Sexually dimorphic nociception and opioid antinociception is very pervasive but poorly understood. We had demonstrated that spinal morphine antinociception in females, but not males, requires the concomitant activation of spinal μ-and κ-opioid receptors (MOR and KOR, respectively). This finding suggests an interrelationship between MOR and KOR in females that is not manifest in males. Here, we show that expression of a MOR/KOR heterodimer is vastly more prevalent in the spinal cord of proestrous vs. diestrous females and vs. males. Cross-linking experiments in combination with in vivo pharmacological analyses indicate that heterodimeric MOR/KOR utilizes spinal dynorphin 1-17 as a substrate and is likely to be the molecular transducer for the female-specific KOR component of spinal morphine antinociception. The activation of KOR within the heterodimeric MOR/KOR provides a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves. Spinal cord MOR/KOR heterodimers represent a unique pharmacological target for female-specific pain control.estrous cycle | sexual dimorphism | sex steroids | signaling complexes | estrogen and progesterone S exual dimorphism in nociception and opioid antinociception has been extensively documented in humans (1-4) and laboratory animals (5-9). Nevertheless, underlying molecular mechanisms causally associated with sex-dependent nociception and opioid antinociception remain enigmatic. For example, there is little mechanistic understanding of why women are more likely than men to experience myriad chronic pain syndromes (1-3) as well as recurrent pain, more severe levels of pain, and pain of longer duration (10). Similarly, reports of more robust κ-opioid receptor (KOR) antinociception in females vs. males (11)(12)(13)(14) are not accompanied by compelling mechanistic rationales.In addition to proposed genetic contributions (15), the milieu of ovarian sex steroids is thought to contribute to sex-dependent nociception (5, 6) and opioid antinociception (5, 16). However, sex steroid molecular targets and their altered functionality that are relevant to sex-dependent nociception and opioid antinociception are not defined. This laboratory reported (17) that the antinociception produced by intrathecal (i.t.) morphine results from the sex-based differential recruitment of spinal analgesic components. In males, spinal morphine antinociception results from the exclusive activation of spinal μ-opioid receptor (MOR). In contrast, in females, spinal morphine antinociception requires the concomitant activation of spinal MOR and KOR (17). The most parsimonious explanation for this sex-dependent dichotomy would be the female-specific recruitment of spinal MOR/KOR heterodimers.We investigated the hypothesized sexually dimorphic expression in spinal cord of MOR/KOR heterodimers by comparing their presence in the spinal cord of male, proestrous and diestrous rats as well as rats subjected to ovariectomy. Here, ...

Maternal pain thresholds in rats were determined during various stages of pregnancy and parturition by measuring the intensity of electric shock that elicited reflexive jumping. There was a gradual rise in the pain threshold between 16 and 4 days prior to parturition and a more abrupt rise 1 to 2 days before that event. This increase was abolished by long-term administration of the narcotic antagonist naltrexone. The endorphin system is thus an important component of intrinsic mechanisms that modulate responsiveness to aversive stimuli. The data also demonstrate the activation during pregnancy of an endorphin system that is apparently quiescent in nonpregnant female rats treated the same way.

We previously demonstrated that the spinal cord κ-opioid receptor (KOR) and μ-opioid receptor (MOR) form heterodimers (KOR/MOR). KOR/MOR formation and the associated KOR dependency of spinal morphine antinociception are most robust during proestrus. Using Sprague Dawley rats, we now demonstrate that (1) spinal synthesis of estrogen is critical to these processes, and (2) blockade of either estrogen receptor (ER) α-, β-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception. Effects of blocking ERs were manifest within 15 min, whereas those of PR blockade were manifest after 18 h, indicating the requirement for rapid signaling by estrogen and transcriptional effects of progesterone. Individual or combined blockade of ERs produced the same magnitude of effect, suggesting that they work in tandem as part of a macromolecular complex to regulate KOR/MOR formation. Consistent with this inference, we found that KOR and MOR were coexpressed with ERα and G-protein-coupled ER1 in the spinal dorsal horn. Reduction of KOR/MOR by ER or PR blockade or spinal aromatase inhibition shifts spinal morphine antinociception from KOR dependent to KOR independent. This indicates a sex steroid-dependent plasticity of spinal KOR functionality, which could explain the greater analgesic potency of KOR agonists in women versus men. We suggest that KOR/MOR is a molecular switch that shifts the function of KOR and thereby endogenous dynorphin from pronociceptive to antinociceptive. KOR/MOR could thus serve as a novel molecular target for pain management in women.

