Biomedical subjects

U S Ryan

Publications and source records attributed to U S Ryan.

At least 37 records · Page 2Linked to original sources

PubMed1993

Evaluation of activity of putative superoxide dismutase mimics. Direct analysis by stopped-flow kinetics.

By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.

Cyclic N-Oxides↗

PubMed1993

Inhibition of lung inflammatory reactions in rats by an anti-human IL-8 antibody.

IL-8 belongs to the family of chemotactic cytokines and may play an important role in the inflammatory response. In the current studies, a murine mAb (DM/C7) to human rIL-8 was found to have protective effects in inflammatory lung injury in rats. DM/C7 was nonreactive with the rat cytokine-induced neutrophil chemoattractant peptide. In vivo, DM/C7 blocked the glycogen-induced accumulation of neutrophils in rats and was highly protective against lung and dermal vascular injury after deposition of IgG immune complexes. The latter model of injury has recently been shown to be E-selectin dependent. The protective effects of DM/C7 correlated with reduced tissue accumulation of neutrophils, as measured by myeloperoxidase content. DM/C7 reacted with an epitope expressed by TNF-alpha-stimulated rat pulmonary artery endothelial cells and with the pulmonary vascular endothelium after intrapulmonary deposition of IgG immune complexes. In the model of IgG immune complex-induced lung injury, the protective effects of DM/C7 were abolished by prior absorption of the antibody with human rIL-8. Polyclonal antibody to cytokine-induced neutrophil chemoattractant peptide failed to protect against IgG immune complex-induced vascular injury even though this antibody blocked the in vitro chemotactic activity of cytokine-induced neutrophil chemoattractant. In the model of rapidly developing lung injury due to systemic activation of C after infusion of cobra venom factor, DM/C7 was not protective. As well, in the neutrophil-independent model of IgA immune complex-induced lung injury, treatment with DM/C7 was not protective. These data indicate that in inflammatory lung injury that is linked to E-selectin-dependent recruitment of neutrophils in rats, antibody to human IL-8 also blocks recruitment of neutrophils and thereby affords protection against lung injury. The data suggest the presence of an IL-8-like product in this model of lung injury.

Animals↗

PubMed1993

The diffusional transport of water and small solutes in isolated endothelial cells and erythrocytes.

The diffusional permeability coefficients, PD, for tritiated water (3HHO) 14C-antipyrine (AP) and 14C-iodoantipyrine (IAP) in isolated calf pulmonary artery endothelial cells and dog erythrocytes are measured with the linear diffusion technique at 11.5, 15, 20 and 37 degrees C. The PD values for both cell populations follow the sequence 3HHO > IAP > AP at each of the temperatures. PD for water is higher in the erythrocyte compared to the endothelial cells. The differences in PD for AP and IAP in the erythrocytes and endothelial cells are not dramatic and are similar to the differences seen in comparing permeation of the same solute through bilayers of different composition. A comparison of the values of PD calculated for the endothelial cells with those for isolated capillaries and the structured endothelium in whole lungs validates the use of the isolated cells as models for the endothelial cells in situ. Incubation of the endothelial cells with cis-vaccenic acid or cholesterol produces a reduction in PD for water and antipyrine. These data are analyzed in terms of Stokesian and non-Stokesian diffusion. The interpretation which best accommodates the data is that the phospholipid area of the membrane, rather than the hydrocarbon core, provides the greatest resistance to permeation for these solutes.

Alkanes↗

PubMed1993

Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells.

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet-derived growth factor (PDGF)-mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin-binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an alpha v-containing integrin receptor, but not alpha v beta 1 or alpha v beta 3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50-100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation.

Animals↗

PubMed1993

Intestinal ischemia-reperfusion injury causes pulmonary endothelial cell ATP depletion.

