Jack P. Smith

Subcellular distribution of sodium-potassium adenosine triphosphatase, acetylcholine and acetylcholinesterase in developing rat brain.

Summary The normal pattern of appearance of acetylcholinesterase, acetylcholine and sodium-potassium adenosine triphosphatase has been determined in the synaptosomal and microsomal fractions during the development of rat brain. Changes in the concentrations of acetylcholinesterase and acetylcholine were found to be gradual with development whereas that of sodium-potassium adenosine triphosphatase increased dramatically around birth and levelled off during the first 2 weeks after birth. The pattern of appearance of both enzyme systems was different in both subcellular fractions and further studies on the effect of activators and inhibitors on the two enzymes showed no relationship between their activities. The possible interrelations between the synaptosomes and microsomes were discussed.

In vivo incorporation of choline, glycerol and orthophosphate into lecithin and other phospholipids of subcellular fractions of rat cerebrum

1. 1. The turnover of lecithin of the membranes of synaptosomes, microsomes and 15-K fraction, isolated from rat cerebra during the stage of active myelination, has been measured by injecting intracranially [14C]choline, [14C]glycerol or [32P]orthophosphate over a period ranging from 30 min to 14 days and the average biological half-lives of lecithin from all the subcellular fractions studied for the three labeled precursors was found to be 11.7 days, 2.9 days and 18.6 days, respectively. 2. 2. The half-life of lecithin from microsomes was slightly lower than that from the other two subfractions. 3. 3. These results suggest that the metabolism of phospholipids in the nervous system is a heterogeneous process. A number of explanations for these differences in half-lives of lecithin were offered. 4. 4. All subfractions studied were found to incorporate the three labeled precursors into their respective major phospholipids. 5. 5. The rate of incorporation of the radioactive precursors into lecithin of the various subcellular fractions was in the following order : microsomes > 15-K > synaptosomes. 6. 6. These data suggest that at least part of the phospholipids can be synthesized at the synapse and this synthesis is probably independent of the cell body of the neuron.

Effects of dl-propranolol on the synthesis of glycerolipids by rabbit iris muscle

Abstract Propranolol (0.03−0.3 mM), an amphiphilic cationic drug which is used therapeutically as a β-blocker, was found to alter significantly the incorporation of [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate, 32 Pi, [ 3 H]cytidine, [ 3 H]inositol, [ 14 C]choline, [ 14 C]ethanolamine and [ 14 C]serine into phospholipids of the iris muscle. Furthermore, it was found to exert a stimulatory effect on the [ 14 C]serine incorporation into phosphatidylserine of the muscle and microsomes. In contrast, sotalol, another β-blocker-but lacking the hydrophobicity of propranolol-exerted no effect on lipid metabolism. Whereas norepinephrine stimulated only the turnover of the phosphate moiety of phosphatidic acid and phosphatidylinositol, in general propranolol caused the following changes: (a) it stimulated by 2- to 6-fold the labelling of phosphatidic acid and phosphatidylinositol from [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate, 32 Pi and [ 3 H]inositol, (b) it increased by 5- and 38-fold the incorporation of 32 Pi and [ 3 H]cytidine, respectively into CDP-diglyceride, (c) it inhibited appreciably the incorporation of [ 14 C]glucose, [ 14 C]glycerol, [ 14 C]acetate and 32 Pi into phosphatidylcholine and phosphatidylethanoalmine. However, while it inhibited significantly the [ 14 C]choline incorporation into the former, it stimulated by 60 per cent the ethanolamine incorporation into the latter phospholipid. These results indicate that propranolol probably redirects phospholipid synthesis de novo , by inhibiting phosphatidate phosphohydrolase, such that the increase obtained in the biosynthesis of phosphatidylinositol is accompanied by a corresponding decrease in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Propranolol also caused a 250 per cent increase in the [ 14 C]serine incorporation into phosphatidylserine of the iris muscle and 28 per cent increase in that of microsomes. The drug appears to stimulate the Ca 2+ -uptake by muscle and microsomes, which in turn could act to stimulate the Ca 2+ -catalyzed base-exchange reaction. In addition the metabolic pathways involved in the biosynthesis of the major phospholipids of the iris, a smooth muscle, are reported for the first time. These pathways were found to be essentially similar to those reported for other tissues.

Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle.

Some properties of the soluble phosphatidylinositol phosphodiesterase (monophosphatidylinositol inositolphosphohydrolase, EC 3.1.4.10) of rabbit iris smooth muscle are described. Studies on its subcellular distribution showed that in this tissue the phosphodiesterase is not exclusively cytosolic. Thus, under our experimental conditions about 58% of the enzyme activity was found in the soluble fraction and the remainder was particulate. When the latter was treated with deoxycholate about 59% of the enzyme activity, compared to 86% of that of ATPase, was still bound to the particulate fraction. The kinetic properties of the enzyme (30--50% (NH4)2SO4 fraction) were examined. Maximum breakdown was 7.7 mumol/h per mg protein and occurred at pH 5.6. The products of [14C]arachidonic acid-labelled phosphatidylinositol were 1,2-diacylglycerol and a mixture of 86% myoinositol 1-phosphate and 14% myoinositol 1,2-(cyclic)phosphate. The enzyme has an absolute requirement for Ca2+. Addition of Ba2+, La3+, Mg2+, Mn2+, EGTA or EDTA at 0.05--5 mM concentrations; Sr2+ at higher concentrations (greater than 0.25 mM) markedly inhibited the phosphodiesterase activity and this inhibition was completely reversed by Ca2+. The enzyme is specific for the phosphoinositides.

Studies on the incorporation of [l-14C]arachidonic acid into glycerolipids and its conversion into prostaglandins by rabbit iris: Effects of anti-inflammatory drugs and phospholipase A2 inhibitors

The effects of the anti-inflammatory drugs, indomethacin and aspirin, and the phospholipase A2 inhibitors, p-bromophenacyl bromide and mepacrine, on the in vitro metabolism of [1-14C]arachidonic acid by rabbit iris smooth muscle and iris microsomes were investigated. The incorporation of arachidonate into glycerolipids and its conversion into prostaglandins were rapid and time-dependent. About 65% of the total radioactivity was recovered in triacylglycerol, followed by that in phosphatidylcholine (20%), diacylglycerol (6%), phosphatidylethanolamine (5%) and phosphatidylinositol (3%), respectively. Time-course studies on arachidonate release from glycerolipids of prelabelled tissue showed that triacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major source for arachidonate in prostaglandin synthesis in this tissue. Arachidonate release from glycerolipids was not blocked by indomethacin and the effects of the phospholipase A2 inhibitors were nonspecific. p-Bromophenacyl bromide inhibited the labelling of glycerolipids in a dose-dependent manner. Mepacrine stimulated the labelling of phosphatidic acid, phosphatidylinositol and diacylglycerol, and inhibited that of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol. At concentrations under 0.25 mM it stimulated prostaglandin synthesis in microsomes and at concentrations over 0.25 mM it inhibited their synthesis in both muscle and microsomes. Indomethacin and aspirin moderately increased the labelling of glycerolipids; however, both drugs inhibited prostaglandin synthesis by iris and iris microsomes in a dose-dependent manner. Possible explanations for mechanisms underlying these effects were presented. It is concluded that the phospholipase A2 inhibitors and the anti-inflammatory drugs exert profound effects on the incorporation of [1-14C]arachidonate into glycerolipids of the rabbit iris and on its conversion into prostaglandins by both iris and iris microsomes.

