Background
In the nervous system, G protein-coupled Mu-opioid receptors (MORs) drive the initial steps of both the positive effects of opioids (i.e. relief of intense inflammatory pain) and their addictive effects. A desensitization to morphine that last for several days can occur within hours of administering an appropriate single dose [
1] and this is accompanied by some degree of physical dependence [
2]. Both single-dose tolerance and that promoted by repeated exposure to morphine seem to share some certain molecular mechanisms. Indeed, both situations can be modulated by similar pharmacological treatments [
3].
The inactivation of G protein-coupled receptors (GPCRs) commences with the activation of G proteins upon agonist binding, which in turn produces the segregation of GαGTP subunits from the Gβγ dimers. The increased pool of free Gβγ dimers facilitates their binding to the G protein-coupled receptor kinases (GRK) and hence, the interaction between these kinases and the receptors. In this way, the agonist-bound receptors become a GRK substrate, leading to the phosphorylation of critical cytosolic serine/threonine residues in the receptor. This modification enables β-arrestin to bind to these residues if the agonist remains bound to the receptor [
4], setting in motion an endocytic process. Recycling of these internalized receptors to the plasma membrane must occur for the response to agonists to be more rapidly recovered [
5]. However, the proteolytic degradation of the endocytosed receptors in lysosomes promotes the down-regulation of the number of surface receptors and brings about a decreased response to the agonist [
6].
The phosphorylation of serine 375 in the C terminus of the MOR accompanies the agonist-driven internalization process [
7,
8]. Although the endocytosed MORs can be sorted into lysosomes, the majority recycle rapidly to the plasma membrane through a signal-dependent process [
9]. Interestingly, the efficiency of opioid agonists to stimulate MOR endocytosis differs and this is related to their capacity to promote GRK-dependent phosphorylation of cytosolic residues in the MOR [
10,
11]. It is believed that morphine induces a high degree of desensitization because it fails to provoke significant phosphorylation and internalization of the MORs [
12]. Therefore, opioid agonists that efficiently promote MOR endocytosis would not be associated with high opioid tolerance [
13].
It is evident that studies on cells have revealed some critical mechanisms that control the activity of cell surface MORs. However, there is still limited information on the molecular processes that are involved in regulating MORs in the mature nervous system. In this respect, opioid agonists such as etorphine and DAMGO have been shown through immunofluorescence techniques to produce MOR internalization in brain, spinal cord and dorsal root ganglia neurons [
14‐
16]. Notably, and in contrast to what is observed in cultured cells, morphine produces some membrane trafficking of the MORs in dendrites of nucleus accumbens neurons and more extensive MOR internalization in embryonic striatal neurons and ganglia neurons [
17‐
19]. Therefore, although the essential mechanisms of MOR regulation established in cultured cells could apply to neurons, these highly specialized cells also have their own rules to control GPCR function. For example, the expression of certain RGS proteins such as members of RGSZ1, RGSZ2, RGS-R7 subfamily, and of Gαz subunits, is virtually restricted to nervous tissue, and these proteins certainly influence the regulation of neural MORs [
20].
We set out here to evaluate the implication of the phosphorylation, internalization and recycling of MORs on the desensitizing capacity of morphine and DAMGO in the murine nervous system. We show that tolerance to intracerebroventricular (icv) morphine was induced by the stable transfer of part of the MOR-activated Gα subunits to RGS proteins of the R7 and Rz subfamilies [
21,
22], thereby increasing the pool of free Gβγ dimers in the receptor environment. Afterwards, subsequent doses or prolonged exposure to this opioid promoted the GRK phosphorylation of MORs and their internalization and recycling. In these circumstances, the effects that remain after the first dose now desensitized at a much slower rate. DAMGO evaded the first part of this process directly producing the efficient Ser375 phosphorylation and recycling of MORs, which was accompanied by low tolerance to its effects. However, repeated exposure to these opioids led to the incomplete recycling of the MORs and strong tolerance developed.
