Relevance of c-Src and protein phosphatase 2A to aromatase activity: Evidence of an acute self-regulating oestrogenic signalling complex in rat central nervous system.

We previously reported that aromatase protein levels do not parallel aromatase enzyme activity. This suggests that oestrogenic signalling may be modulated via post-translational modification of aromatase protein. The tyrosine and serine phosphorylation state of aromatase are known to influence its activity. To investigate the possible relevance of aromatase phosphorylation to the incongruity observed between aromatase protein and its activity, we explored interactions between aromatase and the tyrosine kinase c-Src and the serine protein phosphatases 2A and 5 (PP2A and PP5), as well as the relationship between levels of tyrosine-phosphorylated aromatase and the extrapolated aromatase activity. We found that (a) hypothalamic aromatase was significantly more heavily tyrosine-phosphorylated than spinal aromatase; (b) aromatase was oligomerized with c-Src and PP2A/PP5, potentially activating aromatase via tyrosine-phosphorylation and serine-dephosphorylation; (c) the associations of c-Src and PP2A/PP5 with hypothalamic aromatase were substantially greater than with spinal aromatase; and (d) aromatase, oestrogen receptor α, PP2A, and c-Src were present in a common membrane oligomer. The existence of c-Src and PP2A in an oligomer that also contains aromatase and membrane oestrogen receptor α (and presumably other signalling molecules) indicates the presence in the CNS of a potentially self-regulating oestrogenic signalling unit. The degree to which such a complex operates autonomously and the regulatory factors thereof are likely to have substantial physiological implications and clinical relevance.

Arbiters of endogenous opioid analgesia: role of CNS estrogenic and glutamatergic systems.

Nociception and opioid antinociception in females are pliable processes, varying qualitatively and quantitatively over the reproductive cycle. Spinal estrogenic signaling via membrane estrogen receptors (mERs), in combination with multiple other signaling molecules [spinal dynorphin, kappa-opioid receptors (KOR), glutamate and metabotropic glutamate receptor 1 (mGluR1)], appears to function as a master coordinator, parsing functionality between pronociception and antinociception. This provides a window into pharmacologically accessing intrinsic opioid analgesic/anti-allodynic systems. In diestrus, membrane estrogen receptor alpha (mERα) signals via mGluR1 to suppress spinal endomorphin 2 (EM2) analgesia. Strikingly, in the absence of exogenous opioids, interfering with this suppression in a chronic pain model elicits opioid anti-allodynia, revealing contributions of endogenous opioid(s). In proestrus, robust spinal EM2 analgesia is manifest but this requires spinal dynorphin/KOR and glutamate-activated mGluR1. Furthermore, spinal mGluR1 blockade in a proestrus chronic pain animal (eliminating spinal EM2 analgesia) exacerbates mechanical allodynia, revealing tempering by endogenous opioid(s). A complex containing mu-opioid receptor, KOR, aromatase, mGluRs, and mERα are foundational to eliciting endogenous opioid anti-allodynia. Aromatase-mERα oligomers are also plentiful, in a central nervous system region-specific fashion. These can be independently regulated and allow estrogens to act intracellularly within the same signaling complex in which they are synthesized, explaining asynchronous relationships between circulating estrogens and central nervous system estrogen functionalities. Observations with EM2 highlight the translational relevance of extensively characterizing exogenous responsiveness to endogenous opioids and the neuronal circuits that mediate them along with the multiplicity of estrogenic systems that concomitantly function in phase and out-of-phase with the reproductive cycle.

Relevance of Mu-Opioid Receptor Splice Variants and Plasticity of Their Signaling Sequelae to Opioid Analgesic Tolerance.

