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Biomedical subjects

U S Ryan

Publications and source records attributed to U S Ryan.

At least 91 records · Page 5Linked to original sources
PubMed1988

Water permeability of isolated endothelial cells at different temperatures.

The endothelial cells provide a potential pathway for water movement across the endothelium. The endothelial cell permeability to water can, therefore, be a factor in regulation of the rate of water movement out of the vasculature. Endothelial cells are isolated from calf pulmonary artery and cultured. The cells are removed from culture, and the diffusional water permeability is determined with the linear diffusion technique. The mean membrane permeability coefficients (PDS) determined at 20, 30, 37, and 41 degrees C are 160, 273, 304, and 387 x 10(-5) cm/s, respectively. The temperature dependence of PD is calculated with the Arrhenius equation to be 7.2 kcal/mol. If these values of PD are compared with values we have reported for the osmotic permeability coefficient (PF), the PF/PD is about one at each temperature. The values of PD in the isolated endothelial cells are compared with PD for endothelial cells estimated from whole organ studies and are similar to recently reported values.

Animals↗
PubMed1988

Influence of vasoactive agents on cytoplasmic free calcium in vascular endothelial cells.

The regulation of cytoplasmic free calcium concentration [( Ca2+]i) in endothelial cells (EC) derived from human umbilical vein, aorta, and pulmonary artery, or from bovine pulmonary artery, was studied by means of the fluorescent Ca2+ indicator indo-1. Histamine and thrombin caused a rapid transient elevation in [Ca2+]i in the EC of all the human blood vessels tested. In aortic EC, [Ca2+]i also rose in response to ATP and bradykinin. It was shown that in bovine pulmonary artery EC [Ca2+]i rises in response to platelet-activating factor (PAF) and thrombin. For a more detailed investigation of the receptor-mediated mechanism of [Ca2+]i increase in EC we used histamine as a stimulating agent. Histamine effects were seen at concentrations ranging from 5 X 10(-7) to 10(-4) M [50% effective dose (ED50) approximately 2-4 microM)] and were mediated by H1-receptors. The histamine-induced increase in [Ca2+]i was not markedly diminished when the extracellular calcium was bound by excess ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The data obtained indicate that the histamine effect is best explained by Ca2+ mobilization from intracellular stores. The histamine-induced increase in [Ca2+]i was not influenced by elevating the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or cyclic guanylic acid (cGMP) by use of isobutylmethylxanthine and forskolin or by nitroprusside preincubation, respectively. However, the protein kinase C stimulator, phorbol myristate acetate (PMA), strongly inhibits [Ca2+]i elevation. It is assumed that a negative feedback mechanism that blocks receptor-mediated [Ca2+]i increase is triggered as a result of the activation of protein kinase C.

Adenosine Triphosphate↗
PubMed1988

Role of calcium in the activation of endothelial cells.

The involvement of calcium in the release of EDRF from cultured endothelial cells is reviewed. It is suggested that both intracellular and extracellular calcium can release EDRF depending not only on the experimental conditions but also on the agonist involved in the release. Calcium enters the cell, following receptor occupation, through a nonselective receptor-operated channel. Intracellular release of calcium following receptor occupation is probably via the production of inositol trisphosphate (IP3). It is suggested that the initial calcium signal for the release of endothelium-derived relaxant factor (EDRF) is of intracellular origin whereas the maintained release of EDRF is due to calcium entry from the extracellular space.

Animals↗
PubMed1988

Tumor necrosis factor enhances susceptibility of vascular endothelial cells to neutrophil-mediated killing.

Tumor necrosis factor has a variety of effects on different types of cells in vitro, including endothelial cells. The current studies show that pretreatment of rat pulmonary artery endothelial cells with tumor necrosis factor increases in a time- and dose-dependent manner their sensitivity to killing by neutrophils stimulated with phorbol myristate acetate or C5a. Similar effects are seen with interleukin-1. These data suggest that tumor necrosis factor and interleukin-1 may have important pro-inflammatory effects on endothelial cells.

Animals↗
PubMed1987

Endotoxin-induced secretion of an active plasminogen activator inhibitor from bovine pulmonary arterial and aortic endothelial cells.

Treatment of bovine pulmonary arterial and aortic endothelial cells for 24 h with 10-20 ng/ml endotoxin appeared to suppress urokinase secretion by over 90%. Immunodepletion of urokinase from conditioned medium revealed high levels of a fully active plasminogen activator inhibitor (PAI). In contrast, medium from aortic cells not treated with endotoxin expressed low levels of a latent PAI. The endotoxin-induced PAI from both cell types was not immunoprecipitated by incubation with IgG directed against the endothelial-type inhibitor, PAI-1, or antiserum directed against the placental-type inhibitor, PAI-2. Endotoxin-induced PAI was inactivated by 0.2-1.0 M of 2-mercaptoethanol, but unaffected by treatment with up to 500 microM of chloramine-T. We conclude that the exposure of bovine endothelial cells to endotoxin causes the secretion of a fully active PAI with properties different from those reported for PAI-1.

Animals↗
PubMed1987

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells.

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.

