Peripheral alpha4beta2 nicotinic acetylcholine receptor signalling attenuates tactile allodynia and thermal hyperalgesia after nerve injury in mice.

AIM: Neuropathic pain is often refractory to conventional analgesics including opioids and non-steroidal anti-inflammatory drugs. Evidence suggests nicotinic acetylcholine receptor ligands regulate pain transmission. Effects of α4ß2 nicotinic acetylcholine receptor activation on pain behaviours after nerve injury were studied. METHODS: Mice were subjected to partial sciatic nerve ligation (PSL). Nicotinic acetylcholine receptor α4 and ß2 subunits localization in injured nerves were evaluated by immunohistochemistry. Neuropathic pain, assessed by tactile allodynia and thermal hyperalgesia, was examined by von Frey test and Hargreaves test respectively. RESULTS: Nicotinic acetylcholine receptor α4 and ß2 subunits were up-regulated in injured nerves and were expressed on F4/80-positive macrophages. When nicotine was perineurally administered daily for 4 days (day 7-10; maintenance phase) after nerve injury, pain behaviours were significantly alleviated. The inhibitory effects of nicotine were reversed by co-administration of mecamylamine (non-selective nicotinic acetylcholine receptor antagonist) and dihydro-ß-erythroidine (selective α4ß2 nicotinic acetylcholine receptor antagonist). Likewise, when α4ß2 nicotinic acetylcholine receptor agonists (TC2559 or ABT418) were administered daily for 4 days (day 7-10) after nerve injury, pain behaviours were significantly attenuated. On the other hand, nicotine administered daily for 4 days (day 0-3; initiation phase) after nerve injury alleviated pain behaviours, which were antagonized by co-administration of dihydro-ß-erythroidine. TC2559 administered daily for 4 days (day 0-3) also attenuated nerve injury-induced pain behaviours. CONCLUSION: The activation of α4ß2 nicotinic acetylcholine receptor expressed on infiltrating macrophages in injured nerves may participate in the relief of PSL-induced neuropathic pain during maintenance and initiation phases.

CC-chemokine ligand 4/macrophage inflammatory protein-1ß participates in the induction of neuropathic pain after peripheral nerve injury.

BACKGROUND: Neuropathic pain is caused by neural damage or dysfunction and neuropathic pain-related symptoms are resistant to conventional analgesics. Neuroinflammation due to the cytokine-chemokine network may play a pivotal role in neuropathic pain. We demonstrate that macrophage inflammatory protein-1ß (MIP-1ß) participates in neuropathic pain. METHODS: Mice received partial sciatic nerve ligation (PSL), and tactile allodynia and thermal hyperalgesia were assessed by von Frey test and Hargreaves test, respectively. Agents were administered into the region surrounding the sciatic nerve (SCN). RESULTS: Using reverse transcription polymerase chain reaction, the mRNA expressions of MIP-1ß and its receptor (CC-chemokine receptor 5; CCR5) in the injured SCN were up-regulated after PSL. MIP-1ß immunoreactivity was localized in macrophages and Schwann cells and increased in the injured SCN on day 1. PSL-induced tactile allodynia on days 4 to 7 was prevented by the administration of MIP-1ß neutralizing antibody (anti-MIP-1ß; on days 0, 3 and 6). PSL-induced up-regulations of inflammatory cytokine-chemokine mRNAs in the injured SCN were suppressed with anti-MIP-1ß treatment on day 7. Administration of CCR5 antagonist, D-ala-peptide T-amide (on days 0, 3 and 6) prevented tactile allodynia and thermal hyperalgesia on days 4 to 14. Single administration of recombinant mouse MIP-1ß (rmMIP-1ß) elicited tactile allodynia. Moreover, rmMIP-1ß increased the mRNA expression of inflammatory mediators in the SCN on day 1 after administration. CONCLUSIONS: These results suggest that MIP-1ß is a novel key mediator, and the peripheral MIP-1ß-CCR5 axis contributes to neuropathic pain. Therefore, investigation of this cascade might be a validated approach for the elucidation of neuropathic pain mechanisms.

Inhibition of morphine tolerance is mediated by painful stimuli via central mechanisms.

