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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). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213-1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374-1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor alpha (ERalpha) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ERalpha by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ERalpha and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ERalpha-ir cells, cells double-labeled for DYN-ir and ERalpha-ir and the proportion of DYN-ir cells coexpressing ERalpha. Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K(+)-evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain.

The concomitant activation of spinal κ- and δ-opioid systems is a prerequisite for the antinociception of gestation and its hormonal simulation [via 17β-estradiol and progesterone administration; hormone-simulated pregnancy (HSP)]. However, it is not known whether the release of κ- and δ-opioids is also concomitantly regulated. This study investigates whether the release of methionine-enkephalin and modulation thereof is altered during HSP, as has been reported for dynorphin. K+-stimulated release of spinal methionine-enkephalin from lumbar spinal tissue obtained from control animals is negatively modulated by nociceptin (orphanin FQ; N/OFQ) in a dose-dependent manner, but not by opioids. Conversely, selective blockade of spinal N/OFQ, but not opioid receptors, augments the K+-induced increase in methionine-enkephalin release, indicating that endogenous N/OFQ also functions as a negative modulator of methionine-enkephalin release. The magnitude of K+-evoked methionine-enkephalin release from spinal tissue obtained from ovarian steroid-treated animals remains unchanged, consistent with the insensitivity of its modulation by N/OFQ to the ovarian sex steroid milieu. These characteristics of methionine-enkephalin release stand in sharp contrast to those previously reported for the evoked release of spinal dynorphin. Dynorphin release is subject to negative modulation by opioid (predominantly δ) as well as N/OFQ, both of which are offset during HSP, resulting in an ≈2-fold increase in the magnitude of its release. These observations reveal that regulation of spinal dynorphin/κ- and methionine-enkephalin/δ-spinal opioid antinociceptive systems is independent, divergent, and not symmetrical and support the formulation that spinal methionine-enkephalin/δ-opioid tone acts in a permissive/facilitative capacity to accentuate spinal dynorphin/κ-activity.

Pregnancy and its hormonal simulation via 17β-estradiol (E2) and progesterone (P) are associated with spinal opioid antinociception, primarily driven by augmented dynorphin/κ-opioid activity. This study addresses the ovarian sex steroid-activated mechanism(s) that underlie this activation using an ex vivo spinal cord preparation. In lumbar spinal cord obtained from control animals, exogenous κ- or δ-opioid agonists (but not μ), as well as nociceptin (orphanin FQ; N/OFQ), dose dependently inhibit the stimulated release of dynorphin. Consistent with these observations, stimulated dynorphin release is enhanced following selective blockade of opioid or N/OFQ receptors, indicating that their endogenous ligands are negative modulators of dynorphin release. In lumbar spinal cord obtained from ovariectomized animals exposed to pregnancy blood levels of E2/P, basal and stimulated rates of dynorphin release increase ≈2-fold. Moreover, evoked dynorphin release is no longer negatively modulated by κ- or δ-opioid agonists or N/OFQ. Interestingly, in these preparations, release can be facilitated by δ-opioid receptor activation, and neither spinal opioid nor N/OFQ receptor blockade enhances evoked dynorphin release. Consistent with these observations, guanosine-5′-O-3-[35S]-thio triphosphate binding analyses indicate a reduction in functional N/OFQ receptors. These data indicate that at least part of the E2/P-induced augmented activity of lumbar dynorphin neurons results from their disinhibition via the removal of negative opioid and N/OFQ modulation. These results underscore the plasticity of spinal opioid and N/OFQ systems and their dependence on the ovarian sex steroid milieu. Ovarian sex steroid-activated antinociception reveals mechanisms that enable sustained opioid activation without concomitant tolerance formation.