Lena Gustavsson

Phosphatidylethanol formation in rat organs after ethanol treatment

An abnormal acidic phospholipid was found in high concentration in kidney and brain, and also in other organs of rats exposed to ethanol by i.p. injection or by a liquid diet. The compound could be identified as phosphatidylethanol. Phosphatidylethanol is probably formed in cell membranes by a phospholipase D-catalyzed transphosphatidylation reaction.

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

Abstract: The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D‐catalyzed transphosphatidylation, was formed. The half‐maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol‐treated cells was accompanied by an inhibition of the TPA‐induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoylglycerol (100 μM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol‐treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.

Phosphatidylethanol formation and degradation in brains of acutely and repeatedly ethanol-treated rats

The formation of the abnormal phospholipid phosphatidylethanol (PEth) was studied in hippocampus, cerebellum and cerebrum of rat brain after intraperitoneal ethanol administration. Prior to analysis by high performance thin layer chromatography PEth was purified. After one injection, PEth levels reached a maximum after 2 h and remained detectable for 14-24 h in all three regions. Repeated injections led to additional accumulation. Maximum in vivo levels of 30-50 nmol/g wet wt. were reached.

Controlled-pH Tissue Cleanup Protocol for Signal Enhancement of Small Molecule Drugs Analyzed by MALDI-MS Imaging

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.

Effects of chronic ethanol exposure on fatty acids of rat brain glycerophospholipids.

The lipid composition was analysed in forebrain subcellular fractions from rats treated with ethanol for three weeks and control rats. Increased proportions of oleic acid and a decrease in palmitic acid were consistently found in total glycerophospholipid fractions after ethanol exposure. The fatty acid compositions of individual phospholipids were also significantly changed. The proportion of docosahexaenoic acid was decreased in brain phosphatidylserine. In contrast to the decrease in the degree of unsaturation in phosphatidylserine, there was an opposite change in phosphatidylcholine wherein the degree of unsaturation was increased. No changes were produced in total cholesterol or phospholipid concentrations. These results point to a high degree of complexity of the mechanisms behind ethanol-induced changes in membrane lipid composition. The decrease in unsaturation in phosphatidylserine is probably an adaptive effect in order to counteract the fluidizing effect of ethanol. There are two possible explanations for the increase in unsaturation in brain phosphatidylcholine. The change may be due to adaptation to other biophysical effects, e.g., expansion of the membrane surface or be secondary to a change in liver lipid metabolism.

Densitometric quantification of individual phospholipids. Improvement and evaluation of a method using molybdenum blue reagent for detection.

A densitometric method for simultaneous quantification of individual phospholipids based on visualization with molybdenum blue reagent is presented. Previously reported problems concerning colour instability have been solved. The method is specific for phosphorus-containing compounds and independent of the degree of fatty acid unsaturation. The molar absorptivities of most analysed phospholipids do not differ more than 10% from that for phosphatidylcholine from egg yolk. For cases of greater deviation (sphingomyelin, lysophosphatidylcholine and phosphatidylserine), analysis is accomplished by use of appropriate standard mixtures. The between-series coefficient of variation is ca. 9%. The method is compared to a preparative thin-layer chromatographic assay and another commonly used densitometric method.

Phosphatidylethanol affects inositol 1,4,5-trisphosphate levels in NG108-15 neuroblastoma x glioma hybrid cells

Abstract: Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108–15 neuroblastoma × glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5‐trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol‐naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5‐h incubation with 30 μM phosphatidylethanol. Phosphatidylethanol was found to cause a time‐and dose‐dependent increase in the basal levels of inositol 1,4,5‐trisphosphate. The effects on inositol 1,4,5‐trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin‐stimulated inositol 1,4,5‐trisphosphate production could be detected. However, the increase in basal inositol 1,4,5‐trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5‐trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol‐induced effects on cellular processes.

