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The effects of dopamine, serotonin and influences associated with meconium on lung liquid production… Chua, Beverly A. 1997

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THE EFFECTS OF DOPAMINE, SEROTONIN A N D INFLUENCES ASSOCIATED WITH M E C O N I U M ON L U N G LIQUID PRODUCTION IN IN VITRO LUNGS F R O M F E T A L GUINEA PIGS (Cavia porcellus) By Beverly A. Chua BSc. University of British Columbia, Canada, 1992 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A January 1997 © Beverly Chua, 1997 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 "Z^QoVogj^ / The University of British Columbia Vancouver, Canada Date r\r\\ , 1^1-DE-6 (2/88) 1. ABSTRACT Lungs from near term fetal guinea pigs (58-67 days of gestation) were supported in vitro for three hours; lung liquid production was monitored by a dye dilution technique. 42 preparations (61 + 1 days of gestation, 81.9 ± 9.5 g (SD) body weight) were used to study the effects of dopamine placed in the outer saline for the middle hour. At concentrations of 10~7 and 10~6 M , dopamine produced significant reductions of fluid production; at 10~5 M , there was complete arrest of production. Dopamine, at 10~4 M , produced fluid reabsorption. The linear log concentration response curve (r=0.99) showed a theoretical threshold at 1.7 x 10~9 M dopamine. Responses from dopamine involved dopaminergic receptors, since they were abolished by haloperidol (10"5 M) (n=24), a non-specific dopamine antagonist. Activation was through D 2 receptors, since responses were abolished by 10~5 M domperidone (D2-receptor antagonist) (n=24), but unaffected by 10" 5 M SCH 23390 (Dj-receptor antagonist) (n=24). Reductions by dopamine were resistant to amiloride (IO"6, 10"5 and IO"4 M) (n=66) and benzamil (10"5 M) (n=24). 36 preparations (61 ± 1 days of gestation, 77.7 + 11.5 g (SD) body weight) treated with serotonin showed about 50% reduction in fluid (10~7, 10"6 and 10"5 M). Below 10"7 M serotonin, there was a linear log concentration response curve (r=0.98), and the line of best fit suggested a theoretical threshold of 5 x IO"9 M . Preparations from 10 more mature fetuses, (65 ± 1 days of gestation, 126.2 + 13.2 g (SD) body weight) showed significantly greater responses. Maximal responses at 10~7 M involved serotonergic receptors since they were eliminated by the general serotonin antagonist, cyproheptadine (IO - 6 M) (n=24). In addition, 10~6 M amiloride abolished effects by serotonin (n=24). 6 untreated lungs from meconium-stained fetuses produced lung fluid consistently throughout 3 hours of incubation. However, rates of lactate production throughout experimentation were higher than rates produced by untreated, meconium-free fetuses. This suggested that these meconium-influenced lungs have increased their glycolytic metabolism to support normal fluid production. Twelve meconium-free preparations treated with 10~3 M 2,4-dinitrophenol (DNP) showed strong reabsorptions, and total loss of lactate increased by 3.8-fold during treatment. These reabsorptions resembled those produced after 2 x 10~4 M DNP; however, total lactate loss during treatment with 2 x 10^ M DNP only doubled. This suggested that reabsorptions can operate on mainly glycolytic processes. In contrast, twelve lungs taken from meconium-stained fetuses and treated with 2 x 10~4 M DNP showed no change in fluid production; however, lactate production again doubled. It appeared that these lungs, although already showing above average glycolytic activity, were still capable of increasing it. Failure to reabsorb fluid may be due to a disruption in the link between metabolic processes and the reabsorptive mechanism. These studies suggest that lungs from meconium-stained fetuses have differences concerning fluid production and reabsorption processes. Furthermore, in the guinea pig, dopamine may play a role in reducing lung fluid production or initiating reabsorption close to birth, but by processes other than N a + transport. Serotonin can slow lung liquid production or cause reabsorption; the effect increases close to term and is due to activation of amiloride-sensitive N a + channels. i v Contents 1. ABSTRACT i i LIST OF FIGURES vii 2. ACKNOWLEDGEMENTS ix 3. INTRODUCTION 1 3.1 Origin of fetal lung fluid 1 3.2 Secretion of fetal lung fluid 2 (a) Composition of fetal lung fluid 2 (b) Fate and role of fetal lung fluid in lung development 2 (c) Ion transport and mechanism of lung fluid secretion 3 3.3 Reabsorption of fetal lung fluid 4 (a) Prenatal reduction in fetal lung fluid 4 (b) Hormonal effects on lung liquid production 4 (c) Mechanics of lung liquid clearance 5 3.4 Fetal lung fluid regulation by neuropeptides 6 3.5 Meconium 7 4. STATEMENT OF THE PROBLEM 9 5. MATERIALS AND METHODS 10 5.1 Animals 10 5.2 Basis of the method 10 5.3 Experimental procedures 11 (a) Dopamine 14 (b) Serotonin 15 (c) Meconium studies 16 (d) 2,4-dinitrophenol (DNP) 16 5.4 Chemical methods 17 5.5 Quantification of results and statistical methods 17 R E S U L T S 6.1 Fluid production by untreated in vitro lungs from fetal guinea pigs Part I: Studies pf effects on lung liquid production using agents found in the neuroepithelial bodies (NEBs) 6.2 Effects of dopamine on lung liquid production (a) The effects of IO"4, IO"5, IO"6, IO"7 and IO"8 M dopamine on lung fluid production (b) The effects of IO - 5 M haloperidol on the reductions seen after 10~6 M dopamine (c) The effects of 10~5 M domperidone on the reductions that follow 10"6 M dopamine (d) The effects of IO"5 M SCH 23390 on the reductions seen after 10~6 M dopamine (e) The effects of 10~6 M amiloride on reabsorptions seen after 10"4 M dopamine (f) The effects of 10~5 M amiloride on reabsorptions that follow 10"4 M dopamine (g) The effects of 10~4 M amiloride on the effects seen after 10~4 M dopamine (h) The effects of 10"5 M benzamil on the reabsorptions after 10"4 M dopamine 6.3 Effects of serotonin on lung liquid production (a) Fluid production by in vitro fetal lungs treated with IO"5, IO"6, IO"7, 5 x IO"8 and IO"8 M serotonin (b) The effects of 10~6 M cyproheptadine on the reductions seen after 10"7 M serotonin (c) The effects of 10~6 M amiloride on the reductions seen after 10"7 M serotonin 6.4 Effects of serotonin on lungs from fetuses more advanced in gestation The effects of 10"5 M serotonin on lung fluid secretion in lungs from fetuses more advanced in gestation v i Part II: Studies of lung liquid production in normal fetuses and those showing meconium staining 62 6.5 Effects of meconium on lung fluid and lactate production 62 (a) Fluid and lactate production by untreated normal in vitro lungs 62 (b) Fluid and lactate production by in vitro lungs from meconium-stained fetuses 65 6.6 Effects of 2,4-dinitrophenol (DNP) on lung liquid and lactate production 68 (a) Effects of DNP on fluid and lactate production by lungs from fetuses free of meconium 71 (i) Studies with DNP at 2 x IO"4 M 71 (ii) Studies with DNP at IO'3 M 74 (b) Effects of DNP on fluid produced by lungs from fetuses showing staining with meconium 77 (c) Effects of 2 x IO"4 M DNP on lactate production by in vitro lungs from meconium-stained fetuses 80 7. DISCUSSION 84 7.1 Effects of dopamine on lung liquid production 85 7.2 Effects of serotonin on lung liquid production 89 7.3 Effects of meconium and 2,4-dinitrophenol (DNP) on lung liquid production 91 General Summary 95 8. R E F E R E N C E S 96 v i i List of Figures 1. Apparatus for the maintenance of the in vitro guinea pig fetal lung preparation 13 2. Lung liquid production over a 3 h period by in vitro lungs from fetal guinea pigs 21 3. Effects of dopamine, on lung liquid production by in vitro lungs from fetal guinea pigs 24 4. The relationship between log concentrations of dopamine and the response of in vitro fetal lungs 27 5. The effect of haloperidol on responses to 10"6 M dopamine 29 6. The effect of domperidone on responses to 10~6 M dopamine 32 7. The effect of SCH 23390 on responses to IO"6 M dopamine 34 8. The effect of 10"6 M amiloride on responses to 10~4 M dopamine 37 9. The effect of 10 - 5 M amiloride on responses to 10"4 M dopamine 40 10. The effect of 10"4 M amiloride on responses to 10~4 M dopamine 42 11. The effect of 10~5 M benzamil on responses to 10~4 M dopamine 45 12. Effects of serotonin on lung liquid production by in vitro lungs from fetal guinea pigs 48 13. The relationship between log concentrations of serotonin and the response of in vitro fetal lungs 51 14. The effect of cyproheptadine on responses to 10~7 M serotonin 53 15. The effect of amiloride on responses to 10"7 M serotonin 56 16. The effect of gestational age on responses to serotonin 59 17. The effect of changes of gestational age on responses to serotonin divided according to age 61 18. Lung liquid and lactate production in untreated in vitro lungs from fetal guinea pigs 64 19. Lung liquid and lactate production in untreated in vitro lungs from fetal guinea pigs stained with meconium 67 20. Lactate production in normal and meconium-stained fetuses 70 21. The effect of 2 x IO"4 M dinitrophenol (DNP) on lung liquid and lactate production in in vitro lungs from fetal guinea pigs 73 v i i i 22. The effect of 10"3 M dinitrophenol (DNP) on lung liquid and lactate production in in vitro lungs from fetal guinea pigs 76 23. The effect of 2 x IO"4 M dinitrophenol (DNP) on fluid production in lungs from fetuses stained with meconium 79 24. The effect of 2 x IO"4 M dinitrophenol (DNP) on lactate production in in vitro lungs from fetal guinea pigs stained with meconium 82 i x 2. ACKNOWLEDGEMENTS I would like to give special thanks to Dr. Anthony M . Perks for his patience and encouragement throughout the course of my graduate work. His help and advice in the preparation of this thesis were invaluable. Also to Sam Doe, David Kojwang and Leslie Chan for the use of some of their data to augment my own. Many thanks to Pawel Kindler for his assistance, Elizabeth Vanderhorst for her help with the lactate analysis and all members of the laboratory for their friendship and companionship during these experiments. Finally, thank you to Lowell McPhail and to my family for their support during my academic life. 1 3. INTRODUCTION Lung development begins in early embryonic life and continues throughout the period of body growth. Mammalian lungs before birth are secretory organs that undergo breathing-like movements but serve no respiratory function (Strang, 1977). However, during the transition from intra- to extrauterine life the lungs must be cleared of fluid. This is important in the timely switch from placental to pulmonary gas exchange so that the newborn can achieve effective pulmonary ventilation (Wallace et al., 1990). 3.1 Origin of fetal lung fluid It was evident as early as 1885 that the fetal lungs contained fluid (Preyer, 1885) and that some component of the lung lining acts as a semipermeable membrane providing a barrier to the net movement of water and solutes from the circulation into the fine air spaces (Lacqueur and Magnus, 1921). The fluid was mostly thought to be amniotic in origin until 1948, when Jost and Policard discovered that ligating the trachea of fetal rabbits caused their lungs to be distended with fluid, implying that the fluid came from within the lungs and was not aspirated from the amniotic sac. The distinction between lung liquid and amniotic liquid was further established by Adams et al. (1963) who found that the composition of liquid drained from the trachea of fetal lambs differed considerably from that of plasma and amniotic fluid sampled from the same animals. Although by 1963 it was clear that fetal lung fluid was formed within the lungs, the mechanism involved in its secretion and reabsorption remained to be investigated. 2 3.2 Secretion of fetal lung fluid (a) Composition of fetal lung fluid Based on the known concentrations of ions, urea, and protein in lung fluid, lymph, plasma, and amniotic fluid (Strang, 1977), it became clear that lung fluid is not merely aspirated amniotic fluid, nor any kind of mixture of amniotic fluid and a plasma filtrate. Further, although lymph contains less protein than plasma, they both share similar ionic concentrations. In lung fluid, however, the amount of protein is very low and the pattern of ionic concentrations distinctly unusual: [Cl"] and [K+] are significantly higher than in plasma, whereas [HC03~], [Ca2+], [phosphates] and pH are lower. This composition is unique to this body fluid. (b) Fate and role of fetal lung fluid in lung development Some of the fluid that passes from the trachea to the oropharynx of the fetus is probably swallowed, but the remainder contributes directly to the formation of amniotic fluid (Gluck et al., 1971) and may play a role in fluid and electrolyte balance (Cassin and Perks, 1982). The fetal lungs produce quantities of fluid at a rate comparable to that of fetal urine production (Strang, 1977). It has been shown that lung fluid can contribute approximately 25% - 50% of the daily production of amniotic fluid in the sheep fetus depending on gestational age (Brown et al., 1983). The upper airway serves as a one-way valve, inhibiting entry of amniotic fluid but allowing outward flow of pulmonary fluid (Harding et ah, 1986). Expansion of the fetal lungs by this liquid is critical for normal lung development (Alcorn et al, 1977). Lung fluid production appears to affect the shape of the developing airways and alveoli, and disruption of this process alters normal development, as studies in fetal lambs with tracheal ligation or chronic drainage 3 of lung fluid have demonstrated (Alcorn et al, 1977; Fewell et al, 1983; Moessinger et al, 1990). (c) Ion transport and mechanism of lung fluid secretion As previously mentioned, the high chloride concentration in lung fluid makes it very unlikely that it is formed by simple filtration. Although the site and the contribution of fetal alveolar cells to fluid production in the fetal lung is uncertain, several studies suggest that active transport of chloride and other ions are responsible (Adamson et al, 1969; Olver et al, 1981; Chapman et al, 1991). Olver and Strang (1974) provided further evidence that fetal lung fluid production depended on epithelial active transport. They showed that secretion can be blocked by intra-alveolar instillation of K C N and that Cl" and K + are actively transported into the future airspace. Cassin et al. (1986) also found that this secretory process can be inhibited by diuretics that block Na+K+lCl"-cotransport. This observation, which was confirmed by others (Carlton et al, 1990; Thorn and Perks, 1990) supports the concept that the driving force for transepithelial movement of Cl" in the fetal lung is similar to the mechanism described for C l ' transport across other epithelia (Frizzell et al, 1979; Gatzy, 1983). According to this idea, C l " enters the epithelial cell across its basolateral surface linked to N a + and K + transport. N a + enters the cell down its electrochemical gradient and is then extruded in exchange for K + by the action of the Na + K + -ATPase located on the basal membrane of the cell. This active process increases the Cl" concentration within the cell so that it exceeds its electrochemical equilibrium. As a result, Cl" passively exits the epithelial cell through specific channels located in the apical membrane, resulting in the movement of liquid from the pulmonary circulation and interstitium into potential airspaces. However, the specific properties of the apical Cl" channels, their distribution on the various cells that 4 make up the fetal respiratory tract epithelium, and signals that regulate various ions through them remain unclear. 