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U S Ryan

Publications and source records attributed to U S Ryan.

At least 55 records · Page 3Linked to original sources
PubMed1992

Interleukin-2 directly increases albumin permeability of bovine and human vascular endothelium in vitro.

The direct effects of interleukin-2 (IL-2) on albumin permeability of cultured bovine pulmonary artery endothelial cell (BPAEC) and human arterial endothelial cell (HAEC) monolayers were studied. BPAEC were exposed to IL-2 (500 to 25,000 U/ml) for 4 h. The steady-state transfer rate of [125I]albumin across the BPAEC monolayer was 3.3 +/- 0.4%/h (n = 10) in control BPAEC (diluent alone), was significantly increased in BPAEC exposed to 500 U/ml of IL-2 (72 +/- 3% above control values, n = 6, P less than 0.02), and further increased in BPAEC exposed to 5,000 U/ml (60 +/- 2% increase above 500 U/ml values, n = 5, P less than 0.02). No further increase was noted after exposure to 25,000 U/ml of IL-2. Additionally, no further increase in [125I]albumin transfer rates was noted in BPAEC exposed to 5,000 U/ml of IL-2 for 24 versus 4 h. Similar changes were found using HAEC. Preincubation of HAEC with an anti-IL-2 low-affinity receptor antibody (anti-IL-2R alpha) inhibited the IL-2-induced permeability increase. Expression of IL-2R alpha receptors in HAEC incubated with 5,000 U/ml of IL-2 for 4 h was also found. Thus, IL-2 appears to have a direct effect on cultural arterial endothelial monolayers not requiring the presence of other cell types or serum proteins. IL-2-induced increases in endothelial macromolecular permeability may play an important role in the pathogenesis of the IL-2-induced vascular leak syndrome seen in vivo.

Albumins↗
PubMed1992

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

Animals↗
PubMed1992

Methylene blue inhibits nitrovasodilator- and endothelium-derived relaxing factor-induced cyclic GMP accumulation in cultured pulmonary arterial smooth muscle cells via generation of superoxide anion.

The mechanism of modulation of cyclic guanosine monophosphate (cGMP) accumulation by methylene blue (MB), a putative inhibitor of soluble guanylate cyclase, was investigated in cultured rabbit pulmonary arterial smooth muscle cells (RPASM). Control or MB-pretreated RPASM were stimulated with sodium nitroprusside (SNP), nitrosothiols or endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial cells, in short-term co-cultures. The putative EDRF, S-nitroso-L-cysteine (CYSNO), a stable deaminated analog of CYSNO, S-nitroso-3-mercaptoproprionic acid (MPANO) and SNP produced concentration-dependent (1-100 microM) increase (1.5- to 12-fold) in RPASM cGMP levels. MB pretreatment inhibited CYSNO and SNP-induced cGMP accumulation by 51% to 100%, but MPANO-mediated responses were not altered by MB. The inhibition profile of MB on nitrovasodilator-induced cGMP accumulation was quantitatively reproduced by extracellular generation of superoxide anion with xanthine (100 microM) and xanthine oxidase (5 mU). Similarly to MB pretreatment, superoxide anion generation had no effects on base-line cGMP levels or cGMP responses elicited by MPANO. Furthermore, MB induced a dose- and time-dependent generation of superoxide anion from RPASM, as evidenced from spectrophotometric determination of cytochrome c reduction. Inhibition of cGMP accumulation in response to CYSNO and SNP by MB was completely prevented by superoxide dismutase but not catalase. Selective pretreatment of endothelial cells with MB before co-culture with untreated RPASM produced a reduction in RPASM cGMP levels of a magnitude comparable with that seen in co-cultures of MB-pretreated RPASM with untreated endothelial cells, and which was partially prevented by superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA.

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.

Amino Acid Sequence↗
PubMed1991

Hydrogen peroxide-induced cell and tissue injury: protective effects of Mn2+.

Recent evidence indicates that under in vitro conditions, superoxide anion and hydrogen peroxide (H2O2) are unstable in the presence of manganese ion (Mn2+). The current studies show that in the presence of Mn2+, H2O2-mediated injury of endothelial cells is greatly attenuated. A source of bicarbonate ion and amino acid is required for Mn2+ to exert its protective effects. Injury by phorbol ester-activated neutrophils is also attenuated under the same conditions. EDTA reverses the protective effects. Acute lung injury produced in vivo in rats by intratracheal instillation of glucose-glucose oxidase is almost completely blocked in rats treated with Mn2+ and glycine. Conversely, treatment of rats with EDTA, a chelator of Mn2+, markedly accentuates lung injury caused by glucose-glucose oxidase. These data are consistent with the findings of others that Mn2+ can facilitate direct oxidation of amino acids with concomitant H2O2 disproportionation. This could form the basis of a new therapeutic approach against oxygen radical-mediated tissue injury.

