Spinal Endomorphin 2 Antinociception and the Mechanisms That Produce It Are Both Sex- and Stage of Estrus Cycle–Dependent in Rats

Abstract

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 kappaopioid 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-Ala²,N-methyl-Phe⁴,Gly-ol⁵]-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.

There is compelling evidence that women experience greater frequency and severity of numerous chronic pain syndromes than do men. Sexually dimorphic expression of idiopathic chronic pain syndromes pertains to migraine headaches,¹¹ irritable bowel syndrome,²⁶ interstitial cystitis,⁵⁹ musculoskeletal pain,⁴⁹ temporomandibular joint disorders,²³ and fibromyalgia.^68,69 Stage of menstrual (estrus) cycle is an important determinant of pain sensitivity in women^44,54,57,60 as well as laboratory animals.^13,18,24,63 The mechanism(s) responsible for sexually dimorphic occurrence of chronic pain states, variability in pain sensitivity across the menstrual (estrus) cycle, and possible linkages between the 2 remains unidentified.

Endomorphins (EMs) are widely considered to be the endogenous ligands for the mu-opioid receptor (MOR), the predominant opioid receptor mediating antinociception. EM1 (Tyr-Pro-Trp-Phe-NH2) and EM2 (Tyr-Pro-Phe-Phe-NH2) produce potent MOR-mediated antinociception following intracerebroventricular^53,70 or intrathecal administration,⁵¹ which is abolished by selective MOR blockade.⁵¹ Notably, spinally applied EMs are also potent antiallodynic agents in neuropathic pain.^45,46 Notwithstanding the inability to identify a precursor protein for either EM1 or EM2,⁵⁸ 1) the wide distribution of EMs in areas of the central nervous system relevant to pain (spinal cord dorsal horn, parabrachial nucleus, periaqueductal gray, medial thalamus, amygdala^{33,34,41,64,65}), 2) the expression of EM2 (the predominant spinal EM) in dense core vesicles of primary afferent terminals in the lumbar dorsal horn,⁵² 3) colocalization with spinal substance P,⁵² and 4) the dramatic decrease in EM2 content in spinal cord ipsilateral to sciatic nerve lesion⁵² collectively strongly suggest that EM2 is relevant to endogenous spinal MOR-coupled antinociception, particularly in the spinal cord where it is more potent in producing antinociception than at supraspinal sites.^15,70

Physiologic, naturally occurring fluctuations in the functional state of the endogenous spinal EM2/MOR analgesic system during the menstrual cycle in women (estrus cycle in laboratory animals) could contribute to cyclic variations in their pain sensitivity, which over the long run could be a risk factor for developing chronic pain. Because EM2 not only is endogenously expressed in the spinal cord but also is the predominant spinal EM,³³ it is reasonable to assume that antinociceptive responsiveness to its exogenous application reflects its ability to modulate nociception when released endogenously. Therefore, sex-dependent and stage of estrus cycle–dependent differences in the ability of intrathecally applied EM2 to elicit antinociception would likely reflect variations in the effectiveness of the endogenous spinal EM2 system to modulate pain.

Accordingly, we compared antinociceptive responsiveness to spinally applied EM2 among male rats, diestrus female rats, and proestrus female rats. Results revealed not only a dramatic dependence of the magnitude of EM2 antinociception on stage of estrus cycle but also a sexual dimorphism in the mechanisms utilized by spinal EM2 to elicit antinociception. The observed absence of antinociceptive responsiveness to intrathecal EM2 during diestrus, the longest stage of the estrus cycle, could potentially amplify the consequences in women of nociceptive events that occur during analogous stages of the menstrual cycle, thus contributing to the greater risk of women than men for developing more severe and frequent chronic pain.

Methods

Animals

Experiments employed Sprague Dawley rats (225–300 g; Charles River, Kingston, NY), which were maintained in an approved controlled environment on a 12-hour light–dark cycle. Food and water were available ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of SUNY Downstate Medical Center.

