Differential effects of exercise on brain opioid receptor binding and activation in rats.

Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

Capsaicin induces reflex scratching in inflamed skin.

We investigated whether capsaicin induces itching in skin with existing inflammation. We induced skin inflammation by intradermal injection of complete Freund's adjuvant (CFA) in the neck of mice. Four days later, we injected capsaicin in the same area and counted the number of scratching bouts for 30 min. We examined potential effects on pain in parallel experiments in which CFA and capsaicin were intradermally injected into hind paws. We used the time spent licking the hind paws during the 15 min after capsaicin injection as an estimate of pain. Capsaicin injection into the skin pretreated with CFA, but not into healthy skin, induced scratching. The scratching behavior was reduced by pretreatment with naloxone or capsazepine, selective antagonists for transient receptor potential vanilloid receptor-1 (TRPV1), but not morphine or mepyramine, selective antagonists for histamine 1 receptor. In animals injected with capsaicin into the hind paws, licking behavior was significantly inhibited via a µ-receptor-dependent mechanism. Our results show that TRPV1 activation, which normally induces pain, evokes an itch-related response in the presence of inflammation. This model may be interesting for future studies to explore the mechanism of a painful stimuli-induced itch observed under pathological conditions.

Antioxidant, antinociceptive, and anti-inflammatory properties of the ethanolic extract of Combretum duarteanum in rodents.

The antioxidant, antinociceptive, and anti-inflammatory activities of the ethanolic extract from leaves of Combretum duarteanum (EEC) were assessed in rodents through in vitro tests. The antioxidant activity was investigated by using thiobarbituric acid reactive species (TBARS), hydroxyl radical-scavenging, and scavenging activity of nitric oxide assays. The antinociceptive activity was investigated by using acetic acid-induced writhing, formalin, and hot-plate tests in mice. The anti-inflammatory activity was assessed in rats by using the carrageenan-induced hind-paw edema test and arachidonic acid-induced paw edema test. EEC possesses a strong antioxidant potential according to the TBARS, nitric oxide, and hydroxyl radical-scavenging assays; it also presented scavenger activity in all in vitro tests. After intraperitoneal injection, EEC (100, 200, and 400 mg/kg) significantly reduced the number of writhes (38.1%, 90.6%, and 97.8%, respectively) in a writhing test and the number of paw licks during phase 1 (30.5% and 69.5%, higher doses) and phase 2 (38.1%, 90.6%, and 97.8%, all doses) of a formalin test when compared with the control group. Naloxone (1.5 mg/kg, intraperitoneally) antagonized the antinociceptive action of EEC (400 mg/kg), and this finding suggests participation of the opioid system. Administration of 200 and 400 mg/kg (intraperitoneally) of EEC exhibited an anti-inflammatory activity in the carrageenin test, which was based on interference with prostaglandin synthesis. This finding was confirmed by the arachidonic acid test. Together, these results indicate that properties of EEC might be further explored in the search for newer tools to treat painful inflammatory conditions, including those related to pro-oxidant states.

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment.

Adult ovariectomized rats were implanted with [D-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52 +/- 11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24-48 h following ENK implantation, expression of ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.

4-Caffeoyl-1,5-quinide in roasted coffee inhibits [3H]naloxone binding and reverses anti-nociceptive effects of morphine in mice.

