Microvascular pericyte contractility in vitro: comparison with other cells of the vascular wall.

Collagen lattices containing bovine retinal pericytes (RPs), vascular smooth muscle cells (VSMCs), pulmonary microvessel endothelial cells (PMECs), or aortic endothelial cells (AECs) were prepared and contraction was quantitated by measuring the resulting change in lattice area. VSMCs were the most efficient at lattice contraction followed by RPs and then PMECs. AECs did not contract the lattices. To document further that these observations represent contraction, cells were grown on inert silicone rubber sheets. Substratum wrinkling was indicative of tension development and quantitated as percent of cells contracted. RPs were more contractile than PMECs, and AECs were incapable of developing tension. VSMCs were less contractile than RPs, unlike the comparative contractility observed with the lattice system. Alteration of actin-containing filaments by cytochalasin B significantly reduced RP contraction of silicone rubber and inhibited their contraction of collagen lattices in a dose-dependent manner. Rhodamine-phalloidin staining of contracting RPs revealed microfilament bundle orientations that suggested their association in the force applied for contraction. RP, VSMC and PMEC contraction of collagen lattices was directly proportional to the concentration of fetal calf serum. Also, RP contraction was greater in calf serum than calf plasma-derived serum, an indication that RPs respond to substances that appear continuously and episodically in blood. These in vitro findings support the theory that pericytes in vivo are contractile but that endothelial cells may also contribute to microvascular tonus.

Thromboxane mediation of cardiopulmonary effects of embolism.

Humoral factors released from platelets during pulmonary embolism may be the cause of several attendant cardiopulmonary abnormalities. This study examines the role of thromboxanes (Tx) after experimental embolism induced with 0.5 g/kg autologous clot in four groups of five dogs: (a) untreated embolized controls; (b) pretreatment with the Tx synthetase inhibitor, imidazole 25 mg/kg . h i.v., starting 30 min before embolization; (c) pretreatment with the cyclooxygenase inhibitor indomethacin, 5 mg/kg, 12 h per os and 1 mg/kg, 1 h i.v. before the experiment; (d) treatment with prostacyclin (PGI(2)) 100 etag/kg . min i.v. for 1 h, 1 h after embolization. Within 30 min, embolization led to increases of 6-keto-PGF(1alpha), the stable hydrolysis product of PGI(2), from 0.11+/-0.08 etag/ml (mean+/-SD) to 0.33+/-0.10 etag/ml (P < 0.005) and TxB(2), the stable product of TxA(2), from 0.10+/-0.04 etag/ml to 0.38+/-0.06 etag/ml (P < 0.001). Increases were observed in total dead space (V(D)/V(T)) from 0.46+/-0.03 to 0.61+/-0.08 (P < 0.025, physiologic shunting (Q(S)/Q(T)) from 16+/-4% to 38+/-9% (P < 0.01), pulmonary vascular resistance (PVR) from 2.27+/-0.59 mm Hg.min/liter to 9.21+/-1.90 mm Hg.min/liter (P < 0.005) and mean pulmonary arterial pressure from 14+/-6 mm Hg to 34+/-1 mm Hg (P < 0.001). Cardiac index (CI) fell from 139+/-11 ml/kg.min to 95+/-17 ml/kg.min in 4 h (P < 0.025). Imidazole pretreatment prevented a rise of TxB(2), but not 6-keto-PGF(1alpha); indomethacin blocked both. Both agents maintained V(D)/V(T) at base line and limited increases in Q(S)/Q(T) and PVR. CI was higher after imidazole pretreatment compared with controls (P < 0.025). Indomethacin led to intermediate levels of CI. PGI(2) lowered TxB(2) (P < 0.025), V(D)/V(T) (P < 0.025), Q(S)/Q(T) (P < 0.025) and PVR (P < 0.05) within 30 min. During PGI(2) infusion, CI was higher than controls. Concentrations of TxB(2) correlated with V(D)/V(T), r = 0.79 and Q(S)/Q(T), r = 0.69 (P < 0.001). Treatment of three dogs with the imidazole derivative ketoconazole, 10 mg/kg IV, 30 min after 0.75 g/kg autologous clot resulted in a lowering of physiologic dead space, but no other improvement of cardiopulmonary function. These results show that a number of cardiopulmonary abnormalities induced by pulmonary embolism are related directly or indirectly to platelet secretions and that V(D)/V(T) is closely allied to TxA(2) levels.