Determination of Stage of Estrous Cycle

Stage of cycle was determined using histology of vaginal smears. Predominance of small leukocytes was indicative of diestrous; a predominance of large round nucleated cells was indicative of proestrous. Disruptions of the estrous cycle that could result from surgery did not confound data interpretation because vaginal smear histology and not predictions that assumed regularity of cycling was used to define diestrous and proestrous cycles.

Intrathecal Administration of Drugs

Each drug was administered in 5 to 10 μL vehicle (water, saline, or 3% dimethyl sulfoxide in saline) over a 60-second period to the subarachnoid space of the lumbar spinal cord via a permanent indwelling intrathecal cannula. Complete delivery was ensured by flushing the cannula with an additional 10 μL of saline. Thereafter, responsiveness to nociceptive stimuli was determined at various intervals and compared with pre-drug values. Sufentanil and [d-Ala², N-Me-Phe⁴, Gly-ol⁵]-enkephalin (DAMGO) were used as nonpeptide and peptide, respectively, prototypic MOR-selective agonists. Intrathecal kappa-opioid receptor (KOR) blocker nor-binaltorphimine (norBNI)⁴³ was applied 18 hours before intrathecal EM2^9,27 in order to ensure that reported interactions between norBNI and spinal MOR¹⁶ did not confound interpretation of results. Neither vehicle (water, saline, or 3% dimethyl sulfoxide saline), norBNI, nor anti-dynorphin antibody had detectable effects on tail flick latency (TFL).

Assessment of Nociceptive Responses

TFL to radiant heat was quantified using a Tail Flick Analgesia Meter (IITC, Woodland Hills, CA). Intensity of the radiant heat was adjusted such that baseline values were 3.0 to 4.5 seconds. A cutoff of 10 seconds latency prevented any untoward consequences to the tail.

Statistical Analysis

Repeated measures analysis of variance (ANOVA) was used to analyze effect of drug treatment at multiple time points after intrathecal administration within each group. Subsequent Dunnett's Multiple Comparison Test was used to locate the time point(s) that showed significant effect (P < .05). Two-way ANOVA was used to analyze the sex, group, or dose by time effects among groups. Subsequent Bonferroni posttests were used to locate differences between groups.

Results

Antinociceptive Responsiveness to Intrathecal EM2 Is Sexually Dimorphic

As expected, in males, intrathecal EM2 produced a robust and dose-dependent antinociception. A 2-way ANOVA (dose × time) revealed a significant dose effect of EM2 [F_(2,60) = 35.50, P < .0001]. Surprisingly, however, in females, intrathecal EM2 failed to produce significant antinociception, even at a dose of 90 nmol. The apparent sexually dimorphic antinociceptive response to spinal EM2 was validated by a 2-way ANOVA (sex × time), which revealed a significant sex effect [F_(1,40) = 36.22, P < .0001, n = 6 for each group; Fig 1].

Figure 1.

Antinociception elicited by intrathecal EM2 is sexually dimorphic. Ascending cumulative dose responsiveness to intrathecally applied EM2 was determined in uncycled female and male rats. Increasing doses were administered at 30-min intervals. TFL was determined at 5-min intervals following intrathecal administration. In males, intrathecal EM2 resulted in a robust and dose-dependent antinociception, peaking at 5 minutes. This antinociception was not manifest in female rats. n = 6 for each group.

Antinociceptive Responsiveness to Intrathecal EM2 Is Dependent on Stage of Estrus Cycle

The specific stage of the estrus cycle was not determined in the initial determination of the sex dependence of spinal EM2 antinociception. Given that diestrus is the longest stage of the estrus cycle in rats, we presumed that most, if not all, of the females that surprisingly failed to manifest spinal EM2 antinociception were in diestrus, not proestrus. This suggested the possibility that failure to mount significant spinal EM2 antinociception might be restricted to diestrus. In order to validate this hypothesis, we compared spinal EM2 antinociception among proestrus and diestrus females and males (Fig 2). A 2-way ANOVA (group × time) revealed a significant group (diestrus, proestrus, male) effect [F_(2,108) = 4.16, P = .026, n = 8–9]. Subsequent Bonferroni comparisons indicated that proestrus females, like males, mounted an antinociceptive response to intrathecal EM2 (45 nmol, 5 minutes after application), both of which significantly differed (P < .01) from that manifest in diestrus females. Notably, the antinociceptive responsiveness to intrathecal EM2 did not differ between proestrus females and males (P > .05). To ensure that differences in analgesic responsiveness to spinal EM2 between males and uncycled females (depicted in Fig 1) were primarily due to lack of responsiveness of diestrus females, we determined the antinociceptive effects of administering to diestrus females 185 nmol EM2 intrathecally. Even this high dose failed to significantly increase TFL (data not shown).

