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The effect of metabolic inhibitors, piretanide, somatostatin and insulin on fluid secretion by in vitro… Ruiz, Teresa 1988

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THE EFFECT OF METABOLIC INHIBITORS, PIRETANIDE, SOMATOSTATIN AND INSULIN ON FLUID SECRETION BY IN VITRO FETAL LUNGS FROM GUINEA PIGS {Cavia porcellus) . By TERESA RUIZ B.Sc. Universidad Nacional Aut6noma de Mexico, 1983 A THESIS SUBMITED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Departement of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1988 ©Teresa Ruiz, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~^.r$ The University of British Columbia Vancouver, Canada Date VP \Q DE-6 (2/88) A B S T R A C T This study introduced the isolated lungs of f e t a l guinea pigs as a new preparation for the investigation of prenatal lung physiology. It established their basic properties by the use of metabolic in h i b i t o r s and the CI - transport i n h i b i t o r , piretanide. In addition, the effects of two naturally occurring peptides, somatostatin and i n s u l i n were investigated. It also extended studies to a species which has received no investigation by modern methods. The f e t a l lungs of fetuses from near term guinea pigs (54 to 67 days gestation) were used. The to t a l volume of lung f l u i d was measured during three hours by dye-dilution of Blue Dextran 2000. In the second hour the lungs were exposed to one of the substances under investigation. Untreated f e t a l lungs continued to secrete in v i t r o for three hours. The average rate of secretion during the f i r s t hour (2.31 ± 0.17 ml/Kg per h; n=104) i s comparable to secretion rates reported in chronically catheterized f e t a l lambs. The combined data of the control groups showed a steady increase in lung f l u i d volume throughout the three hours of experiment. Sodium iodoacetate (10~3M and 10~4M), an i n h i b i t o r of the gl y c o l y t i c pathway, reduced lung f l u i d secretion. Sodium fluoride, an alternative i n h i b i t o r of gl y c o l y s i s , also reduced secretion. The possible importance of the aerobic metabolic pathways was tested by the use of NaCN (10 _ 3M). The results were more variable than those from the g l y c o l y t i c i n h i b i t o r s , and suggest that NaCN s l i g h t l y reduces the secretion rate. Sodium azide was tested as an alternative i n h i b i t o r of the aerobic metabolic pathway with similar results to those from NaCN, confirming that the oxidative pathway has some influence on lung secretion, although i t does not appear as important as the g l y c o l y t i c system. Dinitrophenol was tested as an alternative i n h i b i t o r of oxidative processes. The results suggest that dinitrophenol not only abolishes secretion but also produces reabsorption. Piretanide, a loop d i u r e t i c capable of i n h i b i t i n g Na* /K* /CI- co-transport, reduced lung f l u i d secretion rates at both 10"7M and 10"8M. Somatostatin, a natural i n h i b i t o r of C]~ secretion in some tissues, reduced secretion at both 10-5M and 10"6M (no si g n i f i c a n t effect at 10"7M). Insulin i s known to influence the maturity of fe t a l lungs, and to stimulate Na+ transport in some tissues; Na1 transport i s probably involved i n reabsorption. The results showed that i n s u l i n at 10 - 6 - 10-7M reduced secretion by the isolated lung. This study suggests that the in v i t r o f e t a l lung could be a useful tool for future study. i v T A B L E ! O F " C O N T E N T S Page T i t l e i Abstract i i Table of Contents iv L i s t of Figures v Acknowledgments v i Introduction 1 Origin of Fetal Lung Liquid 1 Nature of Fetal Lung Flu i d 3 Differences Between the Lung F l u i d , Amniotic Fluid and Plasma 4 Secretion of Lung Liquid 5 Absorption of the Lung Fluid 13 Mechanisms of Lung Liquid Absorption 14 Statement of the Problem 19 Methods and Materials 20 Surgical Procedures 20 Experimental Procedures 21 S t a t i s t i c a l Methods 25 Results 29 Untreated Preparations 29 Experimental Groups 29 Sodium Iodoacetate 29 Sodium Fuoride 30 Sodium Cyanide 33 Sodium Azide 33 Dinitrophenol 36 Piretanide 41 Somatostatin 44 Insulin 47 Discussion 53 Secretion Rates by the Fetal Lung 53 Metabolic Aspects of Lung Fluid Secretion 55 Effects of Sodium Iodoacetate and Sodium Fluoride 56 Effects of Cyanide and Azide 57 Effect of Dinitrophenol 59 Effects of Piretanide 62 Effect of Somatostatin 63 Effect of Insulin 65 Stimulation of the Sodium Pump 67 Unmasking of Sodium Pumps Sites 69 De novo Protein Synthesis 70 Relationship of Na* Transport to the Fetal Lung Fluid 70 Bibliography 72 Appendix A: Krebs-Henseleit Saline 86 L I S T O F " 1= I G U R E S Page Figure 1. A c e l l u l a r model of ion transport i n CI- secreting epithelium. Figure 2. Apparatus for the maintenance of the isolated f e t a l lung. Figure 3. Total Volume of Lun Secretion of Untreated Preparations. Figure 4. The effect of different concentrations of iodoacetate on lung f l u i d secretion i n f e t a l guinea pigs. Figure 5. The effect of sodium fluoride on lung f l u i d secretion in f e t a l guinea pigs. Figure 6. The effect of sodium cyanide and sodium azide on lung f l u i d production in f e t a l guinea pigs. Figure 7. The effect of dinitrophenol on lung f l u i d secretion in f e t a l guinea pigs. Figure 8. The effect of different concentrations of piretanide on lung f l u i d secretion in f e t a l guinea pigs. Figure 9. The effect of different concentrations of somatostatin on lung f l u i d secretion i n f e t a l guinea pigs. Figure 10. The log-dose/response relationship for somatostatin. Figure 11. The effect of different concentrations of i n s u l i n on lung f l u i d secretion i n f e t a l guinea pigs. 10 23 27 32 35 38 40 43 46 49 52 V I I wish to thank Dr. Perks for a l l his support, patience and time in the accomplishment of this work. Dr. P h i l i p s and Bob Harris for their guidance i n the understanding of membrane ion transport. Janet Tom, Jennifer Marshall and B i r g i t t a Woods for allowing me to use their data in Figure 1. J also want to thank B i r g i t t a Woods for her help during the long hours of laboratory work. To Miguel Angel, my husband, for his love and impulse and for helping me out with our daughter Tess. I wish to thank my parents for a l l their valuable comments and their interest in my development as a human being. And f i n a l l y , to Elisabeth Vanderhorst I want to express my gratitude for doing the ion analysis. 1 I N T R O D U C T I O N The p o s s i b i l i t y that the f e t a l lung contains l i q u i d was apparently considered as early as 1787 (Wislow: quoted by Preyer 1885, i n Olver & Strang 1974). The trachea and bronchi, during intra-uterine l i f e , are f i l l e d with l i q u i d which for many years was assumed to be aspirated amniotic f l u i d (Adams et a l . 1963a). The presence of lung l i q u i d i n the f e t a l lungs i s almost cer t a i n l y important for lung development (Liggins 1984). I t must determine the shape and volume of peripheral lung units, as well as the growth and form of vascular, e p i t h e l i a l and connective tissue components; f e t a l lung l i q u i d also seems to contribute s i g n i f i c a n t l y to the formation of amniotic f l u i d (Strang 1977a). ORIGIN OF FETAL LUNG LIQUID The f i r s t experimental study that dealt with lung f l u i d was that of Leclard (1815, i n William et a l . 1913) who clamped the neck of a l i v i n g fetus, and on opening the trachea found a f l u i d analogous to amniotic f l u i d . Subsequent investigations by Geyl (1880, i n William et a l . 1913) and Preyer (1885, i n William et a l . 1913) v e r i f i e d Leclard's findings. Addison and How (1913-14, in DeSa 1969) concluded that the l i q u i d of the "trachea, bronchi and lungs" resembles amniotic f l u i d but was not necessarily inspired from the amniotic cavity, and that the pulmonary l i q u i d was absorbed rapidly at b i r t h , when the air-lung i s established (DeSa 1969, Olver & Strang 1974). It was not u n t i l 1941 that the or i g i n of this f l u i d was seriously questioned. Potter and Bohlender (1941 and Avery & Mead 1959, i n Adams et a l . 1963a), presented the f i r s t compelling evidence that the f e t a l pulmonary f l u i d was produced l o c a l l y i n the lung. Later, Whitehead et a l . (1942, i n Carmel et a l . 1965) supported this p o s s i b i l i t y , and suggested that the f l u i d 2 did not originate from the amniotic space, but flowed outward from the lung, and therefore prevented amniotic debris from occluding future airways. At this point the perennial question of amniotic f l u i d aspiration i n utero had been turned round 180 degrees. Soon afterwards, Jost and Policard (1948) produced clear evidence that the l i q u i d inside the lung was formed within the lung. In their study, the fetus was decapitated in utero, and the trachea ligated; this produced a d i l a t a t i o n of the a l v e o l i , presumably due to accumulation of f l u i d . However, because of the severity of the process of decapitation the significance of the results remained dubious (Carmel et a l . 1965). Reynolds (1953) was the f i r s t to attempt to determine the ori g i n of the lung f l u i d i n l i v e intact fetuses (Reynolds 1953, Avery 1968). During his experiments, s i g n i f i c a n t secretion of f l u i d occurred. However he wrongly attributed the secretory a c t i v i t y to the nasopharynx and buccal c a v i t i e s , rather than to the lungs themselves (Adams et a l . 1963a, Olver & Strang 1974) . The p o s s i b i l i t y that secretions from the nasopharyngeal and buccal c a v i t i e s , as well as the lung i t s e l f contribute to the amniotic f l u i d has been considered by Macafee (1950, i n Adams et a l . 1963b), by Bevis (1953, in Adams et a l . 1963b) and more recently by Setnikar and others (Setnikar et  a l . 1959, V i l l e e 1960, i n Adams et a l . 1963b, Bein & Scott 1960, i n Adams §_t a l . 1963b). Setnikar and his co-workers performed experiments on goats and guinea pigs during the last t h i r d of pregnancy. Their findings demonstrated that the f e t a l lung produces f l u i d , and this supported the o r i g i n a l ideas of Whitehead et a l . (1942, i n Carmel et a l . 1965). The same result was obtained by Carmel et a l . (1965), who performed the study under near-physiological conditions and showed that a f l u i d i s produced i n the lung, and that normal lung development occurs i n the absence of amniotic f l u i d aspiration. This was done by l i g a t i n g the trachea of rabbit fetuses several days prior to term and allowing the fetuses to remain i n utero u n t i l term, at which time they were delivered by Caesarean section, and the lung examined (Carmel et a l . 1965, Strang 1977a). Carmel suggested that the f l u i d i s produced i n the f e t a l lung against a hydrostatic pressure. With no escape channel for the f l u i d , the lung would be forced to expand more and more as the f l u i d was produced. This process would continue at least u n t i l the hydrostatic pressure i n the potential a i r spaces counter-balanced the forces producing the f l u i d . Because lung f l u i d i s actively secreted, i t i s unlikely that amniotic f l u i d would enter the lungs except during f e t a l breathing movements. However, there i s evidence that strongly suggests that amniotic f l u i d aspiration does occur with stress or hypoxia. In contrast, in normal conditions the lung f l u i d must move into the pharynx and then pass through the nose and mouth into the amniotic cavity, or i s swallowed (Carmel et a l . 1965) . Adams and his colleagues published three reports (Adams et a l . 1963a, Adams et a l . 1963b, Adams et a l . 1971) that also claimed the pulmonary o r i g i n of f e t a l lung f l u i d and suggested that small volumes of f e t a l pulmonary f l u i d are released p e r i o d i c a l l y from the the lung, and either swallowed or enter the amniotic cavity. They indicated that the pulmonary f l u i d may be a product of u l t r a f i l t r a t i o n , with selective secretion or reabsorption. NATURE OF FETAL LUNG FLUID The physico-chemical features of lung f l u i d in the last t h i r d of pregnancy are very similar to those found i n i n t e r s t i t i a l f l u i d (Hanon et  a l . 1955, i n Setnikar et a l . 1959), p a r t i c u l a r l y i n animals i n which the a l l a n t o i c sac i s s t i l l present during this period, e.g., goats and sheep 4 (Alexander et a l . 1958, in Setnikar et a l . 1959). It was possible that the lung f l u i d , l i k e i n t e r s t i t i a l f l u i d , could be produced by a process of u l t r a f i l t r a t i o n . This production would require an organ with a large surface area supplied by many c a p i l l a r i e s (Meschia 1955, i n Setnikar et a l . 1959). Two organs present such properties: kidneys and lungs. The lung seems to be a source of amniotic f l u i d , perhaps of particular importance i n those animals in which the urethra becomes patent only late i n f e t a l l i f e . In animals i n which the urethra becomes patent e a r l i e r (e.g. man and guinea pig), the kidney also contributes to production of amniotic f l u i d (Setnikar et a l . 