Biomedical subjects

E R Block

Publications and source records attributed to E R Block.

At least 19 recordsLinked to original sources

PubMed1992

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells.

We recently reported that nitrogen dioxide (NO2), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO2 resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO2 exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO2 for 1-24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p less than 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO2-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar1 Ile8] Ang II. Exposure of 5 ppm NO2 for 1-24 hr provoked rapid increases in [3H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO2 increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.

Analysis of Variance↗

PubMed1992

Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury.

The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time-dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate-treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia-enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge.

Adenosine Triphosphate↗

PubMed1992

Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: the combined effects of fatty acid supplementation and oxidant exposure.

Supplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oleic acid (18:1 omega 9) or with an equivalent volume of ethanol vehicle alone (ETOH-0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 microM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemental PAEC were exposed to HBSS or H2O2 either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2-induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2 reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury.

Animals↗

PubMed1992

Pulmonary endothelial cell pathobiology: implications for acute lung injury.

Pulmonary endothelial cells form a continuous monolayer on the luminal surface of the lung vasculature. Until the mid-1970s, the pulmonary endothelium was felt to provide little more than a passive surface for the exchange of gases, water, macromolecules, and some cell traffic. Recent evidence indicates that the pulmonary endothelium is a metabolically active surface, which provides a regulatory interface for the continual processing of blood-borne vasoactive molecules, plays an active role in hemostasis and immunologic and inflammatory events, regulates vascular tone, and interacts with inflammatory cells and neighboring vascular cell types. These metabolic properties are both constitutive and capable of being induced in response to stimuli or injury. Virtually any agent that causes pulmonary endothelial cell injury will lead to impairments in the functional metabolic properties of these cells, resulting in alterations in hemodynamics, hemofluidity, permeability, gas exchange, and intercellular signaling. The net result in the lung is often the clinical picture of acute lung injury with respiratory distress, refractory hypoxemia, diffuse alveolar infiltrates, and respiratory failure.

Acute Disease↗

PubMed1992

Effect of hypoxia on phospholipid metabolism in porcine pulmonary artery endothelial cells.

The effect of exposure of porcine pulmonary artery endothelial cells to hypoxic (0% O2) and normoxic (20% O2) conditions for 24 and 48 h on phospholipid metabolism was studied. Sonicates prepared from endothelial cells that were exposed to 24 h of hypoxia showed significant increases in phospholipase A1 (91%), phospholipase C (75%), and diacylglycerol lipase (57%) activities. Hypoxic exposure of cells for 48 h caused an increase in diacylglycerol lipase activity (54%) only. Hypoxia also caused significant decreases in ATP levels and ATP-dependent arachidonyl coenzyme A (CoA) synthetase activity. Phospholipase A2, lysophosphatidylcholine acyltransferase, and diacylglycerol acyltransferase activities were not influenced by 24 or 48 h of hypoxia. When endothelial cells were prelabeled with [3H]arachidonic acid and then exposed to hypoxia, increased counts were recovered from the free fatty acid fraction of medium and from the cell fatty acid esters, lysophospholipids, diacylglycerols, and triacylglycerols. There was a concomitant decreased recovery of counts from cell phospholipids. These results indicate that hypoxic exposure of endothelial cells altered phospholipid metabolism by activating deacylation pathways and inhibiting reacylation via ATP-dependent arachidonyl CoA synthetase.

Acylation↗

PubMed1992

Serotonin transport in reconstituted endothelial cell plasma membrane proteoliposomes: effect of hypoxia.

