QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) An erratum has been published Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gupta, D. S. Articles by Gintzler, A. R. Search for Related Content PubMed PubMed Citation Articles by Gupta, D. S. Articles by Gintzler, A. R.

NEUROPHARMACOLOGY

Influence of Ovarian Sex Steroids on Spinal Methionine-Enkephalin Release: Comparison with Dynorphin Reveals Asymmetrical Regulation

Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, New York

Received August 1, 2002 ; accepted October 8, 2002.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The concomitant activation of spinal -and -opioid systems is a prerequisite for the antinociception of gestation and its hormonal simulation [via 17-estradiol and progesterone administration; hormone-simulated pregnancy (HSP)]. However, it is not known whether the release of -and -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 ) 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/-and methionine-enkephalin/-spinal opioid antinociceptive systems is independent, divergent, and not symmetrical and support the formulation that spinal methionine-enkephalin/-opioid tone acts in a permissive/facilitative capacity to accentuate spinal dynorphin/-activity.

Spinal dynorphin/-opioid receptor activity is an essential mediator of the antinociception associated with pregnancy and its hormonal simulation (Dawson-Basoa and Gintzler, 1998), the activity of which (Liu and Gintzler, 1999) is augmented during these conditions (Medina et al., 1993a,b, 1995). During HSP, there is approximately a 2-fold increase in both the basal and stimulated rates of spinal dynorphin release. This results from its disinhibition via the removal of negative nociceptin (Orphanin FQ; N/OFQ) and opioid (/)-modulation (Gupta et al., 2001).

Notably, -opioid receptor-mediated inhibition of evoked dynorphin release, predominant in the absence of ovarian sex steroid treatment, is not only offset after simulation of pregnancy levels of E₂/P but also reverses to a facilitation. -Opioid receptor-mediated positive modulation of spinal dynorphin release is also an essential component of the antinociception of HSP (and physiological pregnancy) as evidenced by their abolishment after the individual blockade of spinal - as well -opioid receptors (Dawson-Basoa and Gintzler, 1998).

These data provide a basis for the prerequisite that spinal dynorphin/- and methionine-enkephalin (Met-Enk)/-opioid pathways are activated concomitantly for ovarian steroid-induced antinociception to be manifest. A remaining question, however, is whether the release of independent but interactive opioids, i.e., spinal dynorphin and Met-Enk, is modulated in parallel or subject to divergent regulation. Accordingly, the influence of the pregnancy blood profile of ovarian steroids, N/OFQ, opioids, and interactions thereof on the release of spinal Met-Enk was investigated. The current results, in combination with those from a previous study from this laboratory (Gupta et al., 2001), reveal that the regulation of spinal dynorphin/- and enkephalin/-antinociceptive systems is independent and asymmetrical.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Experimental Animals
Experiments used female Sprague-Dawley rats (Charles River, Kingston, NY; 250–300 g), which were maintained in an approved controlled environment on a 12-h light/dark cycle. Food and water were available ad libitum. All experimental procedures were reviewed and approved by the Animal Care and Use Committee of SUNY Downstate Medical Center.

Ovarian Sex Steroid Administration
The pregnancy blood concentration profile of E₂ and P were simulated in nonpregnant, ovariectomized rats (HSP) via the subcutaneous implantation of Silastic tubing filled with either a solution of E₂ in sesame oil or crystalline P (Bridges, 1984). Controls consisted of implants that contained sesame oil (vehicle for E₂) and empty Silastic tubing (as a vehicle control for P). Day 1 of steroid hormone administration or its vehicle control was initiated at the time of ovariectomy. Pregnancy-like levels of E₂ and P were achieved by changing the concentration of E₂ in the tubing (10 mm tubing/100 g b.wt.) and by altering the number of 45-mm P implants on days 5, 15, and 19 (for details of implantation procedure and comparison with steroid plasma levels of physiological gestation, see Bridges, 1984; Bridges and Ronsheim, 1987).

Spinal Tissue Preparation
On day 19 of HSP or vehicle treatment, the spinal vertebral column was sectioned at the intervertebral spaces above vertebrae T-12 and L-1. The lumbar spinal cord contained within this segment (L-1 to L-5; 200–250 mg) was quickly expelled by injecting ice-cold saline into the caudal end, minced using a McIliwain tissue chopper (0.3 mm in thickness; Mickle Laboratory Engineering Co., Gomshall, Surrey, UK), placed into a chamber (0.35 ml), and superfused (superfusion system; Brandel, Inc., Gaithersburg, MD). The Krebs' solution used for superfusion contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM NaH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 11.1 mM dextrose, and gelatin (saturated with 4 g/l) and was gassed with a 95% O₂,5%CO₂ mixture. Additionally, the Krebs' superfusate used to assess basal and stimulated Met-Enk release contained the protease inhibitors captopril (10 µM), thiorphan (0.3 µM), bestatin (10 µM), and L-leucyl-L-leucine (2 mM) to protect against proteolytic degradation.