Opioid dose escalation to effectively control pain is often linked to the current prescription opioid abuse epidemic. This creates social as well as medical imperatives to better understand the mechanistic underpinnings of opioid tolerance to develop interventions that minimize it, thereby maximizing the analgesic effectiveness of opioids. Profound opioid analgesic tolerance can be observed in the absence of mu-opioid receptor (MOR) downregulation, aggregate MOR G protein uncoupling, and MOR desensitization, in the absence of impaired G protein coupled receptor kinase phosphorylation, arrestin binding, or endocytosis. Thus, we have explored alternative biochemical sequelae that might better account for opioid analgesic tolerance. Our findings indicate that substantial plasticity among upstream and downstream components of opioid receptor signaling and the emergence of alternative signaling pathways are major contributors to opioid analgesic tolerance. An exemplar of this plasticity is our findings that chronic morphine upregulates the MOR variants MOR-1B2 and MOR-1C1 and phosphorylation of their C-terminal sites not present in MOR-1, events causally associated with the chronic morphine-induced shift in MOR G protein coupling from predominantly Gi/Go inhibitory to Gs-stimulatory adenylyl cyclase signaling. The unique feature(s) of these variants that underlies their susceptibility to adapting to chronic morphine by altering the nature of their G protein coupling reveals the richness and pliability of MOR signaling that is enabled by generating a wide diversity of MOR variants. Furthermore, given differential anatomical expression patterns of MOR variants, MOR splice variant-dependent adaptations to chronic morphine could enable mechanistic underpinnings of tolerance and dependence that are CNS region- and cell-specific.

Phosphorylation of unique C-terminal sites of the mu-opioid receptor variants 1B2 and 1C1 influences their Gs association following chronic morphine.

We recently demonstrated in rat spinal cord that a regimen of escalating doses of systemic morphine, analogous to regimens used clinically for chronic pain management, selectively up-regulates the mu-opioid receptor (MOR) splice variants MOR-1B2 and MOR-1C1 mRNA and functional protein. This study investigated the potential relevance of up-regulating MOR-1B2 and MOR-1C1 to the ability of chronic morphine to shift MOR signaling from predominantly Gi /Go inhibitory to Gs stimulatory. Specifically, we tested the hypotheses that chronic morphine induces phosphorylation of carboxyl terminal sites unique to MOR-1B2 and MOR-1C1, and that this phosphorylation is causally related to augmented association of these variants with Gs α. Hypotheses were validated by (i) abolition of the chronic morphine-induced increment in MOR-1C1 and MOR-1B2 association with Gs α by inhibitors of protein kinase A and Casein kinase 2, respectively; (ii) failure of chronic morphine to augment MOR variant Gs α interactions in Chinese hamster ovary cells transiently transfected with either rat MOR-1C1 or MOR-1B2 in which targeted protein kinase A and Casein kinase 2 serine phosphorylation sites, respectively, were mutated to alanine; (iii) abrogation of chronic morphine-induced augmented MOR Gs α association in spinal cord of male rats following intrathecal administration of dicer substrate small interfering RNAs targeting MOR-1B2/MOR-1C1 mRNA. The ability of chronic morphine to not only up-regulate-specific MOR variants but also their carboxyl terminal phosphorylation and consequent augmented association with Gs α may represent a novel component of opioid tolerance mechanisms, suggesting novel potential targets for tolerance abatement.

Estrogens as arbiters of sex-specific and reproductive cycle-dependent opioid analgesic mechanisms.

The organization of estrogenic signaling in the CNS is exceedingly complex. It is comprised of peripherally and centrally synthesized estrogens, and a plethora of types of estrogen receptor that can localize to both the nucleus and the plasma membrane. Moreover, CNS estrogen receptors can exist independent of aromatase (aka estrogen synthase) as well as oligomerize with it, along with a host of other membrane signaling proteins. This ability of CNS estrogen receptors to either to physically pair or exist separately enables locally produced estrogens to act on multiple spatial levels, with a high degree of gradated regulation and plasticity, signaling either in-phase or out-of phase with circulating estrogens. This complexity explains the numerous contradictory findings regarding sex-dependent pain processing and sexually dimorphic opioid antinociception. This review highlights the increasing awareness that estrogens are major endogenous arbiters of both opioid analgesic actions and the mechanisms used to achieve them. This behooves us to understand, and possibly intercede at, the points of intersection of estrogenic signaling and opioid functionality. Factors that integrate estrogenic actions at subcellular, synaptic, and CNS regional levels are likely to be prime drug targets for novel pharmacotherapies designed to modulate CNS estrogen-dependent opioid functionalities and possibly circumvent the current opioid epidemic.

