Specific Radioactivity Of Phosphatidylcholine Research Articles

Synthesis of Phosphatidylcholine Through Phosphatidylethanolamine N‐Methylation in Tissues of the Mussel Mytilus galloprovincialis

We previously demonstrated the importance of upregulation of phosphatidylethanolamine N-methylation pathway in euryhaline fish and crustaceans facing hyperosmotic conditions. In marine molluscs phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine has not been described until now. In vivo labeling of the mussel Mytilus galloprovincialis with [1-(3)H]-ethanolamine showed that the digestive gland is the tissue expressing the highest incorporation into lipids. A sustained increase in lipid labeling was observed up to 72h following label injection with 79-92% of radioactivity concentrated into phosphatidylethanolamine and phosphatidylcholine. A direct correlation (r=0.47, p<0.01) between the specific radioactivities of phosphatidylcholine in plasma and the digestive gland was observed. Moreover, the phosphatidylcholine fatty acid compositions of plasma and the digestive gland were similar but differed from those of phosphatidylcholine purified from other tissues. In vitro incubation of tissues with [1-(3)H]-ethanolamine or L-[3-(3)H]-serine showed that a significant labeling of the choline moiety of phosphatidylcholine was observed in the digestive gland and hemocytes. Pulse-chase experiments with [1-(3)H]-ethanolamine also demonstrated that hemocytes are exchanging the newly formed phospholipids with plasma. Finally, phosphatidylethanolamine N-methyltransferase assays demonstrated salinity-dependent activities in the digestive gland and hemocytes. We conclude that in M. galloprovincialis an active phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine occurs in the digestive gland and hemocytes and that this newly formed phosphatidylcholine is partly exchanged with plasma.

Two separate pools of sphingomyelin in BHK cells

In BHK cells labelled to equilibrium with [ 3H]choline and treated with sphingomyelinase the surface pool of sphingomyelin is degraded very rapidly (half-time 10 min) but the internal pool of sphingomyelin which accounts for about 30% of the total is only degraded slowly (half-time about 80 h) showing that the internal pool does not normally reach the surface. In [ 3H]choline incorporation experiments the internal pool begins to accumulate radioactivity at about the same time as phosphatidylcholine (30 min) but label does not enter the surface pool of sphingomyelin for a further 90 min. The internal and external pools reach the same specific activity only after about 20 h. Pulse-chase analysis with [ 3H]choline shows that radioactivity in each pool of sphingomyelin continues to increase when the specific radioactivity of phosphatidylcholine is decreasing, consistent with both pools being synthesised from a phosphatidylcholine precursor. The results suggest that sphingomyelin in BHK cells is present not only in the plasma membrane but also in a more rapidly labelling pool which does not mix with the surface pool.

Studies on the origin of biliary phospholipid. Effect of dehydrocholic acid and cholic acid infusions on hepatic and biliary phospholipids

The correlation between the secretion of biliary phospholipid (PL) and bile acid suggests a regulatory effect of bile acid on PL secretion. Bile acids may influence PL synthesis and/or the mobilization of a preformed PL pool. The objective of this study was to determine the contribution of these two sources to biliary PL, by using an experimental protocol in which dehydrocholic acid (DHCA) and cholic acid (CA) were infused to manipulate biliary PL secretion. In control rats, there was a steady state in bile flow. PL secretion and the biliary secretion of newly synthesized phosphatidylcholine (PC). The specific radioactivity of PC in bile was significantly higher than in plasma, microsomes and canalicular membranes. DHCA infusion decreased biliary PC secretion rate by 80%, and secretion returned to normal values at the transport maximum of CA. The specific radioactivity of biliary PC was decreased by 30% by DHCA infusion and reached normal values during CA infusion. There were no significant changes in the specific radioactivity of PC in plasma or cellular organelles during infusion of bile acids. These data indicate that: (1) newly synthesized PC contributes a small percentage to biliary PC; thus a preformed pool (microsomal and extrahepatic) is a major source of biliary PL; (2) the contribution of the extrahepatic pool to the biliary PL may be more important than the microsomal pool.

Phosphatidylcholine as the choline donor in sphingomyelin synthesis.

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [methyl-14C]choline resulted in rapid accumulation of essentially all the label (approximately 95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 microM choline), loss of label from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 microM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 microM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9-12 hr until it reached the specific radioactivity of PC. In the presence of 300 microM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

The release of radioactive arachidonate from lipids of red blood cells during prolonged incubations in vitro.