RATIONALE: Cinnamoylquinides are formed from the corresponding chlorogenic acids during coffee roasting. Instant coffee has been shown to displace binding of the mu opioid receptor antagonist, [3H]naloxone, but the putative active agent, feruloylquinide, has not been characterized. OBJECTIVES: The goal was to identify the active agent(s) in coffee by measuring the binding affinity of individual cinnamoyl-1,5-quinides to the human mu opioid receptor, and determine the effects of these compounds on morphine-induced anti-nociceptive behavior in mice. METHODS: Cinnamoyl-1,5-quinides in extracts of decaffeinated instant coffee were quantified by reverse-phase HPLC comparisons with synthetic samples of 3-coumaroyl-1,5-quinide and 4-coumaroyl-1,5-quinide, 3-caffeoyl-1,5-quinide and 4-caffeoyl-1,5-quinide (4-CQL) 3-feruloyl-1,5-quinide and 4-feruloyl-1,5-quinides and 3,4-dicaffeoyl-1,5-quinide (DICAQ). Affinities of the cinnamoyl-1,5-quinides and decaffeinated instant coffee extract were determined by displacement of [3H]naloxone binding in cultured HEK-MOR cells. Inhibition of the anti-nociceptive activity of morphine (1 mg/kg IP) was determined in C57BL/6J mice using the hot plate test at 52 degrees C. RESULTS: Extract of decaffeinated instant coffee produced a displacement K(i) of 42+/-16 mg/l, while the K(i) of a synthetic sample of 4-CQL was 4.4+/-0.4 microM. Compounds with a cinnamoyl substituent in the 4-position of the quinide, i.e. 4-CQL, DICAQ, 3,4-diferuloyl-1,5-quinide, and 3,4-dicoumaroyl-1,5-quinide, had affinities for the mu opioid receptor in the low micromolar range. In the hot plate test, coffee extract, containing 0.78% of 4-CQL, reversed the anti-nociceptive effect of morphine at 10 mg/kg IP. Two cinnamoyl-1,5-quinides found in roasted coffee, DICAQ, and 4-CQL, were active at 1 and 0.1 mg/kg IP, respectively. CONCLUSIONS: These results suggest that the previously reported anti-opioid activity of instant coffee is caused primarily by the presence of 4-CQL, and to lesser extent by other cinnamoyl-1,5-quinides.

Peculiarities of the development of analgesic effect during transcutaneous dynamic electrical stimulation.

The effect of transcutaneous dynamic electrical neurostimulation on the development of analgesia was studied in behavioral and electrophysiological experiments on rats. A 30-min dynamic electrical stimulation elevated the nociception threshold in tail-flick and hot plate tests, increased the threshold of the late nociceptive flexor reflex, and decreased the number of bursts in the response. Intraperitoneal injection of naloxone (2 mg/kg) abolished the analgesic effect of dynamic electrical neurostimulation. It is concluded that the key role in reflex analgesia during dynamic electrical neurostimulation is played by the endogenous cerebral opioid system, which inhibits the nociceptive signals traveling to CNS via unmyelinated C-fiber afferents.

Ginsenoside Rf potentiates U-50,488H-induced analgesia and inhibits tolerance to its analgesia in mice.

In the present study, the effect of ginsenoside Rf (Rf), a trace component of Panax ginseng on U-50,488H (U50), a selective kappa opioid-induced analgesia and its tolerance to analgesia was studied using the mice tail-flick test. In addition, the possible mechanism by which Rf may affect U50-induced analgesia was investigated. Intraperitoneal administration of U50 (40 mg/kg) produced analgesia. Rf (10(-14)-10(-10) mg/kg) on co treatment dose-dependently potentiated the U50 (40 mg/kg)-induced analgesia. Rf (10(-12)-10(-2) mg/ml) did not alter the binding of [3H] naloxone, a opioid ligand and [3H]PN200-110, a dihydropyridine ligand to mice whole brain membrane. Twice daily administration of U50 (40 mg/kg) for six days induced tolerance to its analgesia. Chronic treatment (day 4-day 6) of Rf (10(-14)-10(-10) mg/kg) to U50-tolerant mice, dose-dependently inhibited the tolerance. The inhibition of tolerance to U50-induced analgesia by Rf was not altered by flumazenil (0.1 mg/kg), a benzodiazepine receptor antagonist and picrotoxin (1 mg/kg), a GABA(A)-gated chloride channel blocker on chronic treatment. In conclusion, these findings for the first time demonstrated that ginsenoside Rf potentiates U50-induced analgesia, inhibits tolerance to its analgesia, and suggests that Rf affects U50-induced analgesia via non-opioid, non-dihydropyridine-sensitive Ca(+2) and non-benzodiazepine-GABA(A)ergic mechanisms in mice.

