George Hauser

Regional distribution of polyphosphoinositides in rat brain

Abstract 1. 1. The distribution of polyphosphoinositides was determined in anatomical regions of 34- and 60-day-old rat brain, reproducibly dissected and frozen either immediately after death or after 10 min at room temperature. 2. 2. Average levels of di- and triphosphoinositides (nmoles/g wet weight) in rapidly frozen samples from 34-day-old rats were 112.9 and 103.2 in cortical gray matter and 235.5 and 549.5 in brain stem with intermediate concentrations in whole brain, forebrain, thalamus-hypothalamus and cerebellum. 3. 3. The two lipids were distributed differently with relatively more diphosphoinositide located in regions rich in gray matter. 4. 4. The percent loss of both compounds during the 10-min period post mortem generally paralleled the content of gray matter structures and resulted in levels significantly lower than controls, except in brain stem where the decreases were minimal. 5. 5. The regional distribution of the remaining polyphosphoinositides approached that of galactolipids which substantiates earlier conclusions that a large fraction of these compounds is present in myelin and suggests that the labile portion may be predominantly in other structures.

The effect of thiamine deficiency on the pyruvate decarboxylase system of the central nervous system

Abstract The activity of pyruvate decarboxylase (2-oxo-acid carboxy-lyase, EC 4.1.1.1) has been compared in homogenates prepared from tissues of normal, pair-fed control and thiamine-deficient rats. Despite severe symptoms of deficiency the enzymic activity remained essentially normal in cerebral cortex, cerebellum and spinal cord and decreased by only 20% in brain stem. In contrast, the ability of heart, liver and kidney homogenates to decarboxylate pyruvate decreased by about 60%. In the normal central nervous system enzymatic activity is about the same in cortex, cerebellum and brain stem, but substantially lower in spinal cord. While transketolase ( D -sedoheptulose-7-phosphate: D -glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1), another thiamine-dependent enzyme, appears to be most active in myelinated tracts, pyruvate decarboxylase activity is highest in neuronal aggregates. The former enzyme may reflect oligodendroglial and the latter neuronal oxidative metabolism. In rat brain, transketolase is more susceptible to thiamine deprivation than pyruvate decarboxylase. It is therefore postulated that the principal “biochemical lesion” of the athiaminotic state consists of a failure in the transketolase system which may be responsible for initiating the clinical and pathological changes. The abnormality of pyruvate metabolism may play a secondary role.

Postsynaptic localization of the alpha receptor-mediated stimulation of phosphatidylinositol turnover in pineal gland.

Abstract The phosphatidylinositol effect in rat pineal gland (defined as the enhanced incorporation of P i into phosphatidylinositol which is elicited by agonists) is mediated through alpha-adrenergic receptors. Experiments with glands from animals treated with 6-hydroxydopamine, with dispersed pinealocytes and with a number of relatively specific pre- and postsynaptic alpha-adrenergic agonists and antagonists have shown the receptors involved to be located at postsynaptic sites.

Modulation of rat brain cytosolic phosphatidate phosphohydrolase: Effect of cationic amphiphilic drugs and divalent cations

The effects of three cationic amphiphilic drugs on rat brain cytosolic phosphatidate phosphohydrolase and their mechanisms of action were studied utilizing membrane-bound, emulsified, and emulsified sonicated phosphatidate as substrates. With the membrane-bound substrate, chlorpromazine, desmethylimipramine, and propranolol inhibited the activity in a dose-dependent fashion with an IC50 of 30-50 microM. In the presence of the emulsified substrate, chlorpromazine was a more potent inhibitor than desmethylimipramine or propranolol but 200 microM was needed for 50% inhibition of activity. Addition of heat-inactivated microsomes to the emulsified substrate, to simulate the conditions with the membrane-bound substrate, did not alter this value. Both Mg2+ and Ca2+ stimulated the enzyme activity but only Ca2+ counteracted the effect of chlorpromazine. Kinetic studies indicate that chlorpromazine acts as a noncompetitive inhibitor of the enzyme. Emulsified sonicated phosphatidate was a good substrate at low (less than 10 microM) concentrations. It was a poor substrate at 1 mM, but at this concentration chlorpromazine stimulated the activity instead of inhibiting. This drug altered the integrity of phosphatidate vesicle membranes as visualized by electron microscopy. The different results obtained with the three types of substrate indicate the importance of the configuration of phosphatidate for the expression of enzyme activity and for its susceptibility to the action of cationic amphiphilic drugs.

Improved conditions for the preservation and extraction of polyphosphoinositides

1. 1. Conditions have been determined which permit the recovery of greater amounts of di- and triphosphoinositides from tissues than previously obtained, especially when some time must be allowed to elapse before the extraction procedure can be started. Addition of CaCl2 at the optimal level of 60 μmoles/g of tissue to the chloroform-methanol (1:1, v/v) (at least 15 vol.) used for the extraction of the bulk of the lipids results in the subsequent isolation of larger quantities of polyphosphoinositides and especially diphosphoinositide. When tissue homogenates or subcellular fractions are to be analyzed, the use of an alkaline homogenization medium largely prevents the degradation of these lipids for several hours. Keeping the temperature near 0 °C is desirable. 2. 2. When these conditions were combined with the usual extraction of polyphosphoinositides with acidified solvents and separation by thin-layer chromatography, levels of the two lipids in brains of 1- and 7-day-old rats were similar at both ages, but diphosphoinositide was about 50 % higher than triphosphoinositide. During standing at room temperature levels in 7-day-old brains fell more rapidly than in 1-day-old brains. 3. 3. Levels in gray matter of 34-day-old rats were higher than previously found (Hauser G., Eichberg J. and Gonzalez-Sastre F. (1971) Biochim. Biophys. Acta 248, 87–95) but the marked disappearance during a period of anoxia was confirmed. 4. 4. Adult rat kidney values were increased much less than those for brain when the initial extraction was carried out in the presence of CaCl2.

