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.

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 (mGluR)], 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.

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.

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.

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 G /G inhibitory to G 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 G α.

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.

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.

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

We showed previously that spinal metabotropic glutamate receptor 1 (mGluR) 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 mGluR when it is activated by membrane estrogen receptor alpha (mERα) and is facilitated during proestrus when mGluR 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/mGluR would be antiallodynic during diestrus, whereas during proestrus, mGluR blockade would worsen the mechanical allodynia.

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.

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 (mGluR) signaling, are critical to the transition from an EM2 nonresponsive state (during diestrus) to an analgesically responsive state (during proestrus).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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-, β-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception.

Formation of mu-/kappa-opioid receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia.

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.

Regulation of spinal dynorphin 1-17 release by endogenous pituitary adenylyl cyclase-activating polypeptide in the male rat: relevance of excitation via disinhibition.

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn.

Relationship of spinal dynorphin neurons to delta-opioid receptors and estrogen receptor alpha: anatomical basis for ovarian sex steroid opioid antinociception.

Pharmacological and behavioral studies suggest that spinal delta- and kappa-opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal kappa-opioid receptors or delta-opioid receptors (DOR).

Sexually dimorphic recruitment of spinal opioid analgesic pathways by the spinal application of morphine.

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.

Phospholipase Cbeta1 modulates pain sensitivity, opioid antinociception and opioid tolerance formation.

Phospholipase C (PLC) activity has been implicated in multiple opioid-induced sequelae. The relevance of PLC-linked pathways to opioid actions is isoform-specific. Chronic morphine augments PLCbeta1 signaling while diminishing that of PLCbeta3.