1959). However, more direct evidence for an active secretion of lung l i q u i d , rather than u l t r a f i l t r a t i o n , was provided by Strang (1967, i n Strang 1977a), and his evidence was based on measurements of the flux ratios of cations and anions between lung f l u i d and plasma. DIFFERENCES BETWEEN THE LUNG FLUID, AMNIOTIC FLUID AND PLASMA The lung f l u i d i t s e l f was colorless, turbid and i n i t i a l l y sometimes v i s c i d . Adams, Moss & Fagan (1963) showed that lung l i q u i d differed in composition from both amniotic l i q u i d and plasma in almost every respect (Adams et a l . 1963b, Olver & Strang 1974). This also supported the o r i g i n of the f l u i d i n the lung i t s e l f , rather than in the amniotic cavity. Adamson, Boyd, P i a t t and Strang (1969) obtained the following values for plasma and lung l i q u i d concentrations (protein i n g/100 ml.; ions i n m-equiv/kg H 2 O ) : [Protein] [Na*] [C1-] [HCO3-] pH Plasma 6.27 150 4.8 107 24.0 7.34 Lung l i q u i d 0.03 150 6.3 157 2.8 6.27 The other differences between lung l i q u i d and plasma, noted by Adamson et a l . (1969), were a very low protein concentration (0.027 5 g/lOOml), a low Ca 2 + l e v e l and apparently a lack of phosphates i n the lung l i q u i d . The low protein concentration was explained by Norman et a l . (1970) i n terms of the small dimensions of w a t e r - f i l l e d pores i n alveolar walls which excluded the penetration of plasma proteins. The lower Ca 2* i n alveolar l i q u i d may be explained i n two possible ways: (1) by Ca 2 + binding to proteins in the plasma, or (2) by the low permeability of the alveolar epithelium to Ca 2 +, in keeping with the large hydrated radius (a s 4.1 A), and the narrow radius of pores in the alveolar walls (Normand et a]. 1970). Sim i l a r l y , the large hydrated radius of phosphates (a = 4.1-4.5 A) could account for their low concentration in alveolar l i q u i d (Olver & Strang 1974). F i n a l l y , a number of e a r l i e r workers were impressed by the high C l _ content of the lung l i q u i d (Adams et a l . 1971). A l l these properties, together with the low t o t a l CO2 content and low pH suggest that alveolar l i q u i d i s a special f l u i d elaborated by the f e t a l lung and that i t i s neither a simple u l t r a f i l t r a t e nor a mixture of amniotic l i q u i d with other secretions (Adamson et a l . 1969, Humphreys et a l . 1967, Strang 1977a, Olver & Strang 1974). No other body f l u i d has this composition. (Adamson 1969, Strang 1977a). SECRETION OF LUNG LIQUID: THE ACTIVE CHLORIDE TRANSPORT AND PASSIVE FLUX The lungs of the mature f e t a l lamb contain about 30 ml of lung liquid/kg body weight, and secrete l i q u i d at a rate of between 3 and 5 ml/kg body weight per h (Olver & Strang 1974, Normand et a l . 1971, Olver et a l . 1981b). Thus a f e t a l lamb near term secretes a volume approaching half a l i t e r every 24 hours. The l i q u i d volume contained in the potential airspace appears to have an important role in normal lung growth; interference with lung volume, either by a r t i f i c i a l l y withdrawing l i q u i d or by preventing i t s escape on a long-term basis profoundly alters lung histology and tissue 6 weight (Alcorn et a l . 1977, in Liggins et a l . 1981). The alveolar epithelium carries out two well-documented transport functions. During f e t a l l i f e , the secretion of CI - into the developing a i r space i s the major driving force for the production of lung l i q u i d (Strang 1974, i n Gatzy 1983). In contrast, i n the lung of the adult, surfactant and, presumably, small amounts of associated electrolyte solution are secreted by the e p i t h e l i a l c e l l s onto the luminal surface (Mason et a l . 1977, Gatzy 1983). For a secretory organ to be capable of generating a chemical gradient, a barrier must be present to r e s t r i c t molecular d i f f u s i o n . Normand et a l . (1971) showed that in the f e t a l lung t h i s barrier resides i n the pulmonary epithelium. In their experiments, the c a p i l l a r y endothelium of the f e t a l lung was found to present l i t t l e barrier to molecular d i f f u s i o n , whereas the pulmonary epithelium was r e l a t i v e l y 'tight', completely r e s t r i c t i n g the movement of molecules larger than mannitol. Describing their results in terms of pore theory, the effective pore radius of the c a p i l l a r y endothelium was i n excess of 11 nm, while that of the epithelium was only 0.6 nm. This may explain why lung l i q u i d contains such a small concentration of protein (molecular radius of albumin i s about 3.5 nm) (Walters & Ramsden 1985) . Several years ago S i l v a and co-workers (1977, i n Gatzy 1983) proposed a scheme to explain CI - secretion by the shark r e c t a l gland. This model was l a t e r applied to other CI - secreting and NaCl absorbing e p i t h e l i a ( F r i z z e l l et a l . 1979, i n Gatzy 1983). The hypothesis depended on the active Na+ transport by the epithelium, a l l i e d to a r e c i r c u l a t i o n of the Na+ ion (Figure 1). In CI - secreting e p i t h e l i a Na+ i s assumed to be transported out of the e p i t h e l i a l c e l l s across the basolatera] membranes by a process that requires a Na+,K+, Mg-ATPase and i s ouabain sensitive. Transepithelial Na+ 7 transport results only i f the apical (luminal) c e l l membranes are permeable to Na +. This permeability i s s e l e c t i v e l y inhibited by amiloride. The Na4 and CI - enter the c e l l s across the basolateral membranes by a neutral coupled process that i s driven by the chemical Na+ gradient. Because the c e l l i n t e r i o r i s electronegative with respect to i n t e r s t i t i a l (submucosal), and usually the luminal compartments, some of the accumulated C l " moves down the electrochemical gradient across apical membranes through a conductive path (Walters & Ramsden 1985). The shark rect a l gland appears to share similar mechanisms of C l -transport with a number of other e p i t h e l i a , including the corneal epithelium (Nagel & Reinach 1980, Candia et a l . 1981, i n Welsh 1987), large and small mammalian intestine ( F i e l 1979, F r i z z e l & Heintze 1979), opercular epithelium of teleost (Degnan et a l . 1977, in Welsh 1987), gastric mucosa (Machen & McLenan 1980, in Welsh 1987) and the canine tracheal epithelium, which might be expected to show s i m i l a r i t i e s to the f e t a l alveolar c e l l s (Widdicombe et a l . 1979, Gatzi 1983). Tracheal epithelium contains a Na4,K4-ATPase (Westenfelder et a l . 1980, in Welsh 1987) that has been lo c a l i z e d to the basolateral membrane by autoradiographic ouabain-binding (Widdicombe et a l . 1979, Welsh 1987) . When active Na 4 transport by e p i t h e l i a l c e l l s i s inhibited by ouabain, or K4 removed from the submucosal bathing solution, the Na+ and thus C l - secretion i s also inhibited (Al-Bazzaz & Al-Awqati 1979, Widdicombe et a l . 1979a, Widdicombe et a l . 1979b). Net t r a n s e p i t h e l i a l Na4 movement stops, the i n t r a c e l l u l a r Na4 concentration r i s e s , while c e l l K4 f a l l s , i n t r a c e l l u l a r negativity declines, and C l - enters. Increased c e l l C l - and Na4 would be expected to increase back flow through the coupled c a r r i e r , so that the unidirectional flux of C l - from lumen to i n t e r s t i t i u m might be expected to increase (Walters & Ramsden 1985). 8 The N a 4 , K 4 - A T P a s e f u n c t i o n s i n t h e e p i t h e l i u m i n a mode s i m i l a r t o t h a t o b s e r v e d i n o t h e r c e l l s , e x t r u d i n g N a 4 f r o m t h e c e l l and a c c u m u l a t i n g K 4 . As a r e s u l t , t h e i n t r a c e l l u l a r N a + c o n c e n t r a t i o n i s l o w and t h e K + a c t i v i t y i s h i g h . K + e n t e r i n g t h e c e l l on t h e N a 4 , K 4 pump must be r e c y c l e d a c r o s s t h e b a s o l a t e r a l membrane, s i n c e t h e r a t e o f t r a n s e p i t h e l i a l K 4 s e c r e t i o n i s m i n i m a l ( W e l s h 1 9 8 7 ) . S e v e r a l o b s e r v a t i o n s s u g g e s t t h a t t h e model o f C l - s e c r e t i o n by s u r f a c e e p i t h e l i a l c e l l s o r a i r w a y e p i t h e l i a i s c o m p a t i b l e w i t h t h e model d i s c u s s e d above ( F i g u r e 1 ) . The c e l l s e c r e t e s C l - f r o m t h e s u b m u c o s a l s u r f a c e t o t h e m u c o s a l s u r f a c e and a b s o r b s N a + i n t h e o p p o s i t e d i r e c t i o n ; b o t h p r o c e s s e s a r e e l e c t r o g e n i c . The main f e a t u r e s o f t h e model a r e : 1) . C l - e n t e r s t h e c e l l a c r o s s t h e b a s o l a t e r a l membrane v i a an e l e c t r i c a l l y n e u t r a l N a C l c o t r a n s p o r t p r o c e s s . B e c a u s e C l - i s c o u p l e d t o N a 4 , t h e movement o f N a 4 " d o w n " a f a v o r a b l e e l e c t r o c h e m i c a l g r a d i e n t a c r o s s t h e b a s o l a t e r a l membrane d r i v e s C l - " u p h i l l " a g a i n s t i t s e l e c t r o c h e m i c a l g r a d i e n t . T h e r e b y , C l - i s a c c u m u l a t e d w i t h i n t h e c e l l a t an a c t i v i t y g r e a t e r t h a n p r e d i c t e d f o r e l e c t r o c h e m i c a l e q u i l i b r i u m w i t h t h e e n e r g y p r o v i d e d i n d i r e c t l y by t h e N a 4 e l e c t r o c h e m i c a l g r a d i e n t . 2) . N a 4 , w h i c h e n t e r s t h e c e l l a t t h e b a s o l a t e r a l membrane c o u p l e d t o C l - , e x i t s back a c r o s s t h e b a s o l a t e r a l membrane v i a t h e N a 4 , K 4 A T P a s e . T h i s enzyme k e e p s i n t r a c e l l u l a r N a 4 a c t i v i t y l o w and t h u s s u p p l i e s t h e n o n c o n j u g a t e e n e r g y f o r t r a n s e p i t h e l i a l C l - s e c r e t i o n . K 4 , w h i c h e n t e r s t h e c e l l i n e x c h a n g e f o r N a 4 on t h e N a 4 , K 4 pump, e x i t s p a s s i v e l y a c r o s s t h e K 4 p e r m e a b l e b a s o l a t e r a l membrane. 3) . C l - l e a v e s t h e c e l l p a s s i v e l y , m o v i n g down a f a v o r a b l e e l e c t r o c h e m i c a l g r a d i e n t a c r o s s a C l - c o n d u c t i v e a p i c a l c e l l membrane. S e c r e t a g o g u e s a p p e a r t o r e g u l a t e t h e p e r m e a b i l i t y o f t h e a p i c a l membrane t o 9 Figure 1. A c e l l u l a r model of ion transport in CI - secreting epithelium. Secretory: the c e l l secretes CI - from the submucosal surface (blood) to the mucosal surface (lumen) and absorbs Na+ i n the oposite d i r e c t i o n ; both processes are electrogenic. In CI - secreting e p i t h e l i a Na4 i s assumed to be transported out of the e p i t h e l i a l c e l l s across the basolateral membranes by a process that requires a Na4,K+,Mg-ATPase and i s ouabain sensitive. Chloride accumulates beyond i t s electrochemical equilibrium inside the c e l l as a result of secondary active transport across the basolateral membrane via Na,K/2Cl co-transport system that i s inh i b i t a b l e by loop diuretics such as piretanide and bumetanide. Regulation of ion transport i s achieved by second-messenger gating of apical chloride channels, which when open allow chloride to exit down i t s concentration gradient into the lumen. Absorptive: i n this model, the paracellular path i s r e l a t i v e l y impermeable to sodium, which now moves t r a n s c e l l u l a r l y . The entrance of Na4 through the apical membrane of the c e l l i s s e l e c t i v e l y i n h i b i t e d by amiloride. 10 11 C I - , thus controlling the rate of CI - secretion. 4) . The apical membrane i s also permeable to Na4 ions. Na4 may enter the c e l l passively at the apical membrane driven by a favorable electrochemical gradient. 5) . Accordingly, during secretion, tr a n s e p i t h e l i a l current flow has several components. At the apical membrane, current i s carried by conductive CI - exit and, to a lesser degree, Na4 entry, while at the basolateral membrane, current i s carried by conductive K+ exit and Na+ exit via the Na 4, K4 pump. When furosemide i s added to the submucosal bathing solution i t s p e c i f i c a l l y i n h i b i t s CI - secretion but does not alter the rate of Na4 absorption (Ludens 1982, in Gatzy 1983, Welsh 1987). On the other hand, in the eye, exposure of the corneal surface to a concentration of amphotericin B that increases t r a n s e p i t h e l i a l CI - permeability abolishes the i n h i b i t i o n effects of loop diuretics on CI- secretion (Candia et a l . 1981, in Gatzy 1983) . Consequently, both coupled Na-Cl entry and passive CI - exit may be inhibited by loop diuretics (Gatzy 1983). CI - i s accumulated against an electrochemical gradient of 35 mV in the canine trachea. Possibly some i n t r a c e l l u l a r mediator may regulate the CI - entry process. One reasonable candidate to regulate the CI - entry mechanism i s i n t r a c e l l u l a r cAMP, since i t i s known to increase with stimulation of secretion. Furthermore, cAMP i s known to mediate e l e c t r i c a l l y neutral CI - entry mechanisms i n other e p i t h e l i a , shark r e c t a l gland, (Silva et a l . 1977) and flounder intestine ( F r i z z e l l et a l . 1979, in Welsh 1987). CI - accumulation i s Na4 dependent; removing Na4 i n h i b i t s CI - entry at the basolateral membrane and allows CI - to dis t r i b u t e passively across a Cl~ conductive apical membrane (Welsh 1987). These observations, taken together, indicate that CI - i s accumulated across the basolateral membrane, against i t s electrochemical gradient, via a Na4 dependent, e l e c t r i c a l l y neutral transport process that i s inhibited by loop d i u r e t i c s . While these considerations provide compelling evidence for Na4 coupled Cl- entry, the i d e n t i t y of the transport process i s not known with certainty. There are several possible neutral C l - transport processes that might mediate C l - entry: (1) NaCl cotransport,(2) cotransport of 2 C l " , 1 Na4 and 1 K4, a process found in Ehrlich ascites c e l l s (Geek et a l . 