Sheep lung cytochrome P4501A1 (CYP1A1): cDNA cloning and transcriptional regulation by oxygen tension.

Lung cytochrome P450 activity has been linked to neoplasia and may produce reactive oxidant species and potent arachidonic acid metabolites. In lamb lung, oxygen breathing increases lung P450 activity, and inhibition of lung cytochrome P450 activity reduces oxygen-induced lung injury. The P4501A1 (CYP1A1) isozyme is present in many lung cells, including endothelial cells, and may therefore be involved in the pathogenesis of hyperoxic injury to microvascular endothelium. Therefore, to test the hypothesis that oxygen regulates P4501A1 gene expression in the lung, we cloned the sheep P4501A1 cDNA, and examined its regulation by oxygen breathing significantly increased lung P4501A1 RNA levels and that this increase preceded the increase in isozyme activity. Oxygen exposure also promptly increased P4501A1 RNA levels in cultured lamb lung microvascular endothelial cells but not in endothelial cells isolated from the main pulmonary artery or in lung smooth muscle cells. The oxygen-stimulated increase in P4501A1 RNA levels was not serum dependent, was unaffected by cycloheximide treatment, and could not be mimicked by treatment of the cells with oxygenated medium, conditioned medium, or by chemical oxidants. By nuclear run-on assay in cultured lung endothelial cells, oxygen increased the transcription rate of P4501A1 by almost fourfold after 90 min of oxygen exposure but had no significant effect on P4501A1 RNA stability. We conclude that oxygen tension, but not chemical oxidants, increases P4501A1 gene expression pretranslationally in lung microvascular endothelial cells. We speculate that oxygen induction of P450 activity in these cells may contribute to microvascular injury during oxygen breathing.

Current therapy of bronchopulmonary dysplasia.

In summary, little progress has been made in the past several years with respect to the treatment of the baby with BPD. The conduct of convincing clinical research seems to be a casualty of budget cuts and a rush to learn the tools of molecular biology. To date, there are no clinical trials that have convincingly demonstrated that long-term diuretic, bronchodilator, vasodilator, or antioxidant therapy is effective in the treatment of chronic BPD. Short-term corticosteroid therapy hastens extubation, but long-term outcome is unaffected and serious questions remain about its safety. Multicenter clinical trials should be carefully designed and implemented to address the values of these therapies. In the design of these trials, care should be taken to stratify treatment groups for known risk factors for BPD. What are the future directions for the treatment of BPD? It is hoped that new BPD treatment strategies will be based on an improved understanding of mechanisms of lung repair and inflammation. Enzyme, gene, cytokine, antioxidant, and antiprotease therapies are being developed in animal models of lung injury. In addition, the use of lung transplantation has begun to be explored for severe cases of BPD. It is also possible, as has occurred in many chronic idiopathic diseases, that nonspecific treatment may prove beneficial. Perhaps it is only a matter of time before intravenous immunoglobulin, cyclosporine, methotrexate, or "biological response modifiers" will be administered to infants with severe BPD. For example, there is anecdotal evidence that recombinant human growth hormone may improve respiratory muscle function in adults with chronic obstructive pulmonary diseases. In the absence of convincing clinical trials, the clinician should reserve existing therapies for the ventilator-dependent infant or infants whose high oxygen requirement is prohibiting discharge or resulting in complex home care or frequent rehospitalizations. It should be emphasized that continuous oxygen therapy combined with avoidance of environmental inhalant and infectious hazards have the strongest rationale and widest margin of safety for treatment of the infant with BPD. Ironically, oxygen therapy is frequently underutilized and discontinued too rapidly. Early discontinuation of oxygen therapy with alveolar hypoxia results in feeding difficulty, slow growth, nutrient malabsorption, bronchoconstriction, and pulmonary hypertension. Oxygen therapy, although more cumbersome and certainly less glamorous than other pharmacologic agents, remains the essential element of BPD care.

Prospects for gene therapy in acute lung injury.

Gene transfection is an extremely useful tool with which to explore mechanisms of gene regulation, to examine the molecular physiology of proteins in cultured cells, and to produce transgenic organisms. Several transfection techniques that employ viral and nonviral vectors have been used successfully to transfect functional genes into lung cells in vivo. This report reviews some of the gene transfection techniques that have been applied to the intact lung, an organ that offers unique challenges and opportunities. Results indicate that somatic cell gene therapy is feasible and that gene-based therapies can be developed for acute and chronic lung diseases.