Prostacyclin reversal of lethal endotoxemia in dogs.

Severe endotoxemia, a condition where microembolization and intravascular coagulation are thought to play important roles, was treated experimentally with prostacyclin (PGI(2)). In a study of 24 dogs, 8 control animals injected with 1.75 mg.kg(-1) of endotoxin died within 24 h. Six animals given intravenous aspirin 100 mg/kg, 30 min after endotoxin died. 9 of 10 dogs infused with 100 ng PGI(2).kg(-1).min(-1) for 3 h, given 30 min after the injection of endotoxin survived 24 h (P < 0.025). Injection of endotoxin resulted in a: (a) maximal 62% fall in mean arterial pressure (P < 0.001); (b) transient doubling of mean pulmonary arterial pressure (P < 0.001); (c) initial 70% drop in cardiac index (P < 0.001); (d) decline in blood platelets from 213,700 to 13,700/mm(3) (P < 0.001), and leukocytes from 7,719 to < 750/mm(3) (P < 0.001); (e) depressed urine output (P < 0.001); (f) 34% decrease in blood fibrinogen (P < 0.01) and an increase in fibrin degradation products > 50 mug/ml (P < 0.001); (g) fivefold increase in circulating cathepsin D titer (P < 0.005) and (h) increase in blood norepinephrine (P < 0.005), dopamine (P < 0.005), and epinephrine (P < 0.001). Aspirin treatment led to an increase in mean arterial pressure (P < 0.001) and mean pulmonary arterial pressure (P < 0.005), but cardiac index, urine flow, platelets, leukocytes, fibrin degradation products, and cathepsin D levels remained similar to untreated controls. After infusion of PGI(2) there was a: (a) prompt increase of cardiac index to base-line levels; (b) late increase in mean arterial pressure (P < 0.005) after the discontinuation of PGI(2) treatment (c) restoration of urine output; (d) increase in circulating platelets to levels still below base line but above untreated control animals (P < 0.05); (e) no effect on circulating leukocyte levels; (f) fall in fibrin degradation products to 11.2 mug/ml (P < 0.05); (g) decline in cathepsin D levels to values 60% lower than the untreated controls (P < 0.025); and (h) reduction in plasma norepinephrine levels to base line at 4 h (P < 0.005). Although the mode of PGI(2) action is not clear, it is effective in the treatment of experimental endotoxemia.

Role of thromboxane in interleukin 2-induced lung injury in sheep.

Interleukin (IL)-2 administration leads to respiratory dysfunction due to increased vascular permeability. This study examines the role of thromboxane (Tx)A2 in IL-2 induced lung injury in sheep with chronic lung lymph fistulae. This preparation enables evaluation of permeability prior to the development of gross edema. IL-2, 10(5) units/kg (n = 6), or its excipient control (n = 5) was given as an i.v. bolus over 2 min. After 2 h of IL-2 administration, plasma TxB2 increased from 168 to 388 pg/ml (P less than 0.05) and lung lymph TxB2 from 235 to 694 pg/ml (P less than 0.05). Mean pulmonary artery pressure (MPAP) rose from 13 to 29 mm of Hg (P less than 0.05) at 30 min and remained elevated for 4 h while the pulmonary artery wedge pressure was unchanged at 4 mm of Hg. Arterial oxygen tension (PaO2) fell from 88 to 77 mm of Hg (P less than 0.05). Lung lymph flow (QL) rose from 2.2 to 3.8 ml/30 min (P less than 0.05) at 1 h and to 6.4 ml/30 min at 3 h. This rise coincided with an increase in the lymph/plasma (L/P) protein ratio from 0.67 to 0.77 (P less than 0.05). In contrast, the non-IL-2-infused sheep (n = 3) recruitment of the lung vasculature by left atrial balloon inflation led to a rise in QL from 2.4 to 8.2 ml/30 min, whereas the L/P ratio declined from 0.62 to 0.25, suggesting that the protein-rich lymph flow after IL-2 administration reflected increased microvascular permeability. In further proof of an increase in permeability, IL-2 administration into sheep (n = 2) with an inflated left atrial balloon led, after a pressure-independent L/P protein ratio had been achieved, to an increase in L/P protein ratio and decrease in protein reflection coefficient. At 2 h after IL-2, the blood leukocyte count fell from 8156 to 4375/mm3 (P less than 0.05) primarily due to a 73% drop in lymphocytes. The platelet count declined from 292 to 184 x 10(3)/mm3 (P less than 0.05). Body temperature rose from 38.9-40.3 degrees C (P less than 0.05), and shaking chills were common. Pretreatment with the Tx synthetase inhibitor OKY 046 (n = 7) lowered baseline plasma and lymph TxB2 levels to 22 and 52 pg/ml (P less than 0.05) and prevented the IL-2-induced increase in plasma and lung lymph TxB2 (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium uptake and associated adenosine triphosphatase activity of isolated platelet membranes.