Acute effects of D1- and D2-receptor agonist and antagonist drugs on somatostatin binding, inhibition of adenylyl cyclase activity and accumulation of inositol 1,4,5-trisphosphate in the rat striatum

A recent study carried out by our group demonstrated that exogenous dopamine increases the somatostatin (SS) receptor-effector system in the rat striatum. The present study examined the participation of the D1- and D2-dopaminergic systems in the modulation of the rat striatal SS receptor-effector system by use of the D1-receptor agonist and antagonist SKF 38393 and SCH 23390, respectively, and the D2-receptor agonist and antagonist bromocriptine and raclopride, respectively. In view of the rapid onset of dopamine action, the effect of dopaminergic agents on the SS mechanism of action were studied 3 h after their administration. SKF 38393 (4 mg/kg i.p.) or bromocriptine (2 mg/kg i.p.) administered to male Wistar rats increased the number of 125I-Tyr3-SMS receptors in the striatum (52 and 30%, respectively) without changing the affinity constant. The effect of SKF 38393 on 125I-Tyr3-SMS binding was antagonized by the D1-specific antagonist SCH 23390 (0.25 mg/kg i.p.) whereas the effect of bromocriptine was abolished by the D2-specific antagonist raclopride (5 mg/kg i.p.). No change in binding was produced when SKF 38393 or bromocriptine were added directly to the incubation medium. The acute systemic administration of SCH 23390 or raclopride alone had no effect on the binding of 125I-Tyr3-SMS to its receptors. The increase of the number of 125I-Tyr3-SMS receptor induced by SKF 38393 or bromocriptine was accompanied by an increase in the capacity of SMS 201-995 to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity when compared to the control groups. In addition, the effect of SMS 201-995 on the mass accumulation of inositol 1,4,5-trisphosphate (IP3) was investigated. SKF 38393 as well as bromocriptine increased the capacity of SMS 201-995 to accumulate IP3 in the rat striatum although this effect was only statistically significant in the case of SKF 38393. These results suggest that the activation of D1 and D2 receptors increases the activity of the SS receptor-effector system, the effect being greater in the case of D1 receptors. These findings are consistent with a functional interaction between dopamine and SS in the rat striatum.

Formation of phosphatidylethanol in frozen kidneys from ethanol-treated rats.

We recently identified phosphatidylethanol (Pet) in tissues from ethanol-treated rats. Since phosphatidyl esters are formed artefactually during freezing in plants we wanted to examine if PE was elevated during freezing in animal tissues. Rats were treated with 3 g/kg of ethanol, killed after 3 h and PE was isolated from kidneys at once or after storage at 0, -5, -10, -15, -20 and -80 degrees C for 7 days. Kidneys analyzed at once or after storage at -80 degrees C had Pet equivalent to 0.02 mumol Pet/g. Storage at -10 degrees C and -15 degrees C resulted in increases of Pet to 1.5 mumol Pet/g and 1.2 mumol Pet/g, respectively. Thus, Pet is artefactually elevated during storage of tissues from ethanol-treated rats at lower freezing temperatures, reflecting considerable changes in composition of acidic phospholipids.

Mechanisms of muscarinic receptor-stimulated expression of c-fos in SH-SY5Y cells

In this study, the signal cascade transducing carbachol stimulation into c-fos expression in SH-SY5Y neuroblastoma cells was investigated. 1,2-Diacylglycerol formation and c-fos expression were mediated via stimulation of muscarinic M1 receptors and the first 5 min of receptor stimulation were critical for these events. Application of 1,2-dioctanoylglycerol induced c-fos expression and this, as well as carbachol-stimulated c-fos expression, was inhibited by protein kinase C inhibitors. Increasing the intracellular Ca2+ concentration had only small effects on c-fos expression. There was a dependency on extracellular Ca2+ for maximal c-fos expression and 1,2-diacylglycerol formation. The carbachol-stimulated c-fos expression was potentiated by application of the protein phosphatase inhibitor okadaic acid. These results demonstrate the importance of 1,2-diacylglycerol formation for muscarinic receptor-stimulated, protein kinase C-mediated c-fos expression in the SH-SY5Y cells and that this cascade is counteracted by an okadaic acid-sensitive protein phosphatase.