Red blood cells were isolated from rat blood and incubated in the presence of [3H]arachidonate. A sizeable quantity (18%) of the radioactivity was incorporated into red cell lipids, of which phosphatidylcholine was the most highly labelled. Radioactive arachidonate was found at position 2 of this phospholipid. Free fatty acids were removed by washing the cells in solutions containing fatty-acid-free bovine serum albumin. The labelled red cells were then incubated for up to 16 h at 37 degrees C. After 16 h of incubation in saline-buffer-glucose or rat serum, 20 and 26%, respectively, of the total radioactivity was found in free fatty acids, and there were corresponding declines in the percentage radioactivities found in phosphatidylcholine. In the presence of serum, there was a more rapid release of radioactive fatty acid over the 2- to 16-h time course. There was not a significant drop in the phosphate levels of the total red cell phospholipids or phosphatidylcholine after 16 h of incubation and, as a result, there were large declines in the specific radioactivities of phosphatidylcholine. Diacylglycerols were not highly labelled and the action of phospholipase A2 on labelled phosphatidylcholine was indicated. When white blood cells were added to labelled red cells, there was little evidence of white cell involvement in the release of radioactive fatty acid, suggesting that the red cells themselves may be involved in arachidonate release. Red cells may serve as sources of arachidonate, released following hemorrhage in brain and metabolized to form various biologically active eicosanoids.

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes.

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.

Active labeling of phosphatidylcholines by [1- 14C]docosahexaenoate in isolated photoreceptor membranes

Isolated bovine rod outer segments and photoreceptor disks actively incorporated [1- 14C]docosahexaenoate (22:6) into phospholipids when incubated in the presence of CoA, ATP, and Mg 2+. About 80% of the esterified fatty acid was in phosphatidylcholine (PC). Microsomal and mitochondrial fractions incorporated as much 22:6 as rod outer segments, but it was distributed among various phospholipids and neutral glycerides. The isolated photoreceptor membrane thus contains an acyl-CoA synthetase which activates the fatty acid and a docosahexaenoyl-CoA-lysophosphatidylcholine acyltransferase activity. The specific radioactivity of PC was higher in rod outer segments than in the other subcellular fractions. About 2 3 of the label in photoreceptor membrane PC was in its dipolyunsaturated molecular species and 1 3 in hexaenes. Dipolyunsaturated PCs showed high turnover rates of 22:6 in all three subcellular membranes, especially in mitochondria. Retinal membranes in vitro seem to take up free [ 14C]22:6 from the medium by simple diffusion or partition into the membrane lipid. The ability of these membranes to activate and esterify [1- 14C]22:6 indicates that docosahexaenoate-containing molecular species of retina lipids, including those of photoreceptor membranes, are subject to acylation-deacylation reactions in situ.

Metabolism of phosphatidylcholine in the frog retina

The biosynthesis and the turnover of phosphatidylcholine were studied in the frog retina following either (a) injection into the animal of 32PO 4, 33PO 4, [1,3- 3H]glycerol, [2- 3H]glycerol, or [ methyl- 3H]choline, or (b) incubation of isolated retinas in solutions containing [ methyl- 3H]choline. 1. 1. Examination of the pools of lipid precursors in the retina demonstrated that the choline and phosphate pools are long-lived compared to the glycerol pool, which is metabolically very active and turns over rapidly. 2. 2. The peak in specific activity of phosphatidylcholine synthesized from labeled glycerol occurred earlier, and was higher in the microsomal fraction than in the rod outer segments, which is consistent with synthesis of phosphatidylcholine on the microsomes of the inner segment and subsequent incorporation into the rod outer segments. 3. 3. Autoradiography of retinas incubated in vitro with tritiated choline revealed a diffuse labeling pattern in the rod outer segments. Biochemical studies following injection of labeled glycerol showed an exponential decline in specific radioactivity of phosphatidylcholine in the rod outer segments, which is consistent with a diffuse labeling of these membranes. 4. 4. The half-life of phosphatidylcholine in the rod outer segments synthesized from labeled glycerol was found to be 18–19 days. Based on these values, calculations were made which indicated that phosphatidylcholine in the outer segments is turning over faster than integral disc membrane proteins.

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo.

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [(3)H]plamitate, [(3)H]oleate or [(3)H]arachidonate and [(14)C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total (3)H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [(14)C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of (3)H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [(14)C]glycerol labelling, generally the (14)C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of (3)H-labelled fatty acid/[(14)C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with (3)H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.

