trophil mediated injury. Lung weights increased 53 g and lung lavage albumins were 690 mg/ dl following addition of arachidonic acid. Arachidonic acid-induced injury was also prevented by adding indomethacin or meclofenamate to the perfusate. DISCUSSION
The present study indicates that arachidonic acid or its metabolites may contribute to the development of lung injury produced by neutrophils stimulated with PMA. This impression is based upon studies showing that cyclooxygenase blockers meclofenamate and indomethacin and the phospholipase A2 inhibitor, tetracaine, decreased PMA and neutrophil-mediated lung injury. Further support is gained from the observation that in isolated rabbit lungs perfusion with arachidonic acid alone produced acute edematous lung injury which mimicked the injury that was seen following perfusion with neutrophils and PMA. Previous work with this PMA-neutrophil model has shown that neutrophil-derived oxygen radicals are also involved in the injury.6 Dimethyl thiourea, an oxygen radical scavenger, prevented lung injury from neutrophils and PMA. Furthermore, neutrophils from a patient with chronic granulomatous disease, which fail to produce oxygen radicals, did not cause injury in the isolated lung when stimulated with PMA. Taken together with our results, these findings suggest that arachidonic acid metabolites and oxygen radicals act synergistically, or that oxygen radical injury is mediated by arachidonic acid metabolism. These findings are consistent with a growing number of experimental models 8 •9 in which both arachidonic acid metabolism and neutrophil-derived oxygen radicals participate in the injury. The isolated, perfused lung model
should permit further clarification of the roles of each of these systems in the generation of acute edematous lung injury. REFERENCES
1 Lee CT, Fein AM, Lippman M, Holtzman H, Kimbel P, Weinbaum G. Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory distress syndrome. N Eng) J Med 1981; 304:192-96 2 Fowler AA, Giclas PC, Hyers TM. Protein and cell content of bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome. Am Rev Respir Dis 1981; 123:247 3 Johnson A, Malik AB. Effect of granulocytopenia on ex-
travascular lung water content after micro-embolization. Am Rev Respir Dis 1980; 122:561-66 Flick MR, Perel A, Staub NC. Leukocytes are required for increased lung microvascular permeability after microembolization in sheep. Circ Res 1981; 48 :344-51 Shasby DM, Fox RB, Harada RN, Repine JE. Granulocytes contribute to acute lung injury from hyperoxia. J Appl Physiol (in press) Shasby DM, VanBenthuysen KM, Tate RM, McMurtry IF, Repine JE. Oxygen radicals released from phorbol myristate acetate ( PMA) stimulated granulocytes cause acute pulmonary vascular injury. Am Rev Respir Dis 1981; 123:248 (abstract) Boyun A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation: and of granulocytes hy combining centrifugation and sedimentation at 1 G. Scand J Clin Lab Invest 1968; 21: (Suppl 87):77-89 Williams TJ, Jose PJ . Meditation of increased vascular permeability after complement activation. J Exp Med 1981; 153:136-53
9 Kontos HA, Wei P, Povlishick JT, Dietrich WD, Magiera CJ, Ellis EF. Cerebral arteriolar damage by arachidonic acid and prostaglandin G". Science 1980; 209:1242-45
Robert ]. Mason, M.D.; Mary C . Williams, M.D.; and Jonathan H. Widdicombe, M.D.
non-surfactant proteins; and 5) transport of fluid and electrolytes from the alveolar subphase into the interstitium. In this brief review we will discuss two of these functions, namely: secretion of surface active material and transepithelial fluid transport.
A lveolar type II cells are cuboidal epithelial cells that
Secretion of Surface Active Material
Secretion and Fluid Transport by Alveolar Type II Epithelial Cells*
line parts of the alveolar wall and are readily recognized by their characteristic lamellar inclusions. 1 These inclusions are the predominant intracellular storage form of surface active material. In adult rat lungs, type II cells comprise 14 percent of total parenchymal lung cells and cover 3 percent of the alveolar surface. 2 Physiologic functions of type II cells include: 1) synthesis, storage, and secretion of surface active material; 2) differentiation into alveolar type I cells; 3) hyperplasia and adaptation after lung injury; 4) secretion of °From the Cardiovascular Research Institute and the Departments of Medicine, Anatomy, and Physiology, University of California, San Francisco.