Tolerance to morphine analgesia following repeated administration disturbs the continuation of opioid therapy for severe pain. Emerging evidence suggests that the development of morphine tolerance may be antagonized by painful stimuli. To clarify the detailed mechanisms of these phenomena, we examined the effects of several pain stimuli on morphine-induced tolerance. Subcutaneous (s.c.) injection of morphine (10 mg/kg) produced an analgesic effect, which was evaluated by tail-pinch test. Morphine-induced analgesia was diminished by repeated administration of morphine (10 mg/kg, s.c.) once a day for 5 days, demonstrating the development of tolerance. Morphine analgesic tolerance was suppressed by nerve injury-induced neuropathic pain and formalin- or carrageenan-induced inflammatory pain. Tolerance to serum corticosterone elevation by morphine (10 mg/kg), which was evaluated by fluorometric assay, was also suppressed by formalin-induced inflammatory pain. Moreover, morphine analgesia induced by intracerebroventricular (10 nmol) or intrathecal (5 nmol) injection was diminished by repeated administration of morphine s.c., and this was also suppressed by carrageenan-induced inflammatory pain. These results suggest that morphine tolerance is inhibited by several pain stimuli, including neuropathic and inflammatory pain, through central mechanisms.

Availability of serum corticosterone level for quantitative evaluation of morphine withdrawal in mice.

Physical dependence on morphine is evidenced by the withdrawal syndromes, including body weight loss, which are induced by the discontinuation of morphine exposure or by the treatment with naloxone, an opioid receptor antagonist. The present study was designed to examine whether the elevation of serum corticosterone (SCS) level induced by naloxone-precipitated morphine withdrawal was a useful index to quantify the physical dependence on morphine in mice, which was compared with body weight loss induced by naloxone-precipitated morphine withdrawal. The SCS level was dependent on the dosage and the number of dosing of morphine and challenging dosage of naloxone. Intraplantar injection of formalin, potentially producing inflammatory pain, inhibited both body weight loss and SCS increase induced by naloxone challenge in mice receiving repeated exposure of morphine, indicating that formalin-induced pain attenuated the development of physical dependence on morphine. The magnitude of body weight loss in morphine withdrawal was significantly correlated with the magnitude of naloxone challenge-induced SCS increase. These results suggest that the naloxoneinduced increase in SCS level is a quantitative index of the magnitude of physical dependence on morphine in mice.

Pioglitazone attenuates tactile allodynia and microglial activation in mice with peripheral nerve injury.

To test the possibility of a peroxisome proliferator activated receptor (PPAR) γ agonist to treat neuropathic pain, we examined the effects of pioglitazone, a PPARγ agonist, on tactile allodynia and expression of activated microglia in the dorsal horn of spinal cord using neuropathic pain model. The unilateral sciatic nerve was partially ligated (PSL) in male ICR mice. Pioglitazone (1-25 mg/kg p.o.) was administrated to mice once daily for five days immediately after PSL. We stimulated the footpad of the hind paw of mice using a von Frey filament to estimate tactile allodynia on day 5 of PSL. The activated microglia in the lumbar spinal cord was observed by immunohistochemistry with anti-Iba1 antibody, a marker for activated microglia. The number of Iba1-immunoreactive cells was counted in the dorsal horn spinal cord. On day 5, significant allodynia was developed in PSL mice. Pioglitazone significantly attenuated the tactile allodynia in a dose of 1-25 mg/kg. However, these doses of pioglitazone did not affect nociceptive responses in sham mice. Moreover, on day 6, the number of activated microglia was significantly increased in the ipsilateral dorsal horn of mice. The increase in the number of activated microglia induced by PSL was significantly suppressed by pioglitazone (1-25 mg/kg p.o.). Pioglitazone did not affect the number of activated microglia in sham mice. These results suggest that PPARγ activation inhibits the development of tactile allodynia and the expression of activated microglia in the dorsal horn of spinal cord in mice with PSLinduced peripheral nerve injury.

Actions of domperidone on the opioid system in isolated guinea-pig ileum: differences between dopamine antagonists on opioid system.