1980, i n Welsh 1987); or (3) p a r a l l e l countertransport of Na4-H+ and C I - - H C O 3 - , proposed as the mechanism in the rabbit ileum (Liedtke & Hopfer 1982, in Welsh 1987). The t h i r d alternative seems unlikely since neither removing H C O 3 - from the bathing solution (Al-Bazzaz & Al-Awqui 1979) nor adding substituted stilbenes (which i n h i b i t C I - / H C O 3 - exchange in other c e l l s ) (Cabantchik & Rothstein 1974, i n Welsh 1987) i n h i b i t s C l " secretion in tracheal epithelium (Welsh 1987). Epinephrine stimulates C l - secretion in the tracheal epithelium (Welsh et a l . 1983, in Welsh 1987). The effect of cAMP-mediated secretagogues i s an increase in the apical C l - permeability (Welsh 1987). K4 entering the c e l l on the Na4,K4 pump must be recycled across the basolateral membrane, since the rate of t r a n s e p i t h e l i a l K4 secretion i s minimal. Several observations indicate that K4 exits passively across a basolateral membrane that i s predominantly K4 conductive (Welsh 1987) . As the rate of C l - secretion increases, the rate of Na4 entry into the c e l l (via the Na 4-coupled C l - entry process) w i l l increase; as a r e s u l t , the rate of Na4 extrusion (via the Na4,K4 pump) w i l l increase. Given a constant pump stoichiometry, the rate of K4 entry into the c e l l w i l l also increase. If K4 i s to recycle back across the basolateral membrane, either the driving force for K4 must increase or the basolateral membrane K4 permeability must increase (Welsh 1987). Na+ absorption by tracheal epithelium i s an electrogenic process. Mucosal amiloride i n h i b i t s Na1 absorption (Widdicombe & Welsh 1980). Although a variety of neurohumoral agents mediate the rate of CI - secretion, no endogenous substance has yet been found that stimulates the rate of Na+ absorption. Although the details of the mechanisms of the formation of lung l i q u i d are s t i l l not clear, the system probably operates on the same principles as other CI - secretory e p i t h e l i a (Olver 1977). ABSORPTION OF THE LUNG FLUID Lung l i q u i d continues to be secreted early in labour, but as delivery of the fetus approaches, secretion slows, and absorption of the lung l i q u i d i s observed when a presenting part (the forelimbs) appears at the vaginal outlet (Walters et a l . 1978). The effect appears to be mediated by ^-receptors, since i t i s blocked by propanolol, and a large concentration of noradrenaline has l i t t l e effect, whereas isoprenaline i s even more potent than adrenaline (Walters et a l . 1978). According to Walters & Ramsden (1985) a l l of the slowing of secretion and the absorption of lung l i q u i d observed during labour can be explained by the endogenous release of adrenaline by the fetus. It i s plain that much of the l i q u i d present must be absorbed at the start of breathing. It i s d i f f i c u l t to aspirate more than half of what i s actually present through a tracheostomy, so i t i s unlikely that much f l u i d i s expelled through the mouth during vaginal delivery (and even less would be expected to drain away after Caesarean section. From lung weight and lymph flow measurements, Humphreys et al.. (1967) showed that much of the l i q u i d i s cleared by lymphatics from the i n t e r s t i t i a l spaces of the lung over a period of 5-6 h, but i t seems l i k e l y that i t must be displaced from 14 the alveolar spaces much more rapidly, otherwise e f f i c i e n t gas exchange wou ld not be possible (Olver et a l . 1974). Olver & Strang (1974) found that arrest of lung l i q u i d secretion, brought about by adding KCN to lung l i q u i d , was followed by i t s absorption, but at such a rate that approximately 12 h would be needed to clear the lungs. The slow rate was attributed to the low permeability of the f e t a l alveolar walls to C l - , the pr i n c i p a l anion i n the lung l i q u i d (Egan et a l . 1975). The apparent anomaly here i s that from what we know about alveolar permeability to C l - , the slowest moving of the bulk solutes present i n lung l i q u i d , we would expect complete absorption to take in excess of 10 hours. However , Humphrey and co -wo r ke r s (Humphreys et a l . 1967) have shown t h a t a l l t he a l v e o l a r l i q u i d i s c l e a r e d f rom the lungr. i n 4-6 hou r s i n newborn lambs (about 40% via-lymph and the rest d i r e c t l y into the c i r c u l a t i o n ) , and observations in humans and animals show that a functional residual c a p a c i t y and e f f i c i e n t gas exchange are established within a few minutes o f b i r t h (Olver 1977) . Although there are probably, at the onset of breathing, hydrostatic as well as protein osmotic pressure gradients across the alveolar epithelium favouring absorption, i t was concluded that, whatever their magnitude, the removal rate would be limited by the movement of C l - (and Na+) into the int e r s t i t i u m (Egan et a l . 1975) . Rapid clearance could only be achieved at bi r t h by an increase in pore size or increase in the e p i t h e l i a l area. Egan et a l . (1976) have demonstrated that pore size increases some eightfold at b i r t h , and they postulate that stretching of the epithelium f a c i l i t a t e s the movement of small solutes, and hence water, between c e l l s (Olver 1977). MECHANISMS OF LUNG LIQUID ABSORPTION Absorption of f e t a l lung l i q u i d could be the result of several different mechanisms. For example, the large protein osmotic difference between lung l i q u i d and the blood plasma would be expected to produce passive absorption of l i q u i d i f the active transport of chloride into the luminal space were to be t o t a l l y i n h ibited. In addition, an increase in permeability to ions, but not to protein, could also help f l u i d reabsorption. Indeed, a temporary increase in e p i t h e l i a l permeability of this type, presumably due to stretching of the alveolar surface, has been described in the f i r s t few hours of spontaneous breathing i n neonatal lambs (Egan et a l . 1975). It i s possible that alterations in pulmonary blood flow could affect l i q u i d movement across the pulmonary epithelium by an effect on the hydrostatic gradient. However, Olver and Strang demonstrated that changes in pulmonary vascular pressure s u f f i c i e n t to double lung lymph flow had no effect on the rate of lung l i q u i d secretion (Olver & Strang 1974). Furthermore, adrenaline can induce rapid absorption of lung l i q u i d in a dose that has no measurable effect on pulmonary blood flow, as detected by electromagnetic flow meters placed around the pulmonary artery. Also i t i s known that isoprenaline and adrenaline have opposite effects on the pulmonary vasculature but similar effects on lung l i q u i d secretion (Cassin & Perks 1982). Studies with the d i u r e t i c , amiloride, suggest that p-receptor stimulation of the f e t a l lung appears to stimulate sodium transport from lumen to plasma, an effect which i s dependent on activation of sodium channels i n the apical surface of the e p i t h e l i a l c e l l s . This could be the result of configurational changes of pre-existing channels, or the insertion of preformed channels into the membrane. When an adrenaline infusion i s stopped, the effect on the lung wears off rapidly and the epithelium begins to secrete again within minutes (Walters & Ramsden 1985). Normal gas exchange in the mammalian lung requires v i r t u a l l y f l u i d free airways and a l v e o l i . It i s l i k e l y that these a i r spaces are maintained in their r e l a t i v e l y dry state by barriers that prevent the flow of f l u i d and solutes from the surrounding i n t e r s t i t i a l and vascular f l u i d into the a l v e o l i and airways. Recent reviews (Staub 1974, i n Crandall 1983) have discussed the factors that influence f l u i d balance between the a i r , i n t e r s t i t i a l and vascular spaces in the lung. Many fundamental studies have been performed on mammalian lungs, involving observations of the transfer of water and solutes between f l u i d - f i l l e d spaces, i n t e r s t i t i a l f l u i d (usually represented by lymph), and/or vascular f l u i d (Vangensteen et a l . 1969, in Crandall 1983) . I t has been concluded that the e p i t h e l i a l barrier l i n i n g the a i r spaces of the adult lung i s a major factor in preventing the movement of f l u i d and solutes into the a l v e o l i (Taylor & Gaar 1970, in Crandall 1983). In addition the transfer of macromolecules across the alveolar e p i t h e l i a l barrier i s reduced by the tight junctions (Scheeberger-Keeley & Karnovsky 1968, Scheeberger-Keeley & Karnovski 1971, in Crandall 1983). If any j u s t i f i c a t i o n i s needed for presenting data from the f e t a l lung to those predominantly interested in a i r breathing, i t i s that study of the fetus may give us useful insight into alveolar f l u i d balance i n the adult. Being naturally f l u i d - f i l l e d , the f e t a l lung lends i t s e l f to the i n vivo study of t r a n s e p i t h e l i a l transport of solutes and water in a way that, does not pertain after b i r t h (Olver 1983). Fetal lung f l u i d i s very low in bicarbonate, suggesting that acid secretion takes place (Adamson et a l . 1969). Immediately after b i r t h , the air-blood barrier becomes somewhat leaky, and then tight again (Egan et a l . 1969, in Olver et a l . 1981a), and active sodium transport out of the a l v e o l i may be involved i n clearance of lung f l u i d (Olver et a l . 1981a, Crandall 1983). The overview of the f e t a l lungs, and their f l u i d production, presented here suggests that a more sophisticated understanding of this system i s beginning to emerge. However, studies have been limited almost en t i r e l y to the f e t a l sheep, with a l i t t l e investigation of the f e t a l goat, a few early studies on rabbits, and one investigation of the guinea pig. The results have been applied to human problems at b i r t h , yet there i s no evidence that a l l mammals operate i n the same way. Indeed, ruminant mammals often have a markedly different physiology from other groups. Therefore, i t seemed important to extend research to other mammals, which could possibly resemble the human baby more closely than the sheep. The guinea pig i s p a r t i c u l a r l y useful because i t carries unusually large fetuses for i t s size, and i s more readily available in large numbers than the sheep. Tn addition, the size of the f e t a l lungs presents important new p o s s i b i l i t i e s . While i t i s large enough to investigate by methods already established in f e t a l sheep, i t i s small enough to be maintained in v i t r o with the p o s s i b i l i t y of a continued secretion. Work with the acute goat and chronic sheep present particular d i f f i c u l t i e s in investigating detailed mechanisms of the secretion and control of the f e t a l lung. Many transport i n h i b i t o r s and hormones may be destroyed rapidly in the intact animal, or lost through the placenta to the large reservoir of the mother, or act i n d i r e c t l y , and i t i s d i f f i c u l t to know the concentrations which act on the pulmonary epithelium. In addition, the i n h i b i t i o n may only act. i n d i r e c t l y in the whole animal. More importantly, some drugs are toxic to the whole preparation -for example ouabain w i l l stop the heart and k i l l the intact fetus. Some of these problems could be solved by tissue culture, and this has been attempted by some groups (Moss & S c a r p e l l i 1979). However, this technique involves the loss of the organization of the lung. An in v i t r o preparation could allow the retention of the lung structure, and yet enable toxic drugs and i n h i b i t o r s to be applied in known concentration to the serosal surface cf the a l v e o l i . T h e r e f o r e the work p r e s e n t e d he r e e x t end s s t u d i e s to the gu i n ea p i g , wh i ch has o n l y been i n v e s t i g a t e d i n one e a r l y s t u d y , wh i ch used r e l a t i v e l y s i m p l e methods ( S e t n i k a r et. a l . 1 9 5 9 ) . I t a l s o i n v e s t i g a t e s t he b a s i c p r o p e r t i e s o f the l u n g s e c r e t i o n i n the i s o l a t e d p r e p a r a t i o n . 