Animals↗
PubMed1991

Thrombospondin production and thrombospondin-mediated adhesion in U937 cells.

U937 cells have low levels of surface thrombospondin (TSP) under control conditions but express higher levels after treatment for 1 day with 100 nM phorbol myristate acetate (PMA). Increased surface expression is due, in part, to increased biosynthesis. Untreated U937 cells do not adhere to TSP-coated plastic culture dishes but adhere strongly to TSP after stimulation with PMA. Untreated U937 cells also adhere weakly to endothelial cell monolayers while PMA-treated U937 cells attach strongly to monolayers of rat pulmonary artery endothelial cells. Endothelial cell adhesion appears to be mediated, in part, by TSP since antibodies to TSP partially inhibit.

Animals↗
PubMed1991

Stimulation of rat endothelial cell transforming growth factor-beta production by bleomycin.

This study examines the hypothesis that mediators from lung endothelial cells could promote lung collagen synthesis in pulmonary fibrosis. Since bleomycin induces pulmonary fibrosis in humans and animals, the effects of this drug on endothelial cells were examined. Endothelial cell conditioned media were prepared in the presence of various doses of bleomycin, and tested for their ability to stimulate lung fibroblast collagen synthesis. The results show a dose-dependent stimulation of endothelial cell secretion of collagen synthesis stimulatory activity by bleomycin, which peaked at a dose greater than or equal to 100 ng/ml. Stimulation was selective for collagenous protein synthesis. Gel filtration analysis showed most of the activity to reside in fractions with an estimated molecular mass range of 10-27 kD. The activity was inhibited by anti-transforming growth factor-beta (TGF-beta)antibody, but not by nonimmune control IgG. The presence of TGF-beta was confirmed using the mink lung epithelial cell assay. Northern blotting revealed significant increases in TGF-beta mRNA in bleomycin-stimulated endothelial cells. Thus in vitro stimulation of endothelial cells by bleomycin upregulates TGF-beta production, presumably by increased transcription. In view of the chemotactic and matrix synthesis stimulatory properties of this cytokine, such an increase in TGF-beta production may play an important role in bleomycin-induced pulmonary fibrosis.

Animals↗
PubMed1990

Effects of adenosine and analogs on adenylate cyclase activity in cultured bovine aortic endothelial cells.

We studied the effects of adenosine and analogs on adenylate cyclase (AC) activity in membranes from long-term cultured bovine aortic endothelial cells, using [alpha-32]ATP as substrate and chromatographic separation of [32P]cAMP. Compared to our previous findings in cultured bovine pulmonary arterial endothelial cells (Legrand et al., Biochem Pharmacol 38: 423-430, 1989), the present results were qualitatively and quantitatively comparable between the two cell types. In aortic cells, AC activity was stimulated in a concentration-dependent manner by isoproterenol, forskolin and 5'-guanylylimidodiphosphate (Gpp(NH)p), by 2.6-, 5.2- and 4.8-fold respectively. The A2 adenosine agonist 5'-(N-ethyl)-carboxamidoadenosine induced a smaller (60%) increase of AC activity. Adenosine (10(-3) M) partially inhibited (30%) the Gpp(NH)p-stimulated AC activity. Similarly, adenosine partially reversed, but 2',5'-dideoxyadenosine (DDA) totally blocked (IC50: 540 microM), the forskolin-induced stimulation of AC activity. DDA and 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP) also inhibited the isoproterenol-induced stimulation of AC activity (IC50: 350 and 23 microM respectively). Adenosine-induced inhibition of stimulated AC activity does not appear to involve adenosine A1 receptors since the specific A1 agonist cyclohexyladenosine did not reverse forskolin stimulation of AC activity. Instead, it suggests a direct action of adenosine on the catalytic subunit of the adenylate cyclase (P site). We conclude that membranes from long-term cultured bovine aortic endothelial cells, express beta-adrenergic and adenosine A2 receptors coupled to adenylate cyclase activation. The two P site agonists, DDA and 2'-deoxy-3'-AMP, and, with a weaker effect, adenosine itself, inhibited the activated cyclase at the P site. The natural nucleotide 2'-deoxy-3'-AMP was a strong inhibitor in aortic cell types (as in pulmonary arterial endothelial cells) and may possibly act as a modulator of adenylate cyclase in these cells.

