2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells

Accepted Manuscript Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells

Pichaya Jumnongprakhon, Sivanan Sivasinprasasn, Piyarat Govitrapong, Chainarong Tocharus, Jiraporn Tocharus PII: DOI: Reference:

S0887-2333(17)30031-0 doi: 10.1016/j.tiv.2017.02.010 TIV 3935

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

11 September 2016 8 December 2016 17 February 2017

Please cite this article as: Pichaya Jumnongprakhon, Sivanan Sivasinprasasn, Piyarat Govitrapong, Chainarong Tocharus, Jiraporn Tocharus , Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tiv(2017), doi: 10.1016/ j.tiv.2017.02.010

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Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells Pichaya Jumnongprakhon 1, Sivanan Sivasinprasasn1, Piyarat Govitrapong2,3,4, Chainarong

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Tocharus1, Jiraporn Tocharus 5,* 1

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Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

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Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Bangkok,

Thailand 3

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Center for Neuroscience and Department of Pharmacology, Faculty of Science, Mahidol University,

Bangkok, Thailand 4

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Chulabhorn Graduate Institute, Kamphaeng Phet 6 Road, Lak Si, Bangkok 10210, Thailand

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Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200,

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Thailand

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Correspondence to

Jiraporn Tocharus,

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Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

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Tel.: (6653) 945362. Fax: (6653)945365 E-mail: [email protected]

ACCEPTED MANUSCRIPT 2 Highlights Methamphetamine directly impairs the P-gp transporter in brain endothelial cells.

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Melatonin protects METH-induced toxicity on the P-gp transporter.

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Melatonin and its receptors (MT1/2) protects BBB impairment caused by METH.

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

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ABSTRACT

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Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood–brain barrier (BBB), which is the primary structure that comes into contact

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with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect

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against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell

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homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its

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receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-

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gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses

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were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp

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transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

Keywords: Blood-brain barrier, Melatonin, Melatonin receptors, Methamphetamine, P-

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glycoprotein transporter

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1. Introduction

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Methamphetamine (METH) has been widely known to cause impairment of the central nervous system (CNS) by mediating the dysfunction of neurons, glial cells, and the blood–

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brain barrier (BBB), and thus, causing death (Panenka et al., 2013; Northrop and Yamamoto, 2015; Cheng et al., 2015; Jumnongprakhon et al., 2014, 2015; Castellano et al., 2016). During

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the past decade, BBB impairment by METH has been accepted as an early event and one that

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is closely associated with neurodegeneration (Bank and Erickson, 2010). Impairment of the BBB by METH has been reported, with several lines of evidence reporting that METH

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impaired BBB integrity by enhancing NADPH oxidase 2 (NOX2) activity (Park et al., 2012), oxidative and nitrative stress (reactive oxygen species, ROS; reactive nitrogen species, RNS)

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(Ramirez et al., 2009; Zhang et al., 2009; Fernandes et al., 2015), inflammatory responses via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor erythroid 2-related factor (Nrf2) -dependent mechanisms (Lee et al., 2001; Toborek et al., 2013; Coelho-Santos et al., 2015), cytoskeleton rearrangement (Park et al., 2013), and apoptosis (Abdul Muneer et al., 2011; Ma et al., 2014; Fisher et al., 2015; Cai et al., 2016). Finally, these responses lead to BBB impairment through the mediation of paracellular permeability and the reduction in the transendothelial electrical resistance (TEER >1000

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Ω∙cm2, in vivo), the physiological impedance of the tight junctions (Mahajan et al., 2008; Rosas-Hernandez et al., 2013; Northrop et al., 2016). Previous findings have reported that these responses are associated with the intracellular accumulation of METH and the impairment of the efflux function in the brain endothelial cells, the major component of the BBB (ElAli et al., 2012; Martins et al., 2013). However, the exact mechanisms of the effects

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of METH on the efflux function before mediating negative responses in brain endothelial

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cells are still unclear. A number of adenosine triphosphate-binding cassette (ABC)

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transporters, especially ABCB1, also known as P-glycoprotein (P-gp), actively pump efflux, consuming ATP and transporting a diverse range of neurotoxins out of the brain capillary

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endothelium and the CNS. Disturbance of the P-gp ATPase or the ATP-binding site causes a decrease in the efflux function, leading to the accumulation of neurotoxins in the brain

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endothelium (ElAli and Hermann, 2011; Miller, 2015). Several reviews have documented that exposure to toxins such as amyloid beta (Aβ) or tumor necrosis factor-alpha (TNF-α)

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causes P-gp transporter impairment before the mediation of negative responses and cell

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integrity impairment in brain endothelial cells (Lee et al., 2012; Hartz and Zhong, 2016; Mohamed et al., 2016). According to a previous finding, promotion of the P-gp transporter

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and its functions may be beneficial for protecting against BBB impairment caused by

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neurotoxins such as METH.

