Douglas G. McCartney

Amyloid beta protein (25–35) stimulation of phospholipases A, C and D activities of LA-N-2 cells

[3H]Myristic acid prelabeled LA‐N‐2 cells were exposed to varying concentrations of amyloid beta protein (25–35), from 20 to 250 μg/ml, and the activation of phospholipases A and D estimated. A progressive increase in phosphatidylethanol formation, a measure of phospholipase D activity, and of free fatty acid release, a measure of phospholipase A activity, was observed over a time‐course of 60 min. [3H]Inositol prelabeled LA‐N‐2 cells were exposed to varying concentrations of AβP, from 20 to 125 μg/ml, and phospholipase C activation was measured. There was an increased release of inositol phosphates in the presence of amyloid beta protein as a function of incubation time. The effects of adrenergic, metabotropic amino acid and bombesin antagonists on the AβP mediated stimulation of phospholipase C activity was investigated. Propranolol, a β adrenergic antagonist, 7‐chlorokynurenic acid, a metabotropic amino acid antagonist, and [Tyr4‐d‐Phe12]bombesin, a bombesin antagonist, blunted the AβP stimulation of phospholipase C activity in [3H]inositol prelabeled L‐AN‐2 cells. This suggests that amyloid beta protein activation of phospholipase C may be receptor mediated. The phospholipase C inhibitor U 71322 prevented the activation of phospholipase C by AβP. However, this activation was not effected by tocopherol, propylgallate, or vitamin C.

Regulation of the choline, ethanolamine and serine base exchange enzyme activities of rat brain microsomes by phosphorylation and dephosphorylation

The base exchange enzyme activities of rat brain microsomes were estimated subsequent to preincubations under conditions for either protein phosphorylation or dephosphorylation. Quantitatively the choline base exchange activity was most affected by these treatments. Exposure of the microsomes to alkaline phosphatase resulted in a decrease of all three base exchange activities. Pretreatment with a cAMP‐dependent protein kinase resulted in increases of all 3 enzyme activities. Conditions favoring protein kinase C phosphorylation resulted in stimulation of the choline base exchange activity.

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36‐fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 mM in the presence of 0.75 mM phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF2, AIF3, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.

Phospholipid metabolism in Alzheimer's disease and in a human cholinergic cell

There is evidence available suggesting that membrane alterations occur in Alzheimers disease including the metabolism of membrane phospholipids. We have quantitated in vitro the phospholipase D activity of homogenates from Alzheimers disease brain tissue. There was a significant increase of this enzyme activity as compared to controls. Amyloid beta protein is the predominant protein of the characteristic senile plaques found in Alzheimers disease. Treatment of LA-N-2 cells, a human cholinergic neuroblastoma clone, with amyloid beta protein results in an activation of phospholipases A, C and D.

Alterations of Selected Enzymes of Phospholipid Metabolism in Alzheimer's Disease Brain Tissue as Compared to Non-Alzheimer's Demented Controls

Previous studies have demonstrated elevated brain levels of phosphomonoesters in early stages of Alzheimers disease and elevations of phosphodiesters later in the disease. In addition, preliminary quantitative analyses of the phospholipids of Alzheimers brain reveals either decreases in some phospholipids or elevations followed by decreases in others. This study quantitated the activities of selected enzymes involved in phospholipid and choline metabolism and demonstrated elevated glycerol-3-phosphorylcholine phosphodiesterase and decreased choline kinase activities in Alzheimers disease brain. The former could provide an enzymatic mechanism for the increased phosphorylcholine found in Alzheimers disease brain.

Acidic phospholipids inhibit the phospholipase D activity of rat brain neuronal nuclei

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 μM for PIP2 to 200 μM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

Sphingosine and unsaturated fatty acids modulate the base exchange enzyme activities of rat brain membranes

The base exchange enzymes catalyze the incorporation of L‐serine, ethanolamine and choline into their corresponding phospholipids. The L‐serine base enzyme activity was increased 120% by 0.1 mM sphingosine. There was a modest increase of the ethanolamine base exchange enzyme activity but the choline base exchange enzyme activity was unaffected. Na‐arachadonate, Na‐oleate and Na‐linolenate at 0.2 mM concentration increased the activity of the L‐serine and ethanolamine base exchange enzymes but inhibited the choline base exchange enzyme activity. A model is proposed suggesting that modulations of the L‐serine base exchange enzyme may participate in the regulation of the calcium phospholipid‐dependent protein kinase C.

Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain.

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P2 fraction. The PL-D activity of P2 and P3 fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P2 fraction and the P3 fraction increased after P14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla+pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla+pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.

Modulation of the Serine Base Exchange Enzyme Activity of Rat Brain Membranes by Amphiphilic Cations and Amphiphilic Anions

Abstract: The biosynthesis of phosphatidylserine in mammalian tissues is catalyzed by the serine base exchange enzyme. The activity of this membrane‐bound enzyme can be manipulated by amphiphiles. Amphiphilic cations, such as oleylamine, W‐7, chlorpromazine, and didodecyldimethylamine, stimulate the serine base exchange activity. Amphiphilic anions, such as bis(2‐ethylhexyl) hydrogen phosphate and cholesterol sulfate, inhibit the serine base exchange activity. These effects are more pronounced at pH 7.0 than at the pH optimum of 8.5 for this enzyme. Both the stimulators and the inhibitors alter the Vmax values without changing the Km value for serine, suggesting that their mechanism of action is related to interactions of the membrane‐bound cosubstrate, phosphatidylethanolamine, with the membrane‐bound enzyme. The optimal concentration of stimulator varies with the amount of membrane protein present; however, supraoptimal concentrations cause inhibitions. It is proposed that the amphiphilic cations enhance the interaction of the phosphorylethanolamine moiety of the membrane‐bound cosubstrate with the enzyme and the amphiphilic anions interfere with such an interaction. Some of the pharmacological properties of these amphiphilic cations, employed clinically as antidepressants, may be mediated by modulation of the serine base exchange enzyme activity.

Phospholipase D activity of isolated rat brain plasma membranes

With [14C]oleate‐labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl2, BaCl2, MgCl2 and SrCl2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphosphatidylation activity. GTPγS, an activator of G protein‐mediated events, was an inhibitor of both activities.