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Biomedical subjects

F Antón-Tay

Publications and source records attributed to F Antón-Tay.

At least 19 recordsLinked to original sources
PubMed1998

In vitro stimulation of protein kinase C by melatonin.

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.

Animals↗
PubMed1998

Modulation of the subcellular distribution of calmodulin by melatonin in MDCK cells.

In this paper we describe the interaction of melatonin with calmodulin in MDCK cells. The double staining immunofluorescent method showed that calmodulin in control MDCK cells appeared as fluorescent spots at the cell periphery. In contrast, MDCK cells cultured with 10(-9) M melatonin for 6, 12, 24 hr or for 4 days showed that calmodulin fluorescent spots were distributed throughout the cytoplasm as well as in the nucleus. These calmodulin rearrangements were reversed 6 hr after melatonin withdrawal. Moreover, calmodulin radioimmunoassay showed that in 10(-9) M melatonin-treated cells, membrane-bound calmodulin content was increased by 78% whereas cytosolic calmodulin decreased by 60%. Simultaneous labeling of MDCK cells with specific antibodies against calmodulin and melatonin showed that not only does the indole enter the cell, but that it has the same subcellular distribution as calmodulin. Besides the early responses induced by the melatonin antagonism to calmodulin, the results suggest that the indole may also induce long-term cellular responses by changing calmodulin concentrations in specific cellular compartments.

Animals↗
PubMed1996

In vitro inhibition of Ca2+/calmodulin-dependent kinase II activity by melatonin.

Recent evidence suggests that a melatonin (MEL) mechanism of action may be through modulation of Ca2+-activated calmodulin (CaM). MEL binds to CaM with a high affinity, and has been shown to act as a CaM antagonist. Among the CaM-dependent enzymes, Ca2+/Calmodulin-dependent protein kinase II (CaM-kinase II) is a particularly abundant enzyme in the nervous system. In the brain it phosphorylates a broad spectrum of substrates, thus modulating important neuronal functions. We describe the MEL effect on CaM-kinase II activity in vitro. CaM-kinase II was purified from rat brain by column chromatography, and identified by Western immunoblotting. CaM-kinase II activity was assessed in the presence of Ca2+/CaM by the kinase's ability to phosphorylate the synthetic substrate syntide-2 and by enzyme autophosphorylation. MEL inhibited CaM-kinase II activity, and enzyme autophosphorylation. Inhibition of the enzyme by 10(-9) M MEL was nearly of 30%. Trifluoperazine (10 microM), W7 (10 microM), and compound 48/80 (30 micrograms/ml), inhibited CaM-kinase II activity by 40%, 42%, and 93%, respectively. Both EGTA (5 mM) and MEL (10(-5) M) abolished autophosphorylation. The effect of MEL on CaM-kinase II activity was specific, since neither serotonin, N-acetylserotonin, nor 6-hydroxymelatonin inhibited its activity. Our results support the hypothesis that MEL acts as a CaM antagonist and cellular functions may be rhythmically regulated by MEL modulation of CaM-dependent protein phosphorylation.

Animals↗
PubMed1994

Effects of melatonin on microtubule assembly depend on hormone concentration: role of melatonin as a calmodulin antagonist.

Melatonin may play a key role in cytoskeletal rearrangements through its calmodulin antagonism. In the present work, we tested this hypothesis by studying melatonin effects on both microtubule polymerization in vitro and cytoskeletons in situ. Microtubule assembly is a dynamic process inhibited by Ca2+/calmodulin. Calmodulin antagonists prevent the inhibition by binding to Ca(2+)-activated calmodulin, thus causing microtubule enlargement. In the presence of calmodulin (5 microM) and CaCl2 (1 mM), polymerization at equilibrium was inhibited by 40%. Complete reversal of the Ca2+/calmodulin effect on microtubules was observed with 10(-9) M melatonin or with 10(-5) M trifluoperazine or 1 microgram/ml of compound 48/80. In the absence of Ca2+/calmodulin, melatonin at 10(-5) M inhibited tubulin polymerization like 10(-4) M trifluoperazine does. Melatonin effects on microtubule assembly at both nanomolar and micromolar ranges were corroborated in cytoskeletons in situ. Therefore, it is suggested that at a low concentration (10(-9) M), cytoskeletal melatonin effects are mediated by its antagonism to Ca2+/calmodulin. At a higher concentration (10(-5) M), non-specific binding of melatonin to tubulin occurs, thus overcoming the melatonin antagonism to Ca2+/calmodulin. The results support the hypothesis that under physiological conditions, melatonin synchronizes different body rhythms through cytoskeletal rearrangements mediated by its calmodulin antagonism.

Animals↗
PubMed1993

Calmodulin mediates melatonin cytoskeletal effects.

In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10(-9) M) cytoskeletal effects are mediated by its antagonism to Ca2+-calmodulin. At higher concentrations (10(-5)M) non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca2+-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.

Animals↗
PubMed1993

Binding of 3H-melatonin to calmodulin.

