Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation

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Abstract

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B4 induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT2 and MT3 melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

Introduction

Many biological processes follow diurnal rhythms related to the light–dark cycle. The mice paw thickness during chronic granulomatous inflammation shows a 24-h rhythm with minimal responses during the daily dark period (Lopes et al., 1997). Recently, we demonstrated that this rhythmic response is regulated by melatonin (Lopes et al., 2001), as the diurnal rhythm of inflammation is abolished by pinealectomy and restored by nocturnal replacement of melatonin to pinealectomized animals. Diurnal rhythms are also described in the acute inflammatory responses. Paw edema (Loubaris et al., 1982) and polymorphonuclear leukocytes migration induced by carrageenin (Garrelly et al., 1991) or Bacillus Calmette-Guerin (BCG) (Bureau et al., 1986) varies according to the hour of stimulus administration. Stimulus applied during the dark phase induced a lower inflammatory response than when applied during the light phase. A possible relationship between such rhythms and the nocturnal surge of melatonin hormone has not being described.

The protective effects of melatonin observed in local inflammation Cuzzocrea et al., 1997, Costantino et al., 1998, experimental endotoxic (Maestroni, 1996) or non-septic shock (Cuzzocrea et al., 1998), and ischemia–reperfusion injury in hamster cheek pouch (Bertuglia et al., 1996) were attributed to the putative antioxidant and radical scavenger action of melatonin. The physiological role of endogenous melatonin in these described models has been questioned, as the doses of exogenous melatonin required for inhibition of these responses was higher than the nocturnal maximal serum concentration. However, our recent data demonstrating that melatonin mediates the diurnal rhythm of chronic granulomatous inflammation (Lopes et al., 2001) suggests that doses of melatonin lower than those described as scavengers could modulate inflammatory responses. The effects of melatonin at low doses could be better explained by an action at membrane receptors.

In mammals, melatonin activates at least three distinct high-affinity receptors, the MT1, MT2 and MT3. The melatonin MT1 and MT2 membrane-bound receptors show 60% homology at the amino acid level and are encoded by separate gene. These two melatonin receptors can be distinguished pharmacologically both in vivo and in vitro using the selective MT2 melatonin ligand 4-phenyl-2-propionamidotetralin (4P-PDOT) Dubocovich, 1995, Dubocovich et al., 1997, Dubocovich et al., 1998, Dubocovich and Massana, 1998. Acute inhibition of neuronal firing in the mouse suprachiasmatic nucleus (Liu et al., 1997) and arterial vasoconstriction in the rat Krause et al., 1995, Doolen et al., 1998, Bucher et al., 1999, Ting et al., 1997, Ting et al., 1999 appear to be mediated through activation of the MT1 melatonin receptor. By contrast, activation of the MT2 melatonin receptor by melatonin inhibits dopamine release in retina (Dubocovich et al., 1997) and phase advances circadian rhythms of wheel running activity in the C3H/HeN mouse (Dubocovich et al., 1998).

The putative MT3 melatonin receptor binds to 2-[125I]-iodomelatonin and to the MT3 selective radioligand 2-[125I]-5-methoxycarbonylamino-N-acetyl-tryptamine (2-[125I]-5-MCA-NAT) with nanomolar affinity and shows a pharmacological profile (2-iodomelatonin>prazosin>N-acetylserotonin≥melatonin) distinct from that of the high affinity sites (MT1 and MT2) where melatonin has significantly higher affinity than N-acetylserotonin Dubocovich, 1988, Dubocovich, 1995, Pickering and Niles, 1992, Molinari et al., 1996, Le Gouic et al., 1997. Recently, the MT3 binding sites were suggested to be the enzyme quinone reductase 2 Nosjean et al., 2000, Nosjean et al., 2001, which is a soluble flavoprotein that catalyzes the oxidation of reduced ribosyl nicotinamide by vitamin K3. A protein purified by affinity chromatography using 2-[125I]-5-MCA-NAT and pharmacologically characterized from Syrian hamster kidney was found to be homologous of the human quinone reductase 2. The physiological importance of this enzyme is unknown. Therefore, it is not known if the MT3 is a classical membrane receptor or is an intracellular binding site, but it is accepted that it has a distinct pharmacological profile, being the only with high affinity to N-acetylserotonin when compared to melatonin and the only one responsive to 5-MCA-NAT.

