Morphine-6 beta-glucuronide has a higher efficacy than morphine as a mu-opioid receptor agonist in the rat locus coeruleus

Abstract

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.

Figure 1

M6G and morphine behave as partial agonists in locus coeruleus neurons. In (a) is shown a continuous voltage clamp recording (holding potential: −60 mV) in which outward currents were produced by different increasing concentrations of M6G and a single maximally effective concentration of [Met⁵]-enkephalin. In (b) is shown currents produced by cumulative applications of morphine. Note that the current does not desensitize during a 5 min application of 10 μM morphine and that the current was completely blocked by 1 μM naloxone. In (c) concentration-effect curves for M6G and morphine are shown using data expressed as a percentage of the amplitude of the [Met⁵]-enkephalin maximum. In both cases the maxima for both curves are smaller than the system maximum measured with [Met⁵]-enkephalin. The curves are also vertically and horizontally displaced from one another. The lines through the data are the average logistic fit. Vertical bars show s.e.mean.

Figure 2

Efficacy ratios (M6G:morphine) predicted using dissociation constants obtained from binding studies. Shown is a plot of the efficacy ratio calculated by the equation method of Trzeciakowski (1999a) expressed as a function of the ratio of the affinity constants for M6G and morphine, which were obtained from published binding studies (Table 1). The values assigned to the following variables were estimated from the concentration-effect curves for each agonist: (E_m)_M6G=77.7%, (E_m)_morphine=61%, (EC₅₀)_M6G=19 μM, and (EC₅₀)_M6G=6.2 μM. The filled circle indicates an efficacy ratio of 4.1 calculated when the dissociation constants of M6G and morphine are equal.

Figure 3

M6G and DAMGO currents are antagonized equally by 3-O-methoxynaltrexone. In (a) is shown recording sequences made from a single neuron. In the upper panel are control responses produced by three concentrations of M6G and the selective mu-opioid agonist DAMGO. In the lower panels 3-O-methoxynaltrexone caused a similar reduction in the currents produced by both agonists. In (b) the data for this experiment has been plotted.

Figure 4

M6G currents are occluded by morphine. The effect of M6G is shown on a locus coeruleus neuron voltage clamped at −60 mV. M6G produced an outward current when superfused alone but had no effect when superfused during an application of morphine. Normally the current produced by morphine would reverse slowly during washout but superfusion with naloxone caused an immediate return to the original baseline and also blocked the current response to a third application of M6G.

Figure 5

M6G currents are reduced following desensitization of mu-opioid receptors with [Met⁵]-enkephalin. In the experiment shown following a cumulative application of three concentrations of M6G, a 30 μM concentration of [Met⁵]-enkephalin was applied for 5 min, which causes transient homologous desensitization of mu-opioid receptors. This treatment also caused desensitization of the M6G (3 μM) current.

Figure 6

M3G which does not bind to opioid receptors had no electrophysiological effects in locus coeruleus neurons. In (a) M3G produced no change in current in a neuron voltage clamped at −60 mV and did not antagonize currents produced by M6G. In (b) M3G had no effect on the rate or pattern of firing in a recording made from a spontaneously active locus coeruleus neuron.