Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure

Abstract

1 Oligodendrocytes, the myelin forming cells in the CNS, express muscarinic acetylcholine receptors (mAChR), primarily M3, coupled to various signal transduction pathways. 2 In the present study we have investigated whether mAChR undergo functional agonist-induced regulation in cultured oligodendrocyte progenitors and differentiated oligodendrocytes. 3 The muscarinic agonist, carbachol (CCh) caused a time-dependent desensitization of phosphoinositide (PI) hydrolysis, and the internalization and down-regulation of receptors. Short-time desensitization (5 min) of PI hydrolysis occurred without receptor internalization and reached 54% by 1 h. The same treatment decreased cell surface receptors labelled with the non-permeable ligand [(3)H]-NMS by 47%, while total receptor density ([(3)H]-scopolamine binding) decreased by 30%. Longer CCh treatment down-regulated receptors by 70% and desensitized the PI response by 80%. 4 Although protein kinase C (PKC) activation desensitized mAChR, CCh-mediated desensitization was independent of PKC. 5 Inhibition of receptor endocytosis by low temperature during the pre-stimulation period or in the presence of hyperosmotic sucrose (0.5 M) blocked desensitization, receptor internalization and down-regulation. 6 Recovery of surface mAChR and their functional activity following down-regulation was slow, returning to control levels by 24 h after agonist removal. In progenitor cells, dose-response curves for CCh-mediated PI hydrolysis and c-fos mRNA expression showed that newly synthesized mAChR were supersensitive after recovery. 7 Overall, the present results provide evidence of functional agonist-mediated mAChR regulation in brain oligodendroglial cells.

Figure 1

Effect of short-term CCh pre-treatment on mAChR density and PI hydrolysis. Time course of CCh-induced sequestration of mAChR in progenitors (a) and oligodendrocytes (b). Cells were incubated at 37°C with 1 mM CCh for the indicated times, washed three times with warmed buffer, and mAChRs were measured by radioligand binding at 4°C for 16 h with 1 nM [³H]-NMS or [³H]-scopolamine. Atropine (25 μM) was used to determine non-specific binding. Data are expressed as percentage of untreated control group. (c) Time course of CCh-induced desensitization of PI hydrolysis. Cells were labelled with 1 μCi ml⁻¹ myo-[³H]-inositol for 18 h and 1 mM CCh was added during the labelling period to induce receptor desensitization. Following agonist pre-stimulation, cells were washed three times with warmed buffer and challenged with 1 mM CCh (plus 10 mM LiCl) for 10 min at 37°C. IP were determined as described in Methods. Data are expressed as percentage of maximal IP accumulation determined in the absence of CCh pre-treatment. Results are the means±s.e.mean of four independent experiments performed in triplicate.

Figure 2

Effect of long-term CCh pre-treatment on mAChR density and PI hydrolysis. Time courses of CCh-induced reduction of specific [³H]-NMS and [³H]-scopolamine binding in progenitors (a) and oligodendrocytes (b) and desensitization of IP hydrolysis (c). Cells were incubated at 37°C with 1 mM CCh for the indicated times, washed three times with warmed buffer and IP accumulation and mAChRs were measured as described in Methods. Binding data are expressed as percentage of untreated control group. PI hydrolysis data are expressed as percentage of maximal IP accumulation determined in the absence of CCh pre-treatment. Results are the means±s.e.mean of four independent experiments performed in triplicate.

Figure 3

Time-dependent effect of CCh pre-treatment on mAChR-mediated c-fos mRNA expression in oligodendrocyte progenitors. Cells were pre-treated for 1–24 h with 1 mM CCh, washed three times with buffer and maintained for 1 h in buffer before re-stimulation with 100 μM CCh for 30 min. Such resting period is necessary to decrease c-fos levels to control values, after that time cells are again responsive to CCh re-challenge (Cohen et al., 1996). Levels of c-fos mRNA were detected by Northern blotting as described in Methods. Autoradiographs were analysed by densitometry, and values are expressed as means±s.e.mean of three independent experiments performed in duplicate.

Figure 4

Time dependence of mAChR resensitization and effect of protein synthesis inhibition by cycloheximide. Progenitors and oligodendrocytes were pre-treated with 1 mM CCh for 1 h, washed three times with buffer and allowed to recover for different times in SFM+GF in the absence or presence of cycloheximide (5 μg ml⁻¹). Following the recovery period, [³H]-NMS binding to surface mAChR (a) and IP accumulation (b) were determined as described in Methods. Binding data are expressed as percentage of untreated control group, and PI hydrolysis as percentage of maximal IP accumulation determined in the absence of CCh pre-treatment. Results are the means±s.e.mean of four independent experiments performed in triplicate.

Figure 5

Dose–response relationship for CCh-stimulated IP accumulation in control, CCh-pre-treated and resensitized progenitors and oligodendrocytes. Cells were pretreated for 1 h with 1 mM CCh, washed three times with buffer and allowed to recover for 24 h in SFM+GF. Following the recovery period IP accumulation was determined as described in Methods. Points represent fold increase over basal levels and are mean±s.e.mean values for four separate experiments done in triplicate. Basal values of [³H]-IP (d.p.m. mg⁻¹ protein) were 13545±1612 for control cells and 13163±822 for CCh-pre-treated and recovered cultures, these data indicate no differences in [³H]-IP labelling.

Figure 6

Activation of supersensitive muscarinic receptors by CCh increases c-fos mRNA expression in oligodendrocyte progenitors. Cells were pre-treated for 1 h with 1 mM CCh, washed three times with buffer and allowed to recover for 24 h in SFM+GF. Following the recovery period, cultures were stimulated for 30 min with 25 or 100 μM CCh. Levels of c-fos mRNA were detected by Northern blotting as described in Methods. Autoradiographs were analysed by densitometry, and values are expressed as the means±s.e.mean of three independent experiments performed in duplicate. Densitometric analysis revealed that 1 h CCh pre-treatment but no rechallenge (22±4) did not significantly modify c-fos levels compared to control unstimulated cultures (25±6).

Figure 7

Effect of pre-stimulation of oligodendrocyte progenitors with 1 mM CCh at low temperature or in the presence of hyperosmotic sucrose. Inhibition of mAChR sequestration (a) and desensitization of PI hydrolysis (b) were produced by decreasing the temperature to 4°C or by addition of buffer containing 0.5 M sucrose during the pre-stimulation period (1 mM CCh for 30 or 60 min). Following CCh pre-exposure, cells were washed three times with buffer and challenged with 1 mM CCh (plus 10 mM LiCl) for 10 min at 37°C to determine IP accumulation or incubated for 16 h at 4°C with 1 nM [³H]-NMS. Binding data are expressed as percentage of untreated control group. The 100% value for [³H]-NMS binding was 42±2 fmol mg⁻¹ protein. PI hydrolysis data are expressed as percentage of maximal IP accumulation determined in the absence of CCh pre-treatment. The 100% value was 149,000±17,740 d.p.m. mg⁻¹ protein. Results are the means±s.e.mean of four independent experiments performed in triplicate. Differences with control values: 37°C, 30 or 60 min CCh (P<0.01).