Discussion
The interaction of morphine with neural MORs is the initial step, both in the development of tolerance to this opioid and towards physical dependence. By analyzing MORs during the time-course of opioid antinociception, new aspects of the mechanisms that control these G-receptors in nervous tissue were revealed. The initial exposure to DAMGO or morphine brought about changes at the MOR level that compared satisfactorily with those described in cultured cells. In both systems, DAMGO produces robust Ser375 phosphorylation and internalization of the MORs whereas in contrast, morphine only weakly induces these processes. In addition, following the removal of DAMGO the MORs recycle back to the cell membrane resensitizing the response to the opioid. However, on removal of morphine the cells remain desensitized and exhibit cross-tolerance to DAMGO. Therefore, while DAMGO produces low tolerance to the effects of subsequent opioid administration, morphine yields a high tolerance. Nevertheless, in mature neurons and in contrast to what might be expected if the MORs were to resensitize on withdrawal of the agonists, a second dose of DAMGO had a weaker analgesic effect after an interval of 24 h. This time-dependent desensitization of MORs was more evident when the effect of the second dose of morphine was studied, decreasing rapidly until the interval between doses reached about 6 h. Longer intervals did not increase desensitization and the analgesic activity of morphine is progressively restored after 3 or 4 days [
28]. These observations coincide with the notion that DAMGO and morphine produce low and high tolerance respectively. However, the delayed tolerance to an acute dose of opioid that operates in nervous tissue is known as single-dose tolerance [
1], and this phenomenon is probably related to the permanent transfer of Gα subunits to a subset of signaling proteins specific to this tissue.
There is convincing evidence that relates the ability of DAMGO to promote the Ser375 phosphorylation, internalization and recycling of MORs with its weak desensitizing capacity. Accordingly, when morphine promotes Ser-phosphorylation and internalization of MORs in cells [
8,
11,
32], weak MOR desensitization develops [
8,
13]. In our experimental paradigm, the second dose of 10 nmol morphine spaced 6 h from the first promoted about one third of the analgesic effects of the first dose, coupled with intense phosphorylation and recycling of the MORs. The reduced antinociceptive effects of this second dose of morphine were relatively well reproduced by subsequent administrations of this same dose of morphine but spaced 24 h apart (present work; [
33]). Thus, resensitization of MORs in neurons also requires the recovery of active receptors in the cell membrane. This can be achieved by de novo synthesis, although MOR turnover in the brain takes several days [
28,
29]. Alternatively, and much more rapidly resensitization may occur through the dephosphorylation and recycling of the internalized MORs to the plasma membrane. While the first situation would correspond to the recovery from the first morphine dose, the second applies to the recovery from DAMGO administration or from a second dose of morphine given at least 6 h after the first. Therefore, the use of agonists such as DAMGO could be associated with a reasonable risk of producing tolerance given that the MORs belong to the class of GPCRs that are rapidly dephosphorylated and recycled after internalization, [
12,
34]. Nevertheless, a fraction of these internalized receptors are sorted to lysosomes and undergo proteolytic degradation [
9]. Thus, the repeated administration of DAMGO or morphine could finally desensitize MORs, as observed after administering three consecutives doses of these opioids. It could be argued that agonists that attain their response by activating only a small fraction of MORs would be preferred for the control of severe pain. However, it must be born in mind when used in demanding protocols, these agonists deplete the surface MORs before the novo synthesis can restore the system, which also leads to inescapable desensitization ([
2,
35,
36], present study). Interestingly, even in the demanding protocol used here, the effects that remain after the third dose of morphine or DAMGO were fairly well reproduced by a fourth dose given 18 h later. Obviously, it is difficult to extrapolate this observation to what it is required to effectively drive opioid consumption. However, the biological effects that these opioids conserve after their repeated administration could control physical dependence and therefore, be responsible for the craving behavior.