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200-400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40-60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 microM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5-10 microM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.

Activation of endothelial cell kinin receptors leads to intracellular calcium increases and filamin translocation: regulation by protein kinase C.

Membrane-associated cytoskeletal proteins provide support for endothelial cell (EC) junctional cell adhesion molecules. Nonmuscle filamin is a dimeric actin cross-linking protein that interacts with F-actin and membrane glycoproteins. Both bradykinin and des-Arg9-bradykinin cause filamin redistribution from the plasma membrane to the cytosol of confluent EC. Kinin-induced filamin translocation parallels the dynamics of intracellular Ca2+ increases. Pretreatment with kinin receptor antagonists blocks the Ca2+ response as well as filamin translocation induced by kinins. Protein kinase C activation prior to kinin stimulation attenuates intracellular Ca2+ increases and filamin translocation. BAPTA, a cell-permeable Ca2+ chelator, attenuates bradykinin-induced intracellular Ca2+ increases and filamin translocation. This study demonstrates that bovine pulmonary artery ECs express both kinin B1 and B2 receptors, and that activation of either receptor leads to intracellular Ca2+ increases. This Ca2+ signalling, which is downregulated by protein kinase C activation, is essential for kinin-induced filamin translocation.

Hypoxia reoxygenation-induced injury of cultured pulmonary microvessel endothelial cells.

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.

Filamin redistribution in an endothelial cell reoxygenation injury model.

Ischemia-reperfusion injury increases vascular permeability in part by generating reactive oxygen species that disassemble the endothelial cell actin dense peripheral band. This is followed by an increase in the number and diameter of intercellular gaps. Millimolar concentrations of reactive oxygen metabolites lead to nonspecific endothelial cell injury, but micromolar concentrations activate inflammatory second messenger cascades which produce distributional changes in endothelial cell cytoskeletal proteins. H2O2 (100 microM) causes translocation of filamin, from the membrane to the cytosol within 1 min. Subsequently, gap formation occurs within 10-25 min, which is attributed to rearrangement of the dense peripheral band of F-actin. Plasma membrane blebbing occurs after 90 min and decreases in mitochondrial activity occur after 1-2 h. Deferoxamine (iron chelator) and TEMPO (nonspecific free radical scavenger) inhibit these changes. H2O2 (100-1000 microM) does not increase endothelial cell intracellular Ca2+ through 30 min and pretreating cells with a Ca2+-calmodulin kinase inhibitor or an intracellular Ca2+ chelator does not prevent filamin translocation. Filamin redistribution and actin rearrangement are early events in H2O2-mediated endothelial cell injury that appear to occur through Ca2+-independent pathways.

Solid-phase metal chelate assay for quantifying total protein: resistance to chemical interference.

Recently, we developed reversible metal chelate stains that are fully compatible with immunoblotting and protein sequencing. Membrane supports are incubated in Ferrozine/ferrous complex followed by ferrocyanide/ferric complex (double-metal chelate [DMC] stain). Proteins are quantified by computerized densitometry. In this study, the metal chelate stains are used for routine protein quantitation. Manually applying samples to membranes leads to variable spot spreading. Better results are achieved using a slot-blot apparatus to maintain a constant application area. The Ferrozine/ferrous and DMC assays are compared to colloidal gold and bicinchroninic acid (BCA) assays with respect to chemical interference, protein-to-protein variation, dynamic linear range and sensitivity. The DMC assay provides a superior linear range (100-fold range) and BCA assays (47-fold). Though the colloidal gold assay is more sensitive, it suffers from poor reproducibility, high protein-to-protein variation and lower tolerance to interfering agents. The BCA assay has the least protein-to-protein variation but is also least sensitive and most susceptible to interfering agents.

