Interactions between the antiopiate Tyr-MIF-1 and the mu opiate morphiceptin at their respective binding sites in brain

The interactions between Tyr-MIF-1, a brain peptide with antiopiate activity, and the beta-casomorphins, a family of peptides derived from milk protein with opiate activity, were investigated by in vitro binding assays. Specific binding of 125I-Tyr-MIF-1 to rat brain membranes was displaced with high potency by beta-casomorphin, morphiceptin, and the morphiceptin analog PL017 but not by the analgesically inactive analog D-Pro2-morphiceptin or by several other ligands for classical delta, kappa, or sigma opiate receptors. In addition, Tyr-MIF-1 displaced 125I-morphiceptin from its binding sites in brain with affinities similar to those of unlabeled morphiceptin and PL017. These results, which include the first demonstration of a binding site in brain for labeled morphiceptin, indicate that brain antiopiate Tyr-MIF-1 and the beta-casomorphin derived peptides with opiate activity may share a common binding site or cross-react at each other's site. This suggests a possible mechanism of action for endogenous antiopiate-opiate interactions.     

Ventilatory and cardiovascular responses of the unanaesthetized chicken, Gallus domesticus, to the respiratory stimulants etamiphylline and almitrine

The ventilatory and cardiovascular effects of i.v. administration of the respiratory stimulants etamiphylline and almitrine were investigated in conscious or decerebrate adult female domestic fowl. Infusion of etamiphylline (100 mg . kg-1) or injection of almitrine (2 mg . kg-1) evoked a potent long-lasting stimulation of ventilation in both conscious and decerebrate fowl. The pattern of the respiratory response was characteristically different to that observed in mammals in that the increased minute volume of ventilation was attained by large increases in respiratory frequency accompanied by a reduction in tidal volume. The pattern of respiration following drug-induced stimulation was, in some birds, typical of thermal panting although neither etamiphylline nor almitrine caused significant increases in body temperature. Differences in the pattern of responses of the rate and depth of breathing may be attributed in part to the differences in pulmonary receptor systems involved in the control of breathing in birds and mammals.     

Antisense oligodeoxynucleotide targeting distinct exons of the cloned mu-opioid receptor distinguish between endomorphin-1 and morphine supraspinal antinociception in mice.

Antisense oligodeoxynucleotides (ODN) were used to investigate the supraspinal antinociceptive effects of endomorphin-1, an endogenous peptide whose analgesic profile suggests that it is a ligand at the mu-opioid receptor. To selectively restrict the expression of this receptor, five ODN targeting distinct exons of the gene sequence were injected subchronically by the intracerebroventricular route (i.c.v.) into mice. The antinociception induced by endomorphin-1 was greatly reduced in animals receiving the ODN directed to nucleotides 677-697, which code for a sequence located on the second extracellular loop of the mu receptor. ODN-mu(un), one of the two antisense ODN directed to exon 1, also impaired endomorphin-1 antinociception. ODN targeting exons 2 and 4 were totally inactive. In contrast, all five ODN blocked the antinociception induced by morphine and beta-casomorphin. The analgesic potency of endomorphin-1, morphine, and beta-casomorphin remained unaltered by administration of an ODN to nucleotides 29-46 of the murine delta-opioid receptor gene sequence of a random-sequence ODN. This suggest the existence of diverse molecular forms for the mu-opioid receptor that mediate the antinociceptive effects of endomorphin-1 and morphine/beta-casomorphin.     

