UPTAKE OF ACETYLCHOLINE AND CHOLINE INTO RAT BRAIN CORTICAL SLICES AND SYNAPTOSOMES AS RELATED TO 32Pi, INCORPORATION INTO THEIR PHOSPHOLIPIDS

—The role of ACh-stimulated ³²P_i incorporation into the phospholipids of rat cerebral cortex slices and isolated nerve endings (synaptosomes) has been studied. ACh stimulation is not connected with any carrier-mediated uptake of ACh. Such uptake may occur in slices in the presence of the anticholinesterase Sarin but barely in the presence of eserine. Regardless of the nature of the anticholinesterase used, rat cerebral cortex synaptosomes that respire and show high and low affinity choline uptake do not accumulate ACh against a concentration gradient. At exogenous ACh concentrations of 10^–5m and above, some ACh enters the synaptosomes by diffusion and significantly stimulates ³²P_i incorporation into phosphatidic acid. It is discussed whether, in isolated nerve endings, an increase in cytoplasmic ACh concentration due to diffusion may induce vesicle turnover to keep a balance between ‘free’ and bound ACh or if a presynaptic ACh receptor is responsible for the observed changes in phosphatidic acid. The distribution of accumulated radioactivity derived from exogenous choline and ACh respectively between ACh, choline, phosphorylcholine and betaine has been studied in slices and isolated nerve endings.     

EFFECTS OF ACETYLCHOLINE ON THE IN CORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF SUBSYNAPTOSOMAL MEMBRANES FROM GUINEA-PIG BRAIN

-Synaptosomes prepared from guinea-pig cerebral cortex were incubated with ³²P₁ in a medium with or without 10^?4 M-acetylcholine and 10^?4 M-eserine. They were then subjected to osmotic shock and density-gradient centrifugation for the preparation of subsynaptosomal fractions and the phospholipids of each fraction were separated by two-dimensional thin-layer chromatography. The fraction containing synaptic vesicles and that containing mitochondria were the most highly labelled of the sub-synaptosomal fractions. Phosphatidic acid followed by phosphatidylinositol had the highest specific activity of the phospholipids studied. Acetylcholine caused a marked increase in the specific activity of the vesicular but not of the mitochondrial phosphatidic acid. Phosphatidylinositol specific activity also increased in the presence of acetylcholine but the increase was more reproducible in the fraction containing microsomal membranes than in the vesicle fraction. The other phospholipids were relatively poorly labelled and no effect of acetylcholine on the incorporation of ³²P₁ into these lipids could be detected. Acetylcholine also caused a decrease in the amount of phosphatidic acid in the synaptic vesicles.     

The relationship between the incorporation of 32P into phosphatidic acid and phosphatidylinositol in rat parotid acinar cells

Muscarinic and α-adrenergic stimulation of rat parotid acinar cells increases the turnover of phosphatidylinositol and phosphatidic acid. It is thought that this is initiated by hydrolysis of phosphatidylinositol, which would predict an increase in ³²P incorporation into phosphatidic acid before phosphatidylinositol. We have demonstrated an increase in ³²P incorporation into the former within 1 minute and into the latter by 2 minutes. The initial rapid rate of ³²P incorporation into phosphatidic acid slows, and the ³²P content reaches a steady state after 15 minutes. During the first 2 minutes after the addition of atropine to carbamylcholine stimulated cells, ³²P is lost from phosphatidic acid, and an equal amount is gained by phosphatidylinositol, after which ³²P incorporation equals that of the control. In cells prelabelled with ³²P, carbamylcholine, in the presence of oligomycin stimulated the loss of ³²P from phosphatidylinositol but had no effect on phosphatidic acid.     

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 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.     

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.     

Effects of dopamine, gamma-aminobutyric acid and 5-hydroxytryptamine on incorporation of 32P into phosphatides in slices from the guinea pig brain