Localization and induced expression of fusion genes in the rat lung.

Liposome-mediated gene transfer is useful for DNA transfection into cells in culture. We wondered whether this method could be used to introduce new DNA into the intact lung. Fusion genes containing either the Rous sarcoma virus (RSV) promoter or the mouse mammary tumor virus (MMTV) promoter (which contains glucocorticoid response elements) were linked to the bacterial gene chloramphenicol acetyltransferase (CAT), an enzyme not present in mammalian cells. Plasmids containing the RSV-CAT fusion gene were mixed with cationic liposomes (Lipofectin; BRL, Inc., Grand Island, NY), and single doses were instilled into the cervical trachea of anesthetized rats. Control rats received either liposomes or plasmid. After 24, 48, and 72 h, lungs were perfused free of blood, homogenized, and analyzed for CAT enzyme activity. Liver and kidney tissue were also obtained. We found that rats given either intratracheal liposomes or plasmid had no detectable CAT activity. By contrast, 24 h after instillation of lipid:DNA complexes, lung CAT expression remained elevated for the next 48 h but was barely detectable in liver or kidney. In another group of rats, MMTV-CAT:liposome complexes were instilled intratracheally and then the rats were injected with either dexamethasone or saline. We found that the dexamethasone-treated rats had a 5- to 10-fold higher level of lung CAT expression at 24 and 48 h than the saline-treated controls had; liver and kidney CAT levels were negligible in both groups. Dexamethasone treatment did not increase RSV-CAT expression, indicating that the dexamethasone effect on MMTV-CAT expression was related to the presence of the MMTV promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Double-blind, placebo-controlled trial of alternate-day furosemide therapy in infants with chronic bronchopulmonary dysplasia.

To test the hypothesis that alternate-day administration of furosemide will result in a sustained improvement in pulmonary function without causing alterations in electrolyte or mineral homeostasis, we conducted a randomized, double-blind, placebo-controlled study of 11 hospitalized, oxygen-dependent, spontaneously breathing infants with chronic bronchopulmonary dysplasia. Infants were randomly selected to receive either furosemide, 4 mg/kg in two divided doses on alternate days orally, or placebo for 8 days, followed by crossover to the alternate-therapy for an additional 8-day period. The two study periods were separated by a 48-hour washout period. Dynamic compliance, total pulmonary resistance, the concentration of electrolytes in serum, and the concentrations of calcium and creatinine in urine were measured on nontreatment days. Alternate-day furosemide therapy increased dynamic lung compliance by 76 +/- 112% and decreased total pulmonary resistance by 20 +/- 39%, compared with placebo (both variables p = 0.032). Alternate-day furosemide therapy did not result in increased urine output, electrolyte abnormalities, or increased urinary calcium excretion. We conclude that this simplified treatment regimen may be useful in the management of infants with chronic bronchopulmonary dysplasia. The results support our previous speculation that furosemide improves pulmonary function by mechanisms unrelated to its diuretic properties.

Cimetidine reduces hyperoxic lung injury in lambs.

We previously reported that pretreatment with endotoxin significantly reduced acute pulmonary O2 toxicity in lambs (J. Appl. Physiol. 65: 1579-1585, 1988). One of endotoxin's many effects is to inhibit cytochrome P-450 mono-oxygenation reactions, which are believed to produce toxic O2 species. Therefore, one possible explanation for endotoxin's beneficial effect is that it inhibited P-450-mediated O2 radical production during hyperoxia. To test this hypothesis, we administered a single dose of cimetidine, a noncompetitive inhibitor of P-450 activity, to nine lambs before continuous exposure to greater than 95% O2. Compared with six control O2-exposed lambs, the cimetidine-treated O2-exposed lambs maintained normal gas exchange for a longer period of time (P less than 0.01), accumulated lung water at a slower rate (P less than 0.01), and had normal microvascular permeability after 72 h of O2 exposure. Postmortem levels of antioxidant enzymes in blood-free lung homogenate were not increased in cimetidine-treated lambs. However, the levels of oxidized glutathione were significantly lower in cimetidine-treated lambs, and the ratio of reduced to oxidized glutathione concentrations (GSH/GSSG ratio) was sevenfold higher than the ratio measured in control O2-exposed lambs (P less than 0.001). In four lambs, pretreatment with ranitidine (a drug chemically related to cimetidine but without P-450 inhibitory activity) had no effect either on the time course of O2 injury or on postmortem antioxidants. Microsomes were isolated from blood-free lung of all study animals and P-450 activity of the form 2 isozyme was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of spironolactone-hydrochlorothiazide on lung function in infants with chronic bronchopulmonary dysplasia.

