Effect of alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) on the potency of sodium nitroprusside and nitroglycerine to decrease blood pressure in rats: a dose-response study.

The nitrovasodilators, sodium nitroprusside and nitroglycerine, effect a dose-dependent decrease in mean arterial blood pressure (MABP) by liberating nitric oxide. Alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) is known to bind nitric oxide. We studied the effect of DCLHb on the potency of sodium nitroprusside (n=36) and nitroglycerine (n=36) to decrease MABP in rats which received 1, 10, 100, 1,000, or 10,000 mg/kg of the DCLHb, or normal saline as the Control. Six doses of sodium nitroprusside or nitroglycerine were given to each rat in a systematically varied sequence. For both drugs, in rats given 1, 10, or 100 mg/kg of DCLHb there were no between groups differences in the change in MABP compared to the Control group. For rats that received 1,000 or 10,000 mg/kg of DCLHb, the potency of nitroglycerine and sodium nitroprusside to decrease MABP was less (p<0.05) than the other groups. These data support the hypothesis that small doses of DCLHb effect a minimal change in the potency of nitrovasodilators to reduce blood pressure. However, they suggest that clinically relevant doses of DCLHb attenuate the vasodilatory ability of sodium nitroprusside and nitroglycerine.

Evaluation and validation of virus removal by ultrafiltration during the production of diaspirin crosslinked haemoglobin (DCLHb).

Virus retention during ultrafiltration through A/G Technology filter cartridges was investigated to characterize the removal process and validate the degree of virus titre reduction during the filtration of red blood cell haemolysates performed as part of the production of diaspirin crosslinked haemoglobin (DCLHb). When viruses were suspended in phosphate buffered saline solution, retention was greater with larger sized viruses and smaller filter pore size. Virus titre was maintained at starting levels in the filter retentate circuit during the course of filtration, suggesting that the virus removal mechanism is predominantly size exclusion. Evaluation of specific processing variables indicated that the retention of phiX174 virus was increased in the presence of red blood cell haemolysate or at high membrane crossflow rates and transmembrane pressures, while the retention of EMC virus was less sensitive to variations in these parameters. Using these results to design a validation protocol, log reduction values of >7.9 were demonstrated for the retention of human immunodeficiency virus, pseudorabies virus and bovine viral diarrhoea viruses, 7.6 for hepatitis A virus, and 4.2 for porcine parvovirus. It was also shown that the retention of viruses was maintained during repetitive use of the same filter cartridge.

Mechanism of the acute pressor effect and bradycardia elicited by diaspirin crosslinked hemoglobin in anesthetized rats.

Diaspirin crosslinked hemoglobin (DCLHb) is a chemically stabilized hemoglobin (Hb) that induces an increase in blood pressure and a decrease of heart rate when injected intravenously in some animals. The mechanism by which DCLHb elicits these hemodynamic effects was studied in pentobarbital-anesthetized, vagotomized rats using a variety of drugs known for their inhibitory action towards endogenous hemodynamically active systems. The hypertensive episode elicited by DCLHb (100 or 400 mg.kg-1) was attenuated in animals pretreated with NG-nitro-L-arginine (inhibitor of nitric oxide synthases) throughout the 30-min period of observation, but it was not reduced in those pretreated with a variety of sympatholytic drugs (e.g., prazosin), atropine, BIBP-3226 (neuropeptide Y antagonist), indomethacin, [1-(beta-mercapto-beta,beta-cyclopentanemethylene propionic acid), 2-(0-methyl) tyrosine]-Arg8 vasopressin (vasopressin antagonist), losartan (angiotensin antagonist), bosentan (endothelin antagonist), or L-arginine-(nitric oxide precursor), compared with control animals. With the exception of propranolol and BIBP-3226, none of the aforenamed inhibitors reduced the amplitude of the bradycardia associated with the pressor effect of DCLHb. These results suggest that: (i) the acute (< 30 min) pressor activity of DCLHb in our animal model requires the presence of an endogenous nitric oxide generating system to be expressed; (ii) the bradycardia elicited by DCLHb might involve the participation of neuropeptide Y and (or) its NPY-1 receptors, but it is unlikely to involve a baroreceptor-mediated vagal reflex, at least in our animal model.