Superfusion Paradigm
After an initial equilibration period (20 min), the superfusion buffer was switched to one containing various concentrations of N/OFQ (0, 1, 10, 100, and 1000 nM) or [D-Pen²,D-Pen⁵]-enkephalin (DPDPE), trans-3,4-dichloro-N-methyl-N-[1-pyrroridinyl]cyclohexyl]-benzeneacetamide, methane sulfonate, hydrate (U50,488H), or sufentanil (-, -, or µ-opioid receptor-selective; 1, 10, and 500 nM for each). Responses to N/OFQ were determined with or without blockade of either opioid receptors (via the presence of naloxone at 1 µM) or N/OFQ receptors (NORs) (via the presence of compound 15 at 10 µM, a derivative of the NOR antagoinst 1-[(3R,4R)-1-cycloocrylmethyl-3-hydroxymethyl-4-piperidyl)-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) that does not contain a hydroxymethyl group on the piperidine ring) (Kawamoto et al., 1999) (generously provided by Dr. Lawrence Toll, SRI International, Menlo Park, CA). As reported previously (Gupta et al., 2001), Schild analysis revealed that compound 15 is a competitive antagonist producing a parallel shift in the N/OFQ dose-response curve for stimulation of [³⁵S]guanosine 5'-O-(3-thio)triphosphate binding (pA₂ = 8.48 ± 0.04; K_e = 3.44 ± 0.65 nM; slope = 1.08 ± 0.04). The effect of naloxone or compound 15, individually, on basal and evoked release was separately determined. The basal release of Met-Enk was determined by quantification of the Met-Enk content of spinal superfusate obtained over a period of 18 min (two 3-ml collections). The magnitude of stimulated Met-Enk release was determined by quantification of the rate of its release into spinal superfusate that contained high potassium (K⁺; 50 mM; the content of sodium was proportionally reduced to maintain osmolarity). High K⁺-evoked release of Met-Enk was determined over a 9-min period (3 ml). Basal and stimulated superfusate were collected into prechilled tubes on ice.

Superfusate containing basal release and evoked release were desalted and concentrated using reverse phase C₁₈ cartridges (Sep-Pak; Waters Corporation, Milford, MA). Met-Enk peptide eluted with 70% acetonitrile/0.1% trifluoroacetic acid (TFA) was oxidized using hydrogen peroxide (0.6%, overnight at 4°C) and lyophilized to dryness. Recovery of Met-Enk (1-17) was quantitative (>95%).

Radioimmunoassay (RIA)
Iodination of Met-Enk. Enkephalin (5-L-methionine)[L-tyrosyl-¹²⁵I] was generated by the chloramine T procedure. Briefly, 1 µg of Met-Enk was sequentially mixed with 5 µl of Na[¹²⁵I] (0.5 mCi; Amersham Biosciences Inc., Piscataway, NJ) and 25 µl of chloramine T (25 µg, 50 µl). [¹²⁵I]Met-Enk was separated from nonpeptide bound radioactivity as well as from radioiodinated Met-Enk fragments using C₁₈ Sep-Pak cartridges eluted sequentially with 30 and 70% acetonitrile/0.1% TFA. The 70% acetonitrile eluate was lyophilized to dryness, resuspended in 0.1% TFA containing 2% acetonitrile. Enkephalin (5-L-methionine)[L-tyrosyl-¹²⁵I] was further purified by high-pressure liquid chromatography (HPLC) fractionation using a 15-cm C₁₈ column (5-µm Nova-Pak, Waters, Framingham, MA). The column was eluted at a flow rate of 1 ml/min with a mobile phase containing 0.08% TFA throughout and a linear gradient of acetonitrile ranging from 4% at the start to 70% at 28.6 min. The peak of radioactivity eluting at 14 to 15 min of the fractionation procedure, the time of elution of authentic methionine-enkephalin sulfoxide (generated by incubating Met-Enk with 0.6% hydrogen peroxide) was used as the radioactive tracer.