To test the hypothesis that spironolactone-hydrochlorothiazide (Aldactazide) will improve urine output and lung function in infants with bronchopulmonary dysplasia, we studied 21 hospitalized, spontaneously breathing, oxygen-dependent infants with chronic bronchopulmonary dysplasia. Infants were randomly assigned to receive either a 1:1 mixture of spironolactone and hydrochlorothiazide orally (n = 12) (3 mg/kg/day of both compounds) or no treatment (n = 9) for 6 to 8 days each. Dynamic lung compliance, total pulmonary resistance, and hemoglobin oxygen saturation were measured on the first and last days of each study period. Fluid intake and urine output were measured each day. Although the treatment significantly increased urine output, neither lung mechanics nor oxygenation were improved by the drug. The magnitude of the diuresis achieved with spironolactone-hydrochlorothiazide treatment was comparable to the diuresis achieved in a previous study of furosemide treatment (J Pediatr 1986:109;1034-9). Statistical analysis indicated that a type II error was an unlikely explanation for our failure to detect a beneficial effect. In three patients, doubling the oral dose did not improve lung mechanics or oxygenation. We speculate that diuresis per se is not responsible for lung function improvement during treatment with other drugs with diuretic properties.

Pulmonary O2 toxicity in lambs: physiological and biochemical effects of endotoxin infusion.

Hyperoxic adult rats have prolonged survival and reduced morphological evidence of lung injury when treated with a single dose of bacterial endotoxin; this effect is mediated by an augmentation of antioxidant enzyme activity in lung homogenate. To determine whether endotoxin would prolong survival and influence antioxidant enzyme levels in lambs whose physiological response to O2 breathing can be serially measured, we administered a single intravenous dose of endotoxin (0.75 microgram/kg body wt) to 13 lambs before exposing them to greater than 95% O2 (n = 11) or air (n = 2). Seven additional lambs were placed in O2 after receiving only saline vehicle. All lambs had been instrumented to measure pulmonary vascular pressures and cardiac output, and 10 lambs had lung lymph fistulas. O2-exposed control lambs developed noncardiogenic pulmonary edema and respiratory failure within 85 +/- 10 h (range 76-110 h); antioxidant enzymes were not increased, but reduced glutathione (GSH) levels fell and oxidized glutathione (GSSG) increased, reflecting the oxidant stress of O2 exposure. By contrast, endotoxin-treated O2-exposed lambs had a delayed increase in microvascular permeability to protein, a reduced rate of lung edema formation, normal gas exchange after 72 h in O2, and prolonged survival (136 +/- 15 h; range 90-160 h; all variables P less than 0.05). Despite prolonged survival, postmortem lung water content was no greater in the lambs that received endotoxin. Treatment with endotoxin did not increase antioxidant enzyme levels in lung homogenate, but levels of GSH relative to GSSG were significantly elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of endotoxin pretreatment on the pulmonary vascular response to hypoxia in O2-exposed lambs.