1. To validate the relationship between the dopaminergic, opioidergic and cholinergic nervous systems, we evaluated the effect of domperidone, a dopamine (D2) antagonist, on the opioid system in myenteric plexus-longitudinal muscle preparation isolated from the guinea-pig ileum. 2. One micromolar of domperidone did not affect the 0.1 Hz-evoked (duration 0.5 ms, maximum intensity) twitch response, but concentration dependently inhibited the twitch response between concentrations of 2 and 20 microM, and the inhibition was maximum after 20-30 min at the highest concentration used (20 microM). 3. Acetylcholine-evoked contraction on basal tension was also not inhibited by 1 microM domperidone, but the contraction was concentration dependently inhibited at concentrations of 10-100 microM in a non-competitive manner. 4. One micromolar of domperidone, however, increased post-tetanic twitch inhibition, an indicator of the release of endogenous opioids. This increase was completely antagonized by 1 microM naloxone. Twitch inhibition induced by dynorphin 1-13 (0.1-10 nM) was not affected by 1 microM domperidone, but increased the maximum twitch inhibition caused by morphine (0.1-1 microM). 6. These results might reflect the existence of an interaction between the dopaminergic and opioidergic system without the inhibition of the cholinergic system. Dopamine antagonists increased opioid action, an action that may depend more on the increased release of endogenous opioids than on supersensitivity of the opioid receptor.

Adrenalectomy-induced potentiation of morphine action in guinea-pig ileum: possible decrease in the release of endogenous opioids from opioidergic neurones.

1. The effects of adrenalectomy on exogenous and endogenous opioid actions in guinea-pig isolated myenteric plexus-longitudinal muscle (MPLM) were investigated. 2. A decrease in serum cortisol level to about 37% of the level in the sham-operated group was obtained in adrenalectomized animals (sham: 53.5 +/- 7.2 microg 100 ml(-1); adrenalectomized: 20.0 +/- 3.6 microg 100 ml(-1)). 3. The concentration-response curve of twitch inhibition, which was induced by electrical field stimulation (0.1 Hz, 0.5 ms pulse width, maximum intensity), caused by a low concentration of morphine (5 x 10(-9)-5 x 10(-7) M) was not affected, but at high concentration (10(-6)-10(-5) M) there was an upward shift in the adrenalectomized group compared with the sham-operated control, although the basal twitch contraction was not changed by adrenalectomy. 4. The twitch inhibition induced by a high concentration of morphine (10(-6) M) in the adrenalectomized group was antagonized to the same level as that in sham-operated controls by naloxone (NLX) (3 x 10(-7) M). 5 Post-tetanic twitch inhibition, an indicator of endogenous opioid release, induced by tetanic stimulation (10 Hz, 0.5 ms pulse width, maximum intensity, for 1 min) was inhibited in the adrenalectomized group compared with the sham-operated controls. The antagonism of inhibition in both groups was equivalent to that exerted by NLX (10(-7) M). 6. Acetylcholine-evoked contraction of the muscle was not influenced by adrenalectomy. 7. These results suggested a possible mechanism for the increase in sensitivity of the opioid receptors to morphine by adrenalectomy, resulting from a decrease in the release of endogenous opioids from the opioidergic neurones in the ileum.

Enkephalinergic neurons in the periaqueductal gray and morphine withdrawal.

The effects of opioid (e.g., morphine) withdrawal on levels of endogenous opioid peptides and their mRNA in the various brain regions have been studied. However, the role of this opioidergic mechanism in the mediation of opioid withdrawal is not fully understood. Preproenkephalin (PPE) mRNA in the caudal periaqueductal gray (cPAG), an important brain region in opioid withdrawal, is increased by both opioid antagonist (naloxone)-precipitated and spontaneous morphine withdrawal, but not by various other stresses in rats, indicating a role of endogenous enkephalins in the cPAG in morphine withdrawal. In addition, PPE mRNA levels in the cPAG increase in the course of the dissipation of morphine withdrawal, and they are returned to the control levels after disappearance of morphine withdrawal signs. Local administration of an enkephalin analog or peptidase inhibitors into the cPAG suppresses morphine withdrawal signs. These facts suggest that enkephalinergic neurons in the PAG may have a critical role in the recovery phase of morphine withdrawal. Recently, an involvement of transcription factors in morphine withdrawal has been suggested. Thus, the possible role of transcription factors in the regulation of PPE gene expression in the cPAG during morphine withdrawal is also discussed.

