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Changes in [(3)H]glibenclamide binding to mouse forebrain membranes during morphine tolerance
L G González1, E Portillo, E Del Pozo
1Departamento de Farmacología e Instituto de Neurociencias, Facultad de Medicina, Universidad de Granada, Avda. Madrid 11, E-18012, Granada, Spain.
Abstract:
The characteristics of specific binding of the ATP-sensitive K(+) (K(ATP)) channel blocker [3H]glibenclamide to forebrain membranes (P(2) fraction, 4 degrees C) obtained from morphine-naive and -tolerant mice were evaluated. Morphine tolerance was induced by osmotic minipumps that released 45 mg/kg/day of morphine subcutaneously for 6 days. This treatment enhanced the antinociceptive ED(50) of morphine without changing its E(max). In morphine-naive animals, (1) both the association and the dissociation of [3H]glibenclamide were biphasic; (2) [3H]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide>glipizide&z.Gt;tolbutamide) with pseudo-Hill coefficients lower than unity and biphasic Hofstee plots; and (3) Scatchard plots of saturation experiments were curvilinear, showed a Hill coefficient of 0.81+/-0.04 and suggested the presence of two binding sites with a K(D) of 0.13 and 3.17 nM and a B(max) of 12.30 and 84.47 fmol/mg protein, respectively. By contrast, in membranes obtained from morphine-tolerant animals, (1) the Scatchard plots showed only one population of binding sites with a K(D) of 0.87 nM and a B(max) of 77.99 fmol/mg protein, and the Hill coefficient was very close to unity (0.96+/-0.1); (2) competition experiments (using glibenclamide as displacer) showed a pseudo-Hill coefficient of 0.99+/-0.04; and (3) dissociation experiments showed only one phase of dissociation. These results suggest that [3H]glibenclamide binds to two different sites in membranes obtained from morphine-naive animals, but to only one site in morphine-tolerant animals. Consequently, it seems that morphine tolerance in mice involves adaptive changes in K(ATP) channels.
Insights
Morphine tolerance in mice alters ATP-sensitive K(+) channel binding sites. This suggests adaptive changes in K(ATP) channels are involved in developing morphine tolerance.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- Opioid analgesics like morphine are widely used for pain management.
- Development of tolerance to morphine necessitates higher doses for equivalent pain relief.
- ATP-sensitive potassium (K(ATP)) channels are implicated in opioid action and tolerance.
Purpose of the Study:
- To investigate the binding characteristics of the K(ATP) channel blocker [3H]glibenclamide in the forebrain of morphine-naive and morphine-tolerant mice.
- To determine if morphine tolerance is associated with changes in the number or affinity of K(ATP) channel binding sites.
Main Methods:
- Radioligand binding assays using [3H]glibenclamide on forebrain membrane preparations (P(2) fraction).
- Induction of morphine tolerance via continuous subcutaneous infusion of morphine using osmotic minipumps.
- Analysis of binding kinetics (association, dissociation), competition, and saturation using Scatchard and Hofstee plots.
Main Results:
- In naive mice, [3H]glibenclamide binding was biphasic, indicating two distinct binding sites with different affinities (K(D) 0.13 nM and 3.17 nM).
- In tolerant mice, binding became monophasic, with a single population of sites (K(D) 0.87 nM) and a Hill coefficient near unity.
- Competition studies showed altered affinity for sulfonylureas in tolerant animals, and dissociation was also monophasic.
Conclusions:
- Morphine tolerance in mice is associated with a reduction in the number of [3H]glibenclamide binding sites or a shift in affinity.
- The data suggest that adaptive changes occur in K(ATP) channels during the development of morphine tolerance.
- These findings highlight the role of K(ATP) channels in the neurobiological mechanisms underlying opioid tolerance.