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Notes: Abstract: : The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30–500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater than that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.

Notes: Abstract: We investigated the effect of insulin on phosphatidylinositol (PtdIns) 3-kinase (PtdIns 3-kinase) activity in neuronal cultures to determine if this enzyme is involved with the neurotrophic actions of insulin. Insulin caused a concentration-dependent increase in PtdIns 3-kinase activity in anti-phosphotyrosine immuno-precipitates. The kinase activity was able to phosphorylate PtdIns, PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate. In intact neurons, a 10-min 1 mM insulin treatment in the presence of [32P]orthophosphate increased the levels of both 3-[32P]PtdIns phosphate and 3,4-[32P]PtdIns bisphosphate by 55 and 193%, respectively. This increase was associated with an increase in neurite outgrowth mediated by insulin. Our results indicate that insulin treatment of neuronal cells in primary culture increases PtdIns 3-kinase activity and the formation of the unique d-3-phosphorylated phosphoinositides, suggesting that growth factor-mediated neuronal growth may include the formation of novel phosphoinositide 3-phosphate phospholipids.

Notes: Abstract: We have shown previously that calcium and guanine nucleotides stimulate the activity of a phosphoinositide (PI) phosphodiesterase in membranes from rat cerebral cortex and that their effects are additive. To understand further guanine nucleotide- and calcium-stimulated PI phosphodiesterase activity, we have investigated the pH sensitivity and effects of inhibitors on the two modes of stimulation. NaF stimulates PI hydrolysis in brain membranes with an EC50 of 2 mM and a maximal effect at 10 mM, suggesting that a guanine nucleotide binding protein can regulate PI phosphodiesterase. Neomycin inhibited guanylylimidodiphosphate (GppNHp)-stimulated PI phosphodiesterase activity in a concentration-dependent manner, with 90% inhibition at 0.3 mM. Neomycin was not as effective at inhibiting calcium-dependent PI hydrolysis (32% inhibition at 0.3 mM). Chloroquine also had a greater inhibitory effect against GppNHp-stimulated PI phospho diesterase activity compared to calcium-dependent activity. Guanine nucleotide- and NaF-dependent activations of PI phosphodiesterase were strongly pH-dependent, with greatest stimulation observed at pH 5–6 and inhibition at more alkaline pH. Calcium-stimulated PI hydrolysis was not as sensitive to changes in pH and had a peak of activity at pH 9. Our findings of different pH optima and differential sensitivity to inhibitors suggest that calcium and guanine nucleotides may regulate PI phosphodiesterase in rat cortical membranes through independent mechanisms. Key Words: G-proteins—Calcium—Phosphoinositides—Membranes—Phosphoinositide phosphodiesterase—Brain. Gonzales R. A. and Crews F. T. Differential regulation of phosphoinositide phosphodiesterase activity in brain membranes by guanine nucleotides and calcium. J. Neurochem. 50, 1522–1528 (1988).

Notes: Previous studies have suggested that protein ki-nase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.

Notes: Abstract: The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0–1.0 mM), basal, potassium-stimulated, and norepinephrine -stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-d-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 μM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis. These results also support previous evidence of two separate and distinct pathways for stimulating phosphoinositide-linked phospholipase C (PLC) activity in brain: One pathway appears to involve a phosphoinositide-associated guanine nucleotide binding protein-PLC coupling process, and the other a direct activation of PLC by an increase in intracellular calcium.

Notes: Abstract: Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by α1-adrenergic receptors. Because α1-adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1-day-old rat brains. The active phorbol ester phorbol- 12-myristate-13-acetate (TPA) caused time- and concentration-dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester-induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.

Notes: Abstract: Carbachol and norepinephrine were used as agonists to compare and contrast cholinergic and adrenergic stimulation of inositide breakdown in rat brain slices. Carbachol acts through a muscarinic (possibly M1) receptor and norepinephrine acts through an α1 adrenoceptor. Studies in cerebral cortical slices indicated that both agonists stimulated the production of inositol-1- phosphate and glycerophosphoinositol. Although the initial rates for the stimulation of inositol phosphate release were similar for the two ligands, the response to norepinephrine continued for 60 min and was larger compared with carbachol which plateaued at 30 min. The presence of carbachol did not affect the ED50 for norepinephrine. Concentrations of carbachol near the ED50 in combination with norepinephrine resulted in an additive response whereas maximal concentrations of carbachol and norepinephrine resulted in a less than additive response in the cortex. This negative interaction was also seen in the hippocampus and hypothalamus but not in the striatum, brainstem, spinal cord, olfactory bulb, or cerebellum. Norepinephrine had a larger response than carbachol in the hippocampus, striatum, and spinal cord, but the reverse was true in the olfactory bulb. Manganese (1 mM) stimulated the incorporation of [3H]inositol into phosphatidylinositol (PtdIns) four- to fivefold but not into polyphosphoinositides. The stimulation by manganese of PtDIns labelling increased the monstimulated release of inositol phosphates but did not affect the stimulated release of inositol phosphates by carbachol or norepineph rine. These data suggest that: (a) there may be a common pool of inositides that can be hydrolyzed by cholinergic and/or adrenergic agonists in certain brain areas; (b) manganese may stimulate the labelling of a pool of PtdIns that does not interact with the receptor-coupled pool of inositides; and (c) there may be differences in the basic mechanisms coupling receptor occupation and inositide breakdown for cholinergic and adrenergic receptors.

Notes: Low-leakage, thin-film planar tunnel junctions made of Y1Ba2Cu3O7−x/ native barrier/Pb were fabricated. The Y1Ba2Cu3O7−x films were prepared by in situ molecular beam epitaxy aided with an activated oxygen source. The as-grown, smooth superconducting perovskite film surface exhibits quasi-particle tunneling characteristics very similar to the etched bulk single-crystal data. The results in agreement are a linear dependence of the normal-state conductance on voltage, a gap-like structure at ∼20 mV, asymmetric modulations up to 50 mV, and a finite zero-bias conductance at low temperature. Junctions of lower resistance show, at temperatures below Tc of Pb, the development of a supercurrent at zero bias and associated hysteretic subgap structure, with a typical IcR ∼0.5 mV. Josephson-like behavior occurred in response to applied magnetic field and microwaves.

Notes: The critical currents in high quality thin films of the high Tc superconductor, YBa2Cu3O7−δ, can be controllably reduced by orders of magnitude using ion irradiation. This reduction in critical current occurs without substantial decrease in Tc or increase in room-temperature resistivity. Using this technique, we have fabricated weak links that exhibit an ac Josephson effect at 77 K.

Notes: Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S-adenosyl-l-methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl-N-monomethylethanolamine was found to have a pH optimum of 7.5, a low Km for 5-adenosyl-l-methionine and a partial requirement for Mg2. Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl-N-monomethylethanolamine to phosphatidyl-N, N-dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent Km for S-adenosyl-l-methionine and no requirement for Mg2. Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.