To determine whether changes in the lipid dynamics of the plasma membrane bilayer are responsible for hypoxic stimulation of serotonin (5-hydroxytryptamine [5-HT]) transport in pulmonary artery endothelial cells, we solubilized and isolated phospholipid and protein fractions from plasma membrane vesicles derived from endothelial cells exposed to 20% O2 (normoxia) or 0% O2 (hypoxia) for 24 h. Four different combinations of proteoliposomes were prepared by reconstituting (1) normoxic protein and normoxic phospholipid, (2) normoxic protein and hypoxic phospholipid, (3) hypoxic protein and normoxic phospholipid, and (4) hypoxic protein and hypoxic phospholipid. Fluorescence anisotropy of diphenylhexatriene (DPH), a measure of fluidity, and 5-HT transport were evaluated in each of the four groups of reconstituted proteoliposomes. 5-HT transport by the reconstituted proteoliposomes was saturable, linear with protein (5 to 25 micrograms) and time (15 to 60 s), and optimal with a phospholipid-to-protein ratio of 3:1. There were no significant differences in intravesicular volume, phospholipid-to-protein ratio, and size distribution among the four different groups of proteoliposomes. 5-HT transport was significantly higher and fluorescence anisotropy of DPH was significantly lower in proteoliposomes made from hypoxic phospholipids irrespective of the source of protein. Hypoxia also had a direct effect on the 5-HT transporter since uptake was increased slightly in proteoliposomes from group 3. These results indicate that changes in the plasma membrane phospholipids, and to a much lesser extent changes in the 5-HT transporter, are responsible for increases in the transmembrane transport of 5-HT by hypoxic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Plasma membrane-specific phospholipase A1 activation by nitrogen dioxide in pulmonary artery endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant, alters the plasma membrane structure and function of pulmonary artery endothelial cells through peroxidative injury. Because perioxidative injury can activate membrane phospholipases and alter phospholipid composition of membranes, we evaluated the effects of NO2 exposure on phospholipase A1 (PLA1), phospholipase A2 (PLA2), and diacylglycerol lipase (DG lipase) activities in pulmonary artery endothelial cell plasma, mitochondrial, and microsomal membranes. We also evaluated the effect of NO2 exposure on the phospholipid composition of plasma membranes of these cells. Exposure to 5 ppm NO2 for 48 hr resulted in a significant (p less than 0.01) increase in PLA1 activity in plasma membranes but not in mitochondrial or microsomal membranes of pulmonary artery endothelial cells, whereas PLA2 and DG lipase activities were comparable to controls in all membranes. As a result of PLA1 activation, the total phospholipid content of the plasma membranes of NO2-exposed cells was significantly (p less than 0.01) reduced compared to controls. Phosphatidylethanolamine (PE) content was reduced (p less than 0.05), whereas lyso-PE (LPE), a product of PLA1 hydrolysis of PE, as well as phosphatidylserine (PS) contents were increased (p less than 0.01 for both LPE and PS) in the plasma membranes of NO2-exposed cells. Incorporation of exogenous PS into pulmonary artery endothelial cells mimicked the stimulatory effect of NO2 on PLA1 activity. These results demonstrate that NO2 specifically reacts with the plasma membrane component of pulmonary artery endothelial cells, causing specific activation of PLA1. The NO2-induced increase of PS in the plasma membranes appears to be responsible for the specific activation of PLA1 in pulmonary artery endothelial cells.

Animals↗

PubMed1991

Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells.

Hydrogen peroxide (H2O2) is an important mediator of acute oxidative injury to vascular endothelium. Because the plasma membrane is the initial site of interaction between endothelial cells and extracellular H2O2 produced by stimulated neutrophils or macrophages, we evaluated the effect of H2O2 on the physical state, i.e., fluidity, and function of porcine pulmonary artery endothelial cell plasma membranes. Lactate dehydrogenase (LDH) release, 5-hydroxytryptamine (5-HT) uptake, limiting fluorescence anisotropy (r infinity) for trimethylamino-diphenylhexatriene (TMA-DPH), and conjugated dienes were measured 0.5, 6, and 24 hr after cells were exposed for 30 min to 50-microM H2O2 or Hank's Balanced Salt Solution (control). Compared with control cells, H2O2 caused significant increases in LDH release and in 5-HT uptake 6 hr after exposure. The increase in 5-HT uptake was not blocked by imipramine. H2O2 also caused a significant increase in r infinity for TMA-DPH 0.5 hr after exposure and a significant reduction in r infinity for TMA-DPH 6 hr after exposure. Cellular contents of conjugated dienes were increased 0.5 and 6 hr after exposure to H2O2. Twenty-four hours after exposure LDH release, r infinity, 5-HT uptake, and conjugated dienes had returned to control levels. Preincubation with 50-microM alpha-tocopherol (vitamin E) or 1-mM or 10-mM dimethylthiourea (DMTU) for 1 hr or 24 hr prevented endothelial cell injury, whereas addition of vitamin E or DMTU to the medium 1 hr or 3 hr after H2O2 exposure did not protect against injury. These results indicate that H2O2 causes significant damage to the plasma membrane of pulmonary artery endothelial cells in vitro, leading to alterations in fluidity and leakiness of the membrane. This injury is associated with membrane lipid peroxidation, is reversible, and can be prevented by pretreatment, but not by post-treatment, with vitamin E or DMTU.

