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Objectives/Hypothesis: At present, the optimal treatment for appropriately resuscitated, multiply injured patients includes fixation of long bone fractures within twenty-four hours of injury. This management approach has been shown to decrease the incidence of pulmonary complications, multiple organ failure, and death. Some investigators have hypothesized that acute reamed intramedullary nailing of the femur (RIMNF) may result in pulmonary dysfunction as a result of the pulmonary fat embolization generated during this procedure. Patients with concomitant thoracic trauma may be at particular risk for this potentially severe complication. In an attempt to determine whether RIMNF can be safely carried out regardless of the severity of a pulmonary injury, we monitored the pulmonary effects of RIMNF in sheep in which an acute respiratory disorder (ARDS)-like state had been induced. Our hypothesis was that, if the pulmonary fat embolization that occurs as a result of RIMNF has a clinically significant effect, it would be detectable in an animal model in which a severe lung injury had been induced prior to the start of RIMNF. Study Design: This was an acute experimental procedure performed on yearling sheep. Methods: Reamed intramedullary nailing of the femur was performed in two groups of instrumented sheep. The first group had no pulmonary injuries. The second group had an ARDS-like state induced by intravenous infusion of perilla ketone prior to RIMNF. Perilla ketone increases pulmonary microvascular permeability without changing filling pressures and is used to induce a model of human ARDS. Hemodynamic and oximetric parameters were measured or calculated, as was pulmonary dynamic compliance during the experiment. Results: Infusion of perilla ketone caused a significant pulmonary injury. RIMNF caused no additional significant effect on intrapulmonary shunt, mixed venous oxygen saturation, or dynamic compliance, which are clinically used to assess the severity of pulmonary dysfunction in injured patients. Conclusions: The fat embolization that occurs during RIMNF in an appropriately resuscitated sheep has no clinically significant effect on pulmonary function, even in the setting of a severe pulmonary dysfunction.

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 vasopressin's (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.

The metabolism of cis-epoxyeicosatrienoic acids (EETs), methyl cis-epoxyeicosatrienoates, and cis-epoxyeicosanoic acids by cytosolic epoxide hydrolase was studied to identify substrate structural features important for stereoselective metabolism and chiral diol formation. 14(R), 15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET, the predominant enantiomers present endogenously in rat organs, were metabolized at substantially higher rates than their antipodes. With the exception of 8(R),9(S)-EET (Km = 41 μM), differences in enantiomer hydration rates appear to be caused by Km-independent factors since the apparent Km values for the enantiomers of 14,15-, 11,12-, and 8(S),9(R)-EET were similar (between 3 and 5 μM). Chiral analysis of the diols resulting from enzymatic hydration of homochiral EETs showed that the regio and/or stereochemistry of water addition was EET regioisomer dependent. For the 11,12-EET enantiomers, water addition was nonregioselective; whereas, with both 8,9-EET antipodes water addition occurred predominantly at C9. Importantly, for 14,15-EET the regiochemistry of water addition was enantiomer-dependent. Only with 14(R),15(S)-EET did enzymatic hydration result in regiospecific addition at C15. Hence, enantioselective EET hydration is determined, principally, by enantiomer specific differences in rates of catalytic turnover and/or substrate binding parameters. On the other hand, the chirality of the diol products is determined by EET enantiomer-dependent differences in the regiochemistry of enzymatic oxirane cleavage and water addition. Esterification resulted in an overall reduction in the rates of epoxide hydration for all three EET-methyl esters (59, 89, and 68% of the EET rate for 8,9-, 11,12-, and 14,15-EET-methyl ester, respectively) and in the loss of regioselectivity during methyl 8(S),9(R)-EET oxirane cleavage. Catalytic EET hydrogenation reduced the rates of EET hydration (56, 45, and 23% of the EET rates for 8,9-, 11,12-, and 14,15-epoxyeicosanoic acids, respectively). Compared to 14,15-EET, enzyme catalyzed hydration of 14,15-epoxyeicosanoic acid was less regioselective and yielded products with a substantially lower chiral purity. Based on these data, as well as on the documentation of 14(R),15(R)-dihydroxyeicosatrienoic acid as an endogenous constituent of rat urine we concluded that: (1) cytosolic epoxide hydrolase plays a significant role in the regio- and stereoselective metabolism of endogenous EETs; (2) differences in the affinities and/or turnover rates of the enzyme for the individual EET antipodes may be responsible for enantioselective EET metabolism; and (3) for 14,15- and 8,9-EET, regioselective and/or enantioselective oxirane water addition is responsible for asymmetric diol formation. The protein spatial coordinates responsible for the asymmetry of EET hydration and diol formation must be circumscribed by a highly structured active site capable of recognizing, regio- and stereospecifically, overall substrate polarity, freedom of C C bond rotation, and/or protein-substrate π-π dipole interactions.

