.84 +/- 0.09 (n = 22). Melatonin (10-300 nM) did not modify the spontaneous release of [3H]-norepinephrine but potentiated in a concentration-dependent manner the calcium-dependent release. The competitive melatonin receptor antagonist luzindole did not modify the calcium-dependent release of [3H]-norepinephrine when added alone but completely antagonized the potentiation of release elicited by melatonin, suggesting interaction at the level of a melatonin receptor. We conclude that melatonin potentiates the nicotine-evoked release of [3H]-norepinephrine in the rat vas deferens through activation of melatonin presynaptic heteroreceptors on noradrenergic nerves involved in a positive feedback mechanism. This mechanism may mediate the increase in sympathetic neurotransmission observed with melatonin in the vas deferens. We cannot exclude, however, an effect of melatonin on cellular proteins and enzymes associated with the exocytotic process, which directly or indirectly may lead to the facilitation of release.