THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

The formation and continuous turnover of a fraction of phosphatidic acid on stimulation of NaC1 secretion by acetylcholine in the salt gland

Acetylcholine, which stimulates NaCl secretion in the avian salt gland, causes the rapid formation of a fraction of phosphatidic acid, as measured by ³²P incorporation, which amounts maximally to about 0.18 µmoles per g of fresh tissue. This does not appear to involve synthesis of the diglyceride moiety of phosphatidic acid, as measured by glycerol-1-¹⁴C incorporation. It presumably involves formation of phosphatidic acid by the diglyceride kinase pathway from preformed diglyceride and ATP. The specific activity of the AT³²P of the tissue is not increased in the presence of acetylcholine. At time intervals after addition of acetylcholine during which a full response, measured as increased O₂ uptake, may be observed, phosphatidic acid appears to be the only phosphatide which shows any increase either in total ³²P radioactivity or in net specific acitvity. This responsive fraction of phosphatidic acid undergoes continuous turnover of its phosphate moiety. There is no evidence that this turnover is due to the phosphatidic acid acting as a pool of intermediate for the synthesis of other phospholipids or glycerides. The responsive fraction amounts to not more than 20% of the total phosphatidic acid of the tissue; it does not mix with the other (non-responsive) phosphatidic acid of the tissue. The observations suggest that this phosphatidic acid plays some role in the over-all secretory process.     

THE EFFECT OF N-METHYL-4-PIPERIDYL-DIPHENYL GLYCOLATE ON THE INCORPORATION OF 32P INTO PHOSPHOLIPIDS FROM RAT BRAIN CORTEX SLICES AND ITS SUBCELLULAR LOCALIZATION

—N-Methy-4-piperidy1-diphenyl glycolate (N-methy1-4-piperidy1 benzilate), an anticholinergic drug, was shown to stimulate ³²P-incorporation into total phospholipids of rat brain cortex slices. Analysis of the total phospholipids showed stimulation in phosphatidic acid, phosphatidylinositol and phosphatidylethanolamine. Stimulated ³²P-incorpora-tion was accompanied by a decrease in the Qo₂ (μ1 O₂/mg dry tissue) value. The effects of the drug were compared with those of some other CNS-active drugs known to interfere with the ACh content of brain; tremorine, oxotremorine and atropine; and in the presence of eserine, with that of the neurotransmitter acetylcholine. Increase of the outer K+-concentration resulted in increased Qo₂ and ³²P-incorporation into the slices. The effect of the glycolate and perhaps that of atropine tended to increase in this medium. Subcellular fractionation of slices showed that the glycolate stimulated ³²P-incorporation occurred mainly in the nerve end fraction. The total amount of the individual phospholipids was not changed, but the specific activity had significantly increased in phosphatidic acid and phosphatidylinositol. The possibility that glycolate-induced stimulated ³²P-incorporation into nerve end phospholipids is due to increased glycolysis is discussed.     

EFFECTS OF ACETYLCHOLINE ON THE INCORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF NERVE-ENDING PARTICLES FROM GUINEA PIG BRAIN

Abstract— Guinea pig brain nerve-ending particles (synaptosomes) were incubated with [³²P]orthophosphate in a medium with or without 10⁻⁴M-acetylcholine and 10⁻⁴ M-eserine. Phospholipids were then extracted and separated by chromatography. About 60 per cent of the ³²P was found in phosphatidic acid and about 20 per cent in triphosphoinositide. Acetylcholine significantly increased the specific radioactivity of phosphatidic acid but had no effect on that of phosphatidylinositol or the nucleotide fraction. Labelling of the other phospholipids, including diphosphoinositide and triphosphoinositide, was not altered significantly by acetylcholine. Labelling of the nucleotide fraction and the polyphosphoinositides reached a peak at 40 min, that of phosphatidic acid at 80 min, while that of phosphatidylinositol was still rising at 160 min.     

THE LABELLING OF NERVE ENDING PHOSPHOLIPIDS IN GUINEA-PIG BRAIN IN VIVO AND THE EFFECT OF ELECTRICAL STIMULATION ON PHOSPHATIDYLINOSITOL METABOLISM IN PRELABELLED SYNAPTOSOMES

The intracerebral injection of ³²P_i into guinea-pig cortex resulted in a steady rate of incorporation into all phospholipids over a 20 h period. The specific radioactivities of phosphatidate and phos-phatidylinositol in synaptosomes prepared from cortex prelabelled, in vivo, were at a maximum after 2 h and the respective activities were 3–8 times higher than in whole cortex. This peak in labelling corresponded with the maximum specific activity of the brain ATP. No similar differential labelling pattern was observed for phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine. Electrical stimulation of the prelabelled synaptosomes produced a rapid drop in the specific activity of phosphatidylinositol and phosphatidate and an increase in the specific activity of CDP-diacylglycerol. The specific activity of synaptosomal ATP was not affected. Study of the subsynaptosomal fractions obtained after osmotic rupture of the synaptosomes revealed that the most highly labelled phosphatidylinositol was in the synaptic vesicle fraction (D) and the most active phosphatidate was in a ‘microsomal’ fraction (E). Electrical stimulation caused a loss of phosphatidylinositol radioactivity from fraction D and a loss of phosphatidate radioactivity from fraction E. The specific activity of these lipids in other fractions was not affected. A possible role for presynaptic phosphatidylinositol is suggested.     