Met-Enk Quantification. Met-Enk was quantified in spinal superfusate using an RIA that used an antibody (1:500) highly specific for the oxidized form of this peptide (generously supplied by Dr. Richard Kream, SUNY Downstate Medical Center) (Kumar et al., 1990) as previously used by this laboratory (Xu et al., 1989). A standard curve (1.0–250 pg/assay tube) in which the percentage of inhibition of binding was plotted against the log of unlabeled oxidized Met-Enk in the reaction tube was generated in each assay. To generate a standard curve, synthetic Met-Enk (Peninsula Laboratories, Belmont, CA) was dissolved in acetic acid (0.1 N) and oxidized by hydrogen peroxide (0.3% overnight, 4°C). Bovine serum albumin (0.1%) was included in the assay buffer to minimize nonspecific adherence to the tube surface. After a 2-h incubation at room temperature, radiolabeled Met-Enk (10,000 cpm) was added and the reaction mixture incubated overnight (4°C). Antibody-bound radioactivity was separated from unbound tracer by the addition of ice-cold absolute ethanol (1 ml for 20 min at 4°C) followed by centrifugation (3200g at 4°C). The (antibody-bound) radioactivity contained in the pellet was quantified using a gamma-counter (CliniGamma; PerkinElmer Wallac, Gaithersburg, MD). Values of experimental samples were calculated from the standard curve using Ultroterm software (PerkinElmer Wallac). The minimum detectable concentration was 2 pg/assay tube, which produced 20% inhibition of maximum binding. A 50% reduction in binding was produced by 20 pg/assay tube. Peptide concentrations were derived from RIA analyses of superfusate that produced between 20 and 75% inhibition of binding, the linear and sensitive portion of the standard curve. All standards and experimental samples were run in triplicate.

The chemical identity of Met-Enk-like immunoreactivity was analyzed by combining HPLC fractionation with RIA detection. Spinal superfusate was collected under basal and stimulated conditions. The Met-Enk peptide contained therein was desalted and concentrated using reverse phase C₁₈ Sep-Pak cartridges and oxidized with hydrogen peroxide as described above. The 70% acetonitrile/0.1% TFA eluate was lyophilized to dryness, resuspended in 60 µl of 2% acetonitrile/0.1% TFA, and centrifuged (500g for 5 min). Fractionation by HPLC was accomplished by applying supernatant (20 µl) or the same volume of standard peptide onto a 15-cm C₁₈ column (5-µm Nova-Pak; Waters). The column was eluted at a flow rate of 1 ml/min with a mobile phase containing 0.08% TFA throughout and a linear gradient of acetonitrile ranging from 4% at the start to 70% at 28.6 min. These chromatographic conditions permit authentic Met-Enk to be separated from other opioid peptides (leucine-enkephalin, -endorphin, -endorphin, -endorphin, N-acetylated -endorphin, and dynorphin). HPLC eluates (0.5-ml fractions) were oxidized with hydrogen peroxide (0.6%), lyophilized to dryness, and the content of Met-Enk was determined by RIA as described above. Approximately 84% of the Met-Enk-like immunoreactivity that was contained in spinal tissue superfusate had a retention time comparable with that of standard Met-Enk. The data shown have not been corrected for recovery. Lyophilized Krebs' buffer (3 ml corresponding to basal and evoked release) that was processed as described for tissue superfusate but not exposed to tissue did not produce any appreciable inhibition of binding of radiolabeled Met-Enk to the anti-Met-Enk antibody.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Influence of Hormonal Milieu, Opioids, and N/OFQ on Basal met-Enk Release from Spinal Tissue. Two-way ANOVA was used to compare the basal rate of Met-Enk release from lumbar tissue obtained from either E₂/P- or placebo-treated animals, before and after in vitro blockade of opioid receptors (via naloxone, 1 µM). There was no main effect of hormone (F_1,25 = 0.798, p > 0.3) but a main effect of naloxone was observed (F_1,25 = 18.009, p < 0.01). There was no naloxone by hormone interaction (F_1,25 = 0.351, p > 0.5).