Structural requirements and mechanism of the pressor activity of Leu-Val-Val-hemorphin-7, a fragment of hemoglobin beta-chain in rats.

A rat blood pressure assay was used to perform a structure-activity relationship study (SAR) of Leu-Val-Val-hemorphin-7 (LVV-H7), a fragment of hemoglobin (Hb) beta-chain, elucidate the mechanisms of its cardiovascular effects, and test its potential involvement in the pressor activity of diaspirin crosslinked Hb (DCLHb), a recently developed Hb-based oxygen carrier. The SAR study revealed that the C-terminal-Arg-Phe-amino acid sequence of LVV-H7 contained the main determinants of the pressor activity of this peptide. Drug interaction studies using various inhibitory drugs (e.g., phentolamine, clonidine, etc.) and LVV-H7 showed that the pressor effect and tachycardia elicited by LVV-H7 involved the activation of the sympathetic nervous system (SNS). Additional studies using phenytoin (sodium channel blocker), [Tic7]H7(5-7)-NH2 (putative antagonist of receptors for LVV-H7) and H7(5-7)-NH2, an amidated C-terminal fragment of LVV-H7, suggested that LVV-H7 activated the SNS by interacting with specific receptors functionally coupled with phenytoin-sensitive sodium channels. The pressor effect and tachycardia caused by LVV-H7 were potentiated by captopril, suggesting that the angiotensin converting enzyme may contribute to the inactivation of LVV-H7 in rats. The pressor activity of DCLHb, in contrast to that elicited by LVV-H7, was not affected by animal pretreatment with LVV-H7 fragments shown to inhibit the pressor effect of LVV-H7. We conclude that: 1) LVV-H7 is unlikely to mediate the pressor activity of DCLHb in rats; 2) the pressor and tachycardic activities of LVV-H7 are mediated by the SNS; 3) the C-terminal-Arg-Phe-amino acid sequence of LVV-H7 contains the chemical groups responsible for the pressor effect of this peptide in rats; 4) LVV-H7 and FMRF amide-related peptides may share the same mechanism of pressor activity in rats.

Nimodipine inhibits the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb) in the rat.

Impaired nitric oxide (NO) activity is associated with an increase in blood pressure in rats. Voltage-regulated calcium channels are believed to participate in this hemodynamic event. To further test this hypothesis, we examined the effect of nimodipine and verapamil (calcium antagonists) on the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb), a well-known NO scavenger, in anesthetized rats. Nimodipine, the most potent of the two calcium antagonists used, was also tested against phenylephrine (alpha1-adrenoceptor agonist). The pressor effect of DCLHb was reduced markedly by nimodipine and verapamil, whereas that elicited by phenylephrine, particularly the tonic phase of its pressor response, was resistant to blockade by nimodipine. The bradycardia and tachycardia associated with the pressor effects of DCLHb and phenylephrine, respectively, were not affected by nimodipine. The pressor effect elicited by DCLHb and its alteration by nimodipine were also examined in rats pretreated with 100% O2. This treatment was found to potentiate the pressor effect of DCLHb. However, this synergism did not impair the inhibitory action of nimodipine towards the pressor activity of DCLHb. Altogether these results suggest that the pressor activity of DCLHb in our animal model might involve the participation of voltage-regulated calcium channels.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure.

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Infusion of ovine C5a into sheep mimics the inflammatory response of hemodialysis.

