Doe, S., and A. M. Perks. α-Adrenoreceptor influences on liquid movements by in vitro lungs from fetal guinea pigs.J. Appl. Physiol. 84(2): 746–753, 1998.—Lungs from near-term fetal guinea pigs (60 ± 2 days of gestation) were supported in vitro for 3 h; lung liquid production was monitored by a dye-dilution method. Studies of 30 fetuses showed that untreated preparations produced fluid at 1.34 ± 0.21 ml ⋅ h−1 ⋅ kg body wt−1, but epinephrine at concentrations known at delivery (10−8 and 10−7 M) produced significant reductions or fluid reabsorption (analysis of variance, regression analysis); at high levels (10−6 and 10−5 M), epinephrine had no effect. Maximal responses from 10−7 M epinephrine involved α-adrenoreceptors, since they were abolished by 10−6 M phentolamine (α-antagonist) but were unaffected by 10−6 M propranolol (β-antagonist; n = 36). Activation was through α2-adrenoreceptors, since responses were abolished by 10−4 M yohimbine (α2-antagonist;n = 24) but were resistant to 10−5 M prazosin (α1-antagonist;n = 24). At high levels of epinephrine (10−5 M), where responses did not normally occur, reductions in lung liquid production were large if prazosin was also present (n = 24), and increases were significant if yohimbine was included (n = 24). In guinea pigs, epinephrine appears to activate lung fluid reabsorption through α2-adrenoreceptors; at high concentrations only, it can also increase production through α1-adrenoreceptors. Therefore, species differences appear to exist.
- lung fluid
the fetal lung produces large quantities of fluid by means of a Na+-K+-2Cl−cotransport system, probably in type II alveolar cells (21, 29). However, this fluid is reabsorbed at birth by activation of an amiloride-sensitive, Na+-based reabsorptive system, aided by colloid osmotic effects (21, 29). This reabsorption may be turned on by a number of factors, but an important stimulus is probably epinephrine (4, 31). Although epinephrine appears to act through β-adrenoreceptors in sheep and rabbits, there have been some difficulties in accepting the β-receptor system as the only means of activation (2, 3, 11, 32). First, other systems must exist, probably outside the field of catecholamines, since irreversible β-antagonists and propranolol have failed to stop fluid clearance at birth in both species (8, 19). Second, norepinephrine has been shown to be capable of reducing lung liquid production via α-receptors in fetal sheep (13). Third, initial studies in our laboratory suggested that epinephrine acted through α-, not β-, adrenoreceptors in in vitro lungs from fetal guinea pigs. The studies presented here were carried out at the concentration of epinephrine with maximal effects and were extended to the more specific α1- and α2-antagonists prazosin and yohimbine. This allowed analysis of the nature of the α-adrenoreceptor involved; therefore, the work gave probable reasons for the failure of the action of epinephrine at higher concentrations.
MATERIALS AND METHODS
Pregnant albino guinea pigs of an inbred departmental stock were given food and water ad libitum (Ralston-Purina guinea pig chow supplemented with fresh vegetables and vitamin C). Treatment of the animals was in accordance with the Canadian Council for Animal Care, and conditions were approved by the Animal Care Committee of the University of British Columbia. Studies were performed on fetuses of 60 ± 2 days of gestation (full term = 67 days) and 78.9 ± 13.4 (SD) g body wt.
The rate of lung liquid production was measured by an impermeant tracer technique using blue dextran 2000 (Pharmacia, Dorval, PQ, Canada; molecular mass = 2 × 106 Da, Stoke’s radius = 270 Å; radius of gyration = 380Å). The basis of the method and confirmation of its validity have been reported previously (6, 25).
Pregnant guinea pigs were anesthetized with halothane (Fluothane, Ayers, Montreal, PQ, Canada) until full inhibition of the corneal reflex; euthanasia was achieved by severing the carotid arteries. The fetuses were removed by cesarean section, with their amniotic sacs intact, and transferred to Krebs-Henseleit saline. Ligatures were placed around the amnion at the level of the neck to maintain a pool of amniotic fluid around the head and prevent inhalation of air; no fetal breathing movements were seen. A midline incision through the thorax exposed the lungs and trachea. The trachea was ligated rostrally and cannulated caudally with 1.5–2.0 cm of polyethylene tubing filled with saline (PE-50, Intramedic, Clay Adams, Parsippany, NJ). The cannula was attached to a 1.0-ml tuberculin reservoir syringe via an 18-gauge hypodermic needle and three-way stopcock (model K75, Pharmaseal). The cannula was tied in place with double ligatures just above the bifurcation of the bronchi; this eliminated the trachea itself from study. The trachea was severed rostral to the cannula, then the lungs were separated from their vascular attachments and from the esophagus. Throughout this time the lungs were kept warm and moist by frequent washes with Krebs-Henseleit saline at 37°C. The lungs, with the heart still attached, were transferred to fresh saline, and the heart was removed. The preparations were then suspended in 100-ml baths of Krebs-Henseleit saline at 37°C, oxygenated, and maintained at pH 7.4 with 95% O2-5% CO2. In all studies of catecholamines, the baths were protected from light with aluminum foil. It was important to set up the preparations rapidly (within 3–4 min). Approximately 0.35 ml of lung liquid was withdrawn into the reservoir syringe, and a 10-μl sample was taken from the upper cup of the stopcock with a gastight fixed-volume syringe (model 1701 NCH, Hamilton, Reno, NV); this was a blank for spectrophotometry. One hundred microliters of blue dextran 2000 (50 mg/ml in 0.9% NaCl) were added to the fluid remaining in the syringe and thoroughly mixed in, and the mixture was passed into the lungs. The preparations equilibrated for 30 min, and ∼0.3 ml of lung fluid was withdrawn and returned every 5 min throughout this period to ensure an even distribution of dye throughout the lungs.