Figure 1 illustrates that ovarian steroid hormone treatment did not alter the basal rate of Met-Enk release (61.4 ± 2.6 versus 61.7 ± 1.6 ng/9 min; n = 4–6). It was, however, significantly (albeit slightly) augmented, after naloxone treatment (placebo, 61.4 ± 2.6 versus 68.1 ± 1.6 ng/9 min; steroid-treated, 61.7 ± 1.6 versus 71.8 ± 4.2 ng/9 min; n = 4–6). This increase in Met-Enk release did not differ among lumbar tissue obtained from placeboversus steroid-treated animals. Consistent with this effect of naloxone, exogenous application of the -opioid DPDPE (1–500 nM; n = 4 for each concentration) (but not the -opioid U50,488H) dose dependently attenuated basal Met-Enk release from lumbar spinal tissue obtained from both control and ovarian steroid-treated animals (F_3,18 = 13.06 and F_3,20 = 4.80, respectively, p < 0.02; data shown for only the 500 nM concentration). In contrast to the involvement of -opioid receptors in modulating basal Met-Enk release, blockade of NORs (via compound 15, 10 µM; a derivative of the NOR antagoinst J-113397) (Kawamoto et al., 1999; Gupta et al., 2001) was without effect [t(17) = 1.828, n = 5, p > 0.08].

View larger version (47K): [in this window] [in a new window]   Fig. 1. Influence of E2/P and opioid or N/OFQ receptors on the basal rate of Met-Enk release. Lumbar spinal tissue was obtained from untreated and E2/P-treated animals and processed as described under Materials and Methods. Eluates (collected over 18 min) were desalted, lyophilized, and assayed by RIA for Met-Enk immunoreactivity. Each column represents mean ± S.E.M. obtained from a minimum of four experiments. Control, CTRL. *, p < 0.05 for comparison with basal release in the absence of exogenous antagonist or agonist (solid column).

Effect of Hormonal Milieu and N/OFQ on K⁺-Evoked met-Enk Release from Spinal Tissue. A two-way ANOVA was also used to assess effects of ovarian steroid treatment and exogenous N/OFQ on the fractional rise above the basal rate of Met-Enk release elicited by high K⁺ (50 mM). As was observed for basal Met-Enk release, main effects of hormone were not observed (F_1,33 = 1.078, p > 0.3). In contrast to basal Met-Enk release, however, main effects of N/OFQ were observed (F_4,33 = 19.658, p < 0.001) but there was no N/OFQ by hormone interaction (F_4,33 = 0.862, p > 0.4). Results are illustrated in Fig. 2. High (50 mM) K⁺ significantly increased the rate of Met-Enk release from lumbar spinal tissue obtained from control (80 ng/9 min; 230 ± 12.1%) or HSP animals (67 ng/9 min; 209 ± 12.5%) [t(3) = 10.35 and t(5) = 10.15, p 0.002 for both comparisons]. The numeric difference in the K⁺-evoked increase among the placebo and hormone-treated groups was not statistically significant [t(8) = 1.263, p > 0.2]. K⁺-stimulated Met-Enk release from lumbar spinal tissue obtained from both placebo and ovarian steroid-treated animals was dose dependently inhibited by N/OFQ (1–1000 nM; p < 0.02). An inhibition of 55% could be obtained with the highest concentration used (1000 nM). Neither the magnitude of the K⁺-evoked release of Met-Enk nor its inhibition by N/OFQ was influenced by the hormonal state of the animal. Figure 2 also shows that the inhibition of evoked Met-Enk release by 100 nM N/OFQ was abolished after its coadministration with the selective N/OFQ antagonist compound 15 (10 µM). This, in combination with the inability of opioid receptor agonists and antagonists to influence evoked Met-Enk release (see below), indicates that spinal NORs mediate the negative modulation of evoked Met-Enk release by exogenous N/OFQ.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Activation of spinal N/OFQ receptors dose dependently inhibits the K+-stimulated release of Met-Enk, but this negative modulation is not altered by the ovarian steroid treatment. Lumbar spinal tissue, obtained from control or E2/P-treated animals, was processed, superfusate collected, and its content of Met-Enk determined by RIA, as described under Materials and Methods. Basal and K+-stimulated release of Met-Enk, in the absence or presence of the indicated concentrations of N/OFQ, was determined in spinal tissue obtained from control (F) or E2/P-treated animals (E). Each point represents the mean ± S.E.M. percentage of increase obtained from five to six experiments. , demonstrates that blockade of NORs via compound (cpd) 15 (10 µM) abolishes the 55% inhibition of Met-Enk release produced by 100 nM N/OFQ. S, stimulated; B, basal; CTRL, control (CTRL).

