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Biomedical subjects

J D Pearson

Publications and source records attributed to J D Pearson.

At least 199 records · Page 11Linked to original sources
PubMed1984

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK).

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.

Animals↗
PubMed1984

Purinoceptor mediated stimulation of prostacyclin release in the porcine pulmonary vasculature.

Prostacyclin (PGI2) release from the piglet isolated perfused lung was measured by radioimmunoassay of 6-keto-PGF1 alpha in the venous effluent. Basal release of PGI2 was transiently stimulated up to 30 fold, in a dose-dependent manner, by bolus injections of ATP (0.03-3 mumol). A continuous infusion of ATP also produced a transient response. Dose-response curves for purinergic stimulation of PGI2 release showed that ADP was equipotent with ATP, while AMP and adenosine were virtually inactive. The non-hydrolyzable ATP analogue, ATP-gamma-S, elicited PGI2 release of similar magnitude and duration to that of ATP, suggesting that pulmonary catabolism of ATP is not required to induce PGI2 release. The results suggest that the porcine pulmonary vasculature possesses P2-purinoceptors through which the synthesis and release of PGI2 can be mediated.

6-Ketoprostaglandin F1 alpha↗
PubMed1984

Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK).

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3 h co-culture period. Cell-to-cell communication in either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acid-insoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.

Animals↗
PubMed1983

Stereoselectivity of ectonucleotidases on vascular endothelial cells.

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5'-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5'-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.

5'-Nucleotidase↗
PubMed1983

The control of adenosine concentration in polymorphonuclear leucocytes, cultured heart cells and isolated perfused heart from the rat.

Rat polymorphonuclear leucocytes or neonatal-rat heart cells in culture were treated with 2'-deoxycoformycin and 5-iodotubercidin at concentrations that inhibited adenosine deaminase (EC 3.5.4.4) and adenosine kinase (EC 2.7.1.20) inside the intact cells, and the rate of adenosine accumulation was determined. The basal rate of adenosine formation was 2% (polymorphonuclear leucocytes) or 9% (heart cells) of the maximal activity of adenosine kinase also measured in intact cells. Greatly increased rates of adenosine formation were observed during adenine nucleotide catabolism. This condition also led to a decrease in adenosine kinase activity. When isolated rat hearts were perfused with 5-iodotubercidin alone at a concentration which inhibited adenosine kinase, no increase in tissue or perfusate adenosine or inosine concentration was observed. However, perfusion with hypoxic buffer or infusion of adenosine into the coronary circulation at a rate (20 nmol/min) equivalent to 40% of the activity of adenosine kinase caused large increases in effluent perfusate adenosine and inosine concentrations. These data argue unanimously against the existence of a substrate cycle controlling adenosine concentration. They suggest instead that an increase in the rate of adenosine formation is the principal cause of elevations in adenosine concentration during ATP catabolism.

Adenosine↗
PubMed1983

Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells.

ATP (approx. 1-300 microM) induces dose-dependent prostacyclin secretion from perfused columns of microcarrier beads with cultured endothelial monolayers attached. The response is transient, shows little tachyphylaxis and can reach approx. 100 times control values. 2-Methylthio-ATP is more potent, ADP slightly less potent, AMP much less potent and adenosine is ineffective. These results are consistent with the presence of a purinoceptor on endothelium linked to the prostacyclin synthetic pathway.

Adenine Nucleotides↗
PubMed1983

Prostacyclin release stimulated by thrombin or bradykinin in porcine endothelial cells cultured from aorta and umbilical vein.

We have investigated the responsiveness of porcine endothelial cells, cultured from aorta or umbilical vein, to bradykinin and thrombin. Aortic cells studied in situ, in primary culture and in subculture responded to both agents with an increase in prostacyclin (PGI2) release; the increase was greater with bradykinin than with thrombin. Umbilical vein cells in primary culture or in subculture also responded to bradykinin and to thrombin; bradykinin was again more effective than thrombin. For both cell types there was a rapid quantitative decline in their ability to be stimulated by either agent with increasing passage number, so that by passage 8 stimulation of PGI2 release could no longer be detected from monolayers cultured under conventional conditions. The presence of a stimulatory response was still, however, easily demonstrable in subcultured cells when they were grown on microcarrier beads, packed in small columns, and perfused. Under these conditions greater than 10-fold stimulation above baseline levels was often found, while the conventional monolayer culture technique gave a smaller maximum stimulation even in primary cultures.

Animals↗
PubMed1983

Metabolism of circulating adenosine by the porcine isolated perfused lung.

