enkephalin--ala(2)-mephe(4)-gly(5)- and morphine-3-glucuronide

High doses of intrathecally applied morphine or morphine-3beta-D-glucuronide (M3G) produce allodynia and hyperalgesia. Whole-cell patch-clamp recordings were made from substantia gelatinosa neurons in transverse slices of adult rat lumbar spinal cord to compare the actions of M3G with those of the mu-opioid agonist, DAMGO ([D-Ala(2),N-Met-Phe(4),Gly-ol(5)]-enkephalin), and the ORL(1) agonist, nociceptin/orphanin FQ (N/OFQ). M3G (1-100 microM) had little or no effect on evoked excitatory postsynaptic currents (EPSC) and no effect on postsynaptic membrane conductance. In contrast, 1 microM DAMGO and 1 microM N/OFQ reduced the amplitude of evoked EPSCs and activated an inwardly rectifying K(+) conductance. M3G did not attenuate the effect of DAMGO or N/OFQ on evoked EPSC amplitude. However, 1 to 100 microM M3G reduced the amplitude of evoked GABAergic and glycinergic inhibitory postsynaptic current (IPSC) by up to 48%. This effect was naloxone-insensitive. The evoked IPSC was also attenuated by DAMGO, but not by N/OFQ. Because M3G reduced the frequency of tetrodotoxin-insensitive miniature IPSCs and increased paired-pulse facilitation, it appeared to act presynaptically to disinhibit substantia gelatinosa neurons. This effect, which does not appear to involve mu-opioid or ORL(1) receptors, may contribute to the allodynia and hyperalgesia observed after intrathecal application of high doses of morphine.

Topics: Animals; Central Nervous System Stimulants; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Postsynaptic Potentials; Morphine Derivatives; Nociceptin; Opioid Peptides; Rats; Rats, Sprague-Dawley; Strychnine; Substantia Gelatinosa; Synaptic Transmission

1. The pharmacological properties of the active morphine metabolite, morphine-6 beta-D-glucuronide (M6G), and the parent compound were compared in rat locus coeruleus neurons by electrophysiological recording in brain slices. 2. M6G and morphine activated potassium currents in voltage clamped neurons, which were blocked by the opioid receptor antagonist naloxone. 3. Both M6G and morphine behaved as partial agonists that produced maximal responses smaller than the system maximum, which was measured using [Met(5)]-enkephalin. M6G produced a larger maximal response (78%) than morphine (62%), which we estimated was due to a 2 - 4 fold difference in the relative efficacy of the agonists. 4. 3-O-methoxynaltrexone, which has been reported to behave as a selective antagonist of a M6G preferring receptor, was equally effective at blocking currents produced by M6G and the selective mu-opioid receptor agonist DAMGO. 5. M6G currents were occluded by a prior application of morphine, and were reduced when mu-opioid receptors were desensitized by using [Met(5)]-enkephalin. 6. Morphine-3 beta-D-glucuronide did not affect action potential firing or membrane currents in locus coeruleus neurons and had no effect on currents produced by M6G. 7. These results show that the relative efficacy of M6G is higher than morphine in locus coeruleus neurons, contrary to what has been shown using mu-opioid receptors expressed in cell clones.

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Locus Coeruleus; Male; Membrane Potentials; Morphine; Morphine Derivatives; Naltrexone; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

We investigated the nature of interaction of morphine-3-O-beta-D-glucuronide (M3G) and morphine-6-O-beta-D-glucuronide (M6G) with opioid binding sites at the mu-, delta- and kappa-opioid receptors (mu-OR, delta-OR and kappa-OR) in cerebral membranes. Saturation binding experiments revealed a competitive interaction of M6G with all three opioid receptors. Inhibition binding experiments at the mu-OR employing combinations of morphine and M6G resulted in a rightward shift of the IC50 for morphine proportional to the M6G concentration, thus strengthening the finding of competitive interaction of M6G at the mu-opioid binding site. Data in absence and presence of M6G were included in a three-dimensional model. Compared to a model with one binding site a model with two binding sites significantly improved the fits. This might indicate that different mu-OR subtypes are involved. Hydrolysis of M6G to morphine was investigated and did not occur. Therefore the effects of M6G on binding to the mu-OR were due to M6G and not due to morphine. In contrast, M3G at the three opioid receptors was found to inhibit binding being about 300 times weaker than morphine. This effect was well explained by the amount of contaminating morphine (about 0.3%) identified by HPLC. We conclude that M6G binds to mu-, delta- and kappa-OR in a competitive manner. Some of our results on the mu-OR suggest two binding sites for agonists at the mu-OR and that M6G binds to both sites. Our results suggest that the high potency of M6G as an analgesic is mediated through opioid receptors. In contrast, M3G does not interact with the mu-, delta- or kappa-OR. We therefore doubt that any effect of M3G is mediated via opioid receptors.

Topics: Animals; Binding Sites; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Guinea Pigs; Male; Morphine; Morphine Derivatives; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

Opioid receptor binding of morphine-6-beta-D-glucuronide (M6G), morphine and [3H][D-Ala2,N-methylPhe4-Glyol5]enkephalin (DAMGO) were determined in neonatal guinea pigs. Pontomedullary membranes specifically bound [3H]DAMGO, which was displaced by M6G and morphine. The KI for M6G and morphine were 15.1 nM and 5.0 nM, respectively, and did not change between day 3 and day 7 after birth. KD for [3H]DAMGO binding was constant (1.1nM), however Bmax increased from 62.2 to 88.3 fmol/mg protein between days 3 and 7 (P < 0.01). This 42% increase in mu receptors may play a role in the increased potency of M6G respiratory effects for guinea pigs during the first week after birth.

Topics: Animals; Animals, Newborn; Brain Stem; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Morphine; Morphine Derivatives; Receptors, Opioid, mu