Melatonin (N-acetyl-5-methoxytryptamine) is a hormone from the pineal gland and is known as a neuroprotective agent (Tan et al., 1993; Sarlak et al., 2013; Manchester et al., 2015; Miller et al., 2015; Moretti et al., 2015; Zhao et al., 2015; Alluri et al., 2016). Previous studies have documented that melatonin is capable of protecting neurons, glial cells, and the BBB, both in vitro and in vivo, against impairment caused by neurotoxins, including METH (Shaikh et al., 1997; Chen et al., 2006; Garcia et al., 2014; Perpoonputtana et al., 2013; Jumnonprakhon et al., 2014, 2015; Parameyong et al., 2015). The protective role of

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melatonin in brain cells consists of the activation of its receptors (MT1/MT2) before the induction of negative responses such as inflammation and cell death (Chern et al., 2012; Hutchinson et al., 2014; Singhakumar et al., 2015; Wongprayoon et al., 2015, 2016; Alluri et al., 2016). In the BBB, melatonin reverses negative responses by acting on receptors in the BBB as well (Jumnongprakhon et al., 2016, 2016). Nevertheless, the effects of the activation

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of melatonin receptors by melatonin on the P-gp transporter upon METH administration have

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not yet been reported. Hence, we determined the protective role of melatonin and its receptors

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on P-gp transporter-mediated oxidative and nitrative stress, apoptosis, and BBB integrity impairment upon METH treatment.

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2. Materials and methods

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2.1. Chemicals and reagents

Melatonin, verapamil, FITC-dextran, and rhodamine123 were purchased from Sigma-Aldrich

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Chemical Company (St. Louis, MO, USA). The mouse anti-β-actin monoclonal antibody, the

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Muse® Annexin V & Dead Cell kit, and the Muse® Caspase-3/7 kit were purchased from Merck Millipore (MA, USA). The anti-P-gp antibodies were purchased from Santa Cruz

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Biotechnology (Santa Cruz Biotechnology, CA, USA); the anti-mouse IgG peroxidase-

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conjugated secondary antibody was purchased from Merck Millipore (MA, USA). 2.2. Cultivation of a BBB model in vitro A suitable representation of the BBB in vitro was prepared by following the procedures of a previous study (Liu et al., 2013). Briefly, purified BMVECs obtained from co-culturing with primary mixed glial cells were cultured on collagen/fibronectin-coated (Sigma, St. Louis, MO, USA) 0.4-µm trans-wells and maintained at 37°C in a humidified atmosphere at 5% CO2 until cells reached 100% confluence or transendothelial electric resistance (TEER) values >200 Ω∙cm2 before investigating in all the experiments.

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2.3. Rhodamine 123 (Rho123) intracellular accumulation and efflux assay The BMVECs at a density of 1×105 cells/ml, cultured in trans-wells in 24-well plates coated with collagen/fibronectin, were pretreated with verapamil (100 µM), the P-gp competitive inhibitor, for 30 min. After the inhibition of P-gp, the cells were treated with melatonin (1,

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10, and 100 nM) for 2 h prior to treatment with or without METH (100 µM) for 24 h (Jumnongprakhon et al., 2016, 2016). The cells were then incubated with 10 µM Rho123 in

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an assay buffer containing the following composition: 122 mM NaCl, 25 mM NaHCO3, 3

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mM KCl, 1.2 mM MgSO4, 0.4 mM K2HPO4, 1.4 mM CaCl2, 10 mM HEPES, and 10 mM glucose, at 37°C for 90 min, as described by He et al., 2009 (He et al., 2009). After

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incubation, the cells were washed with ice-cold PBS, followed by permeabilization with 1%

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Triton X-100, and the fluorescence intensity at the excitation/emission wavelengths of 488/525 nm was determined by using the Synergy H4 microplate reader. For the assessment of Rho123 efflux, the cells were incubated in a medium containing 10 µM Rho123 at 37°C

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for 90 min prior to treatment. The amount of Rho123 in the cells was examined as described,

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which was considered the intracellular accumulation assay.