Adenosine uptake was studied in the piglet isolated perfused lung by means of the single-circulation paired-tracer dilution technique. Adenosine was efficiently taken up from the pulmonary vascular bed, and the process was potently inhibited by dipyridamole. Following uptake, adenosine was incorporated into intracellular nucleotides, and at low perfusate concentrations, little or none of the incorporated radioactivity returned to the circulation. At higher concentrations, cellular uptake was saturable and products of intracellular catabolism (inosine and hypoxanthine) were returned to the circulation. Perfusion of low concentrations of adenosine after inhibition of pulmonary adenosine kinase led to a proportional decrease in the retention of nucleotides and to a release of inosine and hypoxanthine. A small proportion of adenosine was metabolised extracellularly by adenosine deaminase; this activity was not released from perfused lungs and is apparently an ecto-enzyme.

Adenine Nucleotides↗
PubMed1982

The development of gamma-glutamyltransferase in a pig renal-epithelial-cell line in vitro. Relationship to amino acid transport.

The pig kidney-cell line, LLC-PK1, possesses gamma-glutamyltransferase with properties similar to those of the purified renal enzyme. gamma-Glutamyltransferase activity increases after cells are seeded at low density to reach values comparable with those found in kidney cortex when the cells are fully confluent. This increase is paralleled by an increase in alpha-methyl D-glucoside uptake. In contrast, the uptakes of L-leucine and L-alanine decrease during this time. These results suggest that gamma-glutamyltransferase is not important as a mediator of the renal transport of neutral amino acids.

Amino Acids↗
PubMed1982

Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells.

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.

Animals↗
PubMed1982

Characterization of neutral amino acid uptake by cultured epithelial cells from pig kidney.

Two transport systems for neutral amino acids have been characterised in LLC-PK, cells. The first, which transports alanine in a sodium-dependent manner, also mediates alanine exchange and is perferentially inhibited by serine, cysteine, and alpha-amino-n-butyric acid. This system resembles the ASC system in Ehrlich ascites and some other cell types. There is only a small contribution of other systems to alanine uptake. The second, which transports leucine with no requirement for sodium and mediates leucine exchange, is blocked by 2-aminonorbornane-2-carboxylic acid and hydrophobic amino acids. This system is similar to the L system described in other cell types. LLC-PK1 cells retain several other features implying renal proximal tubule origin; our results thus suggest that these transport systems may be involved in the reabsorption of neutral amino acids by the nephron in vivo.

Alanine↗
PubMed1982

Effect of sulphasalazine on pulmonary inactivation of prostaglandin F2 alpha in the pig.

1 The metabolism of prostaglandin F2 alpha (PGF2 alpha) 15 nm in 100,000 g supernatant fractions from piglet lung homogenates was inhibited by sulphasalazine with an IC50 value of 25 micrometers. 2 The piglet isolated lung perfused with Krebs solution, containing either albumin or Ficoll 70 to prevent oedema and vascular damage, efficiently metabolized PGF2 alpha given as a bolus injection (1 ng in 0.1 ml; 30 nm). 3 In Krebs solution containing Ficoll 70, sulphasalazine inhibited the pulmonary inactivation of PGF2 alpha in a dose-dependent manner with an IC50 value of 110 micrometers. No inhibition of inactivation by sulphasalazine was found when the perfusion fluid contained albumin, which is known to bind this drug effectively. 4 Analysis of the separated efflux profiles for PGF2 alpha and its metabolites with reference to the dilution curve for an extracellular marker provided evidence that sulphasalazine inhibited PGF2 alpha uptake into lung cells. 5 We conclude that the effect of sulphasalazine on pulmonary prostaglandin inactivation is primarily due to inhibition of prostaglandin transport, and not to inhibition of prostaglandin metabolism.

Animals↗
PubMed1981

Reversed-phase supports for th resolution of large denatured protein fragments.

Large pore (much greater than 300 A), spherical partical silica stationary phases possessing either C18 or C8 hydrocarbon ligands out-performed small pore (60-100A), irregular shaped silicas for the purification of large denatured peptides. Since columns 5 cm in length appeared to be as effective in separating peptides as columns 5 times longer, it is likely that large peptides absorb to the matrix rather than partition between the stationary and mobile phases.

Chromatography, High Pressure Liquid↗
PubMed1981

Lymphocyte traffic through antigen-stimulated lymph nodes. II. Role of Prostaglandin E2 as a mediator of cell shutdown.

Prostaglandin E2 (PGE2) can mediate changes in lymphocyte traffic through lymph nodes. By measuring PGE2 levels in efferent lymph from cannulated sheep lymph nodes it was shown that increased synthesis of PGE2 always correlated with cell shutdown; infusion of PGE2 alone induced cell shutdown and inhibition of PGE2 synthesis by the node abolished cell shutdown induced by antigen.

Animals↗