19 S T A T E M E N T O F T H E P R O B L E M The objective of this study was to introduce the isolated lungs of the f e t a l guinea pig as a new preparation for the study of the physiology of the lung prior to b i r t h , and to establish i t s basic properties by the use of metabolic i n h i b i t o r s (iodoacetate, flu o r i d e , cyanide, azide, dinitrophenol) and of the C l - transport i n h i b i t o r (piretanide). In addition, the effects of two naturally occurring peptides, somatostatin and i n s u l i n were investigated. It also extends studies to a species which has received no investigation by modern methods. M E T H O D S A N D M A T E R I A L S Near term guinea pigs, between 54 and 67 days gestation (normal term = 67 days) were used in the experiments carried out i n this study. Whenever possible, ages were based on the previous delivery data, since guinea pigs go into oestrus and w i l l mate immediately after p a r t u r i t i o n . In some cases, ages were estimated from f e t a l weights, using the average weight of each l i t t e r correlated to the number of fetuses (average l i t t e r size 4; range, 3-10), after the method of Ibsen (1928) . The estimation was modified from age/weight data obtained from animals of known gestation age from our own stock. The guinea pigs were kept under optimal conditions i n a vivarium, according to Canadian Guidelines for Animal Care. They were fed Purina rat chow and given free access to water. SURGICAL PROCEDURES The pregnant guinea pig was placed in a container which had a layer of ether-drenched shavings. The animal was l e f t there for approximately ten minutes, u n t i l the blink reflex was extinguished. Then she was removed from the container and placed in a dissection tray. A mid-line i n c i s i o n was made from the sternum through the abdomen to the bottom of the uterus. The uterus was then removed and the fetuses taken out i n d i v i d u a l l y , taking care that the amnion remained intact around the head. A single tight ligature was placed around the amnion and neck; this procedure e f f e c t i v e l y prevented the fetus from breathing. At this point the umbilical cord was cut, and each fetus was weighed rapidly and placed i n a beaker containing Krebs-Henseleit saline at 37°C (see Appendix A). A mid-line i n c i s i o n was made from the abdomen to the top of the neck of the fetus. The sternum was cut, with care not to damage the lungs. The r i b cage was then opened to expose the lungs and trachea. The lungs and trachea were rinsed repeatedly with warm Krebs-Henseleit saline to keep them 21 moist and to prevent any accumulation of blood during dissection. Two ligatures were passed under the trachea; the f i r s t was used to t i e off the trachea as far r o s t r a l l y as possible, the second to secure a cannula. The cannula was inserted through a s l i t placed below the upper liga t u r e . The r o s t r a l end of the cannula was connected to a 3-way valve with a 1 cc glass tuberculin syringe. The cannula and the 3-way valve had previously been f i l l e d with warmed Krebs-Henseleit saline, taking care to remove any bubbles from the system. The 3-way valve allowed the f l u i d to be withdrawn and returned to the lung. In addition, f l u i d could be passed into the t h i r d cup of the stop-cock, and th i s allowed the addition of dye or the withdrawal of samples. To remove the lungs together with the cannula system, the trachea was cut above the cannula. A sl i g h t tension was put on the trachea to allow the connective tissue to be cut beneath the preparation. The preparation was then separated from the body, and the heart was removed. Throughout the dissection, the lungs were rinsed with Krebs-Henseleit saline to keep the organ warm and moist. The lungs were then placed in a 50 ml container with Krebs-Henseleit saline bubbled with 95% 02/5% CO2, and kept at a constant temperature of 37° C i n a water bath (Figure 2). EXPERIMENTAL PROCEDURES 1) . Basis of the method The rate of lung l i q u i d secretion was measured by a dye d i l u t i o n technique, using Blue Dextran 2000, and based on the methods of Normand et a l . (1971), and Perks & Cassin (1985b). 2) . Methods After placing the lung preparation in the 50 ml container of Krebs-Henseleit saline, lung f l u i d was withdrawn into the syringe and a 10 mic r o l i t r e sample of the lung f l u i d was taken, as a blank, at zero time. 0.1 22 Figure 2. Apparatus for the maintenance of the isolated f e t a l lung. 23 ml of freshly dissolved Blue Dextran 2000 (50 mg/ml of 0.9% NaCl; Pharmacia, Uppsala Sweden) was mixed thoroughly into the remaining lung f l u i d and reinfused into the lung. At 5 minute in t e r v a l s , lung f l u i d was gently mixed by withdrawing f l u i d and inj e c t i n g i t back into the lung 3 times with the tuberculin syringe. The mixing of the dye also helped to open up any pockets of the lung which the dye had not reached. Additional mixing was provided by the agitation of the preparation by the bubbles of the oxygen supply. The lung preparation was allowed to equilibrate for 30 minutes i n saline solution; the outer saline was replaced at 15 and 30 minutes, to remove any blood or other contaminants released by the dissection procedure, and to renew necessary materials. Samples were taken every 10 minutes by drawing the lung f l u i d into the tuberculin syringe (after mixing) and then closing off the valve to the lung. The f l u i d could then be injected into the top part of the 3-way valve, where a sample could be taken by means of the 10 mi c r o l i t r e fixed volume syringe. Samples were then placed into 0.25 ml p l a s t i c microcentrifuge tubes and diluted 20 times with 0.19 ml of d i s t i l l e d water, using a fixed volume syringe. The samples were then mixed by putting them on a vortex mixer (Vortex Genie Fisher s c i e n t i f i c ) for fiv e seconds. The samples were then centrifuged at approximately 1500 rpm for 10 minutes (Model 1221, International Equipment Co.) The samples from the f i r s t hour after the equilibration period gave the basic rate of secretion of the lung preparation (control period). A l l samples were estimated for Blue Dextran 2000 by spectrophotometer (model 250, G i l l f o r d Instruments; used at 620 nm with 0.10 ml microcells). Readings on the spectrophotometer were taken nine times per sample, and then averaged. In the second hour the lung was transferred to a bath which contained one of the various substances used in this study. The test materials used were: Insulin 10 - 6 M, Insulin 10 - 7 M, Piretanide 10 - 7 M, Piretanide 10 - 8 M, Somatostatin 10 _ s M, Somatostatin 10 - 6 M, Somatostatin 10"7 M, Iodoacetate 10~3 M, Iodoacetate 10-" M, Dinitrophenol 2xl0-« M, Sodium Chloride 10~3 M, Sodium Fluoride 10"3 M, Sodium Azide 10~3 M, Sodium Cyanide 10"3 M. After 60 minutes i n the test solution the lung preparation was tranferrred again to a fresh bath of Krebs-Henseleit saline to find out whether the lung would recover from the treatment. This procedure constituted an ABA experimental design. The control periods were the hour before and the hour after treatment. In addition, external controls were tested, since in studies of metabolic i n h i b i t o r s , recovery might be slow or absent. In the external controls, the lungs were tranferrred in the same manner, but received saline alone i n the middle period (Figure 3). STATISTICAL METHODS F l u i d production rates were calculated from the f a l l in concentration of Blue Dextran. They were estimated from plots of the t o t a l volume of f l u i d against time, with readings recorded every ten minutes; the to t a l volume of f l u i d was the sum of that within the lungs and that removed for study. The rates of production of f l u i d over one hour intervals were calculated from the volume plots, using the slopes of their linear regressions, f i t t e d by the method of least squares (Steel and Torrie 1970, in Cassin & Perks 1982; Apple I I computer). The significance of changes i n rate were estimated from changes in slope, analysed by a t-test for the difference between two regressions (Steel & Torrie 1970; for details see Cassin & Perks 1982). Significance was accepted at p< 0.05. Where results from different fetuses were combined, the volumes for' each fetus were 26 Figure 3. Total Volume of Lung Secretion of Untreated Preparations. Abscissa: t o t a l volume of lung secretion, expressed as a percentage of that present at the end of the f i r s t hour. The slopes represent the secretion rates; the values below the lines give the average rates in ml/Kg per h of 24 fetuses. The points are graphed with their associated standard errors. TOTAL V O L U M E of L U N G SECRETION (%). expressed as a percentage of the volume- at the start of the treatment; the corresponding values were averaged. A l l mean values are given with their standard errors. Na* and K+ ions were measured by flame photometer (Instrumentation Laboratories, Inc. Model 343), and C l - ions by a Buchler-Cotlove Chloridometer. The rates of secretion of ions were calculated by the same methods used for secretion of f l u i d , where t o t a l milliequivalents replaced t o t a l volume. Rate of secretion of fluid by fetal guinea pig lungs in .vitro Untreated Preparations The control groups consisted of six or twelve guinea pigs. The secretion rates of each group were averaged for each of the three experimental hours to obtain combined data. A l l the control experiments showed a steady increase in the volume of lung f l u i d throughout the experiments; there were no si g n i f i c a n t differences in the rates in the different hours. One combined group i s shown in Figure 3; the average secretion rates were 1.71 ± 0.32 ml/Kg per h for the f i r s t hour, 1.54 ± 0.27 ml/Kg per h for the second hour and 1.56 ± 0.26 ml/Kg per h for the th i r d hour, and there were no sign i f i c a n t changes between any of the periods. The results show that the- in v i t r o lung continues to secrete for at least 3 hours, with l i t t l e change, and the rates are comparable to those found in in vivo preparations of sheep and goats (see Discussion). Experimental Groups a). Sodium iodoacetate Iodoacetate i s a well-known i n h i b i t o r of the g l y c o l y t i c pathway, via the i n h i b i t i o n of frutose diphosphate aldolase (Lehninger 1975) . Sodium iodoacetate at 10 _ 3K A l l six individual experiments showed a similar reduction in secretion after iodoacetate. In a l l cases, the reduction was seen in the f i r s t hour of treatment, where the change was si g n i f i c a n t in 4 preparations (p<0.05-0.001); i n 3 experiments secretion changed to reabsorption. In the f i n a l hour a l l reductions but one were s i g n i f i c a n t (p<0.05-0.01), and reabsorption occurred in two preparations; c l e a r l y the effect of iodoacetate 30 persisted after i t s removal from the outer saline. The results are combined in Figure 4. The figure suggests that iodoacetate abolishes secretion by the isolated lung, with an average depression from 2.65 ± 0.54 ml/Kg per h to 0.34 ± 0.29 ml/Kg per h i n the hour of the treatment and a sl i g h t reabsorption of - 0.002 ± 0.015 ml/Kg per h i n the f i n a l hour. Both these reductions were s i g n i f i c a n t at p<0.001. Sodium iodoacetate at 10-4M Five out of six experiments showed a similar reduction i n secretion at a lower concentration of iodoacetate. The reduction was seen i n the f i r s t hour of treatment, where the change was si g n i f i c a n t i n two preparations (p<0.05-0.001); in one preparation secretion changed to reabsorption; however, in one experiment the secretion rate increased s i g n i f i c a n t l y . In the f i n a l hour a l l secretion rates decreased, but only two reductions were si g n i f i c a n t (p<0.02). The effect of iodoacetate continued after i t s removal from the outer saline. The combined results are presented in Figure 4. The figure suggests that iodoacetate reduces secretion by the isolated lung, with an average depression from 2.31 ± 0.44 ml/Kg per h to 1.03 ± 0.57 ml/Kg per h i n the hour of treatment and no recovery in the f i n a l hour, when the secretion rate was 0.81 ± 0.32 ml/Kg per h. Both reductions were si g n i f i c a n t (p<0.05-0.001). A rough estimate from the re s u l t s , by use of their log dose/response relationship, suggests a threshold l e v e l at 4.1xlO _ 6M iodoacetate. The results suggest that glycolysis i s necessary for the production of lung f l u i d i n the isolated preparation. b). Sodium Fluoride These results were checked by the use of an alternative i n h i b i t o r of gl y c o l y s i s , sodium fluo r i d e , which i n h i b i t s a different enzyme, enolase (Baldwin 1952, Kessler 1966). 31 Figure 4. The effect of different concentrations of sodium iodoacetate on lung f l u i d secretion in f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) Na iodoacetate at 10-"M, 0.8678 ± 0.1689 ml; (b) Na iodoacetate at 10-3M, 0.8865 ± 0.1109 ml; (c) control, 0.08826 ± 0.0866 ml. Ordinate: time i n hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, in Cassin & Perks 1982). The slopes represent the secretion rates; the values below the li n e s give the average rates i n ml/Kg body weight per hour. Asterisks above the l i n e show si g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . A l l graphs are averages based on s i x fetuses. 32 120i 100 LU DC O LLI (/) o o LU 70 100 70 140 100H < I -O 80 . 1 (a) n = 6 (b) n = 6 2.65 ± 0 . 5 4 (c) n = 6 2.23 ± 0 . 4 3 t i d 3 M N a I O D O A C E T A T E | C o n t r o l s TIME in HOURS -0.002 ± 0 . 