Wild ginseng attenuates anxiety- and depression-like behaviors during morphine withdrawal

The purpose of this study was to evaluate whether wild ginseng (WG) administration could attenuate anxiety- and depression-like behaviors and expression of corticotrophin-releasing factor (CRF) and neuropeptide Y (NPY) following withdrawal from repeated morphine administration in rats. Male rats were administered daily doses of WG (50, 100, or 200 mg/kg, i.p.) for 5 days, 30 min before morphine injection (40 mg/kg, s.c). The anxiety- and depression-like behavioral responses were measured 72 h after the last morphine injection using an elevated plus maze (EPM) and forced swimming test (FST), respectively. Changes in hypothalamic CRF and NPY expressions were also examined by analyzing their immunoreactivities in the hypothalamus. Daily administration of WG significantly reduced anxiety-and depression-like behavior, and elicited the suppression of CRF expression and the stimulation of NPY expression in the hypothalamus. Our results demonstrated that WG extract might be effective at inhibiting the anxiety and depression responses due to morphine withdrawal by possibly modulating the hypothalamus CRF and NPY systems. Furthermore, these findings imply that WG extract can be used for developing new medication to cure or alleviate morphine withdrawal symptoms and to prevent relapses of morphine use.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Sex differences in morphine-induced analgesia of visceral pain are supraspinally and peripherally mediated

Increasing evidence suggests there is a sex difference in opioid analgesia of pain arising from somatic tissue. However, the existence of a sex difference in visceral pain and opioid analgesia is unclear. This was examined in the colorectal distention (CRD) model of visceral pain in the current study. The visceromotor response (vmr) to noxious CRD was recorded in gonadally intact male and female rats. Subcutaneous injection of morphine dose-dependently decreased the vmr in both groups without affecting colonic compliance. However, morphine was significantly more potent in male rats than females. Because systemic morphine can act at peripheral tissue and in the central nervous system (CNS), the source of the sex difference in morphine analgesia was determined. The peripherally restricted mu-opioid receptor (MOR) antagonist naloxone methiodide dose-dependently attenuated the effects of systemic morphine. Systemic administration of the peripherally restricted MOR agonist loperamide confirmed peripherally mediated morphine analgesia and revealed greater potency in males compared with females. Spinal administration of morphine dose-dependently attenuated the vmr, but there was no sex difference. Intracerebroventricular administration of morphine also dose-dependently attenuated the vmr with significantly greater potency in male rats. The present study documents a sex difference in morphine analgesia of visceral pain that is both peripherally and supraspinally mediated.     

Postnatal development of the Hering-Breuer reflex in the rat

The intensity of the Breuer-Hering inflation reflex was studied in newborn (1 day old), in young (8 days old) and in adult rats (90 days old) under urethane general anaesthesia (1.3 g/kg i.p.). The inflation pressure was adjusted with the aid of a water-valve. The reflex was present in all 3 age groups. An inflation pressure of 0.2 kPa applied in the course of expiration produced a long lasting apnoea in newborn rats which lasted 48 normal respiratory cycles. An inflation pressure of 0.5 kPa in young rats induced an apnoea lasting for only 3 normal respiratory cycles, whereas a pressure of 1 kPa in adult rats led to an apnoea which lasted for 20 normal respiratory cycles. The compliance of the respiratory system in relation to lung weight is approximately 5 times higher in adult rats compared with that of newborn rats. It is approximately double in comparison with young rats. The pressures of inflation mentioned in the 3 age categories can be considered as equieffective from the point of stimulation pulmonary stretch receptors. It can be concluded from these findings that the reflex of Breuer-Hering in newborn rats is more potent in comparison with adult rats, but it is lower in young rats at the age of 8 days. It is suggested that the differences observed are due to functional and anatomical maturation.     

Dynorphin: potent analgesic effect in spinal cord of the rat

Evidence is presented to show a strong and long-lasting analgesic effect after injection of dynorphin into the subarachnoid space of the spinal cord of the rat. Calculating on a molar basis dynorphin was 6-10 times more potent than morphine and 65-100 times more potent than morphiceptin, the specific mu receptor agonist. Dynorphin analgesia was completely reversed by intrathecal injection of anti-dynorphin IgG and partially reversed by naloxone. Acute tolerance to morphine analgesia did not affect the occurrence of dynorphin analgesia. Evidence from different lines of approach suggest that dynorphin may bind with kappa receptors in the spinal cord to exert its analgesic effect.     

Involvement of vagal opioid receptors in respiratory effects of morphine in anaesthetized rats.

