Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells

Abstract

Lipid rafts are small cholesterol- and glycosphingolipid-enriched membrane subdomains. Here we compared the mu opioid receptor (MOR)-lipid rafts relationship in the rat brain, where neurons have non-caveolae rafts, and in CHO cells stably transfected with HA-rat MOR (CHO-HA-rMOR), which are enriched in caveolae. Membranes of rat caudate putamen (CPu) and thalamus or CHO-HA-rMOR cells were homogenized, sonicated in a detergent-free 0.5 M Na(2)CO(3) buffer and fractionated through sucrose density gradients. Western blot and [(3)H]diprenorphine binding showed that approximately 70% of MOR in CHO-HA-rMOR was present in low-density (5-20% sucrose) fractions enriched in cholesterol and/or ganglioside M1 (GM1) (lipid rafts) in plasma membranes, whereas about 70% and 45% of MOR in CPu and thalamus, respectively, were associated with lipid rafts. Incubation with a saturating concentration of etorphine or morphine at 37 degrees C for 30 min failed to change the MOR location in rafts in CHO-HA-rMOR, indicating that the internalized MOR does not move out of rafts, in contrast to the delta opioid receptor. In vivo, rafts association of MOR in CPu and thalamus was not affected significantly in rats implanted with two 75-mg morphine pellets for 72 h. In addition, cholesterol reduction by methyl-beta-cyclodextrin (MCD) disrupted rafts and shifted MOR to higher density fractions in both CHO-HA-rMOR and CPu membranes. However, MCD treatment had opposite impacts on MOR signaling in the two tissues: it attenuated MOR-mediated [(35)S]GTPgammaS binding in CPu but enhanced it in CHO-HA-rMOR.

Fig. 1

Localization in lipid rafts of the mu opioid receptor (MOR) in membranes of the rat caudate putamen (CPu) and CHO-HA-rMOR cells. CPu membranes prepared from 6 frozen meninges-stripped rat brains or three 100-mm dishes of CHO-HA-rMOR cells were sonicated in 0.5 M sodium carbonate buffer (pH 11) and then fractionated through a discontinuous sucrose gradient (5%/35%/45%) by ultracentrifugation as described in Experimental procedures. Twelve 1-ml fractions were collected and each fraction was subjected to (A and C) [³H]diprenorphine (~1 nM) bindings using naloxone (10 μM) to define nonspecific binding. Two 100-μl aliquots from each fraction were used in binding in duplicate as described in Experimental procedures. Data are expressed as percent of total specific [³H]diprenorphine binding. (B and D) Immunoblottings with anti-μC, anti-HA(HA.11), anti-flotillin-1 and anti-caveolin-1antibodies, respectively. Data shown in this image are representatives of the two experiments performed with similar results.

Fig. 2

Lipid rafts distribution of HA-rMOR expressed in CHO cells was not affected by acute agonist treatment. CHO-HA-rMOR cells were incubated at 37 °C in the absence and presence of 1 μM etorphine or 10 μM of morphine for 30 min. Cells were collected and sonicated in 0.5 M sodium carbonate buffer (pH 11) and then fractionated through a continuous sucrose gradient (fractions 1–8: 5–35%; fractions 9–12: 45%) by ultracentrifugation as described in Experimental procedures. Twelve fractions from continuous sucrose gradients were collected and each fraction was subjected to (A) immunoblotting of HA-rMOR (the upper panel) with a monoclonal anti-HA antibody (HA.11) and caveolin-1 (the lower panel) with an anti-caveolin-1 monoclonal antibody. Each image represents one of the two experiments performed with similar results. (B) [³H]diprenorphine (~1 nM) binding and data were expressed as percent of total specific [³H]diprenorphine binding. Three 100-mm dishes of confluent CHO-HA-rMOR cells were used for fractionation and two 100-μl aliquots of each 1-ml fraction were used in binding in duplicate. The average [³H]diprenorphine-specific binding in the fraction 1 was ~6300 dpm/100 μl. Data are shown as mean±SEM from three independent experiments.

Fig. 3

In CHO-HA-rMOR cells, the MOR was internalized by acute treatment of etorphine, but not morphine. (A) Immunofluorescence imaging by use of the anti-HA monoclonal antibody, HA.11, as described in the Experimental procedures. (B) Quantification of intracellular receptors by intact cell binding assay as described in the Experimental procedures.

Fig. 4

Lipid rafts distribution of the MOR in rat CPu and thalamus membranes was not affected by chronic in vivo morphine treatment. Three adult male Sprague–Dawley rats (250–275 g) were implanted subcutaneously with two control (placebo) or 75-mg morphine pellets in the middoral region under light isoflurane anaesthesia. The animals were decapitated at 72 h after implantation and the CPu and thalamus tissues collected. Brain membranes were prepared from each region and fractionation for lipid rafts was performed as in Fig. 2 legend. Twelve fractions from continuous sucrose gradients were collected and each fraction was subjected to immunoblotting of the MOR in rat CPu (A) and thalamus (C) with anti-μC antibody against a synthetic peptide corresponding to rMOR (381–398). The result represents one of the two experiments performed with similar results. [³H] diprenorphine (~1 nM) binding of rat CPu (B) and thalamus (D) using naloxone (10 μM) to define nonspecific binding. Two 100-μl aliquots from each fraction were used in binding in duplicate as described in Experimental procedures. Data are expressed as percent of total specific [³H]diprenorphine binding. Data are shown as mean±SEM from three independent experiments.

Fig. 5

Effects of MCD treatment on MOR and caveolin-1 distribution in CHO-HA-rMOR cells. Cells were treated with (A) serum-free medium or (B) 2% MCD for 1 h at 37 °C as described in Experimental procedures, then collected and subjected to lipid rafts preparation through continuous sucrose gradients as described in Fig. 2 legend. Twelve 1-ml fractions were collected and for each fraction, HA-rMOR and caveolin-1 were detected by immunoblottings with the anti-HA antibody, HA.11 and an anti-caveolin-1 monoclonal antibody. Each image represents one of the two experiments performed with similar results.

Fig. 6

MCD pretreatment has different effects on agonist-induced [³⁵S]GTPγS binding to membranes of (A) rat brain CPu and (B) CHO-HA-rMOR cells. Cells or rat CPu membranes were incubated with vehicle or 2% MCD, membranes were prepared and [³⁵S]GTPγS binding experiments were performed as described in Experimental procedures. Data are normalized as percent of basal [³⁵S]GTPγS binding (in the absence of agonists) and shown as mean±SEM of 3–5 experiments performed in duplicate and summarized in Table 1. The basal [³⁵S]GTPγS binding was ~2000 dpm for non-treated membranes from both CHO cells and CPu. After MCD treatment, the basal binding for CHO cells remained the same, however, the basal binding for CPu was only ~50% of that for the non-treated sample.