Figure 2.

In females, intrathecal EM2 can elicit antinociception, but it is linked to specific stages of the estrus cycle. Antinociceptive dose responsiveness to intrathecal EM2, determined as described for Fig 1, was compared among diestrus and proestrus female and male rats. Robust antinociception to spinal EM2 was observed in proestrus females, comparable in magnitude and time course (for 45 nmol) to that manifested by males, which was not observed in diestrus females. n = 8–9.

To confirm that the spinal EM2 antinociception we observed in proestrus females and males was mediated via spinal MOR, we determined the effect of the longlasting MOR1 antagonist naloxonazine.^39,40,67 We selected naloxonazine because it was shown to be selective for spinal EM2 vs EM1 (or DAMGO).⁵¹ As expected, intrathecal naloxonazine (2 hours, 10 nmol) blocked spinal EM2 antinociception in both male and proestrus female rats [males: F_(1,48) = 6.85, P = .012; proestrus females: F_(1,45) = 5.80, P = .029; n = 6–8] (data not shown), confirming MOR mediation of spinal EM2 antinociception in females as well as males.

Mechanisms Mediating Spinal EM2 Antinociception Are Sexually Dimorphic

Dependence of spinal EM2 antinociception on stage of estrus cycle could suggest that in addition to MOR, females require additional signaling components, the spinal content of which varies across the estrus cycle. In the spinal cord, MOR heterodimerizes with KOR.⁴ The levels of heterodimeric KOR/MOR are dramatically elevated not only in proestrus vs diestrus females but also in proestrus females vs males.⁴ This suggested the possibility that expression of spinal EM2 antinociception in proestrus females (but not males) might require dynorphin and KOR activity, as had been reported earlier for intrathecal morphine.²⁸ In order to test this hypothesis, we investigated the effects of intrathecal anti-dynorphin antibodies and the KOR blocker norBNI⁴³ on spinal EM2 antinociception (Fig 3). As we had hypothesized, the effect of intrathecal treatment with either anti-dynorphin antibodies or norBNI on spinal EM2 antinociception was strikingly sexually dimorphic. Both intrathecal treatments abolished spinal EM2 antinociception in proestrus females [F_(1,36) = 5.44, P = .045, and F_(1,36) = 6.844, P = .028, for intrathecal anti-dynorphin antibody and norBNI, respectively]. In contrast, the identical treatment had no effect in males [F_(1,60) = .001, P = .961, and F_(1,60) = .095, P = .762, for intrathecal anti-dynorphin and norBNI, respectively]; n = 4 for anti-dynorphin antibody- or norBNI-treated group.

Figure 3.

Spinal EM2 antinociception in proestrus females required spinal dynorphin and KOR activity, which was not required for spinal EM2 antinociception elicited in males. Proestrus female and male rats were treated with either intrathecal anti-dynorphin antibodies (DynAb, 300 ng; 30 min) or intrathecal norBNI (26 nmol; 18 h) preceding intrathecal EM2. Antinociception in response to 45 nmol intrathecal EM2 was then determined at the indicated times using TFL. Both intrathecal DynAb and norBNI abolished spinal EM2 antinociception in proestrus female but not male rats. n = 5 for norBNI or DynAb.