The in vivo responses to staphylococcal alpha toxin are reported for 15 chronically instrumented awake yearling sheep. The data obtained from a total of 30 experiments are grouped into four categories of response: no response, noted in seven experiments done on 5 sheep; pressor response, obtained seven times in 4 sheep; fluid and solute exchange, noted on six occasions in 3 sheep; and acute heart failure and death, which occurred in 10 of the 15 sheep. "No response" denoted no change in any of the measured outcome variables. The group of sheep labeled as showing "pressor response" responded to alpha toxin infusion with an increase in pulmonary artery pressure, unaccompanied by changes either in lung lymph flow or in lung mechanics. "Changes in lung fluid and solute exchange" involve increases in lung lymph flow. The harbinger of the last category, acute left heart failure leading to death, was a marked elevation in left atrial pressure. The threshold response dose in sheep is approximately 21 micrograms/kg. A very steep dose-response curve is observed, with only a narrow window of doses, 15 to 25 micrograms/kg, between the group showing no response and the group showing death from acute heart failure. The data obtained in these studies indicate that the lethal effects of alpha toxin in sheep include acute heart failure, which may be due to direct toxicity to heart muscle and/or the coronary vasculature endothelium.

We have recently developed an open-chest sheep model to monitor and study the effects of major orthopedic procedures on pulmonary physiology. In this pilot study, we focused on reamed intramedullary femoral nailing in animals without pulmonary injury. Details of the model are described herein. The control group consisted of sheep that underwent thoracotomy and invasive monitoring only, while the study group also underwent femoral osteotomy, reaming, and intramedullary nailing. Baseline, postthoracotomy, and post-reaming/nailing values were recorded for mean pulmonary arterial pressure, central venous pressure, left arterial pressure, dynamic compliance, arterial blood gas, mixed venous O 2 , cardiac index, and mean arterial pressure so that hemodynamic and oxygen transport data could be calculated. Postprocedure values were recorded at hourly intervals for 4 h. A physiologically stable, reproducible model was created. No statistically significant differences were found between the control and experimental groups, indicating no adverse effect of femoral reaming/nailing. In one animal, using echocardiography, pulmonary embolization was documented while reaming and inserting the intramedullary nail. Reamed femoral intramedullary nailing is not detrimental to sheep with otherwise normal lungs. This finding suggests that femoral reaming and nailing in trauma patients without associated pulmonary injuries and otherwise normal lungs may be carried out without risk of inducing significant respiratory complications.

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.

The role of thromboxane A2 in sheep endotoxemia, an animal model of the adult respiratory distress syndrome, was investigated by a combined biochemical and pharmacological approach. Endogenous thromboxane biosynthesis was assessed by gas chromatographic-mass spectrometric analysis of urinary (thromboxane B2, 2,3-dinor-thromboxane B2) and plasma (11-dehydrothromboxane B2) metabolites that demonstrated a significant stimulation by endotoxin. The functional relevance of thromboxane A2 was probed with a specific thromboxane-prostaglandin endoperoxide receptor antagonist, SQ 29548. The antagonist significantly blunted the increase in pulmonary arterial pressure, pulmonary vascular resistance, lung lymph flow, and lymph protein clearance induced by endotoxin. Whereas the reduction in lung compliance caused by endotoxin was abolished, the augmented airway resistance was unaffected. From the simultaneous increase in thromboxane biosynthesis and effects of receptor blockade, it was concluded that thromboxane A2 mediates the early pathophysiological changes of sheep endotoxemia. Thromboxane receptor antagonism may offer a potential therapeutic approach to patients at risk of the adult respiratory distress syndrome.

