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J D Pearson

Publications and source records attributed to J D Pearson.

At least 163 records · Page 9Linked to original sources
PubMed1989

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column. 2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 +/- 2%), L-phenylalanine (73 +/- 2%), L-serine (40 +/- 4%), L-arginine (42 +/- 3%) and L-ornithine (26 +/- 3%). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10%), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 3. Endothelial cell transport of L-leucine was markedly inhibited during perfusion with 1 mM-BCH (beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, system L analogue), L-leucine, D-leucine, L-phenylalanine, L-methionine and L-DOPA. 2-MeAIB, L-cysteine, glycine, L-proline, hydroxy-L-proline, L-aspartate and beta-alanine were poor inhibitors, while L-serine and the cationic substrates L-lysine and L-arginine inhibited uptake by 10-35%. 4. When the kinetics of L-leucine transport were examined over a wide range of substrate concentrations (0.025-1 mM) transport was saturable. A single entry site analysis gave a half-maximal saturation constant Kt = 0.24 +/- 0.08 mM (mean +/- S.E.M., n = 5) and a Vmax = 27.8 +/- 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L-leucine, L-serine and L-arginine by respectively 20 +/- 5% (n = 3), 77 +/- 5% (n = 3) and 35 +/- 4% (n = 3). 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na+-dependent and Na+-independent L-arginine uptake is of interest in view of recent reports that this cationic amino acid may be the physiological precursor for nitric oxide released by endothelium.

Amino Acids↗
PubMed1989

Kinetics of adenine nucleotide catabolism in coronary circulation of rats.

We have used the rat isolated, perfused heart to study the metabolism of adenine nucleotides on a single passage through the coronary circulation. Low doses (3-30 nmol) of ATP, ADP, or AMP injected as a bolus were extensively catabolized by ectoenzymes. Increasing doses of each nucleotide demonstrated saturability of catabolism that occurred at significantly lower doses of AMP than of ADP or ATP. The patterns of catabolites formed in each case were consistent with the major pathway of metabolism being sequential dephosphorylation of ATP----ADP----AMP----adenosine, although from experiments in which [3H]ATP was co-injected with unlabeled ADP, it appears that some direct conversion of ATP----AMP can occur. Furthermore, particularly in the presence of excess unlabeled ATP, [3H]ADP was phosphorylated to [3H]ATP, indicating that ectoenzymes capable of interconverting nucleotides are present. By evaluating recovery and metabolism in serial samples collected rapidly after bolus injection, we were able to use the integrated form of the Michaelis-Menten equation as developed by Bronikowski et al. (Math. Biosci. 61: 237-266, 1982) to derive Michaelis constant (Km) and maximum velocity times capillary plasma volume (Amax) values for adenosinetriphosphatase, adenosine diphosphatase, and 5'-nucleotidase (450, 300, and 93 microM; and 5.3, 5.9, and 1.7 mumol/min, respectively). This analysis also indicated that there is a high degree of heterogeneity of path lengths within the coronary circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1989

Metabolism of adenine nucleotides in human blood.

Biologically active concentrations of potently vasoactive and platelet-active adenine nucleotides are generated in plasma by a variety of pathophysiological mechanisms. Although there is evidence that ATP and ADP are inactivated by endothelial ectonucleotidases, there has been little attempt to study the metabolic routes of their catabolism in blood or to assess the contribution of this process to their clearance in vivo. Therefore, we have studied the rates and patterns of catabolism of ATP, ADP, and AMP in whole blood, plasma, and isolated blood cells. Rates of degradation of each nucleotide in cell-free plasma ranged from 0.07-0.32 nmol/min/ml with 1 microM substrates to 1.1-3.6 nmol/min/ml with 100 microM substrates. The pattern of catabolism indicated that sequential dephosphorylation from ATP----ADP----AMP----adenosine occurs. In whole blood, the pattern was similar although ATP and ADP (but not AMP) breakdown was more rapid. This was due to leukocyte ectonucleotidase activity. The use of selective inhibitors demonstrated that catabolism was not due to nonspecific phosphatase activity and that plasma 5'-nucleotidase is distinct from ATPase or ADPase. In leukocytes, ATPase and ADPase activities were distinguishable, and each contributed substantially to the rates of catabolism in whole blood. Leukocyte 5'-nucleotidase did not measurably contribute to AMP dephosphorylation in blood. By comparison, ecto-ATPase and ecto-ADPase activities on cultured human umbilical vein endothelial cells were similar to those on leukocytes while endothelial 5'-nucleotidase per 10(6) cells was equivalent to the soluble activity in 1 ml of blood or plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1989