We recently reported that endotoxin infusion before O2 exposure significantly reduced or delayed the onset of pulmonary edema formation and respiratory failure by reducing the oxidant stress of O2 exposure. Despite these beneficial effects of endotoxin treatment, lung microvascular permeability eventually increased, but postmortem lung water content was less than expected. Prolonged O2 breathing blunts or abolishes the pulmonary constrictor response to alveolar hypoxia in some species, and it is possible that the loss of this response could contribute further to edema formation. To determine whether the reduction in lung edema observed in endotoxin-treated, O2-exposed lambs was linked to the preservation of hypoxic pulmonary vasoconstriction (HPV), we measured pulmonary vascular resistance before and after 8 min of isocarbic hypoxia (inspired O2 fraction 0.12) during each day of O2 exposure. In six control lambs, the pressor response to hypoxia was abolished after 72 h in O2, and the lambs developed respiratory failure shortly thereafter. In six endotoxin-treated lambs, HPV was preserved for as long as 144 h of O2 exposure. In two control O2-exposed lambs in whom HPV was abolished, the infusion of either angiotensin or prostaglandin H2 analogue increased pulmonary vascular resistance by greater than 75%. We conclude that in lambs 1) hyperoxia abolishes the pulmonary vascular response to hypoxia, 2) endotoxin pretreatment reduces acute O2-induced lung injury and preserves the pulmonary constrictor response to hypoxia, and 3) the loss of HPV during O2 exposure may be the result of oxidant-mediated injury to the hypoxia response itself and not the result of diffuse damage to the vasoconstrictor effector mechanism.

Effect of hypoxia on lung lymph flow in newborn lambs with left atrial hypertension.

To determine the effect of left atrial hypertension on the vascular response to hypoxia in the newborn lung, we measured pulmonary artery and left atrial pressures, lung blood flow and lymph flow, and concentrations of protein in lymph and plasma of 13 lambs that spontaneously breathed air for 2-6 h (control period), followed by 8-11% O2 mixed with 3-5% CO2 and N2 for 2-4 h (experimental period). In eight studies, the lambs were made hypoxic first, after which we elevated their left atrial pressure by 10-12 Torr for 2-3 h. In 10 additional studies, we reversed the sequence by raising left atrial pressure first followed by addition of hypoxia. In lambs with normal left atrial pressure, alveolar hypoxia increased both pulmonary blood flow and lymph flow, with an associated reduction in lymph-to-plasma protein ratio (L/P). When left atrial pressure was increased in the presence of hypoxia, lymph flow increased by a small amount and L/P decreased further. In lambs with preexisting left atrial pressure elevation, addition of alveolar hypoxia increased both blood flow and lymph flow with no significant change in L/P. These results suggest that in newborn lambs with normal left atrial pressure, alveolar hypoxia increases lung lymph flow mainly by increasing microvascular filtration pressure, whereas in lambs with elevated left atrial pressure, hypoxia increases lymph flow by another mechanism, perhaps by increasing the perfused surface area for fluid filtration.

Control of water balance in infants with bronchopulmonary dysplasia: role of endogenous vasopressin.

Babies with chronic bronchopulmonary dysplasia (BPD) can sometimes develop pallor, systemic and pulmonary edema, oliguria, and hyponatremia not attributable to cardiopulmonary or renal impairment. These signs and symptoms might, however, be explained by inappropriate control of vasopressin secretion. To test this hypothesis, we measured plasma vasopressin and osmolality, serum sodium and potassium concentrations, urine output and osmolality, and free water clearance in 26 normoxic infants with BPD aged 1-4 months. All of these infants required supplemental oxygen (FiO2 0.41 +/- 0.03, mean +/- 1 SE) to maintain O2 saturation of greater than 88%, and six infants also required mechanical ventilation. As controls, 10 infants of similar age but without BPD were also studied. None of the infants had been discharged from the nursery and was receiving any medications, and all were clinically stable when studied. Compared to control infants, infants with BPD had significantly elevated plasma vasopressin concentrations (control 5.2 +/- 0.9 pg/ml; BPD 42.4 +/- 5.1; mean +/- SE, p less than 0.05). Moreover, infants with BPD had hyponatremia and hypotonic plasma, and both urine output and free water clearance were significantly reduced. These data suggest that some infants with chronic BPD have elevated vasopressin levels that are functionally significant. We speculate that excessive stimulation of vasopressin secretion may explain some of the pulmonary and nonpulmonary signs and symptoms in infants with chronic BPD.

Alveolar hypoxia increases small pulmonary wedge pressure in awake young lambs.

