Large number of extracellular signals is received by plasma membrane receptors which, upon activation, transduce information into the target cell interior via trimeric G-proteins (GPCRs) and induce activation or inhibition of adenylyl cyclase enzyme activity (AC). Receptors for opioid drugs such as morphine (micro-OR, delta-OR and kappa-OR) belong to rhodopsin family of GPCRs. Our recent results indicated a specific up-regulation of AC I (8-fold) and AC II (2.5-fold) in plasma membranes (PM) isolated from rat brain cortex exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Increase of ACI and ACII represented the specific effect as the amount of ACIII-ACIX, prototypical PM marker Na, K-ATPase and trimeric G-protein alpha and beta subunits was unchanged. The up-regulation of ACI and ACII faded away after 20 days since the last dose of morphine. Proteomic analysis of these PM indicated that the brain cortex of morphine-treated animals cannot be regarded as being adapted to this drug because significant up-regulation of proteins functionally related to oxidative stress and alteration of brain energy metabolism occurred. The number of delta-OR was increased 2-fold and their sensitivity to monovalent cations was altered. Characterization of delta-OR-G-protein coupling in model HEK293 cell line indicated high ability of lithium to support affinity of delta-OR response to agonist stimulation. Our studies of PM structure and function in context with desensitization of GPCRs action were extended by data indicating participation of cholesterol-enriched membrane domains in agonist-specific internalization of delta-OR. In HEK293 cells stably expressing delta-OR-G(i)1alpha fusion protein, depletion of PM cholesterol was associated with the decrease in affinity of G-protein response to agonist stimulation, whereas maximum response was unchanged. Hydrophobic interior of isolated PM became more "fluid", chaotically organized and accessible to water molecules. Validity of this conclusion was supported by the analysis of an immediate PM environment of cholesterol molecules in living delta-OR-G(i)1alpha-HEK293 cells by fluorescent probes 22- and 25-NBD-cholesterol. The alteration of plasma membrane structure by cholesterol depletion made the membrane more hydrated. Understanding of the positive and negative feedback regulatory loops among different OR-initiated signaling cascades (micro-, delta-, and kappa-OR) is crucial for understanding of the long-term mechanisms of drug addiction as the decrease in functional activity of micro-OR may be compensated by increase of delta-OR and/or kappa-OR signaling.

• MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• fluidita membrány fyziologie   MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• membránové lipidy metabolismus   MeSH
• mozková kůra metabolismus   ultrastruktura   MeSH
• opioidní analgetika metabolismus   MeSH
• receptory opiátové metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

With the aim to understand the onset of expression and developmental profile of plasma membrane (PM) content /density of crucial components of GABA(B)-R signaling cascade, GABA(B)-R1a, GABA(B)-R1b, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta subunit proteins were determined by quantitative immunoblotting and compared in PM isolated from brain cortex of rats of different ages: between postnatal-day-1 (PD1) and 90 (PD90). PM density of GABA(B)-R1a, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was high already at birth and further development was reflected in parallel decrease of both GABA(B)-R1a and GABA(B)-R2 subunits. The major decrease of GABA(B)-R1a and GABA(B)-R2 occurred between the birth and PD15: to 55 % (R1a, **) and 51 % (R2, **), respectively. Contrarily, PM level of the cognate G-proteins G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was unchanged in the course of the whole postnatal period of brain cortex development. Maturation of GABA(B)-R cascade was substantially different from ontogenetic profile of prototypical plasma membrane marker, Na, K-ATPase, which was low at birth and further development was reflected in continuous increase of PM density of this enzyme. Major change occurred between the birth and PD25. In adult rats, membrane content of Na, K-ATPase was 3-times higher than around the birth.

• MeSH
• buněčná membrána metabolismus   MeSH
• heterotrimerní G-proteiny metabolismus   MeSH
• krysa rodu rattus MeSH
• mozková kůra růst a vývoj   metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotka Gi2 metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky metabolismus   MeSH
• receptory GABA-B metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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 β 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.

• MeSH
• abstinenční syndrom MeSH
• adenylátcyklasy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• izoenzymy metabolismus   MeSH
• krysa rodu rattus MeSH
• morfin škodlivé účinky   MeSH
• potkani Wistar MeSH
• receptory opiátové delta metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH