Anti-VEGF Signalling Mechanism in HUVECs by Melatonin, Serotonin, Hydroxytyrosol and Other Bioactive Compounds.

Angiogenesis drives evolution and destabilisation of atherosclerotic plaques and the growth and expansion of tumour cells. Vascular endothelial growth factor (VEGF) is the main endogenous pro-angiogenic factor in humans. The aim was to provide insight into the anti-VEGF activity of bioactive compounds derived from aromatic amino acids (serotonin, melatonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol). Experiments involved endothelial cell migration (wound-healing assay), the molecular mechanisms (ELISA assay) and the downstream effects (phospholipase C gamma 1 (PLCγ1), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) by Western blot) on human umbilical vein endothelial cells (HUVECs). The data suggest for the first time that hydroxytyrosol interacts with surface components of the endothelial cell membrane (, preventing VEGF from activating its receptor. Serotonin and 5-hydroxytryptophol significantly inhibited HUVEC migration (98% and 50%, respectively) following the same mechanism. Conversely to other bioactive compounds, the anti-angiogenic effect of melatonin, serotonin, 3-indoleacetic acid and 5-hydroxytryptophol is not mediated via PLCγ1. However, hydroxytyrosol inhibits PLCγ1 phosphorylation. Additionally, melatonin and serotonin maintained eNOS phosphorylation and hydroxytyrosol significantly activated eNOS-all via Akt. These data provide new evidence supporting the interest in melatonin, serotonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol for their further exploitation as anti-VEGF ingredients in food.

Modulation of midazolam 1-hydroxylation activity in vitro by neurotransmitters and precursors.

OBJECTIVE: The aim of this study was to find whether endogenous substances could modulate CYP3A activity. There is evidence that CYP3A, a major phase-I xenobiotic metabolizing enzyme, is present in human brain but, at the present time, endogenous substrates for such an enzyme remain to be identified. A possible linkage between the CYP2D6 enzyme and serotonergic transmission has been recently reported by our group. In the same manner, structurally related enzymes such as CYP3A could also be related to endogenous compounds. METHODS: CYP3A activity was measured using the enzyme-specific substrate midazolam in human liver microsomes. Several neurotransmitters, precursors, and their metabolites, corresponding to three different metabolic routes, were assayed as putative modulators of CYP3A enzyme activity. These comprised serotonergic, catecolaminergic, and GABAergic transmitters and precursors. The inhibitory capacity of ketoconazole, a competitive inhibitor of CYP3A, was also analyzed for comparison. RESULTS: The kinetic analysis of the midazolam 1-hydroxylase activity measured in microsomes from five human liver samples indicated Km values (mean +/- SD) of 5.8 +/- 4.9 microM, and Vmax values of 1.7 +/- 1.4 nmol min(-1) per mg microsomal protein in all the samples used in the study. Of the 14 substances analyzed, adrenaline, serotonin, and 5-hydroxytriptofol were full inhibitors of CYP3A enzyme activity (Ki values of 42.3, 26.4, and 43 microM, respectively). The remaining substances were weak inhibitors or had no inhibitory effect. CONCLUSION: Brain CYP3A activity could be modulated by some neurotransmitters and precursors.

Anxiolytic-like actions of melatonin, 5-metoxytryptophol, 5-hydroxytryptophol and benzodiazepines on a conflict procedure.

Male Wistar rats (120-230 g) were used in these experiments. Some rats were deprived of water for 48 h before testing in a conflict procedure. Then, after 20 licks from a water bottle with a metal drinking tube, the animal received an electric shock (0.5 mA/500 msec). The effects of two classical anxiolytic drugs, DIAZ (1.0, 2.0 and 4.0 mg/Kg b.wt), and CDP (5.0 and 10.0 mg/Kg b.wt) were compared to those produced by MEL (0.1, 0.2, 0.5, 1.0 and 2.0 mg/Kg b.wt), 5-MTOPHOL (1.0 and 2.0 mg/Kg b.wt), 5-HTOPHOL (1.0 and 2.0 mg/Kg b.wt) and vehicle solution. Anxiolytic drugs as well as MEL produced a dose-dependent increase in the number of shocks received. The results suggest that the three pineal indoles are involved in the modulation of the stress responses. MEL showed a higher potency than the other indoles.

Vascular responsiveness to serotonin metabolites in mineralocorticoid hypertension.

This study characterizes vascular responsiveness to serotonin and its metabolites and to several monoamines that are structurally related to serotonin in deoxycorticosterone acetate (DOCA)-salt hypertension. Mesenteric arteries from normotensive and hypertensive rats were excised and cut into helical strips for isometric force recording. Dose-response curves to serotonin in arteries from hypertensive rats were shifted significantly to the left compared with those in arteries from normotensive rats (ED25: DOCA-treated = 2.4 X 10(-8) M; control = 17.1 X 10(-8) M). Contractile responses to 5-hydroxyindole acetic acid and 5-hydroxytryptophol were greater in mesenteric arteries from hypertensive rats, whereas reactivity to 5-methoxytryptamine and melatonin in arteries from hypertensive rats did not differ from that in arteries from normotensive rats. Mesenteric arteries from both rat groups were unresponsive to the serotonin metabolite N-acetylserotonin. Contractile responses to 5,6-dihydroxytryptamine and 6-hydroxytryptamine were greater in mesenteric arteries from hypertensive rats, whereas responsiveness to 3-hydroxytryptamine in hypertensive arteries did not differ from normotensive values. Contractile responses to serotonin and its metabolites and to the structurally related monoamines were inhibited by the serotonergic antagonist ketanserin. These results demonstrate that vascular sensitivity to serotonin is increased in DOCA-hypertensive rats. Based on the experiments with serotonin metabolites and with other monoamines, the increased responsiveness to these compounds appears to be related to the structural location of hydroxyl and amine moieties.