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Secretion of surface active material has been difficult to study because of the inaccessibility of the secretory product in vivo, the complex cellular heterogeneity of lung tissue, and the nature of the secretory product. A convenient means of reproducibly sampling alveolar contents in vivo without perturbing the system has not been reported. Alveolar lavage itself may stimulate secretion of surface active material. 3 The cellular heterogeneity of the lung can be avoided by using isolated type II cells, but in the process of isolating type II cells, many physiologic control factors, eg, cell-cell interactions, neurohumoral regulation, and cell receptors, may
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be altered. Surface active material has several different physical and chemical forms during the cycle of secretion, adsorption, compression of the surface monolayer, and catabolism or reutilization. Most investigators have avoided the issue of heterogeneity of surface active material and simply measured the secretion of saturated phosphatidylcholine, the main surface active phospholipid. Although we know the chemical composition of surface active material purified from lavage, we do not know the precise chemical composition, ie, lipids and protein, of material directly secreted by type II cells. In spite of these difficulties, some of the factors that regulate secretion have been defined. Studies in vivo and with the isolated perfused lung in vitro have demonstrated that there is increased secretion of surface active material with hyperventilation and with beta adrenergic stimulation. •- 7 The mechanism of increased stimulation with hyperventilation is not currently known, but is obviously complex since it can be inhibited by atropine, propranolol, and inhibitors of prostaglandin synthesis. 7 Secretion in vivo has also been reported to be increased by cholinergic stimulation. 4 Stimulation of secretion of saturated phosphatidylcholine by isolated type II cells in vitro has been reported for beta adrenergic agonists, cholera toxin, tetradecanoyl phorbol acetate, the calcium ionophore A23187 and analogues of cyclic AMP, but not cyclic GMP.t.S-to Cholinergic agonists do not stimulate secretion by isolated type II cells. 9 • 10 Hence, although there is agreement in vivo and in vitro that beta adrenergic agonists can stimulate secretion, the actual physiologic regulation of surfactant secretion during quiet breathing and during hyperventilation are not known.
Transepithelial Fluid Transport Gas exchange is facilitated by minimizing the diffusion distance between alveolar gas and blood, a condition which suggests that alveolar fluid volume should be minimized. The regulation of the alveolar subphase is not known and the magnitude of passive forces that govern fluid movement across the epithelium are controversial. 11 • 12 Investigators agree that there is a force derived from surface tension that is directed to drawing fluid into the alveolus. The magnitude of this force has been estimated to be from 4 to 12 mm Hg, 11 • 13 but may be much larger in the comers of the alveoli where radius of curvature is very short. 14 The shorter the radius, the greater the force. In electron micrographs of alveolar corners fixed by vascular perfusion, there are collections of alveolar fluid with radii as small as 0.5 micron. 14 Mathay and Staub 15 have recently shown that fluid can be resorbed from the alveolus against a large oncotic pressure gradient and concluded that the interstitial pressure is very negative or there is active resorption of fluid. In the fetal sheep, recent studies have demonstrated that there is net fluid resorption during labor before birth and that resorption rate was increased by beta adrenergic agonists. 16 • 17 The cellular site for fluid resorption in adult and fetal lung is not known.