Biochemical characterisation of polycation-induced cytotoxicity to human vascular endothelial cells.

Pro-inflammatory effects of cationic proteins secreted by human granulocytes include induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. We have shown previously that a series of synthetic polycationic amino acids produce a dose-, time- and Mr-dependent inhibition of [3H]leucine or [3H]thymidine incorporation into macromolecules by human umbilical vein endothelial cells, and that the extent of inhibition was correlated with changes in cell morphology, with release of cytoplasmic constituents and was irreversible. The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations. We have found that the extent of inhibition is related to both the identity of the monomer, for polymers of Mr 40,000 the order is ornithine greater than lysine greater than arginine, and to its configuration; poly-D-lysines are more potent inhibitors than poly-L-lysines of similar Mr. Only brief exposure to the agonist is required, 90% inhibition occurred after 10 min of exposure to poly-L-lysine (Mr 90,000). Treatment of endothelial cells with neuraminidase, heparinase, hyaluronidase, chondroitinase or trypsin did not reduce their susceptibility to polylysine. Inhibition of microtubule or microfilament formation also had no effect on polylysine cytotoxicity, indicating that internalisation of the polymer was not a prerequisite for the effect. Inhibition of protein synthesis or pretreatment with simple sugars likewise failed to block the effects of polylysine treatment. Natural cationic proteins exerted similar effects on endothelial cells, the extent of the effect apparently being related to the pI of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells.

Prostacyclin (PGI2) release was studied in perfused columns of human umbilical vein endothelial cells cultured on microcarrier beads. Substantial homologous desensitization of PGI2 release occurred when cells were exposed to agonist for 2 min after a previous exposure; the extent depended on the concentration and duration of the first challenge. Recovery from exposure to ATP or bradykinin was complete in less than 80 min; recovery from thrombin was incomplete after greater than 80 min, and this was apparently related to its proteolytic activity. Experiments with ibuprofen, a reversible inhibitor of cyclo-oxygenase, demonstrated that homologous desensitization did not involve inactivation of cyclo-oxygenase. ATP and bradykinin did not induce heterologous desensitization. Thrombin and trypsin induced cross-desensitization, but neither agonist significantly reduced responses to ATP or bradykinin, suggesting that a common proteolytic mechanism is responsible for their ability to induce PGI2 synthesis. We conclude that desensitization of PGI2 release in response to physiological agonists is generally agonist-specific and involves modulation of molecular events at or close to the receptors involved, rather than inactivation of prostanoid biosynthesis.

Adenosine Triphosphate↗
PubMed1988

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase.

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.

Adenosine Triphosphate↗
PubMed1988

Determination of monosaccharides in glycoproteins by reversed-phase high-performance liquid chromatography on 2.1-mm narrowbore columns.

The monosaccharide composition of glycoproteins was determined by using a Supelcosil LC-18 150 x 2.1 mm narrowbore column under reversed-phase conditions. Methodology was developed for sample hydrolysis, N-acetylation, benzoylation, and cleanup prior to high-performance liquid chromatography. Monosaccharides were identified and quantified at picomole levels. Various glycoproteins were then analyzed, including ribonucleases A and B, Mucin, and fetuin. In addition, the monosaccharide content of Chinese hamster ovary cell-expressed recombinant human renin was determined.

Chemical Phenomena↗
PubMed1988

Determination of [32P]phosphoamino acids in protein hydrolysates by isocratic anion-exchange high-performance liquid chromatography.