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2.4. Western blot analysis

After the cells were exposed to the treatment conditions, equal amounts of protein (50 µg) were subjected to electrophoresis in 10% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA). The blotted membrane was then probed with anti-P-gp overnight. After extensive washing with Tris-buffered saline and Tween-20 (TBST), the membrane was incubated with anti-mouse IgG peroxidase-conjugated secondary antibodies (Millipore, MA, USA). Finally, the blot was incubated with Immobilon Western

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(Millipore, MA, USA) and exposed to an X-ray film. The densitometry was analyzed using the Image-J® software. β-actin was used for normalization. 2.5. Determination of ROS and RNS levels The cultured BMVECs at a density of 1×105 cells/ml in 96-well microplates were pretreated

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with verapamil (100 µM), the P-gp competitive inhibitor, for 30 min. After the inhibition of P-gp, the cells were treated with melatonin (100 nM) for 2 h prior to treatment with or

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without METH (100 µM) for 24 h (Jumnongprakhon et al., 2016, 2016). After the cells were

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exposed to this condition, H2DCFDA was added for determining the ROS level, and DAF-

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2DA was added for determining the RNS level. The cells were then incubated further for 2 h at 37°C in the dark. Finally, the fluorescence was measured at excitation/emission

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wavelengths of 485/535 nm using the Synergy H4 microplate reader (Biotek, VT, USA).

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2.6. TEER determination

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The EVOM2 voltometer with STX-2 electrodes was used to determine the TEER values. The cultured BMVECs were seeded in collagen/fibronectin coated trans-wells in24-well plates at a density of 1×105 cells/ml at 37°C until 100% confluence or until TEER >200Ω∙cm2. Then,

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again.

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the cells were exposed to the treatment conditions, and the TEER values were measured

2.7. Paracellular permeability assay After the cultured BMVECs at a density of 1×105 cells/ml were inserted into collagen/fibronectin coated trans-wells in 24-well plates and exposed to the treatment conditions, 5 mg/mL FITC-dextran in assay buffer containing the following composition: 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.2 mM MgSO4, 0.4 mM K2HPO4, 1.4 mM CaCl2, 10 mM HEPES, and 10 mM glucose, was added to the upper chamber at 37°C in the dark for

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2 h. After incubation, the medium fluorescence intensity of the upper and lower chambers was determined at excitation/emission wavelengths of 488/525 nm using the Synergy H4 microplate reader. 2.8. Flow cytometry

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Flow cytometry was performed to detect the number of apoptotic cells and the caspase-3 level. The percentage of cells undergoing apoptosis after treatment was determined using the

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MuseTM Annexin-V & Dead Cell Assay kit and the MuseTM Caspase-3/7 Assay kit (EMD

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Millipore Bioscience). The MuseTM Cell Analyzer was used to perform the analysis

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according to the manufacturer’s protocol.

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2.9. Statistical analysis

The data are reported as the means±SEM of three independent experiments. The significant difference was analyzed using one-way analysis of variance (ANOVA), followed by a post

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hoc Dunnett’s test to compare the significance between the individual groups. A value of P<0.05 was considered statistically significant.

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3. Results

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3.1. Melatonin directly promotes the function of the P-gp transporter inhibited by METH BBB endothelial cells contain high expression levels of the ATP-binding cassette superfamily of drug efflux transporters, such as P-gp, that prevent penetration of the brain and accumulation of various toxic substances including METH. Hence, we examined whether melatonin could protect against the impairment in P-gp function caused by METH. P-gp function was assessed by the efflux of Rho123, a P-gp substrate. The expression of P-gp was determined by western blotting. To verify P-gp function, verapamil (the competitive inhibitor of P-gp) was used. In the presence of verapamil (100 µM, the maximal non-toxic

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concentration on cell viability determined from an MTT assay (data not shown)), treatment with METH significantly decreased the P-gp-mediated transport of Rho123 (Fig. 1A) and increased the intracellular accumulation of Rho123 (Fig. 1B) compared to that observed in the presence of METH alone (P<0.001). These results suggest that METH markedly impaired the P-gp-mediated transport of Rho123 and that melatonin could reverse these

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effects directly via the P-gp transporter, which was confirmed by the impairment of P-gp in