Altered adenylyl cyclase responsiveness subsequent to point mutations of Asp 128 in the third transmembrane domain of the delta-opioid receptor.

delta-Opioid receptors belong to the superfamily of G protein-coupled receptors, characterized by seven putative transmembrane domains, and have been shown to interact with a host of effector systems. It has been suggested that the charge on the conserved aspartic acid residue at position 128 in transmembrane domain 3 of the delta-opioid receptor contributes to both the conformation of the receptor binding pocket and the molecular rearrangements which accompany the establishment of high-affinity states of the receptor. In light of this, we used site-directed mutagenesis to determine whether this residue participates in the transmission of signals to adenylyl cyclase, the effector with which opioid receptors have been classically associated. Substitution of this aspartic acid (D128) for the neutral amino acid alanine, or the protonated amino acids lysine and histidine, constitutively couples the receptor to adenylyl cyclase, as evidenced by a curtailed response to forskolin stimulation in transfected cells. In addition, this constitutive activity can be blocked by pretreatment of the transfected cells with pertussis toxin. Interestingly, naloxone blocks this effect in cells expressing the D128A mutant, but acts as an agonist at the D128K mutant. Our findings support the hypothesis that the interaction between agonist and receptor promotes conformational changes that may be mimicked, at least in part, by mutation of the aspartate residue at position 128. Furthermore, these changes appear to be involved not only in receptor activation, but also in the functional discrimination between agonists and antagonists.

Intraarticular and periarticular opioid binding in inflamed tissue in experimental canine arthritis.

UNLABELLED: Small-dose (1 mg) intraarticular morphine has been used successfully in many studies to provide long-lasting analgesia after arthroscopic knee surgery. We used radioligand binding to determine whether these effects could be mediated by opioid binding sites in the joint, particularly after the induction of inflammation. Inflammation was induced by the injection of oleyl alcohol (20 microL) in sterile peanut oil (0.25 mL) into the left radiocarpal joint of 27 dogs, and the dogs were euthanized at 12 h. The articular and periarticular tissues from both treated and control joints were collected, and membranes were prepared for equilibrium binding assays. The density of specific opioid binding was markedly enhanced (P < 0.05) in homogenates prepared from the treated relative to those from the control joint. The binding affinities (KD values) for morphine and naloxone (mean +/- SEM) were approximately one one-hundredth (79 +/- 17 nM and 124 +/- 5.5 nM, respectively) that of the corresponding published affinities in brain tissue. However, the binding site densities were approximately one hundred times larger (Bmax = 1032 +/- 265 and 543 +/- 51 fmol/mg of protein) than the respective published values in brain tissue. These findings imply that the opioid binding sites, found in the inflamed articular and periarticular tissues in this study, are similar to those of putative mu 3-opioid binding sites that appear to be present on cultured thymocytes and in the airways of rats and humans. IMPLICATIONS: The high density of opioid binding sites found in inflamed canine joint tissue supports the clinical use of intraarticular opioids in the treatment of postoperative and chronic inflammatory joint pain.

Hypericum perforatum inhibits the binding of mu- and kappa-opioid receptor expressed with the Semliki Forest virus system.

The effects of Hypericum perforatum extracts on in vitro [3H]naloxone binding to the human mu- and rat kappa-opioid receptors were studied in chinese hamster ovary (CHO) cells using the Semliki Forest virus (SFV) expression system. Binding of [3H]naloxone to the mu- and kappa-opioid receptor was inhibited in the presence of Hypericum extracts showing IC50 values of approximately 25 and 90 micrograms/ml, respectively. In contrast, extracts of Valeriana officinalis did not inhibit binding to the mu-opioid receptor. Also, single constituents of H. perforatum like the flavonoids quercetin and kaempferol and the glycosilated flavonoid quercitrin did not inhibit [3H]naloxone binding to the mu-opioid receptor up to a concentration of 10 microM. The present in vitro data may suggest a new possible mechanism for the anti-depressant effect of H. perforatum.

Effects of Psychotria colorata alkaloids in brain opioid system.