In this work we intend to define the effects of physiological concentrations of melatonin on the first steps of acute inflammation. Furthermore, we show the pharmacologically profile of the receptors involved.

Initial inflammatory responses involve three major changes in the microcirculation. First, a transient vasoconstriction of arterioles followed by a vasodilation leads to an increase in local blood flow. Then, an increase in vascular permeability causes a slowing of the circulation. These first two events allow leukocytes, mainly neutrophils, that usually travel at the middle of the vessels, to marginate entering in contact with endothelium and initiating leukocyte recruitment.

Vascular reactivity is affected by melatonin. Melatonin enhances vascular tone in both peripheral and cerebral arteries Ting et al., 1997, Geary et al., 1995 and potentiates neurogenic induced contraction in rat caudal artery Krause et al., 1995, Geary et al., 1998, Lew and Flanders, 1999. Therefore, a potential mechanism by which melatonin may inhibit inflammatory responses is by decreasing blood flow due to arteriolar contraction.

Another possibility is that melatonin could modulate acute inflammatory responses by interfering with leukocyte recruitment. Migration of leukocytes to the adjacent tissue shows diurnal variation Garrelly et al., 1991, Bureau et al., 1986. Migration of leukocytes from the vessels to the tissue occurs mainly at postcapillary venules as a result of interactions between leukocytes and endothelium (Granger and Kubes, 1994). As leukocytes marginate, they initiate a process called rolling, which is described as a low-affinity adhesive interaction with the endothelium whereby the force of blood flow acts on the leukocyte to induce a rotational motion. A class of adhesion molecules named selectin (Granger and Kubes, 1994) mediates leukocyte rolling. A stronger interaction of leukocytes to endothelial cells that precede transmigration is called adhesion, which is mediated by another class of adhesion molecules (integrin and immunoglobulin) (Granger and Kubes, 1994). Integrins are constitutively expressed in leukocytes in a low affinity state. changing toward a high affinity state in response to mediators such as leukotriene B4 (Dahlen et al., 1981).

The aim of this study was to investigate if melatonin and its precursor N-acetylserotonin could play a direct role on arteriolar contraction, blood flow, leukocyte rolling and adhesion in rat microcirculation. We demonstrated that activation of melatonin receptors by melatonin inhibited both leukocyte rolling and adhesion, and by N-acetylserotonin inhibited only leukocyte adhesion. The effects herein described were obtained with doses of melatonin and N-acetylserotonin that are in the range of the nocturnal surge, providing the first evidence of a possible physiological role of these hormones in acute inflammation.

Section snippets

Melatonin receptor nomenclature and classification

Here, we use the official nomenclature for melatonin receptors approved by the Nomenclature Committee of the International Union of Pharmacology (IUPHAR) Dubocovich et al., 1998, Dubocovich et al., 2000. The designation “mt1” and “mt2” corresponds to that of the recombinant melatonin receptors previously known as Mel1a (Reppert et al., 1994) and Mel1b Reppert et al., 1995, Reppert et al., 1996, respectively. These receptors should be referred in upper case, i.e., MT1 and MT2, to reflect the

Arteriolar diameter and blood flow rate

Noradrenaline (1 μM) reduced arteriolar diameter but did not affect blood flow. Melatonin (10 nM) alone or added together with noradrenaline did not change arteriolar diameter or venular blood flow rate (Table 1).

Leukocyte rolling

Leukocyte rolling in a selected postcapillary venule measured 10 min before drug administration was taken as basal value. Melatonin inhibited leukocyte rolling in a concentration dependent manner (−log EC50=11.47, ±0.37, n=3 to 4 animals per group) (Fig. 1). The selective melatonin MT2

Discussion

In this study, melatonin and its precursor, N-acetylserotonin, inhibit acute inflammatory responses in the microcirculation by activation of pharmacologically distinct melatonin receptors. The inhibition of leukocyte rolling and adhesion is not mediated through changes in arteriolar contraction or blood flow rate in the rat microvasculature. We demonstrated that activation of a high affinity melatonin receptor (MT2) mediates inhibition of leukocyte rolling, while the MT3 binding site melatonin

Acknowledgements

We gratefully acknowledge Débora Aparecida de Moura for technical assistance. This work was supported by the grant FAPESP 96/04497-0 to RPM.

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