RESPIRATION <i>IN VITRO</i> OF SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

Bradford, H. F.

doi:10.1111/j.1471-4159.1969.tb06444.x

Cited by 187 publications

(94 citation statements)

References 21 publications

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“…As can be seen from Bradford (1969) and containing 25,uCi of 32P1 in a final volume of 1 ml. One of the pair was washed four times with excess of unlabelled cold medium and incubated for 10min in the same medium.…”

Section: Resultsmentioning

confidence: 99%

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate

Abdel‐Latif¹,

Akhtar²,

Hawthorne³

1977

Biochemical Journal

254

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1. Paired iris smooth muscles from rabbits were incubated for 30min at 37°C in an iso-osmotic salt medium containing glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37'C for 10min in unlabelled medium containing lOmM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71 % and inhibited further 32p incorporation from ATP into triphosphoinositide by almost 30 %. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32P1 was replaced by myo-[3H]inositol in the incubation medium.4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32p incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32p labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32p label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction.

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Section: Resultsmentioning

confidence: 99%

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate

Abdel‐Latif¹,

Akhtar²,

Hawthorne³

1977

Biochemical Journal

254

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“…This is a value which is comparable with the rate of respiration of more highly purified synaptosomal preparations (Bradford, 1969). Table 1 presents values for the radioactive labelling of the crude synaptosomal fraction (P2) after 7.5 and 15min at 37°C in vitro and the corresponding rate of incorporation of radioactivity into the phospholipids of the D fraction after 15min, based on the specific radioactivities of the labelled compounds used.…”

Section: Radioactive Labelling Ofp2 Fractionsmentioning

confidence: 71%

“…This was a reasonable finding, as extrasynaptosomal microsomal and mitochondrial fractions were deprived of substrates and of cofactors which are essential for phospholipid turnover, by the rudimentary nature of the Krebs glucose incubation medium used. In the absence ofsuitable substrates for mitochondrial respiration and because of the concentration of Na+ in the medium (Bradford, 1969), no significant ATP generation outside the synaptosomes would also be expected.…”

Section: Discussionmentioning

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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“…Recently, numerous studies have shown that synaptosomes (pinched-off nerve ter minals isolated from brain homogenate) resemble intact tissue in transport (2,3) and metabolic processes (4,5), and thus syn aptosomes should afford on excellent model system for examining calcium disposition at the nerve endings.…”

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confidence: 99%

Effect of 1,1-diphenyl-3-piperidinobutanol hydrochloride (aspaminol) on the stimuli-induced calcium uptake in rat brain synaptosomes.

Konno

Takayanagi

1981

Jpn.J.Pharmacol

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Abstract-Effects of 1,1-diphenyl-3-piperidinobutanol hydrochloride (Aspaminol), a nonspecific smooth muscle relaxant, on the uptake of calcium in the synaptosomes of rat brain were studied, using two stimuli i.e. high KCl and ATP. We also studied the effect of Aspaminol on the analgesic response of rat hindpaw using the Randall-Sellito test. The addition of Aspaminol inhibited the stimuli-induced calcium uptake in a concentration dependent manner. Aspaminol inhibited the high KCl induced calcium uptake competitively against the external calcium concen tration. These findings suggest that Aspaminol may act on the surface of the synaptosomal membranes. In case of ATP stimulated calcium uptake, Aspaminol reduced the rate of uptake and maximal level of the synaptosomal calcium, and did not appear to have any effect on the rate of calcium release. These findings suggest that the inhibitory effect of Aspaminol can be mainly attributed to inhibition of the calcium influx, but not to acceleration of calcium efflux. In the five weeks old rats, Aspaminol did not influence the responses in the paw pinch test, but, in the weanling rats (about three weeks old), Aspaminol had a slight effect on the responses in the paw pinch test. This discrepancy may be due to difficulties in passing through the blood-brain barrier. Our findings suggest that a certain relationship may exist between the analgesic effect and the calcium uptake in the synaptosomes.

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RESPIRATION IN VITRO OF SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

Cited by 187 publications

References 21 publications

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate

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

Effect of 1,1-diphenyl-3-piperidinobutanol hydrochloride (aspaminol) on the stimuli-induced calcium uptake in rat brain synaptosomes.

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