Previous studies in our group have explored the inflammatory response in sheep to dialysis with a variety of different hemodialysis membranes. In the present study we investigated the potential role of C5a in mediating inflammatory responses that have been attributed to complement activation in the extracorporeal setting. Sheep C5a was infused into sheep in a manner that simulated exposure to this anaphylatoxin during dialysis. C5a infusion into sheep was shown to produce a dose-dependent neutropenia that was quantitatively and temporally identical to the response of sheep undergoing dialysis with complement-activating membranes. The two lowest doses used (0.25 and 0.50 micrograms/kg), which resulted in concentrations below the detectable limits of current assays (10 ng/ml), produced significant neutropenia (21.8% and 78.1%, respectively). The ability of the neutrophils (PMNs) to bind fluorescein isothiocyanate-C5a or initiate a respiratory burst in response to phorbol myristate acetate were also affected in a dose-dependent manner. In contrast, C5a alone was not able to produce significant release of lactoferrin, a specific granule constituent, suggesting that degranulation of PMN-specific and primary granules requires secondary stimuli. The production of thromboxane A2 and thromboxane's consequent cardiopulmonary effect of increasing mean pulmonary artery pressure were both observed in a dose-dependent fashion. However, larger amounts of C5a were required to elicit these latter responses as compared with the PMN activities. These results suggest that C5a may be a primary mediator of complement-dependent events that occur during extracorporeal therapies such as hemodialysis, and they also suggest that very little complement activation is necessary to activate leukocytes, whereas higher thresholds are required to produce cardiopulmonary responses.

The effects of diaspirin cross-linked hemoglobin in sepsis.

We tested the hypothesis that diaspirin cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp.) would improve blood pressure, organ perfusion, and mortality during sepsis. Rats were catheterized to assess general hemodynamics (protocol 1) or regional blood flow (protocol 2). Sepsis was induced by intraperitoneal introduction of a cecal slurry (100 mg/kg). In protocol 1, rats received either 100 or 250 mg/kg DCLHb, or albumin at 1, 2, or 4 h after sepsis induction. Hemodynamics were recorded at these times and daily for 72 h. DCLHb increased blood pressure, prevented 72 h leukocytosis, and reduced mortality, but the timing of DCLHb administration was crucial. In protocol 2 only moribund septic animals received 100 mg/kg DCLHb or iso-oncotic albumin i.v. Hemodynamics and regional organ blood flows were measured at baseline, immediately before and after treatment, and at 24 h. DCLHb immediately increased blood pressure with no changes in cardiac output, heart rate, or regional perfusion. DCLHb increased regional perfusion to vital areas at 24 h (compared to albumin group). Distribution of cardiac output in albumin-treated rats was significantly skewed toward skeletal muscle at a time when cardiac output was significantly lower as compared with DCLHb treated animals. In conclusion, DCLHb safely elicited a pressor response, and improved regional perfusion to selected tissues. However, DCLHb benefits were best obtained when given within a specific time frame.

Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits.

BACKGROUND AND PURPOSE: Hemodilution using modified hemoglobin solutions may reduce ischemic central nervous system injury. Purified diaspirin cross-linked hemoglobin (DCLHb) is a cell-free hemoglobin that is intramolecularly cross-linked between the two alpha subunits, resulting in enhanced oxygen offloading to tissues and increased half-life. In the present experiments, we evaluated the ability of DCLHb to reduce neurological damage in two rabbit stroke models. METHODS: In a reversible spinal cord ischemia model, ischemia of the caudal lumbar spinal cord was produced by temporary occlusion of the abdominal aorta. In an irreversible model of cerebral ischemia, plastic microspheres (50 microns) were injected into the internal carotid artery and lodged in the cerebral microvasculature. DCLHb was administered 5 minutes after initiation of ischemia as either a 10-mL/kg infusion, 10-mL/kg exchange transfusion, or a 20-mL/kg infusion. Control animals received human serum albumin that was oncotically matched to the DCLHb. RESULTS: In the spinal cord model, DCLHb significantly increased the duration of ischemia required to produce permanent paralysis from 27.33 +/- 8.71 minutes (mean +/- SD) in controls to 42.59 +/- 10.10 minutes in the 10-mL/kg exchange transfusion group and to 40.82 +/- 18.16 minutes in the 20-mL/kg infusion condition (P < .05). DCLHb did not significantly reduce neurological damage in the microsphere embolization model. CONCLUSIONS: These data suggest that cross-linked hemoglobin reduces neurological damage after reversible central nervous system ischemia and that this is not attributable to hemodilution or hypervolemia only.

Effect of stroma-free hemoglobin and diaspirin cross-linked hemoglobin on the regional circulation and systemic hemodynamics.