(1) Dopamine–In slices from guinea pig corpus striatum, dopamine significantly inhibited incorporation of ³²P into phosphatidylethanolamine-plus-phosphatidylserine at a concentration of 0001 mM, and into phosphatidylinositol and phosphatidylcholine at 001 mM. In eight areas of the guinea pig brain in which the effects of 01 mM-dopamine were studied, the only significant increase in incorporation of ³²P into phosphatides was into phosphatidic acid in the hypothalamus; there was significant inhibition of incorporation of ³²P into phosphatidylcholine in cerebellar cortex and thalamus, and into phosphatidylethanolamine-plus-phosphatidylserine in the olfactory bulbs. (2) Gamma-aminobutyric acid—In slices of guinea pig cerebral cortex, GABA (1 mM) significantly inhibited incorporation of ³²P into only phosphatidic acid, diphosphoinositide and phosphatidylinositol and did not significantly affect the level or the specific activity of the nucleotide ～P. GABA (10 mM), significantly inhibited incorporation of ³²P into diphosphoinositide, phosphatidylinositol and phosphatidylcholine, and significantly lowered the specific activity of the nucleotide ～P. (3) 5-Hydroxytryptamine—In slices of guinea pig cerebral cortex, 5HT, (1 mM) significantly increased incorporation of ³²P into phosphatidic acid; in a concentration of 10 mM, 5HT increased incorporation of ³²P into phosphatidic acid four-fold and into both diphosphoinositide and phosphatidylinositol two-fold; other phosphatides were not significantly affected and the specific activity of the nucleotide ～P was not significantly different. In eight brain areas studied, 5HT (10 mM) significantly increased incorporation of ³²P into phosphatidic acid in all areas; into phosphatidylinositol in six areas (excepting cerebellar cortex and hypothalamus); and into diphosphoinositide in the olfactory bulbs, cerebral cortex, hypothalamus and corpus striatum. Incorporation of ³²P into triphosphoinositide was not significantly affected in any area. Incorporation of ³²P into phospha-tidylethanolamine-plus-phosphatidylserine was significantly greater than the control in the olfactory bulbs and incorporation of ³²P into phosphatidylcholine was significantly less than the control in the cerebellar cortex, olfactory bulbs and hypothalamus. (4) The possibility is discussed that increased incorporation of ³²P into phosphatidic acid and/or phosphatidylinositol in response to neurotransmitters might be associated with excitatory, but not inhibitory, neurotransmission; and that inhibition of incorporation of ³²P into various phosphatides may be associated with inhibitory neurotransmission or neuromodulation.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Microwave induced stimulation of32Pi incorporation into phosphoinositides of rat brain synaptosomes

Summary Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the³²Pi-incorporation into phosphoinositides. The extent of³²Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP₂) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate³²Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP₂, PIP and PI and increased PA labeling. Li⁺ also inhibited the microwave stimulated PIP₂, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A₂₃₁₈₇, inhibited the basal and microwave stimulated³²Pi labeling of PIP and PIP₂, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP₂ labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.     

Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[³H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [³²P]P_i, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [¹⁴C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca²⁺ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [³²P]P_i and [¹⁴C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [³²P]P_i labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A₂ and C. Whereas mobilization of cellular Ca²⁺ appears sufficient to cause activation of phospholipase A₂ and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca²⁺ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A₂ and C and their specific physiological roles in pancreatic secretion remain to be elucidated.     

PHOSPHOLIPID METABOLISM IN CULTURED NEUROBLASTOMA AND GLIOMA CELLS INCUBATED WITH CARBAMYLCHOLINE AND NOREPINEPHRINE

Cultures of cloned neuroblastoma cells (N1E) in stationary phase and cloned glioma cells (C2₁) in confluency showed substantial differences in phospholipid composition. As a percentage of lipid P, N1E contained more phosphatidylcholine, less ethanolamine phosphoglycerides and much less sphingomyelin than C2₁. When incubated with ³²P_i both cell lines incorporated comparable amounts of radioactivity into total phospholipids. In NIE, phosphatidylcholine contained much more and phosphatidylinositol and phosphatidic acid somewhat less label as compared to C2₁. The presence in the incubation medium of either norepinephrine or carbamylcholine failed to elicit stimulation of ³²P incorporation into any phospholipid class.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

Studies on the effects of acetylcholine and antiepileptic drugs on32Pi incorporation into phospholipids of rat brain synaptosomes

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated³²P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated³²P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the³²P labeling of phospholipids nor on the specific radioactivity of [³²P]ATP. Omission of Na⁺ drastically reduced both the³²P labeling of synaptosomal phospholipids and the specific radioactivity of [³²P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate Na⁺ and Ca²⁺ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca²⁺ and Na⁺ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.Abbreviations used ACh Acetylcholine - PA phosphatidic acid - PI phosphatidylinositol - poly PI polyphosphoinositides (diphosphoinositide and triphosphoinositide) - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - S.A. specific radioactivity     

Phospholipid metabolism in the brown alga, Fucus serratus

The metabolism of phospholipids in the brown alga, Fucus serratus was studied. The major phospholipids of this alga are phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and phosphatidylcholine. When the time-course of labelling of the lipids from [³²P] orthophosphate was studied, total labelling was approximately linear for 8 hr. All the major classes of phospholipid were labelled. The extent and pattern of labelling were not affected by the presence of proteins synthesis inhibitors phosphatidic acid was highly labelled at short time intervals. Phosphatidylcholine was relatively poorly labelled. The extent and pattern of labelling were not affected by the presence of protein synthesis inhibitors indicating that the enzymes involved in phospholipid synthesis have a rather slow turnover. Incorporation of radioactivity into phosphatidylglycerol was stimulated significantly by light.     