Selective propagation of retinal pericytes in mixed microvascular cell cultures using L-leucine-methyl ester.

Endothelial cell (EC) propagation has been simplified by developing cell-specific selection criteria. Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue, (ii) differential plating of cells on extracellular matrices, (iii) lectin affinity isolation of cell populations and (iv) fluorescence-activated cell sorting of cells labeled with a carbocyanine dye of acetylated low-density lipoprotein (DiI-Ac-LDL). Few criteria for selectively propagating pericytes (PC) are currently available. Nonspecific esterases exhibit a high degree of multiplicity when compared with other mammalian isozymes and may be suitable for the identification and selective propagation of cells of the microvasculature. Evaluation of esterase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing esterases as well as dipeptidyl peptidase I, while EC only contain alpha-naphthyl acetate esterase. The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring vital dye uptake and by light microscopy. Several amino acid esters are cytotoxic to both cell types, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC. This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cultures. Analysis of protein composition by two-dimensional gel electrophoresis indicates that L-Leu OMe does not stimulate expression of stress response proteins in PC. Thus, L-Leu OMe can be utilized to cultivate PC selectively from mixed cell populations.

Effect of sodium chloride on limulus amebocyte lysate. Inhibition of endotoxin activation of procoagulase.

Concentrations of sodium chloride up to 3 M increase the time necessary for the clot formation from Limulus amebocyte lysate (LAL) induced with endotoxin. Sodium chloride at a concentration of 4 M prevents clot formation by either precipitation or denaturation of procoagulase. The time necessary for the activation of procoagulase by endotoxin is increased by a change in the sodium chloride concentration from 0.15 M to 0.588 M. No effect on the proteolytic phase or the polymerization phase of the clotting reaction is detected by the increase in sodium chloride concentration from 0.15 M to 0.588 M. The authors conclude that increased sodium chloride concentrations may aid the isolation of procoagulase.

Calcium electrode--a method for the continuous monitoring of the platelet release reaction.

The calcium electrode is a convenient, inexpensive, and non-traumatic method for measuring changes in the extracellular calcium which accompanies a platelet release reaction. With this instrument, the temporal pattern of release by platelets in a buffered-saline medium following thrombin stimulation was observed as follows: an initial time lag phase, followed by a maximum release phase, and finally a slow release phase.

Ornithine decarboxylase activity in cultured endothelial cells stimulated by serum, thrombin and serotonin.

Ornithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10(-5) M) or Lilly 110140 (10(-6) M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Anti-selectin therapy modifies skeletal muscle ischemia and reperfusion injury.

Restoration of blood flow to ischemic skeletal muscle results in a reperfusion injury characterized by permeability edema in part mediated by neutrophils that adhere via the selectin family of adhesion molecules. Rats underwent 4 h of hindlimb tourniquet ischemia followed by 4 h reperfusion. The role of neutrophils was determined by rendering one group of animals neutropenic before ischemia. In additional experimental groups, selectins were blocked with either a soluble form of the selectin counter-receptor, sialyl-Lewis X (SLX) or a monoclonal antibody directed against P-selectin (PB1.3). Neutrophil depletion resulted in a 36.1% reduction in hindlimb permeability (p < .05). SLX reduced hindlimb permeability index (PI) 23.9% at 1 mg/kg and 36.1% at 10 mg/kg compared to a nonfucosylated oligosaccharide, sialyl-N-acetylactosamine (p < .05). SLX also reduced neutrophil sequestration by 48.6% (p < .05). PB1.3 reduced hindlimb injury by 26.5% (p < .05) but did not reduce leukosequestration. We interpret these data to indicate that ischemia and reperfusion lead to selectin-mediated neutrophil sequestration. The oligosaccharide SLX, while moderately effective in limiting neutrophil sequestration was as effective as neutrophil depletion in reducing hindlimb permeability. The lack of concordance between the ability of SLX and PB1.3 in limiting neutrophil sequestration and permeability indicate mechanisms of action of these two agents that are in addition to the blocking of adhesion.

