Regulation of leukocyte function by nitric oxide donors: the effect of S-nitroso-thiol complexes.

The aim of this study was to evaluate the effectiveness of a novel protein-bound S-nitroso-thiol, S-nitroso-albumin (S-NO-alb), in modulating neutrophil-endothelial cell adhesion, activation, and interactions. Due to the highly variable kinetics of NO release from the low-molecular-weight thiol adducts S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-glutathione (GSNO), we expected S-NO-alb to be a more effective modulator of inflammatory interactions through its slow, steady, and prolonged release of NO. Human umbilical-vein endothelial cells (HUVECs) challenged with lipopolysaccharide (LPS) demonstrated upregulated adhesion of neutrophils that was significantly attenuated by pretreatment with S-NO-alb (1.0-100 microM) (p < .05), but not SNAP or GSNO. Pretreatment with S-NO-alb, SNAP, or GSNO attenuated tumor necrosis factor-alpha primed *O2- release from neutrophils and increased neutrophil cGMP accumulation. On a molar basis, S-NO-alb expressed a 10-fold greater potency than SNAP or GSNO at modulating these effects. Kinetics studies confirmed the relative stability of spontaneous NO release from S-NO-alb compared with highly variable kinetic profiles of SNAP and GSNO. Our results demonstrate that S-NO-alb more effectively modulates endothelial-cell and neutrophil immunoinflammatory responses versus its related low-molecular-weight thiol complexes.

A comparison of transdermal fentanyl versus epidural morphine for analgesia in dogs undergoing major orthopedic surgery.

Postoperative analgesia provided by transdermal fentanyl was compared with that provided by epidural morphine in dogs undergoing major orthopedic surgery. Dogs randomly were assigned to receive either a 100 microg per hour transdermal fentanyl patch 24 hours prior to surgery (n=8) or epidural morphine (0.1 mg/kg body weight) administered following induction of anesthesia (n=10). Temperature, heart rate, respiratory rate, and pain score were recorded prior to surgery and zero, six, 18, 30, and 42 hours after surgery. Blood samples were collected from the dogs in the transdermal fentanyl group beginning 24 hours preoperatively to 42 hours postoperatively. Fentanyl concentrations were determined by radioimmunoassay. When all time periods after surgery were combined, dogs in the transdermal fentanyl group were experiencing significantly less pain after surgery than dogs given epidural morphine. The transdermal fentanyl provided analgesia after major orthopedic surgery greater than or equivalent to that of epidural morphine.

Low molecular weight heparin alters porcine neutrophil responses to platelet-activating factor.

Because platelet-activating factor (PAF) is an important mediator of inflammation and heparin has anti-inflammatory effects, we hypothesized that low molecular weight heparin (LMWH) would inhibit PAF-induced activation and chemotaxis in porcine neutrophils. Citrated blood was obtained from pentobarbital-anesthetized pigs, and neutrophils were isolated over a 55%/65% Percoll gradient. The effect of LMWH on basal phorbol myristate acetate (PMA)-induced superoxide (SO) release, as well as its effect on PAF priming for PMA-induced SO release, were investigated. Additionally, the effect of LMWH on PAF-induced chemotaxis of neutrophils across transwell membranes was evaluated. Baseline SO release in response to PMA was .351+/-.046 nmol/10(6) cells/min, and this was decreased to .289+/-.034 nmol/10(6) cells/min by pretreatment with 50 U/mL LMWH. PMA-induced SO production was increased by .240+/-.042 nmol/10(6) cells/min when cells were primed with 10 microM PAF. This priming effect of PAF was reduced significantly by pretreatment of neutrophils with LMWH at 10 and 50 U/mL. Chemotaxis of neutrophils in response to 100 microM PAF was significantly decreased to 70.02+/-6.4% (n = 8) of the control response by pretreatment of cells with 50 U/mL LMWH. We conclude that LMWH has anti-inflammatory effects on porcine neutrophils, which includes attenuation of cell activation and chemotaxis in response to the lipid-derived inflammatory mediator, PAF.