Harnessing endogenous opioids for pain relief: Fantasy vs reality.

OBJECTIVE: To review evidence demonstrating efficacy and feasibility of harnessing the activity of endogenous opioid analgesic systems for pain management. METHODS: The authors sought to summarize a wealth of data that establish proof of concept that the analgesic activity of endogenous opioids can be exploited to clinically benefit from the enormous pain-relieving abilities of these peptides without contributing to the current crisis of death by synthetic opioid overdose. RESULTS: There is a plethora of studies demonstrating that not only can endogenous opioids mediate placebo-induced antinociception but they are also active in modulating clinical pain. Earlier studies convincingly demonstrate the effec-tiveness of psychological strategies to coopt endogenous opioid analgesic systems to produce pain relief. The challenge is to define pharmacological targets for activating endogenous opioid analgesia reliably in a clinical setting. Based on insights gleaned from mechanisms underlying the ebb and flow of analgesic responsiveness to the spinal application of endomorphin 2, multiple signaling proteins were identified that activate endogenous spinal opioid analgesia. Notably, this was achieved in the absence of any exogenous synthetic opioid. CONCLUSIONS: Utilization of drugs that harness endogenous opioid antinociception in accordance with varying physiological states represents a novel approach for effective pain management while mitigating the present epidemic of death by synthetic opioid overdose.

Pharmacological Modulation of Endogenous Opioid Activity to Attenuate Neuropathic Pain in Rats.

We showed previously that spinal metabotropic glutamate receptor 1 (mGluR1) signaling suppresses or facilitates (depending on the stage of estrous cycle) analgesic responsiveness to intrathecal endomorphin 2, a highly mu-opioid receptor-selective endogenous opioid. Spinal endomorphin 2 antinociception is suppressed during diestrus by mGluR1 when it is activated by membrane estrogen receptor alpha (mERα) and is facilitated during proestrus when mGluR1 is activated by glutamate. In the current study, we tested the hypothesis that in female rats subjected to spinal nerve ligation (SNL), the inhibition of spinal estrogen synthesis or blockade of spinal mERα/mGluR1 would be antiallodynic during diestrus, whereas during proestrus, mGluR1 blockade would worsen the mechanical allodynia. As postulated, following SNL, aromatase inhibition or mERα/mGluR1 blockade during diestrus markedly lessened the mechanical allodynia. This was observed only on the paw ipsilateral to SNL and was eliminated by naloxone, implicating endogenous opioid mediation. In contrast, during proestrus, mGluR1 blockade worsened the SNL-induced mechanical allodynia of the ipsilateral paw. Findings suggest menstrual cycle stage-specific drug targets for and the putative clinical utility of harnessing endogenous opioids for chronic pain management in women, as well as the value of, if not the necessity for, considering menstrual cycle stage in clinical trials thereof. PERSPECTIVE: Intrathecal treatments that enhance spinal endomorphin 2 analgesic responsiveness under basal conditions lessen mechanical allodynia in a chronic pain model. Findings provide a foundation for developing drugs that harness endogenous opioid antinociception for chronic pain relief, lessening the need for exogenous opioids and thus prescription opioid abuse.

Exploiting endogenous opioids: Lessons learned from endomorphin 2 in the female rat.

Effective management of chronic pain is demanded by ethical as well as medical considerations. Although opioid analgesics remain among the most effective pharmacotherapies for ameliorating many types of pain, their use is clouded by concerns regarding their addictive properties, underscored by the current epidemic of prescription opioid abuse and attendant deaths. Medicinal harnessing of endogenous opioid antinociception could provide a strategy for continuing to take advantage of the powerful antinociceptive properties of opioids while avoiding their abuse potential. Based on our studies of endogenous mechanism that suppress and facilitate spinal endomorphin 2 antinociception over the rat reproductive cycle, we identified multiple signaling molecules that could serve as targets for activating endogenous opioid analgesia for chronic pain management in women. Our findings emphasize the need for a precision medicine approach that includes stage of menstrual cycle as an important determinant of drug targets for (activating/harnessing) endogenous opioid antinociceptive systems/ capabilities. Utilization of drugs that harness endogenous opioid antinociception in accordance with varying physiological states represents a novel approach for effective pain management.