Animals↗

PubMed1991

The effects of endotoxin on glutamine transport by pulmonary artery endothelial cells.

The effects of endotoxin on glutamine transport by cultured pulmonary artery endothelial cells (PAECs) were studied in order to gain further insight into the regulation of the altered lung glutamine metabolism that characterizes severe infection. Incubation of PAECs with endotoxin (1 micrograms/ml) resulted in a significant increase in System ASC-mediated glutamine transport which did not occur for 8 hr and was maximal after 12 hr of exposure. Kinetic studies indicated that the increase in carrier-mediated activity was not due to a change in Km (101 +/- 6 microM in controls vs 97 +/- 4 microM in endotoxin-treated cells, P = NS), but rather to a 73% increase in Vmax (840 +/- 60 pmole/mg protein/30 sec in controls vs 1450 +/- 80 in endotoxin-treated cells, P less than 0.001). The increase in glutamine uptake by PAECs was completely blocked by actinomycin D and cycloheximide, indicating that the accelerated glutamine transport was most probably due to an increase in transporter synthesis. Endotoxin stimulates glutamine uptake by PAECs, either directly or indirectly, an adaptive response which may be necessary to support cellular metabolism, structure, and function.

Animals↗

PubMed1991

Characterization of L-glutamine transport by pulmonary artery endothelial cells.

This study characterized the transport of L-glutamine by porcine pulmonary artery endothelial cells (PAECs). Uptake of 50 microM glutamine was determined and found to be linear for at least 45 min. The sodium-dependent velocity represented greater than 95% of the total uptake at all time points. Kinetic studies of glutamine uptake at concentrations between 0.005 and 10 mM showed a single saturable high-affinity carrier with a Michaelis constant of 100 +/- 6 microM and a maximal transport velocity of 1.0 +/- 0.08 nmol.mg protein-1.30s-1. Glutamine uptake by PAECs was markedly inhibited in the presence of L-cysteine, L-threonine, or L-alanine; lesser degrees of inhibition occurred when histidine and arginine were added. 2-Methylaminoisobutyric acid and 2-aminobicyclo [2,2,1]heptanedicarboxylic acid had little effect on glutamine uptake. Lithium did not substitute for sodium, strongly suggesting that L-glutamine was not transported by system N. Furthermore, transport of glutamine was not affected by hormones or by changes in external pH. Based on the intolerance of this high-affinity carrier to N-methylated substrate, its insensitivity to pH and hormonal regulation, and the failure of lithium to substitute for sodium, as well as its inhibition by alanine and cysteine, we conclude that in porcine pulmonary artery endothelial cells L-glutamine is predominantly taken up through system ASC.

Amino Acids↗

PubMed1991

Regulation of manganese superoxide dismutase in porcine pulmonary artery endothelial cells.

Bacterial lipopolysaccharide (LPS) was shown to produce an induction of manganese superoxide dismutase (Mn SOD) mRNA levels in porcine pulmonary artery endothelial cells (PAEC). Additional studies in porcine PAEC also demonstrated induction of Mn SOD mRNA in response to the inflammatory mediators interleukin 1 and tumor necrosis factor. On the other hand, we observed no change in Mn SOD mRNA within 24 h of a hyperoxic exposure. The induction of Mn SOD by LPS was blocked by both a RNA synthesis inhibitor, actinomycin D, and a protein synthesis inhibitor, cycloheximide. The data implicate the involvement of Mn SOD in the acute phase response of pulmonary endothelial cells.