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.     

Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [³²P]P_i into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [³²P]P_i into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [³²P]P_i into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.     

NOREPINEPHRINE-STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE OF RABBIT IRIS SMOOTH MUSCLE: EFFECTS OF SURGICAL SYMPATHETIC DENERVATION AND IN VIVO ELECTRICAL STIMULATION OF THE SYMPATHETIC NERVE OF THE EYE

Abstract— Paired iris smooth muscles from rabbits were prelabelled either in vitro by incubation for 30 min at 37°C in an iso-osmotic salt medium containing glucose, inositol, cytidine and ³²Pi, or in vivo by administration of the isotope intracamerally into each eye 1 h before death. One of the pair was then incubated at 37°C for 10 min in an unlabelled medium containing 10 mm of 2-deoxyglucose and the other was incubated in the presence of norepinephrine (NE) or other adrenergic agents. Triphosphoinositide (TPI) was found to contain more ³²P than any other phospholipid (almost 39% of total lipid radioactivity) in both the in vitro and in vivo experiments. NE (50 μm ) increased the loss of ³²P from TPI (the TPI effect') by 28–30% in the ³²P-labelled muscle. The TPI effect was accompanied by a significant increase in ³²P labelling of phosphatidic acid (PA) and phosphatidylinositol (PI) but not phosphatidylcoholine. In this tissue the TPI effect was found to be mediated through α-adrenergic receptors. At 14 days after surgical sympathetic denervation, incorporation of ³²P into phospholipids of the denervated muscle increased by an average of 6% over that of the normal muscle. The increase in TPI, PI and PA was 7%, 4% and 9% of that of the control respectively. There was little change in phospholipid content of the denervated muscle. The increase in sensitivity to NE (12.5 μm ) caused by denervation produced about 18% increase in the TPI effect and a 25% increase in the ³²P labelling of PA, but not PI. In view of our previous findings on the requirement of the TPI effect for Ca²⁺, this observation could suggest that an increase in Ca²⁺ influx, following the interaction between the neurotransmitter and its receptor could stimulate TPI-phosphodiesterase, thus leading to increased PA via increased diglyceride. This denervation-induced supersensitivity to NE appears to be postsynaptic in nature. ³²Pi was injected intracamerally into each eye 1 h before electrical stimulation of one of the sympathetic trunks. After stimulation for 30 min there was a significant loss of ³²P from TPI and a significant increase in the labelling of PI and PA of the stimulated muscle. It is concluded that TPI and its enzymes could play an important role in neurotransmission at the neuromuscular junction of smooth muscle.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.     

Cholinergic-stimulated alkaline phosphatase secretion and phospholipid synthesis in guinea pig seminal vesicles

Incubation of slices of seminal vesicles of the guinea pig with cholinergic drugs led to an enhanced secretion of alkaline phosphatase. Incubation with carbamylcholine also stimulated the incorporation of P³² into the phospholipid fraction. Both cholinergic effects required a supply of energy since dinitrophenol was inhibitory. The stimulation of enzyme secretion and phospholipid synthesis by carbamylcholine was completely abolished by atropine. Omission of calcium ions from the incubation medium and pre-treatment of the slices with ethylenediaminetetraacetic acid (EDTA) caused a marked reduction in alkaline phosphatase secretion induced by carbamylcholine but had no effect on incorporation of P³² into phospholipids. Adenergic agents such as epinephrine, norepinephrine and isoproterenol did not influence these two processes. Addition of cyclic AMP, dibutyryl cyclic AMP and a phosphodiesterase inhibitor was also ineffective. The incorporation of P³² into the various phospholipids of the seminal vesicle was examined. In the presence of carbamylcholine, there was a marked increase in the P³²-specific activities of phosphatidylinositol (nearly 6-fold) and of phosphatidylserine (about 1.5-fold). These observations indicate that the guinea pig seminal vesicle, a large hollow organ composed of a single layer of epithelium, is ideally suited for studies concerning the biochemistry of macromolecular secretion.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.