62S 24TH ASPEN LUNG CONFERENCE
We became interested in active transport of fluid and electrolytes by isolated rat alveolar type II cells when we observed dome formation in vitro. Cells that transport fluid such as epithelial cells from the kidney, mammary gland, and choroid plexus form domes or hemicysts in monolayer culture. Dome formation is thought to be specific for transporting epithelium and apparently requires net fluid transport to produce a hydrostatic pressure gradient to lift the monolayer off of the surface of the culture dish, tight junctions between the epithelial cells, to prevent fluid leaking between the cells and some persistent attachment points to the culture dish to permit domes to form. 18 ·'9 Type II cells maintained on plastic culture dishes produced a few small domes, but type II cells maintained on plastic culture dishes covered with an extracellular matrix produced by corneal endothelial cells 20 formed numerous large domes. The formation of domes was stimulated by sodium butryate ( 3 mM), and inhibited by amiloride ( IO·•M) and ouabain ( I0· 3 M). Transmission electron microscopy of the domes showed that the epithelial cells had flattened out but still retained lamellar inclusions near the nuclei, that the epithelial cells are joined by tight junctions, and that the polarity of the epithelial cells was maintained with microvilli on the apical surface facing the culture medium. To define the transport system more completely, we maintained type II cells on collagen-coated millipore filters and then studied these monolayers in an Ussingtype chamber. 21 The transepithelial potential difference was 0.7 ± 0.1 mv ( 24 filters, 7 experiments) apical side negative and the resistance was 217 ± 11 ohm cm. 2 Terbutaline ( I0- 5 M) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride ( IO-•M) completely abolished the potential difference when it was added to the apical side but not to the serosal side, whereas ouabain ( I 0- 3 M) markedly inhibited the potential difference when added to the serosal side but not the apical side. We conclude from these observations that type II cells form a polarized epithelium in culture and have the ability to transport fluid and electrolytes. The effect of amiloride suggests that the main active transport process is sodium absorption. The data strongly imply that type II cells have the ability to resorb fluid from the alveolar subphase and transport it into the interstitium. Fluid balance in the alveolus probably involves active forces as well as passive forces. REFERENCES
1 Mason RS, Dobbs LG, Greenleaf RD, and Williams MC. Alveolar type II cells. Fed Proc 1977; 36:26972702 2 Haies DM, Gill J. Weibel ER. Morphometric study of rat lung cells. 1. Numerical and dimensional characteristics of parenchymal cell population. Am Rev Respir Dis 1981; 123:533-41 3 Hildebran JN, Goerke J, Clements JA. Air inflation re-
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leases phospholipids ( PL) into the air-spaces of excised rat lungs. Fed Proc 1975; 34:387 ( abs) Oyarzun MJ, Clements JA. Ventilatory and cholinergic control of pulmonary surfactant in the rabbit. J Appl Physiol 1977; 43:39-45 Nicholas TE, Barr HA. Control of release of surfactant phospholipids in the isolated perfused rat lung. J Appl Physiol 1981; 51:90-98 Klass OJ. Dibutyryl cyclic GMP and hyperventilation promote rat lung phospholipid release. J Appl Physiol 1979; 47:285-89 Oyarzun MJ, Clements JA. Control of lung surfactant by ventilation, adrenergic mediators and prostaglandins in the rabbit. Am Rev Respir Dis 1978; 117:879-91 Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats. Release of phosphatidylcholine in response to ,B-adrenergic stimulation. J Clin Invest 1979; 63:378-87 Dobbs LG, Mason RJ. Stimulation of secretion of disaturated phosphatidylcholine from isolated alveolar type II cells by 12-0-tetradecanoyl-13-phorbol acetate. Am Rev Respir Dis 1978; 118:705-12 Brown LAS, Longmore WJ. Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture. J Bioi Chern 1981; 256:66-72 Guyton AC, Moffat DS. Role of surface tension and surfactant in the transepithelial movement of fluid and in the development of pulmonary edema. Prog Resp Res 1981; 15:62-75