3.3 Reabsorption of fetal lung fluid (a) Prenatal reduction in fetal lung fluid The rate of formation and the intraluminal volume of fetal lung liquid decreases near term. Several studies have shown that secretion and the volume of fluid in the lung lumen diminishes before the onset of labor in lambs (Kitterman et al, 1979; Dickson et al., 1986; Chapman et al., 1994). The process of labor further accelerates clearance of lung water content in both rabbit pups and lambs (Bland et al., 1979, 1980, 1982; Brown et al, 1983). (b) Hormonal effects on lung liquid production The mechanism responsible for the reduction in fetal lung liquid secretion and reabsorption remains unknown, but this adaptive process may be the result of hormonal changes that occur in the fetus late in gestation. Several investigators have examined the influence of catecholamines on fetal lung liquid volume. In studies with rabbit pups and lambs, epinephrine or P-adrenergic agonists reduced the amount of water in the lungs or caused reabsorption (Enhorning et al, 1977; Lawson et al, 1978; Walters and Olver, 1978; Chapman et al, 1991). In addition, propranolol, a p-adrenergic antagonist blocked the effects of epinephrine in causing fluid reabsorption in fetal lambs (Walters and Olver, 1978). Further, Olver et al. (1986) found that intraluminal administration of amiloride blocked epinephrine's reabsorptive effects, suggesting that (3-adrenergic agonists stimulate N a + uptake by the lung epithelium. 5 Intracellular mediation of the p-adrenergic effect and its late gestational increase is likely mediated via adenosine 3',5'-cyclic monophosphate (cAMP), since tracheal instillation of membrane-permeant cAMP analogues causes reabsorptions in fetal lung liquid in sheep late in gestation (Barker et al., 1988; Walters et al, 1990). A more recent study by Kindler et al. (1992) confirmed these findings in the guinea pig. These studies support the view that conditions that stimulate release of cAMP in the lung may contribute to a reduction of lung liquid and possibly trigger reabsorptions. Furthermore, the inhibitory effects of both p-agonists and cAMP in the lamb is critically dependent on the state of lung maturity (Brown et al, 1983; Perks and Cassin, 1982; Walters et al, 1990). Other hormones secreted around the time of birth also may influence production of fetal lung fluid. Several studies have shown that arginine vasopressin (AVP) decreased the rate of fluid secretion in goats and lambs (Perks and Cassin, 1982; Ross et al, 1984; Perks and Cassin, 1989; Wallace etal, 1990). More recently, A V P was found to reduce lung liquid production or produce reabsorption in the guinea pig lung (Perks et al, 1993). Furthermore, Hooper et al. (1993) and Perks et al. (1993) have suggested that this reduction results from augmented N a + absorption since they showed that amiloride is capable of abolishing the effects of A V P in fetal sheep and guinea pigs, respectively. (c) Mechanics of lung liquid clearance Although some fetal lung fluid may be cleared from the lumen at birth by mechanical compressive forces squeezing fluid out of the trachea and mouth, it is likely that the majority of fluid moves across the epithelium into the interstitium as a result of active ion transport processes (O'Brodovich, 1991). The stimulus for lung fluid absorption near birth is not clear; however, studies performed with fetal sheep (Olver et 6 al, 1986; O'Brodovich etai, 1990; 1991; Chapman etai, 1991) indicate that active Na+ transport across the mature pulmonary epithelium drives liquid from the lung lumen into the interstitium. The issue of whether Cl" secretion disappears or is merely decreased and overwhelmed by a Na+-absorptive process remains unresolved. There has been evidence that significant Cl" secretion does not continue to occur (Nielson, 1988; Cotton et al, 1988a, 1988b). Thus, it appears that the lung epithelium likely switches from a prenatal Cl"-secreting membrane to a postnatal Na+-absorbing membrane. 3.4 Fetal lung fluid regulation by neuropeptides Pulmonary neuroendocrine epithelial (NEE) cells are specialized epithelial cells found in the airways of many species including humans. They are the first pulmonary cell type to differentiate at about 10 weeks gestation (in humans) (Jeffery et al, 1992). These cells occur either as single cells sparsely distributed throughout the epithelium of the tracheobronchial tract or in small, well-defined clusters which are often autonomically innervated. The latter are referred to as neuroepithelial bodies (NEBs) and are found scattered from the trachea to the bronchiolo-alveolar junctions, frequently located at airway bifurcation points (DiAugustine and Sonstegard, 1984). NEE cells possess many of the common histochemical and ultrastructural characteristics of A P U D (Amine Precursor Uptake and Decarboxylation) cells (Pearse, 1969), thereby linking their cytochemical characteristics with the production of catecholamine and peptide hormones. Further studies have demonstrated that NEE cells of human and various species contain fluorogenic amines (Lauweryns et al, 1970; Cutz et al, 1975) later identified as serotonin and dopamine (Hage, 1972; Lauweryns et al, 7 1973, 1982). Bombesin-like immunoreactivity was first localized in the NEE cells of the human neonate by immunochemistry (Wharton et al, 1978). Since then, two other peptides, calcitonin (Becker et al, 1980) and leu-enkephalin (Cutz et al, 1981) have also been found in such cells in human lung tissue. Solitary NEE cells and NEBs may have a paracrine function or local secretory action, transmitting signals to neighboring cells (Scheuermann, 1991). Several studies on developing lungs (Sorokin et al, 1982; Cutz et al, 1984; Cho et al, 1989) have revealed that these solitary cells and clustered bodies are present in highest levels just prior to birth and decline postnatally, such that they are present only in extremely low numbers in the adult. Since the decrease may be the result of depletion of the secretory substances, some authors have assigned a more important role for these cells during fetal lung development rather than in the mature lung. These suggestions include: 1) control of development of the fetal lung (Hoyt et al. 1990, 1991); 2) release of substances in response to increasing fetal hypoxia near term in order to maintain vasoconstriction in the pulmonary circuit (Hernandez-Vasquez et al, 1978; Lauweryns et al, 1985) and 3) neonatal respiratory adaptation (Redick and Hung, 1984). 3.5 Meconium The fetal lungs can be affected in various ways by a substance termed meconium. Meconium is a viscous green-yellowish liquid that first appears in the human fetal ileum between the 10th and 16th week of gestation. It is composed of gastrointestinal secretions, cellular debris, bile, pancreatic juice, mucus, blood, swallowed lanugo, and vernix (Holtzman et al, 1989). The passage of small quantities of meconium into the amniotic fluid may represent a normal physiologic event in the maturation of the fetal gut. However, numerous investigators have concluded that presence of meconium in the 8 amniotic fluid is a response to fetal asphyxia (Wiswell and Bent, 1993). Response to hypoxia could precipitate vasoconstriction in the fetal gut and cause hyperperistalsis, resulting in the relaxation of the anal sphincter (Miller and Read, 1981). Once meconium is in the amniotic fluid, the fetus is at risk for aspiration of this substance as a result of fetal or neonatal gasping. The aspiration of meconium can then lead to meconium aspiration syndrome, which is often characterized by a clinical state of respiratory distress and neonatal hypoxemia that may be accompanied by multiple air leaks and persistent pulmonary hypertension and hypoxic damage to multiple organ systems (Houlihan and Knuppel, 1994). As with human fetuses, meconium-stained amniotic fluid is sometimes found in fetal guinea pigs. Like humans, heavy passage of this substance into the amniotic fluid may be a sign of fetal distress as a result of fetal hypoxia and asphyxia. Early studies with lungs from meconium-stained fetuses (Chan and Perks, unpublished results) suggested that the underlying mechanisms behind lung liquid production in these fetuses were different from those of normal ones; in particular, the metabolic processes used for lung liquid secretion and reabsorption had been changed. (The word "normal" used throughout the text will be taken to mean fetuses that showed no meconium.) As a result, a dye dilution technique first performed by Perks et al. (1990) on in vitro lungs from fetal guinea pigs was used to investigate the energy sources for lung liquid production in fetuses stained with meconium and to compare them to normal lungs. Because the incidences of finding meconium-stained litters were infrequent and unpredictable, other studies were meanwhile carried out; these studies examined the effects of dopamine and serotonin, abundant in neuroepithelial bodies, on lung fluid production and reabsorption. 9 4. STATEMENT OF THE PROBLEM This study has two objectives. The first is to examine the effects of dopamine and serotonin on fetal lung liquid production. Since both these agents are found in the neuroepithelial bodies (NEBs) of the fetal lung, and these bodies are highly developed at birth, and are thought to release their contents at this time, it was postulated that both these agents could reduce lung liquid production or cause reabsorption in the late-term guinea pig. The second objective is to examine the possible relationship between the presence of meconium and changes in the mechanisms of fluid production or reabsorption, since earlier studies by Chan and Perks (unpublished results) suggested that the metabolic processes underlying liquid movements had been modified in fetuses stained with meconium. In the studies presented here, 2,4-dinitrophenol (DNP) and estimations of lactate production were used to analyse the nature of the changes in metabolism and lung fluid production seen in fetuses stained with meconium. 10 5. MATERIALS AND METHODS 5.1 Animals Pregnant albino guinea pigs of an inbred departmental stock received food and water ad libitum (guinea pig chow, Ralston Purina, supplemented by fresh vegetables and vitamin C). Experimental studies were carried out on fetuses of 58 - 67 days of gestation (term = 67 + 2 days), with body weights of 58.0 - 159.40 grams. In most cases, fetal ages were known from breeding dates, but where necessary they were estimated from the litter size and average body weight of the fetuses, according to Ibsen (1928), but using data from our own stock. 5.2 Basis of the method Lung fluid production rates were measured by an impermeant tracer technique capable of measuring both fluid production and reabsorption. The tracer used was Blue Dextran 2000 (Pharmacia, Dorval, Quebec; molecular mass, 2 000 000 Da; Stokes radius, 270 A ; radius of gyration, 380 A) and the method based on Normand et al., (1971), Martins et al., (1975) and Liu and Chiou, (1981). It has been used previously for in vivo studies of lung liquid secretion in fetal sheep and goats, and in in vitro work on guinea pigs and has been checked by simultaneous use of I 1 2 5 albumen (Perks and Cassin, 1985a; 1985b; 1989; Perks et al, 1990). 11 5.3 Experimental procedures Pregnant guinea pigs were anesthetized with Halothane (Fluothane, Ayers, Montreal, Quebec) until the corneal reflex was extinguished. The carotid artery was then severed and the fetuses removed by caesarean section with their amniotic sacs intact, weighed and transferred into Krebs-Henseleit saline at 37°C. The trachea was exposed, ligated rostrally and cannulated caudally with 1.5 - 2.0 cm of polyethylene tubing (PE-50, Intramedic, Clay Adams, Parsippany, NJ) filled with saline and attached to a 1.0 ml tuberculin reservoir syringe by means of an 18-gauge hypodermic needle and a three-way stopcock (K75 Pharmaseal, Puerto Rico). The cannula was positioned just above the bifurcation of the bronchi to eliminate secretions from the trachea and tied in place with two ligatures. The lungs were then exposed, the trachea rostral to the cannula was severed, and the esophagus and vascular attachments cut during which the lungs were kept warm and moist with frequent washings of Krebs-Henseleit saline at 37°C. The heart was removed and the lungs were again rinsed with warm saline to remove blood. The preparation was then suspended in a 100 ml bath of Krebs-Henseleit solution, maintained at 37°C and pH 7.4, and oxygenated with 95% 0 2 and 5% C 0 2 (Fig. 1). The time from start of dissection to placement in the bath was 3-4 minutes. Next, lung fluid was withdrawn into the reservoir syringe and 100 ul of Blue Dextran 2000 (50 gl"1 in 0.9% NaCl) were added. The two were well mixed and the solution gently returned to the lungs. The preparation was allowed to equilibrate for 30 minutes and the outer saline was replaced at 15 and 30 minutes. During this time, fluid was withdrawn and returned every 5 minutes to ensure an even distribution of the dye throughout the lungs. After equilibration, lung liquid was withdrawn into the reservoir syringe at 10 minute intervals and 10 ul samples were taken. The remaining fluid was returned to the lungs. Proper mixing was ensured by withdrawing and returning the fluid to the lungs Figure 1: Apparatus for the maintenance of the in vitro guinea pig fetal lung preparation. 