An ethnopharmacological survey showed that home remedies prepared with flowers and fruits of Psychotria colorata are used by Amazonian peasants as pain killers. Psychopharmacological in vivo evaluation of alkaloids obtained from leaves and flowers of this species showed a marked dose-dependent naloxone-reversible analgesic activity, therefore suggesting an opioid-like pharmacological profile. This paper reports an inhibitory effect of P. colorata flower alkaloids on [3H]naloxone binding in rat striata as well as a decrease in adenylate cyclase basal activity. The alkaloids did not affect [3H] GMP-PNP binding. These findings provide a neurochemical basis for the opioid-like activity previously detected in vivo and point to Psychotria alkaloids as a potential source of new bioactive opiate derivatives.

Nuclear [3H]naloxone binding in rat hypothalamus.

High affinity (Kd approximately 1 nM) and low capacity [3H]naloxone binding sites were detected in the nuclear fraction of rat hypothalamus. The profile of this binding changed with age. In immature rats from 11 days of age until vaginal opening (approximately 33 day) the Scatchard plots of saturation data were linear and the Hill coefficient was 1, while just after vaginal opening (< 6 h) Scatchard analysis of [3H]naloxone binding gave a curvilinear component, with the Hill coefficient approximately 2, followed by an increase in binding sites and a decrease in Kd. In adults, after ovariectomy, the pattern of [3H]naloxone binding was similar to that observed in immature rats before vaginal opening. Following oestradiol treatment, binding sites with linear Scatchard plots disappeared and returned at least 24 h after hormone administration.

Age-related changes in biogenic amines, opiate, and steroid receptors in the prepubertal bull calf.

Events leading to the increase in pulsatile LH secretion during prepubertal development in the bull calf may include removal of inhibitory or the development of stimulatory mechanisms affecting the hypothalamic release of GnRH. To examine possible contributing systems, serial blood samples were collected from Holstein bull calves at 2, 5, and 10 wk of age one day prior to receiving either no treatment (controls) or two injections of alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of catecholamine synthesis. Blood was sampled every 10 min for 5 h and serum was analyzed for LH by RIA. Following treatment, animals were killed and hypothalamic and pituitary tissues were removed for analysis of total opiate receptors, mu-opiate receptors, estrogen and androgen receptors and concentrations of monoamines: dopamine, the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DO-PAC), norepinephrine, serotonin, and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA). Pulses of LH increased from non-detectable at 2 wk to nearly 1.5 pulses per sampling period at 10 wk. Pulse height rose to 0.95 +/- 0.16 ng/ml at 10 wk. Total opiate receptor number as determined by binding to naloxone was unchanged in all tissues between 2 and 10 wk. In contrast, mu-opiate receptors (DAGO binding) increased 2-fold in the preoptic-anterior hypothalamic area between 5 and 10 wk. No age-related changes in estrogen receptor concentrations were observed in any tissue except the anterior pituitary in which binding increased 3.2-fold between 2 and 10 wk. A similar increase was not noted for androgen receptors in the pituitary; however, testosterone binding in the preoptic-anterior hypothalamic area was 4.6-fold higher at 5 wk compared to levels at 2 and 10 wk.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous intrathecal opioid treatment abolishes the regulatory effects of magnesium and guanine nucleotides on mu opioid receptor binding in rat spinal membranes.