Effect of indolamines on beta-endorphin release by rat anterior pituitary cells.

The effect of indolamine derivatives on beta-endorphin (beta-end) release has been studied in vitro using rat anterior pituitary cells. Incubation of primary cultures for 2 h with 100 nmol/l of melatonin, serotonin or 5-methoxytryptamine significantly increased the beta-end release in response to 20 nmol/l of ovine corticotropin-releasing factor (oCRF). Incubation of the cultures with 100 nmol/l of L-tryptophan, 5-hydroxy-L-tryptophan, 5-hydroxytryptophol or 5-methoxytryptophol had no effect on basal or CRF-induced beta-end release. The effect of serotonin and melatonin was further tested in a superfusion system of dispersed rat anterior pituitary cells. Superfusion with oCRF (200 nmol/l) for 4 min elicited an immediate rapid increase in beta-end release which lasted 30-40 min. Simultaneous superfusion with melatonin (1 mumol/l) or serotonin (1 mumol/l) significantly increased the effect of oCRF pulses on beta-end release. We conclude that melatonin and serotonin are able to act directly on anterior pituitary cells to potentiate the effect of oCRF on beta-end release.

Electrical responses of pineal cells to pineal indoles and putative transmitters in intact and blinded pigeons.

The effects of micro-electrophoretically applied pineal indoles, melatonin (MEL), 5-methoxytryptophol (MTL) and 5-hydroxytryptophol (HTL) and of the neurotransmitters, noradrenaline (NOR), acetylcholine (ACH) and gamma-aminobutyric acid (GABA) on the electrical activity of pineal cells were studied in anaesthetized pigeons. Recordings were made from both sighted and blinded birds during both the day- and night-time. Both excitatory and inhibitory responses to the application of all substances tested were observed, while in all cases unresponsive cells were also found. The pattern of responses to MEL, MTL and to NOR varied significantly depending on whether the cells in the sighted pigeons were tested during the day or at night. This day/night rhythm of responses to MEL and to MTL also occurred in the blinded pigeons, although this was not the case for the effects of NOR. Units which were orthodromically excited by electrical stimulation of the habenular complex were also excited by application of GABA. The activity of these units was also likely to be enhanced by application of ACH. The results suggest that the pigeon pineal organ exhibits intrinsic circadian rhythmicity which does not require entrainment signals from the retina. They also indicate that the activity of pineal cells could be influenced by neurotransmitter input provided from within the gland or via its innervation.

Alterations in the spontaneous activity of cells in the guinea pig pineal gland and visual system produced by pineal indoles.

The indoles serotonin (SER), melatonin (MEL), 5-methoxytryptophol (5-MTL) and 5-hydroxytryptophol (5-HTL) were administered during daytime microelectrophoretically to 240 cells in the pineal gland of the guniea-pig. The action of SER and 5-HTL was predominantly depressant on the electrical activity, MEL and 5-MTL caused an excitation in most of the units. Although MEL and 5-MTL caused fairly similar reactions on average, they appear to act on different cells. The effects of microelectrophoretically applied MEL and 5-MTL on the spontaneous or evoked activity in the visual system (retinal ganglion cells, optic tract, lateral lateral geniculate body, superior colliculus) of the guinea-pig were also investigated. Of the 76 cells tested in the visual system 25 of the ON- and OFF-cells increased the rate of discharge when the two indoles were applied. Cells in the optic layer of the superior colliculus showed no measurable response to the application of the two substances. 5-HTL caused no effect on cells in the visual system.

Effects of 5-hydroxytryptophol, a 5-hydroxytryptamine metabolite, on isolated cerebral arteries of the dog.

5-Hydroxytryptophol (5-HTOL) caused a dose-dependent contraction of helically-cut strips of dog cerebral arteries. The 5-HTOL-induced contraction was suppressed by cinanserin, as was the contraction induced by 5-hydroxytryptamine (5-HT). Treatment with 5-HTOL shifted the dose-response curve for 5-HT to the right and downward in a dose-dependent manner, but did not attenuate the contractile response to prostaglandin F2a. It may be concluded that 5-HTOL elicits cerebroarterial contractions by activating tryptaminergic receptors and also interferes with the action of 5-HT on the receptors.

Changes in rodent thyroid hormones and cyclic-AMP following treatment with pineal indolic compounds.

Treatment of rats with pineal indolic compounds 5-methoxytryptophol, 5-hydroxytryptophol and serotonin brought about a significant increase in serum thyroxine levels, while serotonin and melatonin caused an increase in thyroid cAMP content with corresponding decrease in the gland's hormones. The total quantity of cAMP in the thyroid was also increased by melatonin in the organ culture system. All these findings would indicate that some of the pineal indoleamines elicit a direct action on the thyroid by stimulating the adenyl cyclase activity and intrathyroidal cAMP, bringing about increased release of thyroxine into the blood stream, and that this is usually not accompanied by adequate synthesis in the gland. Our observation that continuous darkness, which stimulates pineal activity, also brought about an increase in cAMP, concours with our finding of a stimulatory effect of the indolic compounds on thyroid hormone release.