Low molecular weight heparin prevents the pulmonary hemodynamic and pathomorphologic effects of endotoxin in a porcine acute lung injury model.

Tumor necrosis factor alpha (TNF-alpha) activity, platelet and neutrophil degranulation and margination, and increased vascular permeability are central to the pathophysiology of endotoxin-mediated acute lung injury. Nonanticoagulant activities of low molecular weight heparin (LMWH) include solubilization of the TNF-alpha receptor protein, inhibition of neutrophil adhesion, and regulation of thromboxane B2 (TXB2) biosynthesis. In this study, we evaluated the ability of LMWH to modulate TNF-alpha and TXB2 activity during endotoxemia and the subsequent effects on pulmonary hemodynamics. Domestic pigs 8-10 weeks old were anesthetized and catheterized for standard cardiopulmonary measurements and the lungs harvested for cuff:vessel ratio, myeloperoxidase activity, and permeability index. Pigs were randomly assigned to one of four groups: lipopolysaccharide (LPS) (n = 6), given .5 microg/kg/h Escherichia coli LPS intravenously for 6 h; saline control (n = 5); LMWH (n = 5), given .5 mg/kg LMWH for 30 min, followed by .5 mg/kg/h; and LMWH + LPS (same dosages, n = 6). Administration of LPS resulted in increased plasma TNF-alpha and TXB2 activity; increased pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen tension; decreased systemic arterial oxygen tension; and pulmonary edema. The cardiopulmonary parameters for the LMWH-treated pigs did not differ from those of the saline-treated control pigs. Pretreatment with LMWH attenuated the LPS-mediated TNF-alpha and TXB2 activity and attenuated LPS-mediated pulmonary hypertension, hypoxemia and neutrophil emigration, and edema formation. In conclusion, the data show that the protective effects of LMWH in this model of acute lung injury are associated with altered neutrophil adhesion and TNF-alpha and thromboxane activity.

Role of lipid-derived mediators in tumor necrosis factor-induced endothelin-1 release in vivo.

We hypothesized that endothelin (ET) may be released in response to tumor necrosis factor-alpha (TNF) and that platelet-activating factor (PAF) and cyclooxygenase products modulate TNF-induced ET-1 release in vivo. Anesthetized and instrumented pigs were randomly assigned to receive: 1) saline + TNF (n = 8); 2) saline + heat-inactivated TNF (control group, n = 5); 3) WEB 2086 (PAF receptor antagonist) + TNF (n = 7); or 4) indomethacin + TNF (n = 6). Infusion of TNF was associated with increases in mean aortic, mean pulmonary artery, and intratracheal pressures, increases in systemic and pulmonary vascular resistances, and elevated plasma thromboxane B2 concentration. Plasma ET-1 concentrations were unchanged in controls and significantly increased in TNF-treated pigs at 2 to 4 h. WEB 2086 did not modify plasma levels of ET-1 during exogenous infusion of TNF. In contrast, the cyclooxygenase inhibitor, indomethacin, mildly, but not significantly, reduced plasma ET-1 levels. In addition, indomethacin (but not WEB 2086) blocked or attenuated the TNF-induced increases in mean aortic pressure, systemic vascular resistance, mean pulmonary artery pressure, pulmonary vascular resistance, and intratracheal pressure. We conclude that in the pig, cyclooxygenase products modulate some of the cardiovascular responses to TNF and may mildly affect ET-1 biosynthesis. On the other hand, PAF neither significantly influences TNF-induced biosynthesis of ET-1 nor its associated cardiovascular responses.

Pulmonary and neutrophil responses to priming effects of platelet-activating factor in pigs.