The regulatory effects of cations and guanine nucleotides on mu receptor binding after opioid drug and pertussis toxin treatment were studied in the rat spinal cord model. Continuous intrathecal (i.t.) infusion with PL017 for 5 days induced tolerance in a dose-dependent manner. Maximal tolerance was observed at day 2. A single i.t. dose (1 microgram) of pertussis toxin also induced tolerance to opioid. When mu receptor binding of the high-affinity sites was determined by 125I-FK33824, spinal membrane preparations from morphine- and pertussis toxin-induced tolerant animals demonstrated approximately 30% less binding than control membranes. Analysis of equilibrium competition binding of FK33824 against [3H]naloxone under a variety of experimental conditions (i.e., cations and guanine nucleotides) revealed differences among control and treated membranes. With Na+ (100 mM) + GDP (100 microM) pretreated membranes and binding assays conducted in the presence of Mg++, all mu receptors were observed to be in a high-affinity state in control membranes, whereas about 30% of receptors were in the low-affinity state in membranes from opioid- and pertussis toxin-treated animals. The increase in the proportion of low-affinity sites was dependent upon the infusion dose of PL017, and the increase correlated well with the degree of opioid tolerance developed. The regulatory effect of 5'-guanylylimidodiphosphate on opioid agonist binding was reduced in membranes from pertussis toxin- or opioid-treated animals. In binding assays conducted in the presence of Na+ (100 mM) + Mg++ (5 mM) + 5'-guanylylimidodiphosphate (30 microM) or Na+ (100 mM) + GDP (100 microM), all mu receptors in control membranes were in a low affinity-state, while those from opioid- or pertussis toxin-treated animals existed in both the high- and the low-affinity states. Continuous i.t. infusion with PL017 at the high dose of 1 microgram/hr for 5 days also decreased significantly (about 40%) the total number of receptors. These studies indicate that continuous opioid infusion and pertussis toxin treatment results in impairment in the receptor-G-protein coupling. This is reflected by the decreased regulatory effects of Mg++ and guanine nucleotides. Thus, in addition to receptor down-regulation, which is induced by PL017 at high doses, receptor-G-protein uncoupling may play a role in opioid tolerance induced by continuous infusion with morphine and PL017.

In vivo and in vitro studies on the opioidergic control of the secretion of gonadotrophin-releasing hormone and luteinizing hormone in sexually immature and adult male rats.

In vivo and in vitro methods have been used to compare the effects of opioid receptor blockade on the functional activity of the hypothalamo-pituitary-gonadal axis in adult (200 g) and sexually immature (50 g) male rats. In the adult, a single injection of the mu-receptor antagonist, naloxone (500 micrograms/100 g body weight, s.c.), produced hypersecretions of luteinizing hormone (LH) and testosterone. Maximal serum concentrations of the two hormones were attained within 20 and 60 min respectively. In contrast, neither ICI 174864 (100 micrograms/100 g body weight, s.c.) nor MR2266 (150 micrograms/100 g body weight, s.c.), which block delta- and kappa-receptors respectively, stimulated pituitary-gonadal activity; indeed, like the saline vehicle, both tended to depress the serum LH concentration. The injection procedure was sufficient to activate the hypothalamo-pituitary-adrenal axis and, thus, the vehicle-treated controls exhibited significant increases in the plasma adrenocorticotrophin and serum corticosterone concentrations. These effects were enhanced by naloxone (500 micrograms/100 g body weight, s.c.) and by the kappa-opioid receptor (MR2266, 150 micrograms/100 g body weight, s.c.) but not by the delta-opioid receptor antagonist (ICI 174864, 30-100 micrograms/100 g body weight, s.c.). The increases in serum corticosterone and LH concentration induced by naloxone in adult rats were not apparent in the sexually immature (50 g) animals. To the contrary, in the young rats naloxone (250 and 500 micrograms/100 micrograms body weight, s.c.) attenuated, in a dose-dependent manner, the pronounced hypersecretion of corticosterone induced by the vehicle injection. The higher dose of the antagonist (500 micrograms/100 g body weight, s.c.) also overcame the significant reductions in serum LH evident 20 (p less than 0.05) and 40 (p less than 0.01) min after the saline injection but the lower dose (250 micrograms/100 g body weight, s.c.) was ineffective in this respect. In vitro, hypothalami from both adult and sexually immature rats responded to the addition of naloxone (10(-8)-10(-6) M) to the incubation medium with significant (p less than 0.01) concentration-dependent increases in the release of gonadotrophin-releasing hormone (GnRH). In contrast, ICI 174864 (10(-7)-10(-6) M) and MR2266 (10(-7)-10(-6) M) had little effect on the secretion of the releasing hormone by hypothalami from rats of either group although, at the highest concentration tested, MR2266 (10(-6) M) precipitated a small increase in GnRH release from hypothalami from adult rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Opioid receptors are present in the hypothalamus but not detectable in the anterior pituitary of the developing ovine fetus.