Stage of Estrus Cycle Dependence of Spinal EM2 Antinociception Does Not Generalize to DAMGO or Sufentanil

In order to determine the generality among MOR agonists of dependence on stage of estrous cycle, we compared antinociceptive responsiveness to the intrathecal administration of the prototypic peptide and nonpeptide MOR agonists DAMGO and sufentanil, respectively (Fig 4; n = 4 for each group). Repeated measures ANOVA confirmed, as expected, that intrathecal DAMGO (.33 and 1 nmol; Fig 4A) produced antinociception in proestrus females [F_(5,15) = 19.99, P < .0001, for .33 nmol and F_(7,21) = 2.591, P =.043, for 1 nmol] and males [F_(5,15) = 9.11, P = .0004, for .33 nmol and F_(7,21) = 8.435, P = .0004, for 1 nmol]. Notably, however, the same doses of DAMGO also produced antinociception in diestrus females [F_(5,15) = 2.98, P = .046, for .33 nmol and F_(7,21) = 4.34, P = .0041, for 1 nmol]. Strikingly, no differences were observed in the magnitude of spinal DAMGO antinociception among male, diestrus female, and proestrus female groups [F_(2,48) = .13, P = .884, for .33 nmol and F_(2,56) = .17, P = .850, for 1 nmol]. Analogous results were obtained for sufentanil (.35 and .7 nmol; Fig 4B). Both high and low doses produced spinal antinociception in diestrus females [F_(6,18) = 2.78, P = .043, for .35 nmol and F_(6,18) = 5.337, P =.0026, for .7 nmol] as well as proestrus females [F_(7,21) = 3.12, P = .021, for .35 nmol and F_(6,18) = 4.651, P = .0051, for .7 nmol] and males [F_(5,15) = 4.37, P = .012, for .35 nmol and F_(6,18) = 12.42, P = .0002, for .7 nmol]. As was observed for DAMGO, antinociception produced by intrathecal sufentanil did not differ among proestrus and diestrus females or males [F_(2,42) = 1.50, P = .2866, for .35 nmol and F_(2,56) = .92, P = .4433, for .7 nmol]. Furthermore, neither intrathecal anti-dynorphin antibody nor intrathecal norBNI altered the antinociception produced by sufentanil or DAMGO [anti-dynorphin antibody: F_(1,18) = .696, P = .436, for sufentanil, and F_(1,18) = .075, P = .793, for DAMGO; norBNI: F_(1,18) = .842, P = .394, for sufentanil, and F_(1,18) = .722, P = .428, for DAMGO, n = 4 for each group; data not shown].

Figure 4.

Spinal antinociception elicited by intrathecal DAMGO or sufentanil was neither dependent on stage of estrus cycle nor sexually dimorphic. The time course for antinociception in response to intrathecal DAMGO (.33, 1 nmol) or sufentanil (.35, .7 nmol) was determined in diestrus and proestrus female and male rats using TFL. No differences in antinociception were observed among groups. n = 4 for each group.

Discussion

Results reveal a fundamental dichotomy between females and males in their utilization of spinal EM2 (summarized in Fig 5). This sexual dimorphism includes 1) female-specific plasticity of the antinociceptive effectiveness of spinal EM2 that is linked to physiological state and 2) female-specific antinociceptive mechanisms that EM2 recruits. Major specific findings of this study are as follows: 1) in females, the spinal EM2 antinociceptive system oscillates between analgesically active and inactive states, which are synchronized with the estrus cycle; 2) spinal EM2 antinociceptive responsiveness parallels circulating levels of estrogens, being robust during proestrus and minimal during diestrus; 3) in proestrus females, spinal EM2 antinociception requires, in addition to MOR, both spinal dynorphin and spinal KOR activation, neither of which contributes to EM2 antinociception in males; and 4) in proestrus females, the mechanisms mediating spinal EM2 antinociception are seemingly unique to EM2; neither spinal dynorphin nor KOR contributes to the spinal antinociception produced by either DAMGO or sufentanil.

Figure 5.

