James R. Snapper

Effect of N-acetylcysteine on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function.

Oxygen free radicals released during endotoxemia may contribute to the lung injury of the adult respiratory distress syndrome (ARDS). As this syndrome occurs frequently after gram-negative sepsis in humans, we studied the effect of intravenous N-acetylcysteine (NAC), a free radical scavenger, upon the endotoxin (E)-induced model of ARDS in awake sheep. In vivo studies demonstrated that NAC attenuates the endotoxin-induced rise in pulmonary artery pressure (62 +/- 3 torr with E control vs. 43 +/- 3 torr for E + NAC), and markedly diminishes the rise in lymph flow at 1 h (8.5 +/- 1.2 vs 4.5 +/- 0.6 ml/15 min) and 4 h (5.0 +/- 0.6 vs. 3.3 +/- 0.4 ml/15 min), respectively, for E control vs. E + NAC. NAC also markedly attenuated the alterations in lung mechanics after endotoxemia. Dynamic compliance at 2 h after endotoxemia was 44 +/- 6% of base line for E vs. 76 +/- 10% of base line for E + NAC. Resistance to airflow across the lung at 1 h postendotoxin was 811 +/- 280% of base line for E vs. 391 +/- 233% of base line for E + NAC. NAC substantially reduced the 1 h postendotoxin rise in lymph concentrations of thromboxane B2 (8.29 +/- 3.28 vs. 2.75 +/- 1.93 ng/ml for E vs. E + NAC) and 6-keto-prostaglandin-F1 alpha (0.91 +/- 0.27 vs. 0.23 +/- 0.12 ng/ml for E vs. E + NAC). In addition, in vitro studies were performed which revealed NAC to be a potent free radical scavenger in both biologic and nonbiologic free radical generating systems. NAC decreased phorbol-stimulated granulocyte aggregation in a concentration-dependent manner in vitro. Minimal effects were observed, however, upon leukocyte degranulation at the concentrations of NAC achieved during the in vivo tests. Thus, NAC significantly attenuated all monitored pathophysiologic changes in the endotoxin model of ARDS in sheep, possibly by its ability to scavenge toxic oxygen free radicals. A direct impairment of the ability of inflammatory cells to generate oxygen radicals cannot be ruled out.

Effects of cyclooxygenase inhibitors on the alterations in lung mechanics caused by endotoxemia in the unanesthetized sheep.

The effects of Escherichia coli endotoxin on lung mechanics, hemodynamics, gas exchange, and lung fluid and solute exchange were studied in 12 chronically instrumented unanesthetized sheep. A possible role for cyclooxygenase products of arachidonate metabolism as mediators of the endotoxin-induced alterations in lung mechanics was investigated by studying sheep before and after cyclooxygenase inhibition with sodium meclofenamate and ibuprofen. Sheep were studied three times in random order: (a) sodium meclofenamate (or ibuprofen) infusion alone; (b) E. coli endotoxin alone; and (c) meclofenamate (or ibuprofen) and endotoxin. Meclofenamate alone had no effect on any of the variables measured. Endotoxin alone caused early marked changes in lung mechanics: resistance to airflow across the lungs (RL) increased 10-fold, dynamic lung compliance (Cdyn) decreased 80% and functional residual capacity (FRC) decreased by greater than 30%. The alveolar-to-arterial oxygen difference (delta AaPO2) increased markedly following endotoxemia. In the presence of sufficient meclofenamate to inhibit accumulation of thromboxane-B2 and 6-keto-prostaglandin F1 alpha in lung lymph, endotoxin caused no increase in RL, Cdyn decreased by less than 40%, and FRC decreased by only 6%. Meclofenamate significantly attenuated the hypoxemia and early pulmonary hypertension caused by endotoxemia but had no effect on the late increases in lung fluid and solute exchange. Ibuprofen had similar effects to those observed with meclofenamate. We conclude that both the pulmonary hypertension and changes in lung mechanics observed after endotoxemia may be mediated, at least in part, by constrictor prostaglandins or thromboxanes and that gas exchange may be improved by preventing endogenous synthesis of these mediators.