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the verapamil treatment groups. Importantly, the results demonstrated that melatonin

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treatment alone did not change the efflux and accumulation of Rho123 when compared to that observed in the group without any treatment. Thus, melatonin possibly only protected the

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P-gp transporter in the presence of METH and did not promote over-activity of the P-gp transporter in normal conditions. Next, we investigated whether the improvement of the P-gp

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function by melatonin was accompanied by a parallel increase in the expression of P-gp. The expression of the P-gp proteins was investigated by western blot analysis at various durations

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of METH treatment, including 0, 3, 6, 9, 12, 24, and 48 h. The results demonstrated that the

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change in expression of the P-gp transporter induced by METH was prevalent at 3 h until 12 h and then significantly declined at 24 h (P<0.001) (Fig. 1C). We then used a 24-h exposure

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period in the subsequent experiments. Melatonin pretreatment significantly increased P-gp expression compared to treatment with METH only (P<0.001), and there was no change in

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P-gp expression in cells treated with melatonin alone. In contrast, verapamil treatment did not restore the expression of P-gp induced by METH (Fig. 1D). From these data, it can be inferred that melatonin markedly attenuated the METH-induced impairment of P-gpmediated transport by increasing both its activity and expression. 3.2. Melatonin attenuates METH-mediated toxicity at least partially through P-gp We further investigated whether METH impaired P-gp function and, thereby, enhanced oxidative stress and apoptosis and impaired BBB integrity. Blocking P-gp with verapamil,

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METH progressively increased oxidative stress, apoptosis, and tight junction impairment compared to that observed in the METH-only treatment group (P<0.001), indicating that Pgp was involved in BBB impairment from METH administration. Pretreatment of the BMVEC cells with melatonin in the presence of METH and verapamil significantly decreased oxidative stress (Fig. 2A and B), apoptosis (Fig. 2E and F), and paracellular

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permeability (P<0.001) (Fig. 2C and D). These results clearly demonstrate that the protective

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effects of melatonin against METH-induced oxidative stress, apoptosis, and paracellular

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permeability are at least partially but not mainly mediated through P-gp function. 3.3. Melatonin abolishes the METH-induced impairment of the P-gp transporter by

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activating MT1/2 receptors

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We further investigated the effect of melatonin on the METH-impaired P-gp transporter regarding whether or not it was mediated by the interaction with the melatonin receptors

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(MTR1/MTR2). Luzindole, a nonselective MTR1/MTR2 antagonist, completely abolished

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the effect of melatonin on the P-gp transporter by significantly decreasing the P-gp-mediated transport of Rho123 (Fig. 3A) and increasing the intracellular accumulation of Rho123 (Fig.

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3B). These results clearly indicate that the activation of melatonin receptors with melatonin could protect against the METH-mediated impairment of the P-gp transporter in brain

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endothelial cells. 4. Discussion

Impairment of the BBB in the cases of both short-term and long-term METH administration has been reported to be closely associated with diseases of neurodegeneration such as Alzheimer’s and Parkinson’s disease (Radfar and Rawson, 2014; Yu et al., 2015). Previous studies have reported that BBB impairment by METH causes dysregulation of brain homeostasis before the induction of neuron and glial cell dysfunction and death (O’Shea et

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al., 2014; Urrutia et al., 2014; Sajja et al., 2015). The mechanistic effects of METH treatment on the BBB have been published in several articles: (1) hyperactivity of NOX-2 (Park et al., 2012); (2) over-activation of oxidative and nitrative stress (Ramirez et al., 2009; Zhang et al., 2009; Fernandes et al., 2015); (3) modulation of inflammatory responses through NF-κB, mitogen-activated protein kinases (MAPK), and Nrf2; (4) cytoskeleton-related tight junction

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rearrangement (Lee et al., 2001; Toborek et al., 2013; Coelho-Santos et al., 2015; Yu et al.,

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2015);(5) cell proliferation and autophagy dysregulation (Ma et al., 2014; Fisher et al.,

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2015); and (6) apoptosis (Abdul Muneer et al., 2011; Cai et al., 2016). Moreover, these reports have been consistent with regard to the close association between the accumulation of

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METH and the efflux transporter in brain endothelial cells (ElAli et al., 2012; Martins et al., 2013). However, the relationship of these negative responses in the BBB upon METH

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administration with the efflux function is still unclear. Previous studies have demonstrated that METH itself is transported across the BBB, mediated by several pathways, including the