Intestinal ischemia-reperfusion is a common clinical event associated with both clinical and experimental distant organ injury. In particular, the pulmonary microvasculature appears to be susceptible to injury resulting from systemic inflammatory mediator activation. This study was designed to evaluate the hypothesis that noncellular humoral factors associated with intestinal ischemia-reperfusion result in pulmonary endothelial cell adenosine triphosphate (ATP) depletion. Male Sprague-Dawley rats had intestinal ischemia induced by microvascular clip occlusion of the superior mesenteric artery (SMA) for 120 minutes. Reperfusion resulted from superior mesenteric artery clip removal. After reperfusion for 0, 15, or 30 minutes, plasma samples were obtained from the portal vein. Monolayers of cultured rat pulmonary artery endothelial cells then were incubated with the plasma samples. Adenosine triphosphate levels were determined using a luciferin-luciferase assay. A 51Cr-release assay using labeled endothelial cells was performed under identical conditions to assess cytotoxicity. Potential mechanisms of ATP depletion were evaluated by analysis of cellular energy charge and assessment of microfilament architecture. Endothelial cell ATP levels decreased from 2.23 +/- 0.16 x 10(-11) moles/microgram DNA in sham preparations to 1.23 +/- 0.09 x 10(-11) moles/microgram DNA (p < 0.001) after 4 hours in plasma from animals undergoing 120 minutes of intestinal ischemia. For plasma obtained after 15 minutes of reperfusion, the decrease in cellular ATP concentration persisted (1.23 +/- 0.27 x 10(-11) moles/microgram DNA, p < 0.001 vs. sham). After 30 minutes' reperfusion, cellular ATP levels increased only slightly after the 4-hour incubation (1.39 +/- 0.26 x 10(-11) moles/microgram DNA, p < 0.005 vs. sham). No significant cytotoxic injury occurred in any group when compared with controls. Cellular energy charge was unchanged, and microfilament architecture was preserved. These data confirm the hypothesis that humoral factors, independent of the neutrophil, result in endothelial cell ATP depletion without metabolic inhibition or cell death. Depletion of energy stores by noncellular humoral factors may represent an early event that predisposes the cell to more severe injury by other mediators of the endogenous inflammatory response.

Actin Cytoskeleton↗

PubMed1993

Regulation of endothelin-1 expression in normal and transfected endothelial cells.

Calcium phosphate precipitation and retrovirus-mediated infection methods were used to stably infect bovine pulmonary artery endothelial cells (BPAECs) with mammalian expression vectors bearing human prepro-ET-1 cDNA. The calcium phosphate precipitation method afforded a stably transfected cell line that expressed approximately four times higher ET-1 than untransfected BPAEC by radioimmunoassay and at the mRNA level. The retrovirus-mediated transfection method yielded stably infected clones that secreted eightfold to 10-fold higher ET-1 than the nontransfected BPAECs; one clone continued to produce 10-fold higher levels after continuous assay for 1 year. Both transfected and nontransfected cells showed an increase (approximately twofold) in ET-1 production in response to thrombin (10 U/ml). Downregulation of ET-1 production was exhibited by both transfected and nontransfected cells in response to nitric oxide (NO) donors: sodium nitroprusside (NOPr), S-nitroso-N-acetoxy penicillamine (SNAP), and acetoxime. The potentiation of NO by superoxide dismutase (SOD) also downregulated ET-1 production. These studies show that an exogenous gene introduced into a cell type that normally expresses that gene product can be regulated by agonists and antagonists in a manner similar to the normal gene regulatory mechanisms for that cell type. This is of potential importance in gene therapy experiments, where mechanisms for regulation of expression remain elusive.

Animals↗

PubMed1993

Effect of endothelin-1 infusion on the development of intimal hyperplasia after balloon catheter injury.

Previous studies have shown endothelin-1 (ET-1) to be mitogenic for smooth-muscle cells. We explored in vivo the ability of high ET-1 levels to worsen angioplasty restenosis. Left carotid artery balloon endothelial denudation was performed on 14 rats. ET-1 was delivered via osmotic pump at a rate of 5 pmol/kg/min. Intimal development and plasma ET-1 levels were assessed at 2 weeks. Blood pressure and heart rate were measured throughout the study. For analysis, the animals were divided into three groups based on ET-1 levels at harvest: control, 4.3 +/- 0.5 pmol/ml (n = 6); low ET-1, 5.2 +/- 0.9 pmol/ml (n = 4); and high ET-1, 23.1 +/- 5.9 pmol/ml (n = 4). Although ET-1 infusion caused blood pressure elevation in both ET-1 groups, this was more marked and prolonged in the group with a high ET-1 level at study conclusion. Evaluation of the intimal:medial area ratio showed a marked increase in intimal thickness in the high ET-1 group versus control (1.13 +/- 0.23 versus 0.35 +/- 0.11; p < 0.05). We conclude that ET-1 infusion in responsive animals can cause worsening of the intimal hyperplastic response after mechanical injury. Further study is required to elucidate whether this is entirely caused by a direct effect of ET-1 on smooth-muscle cell mitogenesis or is also by the hemodynamic effects of ET-1-induced hypertension, or an effect of another mediator released in response to the ET-1 (e.g., angiotensin II).