Inhibition of evoked release of spinal Met-Enk by exogenous N/OFQ suggests that this peptide might also act as an endogenous negative modulator of Met-Enk release. To explore this possibility, the effect of spinal NOR blockade on evoked spinal Met-Enk release and the influence of ovarian steroids thereon was investigated (Fig. 3). A two-way ANOVA indicated a main effect compound 15 (F_1,25 = 31.06, p < 0.001) but no hormone by compound 15 interaction (F_1,25 = 0.019, p > 0.8). Consistent with the negative modulation of evoked spinal Met-Enk release by exogenously applied N/OFQ, compound 15 (10 µM) augmented (78%) the K⁺-evoked release of Met-Enk. The magnitude of this augmentation did not differ in lumbar spinal tissue obtained from placeboversus ovarian steroid treated-animals.

View larger version (35K): [in this window] [in a new window]   Fig. 3. In vivo exposure to the pregnancy blood profile of E2/P does not diminish the enhancement of evoked Met-Enk release produced by NOR blockade. Lumbar spinal tissue, obtained from control or E2/P-treated animals, was processed, superfusate collected and its content of Met-Enk determined by RIA, as described under Materials and Methods. K+-stimulated release of Met-Enk was obtained in the absence or presence of either naloxone (NX, 1 µM) or compound (cpd) 15 (10 µM). Each column represents mean ± S.E.M. percentage of increase obtained from a minimum of four experiments. S, stimulated; B, basal; CTRL, control.

Effect of Hormonal Milieu and Opioids on K⁺-Evoked met-Enk Release from Spinal Tissue. In contrast to compound 15, Fig. 3 illustrates that blockade of lumbar opioid receptors via naloxone (1 µM) did not alter K+-stimulated Met-Enk release from spinal tissue obtained from either placeboor hormone-treated animals [t(6) = 0.7083 and t(10) = 0.5763, respectively, p > 0.5 for both]. This is consonant with the inability of exogenous opioids (DPDPE, U50,488H, and sufentanil; 1–500 nM of each agonist) to attenuate the K⁺-evoked release of Met-Enk (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Evoked release of spinal Met-Enk is not modulated by spinal opioid receptors. Lumbar spinal tissue was obtained from control or E2/P-treated animals and the release of Met-Enk determined by RIA, as described under Materials and Methods. Basal and K+-stimulated release of Met-Enk was determined in the absence or presence of the indicated concentrations of sufentanil, DPDPE, or U50,488H (1, 10, and 500 nM for each). Because ANOVA failed to reveal a drug by hormone treatment interaction, values have been collapsed across treatment groups. Each point represents the mean ± S.E.M. percentage of increase obtained from eight experiments. S, stimulated; B, basal.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study used an ex vivo lumbar spinal cord preparation identical to that previously used for the pharmacological analysis of spinal dynorphin release (Gupta et al., 2001). Thus, the characteristics of the release of both opioid peptides are directly comparable. Although the (concomitant) activity of both spinal - and -opioid receptors is a prerequisite for the manifestation of E₂/P-induced antinociception (as well as that of physiological pregnancy) (Dawson-Basoa and Gintzler, 1998), modulation of the release of their respective cognate peptide during both control and HSP conditions differs dramatically.

In lumbar spinal tissue obtained from either placeboor E₂/P-treated animals, neither exogenous nor endogenous opioids seem to constitute predominant negative modulators of the activity of lumbar spinal Met-Enk neurons. This is reflected by the inability of spinal opioid receptor blockade (via naloxone) to enhance and spinal opioid receptor activation (via DPDPE, U50,488H, or sufentanil) to attenuate rates of the K⁺-stimulated release of Met-Enk from control or ovarian steroid-treated preparations. This stands in striking contrast to the regulation of spinal dynorphin release from control spinal tissue, the K⁺-induced increase of which is markedly facilitated (2-fold) by opioid receptor blockade and substantially attenuated by DPDPE or U50,488H (80 and 40%, respectively) (Gupta et al., 2001). Consequently, although the E₂/P-induced offset of the - and -opioid receptor-coupled negative modulation of evoked spinal dynorphin release contributes to its augmentation during HSP (Gupta et al., 2001), this modality of modulation is not available to spinal Met-Enk neurons.

Naloxone did increase basal rates of Met-Enk release from both preparations, as was observed previously for dynorphin (Gupta et al., 2001). However, the increase in basal Met-Enk release produced by naloxone was small (11%), which stands in contrast to the 44% increase previously observed for dynorphin (Gupta et al., 2001). Thus, opioid receptor-coupled processes seem to be less relevant to the regulation of spinal Met-Enk neuronal activity than to their dynorphin counterparts.