Synergism between leukotriene B4 and thromboxane A2 in mediating acid-aspiration injury.

Acid aspiration leads to thromboxane-dependent lung neutrophil sequestration associated with microvascular permeability increase. Leukotriene B4 (LTB4) is postulated to be a cofactor in the thromboxane-induced inflammatory response. This study tests the interaction between LTB4 and thromboxane, focusing on LTB4 induction of thromboxane-dependent lung neutrophil sequestration after acid aspiration. Anesthetized rats underwent tracheostomy and insertion of a cannula in a left lung segment. This was followed by instillation of either 0.1 ml 0.1N hydrochloric acid (n = 18) or 0.1 ml saline in control rats (n = 18). When assayed at 3 hours, acid aspiration led to increased plasma levels of LTB4 and thromboxane B2 (TxB2), higher than control values (p less than 0.05). The rise in plasma LTB4 was correlated (p less than 0.05; r = 0.83) with sequestration of neutrophils in the nonaspirated lung. The entrapment of thromboxane-dependent lung neutrophil was associated with an increase in protein concentration in bronchoalveolar lavage of the aspirated and nonaspirated sides and an increase in lung wet to dry weight ratio. Pretreatment of other rats (n = 18) with the lipoxygenase inhibitor diethylcarbamazine IV prevented an aspiration-induced rise in plasma LTB4 and TxB2. Further, there was an attenuation of lung leukosequestration and protein leak in bronchoalveolar lavage and lung edema (all p less than 0.05). Pretreatment of other rats (n = 12) with the leukotriene receptor antagonist FPL 55712 IV did not prevent the aspiration-induced rise in LTB4 or TxB2, but otherwise was as effective as diethylcarbamazine in preventing injury. Finally, other hydrochloric acid-aspirated rats (n = 8) were pretreated intravenously with the thromboxane synthetase inhibitor OKY 046 or the thromboxane receptor antagonist SQ 29548. Both agents limited the aspiration-induced rise in plasma LTB4 (p less than 0.05). The data indicate that localized acid aspiration induces synthesis of LTB4 and thromboxane A2. Inhibition of either leukotriene or thromboxane will limit PMN adhesion and increased lung permeability.

Prostacyclin and thromboxane A2 moderate postischemic renal failure.

Since prostacyclin (PGI2) is known to regulate renal cortical blood flow and since ischemia stimulates thromboxane (Tx) A2 synthesis, the role of these prostanoids in moderating the response to renal ischemia was studied in the rat. At baseline, plasma TxB2 concentration in untreated animals (n = 13) was 357 pg/ml. The left renal pedicle was clamped for 45 minutes after a right nephrectomy (n = 16), which led after 5 minutes of reperfusion to a rise in TxB2 to 2825 pg/ml (p less than 0.001), but there was no change in 6-keto-PGF1 alpha. After 24 hours creatinine levels rose from 0.4 to 3.0 mg/dl (p less than 0.001), and left renal weight rose from 94% to 117% (p less than 0.001) relative to the weight of the right kidney. In nephrectomized but nonischemic sham control rats (n = 7), creatinine level was 0.9 mg/dl and kidney weight 91% after 24 hours. Pretreatment with OKY 046 (n = 13) (2 mg/kg administered intravenously) blocked ischemia-induced TxB2 synthesis, while 6-keto-PGF1 alpha levels rose from 96 to 302 pg/ml (p less than 0.001). There was no increase in creatinine levels or kidney weight relative to the sham group. Pretreatment with ibuprofen (n = 10) (12 mg/kg) or OKY 046 and ibuprofen (n = 9) inhibited TxB2 and 6-keto-PGF1 alpha synthesis, but creatinine levels and renal weight rose (p less than 0.001). Renal histology in OKY 046-pretreated animals was equal to that in nephrectomized controls, while all other ischemic groups showed tubular necrosis. Results indicate that a high PGI2/TxA2 ratio protects against renal ischemia.