Animals↗

PubMed1993

Sulfhydryl-depleting agents, but not deferoxamine, modulate EDRF action in cultured pulmonary arterial cells.

The potential role of intracellular sulfhydryls and iron on the biological activity of endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial (BPAE) cells was investigated in a cultured cell bioassay system, by measuring N omega-nitro-L-arginine-sensitive guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rabbit pulmonary arterial smooth muscle (SM) cells. The role of sulfhydryls in the biosynthesis of EDRF was studied by selectively exposing the endothelial cells to thiol-depleting agents. Both N-ethylmaleimide (NEM) and maleic acid diethyl ester (DEM) inhibited EDRF-induced cGMP accumulation in a dose-dependent manner. To study the requirement of SM thiols in the metabolism of EDRF to a stimulator of cGMP formation, SM were selectively exposed to NEM and DEM before bioassay with control, untreated BPAE. DEM and NEM inhibited cGMP formation in response to EDRF by 30 and 68%, respectively. The requirement of SM sulfhydryls was further investigated in the stimulation of SM cGMP accumulation elicited by nitrosothiols [S-nitroso-L-cysteine, S-nitroso-mercaptoproprionic acid, and sodium nitroprusside (SNP)]. NEM pretreatment of SM cells abolished cGMP responses to all vasodilators; DEM did not affect the nitrosothiol responses but reduced by 30% the cGMP accumulation to SNP. The role of iron in the endothelial synthesis of EDRF was assessed by chelating endothelial low-molecular-weight iron compounds. Exposure of BPAE to deferoxamine mesylate had no effect on cGMP accumulation in SM, suggesting that deferoxamine-available iron is not necessary for the endothelial stimulation of SM cGMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells.

IL-1 and TNF are important mediators in the inflammatory response, and have been associated with endothelial cell damage in the lung. TNF and IL-1 cell-mediated injury has been proposed to occur through an increase in intracellular oxygen free radical production. However, these cytokines have also been shown to protect the lung from hyperoxia-mediated oxidant injury. In this paper we evaluated the response of the antioxidant enzymes, MnSOD and Cu/ZnSOD to IL-1, TNF, and LPS in both rat pulmonary artery and microvascular endothelial cells. These mediators produced an increase in MnSOD but not Cu/ZnSOD expression in both rat pulmonary endothelial cells. An additive effect was observed with co-treatment by the cytokines with LPS. The MnSOD mRNA induction is dependent upon a transcriptional event, but did not require de novo protein synthesis.

Animals↗

PubMed1992

The synergistic effect of hypercholesterolemia and mechanical injury on intimal hyperplasia.

In an attempt to clarify data obtained from animal models of intimal hyperplasia, we used New Zealand white rabbits, a standardized balloon catheter injury model, and a 0.25% cholesterol supplemented diet. The effects of mechanical injury and hypercholesterolemia separately and combined were determined at the carotid and iliac positions at 12 weeks. En-face planimetry of lesioned intima and measurement of transverse intima-to-media thickness were taken as indices of intimal hyperplasia. No animals received antiplatelet agents or postoperative anticoagulation and all vessels remained patent. Neither procedure alone resulted in statistically significant lesion increase. However, combinations of injury and cholesterol resulted in statistically significant and synergistic lesion enhancement. The quantitative data, coupled with distinctive features noted on scanning electron microscopy and transmission electron microscopy, showed separate and synergistic effects of mechanical injury and cholesterol diet on intimal lesions in this model. Additionally, these effects must be considered in evaluation of animal models of intimal hyperplasia and atherosclerosis. Furthermore, this may help dissect mechanisms of failed revascularizations.

Animals↗

PubMed1992

Pulmonary endothelial cell ATP depletion following intestinal ischemia.