We have developed a simple method for phosphoamino acid analysis of 32P-labeled phosphoproteins using anion-exchange high-performance liquid chromatography (HPLC). Phosphoproteins undergo partial acid hydrolysis and the resulting hydrolysate is injected directly onto a column. The sample is then isocratically eluted from the column by 35 mM phosphoric acid at pH 3.0 with collected fractions analyzed by Cerenkov counting. Phosphoamino acid identification is accomplished by the comparison of the retention times of 32P-labeled peaks to retention times of phosphoamino acid standards which had been monitored at 206 nm. This method has greater sensitivity and is more reliable than cellulose thin-layer electrophoresis and the results obtained by high-efficiency Cerenkov counting can be evaluated immediately, instead of waiting days or weeks for autoradiographic development of cellulose plates. This HPLC protocol is an improvement over other published HPLC protocols in that there is no need for pre- or post-column derivatization and the free [32P]phosphate elutes long after the phosphoamino acids. Thus sensitivity is increased as there is no interference from the free phosphate. Selection of an HPLC anion-exchange column is critical for this separation. Only two of the four columns that we tested performed well. We present data from several phosphoproteins including calcium-calmodulin dependent protein kinase, the beta-subunit of the insulin receptor, and phosphorylated calmodulin to demonstrate the utility of this procedure.

Buffers↗
PubMed1988

Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production.

Endothelial cells are known to release prostacyclin (PGI2) in response to agonists, and this has generally been assumed to be caused, at least in part, by activation of a phospholipase A2 by elevated concentrations of cytoplasmic free calcium ([Ca2+]i). However, it has been shown in the blood platelet that agonists can cause arachidonate release without elevating [Ca2+]i. In the present study, rigorous analysis is made of the [Ca2+]i-dependence of PGI2 production in the human umbilical-vein endothelial cell. Thrombin caused a rapid increase in [Ca2+]i from the resting basal value of 0.1 microM to a peak, within 10-15 s, of approx. 2 microM. In the absence of extracellular Ca2+, [Ca2+]i then declined back to the resting value within 2-3 min. In the presence of extracellular Ca2+, [Ca2+]i partly decreased to a new steady-state value of approx. 1 microM. The elevated [Ca2+]i was maintained while the stimulus and the source of extracellular Ca2+ were present, suggesting that it was dependent on influx of Ca2+ across the plasma membrane. Thrombin stimulated the production of PGI2 in the presence or in the absence of extracellular Ca2+. However, the production of PGI2 was more prolonged in the presence of extracellular Ca2+. Total accumulated amounts of 6-oxo-prostaglandin F1 alpha on stimulation with thrombin without extracellular Ca2+ were only 65% of those accumulated with extracellular Ca2+ present. Cells depleted of extracellular and intracellular sources of Ca2+ by incubation with 1 mM extracellular EGTA and exposing them to ionomycin to discharge intracellular stores produced no elevation of [Ca2+]i on stimulation with thrombin or production of PGI2. The threshold [Ca2+]i required to support the production of PGI2 was measured to be 0.8-1.0 microM by using different doses of ionomycin selectively to increase [Ca2+]i. This relationship between [Ca2+]i and PGI2 production was similar to that produced by using different doses of thrombin. Our results show that the major and probably exclusive intracellular stimulus for the production of PGI2 by the vascular endothelial cell in response to thrombin is the elevation of [Ca2+]i.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Stimulation of endothelial cell growth by sera from diabetic patients with retinopathy.

An in-vitro proliferation assay has shown that sera from patients with diabetic retinopathy, particularly those with the proliferative form, are two to four times more effective than sera from non-diabetics at stimulating 3H-thymidine incorporation into both human umbilical vein and human omental microvascular endothelial cells, but not at stimulating incorporation into human dermal fibroblasts or 3T3 cells. The factor(s) is heat stable and of molecular weight greater than 15,000, and its presence is unrelated to metabolic control. It is not present in patients with other forms of diabetic vascular disease, which suggests that it is not related to carbohydrate or lipid metabolism. These results provide evidence against the hypothesis that metabolic disturbances are central to the development of diabetic microvascular disease, and raise possibilities of novel forms of therapeutic intervention.