Lavage with leukotriene B4 induces lung generation of tumor necrosis factor-alpha that in turn mediates neutrophil diapedesis.

In experimental models of acute respiratory failure, leukotriene (LT) B4 is generated in the lungs, followed by a 2- to 3-hour delay before there is substantial neutrophil (PMN) accumulation and increased permeability. This study tests whether lavage with LTB4 induces tumor necrosis factor (TNF) synthesis by the lungs that in turn mediates PMN diapedesis. Anesthetized rats underwent lavage with 0.1 ml LTB4 (10(-6) mol/L) into a lung segment. This led to localized TNF synthesis measured in bronchoalveolar lavage fluid with peak concentrations of 580 pg/ml after 1 1/2 hours and 120 pg/ml after 3 hours. These values were higher than after lavage with 0.1 ml saline solution: 0.7 and 4.3 pg/ml, respectively (both p < 0.05). There was a delay before PMN accumulated in bronchoalveolar lavage fluid (x 10(4)). After 30 minutes, the numbers were 2.2 PMN/ml, whereas at 4 hours there was a rise to 40 PMN/ml and at 5 hours 60 PMN/ml, higher than after saline lavage (all p < 0.05). Pretreatment of rats by lavage into airways with actinomycin D, 12 ng in 0.1 ml, minimized LTB4-induced TNF synthesis after 1 1/2 and 3 hours (38 and 51 pg/ml), as well as the delayed diapedesis after 4 hours (12 PMN/ml) (all p < 0.05). Similarly, pretreatment of other rats by lavage with TNF-alpha antiserum (rabbit antimurine), but not normal serum, limited LTB4-induced diapedesis (13 PMN/ml) (p < 0.05). Interestingly, administration of the protein synthesis inhibitor actinomycin D by lavage 10 minutes after LTB4 did not prevent TNF generation after 1 1/2 or 3 hours (490 and 440 pg/ml). However, this agent did limit PMN diapedesis after 4 hours (14 PMN/ml) (p < 0.05), an event possibly caused by limiting later synthesis of endothelial adhesion proteins, a thesis consistent with the findings that pretreatment of rats by lavage with actinomycin D was without any effect on N-formyl-methionyl-phenylalanine (10(-8) mol/L)-induced diapedesis. This agent is known to induce PMN migration without need for synthesis of endothelial adhesion proteins. The data indicate that lavage with LTB4 induces local TNF-alpha generation that in turn mediates a delayed PMN diapedesis. This event is likely regulated by endothelial synthesis of adhesion proteins.

Oxygen free radicals are required for ischemia-induced leukotriene B4 synthesis and diapedesis.

Hind limb ischemia and reperfusion have been shown to result in high plasma levels of leukotriene B4 (LTB4) and polymorphonuclear neutrophil (PMN) sequestration in the pulmonary microvasculature. This study tests whether LTB4 is derived from PMNs and its role in mediating ischemic plasma-induced diapedesis. Plasma derived from rabbit hind limbs after 3 hours of tourniquet ischemia and 10 minutes of reperfusion (n = 6) showed an increased LTB4 level of 560 pg/ml, higher than sham plasma values of 106 pg/ml (p less than 0.05). Introduction of ischemic plasma in abraded skin chambers placed on the dorsum of normal rabbits (n = 6) led after 3 hours to PMN diapedesis of 1175 PMN/mm3, associated with a further increase in LTB4 levels to 820 pg/ml (both p less than 0.05). In contrast, ischemic plasma derived from neutropenic animals (n = 4; nitrogen mustard, 2 mg/kg; PMNs less than 30/mm3) contained lower levels of LTB4, 160 pg/ml (p less than 0.05). When introduced in skin chambers in normal rabbits (n = 4), this plasma induced accumulations of only 163 PMN/mm3, accompanied by a smaller increase in LTB4 levels in the blister fluid after 3 hours, 397 pg/ml (both p less than 0.05). A correlation was found between LTB4 levels in ischemic plasma and PMN accumulations in blister fluid (r = 0.92; p less than 0.05). Intravenous pretreatment of rabbits (n = 4) used in the blister chamber bioassay with the LT receptor antagonist FPL-55712, 40 micrograms/kg/hr, attenuated diapedesis induced by ischemic and ischemic-neutropenic plasma, 103 and 35 PMN/mm3, respectively (both p less than 0.05). Pretreatment with superoxide dismutase, 1500 units/kg, and catalase, 5000 units/kg, both conjugated to polyethylene glycol (n = 4), prevented ischemic plasma-induced LTB4 synthesis, as well as ischemic plasma-induced diapedesis, 12 PMN/mm3 (p less than 0.05). Finally, pretreatment with allopurinol, 25 mg/kg, was similarly effective in preventing LTB4 synthesis and PMN migration. These data suggest that oxygen free radicals are essential for ischemia-induced PMN synthesis of LTB4 that in turn mediates their diapedesis.