In contrast to opioid receptor blockade, acute treatment with a selective N/OFQ antagonist failed to enhance basal release but did significantly enhance the K⁺-induced release of Met-Enk from spinal tissue obtained from placebo-treated animals. Moreover, the K⁺-evoked increase in Met-Enk release was dose dependently reduced by exogenously applied N/OFQ, underscoring that the endogenous peptide acts as a negative modulator of stimulated spinal Met-Enk release. This is consonant with the anatomical distribution of N/OFQ and its receptor, which manifest considerable overlap with their opioid counterparts (for review, see Harrison and Grandy, 2000). In particular, N/OFQ-like immunoreactive fibers and functional NORs are particularly concentrated in the superficial laminae of the dorsal horn and the area surrounding the central canal, areas also rich in dynorphin, enkephalin, and their respective receptors. Modulation of stimulated but not basal Met-Enk release by N/OFQ parallels its ability to modulate dynorphin release from control lumbar tissue (Gupta et al., 2001).

Although ovarian steroid treatment abolished the negative modulation of stimulated dynorphin release by N/OFQ (Gupta et al., 2001), its ability to similarly modulate Met-Enk release was not diminished by E₂/P treatment. The absence of N/OFQ disinhibition of spinal Met-Enk neurons after ovarian steroid treatment is consistent with the constancy of both basal and stimulated Met-Enk release from spinal tissue obtained from control versus HSP animals. Thus, although spinal -opioid receptor activity is a prerequisite for gestational and ovarian steroid-induced antinociception (Dawson-Basoa and Gintzler, 1998), spinal Met-Enk neurons are not subjected to ovarian steroid-dependent presynaptic regulation, as are spinal neurons containing dynorphin. This would explain the ability of i.t. N/OFQ to abolish the antinociception of HSP (as well as that of physiological gestation) (Dawson-Basoa and Gintzler, 1997), despite its attenuated ability to inhibit stimulated spinal dynorphin release during this condition (Gupta et al., 2001), i.e., its i.t. application during pregnancy, and HSP continues to inhibit the release of Met-Enk and thus removes facilitative spinal -opioid tone.

During physiological gestation and parturition, the content of spinal Met-Enk is uniformly augmented throughout the spinal cord, whereas augmented levels of dynorphin are restricted to the lumbar region (Medina et al., 1993b) (the area receiving pelvic afferents; Baljet and Drukker, 1980; Steinman et al., 1992). This could indicate that spinal Met-Enk subserves an "activational" role that is permissive to transducing spinal -opioid receptor antinociception. Such events would not necessarily require enhanced release of Met-Enk but instead its modulated postsynaptic activity. In this regard, it is relevant to note that after E₂/P treatment, there is a qualitative as well as quantitative change in -opioid receptor-mediated modulation of dynorphin release. The loss of -opioid negative modulation of dynorphin release is accompanied by the emergence of its ability to facilitate such release within a defined concentration range. In this scenario, ovarian steroid-induced (and presumably gestational) antinociception is predominantly dynorphin/-opioid receptor driven with Met-Enk/-opioid tone subserving a permissive/facilitative role.

The offset of the negative modulation by N/OFQ of spinal dynorphin, but not Met-Enk release after ovarian steroid treatment reveals that the regulation of these two spinal opioid antinociceptive systems is independent and not symmetrical. Furthermore, the ability of - and -selective opioids to inhibit the stimulated release of dynorphin, but not Met-Enk from control spinal tissue indicates the presence of divergent regulation of the release of these two opioid peptides as well. The independent and divergent regulation of spinal Met-Enk and dynorphin release would maximize the ability to fine-tune their relative contributions to antinociceptive processes. This seems to be particularly relevant to the analgesia of gestation and its hormonal simulation because both require not only the concomitant activity of spinal dynorphin/- and enkephalin/-pathways but also their synergistic interactions (Dawson-Basoa and Gintzler, 1998).