OBJECTIVE: To investigate whether platelet-activating factor (PAF) primes the porcine pulmonary response to lipopolysaccharide (LPS), and what effect PAF priming has on porcine neutrophil superoxide (SO) release. ANIMALS: 8- to 10-week old pigs. PROCEDURES: After pigs were anesthetized with sodium pentobarbital and instrumented for standard cardiopulmonary hemodynamic measurements, they were randomly assigned to receive PAF (0.1 ng/kg of body weight/min, 0 to 2 hours) plus saline solution (2 to 6 hours), saline solution (0 to 2 hours) plus LPS (0.25 microgram/kg/h, 2 to 6 hours), or PAF plus LPS. Cardiopulmonary variables were measured throughout the study. Neutrophils were isolated from saline- or PAF-treated pigs at 0 (baseline) and 2 hours, and the effect of in vivo PAF exposure on ex vivo phorbol myristate acetate (PMA)-induced SO release was measured. Additionally, neutrophils isolated from immune-naive pigs were primed in vitro for 10 minutes with 10 microM PAF, and PMA-induced SO release was measured. RESULTS: PAF infusion significantly enhanced the increase in mean pulmonary arterial pressure, pulmonary vascular resistance, and hypoxemia associated with LPS administration. The infusion increased ex vivo neutrophil SO release, and in vitro PAF exposure primed neutrophils for enhanced SO release that was inhibited by pretreatment of cells with indomethacin. CONCLUSIONS: PAF primes the porcine pulmonary system for the response to LPS. It primes porcine neutrophils in vivo and in vitro for PMA-induced SO release, and in vitro priming is mediated by cyclooxygenase products of arachidonic acid metabolism. CLINICAL RELEVANCE: PAF may modulate the porcine inflammatory response by acting as a priming agent, making pigs more responsive to the negative effects of bacterial LPS.

Craniad migration of differing doses of new methylene blue injected into the epidural space after death of calves and juvenile pigs.

OBJECTIVE: To determine the relation between epidural injectate volume (ml/kg of body weight) and its craniad migration in calves and pigs. ANIMALS: 23 neonatal calves and 26 feeder pigs. PROCEDURE: Animals were randomly assigned to receive different volumes of new methylene blue (NMB, 1.2 mg/ml in 0.9% saline solution). Injections were made into the sacrococcygeal intervertebral space in calves and the lumbosacral intervertebral space in pigs, immediately after euthanasia. Sagittal sections of the spine were made at necropsy, and craniad migration of NMB was determined and rounded to the nearest intervertebral space. RESULTS: In calves treated with 0.05, 0.1, or 0.15 ml of NMB/kg, mean +/- SEM number of stained spinal segments was 5 +/- 0.3, 8 +/- 0.6, and 8 +/- 0.6, respectively. Craniad migration of NMB was significantly greater for 0.15 and 0.1 ml/kg volumes versus 0.05 ml/kg. In pigs treated with 0.05, 0.1, 0.2, of 0.3 ml of NMB/kg, mean number of stained spinal segments was 8 +/- 1.1, 8 +/- 0.9, 10 +/- 1.2, and 18 +/- 2.0. Craniad dye migration was significantly greater in the 0.3 ml/kg group versus the 3 lower volume groups. Linear regression performed on both sets of data after logarithmic transformation of spaces migrated to correct for non-normality was significant (P < 0.05), and R2 values of 0.49 and 0.55 were obtained for calves and pigs, respectively. CONCLUSIONS: There is a significant correlation between volume (ml/kg) of NMB injected in the epidural space and its craniad migration in calves and pigs. CLINICAL RELEVANCE: Results provide a basis for determination of volume of injectate to be given to reach a minimal desired level and should be a useful baseline for future investigations of epidural drug administration.

Morphometric analysis of oleic acid-induced permeability pulmonary edema: correlation with gravimetric lung water.

The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.

Management and emergency intervention during anesthesia.

Proper management and monitoring during anesthesia minimizes the need for emergency intervention. Monitoring of circulation, ventilation, and oxygenation allows for early recognition of problems such as airway obstruction, hypotension, acid-base disturbances, or cardiac arrest, and is discussed. Appropriate therapies are instituted early for best success and are described in this article.