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P-gp transporter (Hayashi et al., 2005; Tournier et al., 2010). The ABC transporter is an ATP-

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dependent efflux transporter that has a wide variety of substrates on the luminal membrane, and such transporters are also known as Mdr1, ABCB1, or P-gp transporters. P-gp is the

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major efflux transporter at the BBB, where it limits the penetration of the brain by neurotoxins (Hsiao and Unadkat, 2014; Chufan et al., 2015). Impairment of P-gp in the BBB

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is a major phenomenon observed in brain damage in patients with neurodegenerative diseases (Abuznait and Kaddoumi, 2012; Qosa et al., 2015). To investigate the P-gp functional activity in the BBB, the accumulation and efflux of Rho123 were determined. Our results indicate that METH significantly attenuated P-gp functions, as demonstrated by the reduction in the efflux of Rho123 in the BMVECs. To confirm the effect of METH on P-gp function, verapamil, the P-gp competitive inhibitor was used. In the presence of verapamil, METH also decreased the efflux of Rho123 with a higher potency than that of treatment with METH

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alone (similar to verapamil alone). Thus, we suggested that the reduction in the efflux of Rho123 in METH treatment was mediated by the P-gp transporter. We further examined the change in expression of the P-gp protein caused by METH, and we found that METH transiently increased the expression after 3–12 h of METH exposure prior to a significant decrease in expression at 24 h of exposure (Fig. 1). As suggested by the results, the disruption

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of the P-gp function by METH was due to its attenuation of the expression of the P-gp

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protein. A previous study demonstrated that an injection of METH in C57BL/6J mice caused

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a transient increase in the P-gp expression at 3 h and a decrease at 24 h. The transient increase in P-gp expression before 24 h is an indicator of the efflux function performed to

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eliminate METH, but long-term exposure to METH (>24 h), resulted in a reduction in this protein, which correlated with the decrease in cell viability and might be correlated with the

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overactivation of the ubiquitin-proteasome system (ElAli et al., 2012; Hartz et al., 2016). Thus, it can be suggested that long-term METH exposure caused P-gp impairment by

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reducing P-gp expression. The next objective was to investigate the mechanistic evidence that

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METH impaired the BBB by inhibiting P-gp function, thereby inducing oxidative stress (ROS and RNS production), apoptosis (caspase 3/7 expression and apoptotic cells), and

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impairment of BBB integrity (TEER and paracellular permeability), as shown in Fig. 2. Our results showed that in the presence of verapamil, METH caused a significant increase in

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oxidative stress, apoptosis, and tight junction leakage that was more potent than that observed with treatment of METH alone (Fig. 2). These results suggest that METH impairs the BBB by decreasing the P-gp efflux function, which subsequently increases intracellular METH accumulation, thereby enhancing oxidative stress, tight junction leakage, and apoptosis. These results suggest that METH might primarily have an effect on the P-gp function prior to mediating oxidative stress and apoptosis that causes the impairment of cellular integrity.

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Melatonin is a hormone that has high potency for protecting against METH toxicity in neuronal tissue damage, including that of the BBB (Esposito and Cuzzocrea, 2010; Reiter et al., 2010; Acuna-Castroviejo et al., 2014). Previous studies have reported that melatonin ameliorated METH-induced toxicity in brain endothelial cells via several mechanisms (Jumnongprakhon et al., 2016, 2016). The present study demonstrated that melatonin directly

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increases P-gp function (as demonstrated by the Rho123 efflux), decreases intracellular

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Rho123 accumulation and increases the P-gp expression inhibited by METH. Moreover, we

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suggested that the increase in the P-gp function and expression might be related with the ability of melatonin to prevent the decline in cell viability reported in our previous finding.

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Interestingly, we also found that the ability of melatonin to protect against oxidative and nitrative stress, apoptosis, and impairments in cell monolayer integrity was at least partially

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via P-gp (as demonstrated by the reversing of the negative responses with verapamil treatment). Therefore, these results suggest that melatonin directly promotes P-gp transporter

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function in BMVECs, but the negative responses that were caused by METH were observed

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via other mechanisms such as directly reducing ROS-induced and RNS-induced oxidative and nitrative stress, inhibiting NOX-2-induced inflammation via NF-κB, and suppressing

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Nrf2 before undergoing apoptosis and tight junction impairment (Jumnongprakhon et al., 2016, 2016). However, whether the protective role provided by melatonin to P-gp was

