Effects of neurotransmitters and other pharmacological agents on 32Pi incorporation into phospholipids of the iris muscle of the rabbit

The effects of norepinephrine, other catecholamines, α- and β- adrenergic receptor blocking agents and acetylcholine on the incorporation of ³²Pi into phospholipids of the iris muscle of the rabbit were studied $\text{in vitro}$. There was a marked stimulation of ³²Pi into phosphatidic acid (PhA), phosphatidyl inositol (PhI) and to a much lesser extent phosphatidyl choline but not into phosphatidyl ethanolamine. The increase in the ³²P labeling of PhA and PhI in the presence of norepinephrine or acetylcholine, which ranged from 2- to 6-fold, was found to be time- and concerntration-dependent. Under our experimental conditions, several adrenergic drugs, including DL-propranolol, phentolamine, isoproterenol, phenylephrine, but not sotalol, increased markedly (nearly up to 5-fold) the ³²Pi incorporation into PhA and PhI of the iris. In contrast, phenoxybenzamine, an α-receptor blocker, blocked completely the stimulatory effects of norepinephrine on phospholipid synthesis. The stimulation of phospholipid synthesis by acetylcholine was completely abolished by atropine. Incorporation of ³²Pi into PhA and PhI was significantly increased in the presence of serotonin, dopamine, epinephrine or histamine. Addition of γ-aminobutyric acid or cyclic AMP was ineffective. These observations suggest that in the iris muscle of the rabbit, which is innervated by cholinergic and adrenergic fibers, the phospholipid effect is probably a membrane effect that is not associated with synaptic transmission.     

Enhancement of the muscarinic synaptosomal phospholipid labeling effect by the ionophore A23187

Addition of either acetylcholine (ACh) or the ionophore A23187 to synaptopsomes resulted in a selective stimulation of 32Pi incorporation into phosphatidate (PhA) and phosphatidylinositol (PhI), while the labeling of phosphatidylinositol phosphate (PhIP) and phosphatidylinositol diphosphate (PHIP2) was reduced. The inclusion of both ACh and A23187 resulted in a synergistic increase in PhA and PhI labeling, and a synergistic decrease in the labeling of the polyphosphoinositides. Added calcium was not required, although inclusion of EGTA prevented the alterations in lipid labeling. The enhanced labeling of PhA and PhI by ACh or A23187 was not the result of either an increase in the radioactivity of the precursor [32P]ATP pool, or increased de novo synthesis of these lipids as judged from the incorporation of [3H]glycerol, [3H]glucose or [3H]myo-inositol. The synergistic alterations in PhA, PhI, and polyphosphoinositide labeling were observed with ionophore only in the presence of selected muscarinic agonists, and with the inclusion of atropine or scopolamine the labeling reverted to a value which approximated that seen with the ionophore alone. Synergistic effects on phospholipid labeling with muscarinic agonists were also obtained with the calcium ionophore, ionomycin, but not with X537A, monensin, or valinomycin. Neither the apparent number of muscarinic receptors present, nor their affinity for the ligand were altered by the presence of A23187. In prelabeling experiments, A23187 accelerated the loss of [32P]label from PhIP and PhIP2, and the rate of loss was further augmented by the addition of ACh. Neither agent produced comparable effects on the breakdown of prelabeled PhA or PhI. It is suggested that phosphodiesteratic cleavage of the polyphosphoinositides might account for both the decrease in labeled PhIP and PhIP2 and increased labeling of PhA and PhI via the availability of resultant diglyceride. In any event, the results demonstrate that the turnover of polyphosphoinositides, in addition to that of PhA and PhI, is linked to the activation of muscarinic receptors.     

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.     

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.     

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.     

Action of aspartate on the32Pi incorporation into phospholipids of cerebral cortex

The effect ofl-aspartate on the³²Pi incorporation of phospholipids, was studied on slices of rat cerebral cortex. This amino acid produced an inhibitory effect in concentrations 0.01–10 mM, which was more evident at 120 min. This effect was not stereospecific and did not imply a change in Pi uptake and in nucleotides P precursors. The inhibition was present in PS, PC, PE and to a lesser extent in PI. On liver slices 1 mMl-aspartate had the opposite effect, stimulating the incorporation of³²Pi into total phospholipids. Our results suggest that the effect ofl-aspartate is by a non-specific mechanism, probably not mediated by a receptor.     

