Enteric glial-mediated enhancement of intestinal barrier integrity is compromised by morphine

Abstract

Background: The opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability.

Materials and methods: Rat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the μ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing.

Results: EGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control.

Conclusions: Morphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.

Keywords: Enteric glia; Enteric nervous system; GDNF; Intestinal barrier integrity; Morphine; Opioids.

Figure 1. EGCs possess the μ-opioid receptor

EGCs were stained with rabbit anti-μ opioid receptor primary antibody and FITC-conjugated goat anti-rabbit secondary antibody.

Figure 2. Enteric glial cell products facilitate tight junction formation in cultured intestinal epithelial cells

IEC-6 cells treated with control media (A) compared with conditioned media from rat enteric glial cells (B). Note organized formation of Occludin staining (green) in panel B. Red represents F-actin staining.

Figure 3 (A,B). EGC-mediated barrier enhancement

A. Control = no cells in either chamber. IEC6 cells alone (plated in transwell insert) or IEC6 + EGCs (EGCs in plate well) were grown to confluence in the apical chamber or plate well, respectively. B. GDNF (100ng/ml) was stimulated to the well of the transwell plate, fluorescence was measured 24 hours after stimulation. * = p < 0.05, n>3 for all experimental groups.

Figure 4 (A,B,C,D). Morphine and EGCs

A. Control = unstimulated IEC6 cells. I+E = IEC6 cultured in plate insert and EGCs cultured in plate well. B. I = unstimulated IEC6 cells. M= morphine (1μM) stimulated in well onto EGCs. ECM = EGC conditioned media added to well of transwell plate without enteric glial cells. C. Morphine (1μM) was always added to the EGCs or empty plate well unless it was treated to only the IEC6 cells shown as M(I). * = p<0.05 (0.0447). D. GDNF = glial derived neurotrophic factor (100ng/ml). In all columns, IECs were placed in the apical chamber of the transwell plate and EGCs were placed in the basal chamber. A= Morphine treatment to IEC-6 cells in the apical chamber. Morphine was always treated to EGCs the well unless designated by Ins. * = p < 0.05, n>3 for all experimental groups.

Figure 5 (A,B,C). Morphine decreases GDNF production

A. Real-time PCR analysis of mRNA levels of GDNF in enteric epithelial cells after 30-hour morphine treatment. **p < 0.01, n=3. B. Cell lysates from control and morphine treatment (+MS) were resolved in SDS-PAGE gel and the blot was probed with antibodies against GDNF (upper panel) and b-actin (lower panel, serving as internal control for equal sample loading). C. Densitometric analysis of GDNF level in control and morphine treatment. The density of each band was determined using ImageJ (NIH). GDNF protein level was normalized to the level of b-actin in each sample. The normalized control was considered as 100%, +MS = morphine treated EGCs (mean = 71.67 +/− 7.71 (SE)). Paired Student’s t-test was performed and graph was plotted using Prizm 4.0 (Graphpad), p = 0.033, n=3, * = p<0.05.

Figure 6 (A,B). Morphine Disrupts EGC-mediated Barrier Enhancement. ECIS

A. Normalized resistance in confluent IECs were stimulated with unstimulated conditioned media (EGC-CM) from confluent EGCs (green) or left unstimulated (black). B. Normalized resistance in confluent IECs cultured in non-Morphine treated EGC-CM (green) or in Morphine treated EGC-CM (black) n=3 in all experimental groups.