PubMed Health⌕ Search

Biomedical subjects

M J Dowdall

Publications and source records attributed to M J Dowdall.

36 records · Page 2Linked to original sources
PubMed1976

Calcium uptake in preterminal central synapses: importance of mitochondria.

Energy dependent 45Ca2+ uptake in the synaptosomal preparation from guinea pig cortex has been investigated. 45Ca2+ uptake was stimulated by ATP, the absolute value of uptake being dependent on the extent of synaptosomal disruption caused by osmotic shock. A quantitative comparison of microsomal and mitochondrial ATP-dependent 45Ca2+ uptake showed that only mitochondria had a large enough capacity to account for the Ca uptake levels observed in the synaptosomal preparation. ATP-stimulated 45Ca2+ uptake in mitochondria, 'intact' and 'shocked' synaptosomes was inhibited by atractyloside, DNP, oligomycin and ruthenium red but unaffected by antimycin A and rotenone. This was interpreted as evidence that mitochondria were responsible for ATP-dependent synaptosomal Ca2+ uptake, the increase in uptake seen on osmotic lysis being due to the deocclusion of intraterminal mitochondria. Synaptosomal and mitochondrial 45Ca2+ uptake was also stimulated by the mitochondrial respiratory substrate glutamate; this uptake was sensitive to antimycin A, DNP, rotenone and ruthenium red but insensitive to atractyloside or oligomycin thus indicating it was of mitochondrial origin. No change in glutamate-dependent 45Ca2+ uptake was seen on osmotic lysis of the synaptosomes as the expected increase due to the release of occluded mitochondria was counterbalanced by the damaging effect of hypo-osmotic shock on the glutamate-stimulated 45Ca2+ uptake process.

Adenosine Triphosphate↗
PubMed1976

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.

Acetylcholine↗
PubMed1975

The involvement of lysophosphoglycerides in neurotransmitter release; the composition and turnover of phospholipids of synaptic vesicles of guinea-pig cerebral cortex and Torpedo electric organ and the effect of stimulation.

(1) Crude synaptosomal fractions (P2) derived from guinea-pig cerebral cortex were incubated in the presence of 50 mM KCl in a Krebs-glucose medium. Torpedo marmorata electric organs were stimulated electrically in vivo at 5 pulses/sec for 30 min by electrodes placed on the electric lobe. Synaptic vesicles were isolated from each source and the phospholipid compositions analysed and compared with vesicles from unstimulated controls. (2) Lysophosphatidylcholine was the only lysophosphoglyceride demonstrable in the synaptic vesicles from either source and its low levels did not increase as a result of chemical or electircal stimulation. In each case there was a close similarity of the phospholipid distributions in the vesicles taken from control and stimulated samples. (3) Control experiments indicated extensive decreases in the acetylcholine content of the vesicles from the stimulated electric organ and smaller decreases in the acetylcholine content of the synaptic vesicles from stimulated crude synaptosomal fractions. These fractions were found to respire linearly in the presence of 10 mM glucose and the vesicle fractions were shown to have low levels of contaiminating membranes as judged by marker enzyme analyses. (4) Crude synaptosomal fractions from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium with labelled fatty acids and [3H]glucose in the presence or absence of 50 mM KCl. Subsynaptosomal fractionation was carried out and specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomes) and H (disrupted synaptosomes). The release of neurotransmitter did not significantly enhance the labelling of phospholipids in any of the fractions studied as compared with phospholipids from unstimulated fractions. This was found after two incubation times and using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose.

Acetylcholine↗
PubMed1974

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.

Acetylcholine↗
PubMed1972

Choline metabolism in the cerebral cortex of guinea pigs. Stable-bound acetylcholine.

1. The turnover of synaptosomal (vesicular-cytoplasmic) and stable-bound (vesicular) acetylcholine isolated from cortical tissue was investigated after the administration, under local anaesthesia, of [N-Me-(3)H]choline into the lateral ventricles of guinea pigs. 2. Radioactive acetylcholine and choline present in acid extracts of subcellular fractions were separated by a combination of liquid and column ion-exchange and thin-layer chromatography. 3. The specific radioactivity and pattern of labelling of acetylcholine present in a fraction of monodisperse synaptic vesicles was found to be essentially the same as that of synaptosomal acetylcholine. 4. The specific radioactivity of stable-bound acetylcholine present in partially disrupted synaptosomes (fraction H) at short times (10-20min) after the injection of [N-Me-(3)H]choline was very variable and inversely related to the yield of acetylcholine in that fraction. 5. Evidence was found for the existence of two small, but highly labelled pools of acetylcholine, one which could be isolated in fraction H and the other which was lost when synaptosomes, after isolation by gradient centrifugation, were left at 0 degrees C or pelleted. 6. It is concluded that the results are best explained by metabolic differences among the nerve-ending compartments (thought to be vesicles) which contain stable-bound acetylcholine. Computer simulation of our experiments supports this possibility and suggests that the highly labelled pool in fraction H is present in vesicles close to the external membrane.

Acetylcholine↗
PubMed1972

Choline metabolism in the cerebral cortex of guinea pigs. Phosphorylcholine and lipid choline.

1. The labelling of phosphorylcholine and choline-containing phospholipids in the subcellular fractions of guinea-pig cerebral cortex after the intraventricular injection of [N-Me-(3)H]choline into conscious animals has been studied. Special emphasis was placed upon the synaptosome fraction and early time-periods after administration. 2. The labelling of phosphorylcholine was rapid compared with that of phospholipid and was confined to two distinct subcellular fractions: the soluble cytoplasmic fraction and the synaptosome fraction. Most of the labelled phosphorylcholine of the synaptosome fraction was readily released by osmotic rupture indicating location in the nerve-ending cytoplasm. The two pools of phosphorylcholine had similar specific radioactivities at all observed times. 3. (3)H-labelled phospholipid was found in all membranous fractions. The labelling was confined to choline-containing phospholipids, notably phosphatidylcholine. 4. The labelling of the different membranous fractions was similar. 5. The half-life of the choline-containing phospholipids in the synaptic vesicle fraction was very much greater than the acetylcholine in this fraction. 6. Evidence is presented that synthesis de novo of phosphatidylcholine at nerve terminals occurs in vivo.

Acetylcholine↗