Mabel Hokin-Neaverson

Acetylcholine causes a net decrease in phosphatidylinositol and a net increase in phosphatidic acid in mouse pancreas

Summary Stimulation of mouse pancreas in vitro with acetylcholine in concentrations of 10−5M or higher led to a net decrease in phosphatidylinositol and a net increase in phosphatidic acid. The phosphatidylinositol level fell from 1.31±0.10 to 0.93±0.08 μmoles per gram of fresh tissue; the phosphatidic acid level rose from 0.45±0.05 to 0.82±0.09 μmoles per gram of fresh tissue. When atropine was added to acetylcholine-stimulated tissue, to cause its return to the unstimulated state, these changes were reversed; the level of phosphatidic acid fell and the level of phosphatidylinositol rose. The levels of the other phosphatides which were measured did not change significantly under these conditions.

Kinetics of phosphorylation of Na+K+-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.

Deficiency of erythrocyte sodium pump activity in bipolar manic-depressive psychosis

Abstract The mean sodium pump activity in erythrocytes of 25 subjects with a history of bipolar manic-depressive disorder was significantly lower than that of 44 control subjects. A lower mean sodium pump activity was manifest during both the active manic phase of the disease and during remission. It was not due to medication with either lithium or phenothiazines. There was a large overlap of individual values with individual control values, indicating that the assay is not, at present, of use for diagnostic purposes.

Acetylcholine increases the level of diglyceride in mouse pancreas

Summary Incubation of mouse pancreas with 10 −5 M or 10 −4 M acetylcholine (plus eserine) resulted in increased tissue diglyceride levels. Diglyceride rose from 0.5 μmoles per gram fresh tissue in unstimulated pancreas to 1.2 and 3.6 μmoles, respectively, in acetylcholine-stimulated tissue. The linear increase in diglyceride level with time at 10 −4 M acetylcholine was blocked and reversed by 10 −5 M atropine. When atropine was added, the level of diglyceride fell to the control, unstimulated value. The increase in diglyceride level was greater than could be accounted for by hydrolysis of phosphatidylinositol or phosphatidic acid.

Inositol is the water-soluble product of acetylcholine-stimulated breakdown of phosphatidylinositol in mouse pancreas.

The water-soluble products of acetylcholine-stimulated breakdown of phosphatidylinositol in mouse pancreas were analyzed by two different and independent procedures. There was an increased formation of free inositol throughout the period of phosphatidylinositol breakdown; no evidence was obtained for acetylcholine-stimulated formation of either inositol 1,2-cyclic phosphate or inositol 1-phosphate under any of the conditions used. The observations suggest that the acetylcholine-stimulated reaction is phosphatidylinositol → phosphatidic acid + inositol. This might occur by either phospholipase D activity, or through complete or partial reversal of the cytidine nucleotide pathway of phosphatidylinositol biosynthesis.

Acetylcholine stimulation of selective increases in stearic and arachidonic acids in phosphatidic acid in mouse pancreas

Abstract During the acetylcholine-stimulated loss of phosphatidylinositol and gain in the level of phosphatidic acid in mouse pancreas, there is a selective increase in stearic and arachidonic acids in phosphatidic acid. The amounts parallel the decrease in phosphatidylinositol, which contains predominantly these two fatty acids. Addition of atropine to stimulated tissue reverses the changes. There is a selective disappearance of the stearoyl, arachidonoyl phosphatidic acid, and phosphatidylinositol increases. The changes support the hypothesis that the 1-stearoyl, 2-arachidonoyl diglyceride backbone of phosphatidylinositol becomes phosphatidic acid during acetylcholine stimulation, and is transformed back to phosphatidylinositol on reversion to the unstimulated state.

Deficient Erythrocyte NaK-ATPase Activity in Different Affective States in Bipolar Affective Disorder and Normalization by Lithium Therapy

This report expands on previously presented evidence for a trait-dependent deficiency of erythrocyte sodium pump activity in bipolar affective disorder. Several parameters of erythrocyte NaK-ATPase activity in different affective states and the effects of lithium therapy were examined. In lithium-free bipolar affective disorder patients, the mean percent differences from the individual sex- and age-matched controls for erythrocyte sodium pump activity were: manic + hypomanic group, -21.5%, n = 16, p less than 0.02; depressed group, -12.4%, n = 14, p less than 0.02; euthymic group, -6.9%, n = 18, p less than 0.10. Ouabain-sensitive potassium ion uptake was less than the controls in the manic group (-25.4%, n = 3, p less than 0.02), and in the combined affectively ill group, manic + depressed (-23.4%, n = 7, p less than 0.02). Cell ouabain binding was less than the controls in the manic group (-19.0%, n = 6, p less than 0.05). NaK-ATPase activity in washed erythrocyte membranes (ghosts) was significantly lower than the controls in the manic group (-14.5%, n = 4, p less than 0.02), and the mean value for the whole group (manic + depressed + euthymic) was lower than the controls (-11.8%, n = 18, p less than 0.05). Values for ouabain binding in ghosts from the bipolar subjects were not significantly different from the matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Sodium Pump Activity in Bipolar Affective Disorder and Other Psychiatric Disorders