13 14 halfway between and during sampling intervals. The samples taken were placed in polyethylene micro test tubes (250 u.1 Eppendorf C3515-7, Brinkmann Instruments (Canada) Ltd., Rexdale, ON), diluted 1:20 with distilled water, sealed and vortexed (Vortex-Genie, Fisher Scientific, NY). Samples were then centrifuged at 250 g for 10 minutes (clinical centrifuge, Model C L , International Equipment Co., Needham Heights, M A ) and the supernatants estimated for Blue Dextran by means of a spectrophotometer (Gilford 250, Oberlin, OH or Beckman DU-8 spectrophotometer, Beckman Instruments (Canada) Inc., Mississauga, ON; both were fitted with 250 (il quartz microcells, type 10972 NSG Precision Cells, Inc., Farmington, NY) at 620 nm. The experiments followed an A B A design and samples were taken for three hours. Samples taken during the first hour following equilibration gave the resting rate of fluid production. The lungs were then transferred to 100 ml of fresh Krebs-Henseleit saline which contained either saline alone (untreated controls) or the experimental compound. After 1 hour of treatment, lungs were then transferred again to 100 ml of fresh Krebs-Henseleit saline and incubated for an additional hour. (a) Dopamine Dopamine was investigated because of its presence in the neuroepithelial bodies (NEBs) in the fetal lung. Concentrations of 10"8, 10"7, 10"6, 10"5 and 10"4 M were used to establish a dose-response relationship (for sources of drugs, see below). 10"6 M dopamine was also tested together with: a) 10"5 M haloperidol, a general dopamine receptor blocker; b) 10"5 M R(+)-SCH 23390 hydrochloride, a Dj-receptor blocker, and c) 10"5 M domperidone, a D2-receptor blocker. Domperidone was initially dissolved in methanol, and then diluted to a final concentration of 10~5 M with Kreb-Henseleit saline; a suitable control of methanol was made in the same saline. In addition, 10"4 M dopamine was tested with IO"6, 10"5 and IO"4 M amiloride hydrochloride, a compound 15 known to inhibit N a + transport on the luminal membrane, to determine whether the effects seen after treatment with dopamine involved N a + channels. Amiloride was placed directly into the lung fluid (apically) just before transfer into the experimental bath. Amiloride was also added to the outer bath to produce the same concentration present in the lung lumen, in order to reduce loss of intraluminal amiloride down any concentration gradient. 10~4 M dopamine was also used with benzamil hydrochloride, an amiloride analogue more specific for blocking N a + channels. Benzamil was also placed into the lung fluid and outer saline to a final concentration of IO"5 M . Suitable controls for 10"6, 10"5 and 10~4 M amiloride and 10"5 M benzamil without dopamine were also carried out. 10 mg ascorbic acid was added per 100 ml bathing saline during the experimental hour for all experiments with dopamine because this agent prevents the rapid degradation of dopamine which can occur in solutions containing sodium bicarbonate at above pH 6.8 (Gardella et al., 1975). Therefore, 10 mg ascorbic acid controls were also performed. (b) Serotonin Serotonin was used because of its abundance in the neuroepithelial bodies in the fetal lung. To establish a dose response relationship, various concentrations were used. First, 10~8, 5 x 10~8, 10"7, 10"6 and 10"5 M were tested on lungs from fetuses of 58 - 62 days gestation. 10"7 M serotonin was also tested with the serotonin receptor antagonist cyproheptadine at 10"6 M to determine if the effects observed were mediated through serotonergic receptors. 10"7 M serotonin was also used with 10"6 M amiloride hydrochloride to establish whether the effects seen after treatment with serotonin involved N a + channels on the luminal membrane. Again, amiloride was placed into the lung fluid to contact the apical side and into the outer bath to a final concentration of 10"€ M , to remove the concentration gradient from lung lumen to outer saline. Control 16 experiments with 10"6 M amiloride but no serotonin were also performed. Further, because of possible age-related effects of serotonin, 10"5 M serotonin was also used on lungs from more mature fetuses (63 - 67 days). (c) Meconium studies The meconium studies involved two groups of fetuses: lungs from fetuses which showed no meconium or lungs from meconium-stained fetuses. There was a wide range of meconium staining of fetuses; therefore, only lungs from heavily-stained fetuses were included for experimentation. These fetuses had dark yellow amniotic fluid and large amounts of meconium staining the fetus. Initial studies were carried out on untreated preparations from both groups. Fluid secretion was monitored, as in earlier experiments. In addition, 20 | l l samples were taken from the outer bath every 10 minute interval for estimation of lactate passage to the outer saline. Tissue dry weights were estimated from a graph correlating fetal weight, tissue wet weight, and tissue dry weight (n=l 13). (d) 2,4-dinitrophenol (DNP) DNP was tested at 10~3 M to investigate whether the effects observed previously by Perks et al. (1993) at the lower dose of 2 x 10"4 M were maximal responses. Furthermore, earlier studies (Chan and Perks, unpublished observations) had suggested that lungs from meconium-stained fetuses behaved differently from normal lungs. As a result, 2 x 10"4 M DNP was also used on meconium-stained fetuses and the values compared to treatment with 2 x 10"4 M DNP on lungs from normal fetuses. In these studies with DNP, samples were also removed from the bathing saline every 10 minutes for lactate estimation. 17 A l l agents were obtained from Sigma (St. Louis, MO) with the exceptions of DNP (Eastman Organic Chemicals, Rochester, NY) and domperidone, SCH 23390 hydrochloride and benzamil hydrochloride (Research Biochemicals International (RBI), Natick, M A ) . 5.4 Chemical Methods Lactate was estimated in both the lung liquid and outer saline by the lactate dehydrogenase procedure (procedure 826, Sigma, (St. Louis, MO); SP8-400 spectrophotometer, Pye Unicam, Cambridge, U.K.; X = 340 nm). 5.5 Quantification of results and statistical methods Fluid production and reabsorption rates were calculated from the change in concentration of Blue Dextran, as described in previous studies (Cassin and Perks 1982; Perks and Cassin, 1989). They were estimated from plots of the total volume of fluid against time, where total volume (in ml) of fluid was that within the lungs and that removed for study. Appropriate sequential adjustments were made every 10 minutes for the addition of Blue Dextran at the onset of the experiment and its carrier saline, and for the removal of both fluid and Dextran during incubation. Slopes from the volume plots were calculated over 1 hour intervals using the slopes of their regressions, fitted by the method of least squares (Steel and Torrie, 1970; Apple II Plus computer). For each hour, the fluid production rates in ml-kg"1 body wt-hr"1 were determined as the change in total volume (in ml) over time relative to the fetus weight. The significance of changes in rate were estimated from the rates in individual hours, analyzed by two-way analysis of variance (ANOVA) and followed by a contrast test (Scheffe's) (Zar, 1984). Finally, results from similar experiments were combined into single graphs by averaging total 18 volumes (in %) at each 10 minute interval, as well as averaging the rates for each hour. A l l mean values are reported with standard errors. Statistical significance was accepted at p<0.05. 19 6. RESULTS 6.1 Fluid production by untreated in vitro lungs from fetal guinea pigs Initial studies showed that lung liquid production could be maintained in in vitro preparations of fetal lungs from guinea pigs. Studies of 30 experimental control (untreated) fetal lungs (61 + 1 days of gestation, 79.7 + 18.4 g (SD) body weight) maintained in vitro in Krebs-Henseleit saline for 3 hours showed that the preparations continued to secrete steadily throughout incubation (Fig. 2). At the start of each hour, the supporting saline was changed. The average rate in the first hour of experimentation was 1.17 + 0.08 rnfkg"1 body weight'hr -1. The average rates during the second and third hours did not change significantly from the first hour, thus demonstrating that lung function was sustained successfully in vitro. Part 1: Studies of effects on lung liquid production using agents found in the neuroepithelial bodies (NEBs) 6.2 Effects of dopamine on lung liquid production Although there is abundant evidence for effects of catecholamines on lung liquid production, dopamine had never been investigated; this was surprising since it is a major component of the highly developed neuroendocrine system of the perinatal lung. Note: Studies using dopamine were all carried out with 0.01% ascorbic acid, because this agent prevents the oxidation and subsequent deactivation of dopamine. 20 Figure 2: Lung liquid production over a 3 h period by in vitro lungs from fetal guinea pigs. Based on 30 fetuses, 61 + 1 days of gestation and 79.7 + 18.4 g (SD) body weight. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, where 100% was 0.82 + 0.25 ml (SD). Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. The dashed lines show the original slope of the first hour, there were no significant changes in slope in the last 2 h (significance accepted at p<0.05). 21 22 (a) The effects of IO-4, IO-5, IO-6, IO-7 and 10"8 M dopamine on lung fluid production Initial studies were carried out on forty-two fetal lungs (61 + 1 d gestation, 81.9 ± 9.5 g (SD) body wt) and the combined results are presented in Fig. 3. Six lungs treated with IO"4 M dopamine showed an overall effect of reabsorption; three preparations produced actual reabsorptions while the remaining three showed complete arrest of fluid production. There was a tendency for recovery in the third hour (Fig. 3a; average rates in successive hours were: before treatment: 1.27 + 0.09 ml kg" 1 body wth" 1 . during treatment: -0.27 + 0.15 (significant reduction of 121.4 + 12.8%, p<0.0005) and after treatment: 0.28 + 0.32 (reduction significant, p<0.005)). Six preparations tested with a lower dose of 10"5 M dopamine almost stopped lung liquid production with a tendency to recover in the final hour; during treatment three lungs stopped fluid secretion completely, two continued to secrete slightly, and one preparation showed reabsorption (Fig. 3b; average rates were: before treatment: 1.23 + 0.22 ml'kg" 1 body wt'h"1, during treatment: 0.10 ± 0.10 (significant fall of 92.1 ± 7.0%, p<0.0005) and after treatment: 0.22 ± 0.07 (reduction significant, p<0.0005)). 10"6 M dopamine produced a large reduction of fluid secretion in six lungs, but there was a tendency to recover after treatment; one lung stopped fluid production completely and no preparation showed reabsorption (Fig. 3c; average rates were: before treatment: 1.37 + 0.15 ml'kg" 1 body wt'h" 1. during treatment: 0.33 ± 0.09 (significant reduction of 75.4 ± 5.9%, p<0.0005) and after treatment: 0.57 ± 0.16 (reduction significant, p<0.0005)). A l l six preparations subjected to 10"7 M dopamine showed significant reductions in fluid production (Fig. 3d; average rates in successive hours were: before treatment: 1.52 + 0.32 ml'kg" 1 body wth" 1 , djjring treatment: 0.87 + 0.15 (significant reduction of 42.6 ± 10.8%, p<0.025) and after treatment: 0.77 ± 0.25 (reduction significant, p<0.01)). 10"8 M dopamine used on six lungs showed no significant changes in fluid secretion during and after treatment (Fig. 23 Figure 3: Effects of dopamine on lung liquid production by in vitro lungs from fetal guinea pigs. Based on 42 fetuses, 61 + 1 days of gestation and 81.9 ± 9.5 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~4 M dopamine; (b) 10"5 M dopamine; (c) 10"6 M dopamine; (d) IO - 7 M dopamine; (e) IO - 8 M dopamine; (f) 0.01% ascorbic acid, or (g) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.94 ± 0.13 ml (SD); (b) 0.97 ± 0.07 ml; (c) 1.09 ± 0.16 ml; (d) 1.08 ± 0.27 ml; (e) 1.04 ± 0.12 ml; (f) 0.90 ± 0.08 ml, and (g) 0.90 ± 0.23 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-fr1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 24 LU CC O LU CO o O LU o > < h-O 120 r 110 -100 90 80 120 110 100 h 90 80 120 110 100 90 80 120 110 100 90 80 120 110 100 90 80 120 r 110 100 90 80 120 110 100 -90 -80 a) 10"4 M Dopamine (n=6) 1.27 + 0.09 b) 10"5 M Dopamine (n=6) 1.23 + 0.22 c) 10"6 M Dopamine (n=6) 1.37 + 0.15 d) 10 7 M Dopamine (n=6) -0.27 + 0.15 0.28 + 0.32 0.10 + 0.10 0.22 + 0.07 0.33 + 0.09 1.52 + 0.32 e) 10"8 M Dopamine (n=6) 1.41 +0.19 f) 0.01% Ascorbic Acid (n=6) 1.23 + 0.11 g) Control (n=6) 1.20 + 0.21 1.14 + 0.09 1.07 + 0.11 1.14 + 0.04 1.01 +0.23 1.10 + 0.13 SALINE 1 TREATMENT SALINE 3 TIME IN HOURS 25 3e). Six ascorbic acid control preparations (10 mg ascorbic acid per 100 ml saline) (Fig. 3f) and six control lungs (Fig. 3g) also showed no significant changes in fluid production. It appeared that the effects which followed dopamine were dose-dependent with reabsorptions seen at 10"4 M . These results are summarized in a log dose response graph (Fig. 4). (b) The effects of 10"5 M haloperidol on the reductions seen after 10"6 M dopamine Haloperidol, a general dopamine receptor blocker, was used to investigate the specificity of the dopamine effects. Studies using lungs from twenty-four fetuses (61 + 1 d gestation, 85.6 + 7.9 g (SD) body wt) were carried out and the results shown in Fig. 5. Effects of 10"6 M dopamine (as in Fig. 3c) are included for comparison (Fig. 5a). 