Studies on choline transport and metabolism in rat brain synaptosomes

Abstract Choline uptake into isolated synaptosomes from rat brain cerebra was investigated by incubating the particles in an iso-osmotic medium containing 14 C-choline and isolating them from the incubation mixture by means of ion-exchange chromatography. Analysis of their radioactive contents by means of high voltage electrophoresis at pH 2.0 showed the per cent distribution of radioactivity for 14 C-choline, 14 C-acetyl-choline, 14 C-phosphorylcholine and 14 C-betaine to be 61,19.8, 14.8 and 4.4 respectively. Incorporation of 14 C-choline into phosphatidylcholine was also studied under the same conditions of incubation and appears to take place at the surface of the synaptosomal membrane through a Ca 2+ -activated base exchange reaction. Intact, but not lysed or sonicated, synaptosomes took up 14 C-choline rapidly. While the choline transport system became saturated at 0.4 mM of 14 C-choline in the external medium, the enzymes involved in its conversion into phosphorylcholine, acetylcholine and betaine leveled off at substrate concentrations lower than 0.2 mM. This uptake is temperature dependent; it is not stimulated appreciably by mono- or divalent ions; it is not influenced by a source of energy or inhibited by 2,4-dinitrophenol (DNP), iodoacetic acid (IAA), KCN or IAA plus KCN, but it is markedly inhibited by p -chloromercuribenzoate (PCMB) or hemicholinium-3. DNP, IAA and KCN inhibited the total uptake of radio-activity into synaptosomes by more than 30 per cent. These metabolic inhibitors were found to exert no effect on the choline pool in the synaptosomes, however, they depressed considerably the radioactivity in the phosphorylcholine and acetylcholine pools. It was concluded that these inhibitors inhibit the uptake of total radioactivity into synaptosomes by inhibiting the formation of labeled phosphorylcholine and acetylcholine, thus preventing the flow of 14 C-choline from the choline pool to the phosphorylcholine and acetylcholine pools. Studies on the time dependence of choline extrusion from 14 C-choline preloaded synaptosomes into a choline-free medium gave half-time values, the time required for loss of 50 per cent of intracellular choline, ranging from 8 to 10 min.

Metabolism of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes from rat brain.

Abstract— Synthesis of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes isolated from rat brain was investigated and compared with the synthesis of these compounds in microsomes and mitochondria. Electron microscopic and marker enzyme studies showed the contaminants in the synaptosomal preparation to consist of a few microsomes and almost no free mitochondria. In synaptosomes, addition of 1,2‐diglyceride exerted no effect on the incorporation of [14C]choline into phosphatidylcholine or on the incorporation of [3H]myo‐inositol into phosphatidylinositol, but it stimulated the incorporation of CDP[1,2‐14C]choline into phosphatidylcholine by more than 50 per cent. The incorporation of the latter in intact synaptosomes, lysed synaptosomes and purified mitochondria was 15‐6, 27 and 9‐9 per cent, respectively, of that in the microsomes. The incorporation of [3H]myo‐inositol into the phosphatidylinositol of synaptosomes and purified mitochondria was 15‐8 and 11‐1 per cent, respectively, of that in the microsomes. Maximal incorporation of [3H]myo‐inositol occurred at pH 7–5 in a medium containing Mg2+ and CTP; it was linear with time and protein concentration and was inhibited by 1 mM Ca2 + but unaffected by the presence of ATP. This incorporation of myo‐inositol appeared to occur through the reversal of the CDP‐diglyceride: inositol transferase reaction. The demonstration of carnitine palmityl transferase in synaptosomes indicated that, as in mitochondrial and erythrocyte membranes, fatty acids can be transported across the synaptosomal membrane. In contrast to mitochondria where maximal incorporation of [14C]carnitine into palmityl carnitine was observed after 20 min of incubation, the incorporation in synaptosomes increased as a function of time up to 60 min of incubation.

ADENOSINETRIPHOSPHATASE AND NUCLEOTIDE METABOLISM IN SYNAPTOSOMES OF RAT BRAIN

—In the presence of synaptosomes prepared from rat brain, only ATP, dATP and ADP but not dADP were active as substrates of phosphatase (ATP phosphohydrolase; EC 3.6.1 4) in the presence of 150mm‐Na+ and 20mm‐K+. An active adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3.) was demonstrated in the synaptosomal fractions by means of paper chromatography, paper electrophoresis and enzymic reactions, so that the high activity with ADP as substrate could represent an activity of an ATPase. Apparently dADP was not a substrate for the kinase; no dATP was formed when dADP was incubated with the synaptosomal fraction in the presence of Na+, K+ and Mg2+. Small amounts of P1 were liberated with dADP, IDP, GDP or CDP, but not UDP, as substrates, but none was produced in the presence of mononucleotides. The adenine‐deoxyribose bond, but not the adenine‐ribose bond, was hydrolysed upon the addition of 5% (w/v) TCA to the reaction mixture.

