Effects of intracerebroventricular β-funaltrexamine on μ and δ opioid receptors in the rat: dichotomy between binding and antinociception

doi:10.1016/0014-2999(91)90715-3

European Journal of Pharmacology

Volume 203, Issue 2, 15 October 1991, Pages 195-202

https://doi.org/10.1016/0014-2999(91)90715-3 Get rights and content

Abstract

The effects of intracerebroventricutar (i.c.v.) β-funaltrexamine (β-FNA) pretreatment at −24 or −6 h were studied on μ and β opioid receptor binding and on antinociception produced by i.c.v. morphine in rats. μ and δ opioid receptor binding in brain membrane preparations was performed with [³H][D-Ala²,MePhe⁴Gly-ol⁵]enkephalin (DAGO) and [³H][D-Pen²,D-Pen⁵]enkephalin (DPDPE) as radiolabeled ligands, respectively. Effects of i.c.v. β-FNA (24 h) on μ, and δ binding depended on dosage. For [³h]dago binding, 3 μg, β-FNA did not affect either the K_d or B_max, whereas 10 μg increased the K_dout changing the B_max· pretreatment for 24 h did not alter [³h]dpdpe binding at 3 μat 10 μ, the K_dincreased with no change in the B_maxeatment with 10 μg β-FNA for 6 h gave similar results to the 24-h treatment in μ binding, but did not change δ binding. When μ binding was performed on various brain regions, pretreatment with 10 μg β-FNA for 24 h increased the K_d in all regions studied (the penaqueductal gray, thalamus, striatum and cortex). However, this pretreatment decreased the B_max only in the periaqueductal gray (by 22%) and cortex (by 14%). Pretreatment of rats with β-FNA (3 or 10 μg at −24 h), which by itself caused some hyperalgesia, greatly antagonized the antinociceptive effect of morphine (10 μg i.c.v.) in the hot-plate test. Our work with β-FNA has revealed an apparent discrepancy between binding and behavioral results. This dichotomy may, in part, be the result of the limited distribution of β-FNA to the periventricular area. It may also be due to the presence of uncoupled receptors and/or may be related to the finding that high affinity [³h]dago binding sites in vitro may not represent functional receptors in vivo.