10"5 M haloperidol appeared to abolish responses due to dopamine. Six lungs treated with both 10"6 M dopamine and 10"5 M haloperidol showed no significant changes in fluid production (Fig. 5b; average rates in successive hours were: before treatment: 1.13 ± 0.17 ml'kg" 1 body wt'hr"1. during treatment: 0.95 + 0.13 and after treatment: 0.86± 0.16 (not significant, both hours)). Six lungs given 10"5 M haloperidol alone (Fig. 5c) and six control preparations (Fig. 5d) showed no significant changes. It was concluded that the effects which followed dopamine were dependent on activation of dopamine receptors. 2 6 Figure 4: The relationship between log concentrations of dopamine and the response of in vitro fetal lungs. Ordinate: responses quantified as the % reduction on resting fluid production (first hour) during the period of treatment with dopamine (second hour). Vertical bars show standard errors of the mean; numbers above the bars are the number of preparations; results are based on 30 fetuses. Abscissa: concentration of dopamine, molarity. 27 28 Figure 5: The effect of haloperidol on responses to 10"6 M dopamine. Based on 24 fetuses, 61 + 1 days of gestation and 85.6 + 7.9 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~6 M dopamine; (b) 10"6 M dopamine with 10"5 M haloperidol; (c) 10~5 M haloperidol, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 1.09 + 0.16 ml (SD); (b) 0.95 ± 0.11 ml; (c) 0.98 ± 0.17 ml, and (d) 1.01 ± 0.17 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 29 120 110 100 90 80 120 Co" ° ^ 110 z Q 100 h 90 80 70 120 110 100 90 80 120 110 100 90 80 a) (n-6) 70 1 ft£ M Dopamine 0.86 + 0.16 1 Cfc5 M Haloperidol 1fc£ M Dopamine 1.06 + 0.22 10^M Haloperidol Control SALINE TREATMENT TIME IN HOURS SALINE 30 (c) The effects of 10'5 M domperidone on the reductions that follow 10"6 M dopamine To establish the possible role of specific dopaminergic receptors (D 1 and D 2 ) in the effects seen after dopamine, domperidone, a D2-receptor blocker was first used. Studies were based on thirty fetal lungs (61 + 2 d gestation, 81.4 + 11.8 g (SD) body wt) and the results are presented in Fig. 6. Effects from 10"6 M (as in Fig. 3c) are inserted for comparison (Fig. 6a). Domperidone appeared to eliminate responses to dopamine. Six preparations tested with the combination of 10~6 M dopamine and 10"5 M domperidone showed no significant changes in fluid production; indeed, the rate appeared to rise during treatment (Fig. 6b; average rates in successive hours were: before treatment: 1.01 + 0.17 ml-kg"1 body wt-hr"1. during treatment: 1.23 + 0.21 and after treatment: 1.07 + 0.19 (both hours not significant)). In six preparations, 10"5 M domperidone alone produced no significant changes in fluid production (Fig. 6c). Because domperidone was dissolved in methanol, studies with 1% methanol were performed. There were no significant changes in the methanol preparations (Fig. 6d) or in six control lungs (Fig. 6e). Therefore, it appeared that blockage of the D 2 receptors and subsequent stimulation of D j receptors (if present) did not affect fluid production rates. (d) The effects of IO"5 M SCH 23390 on the reductions seen after 10-6 M dopamine To further investigate the possible roles of the specific dopaminergic receptors in the effects seen after dopamine, SCH 23390, a Dj receptor antagonist was tested. Twenty-four lungs from fetuses (60 ± 1 d gestation, 79.0 + 11.8 g (SD) body wt) were used and the results are shown in Fig. 7. Results from 10"6 M dopamine (as in Fig. 3c) are 31 Figure 6: The effect of domperidone on responses to 10"6 M dopamine. Based on 30 fetuses, 61+2 days of gestation and 81.4 + 11.8 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10"' M dopamine; (b) 10"6 M dopamine with 10"5 M domperidone; (c) 10"5 M domperidone; (d) 1% methanol, or (e) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 1.09 ±0.16 ml (SD); (b) 0.91 ±0.22 ml; (c) 1.04 ± 0.17 ml; (d) 1.01 ± 0.08 ml, and (e) 1.05 ± 0.28 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 32 120 110 -100 90 30 120 110 100 90 z Q 80 70 120 110 100 90 80 120 110 100 90 80 120 110 100 90 80 70 a) (n=6) 0.33 + 0.09 0.57 + 0.16 1 Q£ M Dopamine 10^ 5 M Domperidone 1 Cfc£ M Dopamine 1frS M Domperidone 1% Methanol Control TIME IN H O U R S SALINE - i 3 33 Figure 7: The effect of S C H 23390 on responses to 10"6 M dopamine. Based on 24 fetuses, 60+1 days of gestation and 79.0 + 11.8 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10"' M dopamine; (b) 10"6 M dopamine with 10' 5 M SCH 23390; (c) 10"5 M SCH 23390, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 1.09 ± 0.16 ml (SD); (b) 0.90 ±0.13 ml; (c) 0.89 ± 0.15 ml, and (d) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight -h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 100 90 80 120 110 100 90 80 70 120 110 100 90 80 L 120 110 100 90 80 1 M Dopamine 0.52 + 0.17 0.25 + 0.12 IQ^MSCH 23390 1 Cfc§ M Dopamine 10^ 5 M SCH 23390 70 SALINE 1 TREATMENT TIME IN HOURS SALINE 35 included for comparison (Fig. 7a). A l l six lungs treated with both IO"6 M dopamine and 10"5 M SCH 23390 produced large reductions in fluid secretion and an overall further reduction in the third hour (Fig. 7b; average rates in successive hours were: before treatment: 1.52 + 0.29 ml-kg"1 body wt-h"1, during treatment: 0.52 + 0.17 (significant fall of 65.5 ± 7.4%, p<0.0025) and after treatment: 0.25 ± 0.12 (reduction significant, p<0.001)). Six preparations given 10"5 M SCH 23390 alone (Fig. 7c) and six control lungs (Fig. 7d) showed no significant changes. It was concluded that the reductions seen with 10"6 M dopamine were probably dependent on the D 2 receptors; the receptors did not appear to be involved. (e) The effects of 10'6 M amiloride on reabsorptions seen after 10"4 M dopamine Amiloride was tested because of its ability to block Na+-dependent reabsorptions. Results are based on thirty lungs (61 + 2 d gestation, 79.9 ± 12.5 g (SD) body wt) and are presented in Fig. 8. Effects from 10"4 M dopamine (as in Fig. 3a) are included for comparison (Fig. 8a). 10"6 M amiloride used with 10"4 M dopamine on twelve fetuses failed to abolish the overall reabsorptions; nine preparations showed reabsorptions and three stopped fluid production completely. There was a tendency towards recovery in the final hour (Fig. 8b; average rates in successive hours were: before treatment: 1.17 + 0.09 ml-kg"1 body wt 'h ' l , during treatment: -0.24 + 0.07 (significant reduction of 120.6 + 8.0%, p<0.0005) and after treatment: 0.27 ± 0.12 (reduction significant, p<0.0005)). Six amiloride controls (Fig. 8c) and six untreated preparations showed no significant changes (Fig. 8d). Therefore, it appeared that the reabsorptions produced by 10"4 M dopamine could not be abolished by the presence of 10"6 M amiloride. 36 Figure 8: The effect of 10"6 M amiloride on responses to 10"4 M dopamine. Based on 30 fetuses, 61+2 days of gestation and 79.9 + 12.5 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~4 M dopamine; (b) 10~4 M dopamine, with 10~6 M amiloride present in the lung lumen and outer saline; (c) 10~6 M amiloride alone, as before, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.94 ± 0.13 ml (SD); (b) 0.81 ± 0.12 ml; (c) 1.06 + 0.31 ml, and (d) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 37 LU rr o LU GO O LL O LU o > o r -110 100 90 80 110 100 90 80 70 120 110 100 90 80 120 110 100 90 80 -0.27 ±0.15 1CH M Dopamine 0.28 + 0.32 c) (n=6) d) (n=6) -0.24 + 0.07 1Q£ M Amiloride 10^ M Dopamine 0.27 + 0.12 1 CH M Amiloride 70 SALINE 1 TREATMENT SALINE TIME IN HOURS 38 (f) The effects of 10"5 M amiloride on reabsorptions that follow 10'4 M dopamine 10"6 M amiloride, as used above, is within the range characteristic for its N a + channel blocking effect; however, 10"5 M amiloride was used with IO"4 M dopamine to reduce the possibility that the lower dose was insufficient to eliminate Na+-dependent transport completely. Eighteen lungs from fetuses (61 + 2 d gestation, 83.6 ± 13.3 g (SD) body wt) were used and the overall effects are presented in Fig. 9. Results from six lungs treated with 10"4 M dopamine (as in Fig. 3a) are inserted for comparison (Fig. 9a). Three lungs were treated with both 10"4 M dopamine and 10"5 M amiloride; all showed reabsorptions that continued into the final hour (Fig. 9b; average rates in successive hours were: before treatment: 1.14 + 0.24 ml-kg"1 body wt'hr"1. during treatment: -0.39 ± 0.09 (significant reduction of 134.0 ± 16.4%, p<0.025) and after treatment: -0.39 ±0 .18 (reduction significant, p<0.025)). Three lungs given 10"5 M amiloride (Fig. 9c) and six untreated preparations (Fig. 9d) showed no significant changes. It was concluded that 10"5 M amiloride also could not abolish the reabsorptions seen after 10"4 M dopamine. (g) The effects of 10'4 M amiloride on the effects seen after 10"4 M dopamine 10"4 M amiloride was used to further confirm that the reabsorptions produced by 10~4 M dopamine at the lower doses of amiloride were not due to an insufficient concentration of the blocker. Studies were based on eighteen fetal lungs (60 ± 2 d gestation, 82.4 ± 14.7 g (SD) body wt) and the effects summarized in Fig. 10. Effects from 10"4 M dopamine (as in Fig. 3a) are shown for comparison (Fig. 10a). Three lungs were treated with a combination of 10"4 M dopamine and 10"4 M amiloride; all three showed strong reabsorptions (Fig. 10b; average rates in successive hours were: before treatment: 0.90 ± 39 Figure 9: The effect of 10"5 M amiloride on responses to 10"4 M dopamine. Based on 18 fetuses, 61+2 days of gestation and 83.6 + 13.3 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~4 M dopamine; (b) 10~4 M dopamine, with 10~5 M amiloride present in the lung lumen and outer saline; (c) 10~5 M amiloride, as before, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.94 + 0.13 ml (SD); (b) 0.96 ± 0.15 ml; (c) 0.81 + 0.12 ml, and (d) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 40 110 100 90 80 110 100 90 80 70 120 110 100 90 80 120 110 100 90 80 70 - i » . -0.27 + 0.15 104 M Dopamine d) (n=6) M Amiloride 10^ M Dopamine M Amiloride 0.28 + 0.32 0.70 + 0.17 SALINE 1 TREATMENT SALINE TIME IN HOURS 41 Figure 10: The effect of 10"4 M amiloride on responses to 10"4 M dopamine. Based on 18 fetuses, 60 + 2 days of gestation and 82.4 + 14.7 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: 10~4 M dopamine; (b) 10~4 M dopamine, with 10~4 M amiloride present in the lung lumen and outer saline; (c) 10~4 M amiloride alone, as before, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.94 ± 0.13 ml (SD); (b) 0.93 ± 0.14 ml; (c) 0.83 + 0.25 ml, and (d) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in mfkg~ l body weighth"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 42 UJ DC O LU CO (D LL O UJ O > < h-o 110 100 90 80 110 100 90 80 70 120 110 100 90 80 120 110 100 90 80 70 ' 1 « -C) (n-3) d) (n-6) -0.27 + 0.15 1 CH M Dopamine 10^ M Amiloride 10=* M Dopamine 1Q^4- M Amiloride 0.28 + 0.32 -0.09 + 0.03 SALINE 1 TREATMENT SALINE TIME IN HOURS 43 0.21 ml-kg"1 body wt-h"1, during treatment: -0.50 + 0.11 (significant reduction of 155.7 ± 37.3%, p<0.005) and after treatment: -0.09 + 0.03 (reduction significant, p<0.01)). There were no significant changes in three preparations given 10"4 M amiloride (Fig. 10c) and six control lungs (Fig. lOd). It was concluded that the reaborptions which followed 10"4 M dopamine were not dependent on amiloride-sensitive N a + channels. (h) The effects of 10"5 M benzamil on the reabsorptions after 10"4 M dopamine Because amiloride failed to block the reabsorptions by dopamine, benzamil, an amiloride analogue more specific for blocking N a + channels was used to investigate further if the reabsorptions that followed 10"4 M dopamine were dependent on N a + transport. Twenty-four fetal lungs (61 + 1 d gestation, 81.9 ± 13.4 g (SD) body wt) were used and the results are summarized in Fig. 11. Results from 10"4 M dopamine (as in Fig. 3a) are included for comparison (Fig. 1 la). Six preparations were given both 10"4 M dopamine and 10"5 M benzamil; the overall effect was reabsorption and this continued into the final hour. Four lungs reabsorbed, one showed a complete stop in fluid secretion, and one preparation continued to secrete slightly (Fig. 1 lb; average rates in successive hours were: before treatment: 1.17 + 0.15 ml-kg"1 body wt-h"1. during treatment: -0.22 + 0.16 (significant reduction of 119.2 ± 13.7%, p<0.0005) and after treatment: -0.08 ± 0.14 (reduction significant, p<0.0005)). Six lungs given 10"5 M benzamil alone (Fig. 11c) and six control preparations showed no significant changes (Fig. 1 Id). It was concluded that the reabsorptions which followed dopamine were not dependent on benzamil-blockable N a + channels. 