Alveolar hypoxia increases pulmonary artery pressure in lambs and sheep but increases lung lymph flow only in lambs; the reasons for this are unknown. To test the hypothesis that hypoxia-induced pulmonary venous constriction could in part explain the fluid filtration response in young lambs, eight young lambs (13 +/- 3 days old) and four older lambs (60 +/- 4 days old) were prepared for chronic studies of pulmonary hemodynamics, gas exchange, and fluid filtration. In all animals, a 5 Fr catheter was intermittently wedged into a distal pulmonary artery to indirectly estimate pulmonary venous pressure. The pressure measured in this manner was termed the "small wedge pressure." Animals were awake, spontaneously breathing, and unsedated, and were exposed to both acute and chronic isocarbic hypoxia (FIO20.12). We found that hypoxia significantly increased both small pulmonary wedge pressure and lung lymph flow in young lambs but that neither of these effects occurred in the older lambs. In young lambs, the hypoxia-induced increase in small wedge pressure could be lowered by sodium nitroprusside administration, and small wedge pressure could be increased by angiotensin II infusion. We conclude that alveolar hypoxia increases pulmonary venous tone and the critical pressure of pulmonary veins in young lambs, and that these effects disappear with maturation. These data suggest that alterations in pulmonary venous tone can have important effects on lung fluid balance in the newborn.

Short- and long-term effects of furosemide on lung function in infants with bronchopulmonary dysplasia.

Although furosemide improves lung mechanics in some infants with broncho-pulmonary dysplasia (BPD), this may not be important unless gas exchange also improves. To determine the relationship between improvement in mechanics and improvement in gas exchange, the short- and long-term effects of furosemide therapy were studied in 16 spontaneously breathing infants with severe BPD who were both oxygen dependent and hypercarbic (mean PCO2 54 +/- 11 torr). Each infant was examined at least three times: before furosemide therapy, 1 hour after the first dose of furosemide, and after a 6- to 10-day course. Ten of the 16 infants were also examined three times during a 7-day control period. Transcutaneous PO2 and PCO2, esophageal pressure, air flow, and tidal volume were measured. Pulmonary resistance, lung compliance, and the alveolar to skin PO2 difference were calculated. After a single dose of furosemide, only compliance significantly improved. After prolonged therapy, compliance, resistance, and oxygenation significantly improved in the group as a whole, but better oxygenation was achieved in only six of 16 infants. tcPCO2 was unaffected by long-term furosemide therapy, but in all infants with decreased tcPCO2 1 hour after a single dose, there was sustained decrease in PCO2 after prolonged therapy. Changes in gas exchange were not explained by changes in lung mechanics. These data indicate that long-term diuretic therapy can improve the mechanical properties of the lungs of spontaneously breathing infants with BPD, but that gas exchange is usually unaffected.

Effect of hypoproteinemia on lung fluid balance in awake newborn lambs.

To study the influence of plasma protein concentration on fluid balance in the newborn lung, we measured pulmonary arterial and left atrial pressures, lung lymph flow, and concentrations of protein in lymph and plasma of eight lambs, 2-3 wk old, before and after we reduced their plasma protein concentration from 5.8 +/- 0.3 to 3.6 +/- 0.6 g/dl. Each lamb underwent two studies, interrupted by a 3-day period in which we drained protein-rich systemic lymph through a thoracic duct fistula and replaced fluid losses with feedings of a protein-free solution of electrolytes and glucose. Each study consisted of a 2-h control period followed by 4 h of increased lung microvascular pressure produced by inflation of a balloon in the left atrium. Body weight and vascular pressures did not differ significantly during the two studies, but lung lymph flow increased from 2.6 +/- 0.1 ml/h during normoproteinemia to 4.1 +/- 0.1 ml/h during hypoproteinemia. During development of hypoproteinemia, the average difference in protein osmotic pressure between plasma and lymph decreased by 1.6 +/- 2 Torr at normal left atrial pressure and by 4.9 +/- 2.2 Torr at elevated left atrial pressure. When applied to the Starling equation governing microvascular fluid balance, these changes in liquid driving pressure were sufficient to account for the observed increases in lung fluid filtration; reduction of plasma protein concentration did not cause a statistically significant change in calculated filtration coefficient. Protein loss did not influence net protein clearance from the lungs nor did it accentuate the increase in lymph flow associated with left atrial pressure elevation.(ABSTRACT TRUNCATED AT 250 WORDS)