Acute dependence on, but not tolerance to, heroin and morphine as measured by respiratory effects in rhesus monkeys.

Acute dependence on and tolerance to heroin and morphine were assessed in rhesus monkeys using measures of respiration. Respiratory frequency (f) and minute volume (V(e)) were measured in monkeys breathing air or 5% CO(2) in air using a pressure-displacement plethysmograph. Cumulative doses of naltrexone (0.0001-1.0 mg/kg, i.m) did not alter these parameters in untreated monkeys. Twenty-four hours after a cumulative dose of heroin (1 mg/kg, i.m.), naltrexone produced an increase in both f and V(e) when monkeys were breathing air or 5% CO(2). Following 1 to 3 days of treatment with heroin (0.5 mg/kg/day, i.m.) or morphine (16 mg/kg/day, i.m.), naltrexone produced an increase in f and V(e) that was related to the dose of naltrexone and the number of days of agonist administration. Two days following termination of heroin administration, naltrexone-induced respiratory stimulation declined and had disappeared completely by the fifth day. In tolerance studies, heroin (0.032-0.5 mg/kg, i.m.) and morphine (1-16 mg/kg, i. m.) were injected cumulatively each day for three consecutive days. These drugs suppressed f and V(e) to nearly the same extent on day 3 as they had on day 1 of administration. These results suggest that dependence to morphine and heroin can be measured under conditions of acute 1 to 3 day administration conditions in primates using f and V(e) as reliable and quantitative indicators of opioid withdrawal. Under these conditions, tolerance does not occur.

Diltiazem enhances the analgesic but not the respiratory depressant effects of morphine in rhesus monkeys.

There is evidence that blockade of Ca(2+) channels can modify the analgesia and respiratory depression produced by opioid drugs. The interaction between Ca(2+) channel blockade and drug-induced analgesia and respiratory depression was examined by administration of the L-type Ca(2+) channel blocker diltiazem together with various analgesic drugs. The antinociceptive effects of the drugs were evaluated using a warm-water (50 degrees C) tail-withdrawal assay in rhesus monkeys, and the respiratory depressant effects were evaluated using a pressure-displacement plethysmograph. Pretreatment with diltiazem (10-40 mg/kg, i.m.) 30 min before administration of morphine (0.3 to 10 mg/kg) or heroin (0.03 to 1.0 mg/kg) produced a dose-dependent potentiation of the opioid-induced analgesia. The analgesic potency of morphine and heroin was increased by approximately 0.5 log unit in the presence of 40 mg/kg diltiazem. However, diltiazem failed to alter the analgesic potencies of the mu-opioid receptor agonists, fentanyl, etonitazene, nalbuphine, the kappa-opioid receptor agonist, U-50,488 [(trans)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide ], or the non-opioid, clonidine. Respiratory frequency, minute volume, and tidal volume were suppressed by morphine, heroin, and fentanyl, but these effects were not modified by pretreatment with diltiazem (40 mg/kg). These results suggest that diltiazem selectively potentiates morphine- and heroin-induced analgesia without modifying the effects of these opioids on respiration.

Buprenorphine and methoclocinnamox: agonist and antagonist effects on respiratory function in rhesus monkeys.

Buprenorphine and methoclocinnamox are partial micro-opioid receptor agonists with potential use in the treatment of opioid abuse. The ability of these drugs to suppress respiration as well as their ability to antagonize the respiratory suppressant effects of morphine and heroin were tested in rhesus monkeys. Frequency (f), minute volume (V(e)) tidal volume (V(t)) in monkeys breathing air or 5% CO(2) in air were recorded using a pressure-displacement plethysmograph. Buprenorphine (0.001-10 mg/kg) produced a dose-dependent decrease in respiratory parameters that plateaued at a dose of 1 mg/kg in both air and 5% CO(2). Methoclocinnamox (0. 032-1 mg/kg) also produced dose-dependent respiratory depression that plateaued at a dose of 0.3 mg/kg in air, and was directly related to dose in 5% CO(2). Respiratory suppression produced by buprenorphine 1 and 10 mg/kg lasted for 3 and 7 days, respectively, whereas the suppression produced by the largest dose of methoclocinnamox (1 mg/kg, the solubility limit) lasted less than 24 h. Buprenorphine and methoclocinnamox antagonized morphine- and heroin-induced respiratory depression, and this antagonist effect was observed concomitantly with, as well as following, the mu-opioid receptor agonist effects of buprenorphine and methoclocinnamox. The mu-opioid receptor antagonist effects of buprenorphine (10 mg/kg) and methoclocinnamox (1 mg/kg) lasted for 2 weeks. These results suggest that buprenorphine and methoclocinnamox have a wide margin of safety in clinical use and that these two compounds have a prolonged, insurmountable, mu-opioid receptor antagonist effect after the disappearance of their agonist effects.