Erythrocyte ouabain-inhibitable sodium pump activity, a measure of NaK-ATPase activity, was studied in 6 diagnostic groups of psychiatric subjects: bipolar affective disorder, unipolar depressive disorder, neurotic depression, chronic alcohol abuse, schizoaffective disorder, and schizophrenia, and in sex- and age-matched normal controls. In the bipolar manic-depressive group, which was restricted to lithium-free subjects, values for sodium pump activity were significantly lower than in the controls (-11.4%, n = 53, p less than 0.001); subgrouping of the bipolar group by sex or age showed a significantly lower sodium pump activity in each of the groups. In the unipolar depressive group, values for sodium pump activity were significantly higher than in the controls (+13.7%, n = 12, p less than 0.01). The difference in direction of changed sodium pump activity between the bipolar and the unipolar groups was also observed in the values for subgroups of subjects in the two categories who were in a depressed state at the time the blood sample was taken. In the chronic alcohol abuse group, values for sodium pump activity were significantly higher than those for the control group (+13.5%, n = 20, p less than 0.05). In the neurotic depression (n = 24), schizoaffective (n = 12), and schizophrenia (n = 35) groups, there were no significant differences in sodium pump activity between the group of psychiatric subjects and their matched controls. These observations indicate that there is a trait-dependent deficiency of NaK-ATPase activity in bipolar affective disorder.

Effects of cholinergic stimulation on levels and fatty acid composition of diacylglycerols in mouse pancreas

1. During 15 min after intraperitoneal injection of 1 mg pilocarpine in vivo in the mouse, the level of diacylglycerol in the pancreas rose from 0.47 to 0.80 mumol per g wet weight. There were increase in the levels of all of the individual fatty acids which were measured in the diacylglycerol pool. The major increases were in palmitic and linoleic acids. These accounted for 65% o the total increase. 2. After in vitro incubation of mouse pancreas for 80 min, the levels of diacylglycerol (expressed in mumol/g wet weight) in unstimulated tissue and in tissue incubated with 10 micrometer or 100 micrometers acetylcholine (plus 100 micrometers eserine) were, respectively, 0.51 1.30 and 3.73. Increases in palmitic, oleic and linoleic acids in the diacylglycerol pool accounted for 75% of the total increase. With 100 micrometers acetylcholine, the proportions of individual fatty acids in diacylglycerol showed a significant enrichment of stearic and arachidonic acid at 40 min and of linoleic and arachidonic acids at 80 min. With the exception of an enrichment of stearic and arachidonic acids, the fatty acid composition of the new diacylglycerol resembled that of the triacylglycerol pool more closely than that of other classes of lipid in the pancreas. 3. After addition of atropine to acetylcholine-stimulated tissue, the level of diacylglycerol fell so that, after 40 min, the levels and proportions of all individual fatty acids in the diacylglycerol were not significantly different from those in unstimulated tissue. 4. In both the in vivo and in vitro experiments, the changes in levels of stearic and arachidonic acids in diacylglycerol indicated that only a small proportion of the total increase in diacylglycerol level could have been derived from the breakdown of stearoyl, arachidonoyl phosphatidylinositol which occurs in response to cholinergic stimulation in the pancreas. The major effect of acetylcholine in diacylglycerol metabolism in mouse pancreas in separate, therefore, from the effect on phosphatidylinositol metabolism.

Synthesis of CDP-diglyceride from phosphatidylinositol and CMP

A reaction in which CDP-diglyceride and inositol are formed from 1-stearoyl, 2-arachidonoyl phosphatidylinositol and CMP occurs readily in dialyzed microsomal preparations from the mouse pancreas. The reaction is Mn2+-dependent, and it is inhibited by each of the two products, CDP-diglyceride and myoinositol. It is presumed to involve the back-reaction of CDP-diglyceride: inositol phosphatidyltransferase (phosphatidyl-inositol synthetase, EC.2.7.8.11.)