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Biomedical subjects

J R Cooper

Publications and source records attributed to J R Cooper.

At least 91 records · Page 5Linked to original sources

The reversible detachment and deposition of surfactant and of pulmonary macrophages during bronchopulmonary lavage in the rat.

A procedure is described for carrying out repetitive bronchopulmonary lavage in the rat, in which a given volume of lavage fluid is introduced into the lungs from a reservoir and then withdrawn from the lungs back into the reservoir, the process being repeated a number of times. During this procedure there is a net release of endogenous surfactant and macrophages from the lungs. [14C] pulmonary surfactant was prepared from rats previously injected intravenously with [1-14C] palmitate, and pulmonary macrophages labelled with 85Sr were prepared from rats which had received by intratracheal injection a suspension of fused clay particles labelled with 85Sr. It was shown by carrying out repetitive bronchopulmonary lavage with 0.15M-NaCl containing either exogenous [14C]-surfactant or [85Sr]-macrophages that the release of endogenous surfactant and macrophages from the lung into the lavage fluid occurred concomitantly with the retention within the lung of radioactive exogenous surfactant and macrophages from the lavage fluid. It is concluded that the process of surfactant and macrophage detachment during bronchopulmonary lavage is reversible and that the exchange processes are of substantial magnitude.

Animals↗

Mammalian small intestinal phytase (EC 3.1.3.8).

Phytase (EC 3.1.3.8) concentration has been measured in the small intestine of rat, rabbit, guinea-pig and hamster. Levels varied from 0.12 units (microgram phosphorus released/min)/mg protein in the rat to 0.03 units/mg protein in the rabbit. The enzyme is localized in the brush border of the small intestine of the rat. It is suggested that the levels and location of phytase are an important factor in the uptake of metals from metal-phytate complexes. Metal ions released in the immediate vicinity of the absorptive surface of the intestine could be absorbed before being rendered insoluble by competing reactions such as hydrolysis.

6-Phytase↗

High affinity choline uptake and calcium-dependent acetylcholine release in proteoliposomes derived from rat cortical synaptosomes.

Proteoliposomes were prepared from rat cortical synaptic plasma membranes that retained high and low affinity choline transport. High affinity transport was inhibited by hemicholinium-3 in a competitive manner and was apparently dependent on membrane potential or ion gradients. Proteoliposomes supplemented with an acetylcholine-generating system were able to synthesize [3H]ACh de novo from [3H]choline taken up by high or low affinity transport; however, we were able to specifically load proteoliposomes derived from cholinergic membranes by using a low choline concentration. [3H]ACh, but not [3H]choline, was subsequently released in a calcium-dependent manner by K+ depolarization. This preparation offers a simplified membrane system for studying functional high affinity choline transport and nonvesicular ACh release.

Acetylcholine↗

High-affinity choline transport in proteoliposomes derived from rat cortical synaptosomes.

Functional high- and low-affinity choline transport processes from rat cortical plasma membranes were reconstituted in phosphatidylcholine bilayer liposomes. The high-affinity choline transporter demonstrated a pharmacological profile and ion dependency that were identical to those of intact synaptosomes. This preparation may be used to further characterize choline transport and, with appropriate supplementation, to investigate the release of acetylcholine in the absence of synaptic vesicles.

Acetylcholine↗

Acetylation and phosphorylation of choline following high or low affinity uptake by rat cortical synaptosomes.

Synaptosomal acetylcholine synthesis was found to be dependent on the presence of Na+-dependent HC-3 sensitive choline transport at low (5.5 mM) and high (35 mM) K+ concentrations. However, at 5, 20, and 100 microM choline, choline phosphorylation was proportional to total choline uptake, in the presence or absence or high affinity transport. Only in the presence of eserine (50 microM) did acetylcholine synthesis increase as the choline concentration was elevated from 20 microM to 100 microM, and this effect was observed at low and high K+ concentrations. Our results suggest that: 1) the synthesis of non-surplus synaptosomal ACh is dependent on high affinity choline transport; and 2) choline is equally likely to be phosphorylated after being taken up by low or high affinity transport.

Acetylation↗

The behaviour of uranium-233 oxide and uranyl-233 nitrate in rats.

233UO2 and 233UO2(NO3)2 in aqueous suspension have been administered to rats by pulmonary intubation. The 233U associated with the fraction of the 233UO2 less than 4 nm in diameter translocates from lungs to blood at the same rapid rate as 233U from 233UO2(NO3)2. Identical reactions with blood plasma and lung fluid were observed whether the 233U was administered as less than 4 nm 233UO2 particles or 233UO2(NO3)2. It is suggested that oxidation of UO2 to UO3 occurs followed by the formation of uranyl ion. In blood plasma, approximately 50 per cent of the 233U is bound to transferrin, 25 per cent to citrate and 25 per cent on bicarbonate.

Animals↗

Neurochemical characterization of an antimony-choline analog in rat cortical synaptosomes.