Physical Linkage of Estrogen Receptor α and Aromatase in Rat: Oligocrine and Endocrine Actions of CNS-Produced Estrogens.

Rapid-signaling membrane estrogen receptors (mERs) and aromatase (Aro) are present throughout the central nervous system (CNS), enabling acute regulation of CNS estrogenic signaling. We previously reported that spinal membrane Aro (mAro) and mERα oligomerize (1). As their organizational relationship would likely influence functions of locally produced estrogens, we quantified the mAro and mERα that are physically associated and nonassociated in two functionally different regions of rat CNS: the spinal cord, which has predominantly neural functionalities, and the hypothalamus, which has both neural and endocrine capabilities. Quantitative immunoprecipitation (IP), coimmunoprecipitation, and Western blot analysis were used to quantify the associated and nonassociated subpopulations of mAro and mERα. Regardless of estrous-cycle stage, virtually all mAro was oligomerized with mERα in the spinal cord, whereas only ∼15% was oligomerized in the hypothalamus. The predominance of nonassociated mAro in the hypothalamus, in combination with findings that many hypothalamic Aro-immunoreactive neurons could be retrogradely labeled with peripherally injected Fluoro-Gold, suggests that a portion of hypothalamic estrogens is secreted, potentially regulating pituitary function. Moreover, circulating estrogens increased hypothalamic Aro activity (quantified by the tritiated water-release assay) in the absence of increased Aro protein, revealing nongenomic regulation of Aro activity in the mammalian CNS. The demonstrated presence of associated and nonassociated mAro and mERα subpopulations in the CNS suggests that their selective targeting could restore impaired estrogen-dependent CNS functionalities while minimizing unwanted effects. The full physiological ramifications of brain-secreted estrogens remain to be explored.

Plasticity of Signaling by Spinal Estrogen Receptor α, κ-Opioid Receptor, and Metabotropic Glutamate Receptors over the Rat Reproductive Cycle Regulates Spinal Endomorphin 2 Antinociception: Relevance of Endogenous-Biased Agonism.

We previously showed that intrathecal application of endomorphin 2 [EM2; the highly specific endogenous µ-opioid receptor (MOR) ligand] induces antinociception that varies with stage of the rat estrous cycle: minimal during diestrus and prominent during proestrus. Earlier studies, however, did not identify proestrus-activated signaling strategies that enable spinal EM2 antinociception. We now report that in female rats, increased spinal dynorphin release and κ-opioid receptor (KOR) signaling, as well as the emergence of glutamate-activated metabotropic glutamate receptor 1 (mGluR1) signaling, are critical to the transition from an EM2 nonresponsive state (during diestrus) to an analgesically responsive state (during proestrus). Differential signaling by mGluR1, depending on its activation by membrane estrogen receptor α (mERα; during diestrus) versus glutamate (during proestrus), concomitant with the ebb and flow of spinal dynorphin/KOR signaling, functions as a switch, preventing or promoting, respectively, spinal EM2 antinociception. Importantly, EM2 and glutamate-containing varicosities appose spinal neurons that express MOR along with mGluRs and mERα, suggesting that signaling mechanisms regulating analgesic effectiveness of intrathecally applied EM2 also pertain to endogenous EM2. Regulation of spinal EM2 antinociception by both the nature of the endogenous mGluR1 activator (i.e., endogenous biased agonism at mGluR1) and changes in spinal dynorphin/KOR signaling represent a novel mechanism for modulating analgesic responsiveness to endogenous EM2 (and perhaps other opioids). This points the way for developing noncanonical pharmacological approaches to pain management by harnessing endogenous opioids for pain relief.SIGNIFICANCE STATEMENT The current prescription opioid abuse epidemic underscores the urgency to develop alternative pharmacotherapies for managing pain. We find that the magnitude of spinal endomorphin 2 (EM2) antinociception not only varies with stage of reproductive cycle, but is also differentially regulated during diestrus and proestrus. This finding highlights the need for sex-specific and cycle-specific approaches to pain management. Additionally, our finding that spinal EM2 antinociception in female rats is regulated by both the ebb and flow of spinal dynorphin/κ-opioid receptor signaling over the estrous cycle, as well as the nature of the endogenous mGluR1 activator, could encourage noncanonical pharmacological approaches to pain management, such as harnessing endogenous opioids for pain relief.