Exogenous opioids stimulate adrenocorticotropic hormone (ACTH) release in fetal sheep after day 125 of pregnancy (term 145 days), but not at day 110. To determine if the different response is due to an alteration in opioid-binding sites and to examine sites of opioid action on ACTH release, we established an in vitro binding assay to study changes and characteristics of opioid receptor binding sites in ovine fetal hypothalamus and pituitary during development. [3H]-naloxone was used as the radiolabelled ligand. The binding of [3H]-naloxone to hypothalamic membrane preparations was specific, saturable with respect to [3H]-naloxone concentration, and linear with the hypothalamic membrane protein content. Binding assays were conducted on tissues collected from fetuses at three gestational ages: days 110-115 and 125-130 and term. In all cases, Scatchard analysis revealed a single class of binding sites with high binding affinity (0.9-1.2 nM) which did not differ significantly with gestational ages. The binding capacity increased significantly from 45.4 +/- 2.5 fmol/mg protein at days 110-115 to 76.8 +/- 2.3 fmol/mg at days 125-130, but did not change further in term fetuses. When anterior pituitaries from the three groups of fetuses were processed and analyzed in a similar manner, no detectable binding was found. These results indicate (1) that endogenous opioid peptides may act at the hypothalamus rather than pituitary to regulate hypothalamic-pituitary-adrenal activity in the ovine fetus, and (2) the developmental changes in the binding capacity may contribute, in part, to the altered ACTH response reported in previous in vivo studies.

Morphine decreases LH secretion in ovariectomized ewes only after steroid priming and not by direct pituitary action.

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogenesis of cell surface mu-opioid ([3H]DAGO) binding sites in rat hypothalamus and ex vivo determination of blood-brain barrier penetration by opioid peptide FK 33-824.

Previous studies have demonstrated that the LH response to naloxone changes during development but the reason(s) for this are unknown. In the present work we have investigated the possibility that variations in cell surface opioid receptor levels (determined in tissue slice/punches) or changes in the ability of opioids to enter the CNS might be responsible. Opioid binding data indicate that both [3H]naloxone and [3H]DAGO-labelled binding sites remain at low levels until 10 days of age after which there is a progressive rise to adult levels at 15 days ([3H]DAGO) and 21 days ([3H]naloxone). Although several peaks and nadirs were observed in this detailed profile of receptor ontogeny, no exact correlation with the time course of LH response to opioid drugs was found. In an adaption of the slice binding assay we are able to quantify drug penetration into the brain (ex vivo binding). Ex vivo binding studies of blood-brain barrier (BBB) ontogeny indicate that there are changes with age in the ability of opioid peptides, injected subcutaneously, to inhibit binding at the mu-receptor. FK 33-824 induced a reduction in [3H]DAGO binding in the mediobasal hypothalamus until 15 days of age. FK in older rats had no effect on [3H]DAGO binding suggesting that formation of the BBB is complete at this age. In contrast, FK injection reduced binding in the median eminence-arcuate nucleus area (outside BBB) until 30 days of age. Surprisingly, this area also became refractory to FK injection after this age.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen and progesterone regulate opiate receptor densities in multiple brain regions.

The negative and positive feedback, actions of estrogen and progesterone on the hypothalamus may be mediated in part by the endogenous opiate system. Estrogen and progesterone suppress physiological responses to the administration of opiate peptides. To determine whether this desensitization to opiates involves receptor down-regulation, we measured the density of naloxone-binding sites in hypothalamic regions regulating gonadotropin release and/or sexual behavior. Using autoradiographic techniques, we measured the density of naloxone-binding sites at various times of the day in intact proestrous, ovariectomized, or ovariectomized rats treated with estrogen or estrogen plus progesterone. Ovariectomy increased naloxone-binding sites compared to intact proestrous rats in all hypothalamic regions examined. Treatment with estrogen decreased and treatment with estrogen plus progesterone decreased even more the densities of naloxone-binding sites. This steroid-induced suppression of opiate receptors appears to have important physiological repercussions and provides a basis for the steroid-induced differential sensitivity to exogenous opiates that has been observed previously.