Nicotine stimulates pulmonary parenchymal thromboxane synthesis.

Smoking increases plasma levels of thromboxane (Tx) B2. Since intravenous nicotine is without effect on platelet TxB2 synthesis, it is likely that lung parenchyma is the site of metabolic importance. This study examines the TxB2 response and functional consequences to the lungs of intratracheal and intravenous instillations of nicotine tartrate. Rat lungs perfused with Krebs-Henseleit (K-H) solution without recirculation were used. After hemodynamic stabilization, the perfusate was either left unaltered or switched to 5 X 10(-4) M nicotine. After 20 minutes of K-H perfusion, effluent levels of TxB2 fell from 41 +/- 6 pg/ml (mean +/- standard error) to 16 +/- 5 pg/ml. A similar decline was noted with nicotine perfusion. K-H perfusion was used throughout the second set of experiments. The lungs were instilled with either saline solution (1 ml/kg body weight) or 5 X 10(-4) M nicotine in saline solution. In the nicotine group, TxB2 levels rose to 86 +/- 5 pg/ml versus 22 +/- 3 pg/ml in saline-instilled controls (p less than 0.05). In addition, pulmonary edema developed in nicotine-instilled lungs. Pretreatment with the Tx synthase inhibitor OKY-046 prevented the rise in TxB2 concentration after nicotine instillation and led to a wet weight/dry weight ratio of 4.0 +/- 0.4 versus 7.5 +/- 1.5 in untreated control lungs (p less than 0.05). Pretreatment with the lipoxygenase inhibitor diethylcarbamazine increased TxB2 levels to 235 +/- 34 pg/ml (p less than 0.05). Diethylcarbamazine also lowered pulmonary artery pressure from 18 +/- 1 mm Hg to 6.1 +/- 0.7 mm Hg in control lungs (p less than 0.05) but did not reduce edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen free radicals mediate ischemia-induced lung injury.

Lower torso ischemia leads during reperfusion to leukocyte (white blood cell)-dependent lung injury. This study tests the intermediary role of oxygen free radicals (OFRs) in mediating this event. Chronically instrumented anesthetized sheep underwent 2 hours of bilateral hindlimb ischemia. In untreated control animals (n = 7), 1 minute after tourniquet release, mean pulmonary artery pressure rose from 13 to 38 mm Hg (p less than 0.05), whereas pulmonary artery wedge pressure was unchanged from 4 mm Hg. The pulmonary hypertension was temporally related to an increase in plasma thromboxane (Tx) B2 levels from 211 to 735 pg/ml (p less than 0.05). At 30 minutes of reperfusion lung-lymph TxB2 levels rose from 400 to 1005 pg/ml (p less than 0.05). This coincided with an increase in lung-lymph flow from 4.3 to 8.3 ml/30 min (p less than 0.05), which remained elevated for 2 hours, an unchanged lymph/plasma protein ratio, and a rise in lymph protein clearance from 2.6 to 4.6 ml/30 min (p less than 0.05). These changes are consistent with increased lung microvascular permeability. White blood cell count fell during the first hour of reperfusion from 6853 to 3793/mm3 (p less than 0.05), and lung histologic findings showed marked leukosequestration relative to sham animals (n = 3). Pretreatment with the OFR scavengers, superoxide dismutase and catalase both conjugated to polyethylene glycol (n = 6) blunted the rise in mean pulmonary artery pressure to 19 mm Hg (P less than 0.05) and prevented the increase in plasma and lymph TxB2 lymph flow, and lymph protein clearance (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)