Summary of differences in spinal antinociceptive responsiveness among EM2, DAMGO, and sufentanil in males vs diestrus and proestrus females. In males, the spinal application of EM2, DAMGO, or sufentanil produced robust antinociception. Female analgesic responsiveness to spinal EM2 differed from that of males, the nature of which depended on stage of estrus cycle. In proestrus females, the magnitude of antinociception elicited by intrathecal EM2 is comparable to that manifest in males but the spinal mechanism(s) mediating the antinociception is sex-dependent. In proestrus females, spinal EM2 antinociception requires the activity of Dyn and KOR in addition to MOR (although the point(s) of intersection between spinal EM2/MOR and Dyn/KOR are not known), whereas in males, Dyn/KOR does not contribute. In contrast to proestrus females and males, diestrus females do not mount an antinociceptive response to intrathecal EM2 despite the fact that MOR functionality is intact as reflected by the responsiveness to DAMGO and sufentanil. Neutralizing those signaling components that are responsible for “clamping” analgesic responsiveness to spinal EM2 during diestrus, the longest estrus (menstrual) cycle stage could represent a novel pharmacologic approach for managing chronic pain in women.

As expected, the intrathecal application of EM2 to male rats produced a robust thermal antinociception, reflected by increased TFL. In order to investigate the putative influence of physiological state on spinal EM2 antinociception, we compared the effect of intrathecally applied EM2 on TFL among proestrus and diestrus female and male rats. Surprisingly, during diestrus, when circulating levels of estrogens are more comparable to those of males than during proestrus, intrathecal EM2 had minimal effect on TFL. However, during proestrus, when female/male differences in circulating sex steroids are the most pronounced, the spinal application of EM2 resulted in robust antinociception, comparable in magnitude to that observed in males. Notably, stage of estrus cycle dependence of spinal EM2 antinociception does not generalize to other MOR-selective agonists such as DAMGO and sufentanil, which are prototypic peptide and nonpeptide MOR-selective ligands, respectively.

The signaling protein(s) by which spinal EM2 produces antinociception in proestrus females is also unique. As expected, in proestrus females as well as males, spinal MOR activation is a prerequisite for spinal EM2 antinociception. However, in proestrus females, but not males, blockade of KOR (via intrathecal norBNI) also abolished the EM2 antinociception. This indicates that activation of spinal KOR, in addition to MOR, is also essential for the observed antinociception. Given that the concentration of EM2 needed to inhibit 50% of the binding of a MOR agonist (Ki) is equal to 360 pM and that EM2 is 15,000-fold more selective for MOR vs KOR,⁷⁰ it is highly unlikely that the KOR component of EM2 antinociception results from a direct interaction of EM2 with KOR. Instead, it is more likely that the KOR component of EM2 antinociception is provided by the ability of spinal EM2 to release dynorphin, which would also explain the observed effect of intrathecal anti-dynorphin antibodies.

It should be noted that neither spinal dynorphin nor KOR contributes to the antinociception produced by intrathecal DAMGO or sufentanil in proestrus females. Spinal morphine antinociception in proestrus females (but not diestrus females or males) does require spinal dynorphin release and KOR activation, in addition to MOR.²⁸ However, in contrast to EM2, spinal morphine continues to produce antinociception during diestrus, albeit exclusively via MOR.²⁸ The molecular basis for the ability of spinal morphine to produce equivalent spinal antinociception during diestrus and proestrus via dynorphin/KOR-independent and dependent pathways, respectively, but restriction of spinal EM2 antinociception in females to proestrus and solely dependent on dynorphin/KOR is not understood. These considerations reveal that EM2 has sex-dependent unique signaling attributes among several commonly used MOR-selective agonists.

The striking emergence of spinal EM2 antinociception during proestrus, when circulating levels of estrogens are high, combined with the requirement for spinal dynorphin and KOR, could indicate that the recently discovered KOR/MOR heterodimer⁴ is critical for spinal EM2 antinociception in proestrus females. In this regard, it should be noted that both EM2 and dynorphin bind to KOR/MOR heterodimers.⁴ Heterodimerized KOR/MOR is 4 to 5 times more prevalent in the spinal cord of proestrus vs diestrus females,⁴ which is dependent on the availability of estrogens and the activity of spinal estrogen receptors.²⁷ Spinal membrane estrogen receptors and locally synthesized estrogens are critical for KOR/MOR formation.²⁷ It remains to be seen if these requirements also pertain to spinal EM2 antinociception in proestrus females.