SK&F 86002, a dual cytokine and eicosanoid inhibitor, attenuates endotoxin-induced cardiopulmonary dysfunction in the pig.

Cytokines and eicosanoids are well documented important mediators of endotoxemia. Bicyclic imidazoles are a novel class of nonsteroidal anti-inflammatory compounds that display unique pharmacological profiles by reducing cytokine production and arachidonic acid metabolism. In this study, we evaluated the ability of the bicyclic imidazole, SK&F 86002, to attenuate endotoxin-induced cardiopulmonary dysfunction. Pigs were randomly assigned to one of four groups: LPS (n = 5), given .5 microgram/kg/h 055:B5 Escherichia coli lipopolysaccharide (LPS) intravenously (i.v.) for 6 h; saline (n = 5); SK&F 86002 (n = 3), given 50 mg/kg SK&F 86002 orally 30 min prior to anesthesia; and SK&F 86002 + LPS (n = 5). Administration of LPS resulted in cardiopulmonary dysfunction characterized by decreased stroke volume and arterial oxygen tension, and increased room air alveolar-arterial oxygen gradient, pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure. Additionally, LPS administration was associated with leukopenia and increased pulmonary myeloperoxidase activity. Pretreatment with SK&F 86002 attenuated LPS induced hypotension, hypoxemia and bronchoconstriction and blocked the pulmonary hypertension. SK&F 86002 blocked the LPS-induced increase in myeloperoxidase activity, indicating a reduction in pulmonary neutrophil infiltration, but had no effect on systemic leukopenia. Pretreatment with SK&F 86002 significantly attenuated LPS-induced increases in plasma thromboxane B2 and tumor necrosis factor-alpha. We hypothesize that ameliorating effects of SK&F 86002 in this endotoxin model of cardiopulmonary dysfunction are related to inhibition of cytokine and eicosanoid biosynthesis.

Cephalad distribution of three differing volumes of new methylene blue injected into the epidural space in adult goats.

Epidural anesthesia and analgesia are popular regional anesthetic techniques in many animal species. However, we have not found any reports of studies in animals that have investigated the extent of cephalad migration and level of sensory blockade achieved based only on the volume of drug injected into the epidural space. The purpose of this study was to determine if there is a relationship between the volume (mL/kg) of an injectate injected epidurally and the extent of its cephalad migration within the epidural space. Twelve adult goats were randomly assigned to three treatment groups based on the volume of 0.12% New Methylene Blue (NMB), 0.1, 0.2, or 0.3 mL/kg, injected into the epidural space. The site and speed of injection, animal position, and direction of needle bevel were held constant. All injections were performed at the lumbo-sacral space immediately following euthanasia. At necropsy, the vertebral columns were transected longitudinally. The extent of cephalad migration of dye within the epidural space was easily determined by staining of the dura. Measurements were rounded to the nearest intervertebral space to which the dye had migrated. The individual making assessments was blinded to all treatments. In goats treated with 0.1, 0.2, or 0.3 mL/kg NMB, the number of stained spinal segments was 3.5 +/- 0.6, 6.5 +/- 0.9, and 8.8 +/- 0.6, (mean +/- SEM), respectively. Linear regression performed on the data was significant (P < .05) with R2 = 0.86. There was a strong linear relationship between volume (mL/kg) of epidurally injected NMB and cranial migration, with the larger volumes producing more cephalad spread within the epidural space. These results provide evidence for the volume of epidural injectate needed to produce a desired level of sensory blockade in adult goats.

Attenuation of endotoxin-induced pathophysiology by a new potent PAF receptor antagonist.