Analgesic, respiratory and heart rate effects of cannabinoid and opioid agonists in rhesus monkeys: antagonist effects of SR 141716A.

This study characterized the antinociceptive, respiratory and heart rate effects of the cannabinoid receptor agonists Delta-9-tetrahydrocannabinol (Delta-9-THC) and WIN 55212 ((R)-(+)-2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol-[1,2,3-de]-1, 4-benzoxazin-6-yl)(1-naphtalenyl)methanone monomethanesulfonate), N-arachidonyl ethanolamide (anandamide) and the mu and kappa opioid receptor agonists heroin and U69593, alone and in conjunction with a cannabinoid receptor antagonist, SR 141716A [N-(piperidin-1-1-yl)-5-(4-chlorophenyl)-1(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and an opioid receptor antagonist, quadazocine, in rhesus monkeys (Macaca mulatta). Using 12 adult rhesus monkeys, latencies to remove the tail from a 50 degrees C water bath, respiration in 5% CO2 and heart rate were measured. When administered alone, SR 141716A (1.8, 5.6 mg/kg i.m.) did not alter nociception, respiration or heart rate. Delta-9-THC (0.1-10 mg/kg i.m.) and WIN 55212 (0.1-10 mg/kg i.m.) dose-dependently increased antinociception and dose-dependently decreased respiratory minute and tidal volumes and heart rate. These antinociceptive, respiratory and heart rate effects were reversed by SR 141716A but not by the opioid antagonist quadazocine (1 mg/kg i.m.). Anandamide (10 mg/kg i.m.) also produced antinociception. Heroin (0.01-10 mg/kg i.m.) and U69593 (0.01-3.2 mg/kg i.m.) also dose-dependently increased antinociception and decreased respiratory and heart rate measures; these effects were antagonized by quadazocine but not by SR 141716A. These results demonstrate selective and reversible antagonism of cannabinoid behavioral effects by SR 141716A in rhesus monkeys.

Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats.

We have previously reported the increase of preproenkephalin (PPE) mRNA in the caudal periaqueductal gray (PAG) of rats during morphine withdrawal. In this study, it was further evidenced that PPE mRNA in the caudal PAG was not increased by various kinds of stressor, suggesting that the increase in PPE mRNA in the caudal PAG is specific to morphine withdrawal. In order to investigate the physiological significance of the increase of PPE mRNA in the caudal PAG, we compared the time course of the increase of PPE mRNA in the caudal PAG with that of naloxone-precipitated or spontaneous morphine withdrawal signs. The increase of plasma corticosterone (PCS: 52 and 52 microg/100 ml; control group, 18 and 15 microg/100 ml) and body weight loss (-6 and -9%; control group, 0 and -1%) were observed but PPE mRNA increase was not detected 1 and 2 h after naloxone in morphine treated rats. PPE mRNA increased by 37 to 56%, while PCS elevation and body weight loss gradually diminished 4 h to 2 days after naloxone. A total of 12 h after spontaneous withdrawal, PCS was prominently increased (51 microg/100 ml; control group, 12 microg/100 ml), but body weight and PPE mRNA were not affected. One day after spontaneous withdrawal, PCS elevation (38 microg/100 ml; control group, 8 microg/100 ml) and body weight loss (-5%; control group, +3%) were observed and PPE mRNA also increased by 42%. Two to 3 days after the final morphine injection, PCS recovered to control level and body weight loss gradually disappeared, while PPE mRNA was still increased by 74 to 46%. These results suggest that PPE gene expression in the caudal PAG is stimulated in the recuperative phase of these morphine withdrawal signs.