The effects of unmodified stroma-free hemoglobin (SFHb) and diaspirin cross-linked hemoglobin (DCLHb) on the regional blood circulation and systemic hemodynamics were studied in rats using a radioactive microsphere technique. SFHb and DCLHb increased mean arterial blood pressure without affecting heart rate. SFHb produced a 24.9% decrease in the cardiac output while DCLHb produced an 44.8% increase in the cardiac output. Stroke volume was decreased (-27.3%) by SFHb and increased (+36.4%) by DCLHb. Total peripheral resistance increased with both SFHb and DCLHb. DCLHb increased blood flow to the heart, spleen, stomach, small intestine and skin, and had no effect on blood flow to the brain, kidneys, liver, mesentery, pancreas, caecum, large intestine and musculo-skeletal system. In contrast, in animals infused with SFHb, blood flow decreased to the kidneys, liver and spleen, increased to the heart, small intestine and skin, and had no effect to the brain, caecum, large intestine and musculo-skeletal system. DCLHb had no effect on vascular resistance in any organ except for an increase in the musculo-skeletal system. In contrast, SFHb increased vascular resistance in the kidneys, liver, spleen, skin, mesentery and pancreas, and had no effect on vascular resistance in the musculo-skeletal system, brain, heart, stomach, small intestine, caecum and large intestine. SFHb had no effect on distribution of cardiac output to the brain, gastrointestinal tract (GIT), kidneys, skin, musculo-skeletal and portal system, while DCLHb significantly decreased the percent cardiac output to the musculo-skeletal system. DCLHb did not affect the distribution of cardiac output to the brain, GIT, kidneys, skin and portal system. SFHb and DCLHb increased the percent cardiac output to the heart. It is concluded that similar concentrations and doses of DCLHb and SFHb produce different effects on the regional blood circulation and systemic hemodynamics.

Biocompatibility of hemodialysis membranes: evaluation in an ovine model.

We studied the cardiopulmonary, hematologic, and inflammatory response to hemodialysis with seven different membranes in sheep. We also compared acetate dialysate with bicarbonate dialysate and evaluated the role of thromboxane in mediating these responses to dialysis with Cuprophan membranes (Baxter Healthcare Corp., Renal Division, Deerfield, Ill.) in sheep. The data generated in these studies indicate that dialyzer membranes can be divided into three major categories, defined by propensity to activate complement. High complement activators such as Cuprophan (low surface-area CF-1511 and high surface-area ST-25 dialyzers) produced dramatic neutropenia and hypoxemia and significant (p < 0.01) increases in the plasma concentration of thromboxane and in mean pulmonary artery blood pressure. The magnitude of these effects appeared to be surface area related. The low-flux Fresenius F-6 polysulfone membrane (Fresenius USA Inc., Concord, Calif.) also resulted in the generation of significant levels of C3a. In contrast, low complement activators such as polyacrylonitrile (AN-69; Gambro Hospal, Inc., Lakewood, Colo.) and cellulose triacetate (CT-110G; Baxter) produced little or no neutropenia, small transient increases in thromboxane, and no rise in mean pulmonary artery pressure. Dialyzers with intermediate complement-activating potential such as cellulose acetate (CA-110; Baxter) and Hemophan (HT-100; Baxter) produced small to moderate degrees of neutropenia and small increases in thromboxane and mean pulmonary artery pressure. Treatment of sheep with sodium ibuprofen before dialysis with Cuprophan CF-1511 membranes prevented the initial increases in mean pulmonary arterial pressure and thromboxane generation and the decrease in arterial oxygen tension, but did not affect the degree of complement activation or neutropenia. In sheep undergoing Cuprophan dialysis, bicarbonate dialysate did not prevent the increase in circulating complement and the associated neutropenia otherwise seen during the early portions of dialysis with acetate dialysate. Bicarbonate dialysate did, however, reduce (not prevent) the initial increases in thromboxane and mean pulmonary arterial pressure, and the magnitude of the hypoxemia seen with the use of acetate dialysate. The results of these experiments therefore indicate that (1) reactions in sheep correlate well with data collected in human beings and the model can be an effective means for comparing novel dialysis membranes and pharmacologic interventions during dialysis and (2) although complement appears to be the transducer of the hematologic and immunologic response, thromboxane appears to be the final effector of the cardiovascular responses to hemodialysis with Cuprophan membranes.