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Antinociception following application of DAMGO to the basolateral amygdala results from a direct interaction of DAMGO with Mu opioid receptors in the amygdala
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Citation Excerpt :
After 7 days of recovery from surgery, the animals were administered with β-FNA (1.00 or 2.00 μg/0.25 μl) [31] or sterile isotonic saline into the BLA. After 24 h [18,31], each animal was TF-tested with DAMGO (0.25 μg/0.25 μl) applied bilaterally to the BLA. Only animals that had recovered 90% or more of their presurgical body weight were used for the TF testing.
Previous studies from our laboratory have shown that application of the mu opioid agonist DAMGO into the basolateral region of the amygdala (BLA) suppresses the radiant heat tail flick (TF) reflex in anesthetized rats. This antinociceptive effect can be blocked by lesions of brainstem regions such as the periaqueductal gray (PAG) or the rostral ventromedial medulla (RVM) or by functional inactivation of neurons in these regions, suggesting the activation of brainstem-descending antinociceptive systems from the amygdala. However, little is known about the direct interaction of DAMGO with mu receptors in the amygdala. In the present series of experiments, the BLA was pretreated with opioid receptor antagonists and a G protein inhibitor prior to TF testing with application of DAMGO into the same site. Rats pretreated with the non-selective opioid antagonist naltrexone (1.25–3.75 μg/0.25 μl per side) or the G protein inhibitor pertussis toxin (0.25 μg) failed to show inhibition of TF reflexes following infusion of DAMGO (0.168–0.50 μg), indicating that DAMGO works through G-protein-coupled opioid receptors in the BLA. Furthermore, pretreatment with the mu antagonist β-FNA (1.00–2.00 μg) attenuated antinociception induced by DAMGO injection, suggesting DAMGO's action on mu receptors in the BLA. Accordingly, we confirm a direct interaction of DAMGO with G-protein-coupled mu receptors in the BLA contributing to induction of opioid antinociception in the amygdala.
Clocinnamox dose-dependently antagonizes morphine-analgesia and [<sup>3</sup>H]DAMGO binding in rats
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Clocinnamox is a long-lasting, nonequilibrium, μ-opioid receptor antagonist in mice and monkeys. The present studies examined the in vivo and ex vivo effects of clocinnamox in rats. Under control conditions, morphine dose-dependently increased tail-withdrawal latencies from 50°C water, with a mean ED₅₀ of 7.3±1.1 mg/kg. Clocinnamox antagonized the antinociceptive effects of morphine. 1.0 mg/kg clocinnamox displaced the morphine dose–response curve 4-fold to the right of the control curve and 10 mg/kg clocinnamox eliminated morphine's antinociceptive effects at doses up to 1000 mg/kg for at least seven days. There was a gradual recovery of the antinociceptive response to morphine; however, the morphine dose–response curve did not return to its original position by five weeks after 10 mg/kg clocinnamox. Whole brain membranes were prepared from separate groups of rats for determination of binding parameters of [ $^{3} H$ ][d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO). Clocinnamox dose-dependently decreased [ $^{3} H$ ]DAMGO binding ex vivo and the decreased binding was a result of changes in B_max. The control B_max for [ $^{3} H$ ]DAMGO was 234±8 fmol/mg protein; in membranes prepared from rats pretreated with 10 mg/kg clocinnamox, the B_max value for [ $^{3} H$ ]DAMGO was 54±2 fmol/mg protein. The B_max values for [ $^{3} H$ ]DAMGO binding after an injection of 10 mg/kg clocinnamox returned towards control values gradually, four weeks after clocinnamox the B_max was 178±10 fmol/mg protein. These results suggest that clocinnamox is a long-lasting, nonequilibrium μ-opioid receptor antagonist in rats.
Involvement of δ<inf>1</inf> and δ<inf>2</inf> opioid receptor subtypes in the development of physical dependence on morphine in mice
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The effects of the highly selective δ opioid receptor antagonists naltrindole (NTI) for δ₁ and δ₂ naltriben (NTB) and naltrindole 5′-isothiocyanate (5′-NTII) for δ₂ and 7-benzylidenenaltrexone (BNTX) for δ₁ on the development of physical dependence on morphine were investigated in mice. Neither NTI (3 mg/kg, sc), NTB (0.5 mg/kg, sc), 5′-NTII (0.5 mg/kg, sc) nor BNTX (0.5 mg/kg, sc) suppressed the antinociception induced by morphine (10 mg/kg, sc). Pretreatment with NTI (3 mg/kg, sc), NTB (0.5, 1.0 mg/kg, sc) or 5′-NTII (0.5, 1.0 mg/kg, sc) during chronic treatment with morphine for 5 days significantly suppressed naloxone-induced body-weight loss in morphine-dependent mice. The incidence of jumping and body shakes in morphine-dependent mice that were pretreated with NTI, NTB or 5′-NTII were significantly lower than with morphine alone. Pretreatment with BNTX (0.5, 1.0 mg/kg, sc) during chronic treatment with morphine also significantly suppressed naloxone-induced body-weight loss in morphine-dependent mice, but this suppression was weaker than that by the antagonists. In contrast to mice that had been pretreated with NTI, NTB or 5′-NTII, the incidence of several withdrawal signs, such as jumping and body shakes, was not significantly affected in morphine-dependent mice that were pretreated with BNTX. These findings suggest that both δ₂ and δ₁ opioid receptors may play important roles in modulating the development of physical dependence on morphine.
The effect of the irreversible μ-opioid receptor antagonist clocinnamox on morphine potency, receptor binding and receptor mRNA
1995, European Journal of Pharmacology
In these experiments, the effect of the irreversible μ-opioid receptor antagonist clocinnamox on the potency of morphine, opioid receptor binding and μ-opioid receptor mRNA was examined. Mice were injected with clocinnamox (0.32–12.8 mg/kg) and the analgesic potency of morphine was examined 24 h later. Clocinnamox produced a dose-dependent decrease in the potency of morphine; and at the higher dose of clocinnamox the maximal analgesic effect was not observed following doses of morphine in excess of 500 mg/kg s.c. In saturation binding studies in brain, clocinnamox (0.32–25.6 mg/kg) dose-dependently decreased μ-opioid ([³H][d-Ala²,MePhe⁴,Gly-ol⁵]enkephalin; DAMGO) receptor B_max with relatively minimal effects on K_d. Binding to δ-opioid receptor ([³H][d-Pen²,d-Pen⁵]enkephalin; DPDPE) and κ-opioid receptor ([³H](5,7,8)-(−)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide; U69,593) was not affected by clocinnamox. The effect of clocinnamox was time-dependent in that the greatest changes in morphine potency and μ-opioid receptor density were observed within 24 h of administration and decreased with time (336 h). Although μ-opioid receptor density was decreased to less than 30% of control 24 h following clocinnamox (12.8 mg/kg) and had increased to 80% by 5 days, a solution hybridization assay for μ-opioid receptor mRNA transcript revealed no changes in the steady-state levels of this mRNA. These studies indicate that clocinnamox is an irreversible antagonist at the μ-opioid receptor since it appears to selectively affect receptor density with minimal effects on affinity. Furthermore, clocinnamox produces time- and dose-dependent changes in B_max and these changes appear to be unrelated to changes in μ-opioid receptor mRNA. It is possible that the repopulation of brain by μ-opioid receptors following clocinnamox is mediated by an existing pool of receptors that are activated following treatment.
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Effects of intracerebroventricular β-funaltrexamine on μ and δ opioid receptors in the rat: dichotomy between binding and antinociception

Abstract

Life Sci.

Pharmacol. Biochem. Behav.

Neurosci. Lett.

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Anal. Biochem.

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Life Sci.

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Eur. J. Pharmacol.

Eur. J. Pharmacol.

Assessment of relative intrinsic activity of mu-opioid analgesics in vivo by using /gb-funaltrexamine

J. Pharmacol. Exp. Ther.

The physiological relevance of low agonist affinity binding at opioid μ-receptors

Br. J. Pharmacol.

Unmasking of magnesium-dependent high-affinity binding sites for [DAla2DLeu5enkephalin after pretreatment of brain membranes with guanine nucleotides

Pre-incubation of guinea-pig myenteric plexus with β-funaltrexamine: discrepancy between binding assays and bioassays

Br. J. Pharmacol.

Unmasking of magnesium-dependent high-affinity binding sites for [DAla₂DLeu₅enkephalin after pretreatment of brain membranes with guanine nucleotides