The purpose of this study was to examine the effects of parenteral and aerosol histamine on lung mechanics in sheep. Eleven sheep were chronically prepared with pulmonary arterial (PA) and left atrial (LA) catheters, a tracheostomy, and a silastic envelope in the pleural space. Unanesthetized sheep were studied in a pressure-compensated integrated-flow whole-body plethysmograph, and measurements of dynamic lung compliance (Cdyn), resistance of the lung (RL), and functional residual capacity (FRC) were made. Histamine was infused through PA or LA catheters or given as an aerosol at increasing concentrations until Cdyn decreased by 35%, RL doubled, or FRC increased by 25%. Parenteral histamine caused a reproducible decrease in Cdyn, a variable increase in RL, and no change in FRC. There were no significant differences between the LA and PA infusions. Aerosol histamine had similar effects to parenteral histamine, except some sheep did show an increase in FRC. Repeat studies in 3 animals showed PA histamine infusions to be highly reproducible, whereas changes associated with aerosol histamine were slightly less reproducible. Among sheep there was a several hundredfold range in responsiveness to either aerosol or parenteral histamine. There was a significant correlation (r = 0.98, p less than 0.001) between the parenteral histamine dose required for a response and the aerosol dose. These data were interpreted as suggesting that airway responsiveness to histamine is an inherent physiologic property.

The effects of elevated left atrial pressure (Pla) on the pulmonary hemodynamic responses to hypoxia and infused prostaglandin-H2 analog (PGH2-A) were studied in 10 chronically instrumented unanesthetized sheep. Sheep were studied with isocapnic hypoxia (fraction of inspired O2, 0.12) or infused PGH2-A (0.2 to 1.0 micrograms X kg-1 X min-1 adjusted to increase pulmonary artery pressure (Ppa) by approximately 15 cm H2O) when Pla was normal or elevated to 10 or 20 cm H2O. The Pla was elevated by inflating a Foley catheter positioned in the mitral valve orifice. Elevation of Pla did not block the increase in Ppa or cardiac output (CO) caused by hypoxia but did block the increase in pulmonary vascular resistance (PVR). When Pla was elevated to 10 or 20 cm H2O, hypoxia caused Pla to increase further, and PGH2-A caused Ppa and PVR to increase whether Pla was elevated or not; PGH2-A did not cause CO to increase or Pla to increase further under any experimental condition. Neither hypoxia nor PGH2-A had any effect on left ventricular end-diastolic pressure under any experimental condition. We hypothesize that when Pla is elevated, the increase in CO may dilate the pulmonary circulation, obscuring hypoxic vasoconstriction. When Pla is elevated, the direct effects of hypoxic pulmonary vasoconstriction cannot overcome the increased intraluminal pressure, and PVR does not increase. The pulmonary vessels are still able to respond to a potent vasoconstrictor such as PGH2-A when Pla is elevated. We conclude that the further increase in Pla caused by hypoxia when Pla is elevated is primarily due to increased flow across a mitral valve behaving as a relatively fixed resistor.

Platelet-activating factor (PAF), a potent proinflammatory ether lipid, has been proposed as a potential mediator of airway hyperresponsiveness. We studied the effect of a single intravascular bolus of synthetic PAF (0.5 microgram/kg) on airway responsiveness to aerosolized histamine in 8 chronically instrumented awake sheep. Lung mechanics were assessed by whole-body plethysmography. Histamine dose-response curves were performed before and 4 h after PAF administration. PAF induced a 60% fall in dynamic compliance and a three-fold increase in resistance to air flow across the lung within 5 min. All animals tested became more responsive to aerosol histamine 4 h after PAF as evidenced by a fall in ED65Cdyn from 9.45 mg/ml before to 4.34 mg/ml. There was no correlation between the initial Cdyn or the Cdyn measured just prior to the second bronchial challenge and the calculated alteration in responsiveness. Animals displaying the greatest initial responsiveness to aerosol histamine also manifested the greatest alteration in lung mechanics after intravascular PAF (r = 0.92). We conclude that PAF consistently alters lung mechanics and increases airway responsiveness in awake sheep, and aerosol histamine responsiveness correlates with the maximal response to intravenous PAF.

The pulmonary and dermal sensitivity of a group of mongrel dogs to an extract of Ascaris suum protein was compared with the pulmonary and dermal sensitivity to histamine in this same group. We found a modest but significant correlation in the entire population between skin test reaction to histamine and to A. suum protein (r = 0.52, p0.05), but when the skin test results were compared with those of aerosol challenge, no significant correlation was found. However, when those dogs with reactions to histamine challenge that fell within a narrow range were considered as a separate group, there was a significant correlation between the reactions of aerosol challenge with A. suum and those of the skin tests with A. suum (r = 0.56, p0.025). These findings were consistent with the hypothesis that aerosol bronchoconstrictor responsiveness and immunologic responsiveness are separate attributes that combine to determine airway responsiveness to a specific antigen.