The role of platelet-activating factor (PAF) as a mediator of endotoxin-induced pathophysiology has been studied in several animal models with conflicting results. We evaluated the effect of a new, potent, and specific PAF receptor antagonist, ABT-299 (Abbott Laboratories) against endotoxin (lipopolysaccharide; LPS)-induced cardiopulmonary dysfunction in a porcine model. In initial experiments, the potency of ABT-299 was confirmed in vitro by its ability to inhibit PAF-induced porcine platelet aggregation at an IC50 of .047 +/- .01 microM, and in vivo by the ability of low doses (.12 mg/kg + .03 mg/kg/h) to block the cardiopulmonary pathologic response to exogenous PAF infusion. To evaluate the effect of ABT-299 administration during endotoxemia, pigs were randomly assigned to one of three groups: controls (n = 7), LPS (n = 9), or ABT-299 + LPS (n =7). ABT-299 was given at 1.0 mg/kg from -0.5 to 0 h plus .3 mg/kg/h from 0 to 6 h. LPS was given at .5 micrograms/kg/hr from 0 to 6 h. ABT-299 reduced the early LPS-induced fall in cardiac index and stroke volume, pulmonary hypertension and vasoconstriction, bronchoconstriction, and hypoxemia. Administration of LPS resulted in 44% mortality (before 6 h), which was blocked by ABT-299. Results with this antagonist indicate that PAF contributes to endotoxin-induced cardiopulmonary dysfunction in the pig, and is associated with mortality in this model.

A comparison of epidural saline, morphine, and bupivacaine for pain relief after abdominal surgery in goats.

The purpose of this study was to determine the analgesic efficacy of bupivacaine, morphine, or saline (control) when injected epidurally into the lumbosacral epidural space in goats after abdominal surgery. Goats received either bupivacaine (0.5%; 1.5 mg/kg in 0.9% sodium chloride solution), 0.9% sodium chloride solution (0.2 mL/kg), or preservative-free morphine (0.1 mg/kg). Total volume injected into the epidural space was 0.2 mL/kg for all groups. The variables evaluated were times to extubation, sternal recumbency, standing, and eating; heart and respiratory rates; and pain score. Only two of the goats in the bupivacaine group were able to stand on their hindlimbs before 6 hours. Time to eating was shorter for the saline group when compared with the bupivacaine group. Heart rate over all time in the saline group (137 +/- 4 beats/min, mean +/- SEM) was higher than the morphine (125 +/- 3 beats/min) and bupivacaine groups (121 +/- 3 beats/min). Respiratory rate over all time was increased in the saline group (26 +/- 1 breaths/min) compared with the bupivacaine (24 +/- 1 breaths/min) or morphine (24 +/- 1 breaths/min) groups. At 50 minutes, the pain score for the saline group was higher than the morphine group. Pain score over all time in the saline group (1.5 +/- 0.10) was higher than the morphine (1.2 +/- 0.07) and bupivacaine (1.2 +/- 0.04) groups. One goat in the saline group required two intravenous injections of flunixin meglumine for pain.

Effect of 5-lipoxygenase and cyclooxygenase blockade on porcine hemodynamics during continuous infusion of platelet-activating factor.

We hypothesized that 5-lipoxygenase and cyclooxygenase products might be mediators of cardiopulmonary and systemic vascular effects induced by a 4 h continuous infusion of platelet-activating factor (PAF, 10 ng/kg/min) in anesthetized pigs. Indomethacin (cyclooxygenase inhibitor) potentiated and CGS 8515 (5-lipoxygenase inhibitor) attenuated PAF-induced increases in total peripheral resistance (TPR) from 2.5 to 4 h. However, the 5-lipoxygenase inhibitor failed to modify pulmonary vasoconstriction and hypertension caused by PAF. Except for a delay in onset (approximately 44 s) and rate of development of pulmonary hypertension during the initial 10 min of PAF infusion, the pulmonary hemodynamic changes were also not attenuated by indomethacin. On the other hand, at 4 h, the PAF-induced pulmonary hypertension and systemic vasoconstriction were completely or partially reversed, respectively, by WEB 2086 (PAF receptor antagonist). The PAF-induced increases in plasma thromboxane B2 (TXB2) were blocked by indomethacin but not by CGS 8515, and at 4 h the 5-lipoxygenase inhibitor potentiated the levels of TXB2 in pigs treated with PAF. The plasma concentrations of 6-keto-PGF1 alpha and leukotriene B4 (LTB4) were not modified by PAF or CGS 8515 + PAF. We conclude that PAF-induced increases in TPR (2.5-4 h) are potentiated by indomethacin and are dependent on 5-lipoxygenase products other than LTB4. Although the early pulmonary vascular response (< 10 min) to PAF is dependent on cyclooxygenase products, the sustained response (after 10 min) cannot be explained by either 5-lipoxygenase or cyclooxygenase products but may be mediated directly by PAF receptors.