Cell Division↗
PubMed1988

Generation of reactive oxygen metabolites by phagocytosing endothelial cells.

We have studied the ability of particulate stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human endothelial cells. Basal release of superoxide (O2-) and hydrogen peroxide from undisturbed monolayers was very low (108 pmol O2- and 75 pmol H2O2 in 3 h from dishes of 3 X 10(5) cells). Addition of 1-micron diameter polystyrene microspheres, which were phagocytosed by the cells progressively, caused a dramatic increase in release of both metabolites; by 3 h, a 13.5- and 6.6-fold increase over controls was observed respectively (P less than 0.001). Addition of formaldehyde-fixed human platelets or chylomicron-size lipid particles also increased production of reactive oxygen species. Similar rises in H2O2 and O2- production were induced by treatment with 10(-7) M phorbol myristate acetate. Pretreatment of endothelial cells with neuraminidase, heparinase or heparitinase to alter their glycocalyx composition substantially enhanced the effect of microspheres on H2O2 and O2- generation. We conclude that the interactions of particles, including platelets and lipids, with endothelial cells leads to the generation of significant pericellular levels of reactive oxygen species. These metabolites can oxidise a wide variety of nearby molecules, leading to cell damage and altered uptake characteristics for lipoproteins containing peroxidized lipids. These effects are exacerbated when endothelial cell glycocalyx composition is disrupted.

Cells, Cultured↗
PubMed1988

Haemoglobin synthesis in K562 erythroleukaemia cells is affected by intimate contact with monolayers of various human cell types.

The haemoglobin content of K562 erythroleukaemia cells was affected by co-culture over monolayers of various human cell types. Haemoglobin synthesis was increased after co-culture with umbilical-cord-derived endothelial cells and most monolayers of bone-marrow-derived macrophages, and inhibited after co-culture with two fibroblast lines, blood-monocyte-derived macrophages, a neuroglial cell line (U-251 MG) and most monolayers of bone-marrow-derived stromal cells. These effects were modified when a thin layer of agar was placed over the monolayers. Cell-free culture media conditioned by all but two of the seven types of monolayer studied inhibited haemoglobin synthesis by K562 cells; those conditioned by blood-monocyte-derived macrophages and two of 11 monolayers of bone-marrow-derived macrophages stimulated haemoglobin synthesis. Thus, the haemoglobin content of K562 cells appeared to be influenced both by intimate contact between K562 cells and the cells of the monolayers and by humoral factors released by the monolayers. The data support the concept that erythroid differentiation is partly dependent on intimate contact between erythroid progenitor cells and microenvironmental cells.

Amniotic Fluid↗
PubMed1988

Cornell Medical Index responses and mortality in a Polynesian population.

The Cornell Medical Index (CMI) is a health questionnaire consisting of a somatic division, divided into 12 sections organized by organ systems, and a psychosocial division. It has been suggested that the CMI taps a dimension of the perception of illness before clinical measurements detect disease. However, this has not been confirmed in nonwestern populations. Our purpose was to determine if the CMI predicted 6-year mortality in a Polynesian population. In 1975, 461 American Samoan men responded to the CMI. Thirty-one died between 1976 and 1981, 13 of cardiovascular disease (CVD). A logistic model was used to predict both total and CVD mortality during the follow-up period. Positive responses to the total, somatic, and psychosocial divisions of the CMI did not significantly improve prediction of mortality compared to models using only age. An analysis using section scores showed that several somatic sections were associated with CVD mortality, but none were associated with total mortality. This study suggests that the CMI is not a strong predictor of mortality and, by extension, of existing health status in American Samoa. Even if the CMI does measure the perception of illness, one cannot assume a priori that it is valid in any given anthropological population. Rather, the instrument must be pretested in each particular cultural setting.

Adolescent↗
PubMed1988

Regulation of P2y-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration.