Estrogens synthesized and acting within a spinal oligomer suppress spinal endomorphin 2 antinociception: ebb and flow over the rat reproductive cycle.

The magnitude of antinociception elicited by intrathecal endomorphin 2 (EM2), an endogenous mu-opioid receptor (MOR) ligand, varies across the rat estrous cycle. We now report that phasic changes in analgesic responsiveness to spinal EM2 result from plastic interactions within a novel membrane-bound oligomer containing estrogen receptors (mERs), aromatase (aka estrogen synthase), metabotropic glutamate receptor 1 (mGluR1), and MOR. During diestrus, spinal mERs, activated by locally synthesized estrogens, act with mGluR1 to suppress spinal EM2/MOR antinociception. The emergence of robust spinal EM2 antinociception during proestrus results from the loss of mER-mGluR1 suppression, a consequence of altered interactions within the oligomer. The chemical pairing of aromatase with mERs within the oligomer containing MOR and mGluR1 allows estrogens to function as intracellular messengers whose synthesis and actions are confined to the same signaling oligomer. This form of estrogenic signaling, which we term "oligocrine," enables discrete, highly compartmentalized estrogen/mER-mGluR1 signaling to regulate MOR-mediated antinociception induced by EM2. Finally, spinal neurons were observed not only to coexpress MOR, mERα, aromatase, and mGluR1 but also be apposed by EM2 varicosities. This suggests that modulation of spinal analgesic responsiveness to exogenous EM2 likely reflects changes in its endogenous analgesic activity. Analogous suppression of spinal EM2 antinociception in women (eg, around menses, comparable with diestrus in rats) as well as the (pathological) inability to transition out of that suppressed state at other menstrual cycle stages could underlie, at least in part, the much greater prevalence and severity of chronic pain in women than men.

Chronic opioid treatment augments caveolin-1 scaffolding: relevance to stimulatory µ-opioid receptor adenylyl cyclase signaling.

Caveolin-1 is the predominant structural protein of caveolae, a subset of (lipid) membrane rafts that compartmentalize cell signaling. Caveolin-1 binds most to G protein-coupled receptors and their signaling partners, thereby enhancing interactions among signaling cascade components and the relative activation of specific G protein-coupled pathways. This study reveals that chronic opioid exposure of µ-opioid receptor (MOR) expressing Chinese hamster ovary cells (MOR-CHO) and chronic in vivo morphine exposure of rat spinal cord augmented recruitment of multiple components of MOR-adenylyl cyclase (AC) stimulatory signaling by caveolin-1. Strikingly, in MOR-CHO and spinal cord, blocking the caveolin-1 scaffolding domain substantially attenuated the chronic morphine-induced increased interaction of caveolin-1 with MOR, Gsα, protein phosphatase 2A (PP2A), and AC. Chronic morphine treatment also increased interactions among the above signaling proteins, thus enabling sufentanil to stimulate (rather than inhibit) cAMP production within lipid membrane microdomains. The latter finding underscores the functionality of augmented interactions among MOR, Gs α, PP2A, and AC. In the aggregate, our data strongly suggest that augmented caveolin-1 scaffolding undergirds the ability of chronic opioids to recruit an ancillary signaling pathway by acting as an organizing template for MOR-Gs α-AC signaling and delimiting the membrane compartment(s) in which it occurs. Since caveolin-1 binds to a wide spectrum of signaling molecules, altered caveolin-1 scaffolding following chronic opioid treatment is likely to pertain to most, if not all, MOR signaling partners. The chronic morphine-induced trigger that augments caveolin-1 scaffolding could represent a seminal perturbation that initiates the wide spectrum of adaptations thought to contribute to opioid tolerance and dependence.

Contribution of Endogenous Spinal Endomorphin 2 to Intrathecal Opioid Antinociception in Rats Is Agonist Dependent and Sexually Dimorphic.