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Simulation of the pregnancy blood concentration profile of 17beta-estradiol (E(2)) and progesterone (P) in nonpregnant ovariectomized rats has been shown to result in a significant elevation of nociceptive response thresholds. The present report demonstrates that spinal opioid antinociceptive responsiveness to these ovarian steroids is not sex-specific. Treatment of orchidectomized sexually mature males with an analogous regimen of E(2) and P also elicits an antinociception, the robustness and temporal profile of which is comparable with that previously observed in females. Neither E(2) nor P, alone, is sufficient to produce antinociception in male rats, as was previously demonstrated in females. Neurobiological substrates and antinociceptive mechanisms underlying ovarian sex steroid antinociception do, however, exhibit sex specificity. In males, the analgesia resulting from ovarian steroid treatment derives from the independent contributions of spinal kappa and mu, not delta, opioid receptor pathways that are additive, not synergistic. Spinal alpha(2)-noradrenergic receptor activity and its attendant analgesic synergy with spinal opioid systems do not contribute to ovarian sex steroid analgesia in males. This is in contrast to the previous demonstrations that ovarian sex steroid-induced antinociception in females results from antinociceptive synergy between activated spinal kappa/delta opioid as well as alpha(2)-noradrenergic receptor systems. The current data reveal that ovarian steroid-activated multiplicative spinal antinociceptive pathways that had been demonstrated in female rats are not manifest in their male counterparts.

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Despite the demonstration that chronic morphine increases phosphorylation of multiple substrate proteins, their identity has, for the most part, remained elusive. Thus far, chronic morphine has not been shown to increase the phosphorylation of any identified effector protein. This is the first demonstration that persistent activation of opioid receptors has profound effects on phosphorylation of adenylyl cyclase (AC). A dramatic increase in phosphorylation of AC (type II family) was observed in ileum longitudinal muscle myenteric plexus preparations obtained from chronic morphine-treated guinea pigs. Analogous results were obtained when AC was immunoprecipitated using two differentially directed AC antibodies. The magnitude of the augmented AC phosphorylation was substantially attenuated by chelerythrine, a protein kinase C-selective inhibitor. These results suggest the potential relevance of increased phosphorylation (protein kinase C-mediated) of AC to opioid tolerant/dependent mechanisms. Because phosphorylation of AC isoforms (type II family) can significantly increase their stimulatory responsiveness to Gsalpha and Gbetagamma, this mechanism could underlie, in part, the predominance of opioid AC stimulatory signaling observed in opioid tolerant/dependent tissue. Moreover, in light of the fact that many G protein-coupled receptors signal through common effector proteins, this effect provides a mechanism for divergent consequences of chronic morphine treatment and could explain the well documented complexity of changes that accompany the opioid tolerant/dependent state.

Current evidence for sex-based nociception and antinociception, largely confined to behavioral measures of pain sensitivity, chronic pain syndromes, and analgesic efficacy, provides little mechanistic insights into biological substrates causally associated with sexual dimorphic pain experience. Spinal cord has been shown to be a central nervous system region in which regulation of opioid antinociceptive substrates manifest sexual dimorphism. This site was therefore chosen to explore whether or not differential mechanisms underlie comparable spinal opioid antinociception in male and female rodents. Intrathecal (i.t.) application of morphine to male and female rats produces a thermal antinociception equivalent in magnitude and temporal profile. Nevertheless, it results from the sex-based differential recruitment of spinal analgesic components. As expected, the spinal -opioid receptor is critical for i.t. morphine antinociception in both sexes. However, in females, but not males, activation by i.t. morphine of spinal -opioid receptors is a prerequisite for spinal morphine antinociception. Furthermore, in females, but not males, i.t. application of antidynorphin antibodies substantially attenuates the antinociception produced by i.t. morphine. This indicates that the antinociception that results from the i.t. application of morphine in females requires the functional recruitment of spinal dynorphin. Female-specific recruitment by i.t. morphine of a spinal dynorphin/-opioid receptor pathway results from organizational consequences of ovarian sex steroids and not the absence of testicular hormones. These observations suggest that sexual dimorphic pain and analgesic mechanisms might be far more pervasive than commonly thought and underscore the imperative for including female as well as male subjects in all studies of pain and antinociception.Sexual dimorphism in nociception and opioid antinociception has been well documented in humans (Ellermeier and Westphal, 1995;Unruh, 1996;Berkley, 1997;Fillingim et al., 1998;Walker and Carmody, 1998) and laboratory animals (Mogil et al., 1993;Coyle et al., 1995Coyle et al., , 1996Kayser et al., 1996;Mogil and Chanda, 2005). Previously, evidence for sexbased nociception and antinociception has been largely confined to behavioral measures revealing differential pain sensitivity, frequency and severity of chronic pain syndromes, and divergent analgesic efficacy of opioid receptor-type-selective agonists. Although underscoring the occurrence of sexual dimorphism in pain processing and its regulation, these studies provide little insight into biological substrates and neuronal organizational parameters that might underlie such sexual dimorphism.The spinal cord has been shown to be a central nervous system region in which components of opioid analgesic pathways and their regulation manifest sexual dimorphism. For example, the density of the -opioid receptor (KOR) and its distribution within axon terminals differs between the spinal cord of male and female rodents (Harris et al....