Effects of intravenous infusions of diaspirin cross-linked hemoglobin (DCLHb) on sheep.

The purpose of this study was to compare the cardiopulmonary, hematologic, and immunologic responses of unanesthetized sheep to single, "topload", intravenous infusions of either 10 mL/Kg or 40 mL/Kg of Diaspirin Cross-Linked Hemoglobin, 10 mL/Kg or 40 mL/Kg of a Human Serum Albumin (HSA) solution oncotically adjusted with human serum albumin to approximately match the oncotic pressure of the DCLHb, or 10 mL/Kg of Erythrocyte Hemolysate solution prepared in a manner similar to that commonly described in the literature and referred to as "stroma free hemoglobin". Solutions were infused at a rate of 1 mL/Kg/minute and animals were monitored for 72 hours after infusion. These studies demonstrated that in sheep infusion of either DCLHb or HSA solutions was well tolerated and did not produce a significant increase in plasma C3a levels, an increase in the plasma concentration of thromboxane B2, or unexpected fluid shifts. In contrast, infusion of the Erythrocyte Hemolysate produced a greater than 10-fold increase in plasma C3a concentrations, a greater than 6000-fold increase in plasma TxB2 concentration, significant fluid shifts, and changes in a variety of other parameters consistent with induction of a dramatic inflammatory response. These results indicate that appropriately prepared and purified DCLHb solutions do not elicit an inflammatory reaction in sheep.

Rapid platelet separation and collection for postoperative autologous transfusion.

The authors describe a technique for rapid, on-line, precardiopulmonary bypass separation and collection of 10-20% of a patient's platelets for storage and postoperative reinfusion. In a three stage process, blood is diverted through a hollow fiber hemofilter and platelets are concentrated near the fiber walls; the core is flushed of whole blood and the concentrated platelets are collected. Process mechanics were investigated in vitro with bovine blood; efficacy was studied with in vivo ovine experiments; and platelet function and viability were examined with in vitro human blood tests. Using commercially available hemofilters with 13,000 polyacrylonitrile fibers, the authors collected 1 x 10(11) to 2 x 10(11) platelets in 250 ml in 3.5 min. With fresh human blood, collected platelets exhibited normal morphology and aggregation response to adenosine diphosphate, collagen, and thrombin for 4 hours after collection.

External catheter immobilization improves wound healing in micropigs.

Recurrent traumatic damage by mechanical action is a major cause of catheter exit site problems in CAPD patients. The purpose of this study was to determine the efficacy of a new external immobilization device (Immobilizer) on wound healing characteristics at the catheter exit site. Sixteen Tenckhoff catheters were surgically implanted in four Yucatan micropigs (two catheters per flank). Immobilization devices were placed on one catheter on each side of each animal; the other catheter was allowed free movement. The devices were changed daily, and exit sites were examined for wound healing progress and infection. Fourteen days following implantation the animals were sacrificed, and the catheter exit sites explanted for histologic examination. The results of this study suggest that immobilization of catheters with this device improves wound healing at the exit site, as evidenced by decreased (p less than 0.05) marsupialization/epidermal down-growth (nonimmobilized 2.9 mm vs. immobilized 1.6 mm), and an increased (p less than 0.05) macrophage/epithelioid cell/giant cell response within the exit tract or in the Dacron cuff (granulomatous response histology score of 2.3 for immobilized vs. a histology score of 1.6 for nonimmobilized). Thus, use of this device may help reduce catheter exit site problems in CAPD patients.

Pulmonary microvascular response to LTB4: effects of perfusate composition.

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.

Role of platelets in maintenance of pulmonary vascular permeability to protein.

