Na+/K+-ATPase level and products of lipid peroxidation in live cells treated with therapeutic lithium for different periods in time (1, 7, and 28 days); studies of Jurkat and HEK293 cells.

Regulation of Na+/K+-ATPase in bipolar disorder and lithium therapy has been investigated for more than 40 years. Contradictory results in this area may be caused by the difference between acute and long-term Li effects on cell metabolism and variance in responsiveness of different cell types. We compared the time-course of Li action focusing on Na+/K+-ATPase and lipid peroxidation in two widely different cell models-Jurkat and HEK293. Na+/K+-ATPase expression level was determined in cells cultivated in the absence or presence of 1 mM Li for different time spans (1, 7, and 28 days) using [3H] ouabain binding and immunoblot assay of α-subunit. In parallel samples, the formation of malondialdehyde (MDA) was quantified by HPLC, and 4-hydroxy-2-nonenal (4-HNE) protein adducts were determined by immunoblot. Cultivation of Jurkat cells in 1 mM Li medium resulted in downregulation of Na+/K+-ATPase (decrease of [3H] ouabain-biding sites and intensity of immunoblot signals) in all Li-groups. In HEK293 cells, the decrease of Na+/K+-ATPase was observed after the acute, 1-day exposure only. The long-term treatment with Li resulted in Na+/K+-ATPase upregulation. MDA and 4-HNE modified proteins were decreased in Jurkat cells in all Li-groups. On the other hand, in HEK293 cells, MDA concentration was decreased after the acute, 1-day Li exposure only; the long-term cultivations, for 7 or 28 days, resulted in a significant increase of lipid peroxidation products. The Li-induced decrease of lipid peroxidation products was associated with the decrease of Na+/K+-ATPase level and vice versa.

Up-regulation of µ-, δ- and κ-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes.

Regulation of µ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express µ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both µ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of µ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized µ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of µ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gß subunits were unchanged. The level of ß-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and ß-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.

Induction of oxidative stress by long-term treatment of live HEK293 cells with therapeutic concentration of lithium is associated with down-regulation of δ-opioid receptor amount and function.

The functional state of δ-opioid receptor signaling cascade in live cells exposed to a therapeutic concentration of lithium for a prolonged period of time (weeks) is not known because the previous studies of Li interference with OR were oriented to µ-OR only. The same applies to the analysis of the prolonged effect of Li on oxidative stress in context with δ-OR function. HEK293 cells stably expressing δ-OR were cultivated in the presence or absence of 1 mM LiCl for 7 or 21 days, homogenized and the post-nuclear (PNS) and plasma membrane (PM) fractions prepared from all four types of cells. Level of δ-OR in PM was determined by specific radioligand [3H]DADLE binding and immunoblot assays; the functional coupling between δ-OR and G proteins was determined as DADLE-stimulated high-affinity [35S]GTPγS binding. In the whole cells, general oxidative stress was monitored by fluorescent dye 2',7'-dichlorofluorescein diacetate (DCF) and results verified by analysis of PNS and isolated PM. Generation of 4-hydroxy-2-nonenal (4-HNE)-protein adducts and malondialdehyde (MDA) level were determined as products of lipid peroxidation. Li-treated cells exhibited the decreased amount of δ-OR. This was evidenced by both [3H]DADLE binding and immunoblot assays. The δ-OR-G protein coupling efficiency was diminished. Simultaneously, in Li-treated cells, the highly increased oxidative stress measured as DCF fluorescence intensity was noticed. Importantly, this result was detected in live cells as well as PNS and PM. Accordingly, production of 4-HNE-protein adducts and MDA was clearly increased in Li-treated cells. The general significance of our work lies in presentation of novel data indicating that prolonged exposure of live HEK293 cells to the therapeutic concentration of Li results in down-regulation of δ-OR protein level and attenuation of δ-OR function in parallel with increased oxidative stress and increased level of lipid peroxidation products.

Determination of µ-, δ- and κ-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal.

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.