The involvement of lysophosphoglycerides in neuro-transmitter release; The composition and turnover of phospholipids of synaptic vesicles of guinea-pig cerebral cortex and torpedo electric organ and the effect of stimulation

1. (1) Crude synaptosomal fractions (P 2) derived from guinea-pig cerebral cortex were incubated in the presence of 50 m M KCl in a Krebs-glucose medium. Torpedo marmorata electric organs were stimulated electrically in vivo at 5 pulses/sec for 30 min by electrodes placed on the electric lobe. Synaptic vesicles were isolated from each source and the phospholipid compositions analysed and compared with vesicles from unstimulated controls. 2. (2) Lysophosphatidylcholine was the only lysophosphoglyceride demonstrable in the synaptic vesicles from either source and its low levels did not increase as a result of chemical or electrical stimulation. In each case there was a close similarity of the phospholipid distributions in the vesicles taken from control and stimulated samples. 3. (3) Control experiments indicated extensive decreases in the acetylcholine content of the vesicles from the stimulated electric organ and smaller decreases in the acetylcholine content of the synaptic vesicles from stimulated crude synaptosomal fractions. These fractions were found to respire linearly in the presence of 10 m M glucose and the vesicle fractions were shown to have low levels of contaminating membranes as judged by marker enzyme analyses. 4. (4) Crude synaptosomal fractions from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium with labelled fatty acids and [ 3H]glucose in the presence or absence of 50 m M KCl. Subsynaptosomal fractionation was carried out and the specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomes) and H (disrupted synaptosomes). The release of neurotransmitter did not significantly enhance the labelling of phospholipids in any of the fractions studied as compared with phospholipids from unstimulated fractions. This was found after two incubation times and using [ 14C]oleate, [ 14C]arachidonate, [ 3H]palmitate and [ 3H]glucose.

Incorporation of ortho[ 32P] phosphate into phosphatidylcholines and phosphatidylethanolamines in rat skeletal muscle

1. 1. The specific radioactivities of individual molecular species of muscle phosphatidylcholine and phosphatidylethanolamine have been measured by a combination of argentation thin-layer chromatography and countercurrent distribution. 2. 2. The specific radioactivities of individual molecular species of muscle phosphoglycerides have been determined 3 h after intraperitoneal injection of ortho[ 32P] phosphate. Under these conditions the specific radioactivities of the species present in rat muscle were found to be measures of the relative turnover times of these molecules. 3. 3. The specific radioactivity of phosphatidylcholine was approx. three times that of phosphatidylethanolamine. The 1-palmitoyl-2-oleoyl and 1-oleoyl-2-linoleoyl phosphatidylcholines had the fastest turnover and the 1-palmitoyl-2-arachidonoyl the slowest. Of the phosphatidylethanolamines, the linoleoyl and the docosahexaenoyl species showed the fastest turnover and 1-stearoyl-2-arachidonoyl the slowest. 4. 4. The results indicate that phosphoglycerides in muscle turn over more slowly and more evenly than do liver phosphoglycerides.

THE SUBCELLULAR LOCALIZATION OF CARBAMYLCHOLINE‐STIMULATED PHOSPHATIDYL INOSITOL TURNOVER IN RAT CEREBRAL CORTEX IN VIVO

Abstract— The intraventricular injection of 40 μCi of 32Pi (carrier free) into adult rats resulted in maximum incorporation of 32Pi into the phosphatidyl inositol of the whole cortex after 20 h. A further intraventricular injection of 2 nmol carbamylcholine plus 0.02 nmol eserine resulted in a 23% decrease in the specific activity of phosphatidyl inositol after 20 min. The specific radioactivities of phosphatidyl choline, phosphatidyl ethanolarmine and phosphatidyl serine were not changed. Cerebral cortex from rats treated in this way was subjected to an extensive subcellular fractionation. It was found that the specific radioactivity of the phosphatidyl inositol of the synaptic vesicle fraction showed a reduction of 60%. No other fractions showed effects of this magnitude.