44 Figure 11: The effect of 10"5 M benzamil on responses to 10"4 M dopamine. Based on 24 fetuses, 61 + 1 days of gestation and 81.9 ± 13.4 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10"4 M dopamine; (b) 10"4 M dopamine, with 10~5 M benzamil present in the lung lumen and outer saline; (c) 10~5 M benzamil alone, as before, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.94 + 0.13 ml (SD); (b) 0.87 ± 0.06 ml; (c) 0.78 + 0.11 ml, and (d) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg - 1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 45 110 100 90 80 110 100 90 80 70 120 110 100 90 80 120 110 100 90 80 70 d) (n=6) -0.27 + 0.15 0.28 + 0.32 110^4- M Dopamine -0.22 + 0.16 105 M Benzamil 1 Cfc4- M Dopamine -0.08 + 0.14 10^ M Benzamil SALINE 1 TREATMENT SALINE TIME IN HOURS 46 6.3 Effects of serotonin on lung liquid production Serotonin was tested because of its abundance in the neuroepithelial bodies of the lung during fetal life; there had been no previous investigation of its possible effects on lung liquid production. (a) Fluid production by in vitro fetal lungs treated with 10'5,10"6,10"7, 5 x 10"8 and 10"8 M serotonin Thirty-six fetal lungs (61 + 1 d gestation, 77.7 + 11.5 g (SD) body wt) were used and the results are summarized in Fig. 12. At all higher concentrations, serotonin reduced lung liquid production; however, the reduction was never beyond approximately 50%. Six preparations given 10"5 M serotonin all showed reductions in lung liquid production and no recovery in the third hour (Fig. 12a; average rates in successive hours were: before treatment: 1.87 + 0.20 ml-kg"1 body wt-h"1. during treatment: 0.89 ± 0.20 (significant reduction of 52.4 + 9.9%, p<0.0005) and after treatment: 0.43 ± 0.17 (reduction significant, p<0.0005)). Six lungs treated with the lower dose of 10"6 M serotonin also showed significant reductions of fluid secretion (Fig. 12b; average rates were: before treatment: 1.25 + 0.22 ml-kg"1 body wt-h"1, during treatment: 0.58 ± 0.10 (significant reduction of 53.5 ± 7.3%, p<0.0025) and after treatment: 0.64 ± 0.13 (fall significant, p<0.005)). Six lungs given 10"7 M serotonin also showed significant reductions and no recovery (Fig. 12c; average rates were: before treatment: 1.15 + 0.13 ml-kg" 1 body wt-h" l , during treatment: 0.56 + 0.10 (significant reduction of 51.0 + 11.6%, p<0.005) and after treatment: 0.27 + 0.12 (reduction significant, p<0.0005)). A l l six lungs given 5 x 10~8 M serotonin also showed reductions in fluid secretion (Fig. 12d; average rates were: before treatment: 1.36 + 0.07 ml-kg"1 body wt-h"1. during treatment: 0.82 ±0.05 (significant reduction of 39.9 ± 4.8%, p<0.01) and after treatment: 0.68 ± 0.18 (reduction 4 7 Figure 12: Effects of serotonin on lung liquid production by in vitro lungs from fetal guinea pigs. Based on 36 fetuses, 61 + 1 days of gestation and 77.7 + 11.5 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~5 M serotonin; (b) 10"6 M serotonin; (c) IO - 7 M serotonin; (d) 5 x 10"8 M serotonin; (e) 10" 8 M serotonin, or (f) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.79 ± 0.18 ml (SD); (b) 0.79 ± 0.11 ml; (c) 0.75 ± 0.14 ml; (d) 0.85 ± 0.10 ml; (e) 0.83 + 0.09 ml, and (f) 0.73 + 0.12 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-fr1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 48 o I-LU rr o LU CO o LL O UJ O > _i o 120 110 100 90 80 70 120 r 110 100 90 80 120 r 110 100 90 80 120 110 100 h 90 80 130 -120 -110 100 90 80 100 90 80 a) 10"5 M Serotonin (n-6) 1.87 + 0.20 b) lO^M Serotonin (n-6) 1.25 + 0.22 c) 10 7M Serotonin (n-6) 1.15 + 0.13 d) 5 x lO^M Serotonin (n-6) 0.56 + 0.10 0.27 + 0.12 0.82 + 0.05 1.36 + 0.07 e) 10"8 M Serotonin (n-6) 0.97 + 0.25 1.24 + 0.19 1.52 + 0.14 f) Control (n-6) 1.02 + 0.14 1.14 + 0.30 ^^lTl7 + 0.23 i i 3 SALINE 1 TREATMENT 2 SALINE 3 TIME IN HOURS 49 significant, p<0.001)). Six additional lungs exposed to IO"8 M serotonin showed no significant reductions in fluid production either during or after treatment (Fig. 12e). Six control lungs also showed no significant changes in fluid production (Fig. 12f). It appeared that serotonin could produce reductions in fluid production. However, there were never periods of reabsorption; the maximal reduction centered around 50%. Maximal responses occured at 10"7 M and did not increase with greater concentrations. These effects are summarized in a log dose response graph (Fig. 13). (b) The effects of 10'6 M cyproheptadine on the reductions seen after 10"7 M serotonin Cyproheptadine, a serotonin receptor antagonist, was used to confirm the specificity of the serotonin effects. Studies are based on twenty-four fetal lungs (60 ± 1 d gestation, 75.0 ± 10.9 g (SD) body wt) and the results are presented in Fig. 14. Effects of 10"7 M serotonin (as in Fig. 12c) are included for comparison (Fig. 14a). 10"6 M cyproheptadine appeared to eliminate responses to serotonin. Six lungs given a combination of 10"7 M serotonin and 10' 6 M cyproheptadine showed no significant changes in fluid secretion during and after treatment (Fig. 14b; average rates in successive hours were: before treatment: 1.82 + 0.13 ml'kg" 1 body wt'hr"1. during treatment: 1.83 ± 0.23 and after treatment: 1.71 + 0.25 (both hours not significant)). Six preparations given 10"6 M cyproheptadine alone (Fig. 14c) and six untreated control lungs (Fig. 14d) showed no significant changes. Therefore, it appeared that the reductions in fluid production which followed serotonin probably involved serotonergic receptors. 50 Figure 13: The relationship between log concentrations of serotonin and the response of in vitro fetal lungs. Ordinate: responses quantified as the % reduction on resting fluid production (first hour) during the period of treatment with serotonin (second hour). Vertical bars show standard errors of the mean; numbers above the bars are the number of preparations; results are based on 30 fetuses. Abscissa: concentration of serotonin, molarity. The curve is that of best fit, estimated by Harvard Graphics 2.1 program. 51 N O i i o n a 3 d % 52 Figure 14: The effect of cyproheptadine on responses to 10"7 M serotonin. Based on 24 fetuses, 60+1 days of gestation and 75.0 + 10.9 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~7 M serotonin; (b) 10"7 M serotonin with 10~6 M cyproheptadine; (c) 10"6 M cyproheptadine, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: 0.75 ± 0.14 ml (SD); (b) 1.06 ± 0.22 ml; (c) 0.81 ±0.14 ml, and (d) 0.78 ± 0.09 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 90 80 120 110 100 90 80 70 L 120 110 100 90 80 120 110 100 90 80 1.15 + 0.13 1QJ- M Serotonin c ) (n=6) d) (n-6) 1fr£ M Cyproheptadine 1 03- M Serotonin 1 M Cyproheptadine Control 70 SALINE 1 TREATMENT TIME IN HOURS SALINE 54 (c) The effects of 10"6 M amiloride on the reductions seen after 10'7 M serotonin Amiloride was tested because of its ability to abolish Na+-based transport in fetal lungs. Twenty-four lung preparations (62 + 2 d gestation, 82.9 ± 13.3 g (SD) body wt) were used and the overall results are summarized in Fig. 15. Effects of IO"7 M serotonin (as in Fig. 12c) are inserted for comparison (Fig. 15a). 10"6 M amiloride appeared to abolish responses due to serotonin. Six lungs tested with both IO"7 M serotonin and 10"6 M amiloride resulted in no significant changes in fluid production either during or after treatment (Fig. 15b; average rates in successive hours were: before treatment: 1.17 ± 0.20 ml'kg" 1 body wt'hr"1, during treatment: 0.97 + 0.10 and after treatment: 0.96 + 0.25 (both hours not significant)). 10"6 M amiloride given alone did not have any significant effect on fluid production (Fig. 15c) and there were no significant changes in six control lungs (Fig. 15d). It was concluded that the reductions in fluid secretion following serotonin application were probably dependent on amiloride-sensitive N a + channels. 6.4 Effects of serotonin on lungs from fetuses more advanced in gestation Previous studies have shown that effects on fluid secretion by agents such as epinephrine (Brown et al., 1983) and A V P (Perks et al., 1993) are related to gestational age. It appeared that the response to these agents was greater as the age or weight of these fetuses increased. Therefore, it was worthwhile to test serotonin on lungs from more mature fetuses to determine whether greater responses could be produced in older fetuses. The fetuses were divided into two groups: less mature (60 - 61 d gestation) or more mature (65 - 67 d). 55 Figure 15: The effect of amiloride on responses to 10"7 M serotonin. Based on 24 fetuses, 62 ± 2 days of gestation and 82.9 ± 13.3 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) 10~7 M serotonin; (b) IO - 7 M serotonin, with 10~6 M amiloride present in the lung lumen and outer saline; (c) 10~6 M amiloride alone, as before, or (d) saline alone. Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.75 + 0.14 ml (SD); (b) 0.96 ± 0.20 ml; (c) 1.06 + 0.31 ml, and (d) 0.94 + 0.25 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml'kg" 1 body weight'h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 56 120 110 100 90 80 120 -110-100 90 80 70 120 110 100 90 80 120 110 100 90 80 70 a) (n=6) 0.27 + 0.12 10=7. M Serotonin 0.96 + 0.25 1 0=£ M Amiloride 10=7- M Serotonin > 10=S M Amiloride Control SALINE 1 TREATMENT SALINE TIME IN HOURS 57 The effects of 10" 5 M serotonin on lung fluid secretion in lungs from fetuses more advanced in gestation Results are based on two groups of fetal lungs and are summarized in Fig. 16. Twelve lungs from less mature fetuses (61 ± 1 d gestation, 88.1 + 21.8 g (SD) body wt) were first used. Six untreated fetal lungs from this group showed no significant changes in fluid production (Fig. 16a). Six additional lungs treated with IO"5 M serotonin (for details see Section 6.3a and Fig. 12a) showed significant reductions in lung liquid production (Fig. 16b). Studies of the second group were based on sixteen more mature fetal lungs (66 ± 1 d gestation, 123.6 + 20.7 g (SD) body wt). The overall result from ten lungs from this group treated with 10"5 M serotonin was a complete arrest in secretion with a tendency for recovery. Five lungs showed reabsorption, and the remaining five showed significant reductions in secretion rates (Fig. 16c; average rates in successive hours were: before treatment: 2.03 + 0.37 ml-kg"1 body wthr" 1. during treatment: 0.00 ± 0.18 (significant reduction of 100.2 ± 8.9%, p<0.0005) and after treatment: 0.84 ± 0.44 (significant fall, p<0.01)). Six untreated older lungs showed no significant changes (Fig. 16d). Comparison of results from Figs. 16b and 16c suggested that there was an apparent increase in response to serotonin between 61 and 66 days gestation. The response was significantly higher in the more mature fetuses (100.2 + 8.9% fall versus 52.4 + 9.9%) (Fig. 17). At 66 days, half the lung preparations reabsorbed; the remaining all showed a greater than 50% reduction in lung liquid production. Therefore, it was concluded that responses to serotonin appeared to be dependent on the maturational age of the fetus. 5 8 Figure 16: The effect of gestational age on responses to serotonin. Based on two groups of fetuses: (i) less mature; n=12, 61 + 1 days of gestation and 88.1 ± 21.8 g (SD) body weight, and (ii) more mature; n=16, 66+1 days of gestation and 123.6 + 20.7 g (SD) body weight. During the middle hour, the preparations were treated with one of the following: (a) less mature fetuses, saline alone (no treatment); (b) less mature fetuses, 10"5 M serotonin; (c) more mature fetuses, 10~5 M serotonin, or (d) more mature fetuses, saline alone (no treatment). Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 1.12 ± 0.19 ml (SD); (b) 0.79 ± 0.18 ml; (c) 1.80 ± 0.41 ml, and (d) 0.89 + 0.28 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in mfkg" 1 body weight'h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 59 120 110 100 90 h 80 £ ^ 120 110 100 90 80 120 110 100 90 80 130 120 110 100 90 80 LU rr o LU CO o Z =3 _ l LL O LU Z ) _1 O > _ i < o a) (n=6) Age = 60-61 d 1.87 + 0.20 c) (n=10) Age = 65 - 67 d d) (n=6) Age = 65 - 67 d Control 0.89 + 0.20 1 Cfc§ M Serotonin 0.00 + 0.18 1Q£ M Serotonin 0.43 + 0.17 0.84 + 0.44 (i) L E S S M A T U R E (ii) M O R E M A T U R E 70 SALINE 1 TREATMENT TIME IN HOURS SALINE 60 Figure 17: The effect of changes of gestational age on responses to serotonin divided according to age. Ordinate: responses quantified as the % reduction on resting fluid production (first hour) during the period of treatment with 10~5 M serotonin (second hour). Striped blocks show standard errors of the means; numbers above the blocks are the number of preparations; results are based on 16 fetuses, 60-67 days of gestation; the asterisk denotes a significant increase in response (p<0.01; A N O V A ) . Abscissa: gestational age, days. 61 62 Part II: Studies of lung liquid production in normal fetuses and those showing meconium staining 6.5 Effects of meconium on lung fluid and lactate production These studies were designed to establish whether lungs from fetuses stained with meconium, and therefore thought to have suffered hypoxic "stress," could still maintain lung liquid production at similar rates to those of normal lungs. Again, the word "normal" used throughout the text will be taken to mean fetuses that showed no meconium. Lactate production was also measured. (a) Fluid and lactate production by untreated normal in vitro lungs Six lung preparations from fetal guinea pigs (61 + 1 days of gestation, 64.9 + 10.6 g (SD) body weight) were maintained in vitro for 3 hours with no treatment (Fig. 18a). During the first hour, fluid was produced at 1.25 ± 0.12 ml'kg" 1 body weight'hr"1, and did not change significantly during the following two hours. In these preparations, the entries of lactate into the lung fluid and outer saline were monitored simultaneously (Fig. 18b, c and d). Average values for lactate entering lung fluid in successive hours were 8.09 ± 0.60 fxM'g"1 dry tissue'h"1, 9.63 + 1.80 (increase not significant) and 4.42 + 0.49 (reduction significant, p<0.025) (Fig. 18b). Lactate entered the outer saline approximately four times faster than it entered the lung fluid. The average rates for lactate entering the outer saline in successive hours were 35.07 + 2.29 jiM-g" 1 dry tissue'h"1, 31.15 + 1.79 (reduction significant, p<0.005) and 30.55 ± 1.09 (reduction significant, p<0.0025) (Fig. 18c). Average rates for the total lactate 63 Figure 18: Lung liquid and lactate production in untreated in vitro lungs from meconium-free fetal guinea pigs. Based on 6 fetuses, 61 + 1 days of gestation and 64.9 + 10.6 g (SD) body weight. Outer saline replaced at 1 and 2 h. Ordinates: (a) total volume of lung fluid; (b) total lactate in the lung fluid; (c) total lactate in the outer saline (on a cumulative basis, with appropriate allowances for the renewal of saline on each hour), or (d) total lactate present in the lung fluid and outer saline. A l l values expressed as percentages of that present at the end of the first hour, where 100% was: (a) 0.61 ± 0.12 ml (SD); (b) 2.76 ± 0.81 | l M ; (c) 7.25 ± 1.31 LiM, and (d) 10.01 + 1.89 (IM lactate. Abscissae: time in hours. Regressions are lines of best fit; slopes represent rates, and rates are shown below the lines in (a) ml lung fluid-kg"1 body weight-h"1, and in (b, c and d) (iM lactate-g"1 dry tissue-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 64 120 110 LU 100 90 LL 80 O 250 200 150 h 100 50 . 0 300 250 CO 2 200 LU h-jf: 150 o < j 100 < O 50 0 300 250 200 150 100 50 a) Control - Fluid movement (n=6). b) Control - lactate to lung fluid (n=6) c) Control - lactate to saline (n-6) 35.07 + 2.29 d) Control - lactate to lung fluid and saline (n-6) 1 30.55 + 1.09 J — i 3 TIME IN HOURS 65 leaving by both routes in successive hours were 43.15 + 2.16 LtM-g"1 dry tissue-h"1, 40.11 ±3 .18 (fall not significant) and 34.60 + 1.47 (reduction significant, p<0.0005) (Fig. 18d). Although there were statistical significances between the successive hours of lactate production entering the outer bath and the total lactate leaving the preparation, this was due to the closeness of the data within the hours. These changes were unlikely to be significant from a physiological point of view (see Fig. 18c and d). In addition, the reductions observed were relatively small, and showed that the preparations were not becoming progressively more hypoxic in the in vitro situation. (b) Fluid and lactate production by in vitro lungs from meconium-stained fetuses Six fetal guinea pig lung preparations from meconium-stained fetuses (61 + 1 d gestation, 96.5 ± 25.9 g (SD) body wt) were incubated in vitro untreated for 3 hours (Fig. 19a). During the first hour, fluid was produced at 0.97 + 0.22 ml kg" 1 body wth" 1 . This rate was not significantly different from that of lungs from normal fetuses. Furthermore, the average rates in the second and third hours did not change significantly from the first. Lactate passage from the lung was also monitored in these six preparations (Fig. 19b, c and d). Lactate entering the lung fluid fell in the final hour of incubation (Fig. 19b). Average values in successive hours were 11.99 ± 1.59 LiM-g"1 dry tissue-h"1, 9.41 + 1.72 (fall not significant) and 4.27 ± 0.88 (reduction significant, p<0.0005). Average rates for lactate entering the outer saline in successive hours were 61.69 ± 9.48 LiM-g"1 dry tissue-h"1, 62.04 ± 8.81 and 59.21 ± 9.14 (reductions not significant, both hours) (Fig. 19c). Average values for total lactate leaving the preparation in successive hours were 73.68 ± 10.60 LtM-g"1 dry tissue-h"1, 66.92 ± 9.21 (fall not significant) and 61.91 ± 9.61 66 Figure 19: Lung liquid and lactate production in untreated in vitro lungs from fetal guinea pigs stained with meconium. Based on 6 fetuses, 61 + 1 days of gestation and 96.5 + 25.9 g (SD) body weight. Outer saline replaced at 1 and 2 h. Ordinates:; (a) total volume of lung fluid; (b) total lactate in the lung fluid; (c) total lactate in the outer saline (as for Fig. 18), or (d) total lactate present in the lung fluid and outer saline. A l l values expressed as percentages of that present at the end of the first hour, where 100% was: (a) 0.81 ± 0.08 ml (SD); (b) 5.10 ± 0.80 | i M ; (c) 18.70 + 4.34 uM, and (d) 23.81 ± 4.15 u M lactate. Abscissae: time in hours. Regressions are lines of best fit; slopes represent rates, and rates are shown below the lines in (a) ml lung fluid-kg - 1 body weight-h -1, and in (b, c and d) u M lactate-g-1 dry tissue-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 67 120 110 100 90 80 200 150 100 50 a) Fluid movement (n=6) Meconium b) Lactate to lung fluid (n=6) Meconium o L 300 250 200 150 100 50 0 300 250 200 150 100 50 c) Lactate to saline (n=6) Meconium d) Lactate to lung fluid and saline (n-6) 73.68 + 10.60 Meconium TIME IN HOURS —' 3 68 (reduction significant, p<0.01) (Fig. 19d). Again, these reductions were small, and it would seem unlikely that these preparations were lacking oxygen in vitro. The first hour rates of the above two groups (no meconium and meconium) were compared, and there were no significant differences found between them. This suggested that lung liquid production was maintained at normal rates in fetuses which showed meconium. The rates of total lactate leaving the preparations were also measured and compared. In the case of lactate production, it was found that the meconium group produced a significantly higher amount of total lactate than the normal group (Fig. 20; average resting rates of 73.68 + 10.60 uJVI-g"1 dry tissue-hr"1 vs. 43.15 ± 2.16, p<0.025). It appeared that lungs from fetuses showing meconium produced a significantly higher amount of total lactate than lungs from fetuses showing no meconium. It is possible therefore, that lungs from meconium-stained fetuses were using more anaerobic energy to fuel their lung liquid production. 6.6 Effects of 2,4-dinitrophenol (DNP) on lung liquid and lactate production DNP was investigated to determine whether the in vitro lungs utilised oxidative metabolism to maintain lung liquid production, since DNP is able to uncouple oxidative phosphorylation (Slater, 1963). It was tested on both normal lungs and on those from meconium-stained fetuses to establish possible differences in their metabolism. 69 Figure 20: Lactate production in normal and meconium-stained fetuses. Ordinate: rate of lactate production during the first hour. Striped blocks show standard errors of the means; numbers above the blocks are the number of preparations; results are based on 12 fetuses; the asterisk denotes significant increase in production (p<0.025; A N O V A ) . 70 71 (a) Effects of DNP on fluid and lactate production by lungs from fetuses free of meconium (i) Studies with DNP at2xlO'4M Twelve lungs from fetal guinea pigs (62 ± 2 d gestation, 100.3,+ 15.7 g (SD) body wt) were treated with 2 x 10"4 M DNP during the middle hour of the 3 hour incubations and the combined results are summarized in Fig. 21a. Reabsorption was produced in every case (Fig. 21a; average rates in successive hours were: before treatment: 1.98 ± 0.56 ml-kg"1 body wt-h"1, during treatment: -0.85 ± 0.35 (significant reduction of 142.9 ± 15.8%, p<0.005) and after treatment: -0.40 ± 0.30 (reduction significant, p<0.001)). In six of these preparations, lactate passage from the lungs was simultaneously monitored (Fig. 21b, c and d). DNP produced a reduction in the lactate entering the lung fluid, although proportionately less than that in fluid production (Fig. 21b; average values were: before treatment: 7.8 +1.0 |0 ,M-g _ 1 dry tissue-h"1, during treatment: 5.4 +1.0 (reduction significant, p<0.05) and after treatment: 2.0 ±2 .1 (reduction significant, p<0.005)). In contrast, lactate entering the outer bath more than doubled during treatment (Fig. 21c; average values were: before treatment: 20.8 ±2 .1 j iM-g" 1 dry tissue-h -1, during treatment: 50.8 ± 4.2 (increase significant, p<0.001) and after treatment: 22.4 ± 3.3 (rise not significant)). The total lactate leaving by both routes doubled during treatment (Fig. 2Id; average values were: before treatment: 28.6 ±2 .0 l iM-g" 1 dry tissue-h"1, during treatment: 55.9 ± 4.9 (increase significant, p<0.001) and after treatment: 24.8 ± 3.6 (fall not significant)). 72 Figure 21: The effect of 2 x 10'4 M dinitrophenol (DNP) on lung liquid and lactate production in in vitro lungs from fetal guinea pigs. Based on 12 fetuses, 62 + 2 days of gestation and 100.3 ± 15.7 g (SD) body weight. DNP was placed in the outer saline during the middle hour only. Ordinates: (a) total volume of lung fluid; (b) total lactate in the lung fluid; (c) total lactate in the outer saline (as for Fig. 18), or (d) total lactate present in the lung fluid and outer saline. All values expressed as percentages of that present at the end of the first hour, where 100% was: 0.87 ± 0.08 ml (SD); (b) 4.45 ± 0.38 |iM; (c) 7.46 ± 0.66 [iM, and (d) 11.81 ± 0.82 (iM lactate. Abscissae: time in hours. Regressions are lines of best fit; slopes represent rates, and rates are shown below the lines in (a) ml lung fluid-kg-1 body weight-lr1, and in (b, c and d) LtM lactate-g"1 dry tissue-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). (Adapted from Perks et al., 1993) _ 120-, (a) Fluid movement n=12 100 2x 10"* M DNP 70-300-(b) Lactate to lung fluid 100-o-500- (c) Lactate to saline 300 2x 10"* M DNP (d) Lactate to lung fluid and saline 200 J 2x10"4 M DNP ~2 2.0 ± 2.1 HOURS 74 The results showed that DNP could convert fluid production to strong reabsorptions accompanied by a doubling in lactate leaving the preparations. This suggested that secretion required aerobic energy, but reabsorption could operate largely on glycolytic energy. (ii) Studies with DNP at 10~3 M DNP at 2 x 10"4 M , as used above, is expected to give a just-maximal effect (Slater 1963); however, a 5-fold increase was tested to investigate whether the reabsorptions seen at 2 x 10"4 M would increase with a higher dose of DNP. Twelve preparations from fetal guinea pigs (62 ± 1 d gestation, 88.6 ± 13.5 (SD) body wt) were treated with IO"3 M DNP and the combined results shown in Fig. 22a. Reabsorptions were produced again in every case (Fig. 22a; average values were: before treatment: 1.07 + 0.58 ml-kg"1 body wt-hr"1 and during treatment: -0.61 ± 0.47 (significant reduction of 157.0 + 15.5%, p<0.0005). There were no data for the last hour because at this high concentration of DNP it was impossible to withdraw fluid during the final hour. Lactate leaving the lungs was also monitored in six of these preparations (Fig. 22b, c and d). The lactate entering the lung fluid increased after treatment with DNP (Fig. 22b; average rates were: before treatment: 9.29 + 0.46 uJVl-g"1 dry tissue-h"1 and during treatment: 14.81 ± 2.64 (significant increase of 59.4 ± 23.1%, p<0.05). The lactate entering the outer saline increased dramatically during treatment hour (Fig. 22c; average values were: before treatment: 28.22 ± 1.98 ^M-g" 1 dry tissue-h"1 and during treatment: 127.27 ± 10.32 (significant increase of 351.0 ±42 .1%, p<0.0005) and similarly the rise in total lactate leaving by both routes was significantly higher during treatment (Fig. 22d; average values were: before treatment: 37.51 ± 1.88 uJVI-g"1 dry tissue-h"1 and during treatment: 141.79 ± 10.54 (significant increase of 278.0 ± 30.2%, p<0.0005)). Lungs 75 Figure 22: The effect of 10"3 M dinitrophenol (DNP) on lung liquid and lactate production in in vitro lungs from fetal guinea pigs. Based on 12 fetuses, 62+1 days of gestation and 88.6 + 13.5 g (SD) body weight. DNP was placed in the outer saline during the middle hour only. Ordinates: (a) total volume of lung fluid; (b) total lactate in the lung fluid; (c) total lactate in the outer saline (as for Fig. 18), or (d) total lactate present in the lung fluid and outer saline. A l l values expressed as percentages of that present at the end of the first hour, where 100% was: (a) 0.62 ± 0.11 ml (SD); (b) 2.71 ± 0.69 | i M ; (c) 6.36 ± 1.86 uJvl, and (d) 9.07 ± 2.29 LtM lactate. Abscissae: time in hours. Regressions are lines of best fit; slopes represent rates, and rates are shown below the lines in (a) ml lung fluid-kg"1 body weight-h"1, and in (b, c and d) (iM lactate-g"1 dry tissue-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). No data were obtained for third hour. 76 77 treated with the higher dose of DNP produced more lactate during treatment than lungs which received the lower dose. It appeared that increasing DNP caused little change in the levels of reabsorption; therefore, the reabsorptions observed at the lower dose of DNP were probably maximal. The increase in lactate production at IO"3 M DNP suggested that although there was a reduction in oxidative metabolism at the lower dose of DNP, oxidative processes were not completely abolished. (b) Effects of DNP on fluid produced by lungs from fetuses showing staining with meconium. In these studies DNP was used only at the lower concentration (2 x 10"4 M), since responses appeared to be maximal, and also the experiments could be followed for the complete 3 hours. Studies were based on thirty-six lungs from guinea pigs (62 + 1 d gestation, 97.3 ± 17.6 g (SD) body wt) and the combined results are shown in Fig. 23. Figure 23a (details presented above) is included for purposes of comparison. It demonstrated that lungs from fetuses stained with meconium could maintain fluid secretion steadily throughout the three hours of experimentation. Twelve lungs from meconium-stained fetuses exposed to 2 x 10"4 M DNP showed an overall reduction in fluid production; three lungs showed an increase in secretion but no preparation reabsorbed (Fig. 23b; average values were: before treatment: 1.15 ± 0.11 ml-kg"1 body wfh" 1 , during treatment: 0.77 ± 0.13 (reduction of 21.5 ± 21.4%, not significant) and after treatment: 0.39 ±0.13 (reduction significant, p<0.005)). The twelve preparations from fetuses free of meconium but treated with DNP all showed the usual reabsorptions (Fig. 23c; average rates were: before treatment: 1.98 + 0.56 ml-kg"1 body wfh" 1 . during treatment: -0.85 ± 0.35 (significant reduction of 142.9 ± 15.8%, p<0.005) and after 78 Figure 23: The effect of 2 x 10"4 M dinitrophenol (DNP) on fluid production in lungs from fetuses stained with meconium. Based on 36 fetuses, 62+1 days of gestation and 97.3 + 17.6 g (SD) body weight. These fetuses were divided into 2 groups: (i) meconium-stained; n=18, and (ii) meconium-free; n=18. During the middle hour, the preparations were treated with one of the following: (a) meconium-stained, saline alone (no treatment); (b) meconium-stained, 2 x IO"4 M DNP; (c) meconium-free, 2 x 10~4 M DNP, or (d) meconium-free, saline alone (no treatment). Ordinates: total volume of lung fluid expressed as a percentage of that present at the end of the first hour, just prior to treatment, where 100% was: (a) 0.81 + 0.08 ml (SD); (b) 0.78 ±0.15 ml; (c) 0.71 ± 0.05 ml, and (d) 0.76 ± 0.24 ml. Abscissae: time in hours. A l l regressions are lines of best