An analog of choline, in which nitrogen was replaced by antimony, was neurochemically characterized in rat cortical synaptosomes. It was found to be a substrate for several cholinergic enzymes, transported by a Na+-dependent, hemicholinium-3-sensitive process, acetylated, and subsequently released by depolarization in a calcium-dependent manner. Sb-choline also completed with choline for Na+-dependent uptake and for acetylation by [14C]acetyl-CoA in synaptosomes. These results suggest that Sb-choline and its acetylated product should be useful substrates for the x-ray microanalytical localization of cholinergic pools in intact nerve terminals.

Acetyl Coenzyme A↗

Cholinergic modulation of the release of [3H]acetylcholine from synaptosomes of the myenteric plexus.

The purpose of these experiments was to determine if cholinergic agents affected the release of acetylcholine (ACh) from a synaptosomal preparation of the guinea pig ileum myenteric plexus. The synaptosomal preparation was first incubated with the precursor [3H]choline; subsequently, release of the stored [3H]ACh was measured. The release was decreased by oxotremorine or exogenous ACh plus hexamethonium and increased by exogenous ACh plus evoked release that was inhibited by nicotinic antagonists or muscarinic agonists. Release was stimulated half-maximally by approximately 2 microM- and maximally by 10 microM-DMPP. Either in the absence of calcium or at 0 degrees C, DMPP was without effect. The effect of 10 microM-DMPP was brief, a significant stimulation occurring only within the first 2 min at 37 degrees C. Tetrodotoxin also inhibited excitation by DMPP but not completely. Thus, the release of [3H]ACh appears to be presynaptically modulated, negatively by muscarinic agonists and positively by nicotinic agonists.

Acetylcholine↗

Modulation of the release of acetylcholine from ileal synaptosomes by adenosine and adenosine 5'-triphosphate.

Low concentrations of either adenosine or adenosine 5'-triphosphate (ATP) inhibited the nicotinically induced release of [3H]acetylcholine from synaptosomes derived from the guinea-pig ileum myenteric plexus. Adenosine and ATP were equipotent in their ability to inhibit the release and the inhibition was reversible by theophylline in both cases. The data suggest that ATP may have acted after initial hydrolysis to adenosine and that the receptor involved may be the P1 or similar R site receptor. High concentrations of ATP caused marked increases in the release of [3H]acetylcholine. This release was neither temperature- nor calcium-dependent. Because the concentrations required were similar, however, to those which have been reported to cause ATP-induced contractions in intact preparations, further studies of the phenomenon were carried out. Lactate dehydrogenase was not released with the [3H]acetylcholine, suggesting that indiscriminate lysis of the membranes had not occurred. The release was not affected by theophylline, indomethacin, tetrodotoxin or adenosine. The ATP analog, adenylyl (beta, gamma-methylene)-diphosphonate did not cause the increase in release, therefore phosphorylation may be required for the effect. The mechanism of increased release remains to be defined, but the data suggest that it is unlikely that a P2 receptor is involved.

Acetylcholine↗

Thiamine as an integral component of brain synaptosomal membranes.

Synaptic plasma membranes were prepared from rat cerebral cortex to determine if thiamine was localized in the membranes. The synaptosomes, prepared by discontinuous sucrose gradient centrifugation, were subjected to osmotic shock at pH 9.5 for 10--15 min and subfractionated on a discontinuous sucrose gradient. The two membrane fractions that were obtained were free of contamination by mitochondrial membrane and soluble fractions. In order to ensure specificity, thiamine was assayed fluorometrically before and after the addition of thiaminase I (thiamin:base 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2). The thiamine content of the two membrane fractions was 9 and 10 pmol/mg protein.

Animals↗

Correlations between Na+-K+ ATPase activity and acetylcholine release in rat cortical synaptosomes.

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.

4-Chloromercuribenzenesulfonate↗

A synaptosomal preparation from the guinea pig ileum myenteric plexus.

Our interest in investigating the presynaptic modulation of acetylcholine release led to the development of a synaptosomal preparation from the guinea pig ileum myenteric plexus-longitudinal muscle. A crude synaptosomal fraction (P2) was obtained by homogenization and differential centrifugation. The preparation exhibited a specific uptake system for choline and for noradrenaline (NA), but not for 5-hydroxytryptamine (5-HT). Synaptosomes were isolated from this P2 fraction by an isoosmotic density gradient prepared from sucrose and metrizamide. The resultant synaptosomal fraction was enriched about sevenfold in both choline uptake and in choline acetyltransferase (ChAT). Choline was transported by a high-affinity system was a Km of 6.5 X 10(-7) M and a Vmax of 41 pmol/mg protein/min. Electron microscopy confirmed the synaptosomal nature of the gradient fraction. Some synaptosomal profiles contained only small, translucent vesicles whereas others also contained large (approx. 100 nm diameter) electron-opaque vesicles. The crude synaptosomal fraction synthesized acetylcholine (ACh) from exogenous choline and it released the synthesized ACh in a calcium-dependent manner.

Acetylcholine↗