Studies in melatonin mechanism of action have suggested that one of them could be the binding of the hormone to calmodulin. We assessed calmodulin-melatonin binding by combining liposome incorporation of calmodulin with separation of free and bound 3H-Melatonin by a rapid ultrafiltration method. Specific binding to calmodulin was saturable, reversible, Ca(++)-dependent, ligand selective, and showed high affinity. Saturation as well as association-dissociation studies revealed that 3H-Melatonin binds to a single site on the calmodulin molecule with a Kd of 188 pM and a total binding capacity Bmax of 35 pM/ug of calmodulin. Displacement experiments showed that the relative order of potency of some compounds for inhibition of 3H-Melatonin was as follows: Melatonin > 6-chloromelatonin > 6-hydroxymelatonin > luzindole > trifluoperazine. The results explain our previously reported melatonin effects such as cytoskeletal rearrangements, inhibition of calmodulin dependent phosphodiesterase activity as well as the modification of Ca(++)-calmodulin electrophoretic mobility. The high affinity of melatonin binding to calmodulin suggests that the hormone is able to modulate cell activity by intracellularly binding to calmodulin at physiologically ranges. Melatonin-calmodulin binding could modulate many intracellular Ca++ functions and thus, the set-point for cell activity will follow the rhythmic circulating levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well preserved compounds, their interaction may represent a primary mechanism for both the regulation and the synchronization of cell physiology.

Animals↗
PubMed1991

Melatonin modifies calmodulin cell levels in MDCK and N1E-115 cell lines and inhibits phosphodiesterase activity in vitro.

The interaction between melatonin and calmodulin was explored. Calmodulin cell levels in MDCK and N1E-115 cells cultured with 10(-9) M melatonin were increased after 3 days but decreased after 6 days. Melatonin inhibited calmodulin-dependent phosphodiesterase and when either melatonin or [3H]melatonin was preincubated with calmodulin and separated by electrophoresis, comigration of calmodulin with the radioactivity as well as modification of the Ca2+ calmodulin shift were observed. The results point out that one of the mechanisms of action of melatonin is a calmodulin-melatonin interaction.

Animals↗
PubMed1990

Melatonin effects on the cytoskeletal organization of MDCK and neuroblastoma N1E-115 cells.

Despite the fact that many physiological and pharmacological actions of melatonin (MEL) have been described, its mechanism of action at the subcellular level remains unclear. It has been suggested that MEL has effects on cellular processes that involve microfilaments and microtubules. In the present study MEL effects on the cytoskeleton were evaluated in MDCK and N1E-115 cells in which the microfilaments have been shown to participate in cell morphology and dome formation (MDCK) and the microtubules in neurite outgrowths. After one day of culture with 10(-11)-10(-7) M MEL MDCK cells showed an increase in the number of elongated cells. After four days with the hormone, an increase in the incidence of MDCK cells contacting neighboring cells through long cytoplasmic elongations was observed. Actin antibody stain showed the appearance of thicker fluorescent fibres beneath the cell membrane and over the nucleus in the MEL treated cells. An increase in dome formation in confluent cells was also observed. In N1E-115 cells MEL (10(-13)-10(-5) M) induced an increase in cell with neurite processes. Neurite outgrowth is clearly seen at 24 h after plating. MEL-treated cells grow in clusters with neurites forming intricate networks. Antitubulin antibody stain showed long fluorescent neurites in the N1E-115 MEL-treated cells. A decrease in N1E-115 neurite formation was observed with either serotonin or 6-hydroxymelatonin (6OH-MEL). However, the number of MDCK cells with cytoplasmic elongations was decreased only after 6OH-MEL. We conclude that MEL action at the cellular level involves a modification of the cytoskeletal organization.

Actin Cytoskeleton↗
PubMed1988

Subneuronal fate of intracerebroventricular injected 3H-melatonin.

The fate of 3H-melatonin after its intracerebroventricular administration was studied both in different brain regions and in subcellular fractions. The rate of disappearance of 3H-melatonin from the brain was found to be multiphasic. Forty-eight h after a 3H-melatonin injection, radioactivity was still present in the brain. Nonlinear regression analysis of the data confirmed a very rapid half-life component and (t1/2 = 3.04 min) a slower one (t1/2 = 36 min). We also found a much slower component (t1/2 = 24 h), however. Considerable metabolism of melatonin was detected since only 36.5% of administered radioactivity remained as melatonin at 45 min. The subcellular distribution of the radioactivity present in the brain at all times studied showed that a major proportion of the radioactivity remained in the cytosol and respectively decreasing proportions in the 900g pellet, mitochondrial pellet, and the microsomes. The radioactivity remaining in the cytosol at 45 min was found to coelute with a macromolecule that was resolved by gel filtration and could be displaced by previous melatonin administration. Purified nuclei retained 0.71% of the radioactivity at 45 min; of this total, 73% was KCl extractable. Our data suggest the presence of a binding site in the cytosol and in the nucleus. The presence of 3H-melatonin up to 48 h after its administration may account for melatonin's long-term effects on brain function.

Animals↗