Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal.

BACKGROUND: Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM). METHODS: Rats were adapted to morphine for 10 days and sacrificed 24h (group+M10) or 20 days (+M10/-M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups-M10 and (-M10/-M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding. RESULTS: ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+M10) when compared with controls (-M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII-ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and ß subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+M10/-M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine. CONCLUSIONS: We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine. GENERAL SIGNIFICANCE: Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.

Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion.

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.

Long-term adaptation to high doses of morphine causes desensitization of mu-OR- and delta-OR-stimulated G-protein response in forebrain cortex but does not decrease the amount of G-protein alpha subunits.

BACKGROUND: The functional activity of trimeric guanine-nucleotide-binding proteins (G-proteins) represents an essential step in linking and regulation of the opioid receptor (mu-, delta- and kappa-OR)-initiated signaling pathways. Theoretical basis and/or molecular mechanism(s) of opioid tolerance and addiction proceeding in the central nervous system were not studied in the forebrain cortex of mammals with respect to quantitative analysis of opioid-stimulated trimeric G-protein activity. MATERIAL/METHODS: G-protein activity was measured in PercollR-purified plasma membranes (PM) isolated from the frontal brain cortex of control and morphine-treated rats by both high-affinity [32P]GTPase and [35S]GTPgammaS binding assays. Exposition to morphine was performed by intra-muscular application of this drug. Control animals were injected with sterile PBS. RESULTS: Both mu-OR (DAMGO)- and delta-OR (DADLE)-responses were clearly desensitized in PM isolated from morphine-treated rats; kappa-OR (U-69593)- and baclofen (GABAB-R)-stimulated [35S]GTPgammaS binding was unchanged, indicating the specificity of the morphine effect. Under such conditions, the amount of G-protein alpha subunits was unchanged. The order of efficacy DADLE>DAMGO>U-69593 was the same in control and morphine-treated PM. Behavioral tests indicated that morphine-treated animals were fully drug-dependent and developed tolerance to subsequent drug addition. CONCLUSIONS: Prolonged exposure of rats to high doses of morphine results in decrease of the over-all output of OR-stimulated G-protein activity in the forebrain cortex but does not decrease the amount of these regulatory proteins. These data support the view that the mechanism of the long-term adaptation to high doses of morphine is primarily based on desensitization of OR-response preferentially oriented to mu-OR and delta-OR.