Respiratory effects of morphine injection to the femoral vein were investigated in urethane and chloralose anaesthetized and spontaneously breathing rats, prior to and after midcervical vagotomy. Bolus injection of morphine HCl at a dose of 2 mg/kg of body weight induced depression of ventilation in all rats, due to the significant decrease in tidal volume and to the decline in respiratory rate both pre- and post-vagotomy. Expiratory apnoea of mean duration of 10.0+/-3.4 s was present in the vagally intact rats only. Bilateral midcervical section of the vagus nerve precluded the occurrence of apnoea. Prolongation of the expiratory time (T(E morphine) / T(E control)), which amounted to 10.7+/-2.2-fold in the intact state, was apparently reduced to 1.5+/-0.3-fold after division of the vagi. Morphine significantly decreased mean arterial pressure (MAP) at 30 s after the challenge, the effect persisted for not less than 1 minute and was absent in vagotomized rats. The respiratory changes evoked by morphine reverted to the control level after intravenous injection of naloxone at a dose of 1 mg/kg. Results of this study indicate that opioid receptors on vagal afferents are responsible for the occurrence of apnoea and hypotension evoked by morphine.     

Naloxone reduces decrease in ventilation induced by hypoxia in newborn infants

The mechanism responsible for the decrease in ventilation during breathing of low fractional concentration of inspired O2 in the newborn infant is poorly understood. The present study tested the hypothesis that endogenous opiates account for this ventilatory decrease. Eleven healthy newborn infants breathed 15% O2, balance N2 for 5 min following an injection of saline and following an injection of naloxone. Neither injection caused a change in minute ventilation (VE) or ventilatory pattern when the infants were breathing room air. However, the decreased ventilation during hypoxia following naloxone was significantly less than that following saline. VE dropped about 14% following saline but only about 4% following naloxone. However, the adult ventilatory response to hypoxemia, i.e., a relatively sustained increase in VE, was not attained. Naloxone had no influence on the occurrence of periodic breathing during hypoxemia. Thus in the healthy full-term newborn infant, endogenous opiates account only for a part of the decreased ventilation during hypoxemia.     

Stimulation of the prostate growth in rats by milk casomorphins]

Thymidine kinase activity of the ventral prostate from adult rats could be stimulated by administration to the animal of casomorphin or casomorphin (morphiceptin). On the other hand addition of both drugs to the culture medium of prostatic cells from immature rats enhanced the cells growth. In both cases, morphiceptin was more efficient than casomorphin. These results suggest than casomorphins have other properties than their morphinomimetic effects previously described.     

Analgesic properties of a systemically-administered synthetic dipeptide of 5-hydroxytryptophan

Synthetic peptides of 5-hydroxytryptophan (5-HTP), including N-acetyl-5-HTP-5-HTP amide (5-HTP-ACETYL-DP), specifically inhibit the binding of serotonin to serotonin binding protein. 5-HTP-ACETYL-DP also produces a long-lasting, opiate-sensitive analgesia following central, but not systemic administration. The present study evaluated an apolar derivative of 5-HTP dipeptide, N-hexanoyl-5-HTP-5-HTP amide (5-HTP-HEX-DP), for its analgesic properties in rats following systemic administration. 5-HTP-HEX-DP (5–50 mg/kg) significantly increased jump thresholds in a dose-dependent manner with peak analgesia occurring at 2.5 hr after injection, and lasting up to 5 hr. In the tail-flick assay, 5-HTP-HEX-DP (20 mg/kg) produced a significant antinociceptive effect at 1 hr post-injection using both high and low intensity levels of radiant heat. While 5-HTP-HEX-DP and morphine each elicited analgesia following acute administration, chronic (14 days) incremental dosing with 5-HTP-HEX-DP or morphine resulted in persistent analgesia in 5-HTP-HEX-DP-treated animals, and a loss of analgesia in morphine-treated rats. Thus, significant tolerance to morphine, but not 5-HTP-HEX-DP analgesia developed using this protocol. Hence, 5-HTP-HEX-DP is a systemically-active analgesic which fails to develop tolerance when administered daily over 14 days.     

Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

Cardiovascular responses to opioid agonists injected into the nucleus of tractus solitarius of anesthetized cats

It has been proposed that various opiate receptor subtypes mediate different cardiovascular responses to centrally administered opioids. We evaluated this hypothesis in chloralose-urethane anesthetized cats by monitoring the cardiovascular and respiratory responses to relative mu [morphine, morphiceptin, D-Ala2, MePhe4, Gly-ol5 enkephalin (DAGO)] and delta [D-Ala2, D-Leu5enkephalin (DADL)] agonists microinjected (0.5 ul/kg) into the caudal region of the Nucleus of Tractus Solitarius (NTS). Dynorphin (1-13), an endogenous opioid which exhibits selective affinity towards the kappa receptor, was also tested. Dynorphin at a dose of 50 nMol/kg did not alter cardiovascular or respiratory variables. Morphine (10-54 nMol/kg) and DAGO (50 nMol/kg) had no effect on blood pressure, heart rate or respiratory rate; morphiceptin (100-320 nMol/kg) caused tachycardia only at the highest dose. DADL (10-100 nMol/kg) elicited a dose-dependent depression of blood pressure. High doses of DADL depressed heart rate and respiratory rate. The depressor effects of DADL were reversed by low doses of naloxone (0.1 mg/kg). This dose of naloxone also elicited pressor responses in cats treated with the other opioids and reversed the morphiceptin-induced tachycardia. These data indicate that opioid agonists differ with regard to their cardiovascular and respiratory effects following microinjection into the NTS of anesthetized cats, with the delta agonist DADL showing greatest activity.     

Demonstration of beta-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans

Healthy young volunteers ingested one liter of cows' milk; then the contents of the small intestine were aspirated through an intestinal tube at various times and assayed for the presence of bovine beta-casomorphin immunoreactive materials. Considerable amounts of beta-casomorphin-7, but no beta-casomorphin-5 and only small amounts of beta-casomorphin-4 or -6 immunoreactive materials were found. Chromatographical characterization showed that most of the beta-casomorphin-7 immunoreactive material was not identical with beta-casomorphin-7, whereas the major part of the beta-casomorphin-4 or -6 immunoreactive materials might be identical with their corresponding beta-casomorphins. Analogous results were obtained for in vitro digestion of bovine milk which had been designed as a rough imitation of the gastrointestinal digestion process. A regulatory influence of beta-casomorphins as "food hormones" on intestinal functions is suggested.     

Local application of somatostatin in the rat ventrolateral brain medulla induces apnea

Local injections of the tetradecapeptide somatostatin (SOM) into the brain stem region were performed in anesthetized and decerebrate rats. SOM administration (0.6-1.8 nmol) into the nucleus paragigantocellularis and the nucleus reticularis lateralis of the ventrolateral medulla oblongata induced ventilatory depression and apnea. The occurrence of apnea was dose dependent and attributed to the anesthetic depth, and it was seen within 60-240 s after injection. In anesthetized rats the apnea was seen as a termination or a continuous decrease in tidal volume while respiratory frequency remained unaltered. SOM-induced apnea was caused by depression of central inspiratory drive. SOM injections into the dorsal medulla were ineffective in eliciting apnea, although a ventilatory depression but no apnea was induced in the awake unanesthetized state. In addition to its effect on basal ventilation, SOM administration in the ventrolateral medulla resulted in a blunted ventilatory response to hypoxic and hypercapnic stimuli in anesthetized rats. We conclude that SOM has potent inhibitory effects on respiration that are specifically located in the nucleus paragigantocellularis and the nucleus reticularis lateralis.     