Morphine is a representative of a particular class of opioid agonists that are useful as analgesics but that are associated with the risk of producing strong tolerance. The limited capacity of morphine to stimulate both Ser375 phosphorylation and MOR internalization could be due to its high off rate from the activated MOR. Thus, morphine will not remain bound to the receptors for long, thereby reducing the probability of GRK phosphorylation and/or the subsequent binding of β-arrestin to the agonist-activated MOR to initiate internalization. The MORs expressed in HEK 293 cells elude internalization upon exposure to morphine, even if the opioid is incubated for long periods of time at high concentrations [
32]. The receptors remain at the cell surface and since G protein coupling is essential to increase their affinity towards agonists but not to antagonists, then phosphorylation and uncoupling from G proteins probably desensitizes MORs [
8]. In contrast, the MORs present in embryonic cultured neurons were internalized upon incubation with morphine [
18]. While an acute dose of morphine produces desensitization without the loss of surface receptors ([
14], present study), the administration of subsequent doses or continuous administration promotes the phosphorylation and internalization of MORs. Therefore, these observations again indicate that different processes regulate MOR activity in mature neurons.
One of such process transfers the control of opioid-activated Gα subunits from the MOR to certain RGS proteins. The internalization of the MORs provokes the return of most of these Gα subunits to re-constitute the G proteins and resensitize the response to the agonist when they again come under the control of the recycled receptors. However, long-lasting transfer (sequestering) of MOR-activated Gα subunits occurs when the effects of morphine reach a certain level [
21]. This phenomenon is mediated by proteins of the RGS-R7/Rz subfamilies, among which RGS9 and RGSZ2 are particularly relevant [
22,
33]. This more persistent interaction seems to be facilitated by post-translational modifications of these RGS proteins, permitting them to bind to the activated Gα subunits but precluding their GAP activity on them. Among such modifications, the phosphorylation of serine residues in the RGS domain of RGS-R7 proteins and the ensuing binding to 14-3-3 proteins appear to be highly relevant [
21], as does the sumoylation of specific sequences in the RGS of RGS-Rz proteins [
22]. The consolidation of this transfer is time-dependent and is probably mediated by the action of certain kinases. Interestingly, PKC has been implicated in MOR desensitization to morphine, but little in the effects of DAMGO [
37]. Moreover, the antagonists of NMDA receptors reduce the development of tolerance to morphine antinociception but have little effect on that promoted by DAMGO [
38]. Thus, the sequestering of morphine-activated Gα subunits at RGS proteins could involve the activation of glutamate NMDA receptors, probably via PKC [
39]. Further efforts will focus on characterizing these mechanisms responsible for the more resolute transfer of morphine-activated Gα subunits to the RGS proteins.
As consequence of impeding the return of GαGDP subunits would be the accumulation of free Gβγ dimers in the environment of the MOR and the improved access of GRKs. Thus, another dose of morphine will promote GRK phosphorylation of the activated MORs. The internalization of MORs produces a reduction in agonist signaling and thus, this RGS-mediated mechanism would exert only a minor effect. Hence, to diminish the signaling of agonists that promote little or no internalization of MORs (e.g. morphine), neural cells would sequester Gα subunits. In this way, the impact of agonist signaling would be reduced and the GRK phosphorylation of MORs would also increase. The influence of such events depends not only on the effect promoted by morphine but also on the interval elapsed after the initial administration of the opioid [
21]. This characteristic could explain why delayed tolerance is only observed when a second dose of morphine is injected within a certain time interval. It could also account for the limited desensitization observed for DAMGO when a second dose was administered 24 h after the first, and no before. At this late interval, moderate sequestering of Gα subunits by RGSZ2 proteins could be consolidated and might provoke the reduction in the antinociceptive response to DAMGO. Mice with reduced levels of RGS9 proteins display both an increase in the analgesic effects of morphine and a poorer single-dose tolerance. Therefore, neural MORs can be regulated at the Gα subunit level, as well as through the associated RGS proteins. Hence, opioid resensitization not only requires MOR internalization but also that the recycled receptors recover control of the G proteins. This knowledge can be complemented with the possibility of delaying the development of tolerance, or even rescuing the system, by influencing regulatory mechanisms that only operate in mature neurons and in which a subset of signaling proteins participates.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MRM and ETM performed the molecular studies, and contributed to the analysis and interpretation of the data. PSB designed and performed the behavioral studies. JG conceived the study, participated in its design, and assisted with the data analysis and interpretation. JG and PSB wrote and revised the manuscript. All authors have read and approved the final manuscript.