We examined the role of platelets in maintenance of pulmonary vascular integrity by inducing thrombocytopenia in sheep using antiplatelet serum (APS). A causal relationship between thrombocytopenia and increase in pulmonary vascular permeability was established by platelet repletion using platelet-rich plasma (PRP). Sheep were chronically instrumented and lung lymph fistulas prepared to monitor pulmonary lymph flow (Qlym). A balloon catheter was positioned in the left atrium to assess pulmonary vascular permeability to protein after raising the left atrial pressure (Pla). Thrombocytopenia was maintained for 3 days by daily intramuscular APS injections. Platelet count decreased from 460,000 +/- 77,000 to 16,000 +/- 4,000 platelets/microliters, without significant changes in the leukocyte count and hematocrit. Studies were made in three groups: 1) control sheep having a normal platelet count; 2) thrombocytopenic sheep; and 3) PRP-infused thrombocytopenic sheep. Elevation in Pla in control sheep increased pulmonary transvascular protein clearance (CL) to 12.1 +/- 2.1 ml/h, whereas the same Pla elevation in thrombocytopenia increased CL to 24.1 +/- 4.0 ml/h, indicating an increase in pulmonary vascular permeability to protein in thrombocytopenic animals. Infusion of PRP restored normal permeability, since it prevented the increase in CL. In studies using cultured bovine pulmonary artery endothelial monolayers, transendothelial permeability of 125I-labeled albumin was reduced 50 and 95%, respectively, when 2.5 X 10(7) or 5 X 10(7) platelets were added onto endothelial monolayers. However, addition of 5 X 10(6) platelets or 5 X 10(7) red blood cells did not reduce endothelial monolayer albumin permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Monohydroxyeicosatetraenoic acids (5-HETE and 15-HETE) induce pulmonary vasoconstriction and edema.

5-, 15-, and 12-HETE (monohydroxyeicosatetraenoic acids) are products of the lipoxygenation of arachidonic acid. We investigated their role as possible mediators of pulmonary vasoactivity and pulmonary edema. Pulmonary artery pressure (Ppa), capillary pressure (Pcap), the change in lung wet weight (delta wt) from baseline, and capillary filtration coefficient (Kf) (as a measure of vascular permeability) were determined following an intravenous injection of each mono-HETE in lungs perfused at constant flow with either a phosphate-buffered Ringer's-albumin solution (PBR) or diluted blood. Injection of 2 micrograms of each compound into the pulmonary artery of lungs perfused with either PBR or diluted blood did not produce any effect. However, in PBR-perfused lungs, 4 micrograms 15-HETE induced increases in Ppa, Pcap, and lung wet weight (p less than 0.05), which were greater than the increases observed after 4 micrograms 5-HETE. Kf increased following both 5- and 15-HETE. The pulmonary vasoconstrictor and edemagenic responses were attenuated by increasing perfusate albumin concentration from 0.5 to 1.5 g%. In contrast, 12-HETE (4 micrograms) had no effect on these parameters. In blood-perfused lungs, the pulmonary vascular responses to all HETE compounds (4 micrograms) were attenuated. In both Ringer's-albumin-perfused and blood-perfused lungs, the relative magnitude of the hemodynamic and fluid filtration responses to each mono-HETE were as follows: 15-HETE greater than 5-HETE greater than 12-HETE. In conclusion, the pulmonary vasoconstrictor and edemagenic effects of 5- and 15-HETE occur independently of blood-formed elements. 15-HETE causes greater pulmonary vasoconstriction and edema than 5-HETE. Both 5- and 15-HETE induce pulmonary edema, probably as a result of increased lung vascular permeability. The results indicate that 5- and 15-HETE are potent pulmonary inflammatory mediators.

Substance P-induced pulmonary vasoreactivity in isolated perfused guinea pig lung.

We examined the effects of the neuropeptide substance P on pulmonary hemodynamic and transvascular fluid filtration in isolated Ringer's-perfused and blood-enriched Ringer's-perfused guinea pig lung and on albumin flux across bovine pulmonary artery endothelial monolayer. Mean pulmonary artery, left atrial, and capillary pressures were determined and used to calculate arterial and venous resistances, and lung weight was continuously monitored. Substance P (0.01-1.0 microM) caused marked increases in pulmonary arterial pressure, capillary pressure, venous resistance, and lung weight within 3-5 minutes after administration. These responses remained elevated above baseline at the end of the 30-minute experimental period in the Ringer's-perfused lungs but not in the blood-enriched Ringer's-perfused lungs. Substance P did not alter the capillary filtration coefficient in isolated lungs and transendothelial albumin permeability in the endothelial monolayer. Substance P resulted in an increase in venous effluent thromboxane B2 concentrations in perfused lungs but had no effect on 6-keto-prostaglandin F1 alpha concentrations. Papaverine (0.27 mM) (a smooth-muscle relaxant) abolished the pulmonary microvascular response to substance P in Ringer's-perfused lungs, and meclofenamate (0.15 mM) (a cyclooxygenase inhibitor) attenuated the pulmonary vasoconstriction and lung weight increase. Pyrilamine (1.0 microM) (a histamine1-receptor antagonist) did not alter the responses to substance P. In conclusion, substance P does not affect pulmonary vascular permeability to water and protein. Substance P induces an intense pulmonary vasoconstriction (due to greater constriction of postcapillary vessels) and an elevation in pulmonary capillary pressure that increases net transvascular fluid filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of heparin on increased pulmonary microvascular permeability after bone marrow embolism in awake sheep.