Multiple organ failure (MOF) is known to follow systemic inflammatory mediator activation associated with intestinal ischemia-reperfusion injury. In particular, the pulmonary microvasculature appears to be susceptible to MOF-related injury. This study was designed to evaluate the hypothesis that non-cellular plasma factors associated with intestinal ischemia without reperfusion also mediate pulmonary endothelial cell injury. Male Sprague-Dawley rats had intestinal ischemia induced by microvascular clip occlusion of the superior mesenteric artery for 30, 60, 90, or 120 min. Following each period of ischemia, plasma samples were obtained from the protal vein. Time-matched sham-operated animals served as controls. Monolayers of cultured rat pulmonary artery endothelial cells were then incubated with the plasma samples and ATP levels determined using a luciferin-luciferase assay. A 51Cr-release assay using labeled endothelial cells was performed under identical conditions to assess cytotoxicity. Endothelial cell ATP levels were 1.99 +/- 0.23 x 10(-11) mole/micrograms DNA in sham preparations. After a 4-hr incubation in plasma from the 90 and 120 min ischemia groups, cellular ATP levels fell significantly to 1.07 +/- 0.23 x 10(-11) mole/micrograms DNA, respectively (P less than 0.005). No significant cytotoxic injury resulted from incubation with plasma from the 120 min group (1.0 +/- 0.4% versus 0.8 +/- 0.4% in sham group, P = NS). All animals survived 24 hr in the sham, 30, and 60 min groups. However, survival was 40 and 0% in the 90 and 120 min groups, respectively (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1992

Cell-cell contact mechanisms.

Exciting new findings link characteristic properties of the inflammatory process previously not linked functionally. For example, it is now clear that oxygen radicals and leukocyte adhesion are intimately related in a carefully transduced and orchestrated series of events that culminates in release of granule contents, but not before the leukocyte has safely transversed the vessel wall. In addition to defining separate heterocellular contacts and homocellular aggregation we must now consider equilibrium events that allow associations among cell-cell partnerships involving different cell types.

Animals↗

PubMed1992

Culture of endothelial cells from baboon and human glomeruli.

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been a failure to isolate and propagate human glomerular capillary endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogeneous monolayers of primate (baboon and human) glomerular capillary endothelial cells. Using selective media and growth factors, the following criteria were identified to optimize the isolation and proliferation of glomerular endothelial cells: (1) collagenase treatment of isolated glomeruli; (2) requirement for 20% serum, endothelial cell growth factor and heparin; (3) requirement of fibronectin as surface matrix; and (4) isolation from donors less than 60 years old, as premature senescence was directly proportional to the age of the human kidney donor. Under these conditions, primary cultures with an endothelial cell composition greater than 70% were reproducibly obtained. Homogeneous endothelial monolayers were developed from 20 of 23 human kidneys, and maintained for 5 to 10 passages, depending on the age of the kidney donor. Purification to homogeneity was achieved by patch cloning or by fluorescence-activated cell sorting. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence, expressed factor VIII-related antigen, angiotensin converting enzyme activity, and endocytosed acetylated low-density lipoproteins. Electron microscopy revealed the presence of intracellular Weibel-Palade bodies and caveolae and microvillous projections on the luminal surface. Glomerular cells also stained positive for Ulex europaeus, a lectin characteristic of human endothelial cells. In addition, preliminary results indicate that human glomerular endothelial cells increase intracellular cyGMP in response to alpha-human 5 to 28 atrial natriuretic peptide and intracellular free calcium in response to thrombin.

Animals↗

PubMed1992

Endothelial cGMP does not regulate basal release of endothelium-derived relaxing factor in culture.

Guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in single and cocultures of calf pulmonary arterial endothelial (CPAE) and rabbit pulmonary arterial smooth muscle cells (RPASM) was investigated to discover whether endothelial cGMP is involved in the feedback regulation of basally released endothelium-derived relaxing factor (EDRF). Endothelial cell-induced increases in smooth muscle cGMP levels were inhibited by competitive inhibitors of endothelial nitric oxide synthesis, NG-monomethyl-L-arginine and N omega-nitro-L-arginine, in both long-term cocultures and short-term bioassay. Such treatment had no effect on endothelial content of cGMP. Coculture cGMP accumulation was stimulated (twofold increases) by endothelium-dependent vasodilators, bradykinin and acetylcholine. Bradykinin and acetylcholine did not elicit cGMP accumulation in single cultures of either smooth muscle or endothelial cells. To investigate the underlying mechanism(s) of dissociation in cGMP accumulation between cocultures and single endothelial cell cultures, the distribution profile of guanylate cyclase isoforms was determined by stimulating CPAE and RPASM cells with vasodilators activating selectively the soluble or particulate isoenzymes. Both nitrovasodilators, sodium nitroprusside and a putative EDRF, S-nitroso-L-cysteine, produced a 20-fold increase in cGMP content of RPASM cells only, having no effect on endothelial cells. Conversely, atriopeptin II caused 80-fold increases in endothelial cells. Exposure of the short-term bioassay system to 100 nM atriopeptin II, which caused 60-fold increases in CPAE cGMP levels, did not affect basal EDRF-induced smooth muscle cell cGMP accumulation, suggesting that a cGMP-mediated negative feedback mechanism does not appear to be involved in the regulation of basally released EDRF in culture.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Superoxide responses of endothelial cells to C5a and TNF-alpha: divergent signal transduction pathways.