Studies on the mechanism of alteration by propranolol and mepacrine of the metabolism of phosphoinositides and other glycerolipids in the rabbit iris muscle

We have investigated the effects and mechanism of action of propranolol and mepacrine, two drugs with local anesthetic-like properties, on phospholipid metabolism in rabbit iris and iris microsomal and soluble fractions. In the iris, propranolol, like mepacrine [A. A. Abdel-Latif and J. P. Smith, Biochim, biophys. Acta 711, 478 (1982)], stimulated the incorporation of [14C]arachidonic acid ( [14C]AA) into phosphatidic acid (PA), CDP-diacylglycerol (CDP-DG), phosphatidylinositol (PI), the polyphosphoinositides (poly PI) and DG, and it inhibited that of phosphatidylcholine (PC), phosphatidylethanolamine (PE), triacylglycerol (TG) and the prostaglandins. Similarly, mepacrine, like propranolol [A. A. Abdel-Latif and J. P. Smith, Biochem. Pharmac. 25, 1697 (1976)], altered the incorporation of [14C]oleic acid, [3H]glycerol, 32Pi and [14C]choline into glycerolipids of the iris. Time-course studies in iris muscle prelabeled with [14C]AA showed an initial decrease in the production of DG and a corresponding increase in that of PA by the drugs, followed by an increase in accumulation of DG at longer time intervals (60-90 min). The above findings are in accord with the hypothesis that these drugs redirect glycerolipid synthesis by inhibiting PA phosphohydrolase. Propranolol and mepacrine stimulated the activities of DG kinase and phosphoinositide kinases and inhibited that of DG cholinephosphotransferase. The drugs had little effect on the activity of DG acyltransferase. It is concluded that propranolol and mepacrine redirect glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in phospholipid biosynthesis. We suggest that these drugs could exert their local anesthetic-like effects by effecting an increase in the synthesis of the acidic phospholipids (PA, PI and the poly PI) and subsequently the binding of Ca2+- to the cell plasma membrane.

Distribution of arachidonic acid and other fatty acids in glycerolipids of the rabbit iris

The phospholipid composition, inclusive of higher inositides, fatty acid composition, and in vivo and in vitro incorporation of [14C]arachidonate into the rabbit iris glycerolipids were determined. (1) The total phospholipid phosphorus was found to range between 13–14 μmol/g of iris. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin constituted about 85% of the total lipid phosphorus. Ethanolamine plasmalogen, choline plasmalogen and inositol-containing phospholipids constituted about 11,4 and 7% of total lipid phosphorus respectively. (2) Analysis of the fatty acids of the various glycerolipids of the iris revealed the major saturates to be 16 : 0 and 18 : 0 and the major unsaturates to be 18 : 1, 18 : 2 and 20 : 4. Ethanolamine-containing phospholipids contained the highest percentage of 20 : 4 followed by choline-containing phospholipids, diacylglycerol and phosphatidylinositol respectively. Sphingomyelin contained only trace amounts of 20 : 4. (3) It was found that the iris can incorporate, both in vivo and in vitro, [14C]arachidonate into its own glycerolipids. In general glycerolipids isolated from the in vitro experiment contained significantly higher radioactivities than those obtained from the in vivo experiment. In vitro about 41% of the [14C]radioactivity was recovered in the neutral lipids and phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contained only 10,2 and 2·8% of the total radioactivity respectively; and in vivo about 25% of the total radioactivity was found in each of phosphatidylcholine and the neutral lipids and 8·4% in phosphatidylethanolamine. (4) No intraocular signs of an inflammatory response were observed 0·5, 1 and 7 hr after intracameral injection of [14C]arachidonic acid (2 μg) -bound albumin (0·1 mg).