In summary (Fig. 5), spinal N/OFQ functions as a negative modulator of the release of both dynorphin and Met-Enk, but the regulation of this modulation differs among them. N/OFQ inhibition of dynorphin, but not Met-Enk, release is abolished as a result of systemic treatment with pregnancy levels of E₂/P. Consequently, the release of dynorphin, but not Met-Enk, is augmented during HSP. Spinal - and -opioids also constrain the release of spinal dynorphin, but not Met-Enk, but this too is offset after simulation of the pregnancy ovarian steroid milieu. Moreover, the negative -opioid modulation of dynorphin release is not only offset but also qualitatively reverses to facilitation, i.e., the invariant release of spinal Met-Enk further accentuates dynorphin release. The combined effect of disinhibition and facilitation of dynorphin release during HSP (and presumably physiological pregnancy) results in spinal -opioid receptor activation of sufficient magnitude to produce the antinociception associated with each condition.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Schematic representation of the regulation of the release of dynorphin and Met-Enk and E2/P-induced alterations thereof. In the absence of pregnancy levels of E2/P, spinal N/OFQ is a negative modulator of both dynorphin and Met-Enk release (control synapse 2 and 3). After simulation of pregnancy levels of E2/P, negative N/OFQ modulation of dynorphin release is offset (HSP synapse 2), but its negative modulation of Met-Enk release persists unabated (HSP synapse 3). In control spinal cord, dynorphin, but not Met-Enk release, is under additional negative constraint mediated via -/-opioid receptors (control synapses 1 and 4). This negative modulation is also offset during HSP. The inhibition mediated via -opioid receptors is abolished (HSP synapse 1), whereas that mediated via -opioid receptors reverses to facilitation (HSP synapse 4). The combined effects of disinhibition and facilitation of dynorphin release during HSP (and presumably gestation) results in a magnitude of -opioid activity that is now sufficient for the antinociception associated with both conditions.

	Footnotes

 
DOI: 10.1124/jpet.102.042689.

ABBREVIATIONS: E₂, 17--estradiol; P, progesterone; HSP, hormone-simulated pregnancy; N/OFQ, nociceptin/orphanin; Met-Enk, methionine-enkephalin; DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; NOR, nociceptin/orphanin receptor; TFA, trifluoroacetic acid; RIA, radioimmunoassay; HPLC, high-performance liquid chromatography; ANOVA, analysis of variance.