Acetylcholine stimulates hydrolysis of 32 P-labeled phosphatidic acid in guinea pig synaptosomes

[³H]-inositol or [³H]-arachidonate was injected intracerebrally into guinea pigs. Labeled nerve endings were incubated with Ach¹ or CCh, both of which stimulate labeling of PhA and PhI from ³²P_i by > 100% and 70% respectively. Their addition did not affect the $\text{in}$$\text{vivo}$ labeled phosphatidyl-[³H]-inositol or [³H]-arachidonyl-diglyceride and -PhI. Enhanced hydrolysis of [³H]-inositol-PhiP and -PhIP₂ in the presence of ACh, CCh or choline was not reversed by atropine. In a two-step experiment, PhA was labeled with ³²P_i, and DNP was added to block further $\text{γ-[}{}^{32}\text{P]-ATP}$ formation. Addition of ACh stimulated an atropine-sensitive $\text{decrease}$ in [³²P]-PhA.     

(14C) acetylcholine synthesis by cortex slices of rat brain

Abstract—

• 1 A procedure has been developed to measure ACh synthesis from [¹⁴C]-precursors. As little as 10^?9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG-50, precipitation and recrystallization of ACh chloroaurate.
• 2 [U^?14C]glucose and [2^?14C]pyruvate formed similar amounts of [¹⁴C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [¹⁴C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden-Meyerhof pathway.
• 3 The amount of ACh detected by bioassay after incubation of cortex slices with [U^?14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose.
• 4 [¹⁴C]choline, either methyl or chain labelled, formed [¹⁴C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose.
• 5 When both [U^?14C]glucose and [¹⁴C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10^?4m -hemicholinium-3 inhibited ACh formation by more than 90 per cent from either [U-¹⁴C]glucose or [Me-¹⁴C]choline.
• 6 Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [¹⁴C]ACh within the slices which reached a maximum during the first hour of incubation. [¹⁴C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm -KCI markedly increased the formation of [¹⁴C]ACh found in the incubation medium. Decreased amounts of [¹⁴C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.

     

Experimental alcoholism in rats: protein synthesis in subcellular fractions from cerebellum, cerebral cortex and liver after long term treatment

Abstract— The incorporation in vivo of [³H]leucine into protein from subcellular fractions was determined in rats chronically ingesting 15 per cent ethanol for 8 months. Mitochondrial, microsomal and cell sap fractions from cerebellum, cortex cerebri and liver were investigated. The results showed a minor over-all depression of protein synthesis in cerebellum and cortex cerebri and a slight stimulation of the incorporation of leucine into protein from liver subcellular fractions. If the animals were abstinent 24 h before injection of the isotope, the incorporation of labelled amino acids into protein was markedly increased in cerebellum and cerebral cortex but not in liver.     

ACETYLCHOLINE SYNTHESIS FROM [2-14C]PYRUVATE IN RAT STRIATAL SLICES

Abstract— Rat striatal slices were incubated with [2-¹⁴C]pyruvate or [6-¹⁴C]glucose as sole carbon source. The method devised to study the accumulation of labelled ACh in tissues and incubating medium in the presence or absence of eserine 200 μM derived from the previous studies of FONNUM (1969) and H emsworth and M orris (1964). Total ACh was estimated by biological assay.
The specific activity of newly synthesized ACh was found to be equal to that of the precursors, even for short incubation times and low substrates concentrations. When slices were incubated with [2-¹⁴C]pyruvate and eserine, the spontaneous release of ACh occurred at a constant rate, was not modified by the addition of 2 mM-choline in the medium, and consisted only of newly synthesized transmitter.
The initial rate of ACh synthesis was found to be independent of choline concentration, but dependent on the [2-¹⁴C]pyruvate concentration, and reached a maximal value corresponding to about 5 per cent of the measured striatal choline acetyltransferase activity.
The appearance of the so called 'surplus ACh' pool, obtained in the presence of eserine, could be detected only after 30 min and represented 26 per cent of the total tissue ACh content after 180 min of incubation.
In the absence of eserine, tissue ACh levels increased six-fold in 80 min and then remained stable until the end of the incubation period (180 min), if sufficient substrate was provided. The maximal ACh accumulation in slices was independent of both excess of choline and [2-¹⁴C]pyruvate.
The 'ACh plateau' represented the attainment of a new dynamic equilibrium, since ACh synthesis could still be stimulated by 30 mM-K⁺. From these results, it was concluded that ACh synthesis is controlled by a negative feed-back regulation.     

Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices

1. Rat cerebral-cortex slices were incubated with ³²P_i, acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.     

Changes in phenolic acids during maturation and lignification of scots pine xylem

The content and fractional composition of alcohol soluble phenolic acids (PhA) in cells with different degree maturation and lignification in the course of early and late wood formation in the pine (Pinus sylvestris L.) stem during vegetation were studied. Phenolic compounds (PhC), extracted by 80% ethanol, were divided into free and bound fractions of PhA. In turn, the esters and ethers were isolated from bound PhA. The contents of all substances were calculated per dry weight and per cell. Considerable differences have been found to exist in both the contents and the composition of the fractions PhA on successive stages of tracheid maturation of early and late xylem. Early wood tracheids at all secondary wall thickening steps contained PhC less and free PhA more than late wood tracheids. Throughout earlywood tracheid maturation, the pool of free PhA per cell declined at the beginning of lignification and then increased gradually while that of bound PhA decreased. The maturation of late wood tracheids were accompanied by the rise of free PhA pool and the diminution of bound PhA pool. In the composition of bound PhA, the ethers were always dominant, and the amount of that in earlywood cells was less than in latewood cells. The cells of early xylem at all steps of maturation contained more of esters. The sum total of free hydroxycinnamic acids, precursors of monolignols, gradually decreased during early xylem lignification as the result of the reduction of the pools of p-coumaric, caffeic, ferulic and synapic acids, while that of their esters rised. In the course of late xylem lignification, the pools of free p-coumaric, ferulic and, especially, synapic acids increased. Simultaneously, the amount of ferulic acid ester and synapic acid ether increased too. According to the data, lignin biosynthesis in early xylem and late xylem occurs with different dynamics and the structure of lignins of two xylem types might be different too.     

Calciotropic hormones raise the chemically detectable [Pi] in UMR 106–06 osteoblast-like cells

Uptake of orthophosphate (Pi) by osteoblast-like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast-like cells (UMR 106-06) that PTH (10^?11 to 10^?7 M ) increases both ³²Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D₃ (1,25D) (10^?12 to 10^?6 M ) and salmon calcitonin (CT) (10^?12 to 10^?6 g ml^?1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular ³²Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy- and 25 hydroxyvitamin D₃. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

Effects of dichloroisopropylarterenol on respiration, nucleotide ∼ P and phospholipids of slices of cerebral cortex

In a Recent report Hokin (1969) showed that norepinephrine stimulated the incorporation of ³²P into phosphatidic acid (PA) of slices of cerebral and cerebellar cortex and into phosphaditylinositol (PI) of slices of cerebral cortex. In the course of our experiments on agents affecting the metabolism of phospholipids, the effects of epinephrine and its antagonist, dichloroisopropylarterenol (DCI), on the labelling of energy-rich nucleotides and phospholipids of cerebral tissue have been measured. Epinephrine had no significant effect but DCI stimulated the incorporation of ³²P into PA by 165 per cent and into PI by 90 per cent.     

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.     

Myocardial phospholipid metabolism in alloxan diabetic rats

Alloxan diabetes in rats was found to decrease the level of phospholipids in the heart. Measurement of specific phosphatides showed that the decrease was restricted only to phosphatidylethanolamine and lysophosphatidylcholine. Study of $\text{in}$$\text{vivo}$ incorporation of ³²Pi indicated an impairment of phosphatidylethanolamine synthesis and conversion of phosphatidylcholine into lysophosphatidyl choline in the heart of diabetic rats. Treatment of diabetic rats with insulin restored the levels of phosphatidylethanolamine and lysophosphatidylcholine and incorporation of ³²Pi into these phosphatides to almost normal.     

The subcellular location of the higher acetylcholine content in rat brain found after killing by the near-freezing method as compared with decapitation

Abstract— We have confirmed the finding of Takahashi & Aprison (J. Neurochem. 11 , 887-898, 1964) that more acetylcholine is found in brains of rats killed by near-freezing compared to decapitation. The radioenzymic assays for acetylcholine and choline of Shea & Aprison (Analyt. Biochem. 56 , 165-177, 1973) and Goldberg & Mc Caman (J- Neurochem. 20 , 1 8, 1973) were used to measure both compounds and gave very similar results. The larger amount of ACh was observed both in powders of frozen rat brain and in homogenates prepared from animals killed by near-freezing. When subcellular fractionation of the homogenates was done in the presence of eserine, the larger amount of ACh was found in the soluble fraction (S₂). These results indicated that with the near-freezing method, an extra amount of ACh is preserved in a form that is originally protected from acetylcholinesterase but that becomes esterase-sensitive on fractionation since no differences were observed in P₁. P₂ or S₂ fractions when no eserine was present. The amounts of choline in homogenates and subcellular fractions were also measured after both methods of killing. Differences in the method of killing and postmortem changes which affect the choline values obtained are described.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.