CGS 8515 and indomethacin attenuate cytokine-induced cardiopulmonary dysfunction in pigs.

We evaluated the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) on pig cardiopulmonary function by intravenously infusing each cytokine individually or in combination (0.5 microgram/kg from 0 to 0.5 h + 5 ng.kg-1 x min-1 from 0.5 to 6 h for each cytokine). The role of eicosanoids in mediating the TNF-alpha + IL-1 alpha-induced cardiopulmonary dysfunction was also investigated by pretreating cytokine-infused pigs with CGS 8515 (5-lipoxygenase inhibitor) or indomethacin (cyclooxygenase inhibitor). Coinfusion of TNF-alpha with IL-1 alpha caused additive increases (P < 0.05) in total peripheral resistance and plasma concentrations of 6-keto-prostaglandin F1 alpha (PGF1 alpha). The increases in mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), alveolar-arterial O2 gradient (AaDO2), alveolar dead space-to-tidal volume ratio (VD/VT), and plasma concentrations of thromboxane B2 were either additive or synergistic. CGS 8515 blocked the TNF-alpha + IL-1 alpha-induced increases (P < 0.05) in mean aortic pressure, total peripheral resistance (4-6 h), VD/VT (5-6 h), and, at 6 h, attenuated the increases in Ppa, PVR, and AaDO2. Indomethacin blocked or attenuated the cytokine-induced increases (P < 0.05) in Ppa, PVR, AaDO2, VD/VT, and plasma concentrations of thromboxane B2 and 6-keto-PGF1 alpha. The 1-to 2-h systemic hypotension, caused by TNF-alpha + IL-1 alpha, was not abrogated by either indomethacin or CGS 8515. The cytokines did not alter plasma concentrations of leukotriene B4 or 5-hydroxyeicosatetraenoic acid. We conclude that coinfusion of TNF-alpha with IL-1 alpha induces physiological responses that are additive or synergistic and that cyclooxygenase and 5-lipoxygenase products (other than leukotriene B4 and 5-hydroxyeicosatetraenoic acid) importantly mediate cardiopulmonary dysfunction in pigs infused with TNF-alpha + IL-1 alpha.

Pertussis toxin attenuates platelet-activating factor-induced pulmonary hemodynamic alterations in pigs.

To determine whether platelet-activating factor (PAF)-induced release of cyclooxygenase products might be dependent on G proteins in vivo, we administered pertussis toxin (PTX) (9.7-10.0 micrograms/kg iv) to conscious pigs approximately 48 h before bolus infusions of PAF (10 ng/kg). Autoradiography of ADP-ribosylated lung cell membrane proteins from PTX-treated pigs demonstrated marked reduction in the amount of radiolabel ([32P]NAD) incorporated, indicating that PTX induced ADP-ribosylation of G proteins in vivo. PAF, infused at hourly intervals from 0-4 h, caused increases in plasma concentrations of thromboxane B2 (TxB2) concomitant with pulmonary hypertension and vasoconstriction in anesthetized pigs. These physiological changes were blocked or markedly attenuated by indomethacin, indicating they were dependent on cyclooxygenase products. In PTX-treated pigs, the PAF-induced pulmonary hypertension and vasoconstriction were modestly attenuated, whereas the increases in plasma TxB2 were markedly attenuated. PTX prevented PAF-induced aggregation of platelets in vivo as evidenced by blockade of thrombocytopenia. However, in vitro, PAF-induced aggregation of platelets was independent of PTX. Moreover, incubation of platelet-rich plasma with 50 microM PAF failed to increase TxB2 levels. These findings suggested that a PTX-sensitive cell other than the platelet was responsible for triggering release of TxA2 and thrombocytopenia in vivo. We conclude that PAF-induced release of TxA2, pulmonary vasoconstriction, and thrombocytopenia in anesthetized pigs are dependent on a PTX-sensitive G protein; however, the residual hemodynamic effects indicate involvement of a PTX-insensitive G protein, or alternatively, G protein independent pathways.