Interestingly, spinal EM2 produces a robust antinociception in males, notwithstanding that the content of heterodimerized KOR/MOR in the spinal cord of males is comparable to those of diestrus females (in which spinal EM2 is not analgesically effective). This suggests that although heterodimerized KOR/MOR may be needed for spinal EM2 antinociception in proestrus females, it would seem not to be prerequisite for spinal EM2 antinociception in males. The molecular mechanisms for this dichotomy are unknown, but this consideration further underscores the sex dependence of the spinal antinociceptive mechanisms recruited by intrathecally applied EM2.

The apparent uniqueness of spinal EM2 antinociception is consistent with the mounting evidence that EMs, but not sufentanil or DAMGO, are β-arrestin–biased ligands, preferentially activating β-arrestin–dependent signaling.^35,48 The function of β-arrestins was originally thought to be restricted to regulating G protein–coupled receptor desensitization, internalization, and receptor recycling. However, there is mounting evidence that β-arrestins also function as G protein–independent signal transducers, simultaneously inhibiting G protein–dependent signaling while promoting the generation of alternative, intracellular signals that result in unique cellular and physiological consequences. The relevance of β-arrestin–biased signaling to the linkage between stage of estrus cycle and spinal EM2 antinociception remains to be established.

We previously proposed^4,27 that the spinal expression of a KOR/MOR heterodimer could provide a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves.^32,61,66 This, in combination with the current data indicating that spinal EM2 analgesic responsiveness is minimized during diestrus, suggests that the aggregate spinal opioid antinociceptive functionality is compromised during diestrus, the longest single stage of the estrus cycle.

Stage of estrus cycle–dependent antinociceptive responsiveness to intracerebroventricular²² and systemic⁵⁶ morphine has been reported. In these studies, in contrast to present findings, major differences were found between estrus and diestrus/proestrus, not between diestrus and proestrus as we report. Shared mechanistic underpinnings between the influence of stage of estrus cycle on supraspinal/systemic morphine antinociception and the spinal antinociception reported for spinal EM2 remains to be established. Similarly, hypothalamic (medial preoptic area, ventromedial nucleus³) and midbrain–medullary (periaqueductal gray–rostral ventromedial medulla)^29-31,37 systems have been shown to mediate both sex and estrus-phase differences in opioid antinociception. The relevance of these supraspinal structures to the currently reported “clamped” antinociceptive responsiveness to spinal EM2 remains to be elucidated. Interestingly, antinociceptive responsiveness to intrathecal EM2 varies in accordance with the estrus cycle, indicating that activational effects of ovarian steroids modulate the magnitude of spinal EM2 analgesic activity. The importance of organizational steroid effects is not known. In contrast, both organizational and activational effects of ovarian sex steroids are relevant to supraspinal morphine antinociception.^2,5,22,25,56

Several of the chronic pain syndromes that are disproportionately manifest in women vs men are responsive to MOR-selective opioids (even if opioids are not the initial drug of choice for their management). These include bladder pain,^19,38,42 migraine,¹² and temporomandibular joint disorders.^10,17,20,50 Furthermore, even in cases in which a female-prevalent chronic pain syndrome— for example, fibromyalgia—is not responsive to opioids, the inability to recruit an endogenous MOR antinociceptive system at critical times could set in motion events that ultimately result in a particular chronic pain syndrome. Accordingly, we hypothesize that the ebb in spinal opioid antinociception during diestrus could be a risk factor for the emergence of chronic pain in women. The inability to harness spinal EM2 antinociception during defined stages of the estrus (and presumably the menstrual) cycle and the impairment of the ability to successfully 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.

Estrogens are well known to influence MOR signaling^4,14,27 and the effectiveness of opioids to relieve pain.^36,55 Additionally, estrogens^1,7,21,36 and progesterone^6,8,47,62 influence pain sensitivity in the absence of opioids. Estrogen/progesterone-dependent functionality and/or expression of signaling proteins critical for “clamping” spinal EM2 antinociception during diestrus could prove to be novel targets for developing female-specific pharmacotherapies for chronic pain management.