Address correspondence to: Dr. Alan Gintzler, Department of Biochemistry, Box 8, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203. E-mail: agintzler{at}netmail.hscbklyn.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Baljet B and Drukker J (1980) The extrinsic innervation of the pelvic organs in the female rat. J Neurophysiol 59: 142–163.
Bridges RS (1984) A quantitative analysis of the roles of dosage, sequence and duration of estradiol and progesterone exposure in the regulation of maternal behavior in the rat. Endocrinolology 114: 930–940.[Abstract/Free Full Text]
Bridges RS and Ronsheim PM (1987) Immunoreactive -endorphin concentrations in brain and plasma during pregnancy in rats: possible modulation by progesterone and estradiol. Neuroendocrinology 45: 381–388.[Medline]
Cogan R and Spinnato JA (1986) Pain and discomfort thresholds in late pregnancy. Pain 27: 63–68.[CrossRef][Medline]
Dawson-Basoa ME and Gintzler AR (1993) 17--Estradiol and progesterone modulate an intrinsic opioid analgesic system. Brain Res 601: 241–245.[CrossRef][Medline]
Dawson-Basoa ME and Gintzler AR (1997) Nociceptin (Orphanin FQ) abolishes gestational and ovarian sex steroid-induced antinociception and induces hyperalgesia. Brain Res 750: 48–52.[CrossRef][Medline]
Dawson-Basoa ME and Gintzler AR (1998) Gestational and ovarian sex steroid antinociception: synergy between spinal and opioid systems. Brain Res 794: 61–67.[CrossRef][Medline]
Gintzler AR (1980) Endorphin-mediated increases in pain threshold during pregnancy. Science (Wash DC) 210: 193–195.[Abstract/Free Full Text]
Gintzler AR and Liu N-J (2000) Ovarian sex steroids activate antinociceptive systems and reveal gender-specific mechanisms. Sex, gender and pain: from the benchtop to the clinic, in Progress in Pain Research and Management (Fillingim RB ed) vol 17, pp 89–108, IASP Press, Seattle, WA.
Gintzler AR, Peters LC, and Komisaruk BR (1983) Attenuation of pregnancy-induced analgesia by hypogastric neurectomy in rats. Brain Res 227: 186–188.
Gupta DS, Kelson AB, Polgar WE, Toll L, Szucs M, and Gintzler AR (2001) Ovarian sex steroid-dependent plasticity of nociceptin/orphanin FQ and opioid modulation of spinal dynorphin release. J Pharmacol Exp Ther 298: 1213–1220.[Abstract/Free Full Text]
Harrison LM and Grandy DK (2000) Opiate modulating properties of nociceptin/orphanin FQ. Peptides 21: 151–172.[CrossRef][Medline]
Iwasaki H, Collins JG, Saito Y, and Kerman-Hinds A (1991a) Naloxone-sensitive, pregnancy-induced changes in behavioral responses to colorectal distention: pregnancy-induced analgesia to visceral stimulation. Anesthesiology 74: 927–933.[CrossRef][Medline]
Iwasaki H, Collins JG, Saito Y, Uchida H, and Kerman-Hinds A (1991b) Low-dose clonidine enhances pregnancy-induced analgesia to visceral but not somatic stimuli in rats. Anesth Analges 72: 325–329.[Abstract/Free Full Text]
Jarvis S, McLean KA, Chirnside J, Deans LA, Calvert SK, Molony V, and Lawrence AB (1997) Opioid-mediated changes in nociceptive threshold during pregnancy and parturition in the sow. Pain 72: 153–159.[CrossRef][Medline]
Jayaram A, Singh P, and Carp H (1995) SCH32615, an enkephalinase inhibitor, enhances pregnancy-induced analgesia in mice. Obstet Anesth 80: 944–948.
Jayaram A, Singh P, Noreuil T, Fournie-Zaluski MC, and Carp HM (1997) RB 101, a purported pro drug inhibitor of enkephalin metabolism, is antinociceptive in pregnant mice. Anesth Analg 84: 355–358.[Abstract]
Kawamoto H, Ozaki S, Itoh Y, Miyaji M, Arai S, Nakashima H, Kato T, Ohta H, and Iwasawa Y (1999) Discovery of the first potent and selective small molecule opioid receptor-like (ORL1) antagonist: 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397). J Med Chem 42: 5061–50633.[CrossRef][Medline]
Kristal MB, Thompson AC, Abbott P, Di Pirro JM, Ferguson EJ, and Doerr JC (1990) Amniotic-fluid ingestion by parturient rats enhances pregnancy-mediated analgesia. Life Sci 46: 693–698.[CrossRef][Medline]
Kumar MSA, Haney M, Becker T, Thompson ML, Kream R, and Miczek K (1990) Effect of early exposure to delta-9-tetrahydrocannabinol on the levels of opioid peptides, gonadotropin and substance P in the adult male rat brain. Brain Res 525: 78–83.[CrossRef][Medline]
Liu N-J and Gintzler AR (1999) Gestational and ovarian sex steroid antinociception: relevance of uterine afferent and spinal ₂-noradrenergic activity. Pain 83: 359–368.[CrossRef][Medline]
Medina VM, Dawson-Basoa ME, and Gintzler AR (1993a) 17--Estradiol and progesterone positively modulate spinal cord dynorphin: relevance to the analgesia of pregnancy. Neuroendocrinology 58: 310–315.[Medline]
Medina VM, Gupta D, and Gintzler AR (1995) Spinal cord dynorphin precursor intermediates decline during late gestation: implications for enhanced processing as an adaptive mechanism in dynorphin neurons. J Neurochem 65: 1374–1380.[Medline]
Medina VM, Wang L, and Gintzler AR (1993b) Spinal cord dynorphin: positive region-specific modulation during pregnancy and parturition. Brain Res 623: 41–46.[CrossRef][Medline]
Sander HW and Gintzler AR (1987) Spinal cord mediation of the opioid analgesia of pregnancy. Brain Res 408: 389–393.[CrossRef][Medline]
Steinman JL, Carlton SM, and Willis WD (1992) The segmental distribution of afferent fibers from the vaginal cervix and hypogastric nerve in rats. Brain Res 575: 25-31.[CrossRef][Medline]
Toniolo MV, Whipple BH, and Komisaruk BR (1987) Spontaneous maternal analgesia during birth in rats (Abstract), in Proceedings of the NIH Centennial MBRS-MARC Symposium 15: 100.
Xu H, Smolens I, and Gintzler AR (1989) Opioids can enhance and inhibit the electrically evoked release of methionine-enkephalin. Brain Res 504: 36–42.[CrossRef][Medline]

This article has been cited by other articles:

				D. S. Gupta, H. von Gizycki, and A. R. Gintzler Sex-/Ovarian Steroid-Dependent Release of Endomorphin 2 from Spinal Cord J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 635 - 641. [Abstract] [Full Text] [PDF]

				P. M. Schmitt and M. P. Kaufman Estrogen's attenuating effect on the exercise pressor reflex is more opioid dependent in gonadally intact than in ovariectomized female cats J Appl Physiol, February 1, 2005; 98(2): 633 - 639. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) An erratum has been published Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gupta, D. S. Articles by Gintzler, A. R. Search for Related Content PubMed PubMed Citation Articles by Gupta, D. S. Articles by Gintzler, A. R.