0 1 5 Sodium Fluoride at 10~3M A l l six experiments showed reduction in secretion after sodium fluo r i d e . Two of the s i x showed reabsorption. The change was si g n i f i c a n t in 5 preparations (p<0.02-0.01). In the f i n a l hour, 4 out of the 6 reductions were si g n i f i c a n t (p<0.02-0.001); reabsorption continued in one preparation. The effect of sodium fluoride continued after i t s removal from the outer sali n e . The results are combined in Figure 5. The figure shows that sodium fluoride abolishes secretion by the isolated lung, with an average depression from 2.96 ± 0.53 ml/Kg per h to 0.2 + 0.35 ml/Kg per h. After the treatment a s l i g h t recovery seems to occur; the secretion rate was 0.31 + 0.31 ml/Kg per h in the f i n a l hour. Both these reductions were sig n i f i c a n t at p<0.001. c) . Sodium Cyanide The possible importance of the aerobic metabolic pathway was then tested by the use of NaCN, which i s known to i n h i b i t the cytochrome system and catalase ( K e i l i n 1936a, K e i l i n & Hartree 1938, Horecker & Stannard 1948). Sodium Cyanide at 10"3M The effects of NaCN 10 _ 3M were more variable than those of the g l y c o l y t i c i n h i b i t o r s . Four preparations showed small reductions in secretion, two of which were si g n i f i c a n t (p<0.001). The combined data suggests that CN- has a small effect on secretion rates, with average values f a l l i n g from 3.25 ± 1.18 ml/Kg per h to 2.06 ± 0.75 ml/Kg per h and to 2.28 + 0.83 ml/Kg per h in succeeding hours. Both reductions were si g n i f i c a n t at p<0.01-0.001. The results suggest the aerobic pathways have some influence on the secretion of lung f l u i d , but the effect i s r e l a t i v e l y small. d) . Sodium Azide Na azide was tested as an alternative to NaCN, since i t also 34 Figure 5 . The effect of sodium fluoride on lung f l u i d secretion i n f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) Na fluoride at 10"3M, 0.9840 ± 0.1199 ml; (b) control, 0.8826 ± 0.0866 ml. Ordinate: time i n hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates i n ml/Kg body weight per hour. Asterisks above the l i n e show si g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . A l l graphs are averages based on s i x fetuses. 115r . 1 0 0 uj 7 0 1 DC 140 120 (b) n = 6 0.2 ±0.35 N a F l u o r i d e 1 0 3 M 0.31 ±0.31 100 801 . 1 Controls TIME in HOURS interferes with the aerobic metabolic pathway by actions on cytochrome oxidase and catalase ( K e i l i n 1933, 1936, K e i l i n & Hartree 1934, Kessler 1966). Sodium Azide at 10-3M The results for Na azide were closely similar to those for NaCN. In fi v e out of s i x experiments, reduction in secretion was found during the period of treatment, and the effect persisted into the f i n a l hour. The effect was s i g n i f i c a n t in two preparations (p<0.01-0.001). One preparation showed a r i s e i n the secretion i n the period of treatment, but th i s was not s i g n i f i c a n t , and i t f e l l in the f i n a l hour. The combined data (Figure 6) suggests an effect closely similar to that of NaCN with average values of 2.93 ± 0.64 ml/Kg per h in the f i r s t hour, 1.2 + 0.27 ml/Kg per h in the second hour and 1.30 + 0.54 ml/Kg per h in the thi r d hour. Both these reductions were s i g n i f i c a n t (p<0.01). The results confirm that the oxidative pathway has some influence on lung secretion, but i t does not appear to be as important as the g l y c o l y t i c system. e). Dinitrophenol Dinitrophenol was tested as an alternative i n h i b i t o r of oxidative processes, since i t causes powerful disruption of the mitochondria which carry the enzymes concerned, and uncouples oxidative phosphorylation (Hainstein 1976, Dall-Larsen et a l . 1976). Dinitrophenol at 2x10-4M A l l s i x experiments showed reduction i n the secretion after dinitrophenol. Four of them showed reabsorption, two continued to secrete at a low rate. The change was si g n i f i c a n t i n 4 preparations (p<0.02-0.001). Clearly the effect of dinitrophenol persisted after i t s removal from the outer saline. The results were combined in Figure 7. The figure suggests 37 Figure 6 . The effect of sodium cyanide and sodium azide on lung f l u i d secretion in f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) Na cyanide at 10"3M, 1.0235 ± 0.1752 ml; (b) sodium azide, 0.9410 ± 0.0726 ml; (c) control, 0.8826 ± 0.0866 ml. Ordinate: time in hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates in ml/Kg body weight per hour. Asterisks above the l i n e show si g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . Averages for (a) and (b) based on six fetuses, average for (c) based on twelve fetuses. 130h (a) n = 6 2.28 ±0.83 6^ z 7 0 O 140 LU DC O LU (/) iooi — B 70| O 1401 LU 3.25 ± 1 ' 1 8 \bla C y a n i d e 10 3 M (b) n = 6 1.2 ±0.27 N a A z i d e 10 M (c) n = 12 100 801 C o n t r o l s TIME in HOURS. 39 Figure 7. The effect of dinitrophenol on lung f l u i d secretion in f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) dinitrophenol at 2xl0" 4, 0.7852 ± 0.0647 ml; (b) control, 0.8826 ± 0.0866 ml. Ordinate: time in hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates i n ml/Kg body weight per hour. Asterisks above the l i n e show sig n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . A l l graphs are averages based on six fetuses. that dinitrophenol abolishes secretion and i n fact produces reabsorption in the isolated lung, with an average depression from 1.5 ± 0.43 ml/Kg per h to -0.6 ± 0.17 in the hour of treatment and continuing reabsorption to -0.78 ± 0.23 ml/Kg per h i n the f i n a l hour. Both these reductions were s i g n i f i c a n t at p<0.001. f ) . Piretanide Piretanide i s a loop d i u r e t i c which i s a potential i n h i b i t o r of coupled Na +/K +/Cl _ entry across the basolateral membranes of a number of CI secreting e p i t h e l i a (Gatzy 1983). Piretanide at 10"7M Five out of s i x experiments showed reduction in secretion after piretanide. Two of them showed reabsorption. Only one experiment continued secreting in the f i r s t hour of the treatment. The change in secretion was s i g n i f i c a n t in 4 preparations (p<0.02-0.001). In the f i n a l hour a l l reductions but one were significant. (p<0.05-0.01) and reabsorption occurred in two preparations. The effect of piretanide persists after i t s removal from the outer saline. The results are combined in Figure 8. The figure suggests that piretanide abolishes secretion by the isolated lung, with an average depression from 1.96 ± 0.24 ml/Kg per h to 0.32 + 0.03 ml/Kg per h in the hour of the treatment and 0.14 ± 0.01 ml/Kg per h in the following hour. Both of these reductions were s i g n i f i c a n t at p<0.01-0.001. Piretanide at 10"8M Five out of s i x of the individual experiments showed a s l i g h t reduction i n secretion with this lower dose of piretanide. In only one experiment i t had no effect on the secretion of the isolated lung. The change in secretion was s i g n i f i c a n t in 4 preparations (p<0.01-0.001). In the f i n a l hour reductions were si g n i f i c a n t (p<0.01-0.001). Clearly the effect of piretanide persisted after i t s removal from the outer saline. The results 42 Figure 8. The effect of different concentrations of piretanide on lung f l u i d secretion i n f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) piretanide 10"7M, 0.8745 ± 0.0627 ml; (b) piretanide at 10"8M, 0.8878 ± 0.1309 ml; (c) control, 0.8826 ± 0.0866 ml. Ordinate: time in hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates i n ml/Kg body weight per hour. Asterisks above the l i n e show si g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . A l l graphs are averages based on s i x fetuses. are combined in Figure 8. The figure suggests that piretanide reduces secretion in the isolated lung with an average depression from 2.4 ± 0.78 ml/Kg per h to 1.18 ± 0.38 ml/Kg per h in the hour of treatment and 0.67 + 0.21 ml/Kg per h in the f i n a l hour. Both reductions were s i g n i f i c a n t at p<0.001. A rough estimate from the res u l t s , by use of their log dose/response relationship, suggests a threshold level at 6.6xlO-10M piretanide. The results suggest that t h i s loop d i u r e t i c has the effect of reducing the production of lung l i q u i d in the isolated preparation. g). Somatostatin Studies of electrolyte secretion by mammalian gastrointestinal tract and pancreas indicate that somatostatin may i n h i b i t secretory transport, and i n h i b i t Cl-secretion (Carter et. a l . 1978, Epstein et a l . 1984). Somatostatin at 10"3M A l l six individual experiments showed a reduction in the secretion rate after somatostatin. In two experiments the secretion changed to reabsorption. In a l l cases the reduction was seen in the f i r s t hour of treatment, when the change was si g n i f i c a n t in 5 preparations (p<0.01-0.001). In the last hour the reductions were also s i g n i f i c a n t (p<0.01-0.05) i n f i v e preparations, and reabsorption occurred in three preparations; c l e a r l y the effect of somatostatin persisted after i t s removal from the outer saline. The results are combined in Figure 9. The figure suggests that somatostatin abolishes secretion by the isolated lung, with an average depression from 3.72 + 0.9 ml/Kg per h to 1.32 ± 0.32 ml/Kg per h in the hour of the treatment and a s l i g h t reabsorption of 0.07 ± 0.02 ml/Kg per h i n the f i n a l hour. Both these reductions were s i g n i f i c a n t at p<0.001. Somatostatin at 10 _ 6M Five of the six individual experiments showed a similar reduction in 45 Figure 9. The effect of different concentrations of somatostatin on lung f l u i d secretion i n f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) somatostatin 10-3M, 1.1675 ± 0.2200 ml; (b) somatostatin 10"6M, 0.8747 + 0.2229 ml; (c) somatostatin at 10"7M, 1.6107 ± 0.1545 ml; (d) control 0.8826 ± 0.0866 ml. Ordinate: time in hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates in ml/Kg body weight per hour. Asterisks above the l i n e show si g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . Average for (a) based on three fetuses, averages for (b), (c) and (d) based si x fetuses. TIME in HOURS J 3 47 the f i r s t hour of treatment. Only in one experiment did somatostatin have no effect i n the secretion of the lung f l u i d . In three preparations the change was s i g n i f i c a n t (p<0.02-0.001). In the f i n a l hour three reductions were si g n i f i c a n t at p<0.001, and reabsorption occurred in one preparation. It i s clear that the effect of these lower doses of somatostatin persisted after i t s removal from the outer saline. The results are combined in Figure 9. The figure suggests that somatostatin diminished secretion by the isolated lung, with an average depression from 3.06 ± 1.06 ml/Kg per h to 1.19 ± 0.54 ml/Kg per h in the hour of treatment and to 0.46 ± 0.49 ml/Kg per h in the last hour. Both of these reductions were si g n i f i c a n t at p<0.001. Somatostatin at 10~7M Only one of the three experiments showed a decreasing secretion after the lowest dose of somatostatin. The change was not s i g n i f i c a n t at p<0.05. In the f i n a l hour only 1 experiment showed a s i g n i f i c a n t reduction of f l u i d with a p<0.05. The results are combined in Figure 9. The figure suggests that at this level somatostatin has no s i g n i f i c a n t effect in the isolated lung. The average secretion rates were 1.73 ± 1.3 ml/Kg per h for the f i r s t hour, 1.83 ± 0.61 ml/Kg per h for the second hour and 1.95 ± 0.48 for the third hour. A rough estimate from the results, by use of the log dose/response relationship, suggests a threshold l e v e l at 9xlO _ 8M, see Figure 10. h). Insulin Insulin i s known to influence the f e t a l lungs, but could act i n opposite ways, since i t maintains immaturity of the lung (Tulchisnky & Ryan 1980), which might promote secretion, but can also stimulate Na* transport, which i s involved i n reabsorption (Macknight et a l . 1980, Scott & Goodman 1981, Hammerman 1985, Guntupalli et a l . 1985, in Epple 1987). Figure 10. The log-dose/response relationship for somatostatin. A rough estimate from the re s u l t s , by the use of log dose/response relationship, suggests a threshold level at 9xlO _ 8M. 50 Insulin at 10-6M A l l six individual experiments showed a similar reduction i n secretion after i n s u l i n . In a l l cases, the reduction was seen in the f i r s t hour of treatment; the change was s i g n i f i c a n t in 5 preparations (p<0.05-0.001). In 2 experiments secretion changed to reabsorption. In the last hour a l l reductions but one were s i g n i f i c a n t at p<0.05-0.001. In the two preparations with the reabsorption effect the isolated lung tended to recuperate in the last hour. In general, however, the effect of i n s u l i n persisted after i t s removal from the outer saline. The results are combined in Figure 11. The figure suggests that i n s u l i n reduces secretion by the isolated lung with an average depression from 2.47 ± 0.68 ml/Kg per h to 0.33 ± 0.09 ml/Kg per h in the hour of treatment and down to 0.28 ± 0.07 ml/Kg per h in the last hour. Both of these reductions were sig n i f i c a n t at p<0.001. Insulin at 10"7M Five of six of the individual experiments showed a similar reduction in secretion after this lower dose of i n s u l i n . One experiment does not show any effect on the secretion rate in the f i r s t hour of treatment. The change was s i g n i f i c a n t in 2 preparations (p<0.01-0.001). In the f i n a l hour 3 reductions were s i g n i f i c a n t at p<0.01-0.001. The effect of i n s u l i n persisted after i t s removal from the outer saline. The results are combined in Figure 10. The figure suggests that at this concentration i n s u l i n reduces secretion by the isolated lung, with an average depression from 2.63 ± 0.84 ml/Kg per b to 1.31 ± 0.42 ml/Kg per h i n the hour of the treatment and to 0.79 ± 0.25 ml/Kg per h in the last hour. Both of these reductions were s i g n i f i c a n t with a p<0.001. A rough estimate from the results, by use of their log dose/response relationship, suggests a threshold level of 4.48xl0"9M of i n s u l i n . 51 Figure 11. The effect of different concentrations of i n s u l i n on lung f l u i d secretion i n f e t a l guinea pigs. Abscissa: the t o t a l volume of lung secretion, expressed as a percentage of that present at the onset of the treatment, where 100% was: (a) i n s u l i n 10"6M, 0.8362 ± 0.1271 ml; (b) i n s u l i n at 10"7M, 0.9248 ± 0.0362 ml; (c) control, 0.8826 ± 0.0866 ml. Ordinate: time i n hours. A l l regressions are lines of best f i t (method of least squares; Steel and Torrie 1970, i n Cassin & Perks 1982). The slopes represent the secretion rates; the values below the l i n e give the average rates in ml/Kg body weight per hour. Asterisks above the l i n e show s i g n i f i c a n t changes (p=0.05 or below) from the o r i g i n a l slope (dotted l i n e s ) . A l l graphs averages based on six fetuses. 