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mediated by its receptors was still unclear. Hence, we then investigated these effects by using luzindole, the non-specific melatonin receptor antagonist. We found that the activation of melatonin receptors (MT1/2) was required for diminishing the effects of METH on the P-gp transporter. 5. Conclusion In summary, the results of this study and our previous findings demonstrate that METH induces BBB impairment through the reduction of the P-gp transporter function and

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the enhancement of oxidative and nitrative stress, apoptosis, and cell integrity impairment; these negative effects were reversed by melatonin and activation of melatonin receptors (MT1/2). Thus, melatonin might be beneficial for protecting against BBB impairment caused by METH.

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Acknowledgments and Conflict of Interest Disclosure This work was supported by the CMU Mid-Career Research Fellowship Program, Chiang

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Mai University, Thailand, as well as by a research grant from the Thailand Research Fund

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(TRF) to JT and a research grant from TRF (DPG5780001) and Mahidol University to PG

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(IRG5780009). The authors have nothing to disclose.

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Figure Legends Fig. 1. Melatonin directly attenuates the METH-induced impairment of the P-gp transporter in BMVECs. The cells were pretreated with verapamil (100 µM), the P-gp inhibitor, for 30 min. After P-gp inhibition, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2

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h prior to being treated with or without METH (100 µM) for 24 h. (A) The cellular efflux was determined by obtaining the efflux levels of Rho123 using the Rho123 assay. (B) The

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intracellular accumulation was determined in order to obtain the Rho123 accumulation using

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the Rho123 assay. (C) The cells were treated with METH (100 µM) for various amounts of time (0, 3, 6, 9, 12, 24, and 48 h). The toxicity of METH on P-gp in the BMVECs was

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evaluated by the P-gp expression using western blot analysis. (D) The cells were pretreated

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with verapamil (100 µM) for 30 min before being treated with melatonin and METH. After P-gp inhibition, the cells were pretreated with melatonin (100 nM) for 2 h prior to being treated with or without METH (100 µM) for 24 h. Equal amounts of cell lysates (50 µg) were

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subjected to electrophoresis and analyzed by western blot for the expression of P-gp and actin for normalization. **P<0.01 and ***P<0.001 compared to the control group. ###P<0.001

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compared to theMETH-only treatment group. n = 3 (independent experiments).

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Fig. 2. Melatonin protects against METH-mediated toxicity through a P-gp-independent and P-gp dependent mechanism in BMVECs. The cells were pretreated with verapamil (100 µM), the P-gp inhibitor, for 30 min. After P-gp inhibition, the cells were pretreated with melatonin (100 nM) for 2 h prior to being treated with or without METH (100 µM) for 24 h. (A) Oxidative stress was determined based on the ROS level using the CM-H2DCFDA assay. (B) Nitrative stress was determined based on the RNS level using the DAF-2DA assay. (C) The formation of the tight junctions determined the TEER values, which were evaluated by

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EVOM2. (D) The paracellular permeability was determined by the ratio of 4 kDa FITCDextran in the permeability assay. (E) Caspase activation was determined by the active caspase-3 level with the Muse® Caspase-3/7 kit by Muse® Cell Analyzer. (F) Apoptosis was measured by the number of apoptotic cells with the Muse® Annexin V & Dead Cell kit by using the Muse® Cell Analyzer. ***P<0.001 compared to the control group. ###P<0.001

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compared to the METH-only treatment group. n = 3 (independent experiments).

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Fig. 3. Melatonin protects against the METH-induced impairment of the P-gp transporter by

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activating MT1/2 receptors in BMVECs. The cells were pretreated with luzindole (1 µM), the nonspecific melatonin receptor antagonist, for 1 h before being treated with melatonin and

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METH. After blocking the MT1/2 receptor, the cells were pretreated with melatonin (100 nM) for 2 h prior to being treated with or without METH (100 µM) for 24 h. (A) The cellular

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efflux was determined by the efflux level of Rho123. (B) The intracellular accumulation was

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determined based on the Rho123 accumulation. ***P<0.001 compared to the control group.

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Fig. 4.The proposed mechanism for the protection against METH-induced impairment of the P-gp transporter by melatonin and its receptors. This diagram shows the possible mechanism by which the interaction of melatonin directly protects the P-gp transporter from the effects of METH treatment and protects, at least partially, against METH-induced toxicity through the P-gp transporter.

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