We examined the alterations in pulmonary transvascular fluid and protein exchange after intravenous infusion of fat emboli, i.e., bone marrow suspension (BMS) in awake sheep prepared with chronic lung lymph fistulas and compared these changes with those observed in sheep pretreated with heparin. The BMS injection (0.2 ml/kg) over 15 min caused rapid, but transient, increases (p less than 0.05) in mean pulmonary artery pressure and pulmonary vascular resistance. These increases were accompanied by significant increases in the lymph concentrations of thromboxane B2 and 6-keto-PGF1 alpha. Pulmonary lymph flow increased by 3.9-fold (+/- 0.8) over baseline by 120 min after BMS with no change in the lymph-to-plasma protein concentration ratio (L/P ratio). Heparin pretreatment (700 U/kg) enhanced the BMS-induced increases in pulmonary artery pressure and pulmonary vascular resistance. Thromboxane B2 concentrations in the lymph increased, whereas there was no change in the concentration of 6-keto-PGF1 alpha. Lung lymph flow increased 4-fold (+/- 1.0) over baseline by 120 min after BMS without a change in L/P ratio. Changes in lung vascular permeability were evaluated by elevating pulmonary microvascular pressure (left atrial balloon catheter inflation) at 120 min after BMS. Lung lymph flow increased 7-fold (+/- 1.1) from baseline, whereas the L/P ratio decreased to a mean value of 0.48 +/- 0.03. The protein reflection coefficient (sigma = 1 - L/P ratio) decreased from a control mean of 0.69 +/- 0.02 to 0.52 +/- 0.03 after the BMS challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary pressor responses in sheep to chemically defined precursors of E. coli endotoxin.

The toxicity of various monosaccharide and disaccharide endotoxin precursors has now been studied in sheep. We measured the early pulmonary arterial pressure responses after injections of the monosaccharides lipid X (2,3-diacylglucosamine 1-phosphate) and MAGP (2-monoacylglucosamine 1-phosphate), of the tetraacyl disaccharide diphosphate precursor of lipid A, IV-A (Federation Proc. 43: 1567, 1984), and of Escherichia coli bacterial endotoxin (lipopolysaccharide). We also measured the response of lipid X after prior administration of indomethacin and MAGP. Lipid X, at a total cumulative dose of 40 micrograms/kg, produced an immediate, but transient dose-dependent pulmonary arterial vasoconstrictive response. MAGP, at a total dose of 40 micrograms/kg, had no pulmonary pressure activity but did increase extravascular lung water and produce some histological changes in the lung. Disaccharide precursor IV-A, at a total dose of 40 micrograms/kg, produced an immediate dose-dependent pulmonary arterial vasoconstrictive response that was prolonged for greater than 2 h. E. coli endotoxin caused a delayed (15-min) increase in the pulmonary arterial pressure but one that also persisted for greater than 2 h. Prior administration of indomethacin blocked the pulmonary pressor activity of lipid X, whereas prior administration of MAGP increased both the magnitude and the duration of the pulmonary pressure response of lipid X. We conclude that the initial pulmonary hypertension seen after lipid X injection may involve cyclooxygenase-dependent formation of prostaglandins and that the genesis of this pulmonary pressor activity is at least in part dependent on the ester-linked hydroxymyristoyl moiety at position 3 of the lipid X molecule.(ABSTRACT TRUNCATED AT 250 WORDS)