UNLABELLED: Interactions between exogenous and endogenous opioids are not commonly investigated as a basis for sexually dimorphic opioid analgesia. We investigated the influence of spinal endomorphin 2 (EM2), an endogenous mu-opioid receptor (MOR) ligand, on the spinal antinociception produced by intrathecally administered opioids. Activation of spinal MORs facilitated spinal EM2 release. This effect was sexually dimorphic, occurring in males but not in females. Although activational effects of testosterone were required for opioid facilitation of spinal EM2 release in males, the absence of this facilitation in females did not result from either insufficient levels of testosterone or mitigating effects of estrogens. Strikingly, in males, the contribution of spinal EM2 to the analgesia produced by intrathecally applied MOR agonists depended on their analgesic efficacy relative to that of EM2. Spinal EM2 released by the higher efficacy MOR agonist sufentanil diminished sufentanil's analgesic effect, whereas EM2 released by the lower efficacy morphine had the opposite effect on spinal morphine antinociception. Understanding antithetical contributions of endogenous EM2 to intrathecal opioid antinociception not only enlightens the selection of opioid medications for pain management but also helps to explain variable sex dependence of the antinociception produced by different opioids, facilitating the acceptance of sexually dimorphic antinociception as a basic tenet. PERSPECTIVE: The male-specific MOR-coupled enhancement of spinal EM2 release implies a parallel ability to harness endogenous EM2 antinociception. The inferred diminished ability of females to utilize the spinal EM2 antinociceptive system could contribute to their greater frequency and severity of chronic pain syndromes.

Estrogens Suppress Spinal Endomorphin 2 Release in Female Rats in Phase with the Estrous Cycle.

BACKGROUND/AIMS: Male and female rats differ in their ability to utilize spinal endomorphin 2 (EM2; the predominant mu-opioid receptor ligand in spinal cord) and in the mechanisms that underlie spinal EM2 analgesic responsiveness. We investigated the relevance of spinal estrogen receptors (ERs) to the in vivo regulation of spinal EM2 release. METHODS: ER antagonists were administered directly to the lumbosacral spinal cord of male and female rats, intrathecal perfusate was collected, and resulting changes in EM2 release were quantified using a plate-based radioimmunoassay. RESULTS: Intrathecal application of an antagonist of either estrogen receptor-α (ERα) or the ER GPR30 failed to alter spinal EM2 release. Strikingly, however, the concomitant blockade of ERα and GPR30 enhanced spinal EM2 release. This effect was sexually dimorphic, being absent in males. Furthermore, the magnitude of the enhancement of spinal EM2 release in females was dependent upon estrous cycle stage, suggesting a relationship with circulating levels of 17ß-estradiol. The rapid onset of enhanced EM2 release following intrathecal application of ERα/GPR30 antagonists (within 30-40 min) suggests mediation via ERs in the plasma membrane, not the nucleus. Notably, both ovarian and spinally synthesized estrogens are essential for membrane ER regulation of spinal EM2 release. CONCLUSION: These findings underscore the importance of estrogens for the regulation of spinal EM2 activity and, by extension, endogenous spinal EM2 antinociception in females. Components of the spinal estrogenic mechanism(s) that suppress EM2 release could represent novel drug targets for improving utilization of endogenous spinal EM2, and thereby pain management in women.

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

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.

Spinal endomorphin 2 antinociception and the mechanisms that produce it are both sex- and stage of estrus cycle-dependent in rats.