Animals↗

PubMed1991

Supplemental fatty acids alter lipid peroxidation and oxidant injury in endothelial cells.

The purpose of this study was to determine the effects of supplemental fatty acids on oxidant injury in cultured endothelial cells. Porcine pulmonary artery endothelial cells (PAEC) in monolayer culture were incubated in culture medium supplemented with 0.1 mM fatty acid or with fatty acid vehicle alone for 3 h. Monolayers were then exposed to oxidant stress (100 microM H2O2 in buffer) or to control conditions (buffer alone) for 30 min. Supplementation with stearic acid (18:0) or oleic acid [18:1(n-9)] reduced H2O2-induced PAEC injury measured as release of intracellular lactate dehydrogenase (LDH). In contrast, supplementation with linolenic acid [18:3(n-6)] or eicosatrienoic acid [20:3(n-3)] enhanced H2O2-induced injury to PAEC. Both supplemental cis-vaccenic acid [18:1(n-7)] and 18:1(n-9) reduced the production of lipid peroxidation products in oxidant-stressed PAEC, whereas supplementation with 18:3(n-6) enhanced lipid peroxidation. Supplementation with 18:1(n-9) protected PAEC from H2O2 as long as 72 h after supplementation despite the intracellular redistribution of [18:1(n-9)] from triglycerides to phospholipids. Saturated and monounsaturated supplemental fatty acids protected PAEC from oxidant injury, but polyunsaturated fatty acids enhanced oxidant injury. These results support the hypothesis that supplemental fatty acids replace resident fatty acids, alter the oxidant reactivity of the cellular lipids, and thereby modify the oxidant susceptibility of PAEC.

Animals↗

PubMed1991

Angiotensin receptor-mediated stimulation of diacylglycerol production in pulmonary artery endothelial cells.

The stimulatory effects of angiotensin (Ang) I, Ang II, and Ang III on production of diacylglycerol (DAG), a second messenger, were examined in porcine pulmonary artery endothelial cells. Ang I, Ang II, and Ang III provoked rapid increases in [3H]glycerol labeling of DAG. The stimulatory effect on DAG production was maximal after 1 and 5 min. Pretreatment of cells with angiotensin-converting enzyme activity inhibitors prevented the stimulatory effect of Ang I on DAG production, indicating that Ang II but not Ang I is responsible for increased DAG production. The stimulatory effects of Ang II and Ang III on DAG production were concentration dependent and were maximal at a 10-nM concentration of both Ang II and Ang III. Data from further experiments revealed that the Ang II- and Ang III-elicited formation of DAG is derived from the coordinated hydrolysis of membrane phosphatidylinositol and phosphatidylcholine by phospholipase C- and phospholipase D-catalyzed pathways. The angiotensin analogue [Sar1 Ile8] Ang II, an Ang II receptor antagonist, blocked the hydrolysis of phosphatidylinositol and phosphatidylcholine and thus the increased production of DAG by Ang II and Ang III. These results indicate that Ang II- and Ang III-induced stimulation of DAG production in pulmonary artery endothelial cells involves multiple pathways of phospholipid hydrolysis and is mediated by angiotensin receptors.

Angiotensins↗

PubMed1990

Exposure of pulmonary artery endothelial cells to nitrogen dioxide activates phospholipase A1.

Phospholipase A1, A2, and C and diacylglycerol lipase activities were measured in cell sonicates after exposing confluent monolayers of porcine pulmonary artery endothelial cells to 5 ppm NO2, a toxic constituent of environmental pollution, for 24 and 48 hr. There was a significant increase (2.25-fold) in phospholipase A1 activity in 24 and 48 hr NO2-exposed cells, whereas activities of phospholipases A2 and C and diacylglycerol lipase were comparable to control cells at both time points. When endothelial cells were prelabeled with [3H]-arachidonic acid and then exposed to NO2 for 48 hr, increased counts were recovered from cell lysophospholipids with concomitant decreased recovery of counts from cell phosphatidylcholine and phosphatidylethanolamine. These results demonstrate that NO2 exposure results in specific activation of phospholipase A1.

