Cellular iron storage and trafficking are affected by GTN stimulation in primary glial and meningeal cell culture

Abstract

A well-balanced intracellular iron trafficking in glial cells plays a role in homeostatic processes. Elevated intracellular iron triggers oxidative stress and cell damage in many neurological disorders, including migraine. This study aimed to investigate the effects of glyceryl trinitrate (GTN), on cellular iron homeostasis, matrixmetalloproteinase (MMP)-9, and calcitonin gene related peptide (CGRP) receptor (CRLR/CGRPR1) production in microglia, astrocyte, and meningeal cell cultures. Primary glial and meningeal cells in culture were exposed to GTN for 24 h. Messenger RNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl's histochemistry. MMP-9 levels in cell culture supernatants were measured using ELISA. Ferritin and CRLR/CGRPR1 proteins were visualized via immunofluorescence staining. Nitric oxide production increased significantly with GTN in meningeal and glial cells. GTN significantly increased the expression of the storage protein ferritin for all three cell types, but ferritin-L for meningeal cells and microglia. Iron trafficking associated with the efflux protein ferroportin and influx protein divalent metal transporter (DMT)1 was affected differently in all three cell types. MMP-9 expression was increased in astrocytes. GTN stimulation increased both CRLR/CGRPR1 expression, and immunostaining was apparent in microglia and meningeal cells. This study showed for the first time that GTN modulates intracellular iron trafficking regulated by storage and transport proteins expressed in meningeal cells and glia. CRLR/CGRPR1 expression might be related to altered iron homeostasis and they both may stimulate nociceptive pathways activated in migraine. These molecules expressed differently in glial and meningeal cells in response to GTN may bring not only new targets forward in treatment but also prevention in migraine.

Keywords: CGRPR; MMP-9; Migraine; astrocyte; ferritin; iron; meningeal cell; microglia.

Figure 1

Summary of the research design.

Figure 2

Cell viability after stimulation with 200 μM GTN in glial and meningeal cell culture (Data have been shown as mean ± SEM) (n = 3).

Figure 3

NO levels measured in microglia, astrocyte, and meningeal cell cultures after incubation with 200 μM GTN for 24 h by ELISA. Data have been shown as mean ± SEM (n = 3) (*P < 0.001).

Figure 4

Iron accumulation showed with modified Perl’s histochemistry in 1) control and 2) GTN induced a) microglia, b) meningeal cells, and c) astrocytes (n = 3) (40× magnification, Scale bars 40 μM in a and b, 20μM in c).

Figure 5

Ferritin-L, ferritin-H, DMT1, and ferroportin expression in primary cell culture of A. microglia, B. meningeal cells, and C. astrocytes after 24-h incubation with GTN (Data have been shown as mean ± SEM) (n = 3) (*P < 0.05).

Figure 6

A. CRLR/CGRPR1 expression in mouse primary cell culture of microglia, meningeal cells, and astrocytes after 24-h incubation with GTN (Data have been shown as mean ± SEM) (n = 3) (P < 0.05). B. Representative fields of CRLR/CGRPR1 immunofluorescence staining in 1) control and 2) GTN induced a) microglia, b) meningeal cells, and c) astrocytes (n = 3) (40× magnification. Scale bars 40 μm). The same field for each different cell type and condition was captured with DAPI used for nuclear staining in all images.

Figure 7

Representative fields of ferritin immunofluorescence staining in 1) control and 2) GTN induced a) microglia, b) meningeal cells, and c) astrocytes (n = 3) (40× magnification. Scale bars 40 μm). The same field for each different cell type and condition was captured with DAPI used for nuclear staining in all images.

Figure 8

MMP-9 levels measured in microglia, astrocyte, and meningeal cell culture after incubation with 200 μM GTN for 24 h. Data have been shown as mean ± SEM (n = 3) (*P < 0.05).