Circling behavior in old rats after unilateral intranigral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Old and young adult rats received unilateral injections of MPTP or saline into the substantia nigra. Unilateral injection of MPTP in old rats induced ipsiversive circling on day 1 and day 7 after the injection; contraversive circling behavior was induced in MPTP-treated rats by systemic administration of apomorphine. Young rats showed ipsiversive circling on day 1 but not on day 7 after the injection; administration of apomorphine did not induce contraversive circling. On day 10 after the injection of MPTP, the concentration of D-2 receptors in the striatum of the injected hemisphere of old rats was increased by about 25% compared with the striatum of old rats with saline injection and of young rats with MPTP or saline injections. These results suggest that MPTP exerts neurotoxic effects on the nigrostriatal dopaminergic system of old rats and produces supersensitive dopamine receptors in the ipsilateral denervated striatum.     

Chronic opioid potentiates presynaptic but impairs postsynaptic N-methyl-D-aspartic acid receptor activity in spinal cords: implications for opioid hyperalgesia and tolerance

Opioids are the most effective analgesics for the treatment of moderate to severe pain. However, chronic opioid treatment can cause both hyperalgesia and analgesic tolerance, which limit their clinical efficacy. In this study, we determined the role of pre- and postsynaptic NMDA receptors (NMDARs) in controlling increased glutamatergic input in the spinal cord induced by chronic systemic morphine administration. Whole-cell voltage clamp recordings of excitatory postsynaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine significantly increased the amplitude of monosynaptic EPSCs evoked from the dorsal root and the frequency of spontaneous EPSCs, and these changes were largely attenuated by blocking NMDARs and by inhibiting PKC, but not PKA. Also, blocking NR2A- or NR2B-containing NMDARs significantly reduced the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs in morphine-treated rats. Strikingly, morphine treatment largely decreased the amplitude of evoked NMDAR-EPSCs and NMDAR currents of dorsal horn neurons elicited by puff NMDA application. The reduction in postsynaptic NMDAR currents caused by morphine was prevented by resiniferatoxin pretreatment to ablate TRPV1-expressing primary afferents. Furthermore, intrathecal injection of the NMDAR antagonist significantly attenuated the development of analgesic tolerance and the reduction in nociceptive thresholds induced by chronic morphine. Collectively, our findings indicate that chronic opioid treatment potentiates presynaptic, but impairs postsynaptic, NMDAR activity in the spinal cord. PKC-mediated increases in NMDAR activity at nociceptive primary afferent terminals in the spinal cord contribute critically to the development of opioid hyperalgesia and analgesic tolerance.     

Dopamine receptor sensitivity after repeated morphine administrations to rats.

It was studied in rats, if chronic morphine treatment induces a supersensitivity of dopamine receptors in brain. The rats were treated twice daily for 8–11 days with single doses of morphine, increasing from 10 to 20 mg/kg i.p. The experiments were carried out 16–20 hours after the last injection of morphine. After chronic morphine treatment, the potency of apomorphine in lowering the striatal dopamine turnover was increased. On the other hand, apomorphine was not more potent in inducing stereotypies (sniffing, licking, gnawing) after chronic morphine administration than in saline controls. Finally, dopamine activated the adenylate cyclase in striatal homogenates of rats after chronic morphine treatment to a similar extent as in homogenates of control rats. The results suggest that a supersensitivity of dopamine receptors in brain is not necessarily involved in symptoms of an increased dopaminergic activity after chronic morphine application.     

Alteration of brain stem auditory evoked potentials after intracerebroventricular administration of met-enkephalin in rabbits.

Brainstem auditory evoked potentials (BAEPs) were elicited by binaural click stimulation and recorded from the rabbits with chronically implanted electrodes and a cannula for intracerebroventricular injection (i.c.v.). 400 BAEPs were averaged off line. The registration was carried out before and after i.c.v. injection of met-enkephalin (2.5 or 25 nmol), naloxone (20 micrograms), or i.v. injection of morphine (1.0, 2.0, 5.0 mg/kg b.w.). Enkephalin caused shortening of interpeak latency time, naloxone caused its lengthening, while the effect of morphine was not unidirectional. Enkephalin caused increase in the surface area below the negative peaks located in the range of 4.5-7.5 ms from the first positive peak, naloxone caused its decrease while the effect of morphine was also in this respect not unidirectional. It is concluded that opiate receptors are involved in the modulation of the auditory brainstem responses.