Effect of PAF receptor antagonism on cardiopulmonary alterations during coinfusion of TNF-alpha and IL-1 alpha in pigs.

We examined the possibility that platelet-activating factor (PAF) might be a mediator of cardiopulmonary alterations induced by a 6-h coinfusion of human recombinant tumor necrosis factor (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) in anesthetized pigs. Our hypothesis was tested by pretreating TNF-alpha + IL-1 alpha-infused pigs with WEB 2086 (3 mg/kg from -0.5 to 0 h + 0.75 mg.kg-1.h-1 from 0-6 h), a specific PAF receptor antagonist. Each cytokine was infused intravenously at 0.5 microgram/kg from 0-0.5 h + 5 ng.kg-1.min-1 from 0.5-6 h. WEB 2086 attenuated the early (0.25 h) cytokine-induced increases in mean pulmonary arterial pressure and pulmonary vascular resistance and blocked or markedly attenuated the later occurring (4-6 h) systemic hypertension and increased systemic vascular resistance. WEB 2086 lessened the severity of TNF-alpha + IL-1 alpha-induced hemoconcentration and airway constriction, but did not modify leukopenia, granulocytopenia, or the cytokine-induced increases in plasma concentrations of thromboxane B2, prostaglandin F2 alpha, and 6-ketoprostaglandin F1 alpha. Microscopically, WEB 2086 did not modify the increased number of granulocytes present in lung tissue derived from pigs infused with TNF-alpha + IL-1 alpha. We conclude that PAF occupies a physiological role in modulating TNF-alpha + IL-1 alpha-induced hemoconcentration, the early changes in pulmonary hemodynamics, and the later alterations in systemic hemodynamics.

Differential effects of WEB 2086 and SRI 63-441 on TNF-alpha-induced alterations in cardiopulmonary function.

We hypothesized that platelet-activating factor (PAF) and cyclooxygenase products might be important mediators of the cardiopulmonary effects induced by tumor necrosis factor (TNF-alpha) in anesthetized pigs. A 6-h infusion of human recombinant TNF-alpha caused hypoxemia, leukopenia, thrombocytopenia, decreased cardiac index (CI), increased pulmonary vascular resistance (PVR) and increased mean pulmonary arterial (Ppa) and intratracheal (Pt) pressures. Administration of the PAF receptor antagonist SRI 63-441 or indomethacin blocked the early (0.25-0.5 h) and attenuated the later increases in PVR and Ppa; indomethacin also attenuated the increase in Pt and hypoxemia associated with TNF-alpha infusion. WEB 2086 did not attenuate the TNF-alpha-induced alterations in CI, PVR, Pt, or PaO2. The in vivo specificity of SRI 63-441 and WEB 2086 was tested by infusing exogenous PAF, prostaglandin (PG) F2 alpha, U-46619 [thromboxane (Tx)A2 receptor mimetic], or arachidonic acid (AA) before and during administration of SRI 63-441 or WEB 2086. Both antagonists blocked the cardiopulmonary effects induced by exogenous PAF. SRI 63-441, but not WEB 2086, significantly attenuated the increased PVR caused by PGF2 alpha, U-46619, and AA. We conclude that SRI 63-441 is a less specific PAF receptor antagonist in vivo compared with WEB 2086 and that cyclooxygenase products, but not PAF, contribute significantly to the cardiopulmonary responses induced by exogenously infused TNF-alpha in pigs.