Choline Metabolism in Discrete Areas of the Brain: the Effect of Hemicholinium-3

When [Me-14C]choline is injected into the brain of the rat it is rapidly phosphorylated to give phosphorylcholine; subsequently the radioactivity appears in the phosphatidylcholine (Ansell & Spanner, 1968). The incorporation into discrete areas of the brain has now been investigated, together with the effect of injecting the labelled choline at different depths from the surface. The object of this was to study the effect of hemicholinium-3, which interferes with the transport and utilization of choline for acetylcholine synthesis. Adult female rat brains were dissected as described by Glowinski & Iversen (1966), and in Table 1 the specific activities of acetylcholinesterase (EC 3.1.1.7) and choline kinase (EC 2.7.1.32) and the concentrations of total phospholipid, phosphatidylcholine and phosphorylcholine are given. Acetylcholinesterase activity was unevenly distributed (cf. cerebellum and striatum) and paralleled acetylcholine distribution (Schmidt et al., 1972), but choline kinase activity, largely present in areas rich in neurons (McCaman, 1962), was, like the phosphorylcholine, evenly distributed (Table 1). When [Me-14C]choline (1 pCi; 0.016pmol) was injected into the region of the lateral ventricle there was an uneven distribution of radioactivity between the six regions of the brain. A distribution of the same order was found for [3H]noradrenaline by Carr & Moore (1969). Of the radioactivity recovered in the brain, 67 % was distributed between the hypothalamus and the midbrain, but there was less than 0.3 % in the striatum. It was therefore decided to vary the depth of injection to test whether this uneven distribution was merely a reflection of the site of injection or was, in fact, a preferential uptake. The choline was injected at the same point of entry as before but the distance from the surface varied from 1.5 to 4mm. After an exchange time of 0.5h the total radioactivity in the brain was slightly higher after the shallower injections than after those at 4mm. The distribution of radioactivity among the regions was very different from that found after the injections at 4mm. For example, after the injection at 1.5mm the percentage of the recovered activity in the hypothalamus was only 8 % compared with 29 % after that at 4mm. However, the cortex contained 35 % compared with 14% after the injection at 4mm. It is noteworthy that the percentage distribution in the midbrain was almost unaffected by the depth of injection. It had been found previously (Ansell & Spanner, 1968) that there was a very rapid phosphorylation of choline after intraventricular injection. This was borne out when the individual regions were examined, each region showing a high percentage of labelling in the phosphorylcholine after 0.5-4.0h of exchange. However, when the labelled choline was injected into the brain tissue above the ventricle there was a very considerable depression of the phosphorylation, i.e. to an average of 15 % after the injection at 1 Smm and of 19.5 % after the injection at 3.0mm compared with over 75 % after that at 4mm. The distribution of choline kinase activity when measured in vitro showed little variation from region to region (Table l), so that the reason for this discrepency has yet to be explained. The ratio of the specific radioactivity of phosphatidylcholine to that of phosphorylcholine in vivo was unaffected. The transfer of the labelled choline and its metabolites from the injected to the uninjected side of the brain was also examined after different depths of injection. The steep gradient found by Carr & Moore (1969) with [3H]noradrenaline and by Fibiger et al. (1972) with ~-[U-'~C]leucine was not so marked with [Me-14C]choline. The side of injection contained 70 % of the isotopic label compared with 30 % in the uninjected side after 1-4h of exchange. This ratio was unaffected by the depth of injection. The cortex, striatum and midbrain showed the highest gradient. The medulla and hypothalamus contained the same percentage of radioactivity on both sides, whereas the cerebellum

Effects of denervation on the incorporation of 32P into phosphatidyl inositol and other phospholipids of rat diaphragm.

Abstract— At 24 h after denervation of the rat hemidiaphragm, incorporation of 32P into phosphatidyl inositol was depressed relative to incorporation of 32P into phosphatidyl choline (measured 75 min after injection of the isotope intraperitoneally). The ratio of the specific radioactivity of phosphatidyl choline to the specific radioactivity of Pi was unaffected by denervation which implies that denervation had depressed incorporation of isotope into phospatidyl inositol. Denervation did not cause a measurable change in the pool size of phosphatidyl inositol relative to that of phosphatidyl choline. The effect of denervation on incorporation of 32P into phosphatidyl inositol was not entirely a direct consequence of the cessation of ACh release at the motor end‐plate since the effect was clearly manifest in strips of muscle not containing motor end‐plates, but the magnitude of the denervation effect was slightly greater in the strips of denervated hemidiaphragm which contained motor end‐plates.

The effect of hemicholinium-3 on the in vivo formation of cerebral phosphatidylcholine

The effect of hemicholinium-3 on the incorporation of [ Me- 18C]choline into the phospholipids of the subcellular fractions of the canine caudate nucleus was studied. A correlation between the amount of hemicholinium-3 administered and increased choline incorporation into phospholipids was obtained. The analysis of the labeled phospholipids revealed that 93% of the radioactivity was in phosphatidylcholine and the remainder in lysophosphatidyl choline and sphingomyelin + choline plasmalogens. The specific radioactivity of phosphatidylcholine (counts/min per μg phosphorus) determined after its isolation by thin-layer chromatography, increased markedly as a result of the administration of hemicholinium. An unequal time-dependence in the labeling of the phosphatidylcholine in the subcellular fraction containing nerve endings, mitochondria and lysosomes, as opposed to the phosphatidylcholine of the microsomal fraction was also noted, particularly after the administration of hemicholinium and choline in a weight ratio of 500. Discontinuous density gradient centrifugation of the fraction containing the nerve endings, the mitochondria and the lysosomes showed that phosphatidylcholine formation was most affected by hemicholinium in the fraction containing the “heavy” nerve endings, followed by the fraction containing the mitochondria + lysosomes. It is suggested that hemicholinium acts by accelerating the intracellular rate of movement of chorine from the water-soluble into the lipid pool of the caudate nucleus where, by virtue of its presence 19, it subsequently also stimulates the incorporation of choline into membrane phosphatidylcholine.