Animals↗
PubMed1987

Effects of colchicine on sulfated glycosaminoglycan production and cell detachment in pre-capillary pulmonary endothelial cells.

The effects of colchicine on the morphology, substrate adhesiveness, and production of glycosaminoglycan (GAG) macromolecules by cultured pre-capillary pulmonary endothelial cell were studied. Colchicine-treated cells demonstrated altered morphology and decreased substrate adhesiveness compared to untreated cells. In addition, [35S]sulfate incorporation into glycosaminoglycans was decreased 33% after treatment with colchicine. Spectrophotometric measurement of total cellular GAG revealed a similar GAG reduction in colchicine-treated cells. The composition of [35S]sulfate radiolabelled GAG was similar in cultures with and without colchicine, consisting of approximately 56% chondroitin sulfate and the remainder heparin/heparan sulfate. The results indicate that colchicine influences the biological behavior of pre-capillary endothelial cells, in part by altering the amount of glycosaminoglycan molecules produced.

Animals↗
PubMed1987

Platelet-activating factor effects on bovine pulmonary artery endothelial cells.

Endothelial cells (ECs) were isolated from bovine pulmonary artery and maintained in long-term culture. On reaching confluency, ECs formed a characteristic "cobblestone" monolayer. One hour after addition of 1 nM platelet-activating factor (PAF) to the growth medium, ECs underwent dramatic changes in shape from their normal polygonal morphology to more elongated spindle-shaped forms. More pronounced effects were evident in the presence of 0.1 nM phorbol-12-myristate-13-acetate (PMA), a potent activator of C kinase. It was found that at concentrations from 10(-11)-10(-7) M, PAF stimulates the phosphoinositide turnover in EC. The half-maximal activation in the release of inositol phosphates was at 10(-9) M. This finding suggested that an increase in intracellular Ca2+ concentration and activation of protein kinase C were involved in the mechanism of action of PAF on EC. The metabolic responses of EC were evaluated by measuring the activity of beta-adrenergic receptor-coupled adenylate cyclase (AC) in a crude membrane fraction and by assay of prostacyclin and thromboxane released by cultured EC. AC from control membranes was activated by isoproterenol in a dose-dependent manner (EC50 = 30 nM) from 0.8-5.5 pmol cAMP/min/mg protein. If the membranes were isolated after preincubation of ECs with 1 nM PAF or 0.1 nM PMA, the AC activity was decreased by 70 and 90%, respectively; in both cases, affinity for isoproterenol was lowered threefold (EC50 = 100 nM). Our data suggest that PAF interaction with EC leads to an apparent beta-adrenergic receptor desensitization that probably acts via a phosphorylation mechanism involving C kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1987

Source of iron in neutrophil-mediated killing of endothelial cells.

Recently we have shown that human neutrophils activated with phorbol ester are cytotoxic for cultured bovine pulmonary artery endothelial cells in an iron-dependent manner. By using the ferric iron chelator deferoxamine mesylate, we have now investigated the source of the iron. Pretreatment of neutrophils with deferoxamine mesylate affected neither their production of O2- nor their cytotoxicity for endothelial cells after addition of phorbol ester. However, similar pretreatment of endothelial cells with deferoxamine mesylate, followed by washing of the cells, resulted in a persistent presence of chelator associated with the endothelial cells and high degrees of protection of endothelial cells from cytotoxicity. The protection was dependent on the amount of chelator used and on the duration of exposure of the endothelial cells to the chelator. These data suggest that iron, which plays an important role in oxygen radical-mediated killing of endothelial cells by neutrophils, is derived from the target (endothelial) cells.

Animals↗
PubMed1986

Endothelial cell permeability to water.

We have calculated diffusional permeability coefficients for tracer water and for [14C]antipyrine in endothelial cells. With these values and those from studies in whole lungs we set a range for diffusional water permeability coefficients of the intact endothelium.

Animals↗
PubMed1986

Reversible alterations in cultured pulmonary artery endothelial cell monolayer morphology and albumin permeability induced by ionizing radiation.

The effects of ionizing irradiation (0, 600, 1,500, or 3,000 rads) on the permeability of pulmonary endothelial monolayers to albumin were studied. Pulmonary endothelial cells were grown to confluence on gelatin-coated polycarbonate filters, placed in serum-free medium, and exposed to a 60Co source. The monolayers were placed in modified flux chambers 24 hours after irradiation; 125I-albumin was added to the upper well, and both the upper and lower wells were serially sampled over 4 hours. The amount of albumin transferred from the upper well/hour over the period of steady-state clearance (90-240 min after addition of 125I-albumin) was 2.8 +/- 0.2% in control monolayers and was increased in monolayers exposed to 1,500 or 3,000 rads (increase of 63 +/- 10% and 61 +/- 10%, respectively, P less than 0.01). No increase was found in monolayers exposed to 600 rads. The increases in endothelial albumin transfer rates were associated with morphologic evidence of monolayer disruption and endothelial injury which paralleled the changes in albumin permeability. Dose-dependent alterations in endothelial actin filament organization were also found. Incubation of the monolayers exposed to 3,000 rads with medium supplemented with 10% fetal calf serum for 24 hours resulted in normalization of albumin permeability, improvement in morphologic appearance of the monolayers, and reorganization of the actin filament structure. These studies demonstrate that ionizing radiation is an active principle in the reversible disorganization of cultured pulmonary endothelial cell monolayers without the need of other cell types or serum components.

