Anxiolytic effects of ethanol are partially related to a reduced expression of adenylyl cyclase 5 but not to µ-opioid receptor activation in rat nucleus accumbens.

Anxiolytic effects of alcohol participate in the reinforcing properties of the drug, in which nucleus accumbens (NAcc) is implicated. The opioidergic system in NAcc is considered a main pathway involved in the emotional responses of animals: rats microinjected with morphine in NAcc and the systemic administration of µ-opioid receptors (MOR) agonists yield low anxiety scores in the elevated plus maze (EPM), a behavioral test of anxiety. However, the specific participation of NAcc MOR in the anxiolytic effect of ethanol has not been studied. AC5, a cAMP-synthezising adenylyl-cyclase, is highly expressed in NAcc; it is negatively coupled to MOR and has been implicated in anxiety levels of animals. We evaluated the anxiolytic effects of an intra-gastric administration of ethanol (2.5 g/kg) in animals subjected to EPM at 1, 4, and 8 h after drug or water exposure. Locomotion was assayed with the open-field test; we also measured accumbal AC5 and MOR mRNA levels by RT-PCR. After 1 h, ethanol-exposed animals showed anxiolytic-like behavior, as well as decreased and increased AC5 and MOR expression in NAcc, respectively. Intra-accumbal injection of ß-funaltrexamine (FNA), a MOR antagonist, did not block ethanol-induced anxiolysis, rather it induced a tendency to increase anxiety levels in the water-exposed group. FNA partially decreased accumbal AC5 expression in ethanol-treated rats. We concluded that AC5 in NAcc is participating in the emotional effects of ethanol; that MOR was not mediating the drug-induced AC5 reduction in NAcc nor the ethanol-induced anxiolysis. MOR only might be involved in basal levels of anxiety of animals.

Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes.

The role of phospholemman (PLM) in taurine and Cl(-) efflux elicited by 30% hyposmotic solution was studied in cultured cerebellar astrocytes with reduced PLM expression by antisense oligonucleotide (AO) treatment. PLM, a substrate for protein kinases (PK) C and A, is a protein that increases an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. Two antisense oligonucleotides (AO1 and AO2) effectively decreased the expression of the PLM protein by 40% and 30%, respectively, and markedly reduced [(3)H]taurine efflux by 67% and 62%. AO treatment also decreased the osmosensitive release of Cl(-), followed as (125)I. The inhibition of Cl(-) efflux (23% for AO1 and 13% for AO2) was notably lower than for [(3)H]taurine. The contribution of PKC and PKA in the function of PLM was also evaluated in astrocytes. Pharmacological activation or inhibition of PKC and PKA revealed that the osmosensitive taurine efflux is essentially PKC-independent while (125)I efflux is reduced by the PKC blockers H-7 (21%) and Gö6983 (41%). The PKA activator forskolin and dbcAMP increased taurine efflux by 66-70% and (125)I efflux by 21-45%. Norepinephrine increased the osmosensitive taurine efflux at about the same extent as dbcAMP and forskolin, and this was reduced by PKA blockers. These results suggest that PLM plays a role in RVD in astrocytes by predominantly influencing taurine fluxes, which are modulated by PKA but not PKC.

Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman.

The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [(3)H]taurine and (125)I efflux induced by hyposmolarity were markedly increased (30-100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50-60%) of [(3)H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [(3)H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.

Inhibition by dihydropyridines of regulatory volume decrease and osmolyte fluxes in cultured astrocytes is unrelated to extracellular calcium.

The 1,4-dihydropyridines (DHP), nimodipine (NMD) and nitrendipine (NTD) were potent blockers of regulatory volume decrease (RVD) and the volume-associated release of [3H]taurine and chloride (measured as 125I) in 2-weeks cultured rat cerebellar astrocytes. The IC50 were 30 microM and 29 microM for taurine efflux and 26 and 27 microM for C1 efflux for NMD and NTD, respectively. Inhibition by DHP was independent of extracellular Ca, as the effect was the same in media with 1 mM Ca or without Ca and 0.5 mM EGTA. DHP did not affect the basal (isosmotic) release of [3H]taurine or 125I inhibition by DHP (measured only on [3H]taurine efflux) was the same in 3-4 weeks cultured cerebellar astrocytes, 2-4 weeks cultured cortical astrocytes and 2-weeks cultured cerebellar astrocytes treated with dibutyril cAMP. Diltiazem (50 microM) and verapamil (100 microM) failed to inhibit RVD or osmolyte efflux.

Inhibition by polyunsaturated fatty acids of cell volume regulation and osmolyte fluxes in astrocytes.

The polyunsaturated fatty acids, arachidonic, linoleic, and linolenic acids, were potent blockers of regulatory volume decrease (RVD) and of the swelling-activated efflux of [3H]taurine, D-[3H]aspartate, [3H]inositol, and 125I (used as marker of Cl) from rat cerebellar astrocytes in culture. The monounsaturated oleic and ricinoleic acids and saturated fatty acids were ineffective. The amino acid and 125I fluxes were similarly inhibited by fatty acids, whereas inositol release was less sensitive. Polyunsaturated fatty acids appear to directly affect RVD in trypsinized astrocytes as the inhibition was immediate and fully reversible. Blockers of the arachidonic acid metabolic pathways, indomethacin (cyclooxygenase), esculetin (lipoxygenases), and metyrapone (P-450 monooxygenases), did not prevent the effect of arachidonic acid, suggesting that further metabolism is not required for displaying the effects of arachidonic acid on RVD and osmolyte fluxes. Some blockers of arachidonic acid metabolic pathways, such as nordihydroguaiaretic acid (lipoxygenases) and naphthoflavone (P-450 monooxygenases), also exhibited marked inhibitory effects on RVD and on osmolyte fluxes. The predominant arachidonic acid metabolite in astrocytes, 12-hydroxyeicosatetraenoic acid, did not affect RVD or osmolyte fluxes. These results suggest that arachidonic acid and other polyunsaturated fatty acids directly inhibit the permeability pathways correcting cell volume after swelling in cultured astrocytes.