Fernando Antón-Tay

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.

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

Huerto‐Delgadillo L, Antón‐Tay F, Benítez‐King G. Effects of melatonin on microtubule assembly depend on hormone concentration: rate of melatonin as a calmodulin antagonist. J. Pineal Res. 1994: 17: 55–62. ©Munksgaard, 1994

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 kinases 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.

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.

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 cytoskel‐eton 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.

Melatonin prevents cytoskeletal alterations and oxidative stress induced by okadaic acid in N1E-115 cells

Progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, both in neural aging and in neurodegenerative diseases. Cytoskeleton in neurodegenerative diseases is characterized by hyperphosphorylated tau assembled in neurofibrillary tangles. Tau protein promotes microtubule enlargement and its hyperphosphorylation inhibits tubulin assembly. Okadaic acid (OA) causes oxidative stress, tau hyperphosphorylation, and altered cytoskeletal organization similar to those observed in neurons of patients with dementia. Since melatonin acts by both enlarging microtubules and as a free-radical scavenger, in this work we studied the effects of melatonin on altered cytoskeletal organization induced by OA in N1E-115 neuroblastoma cells. Optic microscopy, morphometric analysis, and tubulin immunofluorescence staining of neuroblastoma cells incubated with 50 nM OA showed an intact microtubule network following the neurite profile similar to that observed in the vehicle-incubated cells when melatonin was added to the incubation media 2 h before OA. The melatonin effects on altered cytoskeletal organization induced by OA were dose-dependent and were not abolished by luzindole, the mt(1) melatonin antagonist receptor. Also, increased lipid peroxidation and augmented apoptosis in N1E-115 cells incubated with 50 nM OA were prevented by melatonin. The results support the hypothesis that melatonin can be useful in the treatment of neurodegenerative diseases.

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 μM) and protein kinase C inhibitors (100 μM) 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.

Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase

Abstract:  Melatonin increases neurite formation in N1E‐115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho‐associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12‐myristate 13‐acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E‐115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E‐115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

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

Antón‐Tay F, Martínez I, Tovar R, Benítez‐King G. Modulation of the subcellular distribution of calmodulin by melatonin in MDCK cells. J. Pineal Res. 1998; 24:35–42.

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 (t½= 3.04 min) a slower one (t½= 36 min). We also found a much slower component (t½= 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.