Animals↗

PubMed1990

Oxidant injury increases cell surface receptor binding of angiotensin II to pulmonary artery endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant, is known to activate phospholipase A1 and modulate the plasma membrane structure of porcine pulmonary artery endothelial cells. We evaluated the effects of exposure to NO2, purified phospholipase B (which acts as phospholipase A1 and A2), or phospholipase A2 on 125I-angiotensin II (Ang II) receptor binding, internalization, or both in pulmonary endothelial cells. Exposure to 5 ppm NO2 for 48 hr at 37 degrees C or 0.075 U each of phospholipase B or A2 in phosphate-buffered saline (PBS) for 30 min at 24 degrees C resulted in an increase in total Ang II binding (i.e., cell surface bound and internalized) by 45% (p less than 0.05), 50% (p less than 0.05), and 85% (p less than 0.001), respectively, compared to controls. An Ang II receptor antagonist, [Sar1 Ile8] Ang II, competitively displaced Ang II binding to control, NO2-, phospholipase B-, and phospholipase A2-exposed cells. Dissociation of bound Ang II in the presence of PBS was less than 1% of total bound Ang II in control, NO2-, and phospholipase B-exposed cells and was 50% of total bound Ang II in phospholipase A2-exposed cells. In the presence of isotonic acetic acid/NaCl, in excess of 90% of cell surface-bound Ang II was dissociated from control, NO2-, and phospholipase B-exposed cells, and there was less than 2% of Ang II detectable when acid-treated cells were subjected to NaOH solubilization. In cells exposed to phospholipase A2, acetic acid treatment did not release cell-bound Ang II, and the remaining Ang II was recovered in the NaOH solubilized fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1990

Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes.

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.

Animals↗

PubMed1990

Clinical role of bronchoalveolar lavage in adults with pulmonary disease.

BAL remains a powerful investigative tool. In a short span of 20 yr, it has helped tremendously in understanding some of the aspects of the pathogenesis of diseases involving the lower respiratory tract. To realize its full potential in the diagnosis and management of diseases involving the lower respiratory tract, there is a great need for standardization of the technical aspects of BAL as well as processing and analysis of the BAL cellular- and fluid-phase components. Despite these hurdles, BAL has been found to be diagnostic in several infectious and noninfectious diseases involving the lower respiratory tract, and it provides valuable information that may be helpful in characterizing the prognosis and response to therapy in certain interstitial diseases of the lung. It is expected that with future research, in particular long-term prospective epidemiologic and clinical studies in pneumoconioses and in other interstitial lung disease, BAL will prove more valuable in the diagnosis and management of such disease.

Bronchoalveolar Lavage Fluid↗

PubMed1990

Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell plasma membrane vesicles.

To determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin (5-HT), we measured 5-HT transport activity: (1) in plasma membrane vesicles isolated from normoxic and hypoxic endothelial cells, (2) in endothelial cell plasma membrane vesicles that were exposed directly to normoxia or hypoxia, and (3) in endothelial cell monolayers incubated in the presence of 1 x 10(-7) M cycloheximide and exposed to normoxia or hypoxia. A 24-h exposure of endothelial cells to hypoxia resulted in a 40% increase (P less than 0.005) in specific 5-HT transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37 degrees C, a 31% increase (P less than 0.005) in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of 5-HT transport (normoxia = 3.47 microM versus hypoxia = 3.76 microM) but markedly increased the maximal rate of transport (Vmax) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). Cycloheximide alone had no effect on 5-HT transport by normoxic endothelial cells but did block hypoxia-induced increases in 5-HT uptake in endothelial cell monolayers exposed to 24-h hypoxia. These results indicate that hypoxia increases 5-HT transport in pulmonary artery endothelial cells by a direct effect on the plasma membrane, leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for 5-HT, and possibly by an indirect effect involving de novo protein synthesis.

Animals↗