Adenosine↗
PubMed1990

Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation.

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.

Animals↗
PubMed1990

Enhancement of tumor necrosis factor-induced endothelial cell injury by cycloheximide.

Tumor necrosis factor (TNF), a potent polypeptide mediator released by activated monocytes and macrophages, has a number of proinflammatory effects on endothelial cells. TNF is cytotoxic to tumor cells in vivo and in vitro, but TNF-induced toxicity to endothelial cells is less well established. We now report that cycloheximide (CHX), an inhibitor of protein synthesis, renders endothelial cells highly susceptible to TNF-induced lysis. TNF alone did not change the overall rate of protein synthesis by endothelial cells, whereas the addition of CHX completely abolished protein synthesis. Endothelial cells incubated in TNF alone in high concentrations (up to 1,000 U/ml) showed minimal rounding up and release of 51Cr. Likewise, CHX alone (5 micrograms/ml) had no significant effect on endothelial cell morphology and release of 51Cr. However, incubation of endothelial cells in both CHX and TNF caused injury in a dose-dependent manner. Morphological evidence of cell retraction, rounding, and detachment began within 2 h, but specific 51Cr release did not begin to rise until after 4 h. These changes were not observed when endothelial cells were incubated with TNF/CHX at 4 degrees C. The combination of TNF/CHX was lethal to all endothelial cells tested (bovine pulmonary artery, human umbilical vein, and human aorta), with human aortic cells showing the most pronounced changes. We conclude that healthy endothelial cells are resistant to TNF-induced lysis, but inhibition of their ability to make protein renders them highly susceptible.

Animals↗
PubMed1990

Receptors on pulmonary endothelial cells.

Endothelial cells, situated at the interface of blood and the tissues, are ideally positioned to receive circulating and tissue-derived stimuli and equally to affect underlying cell types of the vascular wall. Endothelial cells and smooth muscle cells show a close morphologic association via myoendothelial junctions and also interact via soluble mediators. We have begun to investigate the stimulus-secretion coupling using agonists that elicit the release of endothelium-derived relaxing factors and have particularly focused on the role of Ca2+. In addition, we have examined the effects of endothelium-derived constrictor substances from both large and small pulmonary vessels. On account of the intimate association between microvessels and the airway wall, we have also examined the effects of endothelium-derived constrictors on tracheal smooth muscle and have shown that conditioned media from pulmonary artery and from pulmonary microvascular endothelial cells constrict both coronary artery rings and tracheal smooth muscle strips. The characteristics of the response are similar to the actions of the 21 amino acid peptide endothelin. Thus, endothelial cells regulate the levels of circulating vasoactive substances via their surface enzymes while, via their surface receptors, they are capable of being stimulated to release substances with effects on vascular tone and airway responses.

Animals↗
PubMed1990

Regulation by bradykinin of phosphoinositide metabolism in the endothelial cells of the pulmonary artery.

The effect of the vasodilatory peptide bradykinin on the regulation of phosphoinositide metabolism in endothelial cells was investigated. Activation of phosphoinositide metabolism by bradykinin in the endothelium of the bovine pulmonary artery was not blocked by pertussis toxin, which ADP-ribosylates a membrane protein of molecular mass 40 kDa, but botulinum toxin, which ADP-ribosylates a membrane protein of molecular mass 24 kDa, fully blocked bradykinin-stimulated phosphoinositide metabolism. The effect of bradykinin was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), an activator of GTP-binding proteins, and inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), an inhibitor of GTP-binding proteins. Activation of phosphoinositide metabolism by bradykinin was fully blocked by a B2-receptor antagonist, whereas a B1-receptor antagonist did not affect bradykinin action. It is concluded that the B2-receptor in endothelial cells is coupled to phospholipase C via a GTP-binding protein, which is a substrate for botulinum toxin.

Animals↗
PubMed1990

H2O2-mediated cytotoxicity of rat pulmonary endothelial cells. Changes in adenosine triphosphate and purine products and effects of protective interventions.

