Membrane MT2 Research Articles

Melatonin in Th17/Treg differentiation: the contribution of the hormone's own production by T lymphocytes

The hormone melatonin is involved in regulation of functioning of almost all organs and systems of the organism. In the immune system, T lymphocytes are an important target of melatonin: they express specific melatonin receptors with different affinities – membrane MT1 and MT2 and nuclear RORa, as well as intracellular molecules that nonspecifically bind melatonin at high concentrations. Moreover, many in vitro studies reveal their own production of melatonin by T lymphocytes in response to polyclonal activation and its involvement as autocrine or paracrine factor in the induction of IL-2 and IL-2 receptor (IL-2R) synthesis by T cells, with melatonin receptors involvement in implementation of these effects. Since IL-2/IL-2R-dependent signal is a key event in T lymphocytes proliferative response induction, intrinsic melatonin seems to be directly involved at least in the clonal expansion of these cells. In this work, we investigated the contribution of T cells’ melatonin to regulation of the next stage of T lymphocyte activation, namely, the differentiation of T helper populations Th17 and Treg. It was shown that blockade of both membrane and nuclear melatonin receptors did not cause statistically significant changes in Th17 differentiation, although the trend was fixed for a decrease. Simultaneously, CD4+FoxP3+Т cells level decreased under the nonselective blockade of membrane hormone receptors, and Treg-associated cytokine TGF-b concentration in activated cultures supernatants decreased both in case of MT1/MT2 nonselective blockade and MT2 selective blockade. The data indicate that melatonin produced by T lymphocytes in culture can contribute to the control of naive CD4+T cell differentiation into Treg in vitro, and the hormone effects are mediated by membrane melatonin receptors. The presence of a large number targets with different affinities for melatonin in T lymphocytes determines the key role of the hormone concentration in its effects on these cells. And when interpreting data on melatonin-dependent regulation of Treg, it is important to take into account the hormone’s own production by lymphocytes, since T cells’ melatonin can mask the exogenous hormone effects or interfere with its action due to competitive binding to hormone receptors.

Melatonin Lowers HIF-1α Content in Human Proximal Tubular Cells (HK-2) Due to Preventing Its Deacetylation by Sirtuin 1.

Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O2). Melatonin was applied at 100 μM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced Sirt1 gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.

Melatonin Synergizes the Chemotherapeutic Effect of Cisplatin in Ovarian Cancer Cells Independently of MT1 Melatonin Receptors.

Melatonin (MLT), through the interaction with membrane melatonin receptors MT1, can improve the effectiveness of cytostatic agents, including cisplatin (CP). The aim of this study was to examine the synergistic effect of MLT and CP in three cell lines: IOSE 364, SK-OV-3 and OVCAR-3, as well as to assess the role of MT1 receptors in this mechanism. Using the SRB assay we investigated the effect of different concentrations of CP and MLT on cell viability. Tests, using luzindole - MT1 inhibitor, allowed us to assess the potential involvement of MT1 in the mechanism of MLT action. MLT at certain concentrations demonstrated a synergistic effect in combination with CP. The addition of luzindole did not affect the action of MLT in combination with CP. In summary, the synergistic effect of MLT with CP seems to be independent of membrane MT1 receptors.

The role of melatonin membrane receptors in melatonin-dependent oxytocin secretion from the rat hypothalamo-neurohypophysial system - an in vitro and in vivo approach.

Melatonin exerts its biological role acting mainly via G protein-coupled membrane MT1 and MT2 receptors. To determine whether a response of oxytocinergic neurons to different concentrations of melatonin is mediated through membrane MT1 and/or MT2 receptors, the effect of melatonin receptors antagonists, i.e. luzindole (a non-selective antagonist of both MT1 and MT2 receptors) and 4-phenyl-2-propionamidotetralin (4-P-PDOT - a selective antagonist of MT2 receptor), on melatonin-dependent oxytocin (OT) secretion from the rat hypothalamo-neurohypophysial (H-N) system, has been studied both in vitro and in vivo. For in vitro experiment, male rats served as donors of the H-N explants, which were placed in 1 ml of normal Krebs-Ringer fluid (nKRF) heated to 37oC. The H-N explants were incubated successively in nKRF {fluid B1} and incubation fluid as B1 enriched with appropriate concentration of melatonin, i.e. 10-9 M, 10-7 M, or 10-3 M and luzindole or 4-P-PDOT, or their vehicles (0.1% ethanol or DMSO) {fluid B2}. After 20 minutes of incubation in fluid B1 and then B2, the media were collected and immediately frozen before OT estimation by the RIA. The OT secretion was determined by using the B2/B1 ratio for each H-N explant. During in vivo experiment, rats were given an intracerebroventricular (i.c.v.) infusion of 5 mL luzindole or 4-P-PDOT, or their solvent (0.1% DMSO) and 10 minutes later the next i.c.v. infusion of 5 mL of either melatonin solution (10-7 M) or its vehicle (0.1 % ethanol in 0.9% sodium chloride). Melatonin at a concentration of 10-3 M significantly stimulated, while at a concentration of 10-9 M had no effect on, oxytocin secretion from the rat H-N system in vitro, also when luzindole or 4-P-PDOT was present in a medium. On the other hand, melatonin at a concentration of 10-7 M diminished this neurohormone output from an isolated H-N system and into the blood. Luzindole significantly suppressed such melatonin action, while 4-P-PDOT did not change the inhibitory influence of 10-7 M melatonin on oxytocin release, both in vitro and in vivo. The present study demonstrates that an inhibitory effect of 10-7 M melatonin on oxytocin secretion from the rat H-N system is mediated through a subtype MT1 membrane receptor and its action is independent of subtype MT2 receptor. However, for the stimulatory effect of pharmacological concentration (10-3 M) of the pineal hormone on oxytocin release, probably mechanisms other than membrane MT1/MT2 receptor(s)-dependent are involved. (Endokrynol Pol 2016; 67 (5): 507-514).

Evidence that membrane‐bound G protein‐coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.