Effects of peptidase inhibitors, [D-Ala2, Met5]-enkephalinamide and antiserum to methionine-enkephalin microinjected into the caudal periaqueductal gray on morphine withdrawal in rats.

We examined the involvement of enkephalins in the caudal periaqueductal gray (cPAG) in morphine withdrawal in rats. Rats were treated with increasing doses of morphine (20-30 mg/kg/day, s.c., for 5 days) to develop morphine dependence. Morphine withdrawal was induced by naloxone (5 mg/kg, s.c.) 24 hr after the final morphine injection. The level of preproenkephalin (PPE) mRNA in the cPAG was estimated by quantitative in situ hybridization. PPE mRNA in the cPAG was increased 4-24 hr after naloxone in morphine-treated rats. A mixture of peptidase inhibitors (0.5 microl of a solution of amastatin, captopril and phosphoramidon, 3 x 10(-3) M each) microinjected into the cPAG suppressed morphine withdrawal (a decrease in the number of jumping, chin rubbing, paw rubbing and teeth chattering). Antiserum to methionine-enkephalin (1:10 dilution) microinjected into the cPAG did not significantly aggravate morphine withdrawal with or without the mixture of peptidase inhibitors. However, [D-Ala2, Met5]-enkephalinamide (20 nmol), an enkephalin analog, injected into the cPAG decreased the number of jumping without any influence on the other withdrawal signs. These results suggest that the increase in enkephalins in the cPAG may participate in the alleviation of morphine withdrawal (jumping behavior).

Diltiazem inhibits naloxone-precipitated and spontaneous morphine withdrawal in rats.

The effects of diltiazem, a Ca2+ channel blocker, on naloxone-precipitated and spontaneous morphine withdrawal were studied in male Sprague-Dawley rats. In naloxone-precipitated withdrawal, body weight loss and plasma corticosterone elevation were dose dependently inhibited by diltiazem injected 4 or 2 and 4 h before naloxone, respectively. Three administrations of diltiazem (17, 11 and 5 h before naloxone) did not reduce the above withdrawal signs. Diarrhea was dose dependently inhibited by all schedule of diltiazem treatments. In spontaneous withdrawal, body weight loss and plasma corticosterone elevation were dose dependently inhibited by two (6 and 12 h) or three (6, 12 and 18 h after the last morphine) treatments with diltiazem at 6-h intervals after the last morphine, but not by a single diltiazem injected 18 h after the last morphine.

Increase of preproenkephalin mRNA in the caudal part of periaqueductal gray by morphine withdrawal in rats: a quantitative in situ hybridization study.

Effects of morphine withdrawal on the levels of preproenkephalin (PPE) mRNA in the area from lateral to ventrolateral periaqueductal gray (PAG) were studied in rats by quantitative in situ hybridization. PPE mRNA in the rostral PAG was decreased by naloxone-precipitated morphine withdrawal but not affected by spontaneous morphine withdrawal. PPE mRNA in the caudal PAG was increased by both spontaneous and naloxone-precipitated morphine withdrawal.

Possible involvement of the total amount of morphine infused in the development of acute morphine dependence in rats.

The severity of naloxone-precipitated withdrawal in rats infused intravenously with morphine at the rates of 2.5, 5 and 10 mg/kg/hr over various time periods was investigated. Plasma morphine concentration reached a constant and rate-dependent level at 1 hr after the start of morphine infusion, and this level was maintained until the termination of infusion. Naloxone (2.0 mg/kg, s.c.) was challenged 18 hr after infusion was stopped, and the withdrawal was evaluated by plasma corticosterone (PCS) increase, diarrhea and body weight loss. The incidence of naloxone-precipitated withdrawal signs was related to both the infusion rate and duration of morphine infusion. The duration of morphine infusion (ET50) needed to elicit naloxone-precipitated PCS increase and diarrhea in 50% of the rats was inversely related to the morphine infusion rates, but the total amount of infused morphine (EA50) that elicited naloxone-precipitated withdrawals in 50% of rats was the same at all infusion rates. These results suggest that the total amount of morphine infused may play an important role in the development of acute physical dependence on morphine rendered by continuous intravenous morphine infusion for 1-8 hr.