Albumins↗
PubMed1986

Radiolabelled substrates for angiotensin converting enzyme.

Six [3H]benzoyl-tripeptides were prepared and tested as substrates for angiotensin converting enzyme. Each was prepared first as its [4-iodo]-benzoyl-analog, and an atom of 3H per molecule was introduced by catalytic dehalogenation in 3H2-gas. Kinetic parameters were measured at 37 degrees C using as buffer 0.05 M Hepes, pH 8.0 containing 0.1 M NaCl and 0.6 M Na2SO4. When the substrates were used at concentrations far below their respective Km values, fractional rates of substrate utilization per unit time for constant enzyme concentration were direct function of respective second order rate constants (Kc/Km). Although absolute values of Kc/Km differed for human enzyme as opposed to rabbit enzyme, relative values of Kc/Km were virtually identical. Similarly, relative rates of substrates utilization during passage through lungs of anesthetized rats were similar to relative values of Kc/Km measured in vitro. Thus, there is now a range of ACE substrates usable, in vitro and in vivo, under conditions of first order enzyme kinetics, conditions under which values of V/Km and Ki can be measured directly.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1986

Aggregate anaphylaxis and carboxypeptidase N.

Bradykinin (BK) is widely believed to play a role in the pathogenesis of anaphylaxis. To help clarify any such roles, we examined for effects of inhibitors of kininase II (angiotensin converting enzyme, ACE) and "kininase I" (carboxypeptidase N, CPN), on the early course of egg albumin-induced aggregate anaphylaxis in anesthetized guinea pigs. In this model, pulmonary and systemic arterial blood pressure (BP) rise (unless pulmonary fibrillation occurs), lung wgt increases by approximately 60% and pulmonary microvessels are occluded by cell-rich thrombi, all within 5 min of i.v. antigen. The 30 min mortality rate is approximately 2%. ACE inhibitors (BPP9a, Captopril and MK 422; doses up to 140 mumol/kg) do not make anaphylaxis more nor less severe in terms discernible by changes in BP, lung wgt, EKG or intravascular coagulation. In marked contrast, an inhibitor of CPN (2-mercaptomethyl-3-guanidinoethylthiopropionic acid, 2-MGP; 8-16 mumol/kg) increases the 30 min mortality rate to 94% and lung wgt to 180% of control. The animals die in ventricular fibrillation. Given the enormous BK potentiating effects of BPP9a, Captopril and MK 422, it seems likely that little if any BK is formed in the early min of anaphylaxis. 2-MGP does not potentiate BP effects of BK but markedly potentiates effects of C3a anaphylatoxin. Thus, our data support the views that BK is neither a primary nor secondary mediator of aggregate anaphylaxis, and the adverse effects of 2-MGP are best explained in terms of preservation of anaphylatoxins and not in terms of preservation of kinins.

3-Mercaptopropionic Acid↗
PubMed1986

Receptor-mediated binding of C1q on pulmonary endothelial cells.

Normal undamaged pulmonary endothelial cells appear to be immunologically privileged in that they do not express receptors for the Fc portion of IgG nor for C3b. However, these receptors become unmasked on endothelial cells injured by viral infection or exposure to white cell lysates. We now present evidence to indicate that C1q binds to specific receptors on the surface of normal healthy endothelial cells. The binding is dose-dependent, reversible and saturable. Furthermore our data show that binding of C1q to endothelial cells is via the collagenous portion of the molecule not via the globular head regions. Thus binding of C1q to endothelium would have the effect of exposing Fc receptors that could then bind to IgG of circulating immune complexes. That Fc receptors are in fact exposed is shown by rosette formation with antibody sensitized erythrocytes. With 2C1r-2C1s-associated C1q, no binding occurred using C1 fixation and transfer assays. Our results indicate that C1q binding to endothelium provides a means for localizing immune complexes on pulmonary vessels and may be important in the initiation and progression of the inflammatory response.

Animals↗
PubMed1986

The endothelial surface and responses to injury.

Pulmonary microvascular endothelial cells are known to play an active role in the events that lead to vascular damage in the inflammatory response. The endothelial surface, normally immunologically privileged and very actively antithrombogenic, can respond to certain stimuli, generally injurious, by becoming strongly procoagulant and by expressing Fc and C3b receptors. Like macrophages, activated endothelial cells can provide a common source and substratum for combined hemostatic and complement-linked reactions. Such transformations of endothelial functions may involve alterations in the endothelial glycocalyx and, in addition, may be important for the entrapment and disposal of phagocytosed particulates. What is clear is that it is not simply absence of endothelium that has a bearing on the outcome of inflammatory stimuli but that structural and functional responses of the endothelial surface to injury, resulting in altered expression of hemostatic and immunologic potential, may have an important bearing on the role of the endothelium in the regulation of microvascular permeability.

Animals↗