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H‐7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH‐SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH‐SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH‐SY5Y cells, as well as in rat brain. Protein kinase C‐α and ‐β1 were the most abundant isoforms in SH‐SY5Y cells, and immunoreactive protein kinase C‐δ and ‐ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol or staurosporine, and that protein kinase C‐ɛ is predominantly membrane‐bound. Its localization did not change during differentiation. Western blots of total SH‐SY5Y cell extracts and of subcellular fractions probed with isoform‐specific polyclonal antibodies showed that when SH‐SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C‐α and ‐ɛ decreased, and protein kinase C‐β1, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

Abstract: 5‐Hydroxytryptamine (serotonin or 5‐HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 ± 10‐7M. The phosphoinositide response was blocked by the 5‐HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by ˜30%. The 5‐HT1A agonist 8‐hydroxy‐2 (di‐n‐propylamino)tetralin did not cause stimulation. Incubation with 5‐HT (1 μM) for 1 h increased the incorporation of [2‐3H]myo‐inositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5‐HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI‐4‐phosphate, lyso‐PI, glycerophosphoinositol, and IP, but the presence of 5‐HT again did not cause further stimulation. 5‐HT also stimulated the release of IPs in cells prelabeled with [2‐3H]myo‐inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5‐HT (1 μM). Radioactivity in IP2 and IP3 was very low, was stimulated ˜50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was ˜40 times that in IP3. The EC50 value of 1.8 ± 10‐7M was comparable with that obtained from 32Pi studies measuring labeled PI. Ketanserin and spiperone inhibited 5‐HT‐stimulated [2‐3H]IP release with IC50 values of 3.1 ± 10‐9 and 1.8 ± 10, ‐8M, respectively. This study demonstrates that phosphoinositide hydrolysis is enhanced by 5‐HT in C6 glioma cells and that this phenomenon is linked to 5‐HT2‐like binding sites.

The subcellular distribution of polyphosphoinositides in myelinated and unmyelinated rat brain.

Abstract 1. 1. The distribution of polyphosphoinositides from 7- and 34-day-old rat brain was investigated in subcellular fractions prepared by differentia! and density gradient centrifugation. Fractionation in an alkaline medium produced greatly improved recoveries ofdiphosphoinositide and triphosphoinositide, especially in the synaptosomerich and microsomal fractions. 2. 2. On fractionation of 34-day-old brain, over half of the diphosphoinositide and more than three-quarters of the triphosphoinositide were recovered in myelin-containing fractions. The molar ratio of di- to triphosphoinositide in the myelin-rich band from the density gradient was 0.47. The corresponding ratio in the synaptosome-rich band was 1.96. During myelination, the amount of each polyphosphoinositide did not change in the synaptosomal fraction and declined slightly inthemicrosomes. 3. 3. In primary fractions from 7-day-old brain, more than 60 % of each lipid was recovered in the crude mitochondrial pellet. When this pellet was further fractionated, the greatest amounts of the compounds were found in the synaptosome-rich fraction. Both polyphosphoinositides were most enriched, more than 3-fold on a protein basis as compared to whole homogenate, in a band which migrated to a position on the gradient occupied by a myelin-rich fraction when mature rat brains are used. 4. 4. The distribution of polyphosphoinositides on the density gradient resembled those of acetylcholinesterase and 5′-nucleotidase much more than those of lactic acid dehydrogenase and cytochrome c oxidase. 5. 5. Evidence from marker enzyme data and electron microscopic examination of the density gradient fractions leads us to suggest that in unmyelinated rat brain di-and triphosphoinositides are concentrated in neuronal and glial plasma membranes.

Distinct Mechanisms of Differentiation of SH-SY5Y Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors

Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH‐SY5Y differentiates when exposed to 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron‐specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF‐H). TPA, 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol, and staurosporine, but not DAG or 4‐O‐methyl‐TPA, caused neurite outgrowth. Neuron‐specific enolase was expressed in cells treated with TPA and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol but not with DAG, staurosporine, or 4‐O‐methyl‐TPA. NF‐H increased in the perikarya of cells treated with DAG and 4‐O‐methyl‐TPA, in processes and to varying degrees in perikarya of TPA‐ and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol‐treated cells, but much more in the processes than in the perikarya of staurosporine‐differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH‐SY5Y cells by different mechanisms, and that the five‐membered/seven‐membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

Concentrations and disappearance post mortem of polyphosphoinositides in developing rat brain

Abstract The content of di- and triphosphoinositide in the brains of young rats of different ages was determined. Appreciable quantities of these phospholipids are present as early as two days after birth, possibly in extramyelin structures, and their levels increase during myelination at a rate greater than that for total lipids. Rat brain triphosphoinositide undergoes rapid partial breakdown post mortem after which degradation proceeds only very slowly. The persistence of some brain polyphosphoinositides after death is not due to the irreversible inactivation of triphosphoinositide phosphomonoesterase or triphosphoinositide phosphodiesterase.