Correlation of Oxygenation with Vascular Permeability-Surface Area but Not with Lung Water in Humans with Acute Respiratory Failure and Pulmonary Edema

We used a single-pass multiple tracer technique to measure cardiac output, extravascular lung water (EVLW) and lung vascular [14C]urea permeability-surface area (PSu) in 14 patients with acute respiratory failure and pulmonary edema. All patients had increased EVLW, but EVLW in the 10 surviving patients (0.26 +/- 0.06 SE ml/ml total lung capacity [TLC]) was not significantly different from that in the five patients who died (0.22 +/- 0.05). EVLW did not correlate with intravascular pressures or with alveolar-arterial oxygen pressure difference (A-aDO2). PSu was lower in surviving patients (0.50 +/- 0.16 SE ml/s X liter TLC) than in patients who died (3.44 +/- 0.36; P less than 0.05) and also lower than in previously reported data in patients with normal PSu. PSu correlated significantly with A-aDO2. Serial studies showed that PSu returned from a low value toward normal in a patient who survived but remained high in a patient who died. We conclude that the amount of edema in the lungs measured by indicator methods was not the principal determinant of either the magnitude of oxygenation defect or survival in the patients studied. We interpret the low PSu in surviving patients as decreased surface area and infer that the ability of the lung circulation to reduce perfusion of damaged and edematous areas was important in preserving oxygenation. A high PSu, presumably reflecting perfusion of areas with increased permeability, was a sign of especially poor prognosis. Multiple tracer techniques for measuring lung vascular PSu may help to define the pathogenesis and to evaluate therapies of acute lung injury in humans. Such measurements may be a more useful clinical tool than measurements of lung water in patients with acute respiratory failure and pulmonary edema.

Anti sense DNA down-regulates proteins kinase C-epsilon and enhances vasopressin-stimulated Na+ absorption in rabbit cortical collecting duct.

Hormonal activation of protein kinase C (PKC) is a major signaling mechanism regulating salt and water transport in the distal nephron. We used antisense DNA to down-regulate a PKC isoform in the rabbit cortical collecting duct (CCD) and examined its role in mediating arginine vasopressins (AVP) effect on salt transport in the CCD. Immunoblots demonstrate that PKC-epsilon (diacylglycerol sensitive) and PKC-zeta (diacylglycerol insensitive) are the major PKC isoforms in both freshly isolated and primary cultures of rabbit CCDs. Rabbit CCDs grown on semi-permeable supports, displayed a positive baseline short circuit current (Isc), which was abolished by amiloride, demonstrating active Na+ absorption. Both AVP and 8-chloro-phenylthio-cAMP (8CPTcAMP) transiently increased Isc, however, within 40 min Isc fell below baseline. Down-regulation of PKC-epsilon, as confirmed by immunoblot, was achieved either by treatment with a PKC-epsilon-specific antisense oligonucleotide or 48 h of 1 microM PMA. In PKC-epsilon down-regulated cells, 8CPTcAMP produced a sustained, rather than transient, increase in Isc. We suggest cAMP stimulates Na+ transport, but secondary activation of PKC-epsilon results in the sustained inhibition of Na+ transport seen in response to vasopressin in the CCD.

Effects of platelet depletion on the unanesthetized sheep's pulmonary response to endotoxemia.

The effect of platelet depletion on the unanesthetized sheeps pulmonary response to endotoxemia was studied in eight unanesthetized sheep. Platelets were depleted with rabbit anti-sheep platelet antibodies (APA). Bolus injections of APA alone caused marked pulmonary hypertension (PPA increased from 21 +/- 2 to 62 +/- 5 cm H2O +/- SE) and alterations in lung mechanics (dynamic compliance of the lung [Cdyn] decreased to 38.5 +/- 4.6% and resistance to air flow across the lung [RL] increased to 705 +/- 162% +/- SE of control), which were attenuated by pretreatment with meclofenamate. It was possible to deplete platelets before endotoxemia through a slow continuous infusion of APA without altering base-line values of the measured variables. Platelet depletion did not significantly attenuate the alterations in pulmonary hemodynamics, lung mechanics, lung fluid and solute exchange, or the normal increase in lung lymph concentrations of thromboxane B2 or 6-keto-PGF1 alpha observed following endotoxemia in the sheep. We conclude that normal circulating platelet counts are not required for the full expression of the sheeps response to endotoxemia.

Resolution of dihydroxyeicosanoates and of dihydroxyeicosatrienoates by chiral phase chromatography

A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-vic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vic-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality.