1. ATP and ATP analogues induced prostacyclin (PGI2) secretion from human cultured umbilical vein endothelial cells. 2. The threshold active concentration for ATP was less than or equal to 1 microM. The rank order of potency of analogues was 2-chloroadenosine 5'-triphosphate (2-ClATP) greater than 2-methylthioadenosine 5'-triphosphate (2-MeSATP) greater than ATP greater than ADP, while adenosine 5'-(alpha,beta-methylene)triphosphonate, AMP and adenosine were inactive, indicating the presence of P2y-purinoceptors. 3. In contrast to their actions on P2y-receptors in guinea-pig taenia coli, isopolar analogues of 2-methylthioadenosine 5'-(beta, gamma-methylene)triphosphonate were less effective than ATP. 4. ATP and ATP analogues increased intracellular free calcium ions, [Ca2+]i, giving a rapid transient peak due predominantly to release from intracellular stores, followed by a maintained steady-state elevated level due to influx. 5. The dose-response curves for peak [Ca2+]i induced by ATP, 2-ClATP and 2-MeSATP were very similar to those for PGI2 production. 6. Elevations of [Ca2+]i, above a threshold value of 0.8-1 microM, were necessary for PGI2 production in response to P2y-receptor activation. 7. The dose relationships between PGI2 release and peak [Ca2+]i were equivalent whether [Ca2+]i was raised by ionomycin or via P2y-receptor activation by ATP or 2-ClATP, indicating that elevations of [Ca2+]i provide the major, if not the exclusive intracellular pathway for P2y-purinoceptor-mediated PGI2 synthesis.

Adenosine Triphosphate↗
PubMed1988

Effects of synthetic polycations on leucine incorporation, lactate dehydrogenase release, and morphology of human umbilical vein endothelial cells.

Naturally occurring cationic proteins secreted by human granulocytes have pro-inflammatory effects including induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. Synthetic cationic polyamino acids have been shown to exert similar inflammatory effects in vivo. We have therefore used a range of synthetic polycationic amino acids to investigate the characteristics required to cause endothelial cell damage, assessed by in vitro inhibition of leucine incorporation into macromolecules by human umbilical vein endothelial cells (HUVEC) in culture. Exposure of HUVEC to 20 nM-2 microM cationic polypeptides of similar Mr(av) (approximately 40,000) in the presence of 20% serum produced a dose-dependent inhibition of [3H]leucine incorporation by polymers of ornithine, arginine or lysine. Similar results were obtained using [3H]thymidine. Neutral or anionic polypeptides of similar Mr were without effect. The molar potency of polylysines increased over the range Mr 40,000-120,000, while polylysines of Mr(av) less than 25,000 had no effect. In the absence of serum, inhibition occurred more rapidly and at lower doses. Inhibition of leucine and thymidine incorporation was time-dependent, e.g. exposure to 800 nM-polylysine, Mr(av) 90,000, led to progressively increasing inhibition that was complete after 24 h exposure, and was irreversible. The effects of polycations could not be blocked by pretreatment of the cells with polyanions. Precoating of the culture surface with polylysines had no effect on leucine incorporation by HUVEC or their subsequent response to polylysines in solution. Exposure to the peptide Arg-Gly-Asp-Ser inhibited incorporation by 30% but did not increase susceptibility to polylysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Measurement and characterisation of circulating anti-endothelial cell IgG in connective tissue diseases.

We have detected circulating IgG antibodies that bind with high affinity to human umbilical vein endothelial cells in 74% of patients with systemic lupus erythematosus (SLE), 30% of those with scleroderma and 28% of those with rheumatoid arthritis. IgG binding was F(ab) mediated, and did not involve immune complexes. Anti-endothelial IgG were apparently unrelated to other circulating autoantibodies, including anti-cardiolipin or antiDNA IgG. Bound IgG from SLE or scleroderma patients was displaced by IgG from certain unrelated patients whereas others were ineffective. Anti-endothelial cell IgG from all sera tested were adsorbed by human dermal fibroblasts; erythrocytes and leucocytes each adsorbed a fraction of the activity. Purified IgG did not induce complement-mediated cytotoxicity. We conclude that a discrete group of IgG antibodies is common in connective tissue disease patients, reacts predominantly with endothelial cells and dermal fibroblasts, and may be important in the pathogenesis of vascular damage.

Arthritis, Rheumatoid↗