Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48 h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24-25 h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5-10 min (t = 3 min) initiates a process that results in uncoupling hours later (t = 12 h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of alpha 3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABAinduced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism that negatively regulates GABAergic transmission under physiological conditions in which synaptic GABAA receptors are transiently activated for several minutes. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

This study aimed to determine the damage mechanisms caused by naturally targeted nanoarchaeosomes made of diether lipids from Halorubrum tebenquichense loaded with curcumin (CUR, nATC), which mediated photodynamic therapy (PDT) on A549 cells and on THP-1-macrophages, two cell types found in airway cancers. The effect of nATC- PDT on vessels modeled with a chicken embryo chorioallantoic membrane (CAM), after dropping the formulations on its surface covered with mucins, was also determined. nATCs are known to efficiently trap CUR for at least six months, constituting easy-to-prepare, stable formulations suitable for nebulization. CUR instead, is easily released from carriers such as liposomes made of ordinary phospholipids and cholesterol after a few weeks. Irradiated at 9 J/cm², nATC (made of archaeolipids: Tween 80: CUR at 1:0.4:0.04 w:w, size 180 ± 40 nm, ζ potential −24 mV, 150 μg CUR/15 mg lipids/mL) was phototoxic (3.7 ± 0.5 μM IC₅₀), on A549 cells after 24 h. The irradiation reduced mitochondrial membrane potential (ΔΨm), ATP levels and lysosomal functionalism, and caused early apoptotic death and late necrosis of A549 cells upon 24 h. nATC induced higher extra and intracellular reactive oxygen species (ROS) than free CUR. nATC-PDT impaired the migration of A549 cells in a wound healing assay, reduced the expression of CD204 in THP-1 macrophages, and induced the highest levels of IL-6 and IL-8, suggesting a switch of macrophage phenotype from pro-tumoral M2 to antitumoral M1. Moreover, nATC reduced the matrix metalloproteinases (MMP), −2 and −9 secretion, by A549 cells with independence of irradiation. Finally, remarkably, upon irradiation at 9 J/cm² on the superficial vasculature of a CAM covered with mucins, nATC caused the vessels to collapse after 8 h, with no harm on non-irradiated zones. Overall, these results suggest that nebulized nATC blue light-mediated PDT may be selectively deleterious on superficial tumors submerged under a thick mucin layer.