fit; the slopes represent production rates. The values below the lines give the average rates in ml-kg"1 body weight-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 79 o h-LU DC O LU 00 o L L o LU o > _J < r -o 120 110 100 -90 -80 120 110 100 90 80 70 120-100 Untreated Meconium c) • (n=12) 70-130 120 110 100 90 80 d) (n-6) 2x 1fr*M DNP Meconium 2x10"* M DNP No meconium (i) M E C 0 N 1 U M • S T A I N E D (ii) M E C 0 N 1 U M • F R E E Untreated No meconium 70 SALINE 1 TREATMENT TIME IN HOURS SALINE 80 treatment: -0.40 + 0.30 (reduction significant, p<0.001)). Six normal (no meconium) untreated lungs showed no significant changes in fluid production (Fig. 23d). It appeared that the powerful reabsorptions seen in lungs from normal fetuses following DNP application did not occur in lungs from meconium-stained fetuses. (c) Effects of 2 x 10"4 M DNP on lactate production by in vitro lungs from meconium-stained fetuses Six lung preparations from fetal guinea pigs showing meconium (61 + 1 d gestation, 82.7 + 7.2 g (SD) body wt) were treated with 2 x 10~4 M DNP during the second hour of the 3 hour incubations and followed for both fluid and lactate production (Fig. 24). A l l showed a moderate reduction in fluid production but there were no reabsorptions (Fig. 24a; average values were: before treatment: 1.38 + 0.06 ml-kg"1 body wthr" 1, during treatment: 0.59 ± 0.09 (significant reduction of 56.4 + 7.2%, p<0.01) and after treatment: 0.33 ± 0.22 (fall significant, p<0.001)). In these preparations, the entries of lactate into the lung fluid and outer saline were monitored simultaneously (Fig. 24b, c and d). Lactate entering the lung fluid did not change significantly over three hours (Fig. 24b; average values were: before treatment: 12.37 + 1.50 fiM-g" 1 dry tissue-h"1, during treatment: 11.08 ±0.57 (fall not significant) and after treatment: 7.40 ± 2.50 (fall not significant)). The lactate entering the outer saline increased dramatically in the treatment hour (Fig. 24c; average rates were: before treatment: 55.99 ± 5.24 uJVI-g"1 dry tissue-h"1, during treatment: 152.52 ± 15.45 (significant increase of 176.3 ± 19.6%, p<0.0005) and after treatment: 67.93 ± 9.20 (rise not significant)). Similarly, the total lactate leaving by both routes was significantly higher during treatment (Fig. 24d; average rates were: before treatment: 68.36 ± 5.05 81 Figure 24: The effect of 2 x 10"4 M dinitrophenol (DNP) on lactate production in in vitro lungs from fetal guinea pigs stained with meconium. Based on 6 fetuses, 61 + 1 days of gestation and 82.7 + 7.2 g (SD) body weight. DNP was placed in the outer saline during the middle hour only. Ordinates: (a) total volume of lung fluid; (b) total lactate in the lung fluid; (c) total lactate in the outer saline (as for Fig. 18), or (d) total lactate present in the lung fluid and outer saline. A l l values expressed as percentages of that present at the end of the first hour, where 100% was: (a) 0.83 ± 0.13 ml (SD); (b) 4.66 ± 1.27 u M ; (c) 14.40 ± 3.95 uM, and (d) 19.05 ± 4.80 LtM lactate. Abscissae: time in hours. Regressions are lines of best fit; slopes represent rates, and rates are shown below the lines in (a) ml lung fluid-kg"1 body weight-fr1, and in (b, c and d) \iM lactate-g"1 dry tissue-h"1. Asterisks above the lines show significant changes from the original slope (dashed lines) (significance accepted at p<0.05). 120 GJ 110 CC O UJ 100 90 80 200 150 100 50 0 L 500 -450 -400 350 300 250 200 150 100 50 0 450 400 350 300 250 200 150 100 50 0 a) Fluid movement (n-6) 0.59 t0.09 2x1(HMDNP Meconium 0.33 + 0.22 b) Lactate to lung (n-6) 2 x 1CH M DNP Meconium c) Lactate to saline (n-6) 2x 10^MDNP Meconium d) Lactate to lung fluid and saline (n-6) 75.09 111.03 68.36 + 5.05 2x 10dM DNP Meconium SALINE, ' | 1 | TREATMENT | g C TIME IN HOURS 83 LtM-g"1 dry tissue-h"1, during treatment: 162.73 + 15.09 (significant increase of 139.1 + 13.8%, p<0.0005) and after treatment: 75.09 ± 11.03 (rise not significant)). It appeared that lungs from fetuses showing meconium, and already showing higher lactate production, were still able to increase their production dramatically when treated with 2 x 10"4 DNP. This suggested that these lungs were not operating maximally, since reduction of the availability of oxygen could still increase lactate production. The lack of reabsorptions seen with DNP could not be explained by lack of glycolytic energy; therefore, it appeared probable that either the link between the glycolytic pathway and the Na+-based reabsorptive process had somehow changed or that previous hypoxic "stress" had adversely affected the reabsorptive mechanisms. 84 7. DISCUSSION Earlier research on the fetal lung was performed on either sheep or goats using in vivo techniques. There is almost no information on the guinea pig except for an early study by Setnikar et al. (1959). However, it has been suggested that the guinea pig may be a preferred model for human lung development (Stith and Das, 1982). The series of experiments presented here involve the rarely used guinea pig model. It involves an in vitro technique using a dye-dilution method already established in goats and sheep. This in vitro preparation allows us to treat the fetal lung with hormones and inhibitors at known concentrations and avoid toxic effects to the intact animal. Our in vitro guinea pig lung preparations have produced rates (average 2.14 ± 1.71 (SD) ml-kg^-h"1 (n=450)) similar to those found in early studies involving intact guinea pigs (1.96 ± 0.70 mlkg^-hr 1 ; Setnikar et al., 1959). They are also close to average rates found in acute fetal goat preparations by Cassin and Perks (1982) (1.81 iriTkg^-hr 1) and Perks and Cassin (1989) (2.0 ml-kg^-hr 1) and in intact fetal sheep by Scarpelli etai. (1975) (1.5 + 2.7 iriTkg-i-hr 1), Normand etai. (1971) (2.16±0.02 ml-kg" l-hrl), Platzker et al. (1975) (3.2 ± 1.6 mTkg-J-hr1) and by Perks and Cassin (1985a) (3.1 ±2 .2 ml-kg-i-hr 1). Initial studies have shown that the in vitro preparation remains viable and functional for at least 3 hours. These isolated lungs continue to produce lung liquid at an apparently normal rate and maintain it into the third hour. There was no significant change in fluid composition and the lungs were able to react to hormones like epinephrine (Perks et al, 1990) and A V P (Perks et al., 1993). 85 7.1 Effects of dopamine on lung liquid production It is generally accepted that as birth approaches, there is a reduction in the flow of fluid into the lung lumen. The cause of this change is unknown, but many of the studies indicate that it may be related to alterations in the hormonal levels to which the lung epithelium is exposed late in gestation (Bland, 1990). However, there has been little focus on the possible effects from agents found in neuroepithelial bodies (NEBs) of the fetal lung, agents that are released in significant quantities just prior to birth (Scheuermann, 1991). Dopamine is an endogenous catecholamine that serves not only as a precursor for norepinephrine and epinephrine but also as a neurotransmitter. It has actions on the central and peripheral nervous systems and much of the work involving dopamine has been performed in the anterior pituitary and in the kidney (Jackson and Westlind-Danielsson, 1994). There has been little research on the effects of dopamine in the adult lung and almost no research concerning the fetal lung, with the exception of histochemical demonstrations of its presence. Dopamine has been shown to be stored in the pulmonary NEBs of the neonatal rabbit (Scheuermann et al, 1988). Jelinek and Jensen (1991) have reported an average value of 44 + 6 pg-ml"1 (2 x 10" 1 0 M) dopamine in plasma in the mature fetal guinea pig. In mature fetal sheep, average arterial values in pg-ml"1 plasma are reported as 136 + 47 (7 x 10" 1 0 M ; Reid et al, 1990), 190 ± 60 (IO"9 M) or 498 ± 155 (2 x 10"9 M ; Faucher et al, 1987). The levels reported are lower than those used in the in vitro preparation; however, the NEBs are contained within the fetal lung and release of their active agents could produce high local concentrations of dopamine. 86 The in vitro experiments showed that dopamine (10~7-10~5 M) can profoundly reduce the production of lung liquid; in addition, it is capable of causing reabsorption (10"4 M). Results obtained from the general dopamine antagonist haloperidol indicated that the actions of dopamine are dependent on activation of dopamine receptors. This result also suggests that although dopamine is a precursor for norepinephrine and epinephrine, it is not being converted to either hormone and acting through adrenergic receptors in the lung. Several studies have demonstrated that dopamine receptors are present early in development in the fetal rat (Giorgi et al., 1987; Sales et al., 1989) and are also functional during the prenatal period (Sales et al., 1989; Moody et al., 1993). Receptors for dopamine have been distinguished as Dj and D 2 according to the types of signal transduction pathways they stimulate and the sensitivity to different specific dopaminergic agonists and antagonists (Spano et ai, 1978; Kebabian and Calne, 1979). Tests with SCH 23390 (10"5 M), a D rreceptor blocker, showed that the antagonist was unable to prevent the falls in secretion produced by 10~6 M dopamine. This suggests that dopamine stimulates the D 2 receptors to inhibit liquid production and possibly cause reabsorption. Studies using domperidone (10"5 M), a D2-receptor blocker eliminated responses to dopamine; this confirmed that responses were mediated through the D 2 receptor. The results from the specific dopamine antagonists show that dopamine acts through specific receptors; however the receptor-mediated change in cellular activity was not determined. In the isolated lung preparation, D 1 receptors do not appear to be involved in the regulation of fluid secretion. However, this is unexpected, since these receptors are known to liberate cAMP in many tissues (Kebabian and Calne, 1979), and c A M P is known to produce reabsorption in the lungs of both sheep and guinea pigs (Walters et al., 1990; Kindler et al, 1992). 87 In contrast, D 2 receptors do seem to be concerned in the reductions in lung liquid production produced by dopamine. In strictly physiological circumstances, these receptors are known to operate as inhibitory receptors in many tissues (Vallar et al., 1992). They operate through the G-protein system, and appear to trigger a number of cellular mechanisms, all directed to reducing responses in target cells (Vallar and Meldolesi, 1989). They activate these processes by a number of possible mechanisms, some involving C a 2 + , others by direct effects on cAMP. The mechanisms involving C a 2 + result in a fall in its intracellular level, and can be either by direct effects on its channels, or by indirect effects through K + channels. The effects on the K + channels are of two types. The first involves changes in voltage of K + channels, so that the channels open, and result in a hyperpolarization of the cell; this hyperpolarization results in inhibition of C a 2 + influx (Memo et al, 1992). This system is completely independent of cAMP. The second influence on K + channels can involve activation of Ca2 +-sensitive K + channels, with a resulting loss of K + , and again hyperpolarization of the cell. This system is mediated through a fall in cAMP. However, in the exceptional situation of Ltk-fibroblasts transfected with cDNA of D 2 receptors, effects through the phosphoinositol system have been shown to increase intracellular C a 2 + (Valler et al., 1992). Although the role of C a 2 + ions in the regulation of fetal lung liquid is not yet known, all these studies suggest that C a 2 + could be involved in the responses of the lungs through D 2 receptors. The ability of D 2 receptors to reduce cellular activity by direct inhibition of A C activity is well known (De Camilli et al., 1979). However, this raises a definite problem in explaining the ability of dopamine to reduce lung liquid production through this system. Studies in the guinea pig and sheep have shown that cAMP can influence lung liquid reabsorption but by stimulating it, not inhibiting it (Walters et al, 1990; Kindler et al, 1992). Therefore, it would be expected that dopamine, acting through D 2 receptors 88 would fail to induce fluid reabsorption. However, it is possible that the D 2 receptor is unusual in the fetal lung, and although connected to the adenyl cyclase system, it stimulates rather that inhibits generation of cAMP. This possibility has a clear precedent. It is already known that alpha2-adrenoreceptors, which normally reduce cAMP, produce inhibition of fluid production, and even result in reabsorption in in vitro lungs of fetal guinea pigs (Doe and Perks, submitted). In addition, there is supporting evidence for this possibility. Stimulation of the a-receptors in the lungs of adult guinea pigs has been shown to generate, not reduce levels of cAMP (Palmer, 1971). A similar situation has been seen in isolated tracheal cells from rabbits (Liedtke, 1986). Therefore, it seems possible that the same unusual connection to the A C system exists for both catecholamines, epinephrine and dopamine, despite their acting through different receptors. In the in vitro experiments amiloride (10"6-10"4 M) was unable to eliminate the reabsorption response to dopamine (10~4 M). Benzamil (10"5 M), an amiloride analog known to have effects only on the N a + channel was also unable to stop the reabsorptions produced by dopamine (10"4 M). These results were unexpected and unusual since amiloride is capable of abolishing reduction of lung fluid secretion and/or reabsorptions produced by epinephrine in fetal sheep (Olver et al, 1986) and by A V P in fetal sheep and guinea pigs (Hooper et al., 1993; Cassin and Perks, 1993; Perks et al., 1993). In addition, reabsorptions produced by DNP and expansion in the fetal guinea pig lung preparation have also been abolished by amiloride (10~6 M) (Perks et al., 1993; Garrad Nelson and Perks, 1996), and in further experiments, amiloride used in the same way, in the same preparation, was able to abolish responses to serotonin (see below). It is unlikely therefore, that dopamine acts in a manner similar to epinephrine and A V P , by activating amiloride-sensitive N a + channels. It is possible that the actions of dopamine are linked to a Na+-transport pathway but one that does not involve amiloride-sensitive N a + channels. 