12 Wilson T A. Effect of alveolar wall shape on alveolar water stability. J Appl Physiol 1981; 50:222-24 13 Clements JA. Pulmonary edema and permeability of alveolar membrane. Arch Environ Health 1961; 2:28083 14 Gil ], Bachofen H, Gehr P, Weibel ER. Alveolar volume-surface area relation in air- and saline-filled lungs fixed by vascular perfusion. J Appl Physiol 1979; 47: 990-Hl01 15 Mathay MA, Landolt CC, Staub NC. Effect of 14~ albumin in alveolar fluid on the rate of liquid removal from the airspaces of sheep. Fed Proc 1981; 40:447 (abs) 16 Walters DV, Olver RE. The role of catecholamines in lung liquid absorption at birth. Pediat Res 1978; 12: 239-42 17 Brown MJ, Olver RE, Ramsden CA, Streng LB, Walters DV. Effects of adrenaline infusion and of spontaneous labour on lung liquid secretion and absorption in the fetal lamb. J Physioll981; 313:13P-14P 18 Lever JE. Regulation of dome formation in differentiated epithelial cell cultures. J Supramolecular Structure 1979; 12:259-72 19 Handler JS, Perkins FM, Johnson JP. Studies of renal cell function using cell culture techniques. Am J Physiol 1980; 238:F1-F9 20 Gospodarowicz D, Ill C. Extracellular matrix and control of proliferation of vascular endothelial cells. J Clin Invest 1980; 65:1351-64 21 Widdicombe JH, Ueki IF, Bruderman I, Nadel JA. The effects of sodium substitution and ouabain in ion transport by dog tracheal epithelium. Am Rev Respir Dis 1979; 120:385-92
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The Effects of Arginine Vasopressin and Other Factors on the Production of Lung Fluid in Fetal Goats* Anthony M. Perks, Ph.D.; and Sidney Cassin, Ph.D.
he fetal lungs produce large quantities of fluid which are comparable to those of the kidneys, and probably make a substantial contribution to the amniotic fluid. 1•2 However, it is vital to stop production of fluid at birth, and little is known of any controlling mechanisms. In the work presented here, the possible effects of arginine vasopressin (A VP) and other factors were investigated. This was reasonable, since AVP was known to direct water towards the fetus via the amnion, skin and bladder, 3 and also to enter the blood in large quantities at birth. 4 During this study, the possibility of hormonal controls was greatly strengthened by the demonstration of a reabsorptive effect of epinephrine, by Walters and Olver. 5 METHODS
Acute studies were carried out on 65 fetal goats delivered by Caesarian section under chloralose ( 50 mg/kg, supplemented as needed); control and experimental fetuses were placed on either side of the mother, with their umbilical circulations carefully preserved. Catheters were placed in the tracheas, jugular veins and carotid arteries. An impermeant tracer, blue dextran 2000 ( Pharmacia) was added to the lung fluid at 200 or 300 mg per fetus. Every 10 minutes, the lung fluid was withdrawn into a warm syringe, and a 0.5 ml sample was taken; 3 mixing procedures were carried out between each sample. One hour of equilibration was allowed before collecting data. The rate of secretion was determined by the rate of dilution of the dye (Beckman 25 Spectrophotometer), with values obtained from the slope of the total volume of secretion against time, over 1 hour intervals (all additions and withdrawls were allowed for sequentially). Significance was estimated from changes in slope, using a t-test. Estimates were made of the following : Na + and K + (flame-photometer, Instrument Laboratories, Inc. model 143); Po.., Pco. pH (Instrumentation Laboratories Inc., model 213) . S~dies on 8 chronic sheep were carried out in essentially the same way, except that the fetuses were cannulated under halothane anesthesia in sterile conditions, returned to the uterus, and given 5 days to recover; the tracheal cannula allowed fluid to enter the amniotic sac between experiments. REsULTS
A VP ( Pitressin, Parke Davis) placed directly into the lung fluid of 17 fetal goats ( 135 days-term) produced only short periods of reabsorption ( 10-20 min); •Department of Physiology, College of Medicine, J. Hillis Miller Health Center, University of Florida, Gainesville, and Department of Zoology, University of British Columbia, Vancouver. Supported in part by NIH grant HE 10834 (SC), and Operating Grant A-2584 (AMP), National Science a11d Engineering Research Council of Canada. Reprint requests: Dr. Cassin, Department of Physiology, College of Medicine, ]HMHC, Ben: ]274, Gainesville, Florida 32610
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