52 D I S C U S S I O N 1). Secretion rates by the fetal lung The work presented above shows that the f e t a l lung w i l l continue to secrete in v i t r o for 3 hours. Before these studies, extended observations have been made on the secretion of the f e t a l lung i n vivo. In 1953 Reynolds carried out r e l a t i v e l y simple experiments. He delivered fetuses from ewes by Caesarean section, maintained the placental c i r c u l a t i o n , and prevented breathing by applying a rubber nose-bag over the head of the fetus. Large volumes of f l u i d accumulated in the nose-bag during the period of experimentation, and i t was possible to make rough long-term estima'tes of secretion rates (Reynolds 1953, Avery 1968). Setnikar and co-workers (1959) performed experiments on fetuses of goats and guinea pigs during the last t h i r d of pregnancy. The heads of the fetuses were kept under Ringer solution, and the trachea was cannulated. Displacement of l i q u i d in the respiratory apparatus could be measured by movements of a meniscus in glass tubing, and this allowed measurements of the rate of secretion. However, the values obtained were variable, due to the effects of siphoning and movements of the preparation (Setnikar et a l . 1959). Later workers such as Kitterman et a l . overcame some of these problems by c o l l e c t i n g lung f l u i d in bags s u r g i c a l l y inserted into f e t a l lambs, and measuring the accumulation over 12 hour periods. This method made i t d i f f i c u l t to measure short-term changes, and impossible to detect reabsorption. However, Kitterman et a l . were able to show a progressive decline in secretion rate in the week prior to b i r t h (Kitterman et a l . 1979) . The mechanism of f e t a l pulmonary f l u i d production appears to be dependent on active transport of CI - across the alveolar epithelium. According to Olver & Strang (1974) CI - transport establishes an osmotic gradient, resulting i n movement of water into the alveolus. Olver & Strang (1974) demonstrated that increasing pulmonary blood flow s u f f i c i e n t l y to increase pulmonary lymph flow had no effect on lung l i q u i d secretion (Lawson et a l . 1978). Chronically catheterized near-term f e t a l lambs were used by Cassin et a l . (1986b) who infused different substances into the f e t a l c i r c u l a t i o n . The secretion rate of the f e t a l lung l i q u i d was measured by the dye-dilution technique, which had been used by Normand et a l . since 1971. With this method i t i s possible to measure small changes i n lung l i q u i d secretion. The technique also allows the detection of reabsorption, and i t i s convenient for s t a t i s t i c a l analysis. Average f l u i d production rates were 3.1 ± 0.3 ml/Kg per h in active experiments in chronic f e t a l sheep between 128-145 days gestation. However, production rates changed through gestation. Both the 2.2-fold r i s e to 5.2 ± 0.6 ml/Kg per h at 137-139 days gestation, and the subsequent 83 % f a l l by 143-145 days gestation were s i g n i f i c a n t at p<0.01. These results suggest that lung l i q u i d production in chronic f e t a l sheep f a l l s i n the l a s t week before delivery. However, reabsorption over b i r t h i s far more dramatic (Perks & Cassin 1985). In the experiments of Cassin et a l . (1986a, 1986b) i t was d i f f i c u l t to know the precise concentration affecting the lung; some materials may be rapidly destroyed by the l i v e r and other organs, while some may be lost through the placenta into the large reservoir of the mother (Rudolph 1977). These drawbacks are absent i n the i n v i t r o preparation. In addition, the i n v i t r o preparation i s an excellent experimental system for testing drugs which may be f a t a l to the whole animal. The results presented here allow known concentrations to be applied to the i n v i t r o lung, and overcome some of the problems discussed. The advantages of using guinea pigs as an experimental animal are that they are small, easy to maintain, they breed a l l year round, and their gestation period i s short (67 days), so we can have rapid results based on large numbers. Their main disadvantage l i e s in the small volume of f l u i d which i s available for study. The average secretion rate i n the isolated f e t a l lung during the f i r s t hour of a l l the experiments reported was 2.31 ± 0.17 ml/Kg per h ; n=104. This result i s close to those reported by the group of Normand et a l . (1971) who used the anesthesized f e t a l lamb (2.5 to 4.0 ml per h), and those of Perks & Cassin (1985b) in chronic f e t a l sheep (3.1 ± 2.2 (SD) ml/Kg per h). The result suggests that the in v i t r o preparation secretes at an essentia l l y normal rate. 2). Metabolic aspects of lung fluid secretion The lung i s recognized to be a metabolically active and hormonally responsive organ (Strubbs & A l b e r t i 1980, in Kazuhiro et a l . 1987). Efforts have been made to characterize the metabolic a c t i v i t i e s of the lung and to determine how these a c t i v i t i e s are regulated. Most studies on pulmonary metabolism have used whole lung and focused on glucose and l i p i d metabolism, which are important for the synthesis of pulmonary surfactant (Van Golde 1976, i n Kazuhiro et a l . 1987). Amino acids are of obvious importance for protein synthesis and gluconeogenesis (Ballard et a l . 1969, in Kazuhiro et  a l . 1987). In addition to i n t r a c e l l u l a r proteins, Type I I c e l l s secrete protein components of the basement membrane and the apoproteins of surface-active material. In Type I I c e l l s , amino acids can also be used for lipogenesis, for synthesis of the l i p i d components of surfactant. Amino acid uptake in the lungs has been investigated in lung s l i c e s (Thet et a l . 1977, in Kazuhiro et a l . 1987), in the isolated perfused lung (Besterman et a l . 56 1983, in Kazuhiro et a l . 1987), and most recently i n alveolar Type II c e l l s in tissue culture (Brown et a l . 1985, in Kazuhiro et a l . 1987). Recently Kazuhiro demonstrated that isolated alveolar type II c e l l s have high a f f i n i t y i n s u l i n receptors and that i n s u l i n stimulated glucose transport at physiologic concentrations (Sugahara et a l . 1984, i n Kazuhiro et a l . 1987). Presumably the glucose would be used in metabolic a c t i v i t y , and provide the energy for secretion. Therefore several metabolic i n h i b i t o r s were investigated, mainly to demonstrate that the f l u i d movements into the lungs in the in v i t r o preparation depended on metabolic energy, and were not accidental osmotic s h i f t s . a) . Effects of Sodium iodoacetate and Sodium fluoride Sodium iodoacetate was expected to decrease the secretion rate because this drug abolishes the a c t i v i t y of the fructose diphosphate aldolase dehydrogenase and therefore stops g l y c o l y s i s . Since glycolysis i s one of the main sources of energy in the c e l l i t was important to show that the secretion of lung f l u i d was dependent on this process. Sodium iodoacetate (10 _ 3M and 10-4M) decreased the f e t a l lung f l u i d secretion in the guinea pig. A greater depression of the secretion rate was observed at 10 - 3M sodium iodoacetate and no recovery took place in the last hour of the treatment. At 10 _ 4M the secretion rate was also depressed, and no apparent recovery was seen in the f i n a l hour. This result suggests that at least part of the secretion rate of the lung l i q u i d i s dependent on gl y c o l y s i s , since sodium iodoacetate i s a potent i n h i b i t o r of the secretion of f e t a l lung f l u i d at both dose l e v e l s . Sodium fluoride also had a large effect on the production of lung f l u i d ; the secretion was dramatically depressed. The effect was probably due to i t s known a b i l i t y to i n h i b i t enolase (Baldwin 1952), and therefore stop g l y c o l y s i s . Although i t also has a small i n h i b i t o r y effect on catalase, by forming a well-defined metahemoglobin compound with i t , this i s probably not i t s major effect ( K e i l i n 1936). b). Effects of Cyanide and Azide For many years i t has been known that CN" acts as a powerful respiratory poison, and i t was considered to i n h i b i t by combining with the oxidised "Atmungsferment", or "respiratory enzyme" (K e i l i n 1936). It i s now clear that cyanide may i n h i b i t a number of carriers or associated enzymes in the respiratory chain, mainly by forming stable complexes with their Fe-porphyrin compounds (Stannard 1939, in Potter & Bohlender 1941, Commoner 1939, 1940, in Winzler 1943, Lehninger 1979). Although cytochrome b appeared r e l a t i v e l y insensitive to cyanide ( K e i l i n 1936, Stolz et a l . 1938), small amounts of cyanide (10-"M) could cause cytochrome a and c to remain completely reduced, and i t i s now clear that electron transport from cytochrome a a s to oxygen i s blocked (Dixon 1929, Lehninger 1979). In addition, cyanide can i n h i b i t catalase, the enzyme responsible for the removal of H2O2 (Dixon 1929) generated by the respiratory chain, and i t also i n h i b i t s the indophenol oxidase system ( K e i l i n 1936). In contrast, cyanide does not i n h i b i t aerobic dehydrogenase or systems which react d i r e c t l y with oxygen other than the cytochrome chain (Dixon 1929). The early claim that the "Atmungsferment" accounted for a l l respiration of the c e l l was not born out by la t e r studies, since HCN did not always abolish respiration. In contrast to yeast or Chorella, a considerable number of animal tissues were only inhibited 50-80% by cyanide (Emerson 1927, Dixon & E l l i o t 1929, in Dixon 1929). An example i s the mammalian kidney, where cyanide f a i l e d to abolish the capacity for urine d i l u t i o n or concentration, although i t had a n a t r i u r e t i c action due to interference with oxidative processes concerned i n sodium transport (Matinez-Maldonado 1969). In many tissues i t appears that the cytochrome system does not contribute more than two-thirds of the t o t a l respiration, with the remaining third due to g l y c o l y s i s , or perhaps aerobic dehydrases (Dixon 1929). The maximum i n h i b i t i o n was usually produced by 10 _ 3M HCN, and increasing the concentration 100-fold produced no greater i n h i b i t i o n . The experiments presented here also show that cyanide produced a clear, but only p a r t i a l i n h i b i t i o n of secretion, which might depend, i n part, on oxidative processes. However, before leaving the effects of cyanide, i t must not be forgotten that i t can poison many systems besides the "respiratory enzyme", for instance peroxidase, catecholoxidase, catalase. Some of the i n h i b i t i o n due to cyanide could be due to these other effects (Dixon 1929). In 1933, i t was shown that sodium azide, NaN3, i n h i b i t s c e l l u l a r respiration, the oxidation of "cytochrome", and the indophenol reaction in much the same manner as cyanide. However, i t was less stable than cyanide, and i t s effect on the cytochrome system was only marked at a lower pH. Differences in the cyanide and azide i n h i b i t i o n of tissue respiration have been noted by Stannard (1939 in Winzler 1943) and by Korr (1941 i n Winzler 1943), and led them to postulate the existence of by-pass respiration pathways around the cytochrome system. In these early studies, they suggested the existence of different pathways of respiration in resting and stimulated tissues, with part of the resting respiration insensitive to azide. Armstrong and Fisher (1940) have shown that cyanide and azide behave d i f f e r e n t l y in i n h i b i t i n g the enzymes co n t r o l l i n g the frequency of the embryonic f i s h heart. B a l l (1942 in Winzler 1943) has suggested that the Atmungsferment-azide and the Atmungsferment-cyanide compounds might have different oxidation-reduction potentials which could give them different c a t a l y t i c powers. He pointed out, however, as has also Stolz (1942a in Winzler 1943), that more fundamental knowledge of the mechanism of action of the respiratory i n h i b i t o r s was necessary before any f i n a l interpretations of their effects could be drawn (Winzler 1943). In addition, i t should be remembered that both cyanide and azide also affect the catalase enzyme responsible for peroxide removal along the respiratory chain. However, once again, the mechanisms of the two agents are different ( K e i l i n & Hartree 1934, K e i l i n & Hartree 1936, K e i l i n 1936). In the isolated f e t a l lung preparation cyanide and azide had a depressing effect on the secretion of the lung f l u i d . The effect was bigger with azide, but even in this case the lung was s t i l l secreting u n t i l the end of the experiment. However, i t would appear that part of the normal secretion seen in v i t r o u t i l i z e d the oxidative pathways. c) . Effect of Dinitrophenol The f i r s t agent found to uncouple oxidative phosphorylation, described by Loomis and Lipman in 1948, was dinitrophenol. Today many different uncoupling agents are known. Most are l i p i d s o l u b l e substances containing an acidic group and usually an aromatic ring. These agents do not uncouple g l y c o l y t i c phosphorylation or d i r e c t l y affect c e l l u l a r reactions other than oxidative phosphorylation. The uncoupling agents allow electron transport to continue, but. prevent the phosphorylation of ADP to ATP. Uncoupling agents can promote the passage of H* ions through the mitochondrial membrane, which i s normally impermeable to them (Lehninger 1975) . DNP i s an uncoupler of respiration from oxidative phosphorylation both in vivo and in v i t r o . In early studies, Kotelnikova and co-workers administered DNP to rats and observed a decrease in both ATP levels (Kotelnikova et a l . 