H2O2-mediated cytotoxicity (as measured by 51Cr-release) of rat pulmonary artery endothelial cells was time-dependent and related to the concentration of H2O2 employed. The cytotoxic effects of H2O2 were, as expected, prevented by catalase and the degree of protection was directly related to its time of addition. Endothelial cells were incubated with [14C]adenosine to achieve intracellular labeling of ATP, after which the cells were exposed to H2O2. Based on analysis of cell extracts by high-performance liquid chromatography, there was a time-dependent loss of intracellular radioactivity and ATP with the simultaneous appearance of purine degradation products including xanthine/hypoxanthine. Approximately 50% of the intracellular ATP was lost after 15 minutes of exposure and up to 80% was lost by 30 minutes. The extracellular fluid of cells exposed to H2O2 contained significant amounts of xanthine/hypoxanthine. The ferric iron chelator deferoxamine provided almost complete protection against H2O2-mediated cytotoxicity. Two inhibitors of xanthine oxidase, allopurinol and oxypurinol, were also protective as was deoxycoformycin, an inhibitor of adenosine deaminase. Remarkably, cells protected by these agents showed the same loss of intracellular ATP as unprotected, H2O2-treated cells. These findings demonstrate the dissociation between ATP loss per se and oxidant injury of endothelial cells. ATP breakdown may be an important event leading to cellular injury in that this results in the formation of substrate for xanthine oxidase.

Adenosine Triphosphate↗
PubMed1989

Modulation of adenylate cyclase activity in cultured bovine pulmonary arterial endothelial cells. Effects of adenosine and derivatives.

We studied the effects of adenosine and adenosine derivatives on adenylate cyclase activity in cultured endothelial cells from bovine pulmonary artery. Basal and stimulated enzyme activities were measured in membrane preparations using [alpha-32P]ATP as the substrate and chromatographic isolation of formed [32P]cAMP. Basal cyclase activity was 11 +/- 1 (mean +/- SEM) pmol/mg protein/min. Forskolin, 5'-guanylylimidodiphosphate (Gpp(NH)p) and (-)isoproterenol stimulated adenylate cyclase in a concentration-dependent manner, producing maximal stimulations of three, seven and four times the basal activity respectively. In the presence of adenosine deaminase, cyclohexyladenosine, an A1 agonist, had no effect on basal and forskolin- or Gpp(NH)p-stimulated activities, whereas 5'-(N-ethyl)-carboxamidoadenosine (NECA), an A2 agonist, had a small stimulatory effect (52% increase over basal). In the presence of IBMX, adenosine and two P-site agonists, 2',5'-dideoxyadenosine (DDA) and 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP), inhibited forskolin (30 microM)-stimulated adenylate cyclase activity with an order of potency of 2'-deoxy-3'-AMP greater than DDA greater than adenosine. DDA and 2'-deoxy-3'-AMP were also able to inhibit cyclase activity stimulated by Gpp(NH)p (10(-5)M) or isoproterenol (10(-6)M) with the same order of potency. Only 2'-deoxy-3'-AMP inhibited the stimulated adenylate cyclase activity by more than 50% (IC50 = 19-32 microM). These findings indicate that (1) long-term cultured endothelial cells from bovine pulmonary artery express A2 and beta-adrenergic receptors which stimulate adenylate cyclase activity through Gs transducer proteins, and (2) the natural compound and P-site agonist, 2'-deoxy-3'-AMP, is a potent inhibitor, and possibly a natural regulator, of adenylate cyclase activity in this tissue.

1-Methyl-3-isobutylxanthine↗
PubMed1989

High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors.

By virtue of its immediate contact with the circulating blood, the endothelium provides an attractive target for retroviral vector transduction for the purpose of gene therapy. To see whether efficient gene transfer and expression was feasible, rabbit aortic endothelial cells were infected with three Moloney murine leukemia virus-derived retroviral vectors. Two of these vectors carry genes encoding products that are not secreted: N2, containing only the selectable marker gene neoR, and SAX, containing both neoR gene and an SV40-promoted adenosine deaminase (ADA) gene. The third vector, G2N, contains a secretory rat growth hormone (rGH) gene and an SV40-promoted neoR gene. Infection with all three vectors resulted in expression of the respective genes. A high level of human ADA expression was observed in infected endothelial cell populations both before and after selection in G418. G2N-infected rabbit aortic endothelial cells that were grown on a synthetic vascular graft continued to secrete rGH into the culture medium. These studies suggest that endothelial cells may serve as vehicles for the introduction in vivo of functioning recombinant genes.

Adenosine Deaminase↗
PubMed1989

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells.

The relative importance of intracellular and extracellular Ca2+ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca2+ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC50 of 4 x 10(-10) M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca2+. However, in the absence of external Ca2+, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with 45Ca, bradykinin increased the 45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in 45Ca efflux induced by bradykinin of 1.3 x 10(-9) M. The involvement of an intracellular Ca2+ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca2+,Mg2+ -ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca2+ concentrations showed at least two compartments involved in the uptake of Ca2+. The 45Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the 45Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP3). The IP3-induced release was not affected by apyrase or exogenous GTP. The EC50 for the release of Ca2+ by IP3 was 1.0 microM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