Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine

Abstract

The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction.

Keywords: mu-opioid receptor; opioid tolerance; sexual dimorphism; splice variants.

Figure 1. Sex-dependent effect of chronic morphine on the mRNA expression of MOR variants

Male and female rats were treated with escalating doses of morphine base pellets (see methods). Data are calculated as fold change in MOR mRNA content, where 2^−ΔΔCT represents the change in spinal mRNA content in opioid naïve (Nv) vs. chronic morphine treated (CM) groups relative to that of rMOR-1R. Chronic morphine significantly upregulated two splice variants of MOR, rMOR-1B2 (M-1B2) and rMOR-1C1 (M-1C1) in only males (*= ~ 2 fold; p<0.05). n=5-8).

Figure 2. Effect of chronic systemic morphine on MOR density in spinal cord of male rat

Lumbar spinal tissue obtained from two placebo or chronic morphine-treated male rats were dissected and pooled for each saturation binding experiment using [³H]DAMGO. (A) Mean [³H]DAMGO B_max±SEM was determined in spinal cord from naïve (109.5±3.0 fmol/mg protein) and chronic morphine treated (125.3±4.5 fmol/mg protein) groups (*p<0.05; n=6). Hatched portion indicates the increment in [³H]DAMGO B_max following chronic-morphine treatment. (B) A representative scatchard plot is shown for naïve and chronic morphine-treated spinal cord. No significant difference was observed between [³H]DAMGO K_Ds in naïve (1.49±0.04 nM) and morphine-treated spinal cord (1.60±0.03 nM) group (p>0.05).