SYNTHESIS OF ACETYLCHOLINE IN DIFFERENT COMPARTMENTS OF BRAIN NERVE TERMINALS IN VIVO AS STUDIED BY THE INCORPORATION OF CHOLINE FROM PLASMA AND THE EFFECT OF PENTOBARBITAL ON THIS PROCESS

The time course of the incorporation of choline from plasma into a high and a low molecular weight fraction from mouse brain synaptosomes was studied. The fractions were obtained from lysed synaptosomes by gel filtration on Sephadex G-25. An extremely rapid incorporation of radioactivity into acetylcholine was found in both fractions and in the time interval 0.25-9 min after the intravenous administration of labelled choline, higher specific radioactivities of acetylcholine were found in the high molecular weight fraction than in the low molecular weight fraction. However, the specific radioactivity of choline in the high molecular weight fraction was much lower than that of acetylcholine. It was found that barbiturate anaesthesia caused a marked decrease in the labelling of acetylcholine in the high molecular weight fraction while the incorporation into the low molecular weight fraction was affected to a much smaller extent. Acetylcholine of the high molecular weight fraction showed properties similar to those of vesicle-bound acetylcholine. The recoveries of labelled and endogenous acetylcholine and choline from the brain homogenates were calculated in different steps of the fractionation procedure. In the fraction containing lysed synaptosomes the recovery of radioactive acetylcholine was lower than that of endogenous acetylcholine. This may indicate the presence of two types of bound acetylcholine in the synaptosomes. Different models for the intraneuronal synthesis of acetylcholine are discussed and it is proposed that a site of acetylcholine synthesis in vivo may be closely associated with some constituent of the high molecular weight fraction and directly coupled with the storage of the transmitter.     

ASPECTS OF ACETYLCHOLINE METABOLISM IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA

Abstract— —The synthesis of acetylcholine and its compartmentation were studied in the electric organ of Torpedo marmorata. When electric organ was homogenized in iso-osmotic NaCl-sucrose some 55 per cent of its acetylcholine content was lost unless very potent cholinesterase inhibitors were present. Slices of electric organ incubated in a suitable medium were found to synthesize radioactive-labelled acetylcholine from [ N-Me-³ H] choline. The specific activity of the labelled acetylcholine was higher in the trichloracetic acid extract of the organ slices than in an NaCl-sucrose homogenate. Acetylcholine-containing vesicles isolated from the NaCl-sucrose homogenate contained labelled acetylcholine with about the same specific activity as the parent homogenate. There was thus a fraction of acetylcholine in the incubated tissue of higher specific radioactivity that was lost when the tissue was homogenized. The acetylcholine-containing vesicles lose their acetylcholine when submitted to gel filtration under hypo-osmotic conditions. On standing at 5°C there were only small losses of acetylcholine from the vesicles but at 20°C the losses were substantial. Vesicles containing labelled acetylcholine were studied. On gel filtration under iso-osmotic conditions there was a considerable loss of labelled acetylcholine without a concomitant loss of bio-assayable acetylcholine. The pools of radioactive and bio-assayable acetylcholine are therefore not homogeneous in the vesicles as isolated.     

NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium-3 (HC-3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [³²P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [^l4C]HC-3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC-3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.     

The metabolism of phospholipids in mouse brain slices

1. Slices of mouse brain grey matter were incubated with [³²P]phosphate and [1-¹⁴C]acetate. Doubly labelled phospholipids were extracted from subcellular fractions prepared from the slices in a mixture of metabolic inhibitors, under conditions where there was negligible change in radioactive labelling during the preparation. Two tissue fractions were studied in detail; one contained a high proportion of mitochondria and the other was mainly microsomal. 2. In all tissue fractions the highest incorporations of both [³²P]phosphate and [1-¹⁴C]acetate occurred into phosphatidylcholine. 3. After incubation for 1hr., the ³²P/¹⁴C ratios for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid in the mitochondrial fraction were similar to those in the microsomal fraction. 4. The ³²P/¹⁴C ratios were similar in phosphatidylcholine and phosphatidylethanolamine and much lower than those in phosphatidic acid and phosphatidylinositol.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

