Effects of acetylcholine on incorporation of [<sup>14</sup>C]glucose into phosphatidylinositol and on phosphatidylinositol breakdown in subcellular fractions from cerebral cortex

Lapetina, Eduardo G.; Michell, Robert H.

doi:10.1111/j.1471-4159.1974.tb06949.x

Cited by 14 publications

(8 citation statements)

References 28 publications

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“…However, on the basis of data for the release of unesterified fatty acids in rat brain post mortem (Bazan, 1970) and from incubated cerebral-cortex synaptosomal fractions (Price & Rowe, 1972), the higher rate of incorporation of oleate than of arachidonate into the D fraction phospholipid (Table 1) (Holub et al, 1970;Baker & Thompson, 1972;Breckenridge et al, 1973). However, the relatively high rate of incorporation of radioactivity derived from [3H]glucose (localized in the phospholipid glycerol backbone) was in contrast with results of studies using brain slices and ["Ciglucose, in which phosphatidylcholine was the major contributor (Lapetina & Michell, 1974). This would emnphasize the relatively high rate of synthesis de novo of phosphatidylinositol at the nerve terminal as compared with whole brain and underline the possible functional importance of this phosphoglyceride at the synapse.…”

Section: Discussioncontrasting

confidence: 50%

“…This was also found to be the case in some preliminary studies with [3H] (Miller & Dawson, 1972a) and in vitro (Yagihara et aL., 1973) (Lunt & Lapetina, 1970) with synaptosomal preparations indicated a similar order of labelling of these phosphoglycerides. However, work with [14C]-glucose and brain slices (Lapetina & Michell, 1974) (P1) were incubated in the presence of radioactive compounds for 15 min at 37°C. After the incubation the P2 fractions were washed, hypo-osmotically disrupted and a derived supernatant fraction (Ws) was fractionated by discontinuous-sucrose-gradient centrifugation.…”

Section: Radioactive Labelling Ofp2 Fractionsmentioning

confidence: 99%

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

Baker¹,

Dowdall²,

Whittaker³

1976

Biochemical Journal

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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. fraction D was found to be: phosphatidylinositol > phosphatidylcholine> phosphatidylserine > 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 [(4C]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 0, suggested intrasynaptosomal phospholipid transport.

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Section: Discussioncontrasting

confidence: 50%

Section: Radioactive Labelling Ofp2 Fractionsmentioning

confidence: 99%

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

Baker¹,

Dowdall²,

Whittaker³

1976

Biochemical Journal

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“…The data of Wells and Wells (5) (18)(19)(20). By using methods which allow a more direct measurement of Ptdlns concentration, we were able to show a definite loss of the phospholipid in response to AcCho, strongly suggesting stimulation of the enzyme PtdIns-Ins phosphohydrolase by the neurotransmitter.…”

Section: Discussionmentioning

confidence: 82%

Myoinositol and phosphatidylinositol metabolism in synaptosomes from galactose-fed rats.

Warfield¹,

Segal²

1978

Proc. Natl. Acad. Sci. U.S.A.

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The effects of experimental galactose toxicity on inositol and phosphatidylinositol (PtdIns) metabolism in synaptosomes from 0-to 30day-old rats were investigated. Galactose toxicity was induced by feeding mothers a 40% galactose diet from the 12th day of pregnancy until 19 days postpartum when the offspring were weaned onto the maternal diet. There was no decrease in myoinositol concentrations and only a small decrease in PtdIns in synaptosomes from galactose-fed rats relative to glucose-fed controls. Synaptosomes from rats on the two diets converted equivalent amounts of [ U-14Clglucose to inositol and PtdIns. Acetylcholine stimulated [2-MlHinositol incorporation into PtdIns while producing a net decrease in Ptdlns concentration in synaptosomes from 22-to 30-day-old rats. However, the phospholipid response to acetylcholine in synaptosomes from galactose-fed rats was impaired. Thus, the acetylcholine-stimulated labeling of PtdIns was 40-50% lower in these synaptosomes while the effect on Ptdlns concentration was reduced by a maximum of 55%. The data suggest that galactose-fed rats may have either a deficiency in the number of acetylcholine receptors or a defect in some step between receptor-neurotransmitter interaction and PtdIns breakdown.Since mental retardation appears to be the only incompletely reversible outcome of galactosemia caused by deficiency of the enzyme, galactose-1-phosphate uridylyltransferase, much interest has centered around the cause of the neurotoxicity. Wells and coworkers (1, 2) have reported increased concentrations of galactose and galactitol and a decrease in the concentration of free and lipid-bound myoinositol (Ins) in brain and nerve tissue of several galactosemic infants, and Schwarz (3) has demonstrated an increased concentration of galactose-1-phosphate in the nervous system of such patients. However, the mechanism by which galactose or its metabolites exert their toxic effects on the developing nervous system remains to be elucidated.In rats and chickens, experimental galactose toxicity results in decreased brain development, decreased nerve conduction, and decreased concentrations of glucose and of free amino acids in brain and nerve tissues (4). Wells and Wells (5) reported elevation of galactitol concentration and depression of free and lipid-bound Ins concentrations in brains of developing rats whose mothers were maintained on high galactose diets throughout pregnancy and lactation. In addition, these investigators demonstrated a decrease in the ability of brain slices from young galactose-toxic rats to convert [14C]glucose to Ins or phosphatidylinositol (PtdIns). Kozak and Wells (6) demonstrated decreased labeling of phosphatidic acid (PtdA) and PtdIns in brains of galactose-toxic chickens after intracranial injection of 32P1, although no change in total amount of phospholipid phosphorus was observed.These observations suggesting an impairment in Ins and Ins phospholipid synthesis or metabolism in galactose-toxic animals are of special significance in view of the e...

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“…While InslP, the principal labeled metabolite, could theoretically be derived from the phosphodiesteratic breakdown of PtdIns, three considerations militate against this possibility. First, the mAChR-mediated breakdown of PtdIns has yet t o be demonstrated in brain (Abdel-Latif e t al., 1974;Lapetina and Michell, 1974;Fisher and Agranoff, 1981). Second, there is indirect evidence from nerve-ending preparations that the initial event is the breakdown of PtdIns4P and/or PtdIns(4,5)P2 rather than that of PtdIns (Fisher and Agranoff, 19811, and third, the early release of Ins(] ,4)P, and Ins( 1 ,4,5)P3 following carbamoylcholine stimulation of brain slices has recently been reported (Bemdge et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

Differential Stimulation of Inositol Phospholipid Turnover in Brain by Analogs of Oxotremorine

Fisher

Figueiredo

Bartus

1984

Journal of Neurochemistry

126

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Structural analogs of oxotremorine have been employed to examine the relationship between the binding of agonists to muscarinic receptors in guinea pig cerebral cortex and the enhancement of inositol lipid turnover. Large differences were observed in the ability of the analogs to stimulate inositol phospholipid turnover, as measured both by the increase in labeling of phosphatidate and phosphatidylinositol from 32Pi in a nerve-ending fraction, and by the stimulated release of labeled inositol phosphates from slices of cerebral cortex, a direct measure of inositol lipid breakdown. The quaternary N+ analogs, oxotremorine-M and its N-methylacetamide derivative, were five to thirteen times as effective as oxotremorine. In contrast, methyl substitution of the pyrrolidone ring of oxotremorine resulted in a complete loss of agonist activity. Receptor occupancy data obtained from the displacement of labeled quinuclidinyl benzilate bound to receptors in a nerve-ending fraction indicated that the more efficacious agonists interacted with at least two affinity forms of the muscarinic receptor, whereas the less effective agonists bound to a single affinity form. Dose-response curves obtained in the presence of oxotremorine-M for inositol lipid turnover in both the nerve-ending fraction and slice preparation correlated with the occupancy of a single low-affinity form of the muscarinic receptor. The results suggest that the differential abilities of analogs of oxotremorine to enhance inositol lipid turnover in brain are closely related to the extent of agonist-induced conformational change in the muscarinic receptor.

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Effects of acetylcholine on incorporation of [¹⁴C]glucose into phosphatidylinositol and on phosphatidylinositol breakdown in subcellular fractions from cerebral cortex

Abstract: cional de Investigaciones Cientificas y Tecnicas de la Republica Argentina and the Wellcome Trust for financial 286 Short communication . -Aires. Paraguay 2155, Buenos A i m . 23, Argentina. Biochim. biophys. Acta 318, 306-312.

Cited by 14 publications

References 28 publications

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

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

Myoinositol and phosphatidylinositol metabolism in synaptosomes from galactose-fed rats.

Differential Stimulation of Inositol Phospholipid Turnover in Brain by Analogs of Oxotremorine

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Effects of acetylcholine on incorporation of [14C]glucose into phosphatidylinositol and on phosphatidylinositol breakdown in subcellular fractions from cerebral cortex

Abstract: cional de Investigaciones Cientificas y Tecnicas de la Republica Argentina and the Wellcome Trust for financial 286 Short communication . -Aires. Paraguay 2155, Buenos A i m . 23, Argentina. Biochim. biophys. Acta 318, 306-312.

Cited by 14 publications

References 28 publications

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

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

Myoinositol and phosphatidylinositol metabolism in synaptosomes from galactose-fed rats.

Differential Stimulation of Inositol Phospholipid Turnover in Brain by Analogs of Oxotremorine

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Effects of acetylcholine on incorporation of [¹⁴C]glucose into phosphatidylinositol and on phosphatidylinositol breakdown in subcellular fractions from cerebral cortex