8 9 Recently it has been suggested that the epithelial cell population from the alveolar regions of the fetal rat lung contains two populations of Na+-permeant ion channels (Matalon et al, 1993). The H-type channels have high affinity to benzamil and amiloride and L-type channels possess a lower affinity to amiloride. These investigators found the L-type are more abundant than the H-type, and that most of the N a + uptake occurs through the L-type channels. Further support for the existence of different types of N a + channels within the same cell is derived from patch-clamp studies such as those of Ling et al. (1991), who demonstrated three electrophysically different types of N a + channels in rabbit cortical collecting tubules, two sensitive and one insensitive to micromolar concentrations of amiloride. Furthermore, there appears to be two independent transcellular pathways for N a + in bovine and canine tracheal epithelium, one amiloride sensitive and one amiloride insensitive (Langridge-Smith, 1986; Widdicombe and Welsh, 1980). Other mammalian epithelia have also displayed sizeable amiloride-insensitive Na+-transport pathways, for example the canine lingual epithelium (Mierson et al., 1985) and rat colon (Edmonds and MacKenzie, 1984). The nature of the amiloride-insensitive pathway in all these epithelia is unknown. However, we must also consider the possibility of actions independent of Na + , perhaps involving the large Cl" gradient from lung liquid to the blood. 7.2 Effects of serotonin on lung liquid production Serotonin (or 5-hydroxytryptamine, 5-HT) is a biogenic amine also present in the NEBs of fetal humans and various mammals including the guinea pig (Polak, 1993). In the isolated lung preparations, 5-HT was capable of causing about 50% reduction in fluid secretion. The minimum concentration needed to produce this effect was 10~7 M ; an increase in dose (10"6 and 10~5 M) did not result in greater arrest of liquid production. This suggests that the effects become maximal for this stage of gestation at a 90 concentration of 10~7 M 5-HT. The effect probably does not reflect any non-specific response, such as toxicity, since it was abolished by a 5-HT-receptor antagonist. Evidence for receptor activation comes from the non-specific receptor antagonist, cyproheptadine (10"6 M) , which was capable of abolishing the reductions brought about by 5-HT. Four main families of 5-HT receptors exist: 5-HT,, 5-HT 2, 5-HT 3 and 5-HT 4. Furthermore, 5-HT! receptors are not a homogenous class, but are subdivided further into four subtypes, 5 -HT 1 A , 5 -HT 1 B , 5 - H T l c and 5 - H T 1 D (Brodde, 1990). 5-HT receptor subtypes seem to differ also in their signal-transduction mechanisms. The specific 5-HT receptor(s) through which 5-HT works in the lung preparation is not known; furthermore, the receptor-mediated change in cellular activity was not determined. However, the most likely receptor would be the 5-HT 4 receptor, since these receptors are linked to stimulation of cAMP formation. Stimulation of A C and elevation of c A M P appears to mediate the cellular responses following 5-HT 4 receptor activation in different organs of various mammals (Hoyer et al., 1994) including the brain and ileum of the guinea pig (Dumuis et al, 1988; Craig and Clarke, 1990). It is now known that c A M P is capable of slowing fluid production or causing reabsorption in fetal sheep (Barker et al, 1988; Walters et al., 1990), and also in the guinea pig (Kindler et al., 1992). The responses in the fetal sheep were blocked with amiloride; similarly in this study, amiloride was capable of blocking the effects of 5-HT. 5-HT does not appear to affect the secretory Na+K+2Cl~-cotransport system, since blocking the N a + channels permitted the cells to return to secretion despite the continued presence of the amine. However, the 5 -HTj A receptor is also a possible mediator of these effects, since it is also coupled to c A M P production, although the result can be either stimulation or reduction of cAMP, according to the tissue involved (Brodde, 1990; Zifa and Fillion, 1992). 5 - H T 1 B and 5 - H T 1 D receptors also act through adenyl cyclase; however, they are 91 less likely mediators, since they always cause a fall in levels of cAMP. The remaining receptors, 5 - H T i C and 5HT 2 subtypes are even less likely to be involved since they act through the phosphoinositol system (Hoyer et al., 1994), which, at this time, has not been shown to be concerned in fluid reabsorption. Clearly, work using more specific 5-HT receptor antagonists and/or agonists would be valuable. The general effect of 5-HT was the slowing of fluid production. Reabsorptions were not a feature of 5-HT unless the in vitro lungs were more advanced in gestation. In this set of experiments on more mature fetuses (n=10), half the lungs reabsorbed, the remaining showed a greater than 50% decrease in liquid production. This suggests that important later developmental changes may be necessary for maximal response to 5-HT. This observation has already been suggested for epinephrine (Brown et al, 1983) and A V P (Perks and Cassin, 1982; Perks et ah, 1993). Nevertheless, the work presented here suggests that 5-HT should be considered as a possible agent for promoting reabsorption at birth, and since it is found in the NEBs, which are maximally developed at birth, this further suggests an importance for the internal endocrine system of the lungs. 7.3 Effects of meconium and 2,4-dinitrophenol (DNP) on lung liquid production. Meconium arises initially in the human fetal gastrointestinal tract between the 10th and 16th week of gestation, but only passes in large amounts into the amniotic fluid in exceptional circumstances (Holtzman et ah, 1989). It is commonly believed that although the passage of small amounts of meconium may be a physiological event related to the increasing maturity of the fetus, passage of large amounts is often a result of fetal distress from asphyxia (Katz and Bowes, 1992). Fetal asphyxia primarily occurs as a result of impaired placental exchange. Impaired uterine blood flow, maternal hypoxia, placental insufficiency or abruption and compression of the umbilical cord can all 92 interfere with the transfer of substates to and from the fetus and can lead to asphyxia (Williams et al., 1993). It is believed that in utero hypoxia causes increased intestinal peristalsis and relaxed tone of the anal sphinctor, and this results in meconium passage (Miller and Read, 1981). This finding extends to the guinea pig since meconium-stained amniotic fluid is sometimes found. In the work given here, lungs from meconium-stained fetuses showed remarkable differences from lungs from normal fetuses when treated with DNP. Recently, Perks et al. (1993) studied the effects of DNP on lung liquid secretion in fetal guinea pigs. DNP is able to uncouple oxidative phosphorylation, and convert metabolism to a dependence on glycolysis (Slater, 1963). It was used at a concentration of 2 x 10"4 M DNP, a level expected to have just maximal effects. It was shown not only to stop production but also to produce unexpected, and powerful reabsorptions. It was concluded that this effect, taken in conjunction with effects of other metabolic inhibitors (Perks et al., 1991) suggested that fluid production required both glycolysis and aerobic metabolism, but reabsorption could continue on glycolysis alone. This was supported by the doubling of lactate production during treatment with DNP. In the work carried out here, this concentration of DNP was tested again, but it was also increased to 10"3 M , in order to find out whether a further increase in the responses would occur, and to determine whether lactate production could increase yet again. The studies reported here confirmed the ability of DNP to produce strong fluid reabsorptions, but the earlier conclusions needed some modification. The reabsorptions seen were closely similar at both the high and low concentrations of DNP. However, at the higher concentration of 10~3 M DNP the increases in lactate production were about 3-times greater than at 2 x 10"4 M DNP. This suggested that the lungs tested at the lower level in the previous studies still retained some degree of aerobic metabolism. It would 93 seem probable, but not certain, that aerobic metabolism might be abolished at the higher concentration of DNP, since this concentration is considered too high for routine studies, and indeed, caused problems in sampling during the final hour. However, it can now be concluded that production of fluid requires glycolysis and aerobic metabolism, but that strong reabsorptions can operate on glycolysis, perhaps with a little residual aerobic activity. The studies of lungs from meconium-stained fetuses gave remarkably different results. DNP failed to change their fluid production. Despite this, they resembled other lungs in many ways. When untreated, they continued to produce fluid consistently throughout the three hours of incubation. Furthermore, the resting rates of production were not significantly different from normal lungs. However, lungs from the meconium group did produce significantly greater amounts of lactate throughout the experiments. This suggested that they had compensated for probable hypoxic stress by increasing their glycolytic metabolism to such a degree that their ATP production could maintain a normal secretion of lung liquid. However, glycolytic activity was still not maximal since treatment with 2 x 10~4 M DNP resulted in a doubling in the lactate production. Therefore, their fluid production, although apparently based on an increased glycolysis, was still utilising some aerobic energy. This suggests that their glycolytic processes were not compromised and still capable of increased activity, and leaves their resistance to inhibition by DNP, in terms of fluid production, a distinct problem. Since work on normal lungs showed that strong reabsorption could be supported on almost entirely glycolytic processes, and these meconium-influenced lungs already showed above average lactate production, and could still increase it, it would be expected that reabsorption could continue successfully. This suggests that, although lungs from meconium-stained fetuses have increased their glycolytic activity, they have lost some mechanism which links metabolic processes to the reabsorptive mechanism. 94 The adverse effects of meconium-stained amniotic fluid on lungs have been well documented. It is clear that aspiration of meconium can occur with initial breathing efforts at birth and also by deep breathing in utero. Once in the airways, meconium causes a series of complications for the neonate as a result of the physical obstruction of airways, which makes the transition to air breathing difficult (Tyler et al., 1978; Houlihan and Knuppel, 1994). However, the process by which meconium causes damage to the fetal lung probably involves cellular damage to the lung epithelium. It is possible that some of these adverse effects are due to bile salts, which are present in meconium, and can cause cytotoxicity in type II pneumocytes (Oelberg et al., 1990). Since pulmonary type II cells have been found to exhibit Na+-absorbing properties and are generally thought to be responsible for lung liquid reabsorption (O'Brodovich et al., 1990), injury to these cells could be responsible for the disruption of the reabsorptive process seen in our studies. However, it is still possible that the damage is not a direct effect of meconium on the lung. The passage of meconium and the failure of reabsorption may be common, but independent symptoms of one underlying cause, probably fetal hypoxia. As pointed out before, passage of meconium can be a result of exposure to fetal asphyxia. Fetal asphyxia has been shown to cause edema, necrosis and destruction of alveolar structure of the fetal lung; these changes are related to the length and degree of asphyxia in utero (Jovanovic and Nguyen, 1989). Although it seems most likely that the lungs are influenced by direct effects of hypoxia, there could also be effects due to a limited period of low fluid production, during which a low lung volume might adversely affect the lung (Alcorn et al., 1977; Hooper et al, 1988). Therefore, there remains two main possibilites for the problems of lung liquid reabsorption seen in our experiments: either direct damage due to aspirated meconium, or results from other factors, such as fetal asphyxia. The increased glycolysis shown by untreated, but meconium-influenced fetal lungs could be possibly explained as a response to long-term hypoxia, rather than direct cellular damage due to meconium. However, at this time, these possibilites must remain open. 95 General Summary These studies have shown that lungs from meconium-stained fetuses have different underlying mechanisms concerning production of lung liquid, at least in terms of an increased rate of glycolysis. Furthermore, their lack of ability to show reabsorptions is an important problem since the reabsorptive process is vital to the newborn. In addition, it has been suggested that the NEBs, which are maximally developed at birth and contain specific agents which are thought to be released at this time, have an important role both during fetal life and shortly thereafter. The results here support this idea since dopamine and serotonin, two factors readily found in these NEBs significantly influence lung liquid by reducing it and in some cases, by causing reabsorption. 96 10. REFERENCES Adams FH, Moss AJ , Fagan L. The tracheal fluid in the fetal lamb. Biologia Neonatorum 5:151-158, 1963. 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