THE ORIGIN OF THE ACETYLCHOLINE RELEASED FROM THE SURFACE OF THE CORTEX

—The specific radioactivity of acetylcholine liberated from the surface of the rabbit occipital cortex has been compared with that of the underlying cortical synaptosomal and vesicular acetylcholine at varying times after the administration of [N-Me-³H]choline. Choline was administered by diffusion from solutions placed in cups formed by Perspex cylinders applied to the surface of the cortex. Acetylcholine was collected by diffusion into these cups. The specific radioactivity of the acetylcholine declined progressively. The effect of stimulation of afferent cholinergic pathways was to cause a fall in the specific radioactivity of the released acetylcholine. However this was always higher than that of the synaptosomal or vesicular acetylcholine as represented by fractions P₂ and D of the authors’fractionation scheme. It is concluded that acetylcholine released from the cortex must come from a store or stores more recently synthesized than the endogenous acetylcholine of these subcellular fractions.     

THE METABOLIC TURNOVER OF MOLECULAR SPECIES OF PHOSPHATIDYLINOSITOL and ITS PRECURSOR PHOSPHATIDIC ACID IN GUINEA-PIG CEREBRAL HEMISPHERES

Abstract– The molecular species composition of phosphatidylinositol from guinea-pig cerebral hemispheres was studied and found similar to that of phosphatidylinositol from ox cerebral hemispheres. In both cases the tetraenoic species was predominant. Phosphatidic acid from guinea-pig cerebral hemispheres contained two major molecular species; the monoenoic and hexaenoic (33.4 and 24 mol/100 mol respectively). In order to study the metabolism of molecular species of phosphatidic acid and phosphatidylinositol in the cerebral hemispheres, guinea-pigs were injected intracisternally with ³²P_i and [U-¹⁴C]glucose. After 5 min of isotopic exchange, the specific radioactivity of ³²P in phosphatidylinositol was nearly equal to that in phosphatidic acid, whereas specific radioactivity of ¹⁴C in the glycerol was 1.4 times and in the fatty acids nearly 0.5 times that in the phosphatidic acid respectively, indicating metabolic heterogeneity of both phospholipids. The glycerol specific radioactivity was different in all the molecular species of phosphatidic acid being greatest in the monoenoic and least in the tetranenoic species. When the molecular species were arranged in this way, the order was representative of their relative rates of synthesis by acylation of glycerol-3-phosphate. An almost opposite order was obtained when the molecular species were arranged according to their phosphate/glycerol radioactivity ratios, indicating the relative contribution of the diacylglycerol kinase pathway to their formation. When the specific radioactivity values and ratios of phosphatidylinositol were similarly considered, the orders of the molecular species were, on the whole, similar to that of phosphatidic acid. This indicated that synthesis de novo (Paulus & Kennedy , 1960) was operative in the formation of most of its molecular species, but due to other considerations it was concluded that part of the tetraenoic, and probably the whole of saturated phosphatidylinositol may be formed by transacylation reactions. The results are discussed in terms of the experimental limitations of previous and present techniques for the analysis of phospholipid molecular species.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

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.     

ON THE TURNOVER OF ACETYLCHOLINE IN NERVE ENDINGS OF MOUSE BRAIN IN VIVO

Abstract— Acetylcholine is synthesized and stored in the nerve endings from which the liberation of the nerve transmittor is regulated by the nerve activity. The aim of the present investigation was to measure the in vivo turnover of acetylcholine in this subcellular acetylcholine pool. This has been carried out by injecting labelled choline intravenously and then by measuring at different time intervals the ratio between labelled choline and acetylcholine in the fractions obtained after subcellular fractionation. It was found that the ratio radioactive choline to radioactive acetylcholine was the same (2:1) in whole brain and in the nerve ending fraction 2 to 20 min after injection. Since it was assumed that the same ratio is true also for the endogenous compounds the choline pool in the nerve terminals was considered to make up 13 nmoles/g brain. The results also indicate that plasma choline is rapidly equilibrated with the nerve terminals and transformed to acetylcholine at a rate of about 5 nmoles/g brain/min.     

Effects of norepinephrine and acetylcholine on 32P incorporation into phospholipids of the rabbit iris muscle following unilateral superior cervical ganglionectomy.

The effect of norepinephrine and acetylcholine on the ³²P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of ³²P into the phospholipids of the excised iris muscle. $\text{In vivo}$ thr iris muscle incorporated ³²P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while $\text{in vitro}$ phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when ³²Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the ³²P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated ³²P incorporation into phosphatidic acid appears to be post-synaptic.     

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.     

Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation

Abstract : The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P₃ fraction, but cytoplasmic ACh contained in the S₃ fraction was not significantly affected. The P₃ fraction is known to be heterogeneous ; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K⁺-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [³H]ACh from [³H]choline, the ratio of [³H]ACh in the S₃ to P₃ fractions was not much changed by exposure of tissue to calyculin A or okadaic acid ; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.