UNLABELLED: Endomorphin 2 (EM2) is the predominant endogenous mu-opioid receptor (MOR) ligand in the spinal cord. Given its endogenous presence, antinociceptive responsiveness to the intrathecal application of EM2 most likely reflects its ability to modulate nociception when released in situ. In order to explore the physiological pliability of sex-dependent differences in spinal MOR-mediated antinociception, we investigated the antinociception produced by intrathecal EM2 in male, proestrus female, and diestrus female rats. Antinociception was reflected by changes in tail flick latency to radiant heat. In females, the spinal EM2 antinociceptive system oscillated between analgesically active and inactive states. During diestrus, when circulating estrogens are low, spinal EM2 antinociceptive responsiveness was minimal. In contrast, during proestrus, when circulating estrogens are high, spinal EM2 antinociception was robust and comparable in magnitude to that manifest by males. Furthermore, in proestrus females, spinal EM2 antinociception required spinal dynorphin and kappa-opioid receptor activation, concomitant with MOR activation. This is required for neither spinal EM2 antinociception in males nor the antinociception elicited in proestrus females by spinal sufentanil or [d-Ala(2),N-methyl-Phe(4),Gly-ol(5)]-enkephalin, which are prototypic MOR-selective nonpeptide and peptide agonists, respectively. These results reveal that spinal EM2 antinociception and the signaling mechanisms used to produce it fundamentally differ in males and females. PERSPECTIVE: The inability to mount spinal EM2 antinociception during defined stages of the estrus (and presumably menstrual) cycle and impaired transition from spinal EM2 analgesically nonresponsive to responsive physiological states could be causally associated with the well-documented greater severity and frequency of chronic intractable pain syndromes in women vs men.

Sex, pain, and opioids: interdependent influences of sex and pain modality on dynorphin-mediated antinociception in rats.

The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α(2)-noradrenergic receptor (α(2)-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid-induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α(2)-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.

Importance of sex to pain and its amelioration; relevance of spinal estrogens and its membrane receptors.

Estrogens have a multitude of effects on opioid systems and are thought to play a key role in sexually dimorphic nociception and opioid antinociception. Heretofore, classical genomic actions of estrogens are largely thought to be responsible for the effects of these steroids on nociception and opioid antinociception. The recent discovery that estrogens can also activate estrogen receptors that are located in the plasma membrane, the effects of which are manifest in seconds to minutes instead of hours to days has revolutionized our thinking concerning the ways in which estrogens are likely to modulate pain responsiveness and the dynamic nature of that modulation. This review summarizes parameters of opioid functionality and nociception that are subject to modulation by estrogens, underscoring the added dimensions of such modulation that accrues from rapid membrane estrogen receptor signaling. Implications of this mode of signaling regarding putative sources of estrogens and its degradation are also discussed.

Pleiotropic opioid regulation of spinal endomorphin 2 release and its adaptations to opioid withdrawal are sexually dimorphic.

We studied adaptations to acute precipitated opioid withdrawal of spinal µ-opioid receptor (MOR)-coupled regulation of the release of endomorphin 2 (EM2). The release of this highly MOR-selective endogenous opioid from opioid-naive spinal tissue of male rats is subjected to MOR-coupled positive as well as negative modulation via cholera toxin-sensitive G(s) and pertussis toxin-sensitive G(i)/G(o), respectively. The net effect of this concomitant bidirectional modulation is inhibitory. MOR-coupled pleiotropic regulation of EM2 release is retained in opioid-withdrawn spinal tissue of male rats, but the balance of MOR-coupled inhibitory and facilitatory regulation shifted such that facilitatory regulation predominates. Augmented coupling of MOR to G(s) is causally associated with this change. Strikingly, pleiotropic characteristics of MOR-coupled regulation of spinal EM2 release and adaptations thereof to opioid withdrawal are male-specific. In females, MOR-coupled regulation of EM2 release from opioid-naive and -withdrawn spinal tissue does not have a significant G(s)-coupled facilitatory component, and MOR-coupled inhibition of EM2 release persists unabated in withdrawn preparations. The male-specific adaptations to chronic morphine that shift the relative predominance of opposing dual G protein-coupled MOR pathways provides a mechanism for mitigating inhibitory MOR signaling without losing MOR-coupled feedback regulation. These adaptations enable using endogenous EM2 as a substitute for morphine that had been precipitously removed. The sexually dimorphic functionality and regulation of spinal EM2/MOR-coupled signaling suggest the clinical utility of using sex-specific treatments for addiction that harness the activity of endogenous opioids.

Spinal synthesis of estrogen and concomitant signaling by membrane estrogen receptors regulate spinal κ- and µ-opioid receptor heterodimerization and female-specific spinal morphine antinociception.

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-, ß-, 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.