Pulmonary edema can cause alterations in lung mechanics that directly contribute to clinical morbidity and mortality rates. Both the location of the edema fluid (interstitital versus alveolar pulmonary edema) and the etiology of the pulmonary edema contribute to the severity and type of abnormalities of lung mechanics observed. The alterations in lung mechanics associated with the adult respiratory distress syndrome may involve the direct effects of released mediators, alterations in pulmonary surfactant, and altered airway reactivity, as well as the direct effects of the edema fluid.

The pathophysiology of pulmonary edema formation has been discussed under normal conditions and when pulmonary capillary endothelial (and possibly alveolar epithelial) permeability are increased. The potential anatomic sites for pulmonary edema formation and the clinical relevance of the various sites were discussed. The role of potential "safety factors"--including increased lung lymph flow, increased interstitial hydrostatic pressure, and decreased interstitial protein osmotic pressure--in cardiogenic and fluid overload versus increased permeability pulmonary edema, were addressed. The clinical usefulness of quantitating various variables including pulmonary vascular pressures and lung water were also briefly discussed. Pulmonary edema fluid contains potential mediators that may contribute to the severity and chronicity of the lung injury. It is hoped that the concepts explored here should eventually provide clinically relevant information to guide in the management of critically ill patients with pulmonary edema.

Sixteen anesthetized artificially ventilated open-chest sheep were prepared with retrograde catheters to allow for measurement of dynamic compliance of the lungs (Cdyn), total airflow resistance of the lungs (RL), and central (Rc) and peripheral (Rp) airflow resistance. Twelve sheep received aerosol histamine and 12 sheep received aerosol carbachol. Eight sheep received and responded to both aerosol histamine and aerosol carbachol. Three sheep received both aerosol histamine and aerosol carbachol but failed to respond to both agents. Under base-line conditions, for the 16 sheep, 69% of total RL was located in the peripheral component, Rp, and 31% in the central component, Rc. Aerosol histamine caused only peripheral small airway changes while aerosol carbachol predominantly effected the central large airways. When aerosol histamine responsiveness, defined using Cdyn or Rp, was compared to aerosol carbachol responsiveness using Rc, a correlation was demonstrable (r = 0.84, n = 8, P less than 0.05). It is possible in sheep to cause relatively pure peripheral small airway and relatively pure central large airway changes by using different bronchoconstrictor agents. Aerosol histamine and aerosol carbachol responsiveness correlated with each other in these artificially ventilated anesthetized sheep.

Endotoxemia is used as a model of the adult respiratory distress syndrome in chronically instrumented awake sheep. The toxic effects of endotoxin may be mediated through inflammatory cells and the release of toxic oxidants. N-acetylcysteine (NAC) inhibits the in vivo effects of endotoxemia in chronically instrumented unanesthetized sheep. NAC also functions as a free radical scavenger in vitro. It is possible that the protective effects of pretreatment with NAC result from NAC's ability in vivo to protect against local oxidant injury.

Naturally occurring variations in airway responsiveness to inhaled mediators were correlated with various anatomic indexes thought to be related to airway responsiveness. Specifically, the airways of 2 relatively unresponsive and 3 relatively responsive dogs were compared in terms of epithelial thickness, secretory cell number, smooth muscle thickness, and airway mucous gland number and size. More responsive dogs were found to have thinner epithelium and higher secretory cell counts than less responsive dogs. No significant differences between the 2 groups were noted with respect to smooth muscle thickness or mucous gland number and size. Although these observations identify an association between these anatomic features and airway responsiveness, it does not imply a causal relationship.

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.

Forty-eight patients who presented with acute episodes of asthma were randomized to treatment with subcutaneously administered epinephrine, inhaled isoproterenol, or intravenously administered aminophylline. The patients' couses were followed clinically and with spirometry. Although there were no significant differences between the groups before treatment for any measured variable, at the end of 1 hour, the mean improvement inforced expiratory volume in one second (FEV1) was significantly greater for patients treated with epinephrine (0.76 L) or nebulized isoproterenol (0.79 L) than for those given aminophylline (0.23 L). Similarly, the mean duration of therapy required before discharge from the emergency room was significantly longer for patients receiving aminophylline (5.4 h) than for patients treated with either epinephrine (3.5 h) or isisoproeternol (3.0 h). There was no significant differences between the effects of the 2 beta agonists. These results demonstrated that short-acting sympathomimtic agents produce more rapid and potent bronchodilatation in acutely ill asthmatics than that provided by intravenously administered methylxanthines, and that there are no disadvantages to using an inhaled beta agonist rather than one administered parenterally.

Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung.