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, 29 (2), 307-18

Role of Protein Kinase C and Mu-Opioid Receptor (MOPr) Desensitization in Tolerance to Morphine in Rat Locus Coeruleus Neurons

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Role of Protein Kinase C and Mu-Opioid Receptor (MOPr) Desensitization in Tolerance to Morphine in Rat Locus Coeruleus Neurons

C P Bailey et al. Eur J Neurosci.

Abstract

In morphine tolerance a key question that remains to be answered is whether mu-opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70-80%) of loss of receptor function. Ongoing PKC activity in the MOPr-expressing neurons themselves, primarily by PKCalpha, was required to maintain morphine-induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30-50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in alpha(2)-adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC-dependent component and that this desensitization underlies the maintenance of morphine tolerance.

Figures

Fig. 5
Fig. 5
Converting empirical data of morphine responses to loss of receptor function: morphine-induced profound loss of receptor function. (A) Concentration–response curves constructed from the responses to Met-Enkephalin in the absence (squares) and presence (triangles) of the irreversible MOPr antagonist β-FNA, applied at 30 nm for 30 min. Lines of best-fit were obtained following simultaneous linear-regression analysis using the operational model of agonism. (B) Under conditions resulting in loss of receptor function, τ would be decreased, resulting in a decrease in the response elicited by 30 μm morphine (% morphine max). Empirical data (response to 30 μm morphine following loss of functional receptor normalized to response to 30 μm morphine under control conditions, i.e. % maximum morphine response) could thus be converted to % loss of receptor function. (C) Met-Enkephalin (Met-Enk; 30 μm) for 10 min resulted in rapid MOPr desensitization. The response to morphine in this desensitized state was 13.7 ± 5.3% that of control (n=4; *P < 0.05 vs. control; D), this corresponds to a 95 ± 2% loss of receptor function (E). (F) Empirical data shown in Figs 1 and 4 converted to % loss of receptor function. Black circles, 10 μm DAMGO; black squares, 30 nm DAMGO; open circles, 30 μm morphine. Each point represents mean empirical data converted to % loss of receptor function. (G) Empirical data shown in Fig. 2 converted to percentage loss of receptor function.
Fig. 1
Fig. 1
Prolonged exposure to morphine in vitro results in MOPr desensitization. (A) Concentration–response curve for morphine in rat LC neurons. Morphine responses were normalized to the maximum possible opioid response in each neuron, evoked by a brief (1 min), application of 10 μm Met-Enkephalin (Met-Enk; n=4; error bars represent SEM). (B) Sample current recording from an untreated LC neuron: a receptor-saturating concentration of morphine (30 μm; morph) was applied, followed after 3-min wash by a receptor-saturating concentration of DAMGO (10 μm). The maximum response to morphine was less than that of DAMGO. Following application of the MOPr antagonist naloxone (nalox; 1 μm), a receptor-saturating concentration of noradrenaline (NA; 100 μm) was applied. Scale bars: 50 pA and 5 min. (C) Example recording from an LC neuron, following 6–9 h pre-incubation with 1 μm morphine. Opioid-evoked and NA-evoked currents were elicited using the protocol in (B), by applying 30 μm morphine without washing out the 1 μm morphine in which the slice had been incubated. Naloxone was applied to reveal the baseline current level (dotted line). (D) Example recording following 6–9 h pre-incubation with 30 μm morphine. Note that compared with the NA response, the maximum possible morphine and DAMGO responses were reduced (cf. current traces in B and C). (E) Prolonged treatment with 30 μm, but not 1 μm, morphine reduced MOPr responsiveness as assessed by the maximum response to 30 μm morphine. The effect of 30 μm morphine was time dependent. Pooled data from experiments as described in (C) and (D), displaying responses to 30 μm morphine, normalized to the maximum NA response (*P<0.05 vs. control, Student’s t-test; n=3–6; error bars represent SEM). (F) Responses to NA following pre-incubation with 1 or 30 μm morphine were unchanged (n=3–6; error bars represent SEM).
Fig. 4
Fig. 4