1960, in Fujimoto, et a l . 1964) and turnover (Kotelnikova & Solomatina 1957, in Fujimoto et a l . 1964) in l i v e r tissue (Fujimoto et a l . 1964). Webster in 1953 (in Grenville & Needham 1955) noticed that DNP in high concentrations, increased the ATPase a c t i v i t y of 60 myosin. Therefore he began to investigate this effect in the hope that light might be thrown, ultimately, on the interaction of DNP with an enzyme system (Grenville & Needham 1955). In 1976 Dall-Larsen et. a l . observed that dinitrophenol i n h i b i t s ATP phosphoribosyltransferase, seemingly in competition with ATP. Nitrated phenols and other compounds which share the a b i l i t y to uncouple oxidative phosphorylation have been shown to i n h i b i t several enzymes with adenine-containing substrates or coenzymes (Stockdale et a l . 1975, in Dall-Larsen et a l . 1976). Previous work has demonstrated that DNP acts as a competitive i n h i b i t o r versus ATP in the ATP phosphoribosyltransferase reaction (Dall-Larsen et. a1. 1975, i n Dall-Larsen et a l . 1976). Since oxidative phosphorylation could be important, for secretory processes, DNP could be an i n h i b i t o r of the transport systems involved. In fact, Dinitrophenol, i n h i b i t s sodium transport across a variety of bio l o g i c a l membranes (Bricker & Klahr 1966, Handler et a l . 1966, Huf et al . 1957, in Martinez-Maldonado 1970). DNP also impairs the renal secretion of organic acids such as PAH, phenol red, and diodrast, in the dog (Mudge & Taggart 1950, Stinckler & Kessler 1963, in Martinez-Maldonado 1970). By contrast, in this species, the excretion of sodium does not seem to be affected when the i n h i b i t o r i s infused intravenously (Mudge & Taggart. 1950, in Martinez-Maldonado 1970), or into a renal artery (Fujimoto et a l . 1964, Kessler et a l . 1968, S t r i c k l e r & Kessler 1963, i n Martinez-Maldonado 1970). The lack of a DNP effect on sodium reabsorption in the dog has lead to the proposal that a portion of the energy required for this process i s not derived from adenosine triphosphate, but that i t may come d i r e c t l y from an electron transport system (Kessler 1966). However, in other species the situation appears to be different. Investigators in four different laboratories have shown i n h i b i t i o n of sodium and water reabsorption when DNP i s added t o the l u m i n a l s i d e of the p r o x i m a l nephron o f N e c t u r u s (Schatzmann e t a l . 1958, i n M a r t i n e z - M a l d o n a d o 1970) and of the r a t (Che r t o k e t a l . 1966, G e r t z 1963, Hernandez e t a l . 1969, i n M a r t i n e z - M a l d o n a d o 1970). However i n one s i t u a t i o n ( f r o g musc l e ) DNP has been r e p o r t e d t o a c t i v e l y s t i m u l a t e sod ium t r a n s p o r t (Conway 1960, i n Che r t o k et a l . 1966). I n m i t o c h o n d r i a , wh i ch c a r r y t he o x i d a t i v e m e t a b o l i c s y s t e m , DNP has p o w e r f u l and w i d e s p r e a d e f f e c t s . In t h e p r e s e n c e o f u n c o u p l e r s , m i t o c h o n d r i a l ATP s y n t h e s i s i s r e p l a c e d by ATPase a c t i v i t y , r e s p i r a t o r y c o n t r o l i s a b o l i s h e d , and t h e f r e e ene r g y o f s u b s t r a t e o x i d a t i o n i s d i s s i p a t e d i n t he form of hea t (Poe et a l . 1967, i n H a i n s t e i n 1976). DNP can have o t h e r e f f e c t s : i t i n t e r a c t s w i t h s e v e r a l enzymes t h a t h a n d l e n u c l e o t i d e s , i t i n h i b i t s v a r i o u s k i n a s e s and dehyd rogenase s ( S t o c k d a l e & S e l v i n 197.1, i n H a r r i s e t a l . 1981), i t s t i m u l a t e s the myos in ATPase , ( G r e n v i l l e & Needham 1955), and i n the case o f ATP p h o s p h o r i b o s y l t r a n s f e r a . s e , i t can r e p l a c e ATP as a ' p a r a s i t e ' s u b s t r a t e ( D a l l . - L a r s e n e t a l . 1976). The m i t o c h o n d r i a l ATPase F i , i s a l s o s t i m u l a t e d by d i n i t r o p h e n o l ( Pu l lmann et. a l . 1960, C a n t l e y & Hammes 1973, S e n i o r & Tomtsko 1975, i n H a r r i s e t a l . 1981) i n an i n t e r a c t i o n u n r e l a t e d t o i t s u n c o u p l i g a c t i v i t y ( H a i n s t e i n 1976, H a r r i s et. a l . 1981). In t he i n v i t r o p r e p a r a t i o n DNP not o n l y r educed f e t a l l u n g f l u i d s e c r e t i o n , as seen i n many t i s s u e s , bu t a l s o p roduced a d r a m a t i c r e a b s o r p t i o n d u r i n g t he hour o f t h e t r e a t m e n t and i n t he l a s t hou r of t he e x p e r i m e n t . The p e r s i s t e n c e o f r e a b s o r p t i o n a f t e r DNP i s an u n u s u a l o b s e r v a t i o n . However , e v i d e n c e f rom a d r e n a l i n e e x p e r i m e n t s s u g g e s t s t h a t r e a b s o r p t i o n i s dependent on Na + t r a n s p o r t . T h e r e f o r e i t seems p o s s i b l e t h a t t h e f e t a l l u n g s r e s emb l e t he dog k i d n e y , i n wh i c h DNP d i d no t s t o p Na 4 t r a n s p o r t , a l t h o u g h i t i n h i b i t e d o t h e r p r o c e s s e s . d) . Effects of Pivetanide Chloride transport, i s the dominant process for e p i t h e l i a l transport, in many tissues, including the loop of Henle, cornea, gastric mucosa, marine telost g i l l and anterior intestine (Zeuthen et. a l . 1978) . Apparently the lung f l u i d secretion i s also produced by a chloride transport system in the Type II c e l l s (Olver & Strang 1977). Many of the pumps, channels, and carriers i n a c e l l are amenable to blockade by drugs with varying degrees of s p e c i f i c i t y and potency. The Na +-K +-2C1 _ cotransporter can be inhibited by furosemide, bumetanide, and piretanide. These drugs are c o l l e c t i v e l y referred to as "loop d i u r e t i c s " , because in the kidney they act on the ascending limb of the loop of Henle (Mastella & Quinon 1987). Inhibition by these drugs i s rapid and reversible. They are thought to bind to the cotransport protein at the s i t e normally occupied by CI-. Furosemide i s the most, extensively studied of the three but bumetanide i s now the preferred i n h i b i t o r , as i t i s the more s p e c i f i c and has a potency 50-100 times greater than furosemide (Mastella & Quinon 1987). Piretanide i s an effective i n h i b i t o r of electrogenic chloride transport in f i s h anterior intestine (Zeuthen et a] . 1978) . Gerencer (1984) obtained similar results in the intestine of a mollusc, and these agreed with studies of the gallbladder by Zeuthen et a l . (1978). Although Cassin et.  a l . (1986) showed that bumetanide, and to a lesser extent furosemide, inhibited lung f l u i d secretion in f e t a l sheep, they did not test piretanide. This needed to be investigated. In the in v i t r o f e t a l lung, piretanide had a large effect on the secretion of lung f l u i d . In the two different concentrations that were tested, piretanide slowed the secretion rate in the lower dose and almost stopped i t at. the higher dose l e v e l . This was the f i r s t time that piretanide has been t r i e d on the f e t a l lung. From the results obtained here i t seems 63 that piretanide had a stronger effect than bumetanide and furosemide. We had a reduction in lung f l u i d secretion at 10 _ 8M and a bigger effect, at. 10"7M. Cassin et. a1 (1986) found that bumetanide produced reabsorption in the sheep at 10-"M, and at 10 - 5M i t reduced secretion; however at 10 _ 6M there was no clear effect. Furosemide was less ef f e c t i v e ; at 10 - 3M i t produced an immediate reabsorption, but at 10 _ 4M i t increased secretion s l i g h t l y . The use of "loop d i u r e t i c s " may be p a r t i c u l a r l y important in the treatment of premature babies which suffer from Respiratory Distress Syndrome or Hyaline Membrane Disease, since piretanide and other similar compounds are thought to affect chloride transport and therefore lung f l u i d secretion. 3) . Effects of hormones and related substances on lung fluid Secretion Previous work in the sheep and goat, has shown that, two hormones, adrenaline and AVP, are capable of affecting lung l i q u i d secretion in the intact fetus in utero. These two hormones also affect the in v i t r o guinea pig lungs (Marshall, Woods unpublished observations: see Perks & Cassin, 1987) . In general, the in v i t r o preparation presents a useful and rapid method for screening many other hormones, or hormone related substances. Therefore, in this study, two substances not. previously investigated, somatostatin and i n s u l i n were tested on the in v i t r o f e t a l lung. a). Effect of Somatostatin There are well over a dozen hormonal substances that are known or strongly suspected to be synthesized in normal lungs. These may have important roles i n health and disease. Many of these substances are polypetides. The s i t e of production of most of these agents i s now known (Becker & Gazdar 1984) , but their physiological importance i s not. understood. Somatostatin i s a tetradecapeptide o r i g i n a l l y isolated from ovine and porcine hypothalamus. Tt i n h i b i t s the secretion of growth hormone, TSH and prolactin from the p i t u i t a r y , and can reduce secretion of i n s u l i n and glucagon by a direct effect on the i s l e t s of Langerhans (Robberecht et a l . 1975, Wahren & Feling 1976). Tn addition, somatostatin i s found in the nerves and endocrine c e l l s of the gastrointestinal t r a c t , the endocrine c e l l s of the pancreas, and the p a r a f o l l i c u l a r (C) c e l l s of the thyroid gland (Becker & Gazdar .1984) . The presence of somatostatin within the nervous tissue has led to the presumption that i t has a peptidergic function (Becker & Gazdar 1984) . Somatostatin has been localized throughout the gastrointestinal tract and pancreas, where it. exerts a variety of effects on gastrointestinal function. This peptide has been reported to: a) i n h i b i t amino acid and pepsin secretion and delay gastric emptying; b) i n h i b i t the secretion of pancreatic enzymes and bicarbonate; c) diminish gallbladder contractions; d)decrease splanchnic blood flow; e) reduce i n t e s t i n a l m o t i l i t y : and f) i n h i b i t and/or delay glucose and amino acid absorption in humans. In addition, somatostatin i n h i b i t s the release of and/or blocks the effect of many hormones which affect f l u i d and electrolyte transport in the intestine, e.g. VIP, gastrin, secretin, cholecyst.oki.nin and GIF. Therefore, i t may function as a neurotransmitter or have a paracrine effect in the intes t i n e . In addition somatostatin functions as a c i r c u l a t i n g hormone (Becker & Gazdar 1984) . Somatostatin stimulates net Na+ and Cl~ absorption in the rabbit ileum and appears to do so primarily by stimulating the coupled i n f l u x of Na+ and CI - across the brush border membrane. Somatostatin was able to block the effect of c y c l i c AMP-dependent and c y c l i c AMP-independent secretogogues in the rat "colon without affecting c y c l i c AMP lev e l s , suggesting that i t acts by i n h i b i t i n g a d i s t a l step in the secretory pathway, probably involving a f i n a l common pathway (Dobbins et. a 1. 1981). Somatostatin may also affect water and electrolyte movement by blocking the action of c y c l i c AMP in the rat jejunum (Dharmsathaphorn et a l . 1980). Carter et a l . (1978) found that somatostatin completely blocked VIP-induced i n h i b i t i o n of water absorption and p a r t i a l l y blocked theophylline-induced i n h i b i t i o n of water absorption (in v i t r o everted sac preparation, rat colon). These studies suggest that somatostatin may modulate f l u i d movement and the absorption of nutrients from the intestine (Dharmsathaphorn et al . 1980). Although i t has been reported that somatostatin i s usually undetectable by radioimmunoassay in normal lungs, i t has been found in lung cancer tissue SCCL, and in adenocarcinoma of the lung (Sorenson et a l . 1981, Wood et a l . 198.1, in Becker & Gazdar 1984). However, no studies have been made on f e t a l lungs, which may retain characteristics of the gut from which the lung developed, and therefore might retain higher levels of somatostatin. In addition, cancer c e l l s , which may represent a less differentiated,, and possibly " f e t a l " condition, are known to synthesize somatostatin in the lung (Becker & Gazdar 1984). In the isolated f e t a l lung preparation somatostatin had an effect on the lung secretion rate. The secretion of the lung f l u i d was stopped at the highest dose l e v e l , while the two lower doses reduced secretion. Apparently somatostatin can affect the secretion of C l - and therefore the secretion of the lung f l u i d . b). Effect of Insulin An increased incidence of RDS occurs in infants born to diabetic mothers (Mestyan et a l . 