Pathophysiologic responses of sheep to brief high-level nitrogen dioxide exposure

AbstractThe cardiopulmonary response to short-duration, high-concentration nitrogen dioxide (NO2) exposure was examined in conscious domestic sheep (Ovis aries). Six intubated animals in each of 3 groups were given a 15-min exposure to either air (control) or approximately 700 or 500 ppm NO2− Pulmonary and systemic hemody-namics, pulmonary mechanics, blood gases, and hematologic variables were measured immediately before and after exposure and at 1, 4, and 24 h after exposure. Minute ventilation was monitored during exposure, and a pulmonary histopathologic examination was performed 24 h postexposure. Negligible effects were observed in the control group. Exposure to 100 ppm NO2 caused a modest increase in minute ventilation during exposure, and an increased number of leukocytes was found within interalveolar capillaries upon histologic examination. Exposure to 500 pprn NO2 induced an immediate and profound lung irritant response characterized by an increase in lung resistance, respiratory rate, and minut...

Identification of mediator specific Cardiovascular waveforms using a back propagation neural networks

Abstract A diverse milieu of inhaled substances induce the release of endogenous mediators. These mediators are responsible for the symptoms manifested by a wide spectrum of allergic and toxic scenarios. Platelet activating factor (PAF) and prostaglandin H 2 analog (PGH) are two such mediators. A sheep was exposed to varied doses of PAF and PGH. Five parameters were monitored to characterize the cardiovascular response. A back propagation network was used to analyze the response data. The network was trained using three different groups of 21 of the 28 total data sets. In each case, seven data sets were withheld from training and used to assess the performance of the networks. In each case, the back propagation network (BPN) qualitatively and quantitatively (error

Effects of thromboxane synthase and cyclooxygenase inhibition on PAF-induced changes in lung function and arachidonic acid metabolism

PAF was administered as an intravenous bolus (0.1 micrograms/kg) to eight chronically instrumented awake sheep. The effects of pretreatment with an inhibitor of cyclooxygenase (meclofenamate) on PAF-induced changes in lung function were compared to those observed with a specific inhibitor of thromboxane synthase (DP1904). Each animal was studied four times in varied order: PAF alone, PAF + DP1904, PAF + meclofenamate, and DP1904 alone. Saline alone (control), DP1904 alone, and meclofenamate alone did not cause changes in any of the measured variables. DP1904 and meclofenamate significantly attenuated the PAF-induced fall in lung compliance, elevation in peak pulmonary artery pressure, and increased lung lymph flow. Both drugs abolished the PAF-induced increases in lung lymph thromboxane B2 concentrations. Meclofenamate, but not DP1904, blocked the rise in lymph 6-keto-PGF1 alpha. Although meclofenamate blocked the rise in lymph PGE2, DP1904 resulted in levels 2.7 times higher than PAF alone. We conclude that: (1) inhibition of thromboxane synthase is as effective as inhibition of cyclooxygenase in attenuating PAF-induced changes in lung function, and (2) thromboxane synthase inhibition results in augmented production of PGE2 following PAF administration in vivo.

Direct and indirect effects of leukotriene D4 on the lungs of unanesthetized sheep.

The direct and indirect actions of two active components of slow-reacting substance of anaphylaxis, leukotrienes C4 (LTC4) and D4 (LTD4), were studied in chronically instrumented unanesthetized sheep. Intravenous injection of 3 micrograms of LTD4 caused immediate marked pulmonary arterial hypertension which returned to baseline in 6.5 +/- 1.0 min. Dynamic compliance of the lungs (Cdyn) and left atrial (PLA) and aortic (Paorta) blood pressure fell concomitantly with the increases in pulmonary artery pressure (PPA). PLA and Paorta then increased above baseline and heart rate deceased significantly. LTD4 caused only small increases in lung lymph flow but did increase lung lymph concentrations of thromboxane B2. Lung lymph concentrations of 6-keto-prostaglandin F1 alpha did not increase following LTD4 infusion. The increase in PPA after 3-micrograms injections of LTD4 was greater than that caused by 10-micrograms injections of prostaglandin H2-analog. Injections of 10-30 micrograms of LTC4 caused only minor increases in PPA but did cause bradycardia and delayed increases in PLA and Paorta. The cyclooxygenase inhibitors meclofenamate and ibuprofen inhibited the increases in PPA caused by LTD4 but not the later bradycardia or increases in PLA and Paorta. The thromboxane synthetase inhibitor UK-38485 attenuated the early increase in PPA and moderated the later increases in PLA and Paorta and bradycardia caused by LTD4 injection. The response of unanesthetized sheep to LTD4 is mediated, at least in part, indirectly by stimulation of the cyclooxygenase pathway of arachidonate metabolism.