There is increasing evidence that endothelial cells respond to a variety of mediators. In the current studies rat pulmonary artery endothelial cells (RPAEC) responded to human recombinant C5a and tumor necrosis factor-alpha (TNF-alpha) with the generation of superoxide (O2-). RPAEC responsiveness was dependent on whether cells had been obtained from confluent or subconfluent cell monolayers. RPAEC responded to C5a and TNF-alpha in a dose-dependent manner, with increases in intracellular Ca2+ (Cai2+), formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], and generation of O2-. Optimal O2- responses occurred in cells that had been pretreated with the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate, and O2- responses were allopurinol insensitive. Pertussis toxin pretreatment abolished the ability of C5a to cause increases in Ins(1,4,5)P3 and Cai2+ and formation of O2- but did not inhibit the changes in Cai2+ and formation of O2- after addition of TNF-alpha. The O2- response to C5a but not to TNF-alpha was abolished by pretreatment with the inhibitor of protein kinase C, staurosporine. These data indicate that signal transduction events in response to C5a and TNF-alpha were fundamentally different.

Alkaloids↗

PubMed1992

Microvascular and macrovascular endothelial cells produce different constrictor substances.

The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.

Allosteric Site↗

PubMed1992

Effects of oxidant stress on endothelium-derived relaxing factor-induced and nitrovasodilator-induced cGMP accumulation in vascular cells in culture.

The effects of hydrogen peroxide (H2O2) on the action of basally produced endothelium-derived relaxing factor (EDRF) were investigated by measuring cGMP accumulation in single and cocultures of calf pulmonary artery endothelial cells (CPAEs) and rabbit pulmonary artery smooth muscle cells (RPASMs) as a model for determining the contribution of EDRF dysfunction to altered vascular tone and reactivity frequently associated with oxidant-induced vascular injury. Higher cGMP levels in long-term cocultures (20.4 +/- 1.8 pmol/mg protein/15 min) than in single-cell cultures (CPAE, 9.6 +/- 0.9 pmol/mg protein/15 min; RPASM, 3.7 +/- 0.2 pmol/mg protein/15 min), and CPAE-induced increases (fivefold) in intracellular RPASM cGMP content in short-term cocultures suggest basal release of EDRF. Basal generation and release of an L-arginine-derived endothelial labile factor accounted for the increases in cGMP, since the response was completely blocked by pretreatment of CPAEs with NG-monomethyl L-arginine. Pretreatment of long-term cocultures with H2O2 for 30 minutes resulted in a dose-dependent (0.5-2 mM) decrease in cGMP formation (49-79%). To determine the effects of H2O2 on EDRF synthesis, transport, and RPASM responsiveness, CPAEs or RPASMs were selectively pretreated with H2O2 before establishment of short-term cocultures. In cocultures of H2O2-pretreated CPAEs with untreated RPASMs, RPASM cGMP levels were reduced, suggesting a decrease in EDRF production rather than deterioration of EDRF during transport, because cGMP levels were unaffected by posttreatment with oxygen radical scavengers during coculture. Pretreatment of RPASMs with H2O2 attenuated the untreated CPAE-induced, the putative EDRF S-nitroso-L-cysteine-induced, or the nitroprusside-induced increases in RPASM cGMP levels. This attenuation was prevented by pretreatment with either dimethylthiourea, deferoxamine, or dithiothreitol, suggesting a mechanism of H2O2 action involving iron-catalyzed formation of intracellular hydroxyl radicals and their attack on cellular thiols. H2O2 diminution of cGMP accumulation was not associated with lytic cell injury in the experimental time frame, because morphology and 51Cr release from prelabeled RPASMs and CPAEs were unchanged.

Animals↗