MOPr desensitization induced by DAMGO is PKC independent. (A) Sample current recording showing a 10-min application of DAMGO (30 nm) followed by administration of morphine (morph; 30 μm), 10 μm DAMGO, naloxone (nalox; 1 μm) and noradrenaline (NA; 100 μm) to obtain the maximum opioid and NA responses. Scale bars: 50 pA and 5 min. (B) Pooled data from experiments as shown in (A). DAMGO applied for 10 min at 30 nm or 10 μm caused rapid MOPr desensitization (*P<0.05 vs. control, Student’s t-test; n=3–6). (C) The acute desensitization of the DAMGO-induced response (shown as a percentage of initial peak response) during an 8-min application of 10 μm DAMGO (open circles) was unaffected by inhibition of PKC with 1 μm Go6976 (black squares). (D) Sample current recording following 6–9 h pre-treatment in vitro with 10 μm DAMGO and DAMGO + Go6976 for the last 30–50 min followed by morphine (morph; 30 μm), naloxone (nalox; 1 μm) and noradrenaline (NA; 100 μm) to find the maximum morphine and NA responses. (E) Pooled data from experiments as shown in (D). MOPr desensitization caused by prolonged (6–9 h) DAMGO pre-incubation was not reversed by inhibition of PKC with 1 μm Go6976 (n=3–6; error bars represent SEM).
Fig. 2
Fig. 2
Prolonged exposure to morphine (1 μm) in vitro causes MOPr desensitization only when protein kinase C (PKC) is activated. (A) Sample current recording from an LC neuron incubated for > 6 h with 1 μm morphine (morph) plus 10 μm oxotremorine-M (oxo-M) followed by the drug protocol described in Fig. 3 (scale bars: 50 pA and 5 min). Naloxone (nalox) was applied to reveal the baseline current level (dotted line). (B) Following 6–9 h pre-incubation with 1 μm morphine alone, there was no decrease in the response to morphine (30 μm) shown as a percentage of the maximum noradrenaline (NA) response. However, when LC neurons were pre-treated with 1 μm morphine and either 10 μm oxo-M or 1 μm phorbol 12-myristate 13-acetate (PMA), the maximum morphine response was significantly decreased (*P<0.05, Student’s t-test vs. control; P<0.05, Student’s t-test vs. 1 μm morphine alone). Six–nine hours pre-incubation with oxo-M or PMA alone had no effect on opioid responses (n=3–6; error bars represent SEM). (C) Morphine (1 μm) in the presence of 1 μm PMA did not induce rapid MOPr desensitization. Black squares: 1 μm morphine + 1 μm PMA. Open circles: 30 μm morphine + 1 μm PMA. Error bars represent SEM.
Fig. 3
Fig. 3
Maintenance of MOPr desensitization during prolonged morphine exposure in vitro and in vivo requires ongoing PKC activation. (A) Sample current trace from an LC neuron pre-incubated with 30 μm morphine (morph) for > 6 h, followed by 30 μm morphine + 1 μm Go6976 for 30–50 min. Note that the morphine response was elevated compared with Fig. 1D. Scale bars: 50 pA and 5 min. (B) Pooled data from experiments as shown in (A). The decrease in morphine response following pre-incubation with 1 μm morphine and 10 μm oxo-tremorine-M (oxo-M) for 6–9 h was reversed by the addition of the PKC inhibitor, Go6976 (1 μm) for the final 30–50 min of pre-incubation. Similarly, the decreased morphine response following 6–9 h pre-incubation with 30 μm morphine was reversed by Go6976 (1 μm) applied during the final 30–50 min of pre-incubation (*P<0.05 vs. control; P<0.05 vs. 1 μm morphine + oxo-M; P<0.05 vs. 30 μm morphine, Student’s t-test; n=3–6). (C) Pre-incubation with 30 μm morphine for 6–9 h resulted in a significant loss of MOPr responsiveness, as seen in Fig. 1E. This effect was completely reversed when all of the conventional PKC isoforms were inhibited for the final 30–50 min of pre-incubation by application of 1 μm of the RACK inhibitor of all conventional PKC isoforms. When 1 μm each of the βI, βII and γ isoforms RACK inhibitors were co-applied, loss of MOPr responsiveness was unaffected (n=3–6). All error bars represent SEM (*P<0.05 vs. control pre-incubation; P<0.05 vs. 6–9 h 30 μm morphine pre-incubation). (D) Pooled data from experiments on slices taken from animals pre-treated with morphine for 3 days. Slices from morphine-pre-treated animals were maintained in morphine 1 μm to prevent them going into withdrawal. In these slices the maximum response (i.e. to morphine 30 μm) was significantly decreased compared with that observed in parallel experiments on LC neurons from non-morphine-treated animals. This decrease was significantly reversed by inclusion of Go6976 (1 μm) for the final 30 min before determining the maximum response to morphine. n=4–6, error bars represent SEM (*P<0.05 vs. control; P<0.05 vs. chronic morphine). (E) MOPr desensitization in slices taken from morphine-pre-treated animals is reversed if slices are maintained for 2–4 h after slicing in morphine-free bathing solution. n= 3–5, error bars represent SEM (*P<0.05 vs. control; P<0.05 vs. chronic morphine, morphine in bathing solution). (F) MOPr desensitization caused by in vivo morphine treatment is homologous. In control slices, similar responses are obtained following 30 μm morphine or 1 μm nociception. In slices taken from morphine-pre-treated animals, the nociception response is unchanged, whereas the morphine response is significantly reduced. n=3–5, error bars represent SEM (*P<0.05 vs. control animals). (G) Responses to sub-maximal and maximal concentrations of NA in slices taken from morphine-pre-treated animals (open circles) were not different to those observed in slices taken from control animals (black squares; n=5; error bars represent SEM).

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