1975, in Giannopoulos & Tulchisky 1980), presumably owing to deficiency of pulmonary surfactant at the time of b i r t h . This finding has stimulated interest in the role of i n s u l i n in lung development. Smith and Lumbers (1987) have shown C o r t i s o l stimulates the incorporation of choline into l e c i t h i n in monolayer cultures of mixed fetal lung c e l l s , and that this stimulatory effect of C o r t i s o l i s abolished i f i n s u l i n i s added to the culture medium (Orci et a l . 1975, in Giannopoulos & Tulchisky 1980). This suggests that i n s u l i n antagonizes the glucocorticoid-induced stimulation of pulmonary l e c i t h i n synthesis in the fetus. In other studies (Grasso et a l . 1973, i n Giannopoulos & Tulchisky 1980), i n s u l i n has been shown to stimulate glycogen accumulation and to decrease the number of lamellar bodies in explants derived from 19 old day f e t a l rat lung in short-term culture, indicating that i n s u l i n may delay maturation of the f e t a l lung. The mechanism involved in such a process i s not known, but i t has been s u g g e s t e d that i n s u l i n may i n h i b i t glycogenolysis and deprive the lung of substrate f o r phospholipid synthesis. Alternatively, i n s u l i n may act by stimulating glycogen synthesis from glucose and thereby again divert substrate away from the production of phospholipids (Grajwer et. a l . 1977, in Giannopoulos & Tulchisky 1980). Insulin may play an important role in regulating the metabolism of Type II c e l l s . Clearly, i t w i l l be of great importance to assess the interactions between i n s u l i n and amino acid transport by these c e l l s in abnormal physiological states, especially starvation or diabetes mellitus. These two states have been reported to affect pulmonary surfactant (Gacad & Hassaro 1972, in Kazuhiro 1987), the apoproteins of surface active material (Sugahara et al . 1983, in Kazuhiro 1987), and the ultrastructure of the alveolar type II c e l l (Sugahara et a l . 1981, in Kazuhiro 1987). The perinatal mortality rate for infants of diabetic mothers has been found to be 6.2 % (Lemons et a l . 1981, in Smith & Lumbers 1987), an incidence which i s greater than i n the nondiabetic population. There i s also an increased incidence of unexplained f e t a l death, especially in the last 4 weeks of gestation (White 1974, in Smith & Lumbers 1.987), the etiology of which remains unknown. The fetus of a diabetic woman can be exposed to wide 67 fluctuations in blood glucose levels. Thus, one reason for this unexplained f e t a l death might be the materna] nocturnal hypoglycemia observed by Guillmer et a l . {1975, in Smith & Lumbers 1987). Another explanation could be exposure of the fetus to hyperglycemia. In the normal ovine pregnancy, maternal hyperglycemia caused a r i s e in f e t a l plasma glucose levels and increased f e t a l plasma lactate (Sherlley 1973, in Smith & Lumbers 1987). In addition to i t s effect on glucose metabolism and on the f e t a l lung, i n s u l i n can also affect salt metabolism. Herrera in 1963 was the f i r s t , to observe that i n s u l i n stimulates trans e p i t h e l i a l sodium transport by the isolated skin of the frog, Rana pipens (Herrera 1963, in Cobb et a l . 1981). A similar effect was subsequently described in e p i t h e l i a l tissues of the toad, Bufo marinus (Herrera 1965, Crabbe & Francois 1967, in Cobb et. a l . 1981), notably the urinary bladder, a tissue which has transport characteristics in common with the mammalian d i s t a l nephron. Although a number of other e p i t h e l i a l tissues, including the mammalian kidney (De Fronzn et. al . 1975, De Fronzo et. al . 1976, Nizet et. a l . 197.1, in Cobb et. a l . 1981), responded to i n s u l i n by s i g n i f i c a n t l y increasing sodium transport, the basic mechanisms leading to this effect are unknown (Cobb et. a 1. 1981). However, there are three main theories to account for this effect: 1) a stimulation of the sodium pump; 2) an unmasking of previously unavailable sodium pump s i t e s , and, 3) a de novo synthesis of proteins which increase the a c t i v i t y of the sodium pump: 1). Stimulation of the sodium pump Several laboratories have established that i n s u l i n , when applied to the serosal side or injected into anurans before removal of the e p i t h e l i a , stimulates transport of sodium from the apical mucosa surface to the serosa (blood side) of bladder, skin and intestine. This effect i s probably due to direct action on the sodium pump at the basolateral c e l l membrane (Macknight et a.1 . 1 980, Scott & Goodman 1971, Epple 1 9 8 7 ) . The isolated urinary bladder of both the toad and the frog can actively transport sodium from the mucosal to the serosal bathing medium. Hormones such as i n s u l i n (Herrera 1965, Wiesmann et. al . 1977, in Klahr et a l . 1981), aldosterone (Crabbe 1961, in Klahr et. a l . 1981), and ant i d i u r e t i c hormone (Leaf 1960, in Klahr et a l . 1981), a l l markedly increase this net transport of sodium. The role of i n s u l i n on sodium transport has been demonstrated in several other systems including the amphibian colon and skin, and the isolated perfused mammalian kidney. Studies using stripped amphibian colon and skin have suggested a "d i u r e t i c " stimulation of short-circuit, current, after i n s u l i n was added to the bathing medium; this was similar to the toad urinary bladder. This stimulation was related to increased a c t i v i t y of the sodium pump rather than to changes in the mucosal, permeability of the e p i t h e l i a l c e l l s to sodium. This interpretation has been supported by subsequent observations made by Siegel and Civan (1976) and by Crabbe ( 1 9 8 1 ) . Crabbe, who examined the interactions between ouabain, vasopressin, aldosterone and i n s u l i n on short-circuit, current across the toad bladder, suggested that aldosterone acts primarily to increase sodium entry into the e p i t h e l i a l c e l l s , and that, i n s u l i n acts primarily to increase sodium extrusion. The experiments of Siegel and Civan also provided evidence that i n s u l i n increases sodium transport, at least in part, by d i r e c t l y stimulating the sodium pump (Klahr et a l . 1981). Since i n s u l i n stimulates the sodium pump in amphibian tissues (urinary bladder, colon and skin), and these are considered models for certain parts of the mammalian nephron, a similar action of the hormone on the mammalian kidney could be expected. The stimulation of sodium transport in the nephron and functionally related tissues produced by i n s u l i n deserves interest in that the hormone apparently f a i l s to influence renal glucose metabolism or handling (Crabbe 1 9 8 1 ) . It i s generally agreed that i n s u l i n acts, at least i n i t i a l l y , as a direct stimulant of sodium transport, from the c e l l s to the serosal medium. Evidence includes the fact that i n s u l i n and vasopressin (Wiesmann et a l . 1977 , in Macknight et al . 1 9 8 0 ) , and i n s u l i n and aldosterone (Cox & Singer 1977 , i n Macknight et a l . 1980) together stimulate sodium transport more than supramaximal concentrations of vasopressin or of aldosterone alone. Insulin also stimulates the electromotive force of the sodium pump, Esa, without, affecting c e l l u l a r resistance (Siegel & Civan 1976 , in Macknight et  a l . 1 9 8 0 ) . Extensive studies i n other tissues support this mode of action (Clausen & Hensen 1 977 , Gavrick et al . 1975 , Moore 1973 , Zieler et a l . 1966, in Macknight et a l . 1980) and suggest, that the hormone increases pump a c t i v i t y by increasing the re l a t i v e sodium a f f i n i t y of the pump (Clausen & Hensen 1 977 , Gavrick et a l . 1 975 , Moore 1973 , Hougen et al.. 1978 , in Macknight et al . 1980) rather than by unmasking pump sites ( E r l i j & Grinstein 1976, in Macknight et. a l . 1 9 8 0 ) . In the toad bladder the effect does not. depend on serosal medium glucose (Cox & Singer 1977, Wiesmann et. a l . 1977 , in Macknight et. a l . 1980) nor does i t involve c y c l i c AMP (Wiecsman et. al . 1 977 , in Macknight et al . 1 9 8 0 ) . The rapid onset of the stimulatory effect, of i n s u l i n on sodium transport, together with the f a i l u r e of protein-synthesis i n h i b i t o r s to prevent this effect (Cox & Singer 1.977, in Macknight. et a l . 1980), suggests that i n s u l i n does not stimulate sodium transport by a mechanism involving protein synthesis. In this scenario i n s u l i n produces a rapid increase in tr a n s e p i t h e l i a l sodium transport apparently as a result of a direct effect in the sodium pump at the basolateral membrane (Macknight et al . 1 9 8 0 ) . However, this i s not accepted by a l l workers (see below). 2) . Unmasking of sodium pump sites Evidence for a direct effect of i n s u l i n on sodium transport in muscle has been provided by the studies of Moore (1973, in Klahr et a l . 1981) . Insulin was shown to increase the rate of sodium efflux from, a muscle prelabeled with radioactive sodium, an effect that was blocked by ouabain. The effect could not be explained on the basis of changes in sodium permeability. Grinstein & E r l i j (1974, in Klahr et a l . 1981) demonstrated subsequently that frog muscles exposed to i n s u l i n exhibited increased ouabain binding. These observations suggested that i n s u l i n unmasked sodium-pump sites previously unavailable for binding of the ouabain molecule (Klahr et a l . 1981). 3). De novo protein synthesis Evidence for de novo protein synthesis after i n s u l i n exposure in toad bladder has been presented independently by Benjkamin & Singer (1974, in Klahr 1981) and Cobb et al.. (1981). Cobb et. al.. began their investigation of the mechanism of i n s u l i n regulation of sodium transport in the toad bladder by pursuing the finding of Weismann et a 1. (1976, 1977, in Cobb et  a l . 1981) that actinomycin D i n h i b i t s the prolonged increase in Na+ transport produced by i n s u l i n . Cobb et a 1. (1981) provide evidence for the insulin-induced synthesis cf sp e c i f i c proteins in one of the two principal morphologic c e l l types, the granular (G) c e l l , of the toad urinary bladder. They gave evidence supporting a relationship between induction of these proteins and the prolonged increase in transport caused by i n s u l i n . The effect of i n s u l i n on Na+ transport begins within 15 minutes, but persists for at least 20 hours (Cobb et a l . 1981). The relationship of the reported insulin-induced proteins to each other or to (Na*+K+)-ATPase i s uncertain at this time (Klahr et a l . 1981). c) . Relationship of Na* transport to the fetal lung Evidence produced in sheep studies by Brown et a l . (1983), confirmed by Perks & Cassin (1987) suggests that adrenaline caused reabsorption in the f e t a l lung., and t e s t s w i t h a m i l o r i d e s ugge s t e d t h a t t h i s e f f e c t was due i n part, t o Na* t r a n s p o r t f rom the lumen t o t he b l o o d . S i n c e Na* t r a n s p o r t appea r s t o be i n v o l v e d i n r e a b s o r p t i o n of l u n g l i q u i d , the e f f e c t of i n s u l i n , wh i ch i s i t s e l f a b l e t o i n c r e a s e Na* t r a n s p o r t , i s i n k e e p i n g w i t h ou r u n d e r s t a n d i n g o f f e t a l r e a b s o r p t i o n a t b i r t h . Based on t h i s i n f o r m a t i o n we t h i n k t h a t t h e r e d u c t i o n of l u ng f l u i d s e c r e t i o n o b s e r v e d i n our e x p e r i m e n t a l r e s u l t s i n the p r e s en ce o f i n s u l i n i s due t o the s t i m u l a t i o n o f sod ium t r a n s p o r t . The r e s u l t s d i s c u s s e d above s ugge s t t h a t t he p r o d u c t i o n of f e t a l l u n g f l u i d was a f f e c t e d by the use of the d i f f e r e n t , m e t a b o l i c i n h i b i t o r s . The t r a n spo r t , of C l - was i n h i b i t e d by t h a use of p i r e t a n i d e , a p o t e n t l o op d i u r e t i c i n h i b i t o r . In a d d i t i o n , s o m a t o s t a t i n , a hormonal r e l a t e d s ub s t a n c e wh i ch is known t o a f f e c t C l - t r a n s p o r t , was a l s o found t o r educe l u n g f l u i d s e c r e t i o n . I n s u l i n , a n o r m a l l y o c c u r r i n g hormone, a p p a r e n t l y had no e f f e c t , on the Na* t r a n spo r t , i n t h i s s y s t e m . 72 B I B L I O G R A P H Y A D A M S , F . H . , F U J I W A R A , T . & ROWSHAN,0. 196.3a. The N a t u r e and O r i g i n o f t h e F l u i d i n F o e t a l Lamb L u n g . 6 3 ( 5 ) , 8 8 1 - 8 8 8 . A D A M S , F . H . , M O S S , A . J . & F A G A N , L . 1 9 6 3 b . The T r a c h e a l F l u i d i n t h e F o e t a l Lamb. B i o . N e o n a t . 5 , 1 5 1 - 1 5 8 . A D A M S , F . H . , YANAGISSAWA,M. , K U Z E L A , D . & M A R T I N E K , H . 1 9 7 1 . 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A P P E N D I X A Krebs-Henseleit Saline Method: Stock Solutions 1) . 69.2g NaCl and 3.5g KC1 in 1 l i t e r d i s t i l l e d water 2) . 36.8g CaCl2-H20 in 1 l i t e r d i s t i l l e d water 3) . 15.Og MgSO« i n 1 l i t e r d i s t i l l e d water 4) . 16.Og KH2PO4 in 1 l i t e r d i s t i l l e d water The preparation of saline from the stock solutions. To prepare one l i t e r of saline, mix: 1. 10 ml MgSCU stock solution 2. 100 ml NaCl/KCl stock solution 3. 2.1g NaHCOs 2.0g Glucose F i l l the volumetric flask at least half f u l l with d i s t i l l e d water. Make sure the solids have completely dissolved. 4. 10 ml KH2PO4 stock solution 5. 10 ml CaCl2-H20 stock solution F i l l the flask to 1.0 l i t e r with d i s t i l l e d water, and mix thoroughly. Bubble for at least 20 minutes. 

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