BACKGROUND: Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC. METHODS: Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies. RESULTS: Significant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/-M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats. CONCLUSIONS: In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal. GENERAL SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Substance Withdrawal Syndrome * MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Rats MeSH
• Morphine administration & dosage   adverse effects   MeSH
• Rats, Wistar MeSH
• Prosencephalon metabolism   MeSH
• Receptors, Opioid, delta metabolism   MeSH
• Receptors, Opioid, kappa metabolism   MeSH
• Receptors, Opioid, mu metabolism   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Morphine MeSH
• Receptors, Opioid, delta MeSH
• Receptors, Opioid, kappa MeSH
• Receptors, Opioid, mu MeSH

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

• Keywords
• Cholesterol, Internalization, Plasma membrane, Rab proteins, δ-opioid receptor,
• MeSH
• Cell Membrane chemistry   MeSH
• Cholesterol metabolism   MeSH
• HEK293 Cells MeSH
• Intracellular Membranes chemistry   MeSH
• Humans MeSH
• rab GTP-Binding Proteins metabolism   MeSH
• Receptors, Opioid, delta agonists   metabolism   MeSH
• Cell Membrane Structures chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholesterol MeSH
• rab GTP-Binding Proteins MeSH
• Receptors, Opioid, delta MeSH

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

• MeSH
• Cell Membrane chemistry   drug effects   metabolism   MeSH
• Cetomacrogol pharmacology   MeSH
• Detergents pharmacology   MeSH
• HEK293 Cells MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Humans MeSH
• Pertussis Toxin toxicity   MeSH
• Receptors, Opioid, delta metabolism   MeSH
• Temperature MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cetomacrogol MeSH
• Detergents MeSH
• Pertussis Toxin MeSH
• Receptors, Opioid, delta MeSH

The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-Gi1α (Cys(351)-Ile(351)) fusion protein. Agonist [(3)H]DADLE binding was decreased in the order Na(+) ≫ Li(+) > K(+) > (+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na(+)-responsive sites (r (2) = 0.99). High-affinity Na(+)-sites were characterized by Kd = 7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (Kd = 463 mM). Inhibition of [(3)H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [(35)S]GTPγS binding was increased in a reverse order: Na(+) < K(+) < Li(+). This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to Gi1α(Cys(351)-Ile(351)) within the δ-OR-Gi1α fusion protein, but is also valid for stimulation of endogenous G proteins of Gi/Go family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs(+) < K(+) < Na(+) < Li(+). The results are discussed in terms of interaction of Na(+), K(+) and Li(+) with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl(-), Br(-), and I(-) counter anions in inhibition of both [(3)H]DADLE and [(35)S]GTPγS binding.

• MeSH
• Cell Membrane metabolism   MeSH
• Guanosine 5'-O-(3-Thiotriphosphate) metabolism   MeSH
• HEK293 Cells MeSH
• Enkephalin, Leucine-2-Alanine metabolism   MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Lithium pharmacology   MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go metabolism   MeSH
• Receptors, Opioid, delta metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Sodium metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Guanosine 5'-O-(3-Thiotriphosphate) MeSH
• Enkephalin, Leucine-2-Alanine MeSH
• Lipid Bilayers MeSH
• Lithium MeSH
• GTP-Binding Protein alpha Subunits, Gi-Go MeSH
• Receptors, Opioid, delta MeSH
• Recombinant Proteins MeSH
• Sodium MeSH