No relation of plasma morphine level to the severity of naloxone-induced withdrawal in acute morphine-dependent rats.

Plasma morphine concentration and naloxone-precipitated withdrawal body weight loss and plasma corticosterone (PCS) increase were determined at 12, 18 and 24 hr after i.v. infusion of morphine at a constant rate of 10 mg/kg/hr for 4 hr in Sprague-Dawley rats. Plasma morphine concentration declined 98.0% within 12 hr and further declined 58.8% during 12-24 hr after morphine infusion. There was a significant difference between plasma morphine concentrations at 12 and 24 hr after the morphine infusion. Naloxone (0.5 and 2.0 mg/kg)-precipitated withdrawal, but not spontaneous withdrawal, was elicited at 12-24 hr after the morphine infusion, and the severity of withdrawal precipitated by 2.0 mg/kg naloxone was the same at 12-24 hr after the morphine infusion. Furthermore, there was no significant correlation between plasma morphine concentration and body weight loss or PCS increase. The results suggest that a constant degree of morphine dependence is sustained during 12-24 hr after the morphine infusion and the severity of naloxone-precipitated withdrawal is not related to the plasma morphine concentration at the time of naloxone injection, that is, the rate of morphine removal from its receptor sites.

[3H]naloxone binding sites in porcine ovarian follicles and corpora lutea during the ovarian cycle.

We demonstrated the presence of opioid receptors in the porcine ovary using [3H]naloxone. We also examined the change in the number of opioid receptors during follicular maturation. In addition, we found specific binding of [3H]naloxone in the porcine ovary using naloxone, beta-endorphin, methionine-enkephalin and dynorphin. The binding of [3H]naloxone to porcine granulosa cells and the 2000-g subcellular fraction of corpora lutea was examined to demonstrate the presence of specific [3H]naloxone binding in the porcine ovary. Binding of [3H]naloxone to porcine granulosa cells was displaced by cold naloxone and beta-endorphin but not by dynorphin and methionine-enkephalin. A similar phenomenon was also demonstrated in the 2000 g subcellular fraction of porcine corpora lutea. However, Scatchard analyses revealed a single class of high-affinity (Kd = 28.5 x 10(-9) mol/l) and low-capacity binding sites (Bmax = 30.5 fmol/5 x 10(6) cells) in porcine granulosa cells. Similar binding parameters were obtained in the 2000-g subcellular fraction of porcine luteal tissue (Kd = 28.3 x 10(-9) mol/l, Bmax = 59.3 nmol/kg protein). [3H]Naloxone binding sites in the porcine ovary showed binding characteristics similar to those of opioid receptors in other organs like brain, uterus and placenta. Furthermore, we demonstrated that the specific binding sites of [3H]naloxone in porcine granulosa cells decreased during follicular maturation. Opioid receptors have been detected in the uterus, placenta and Sertoli cell cultures in some species. However, there is no detailed study on opioid receptors in granulosa cells and luteal tissues in any species. Our data suggest a relationship between folliculogenesis and ovarian opioid peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The dopaminergic system modulates the endogenous opioid system in guinea-pig isolated ileal longitudinal muscle.

The effects of the dopamine antagonists haloperidol and sultopride were investigated on the twitch response, evoked by 0.1 Hz stimulation of guinea-pig isolated ileal longitudinal muscle, and on the inhibition of the twitch response induced by 10 Hz stimulation (post-tetanic twitch inhibition) and by application of opioids. Both haloperidol and sultopride concentration-dependently inhibited the twitch response, with threshold concentrations of 2 and 50 microM, respectively, and could also shift the concentration-response curve for ACh-contraction to the right in a non-competitive manner. Haloperidol (1 microM) and sultopride (20 microM) increased post-tetanic twitch inhibition and this could be prevented by naloxone (100 nM). Twitch inhibition induced by morphine and dynorphin 1-13 was not affected by haloperidol (1 microM) or sultopride (20 microM). Prazosin (1 microM) and yohimbine (2 microM) did not affect either the twitch response or the post-tetanic twitch inhibition. These results suggest that dopamine receptors are involved in the modulation of the ileal opioid system, in such a manner as to diminish the release of endogenous opioids by tetanic stimulation.