After equilibration, experiments continued for 3 h. During this time, fluid was withdrawn every 10 min, and 10-μl samples were removed as described above. Fluid was also withdrawn and returned to the lungs midway between sampling; this ensured proper mixing within the lungs. Mixing was also aided by the gentle but continuous movements of the lungs in the bubbled saline. Samples were placed in polyethylene micro test tubes (250-μl Eppendorf C3515–7, Brinkman Instruments, Rexdale, ON, Canada), diluted 1:20 with distilled water, sealed, and vortexed (Vortex-Genie, Fisher Scientific). Samples were then centrifuged at 250 g for 10 min (clinical centrifuge, model CL, International Equipment, Needham Heights, MA). The supernatants were analyzed for blue dextran by spectrophotometer (model 250, Gilford, Oberlin, OH or model DU-8, Beckman Instruments, Mississauga, ON, Canada); spectrophotometry utilized 250-μl quartz microcells (type 10972, NSG Precision Cells, Farmington, NY; wavelength = 620 nm). The experiments followed an ABA design (control-treatment-control). Samples taken during the 1st h after equilibration gave the resting rate of fluid production. The lungs, still attached to their reservoir syringe, were then transferred to fresh Krebs-Henseleit saline, which contained one of the following: 1) epinephrine at 10−8, 10−7, 10−6, or 10−5 M (Adrenalin, Parke-Davis, Scarborough, ON, Canada);2) 10−7 M epinephrine with 10−6 M propranolol;3) 10−6 M propranolol alone;4) 10−7 M epinephrine with 10−6 M phentolamine;5) 10−6 M phentolamine alone;6) 10−7 M epinephrine with 10−5 M prazosin;7) 10−5 M epinephrine with 10−5 prazosin;8) 10−5 prazosin alone;9) 10−7 M epinephrine with 10−4 M yohimbine;10) 10−5 M epinephrine with 10−4 M yohimbine;11) 10−4 M yohimbine alone; and12) Krebs-Henseleit saline with no drugs (untreated controls; these preparations received changes of saline on each hour, as for experimental preparations). All agents were in the form of their hydrochlorides; all drugs were from Sigma Chemical (St. Louis, MO). Concentrations of antagonists were based on the work of Sheppard and Burghardt (26), Starke et al. (27), Dobbs and Mason (9), Han et al. (12), and Takayanagi et al. (30). In all experiments, the preparations were returned to Krebs-Henseleit saline for the final hour.
Quantification of Results and Statistical Methods
The rates of fluid production were calculated from the fall in concentration of blue dextran, as described previously (6, 25). Rates were estimated from plots of the total volume of fluid against time, with readings recorded every 10 min; the total volume of fluid was the sum of that within the lungs and that removed for study. Appropriate sequential adjustments were made every 10 min for the removal of fluid and dextran during incubation. The rates of production of fluid over 1-h intervals were calculated from the volume plots, using the slopes of their regressions, fitted by the method of least squares (28) (Hewlett-Packard program SD-O3A or Apple II Plus computer). In groups of similar experiments, differences in rates in successive hours were analyzed by analysis of variance (ANOVA) and Newman-Keuls test (33). When plots from similar experiments were combined, the volumes were expressed as a percentage of the volume present at the end of the 1st h, just before transfer to test solutions or fresh saline; the values were then averaged (6, 24, 25). The significance of changes in rate was also assessed from the combined graphs by submitting the changes in slope to a test for differences between two regressions (regression analysis) (6, 25); this test utilized all values for volumes from all experiments in the group. Although ANOVA and regression analysis considered the magnitude of the changes seen, ANOVA took into consideration the repeatability of the responses, and regression analysis allowed for scatter around the lines of best fit, a variability not included in ANOVA. All mean values are given with their standard errors, unless otherwise stated. Statistical significance was accepted at or below P < 0.05.
Effect of Epinephrine on Lung Liquid Production
Studies were carried out on 30 fetuses of 61 ± 2 days of gestation and 80.7 ± 12.8 (SD) g body wt. Results are shown in Fig.1. Untreated controls produced fluid with no significant change (Fig. 1 A;n = 6). All preparations treated with 10−8 M epinephrine during the middle hour showed significantly reduced production during treatment (P < 0.001–0.0005; ANOVA, regression analysis), and one showed reabsorption (74.8 ± 11.7% reduction; Fig. 1 B;n = 6). In those treated with 10−7 M epinephrine, production was reduced, and two turned to reabsorption (P < 0.001–0.0005, ANOVA, regression analysis; 91.6 ± 9.8% reduction; Fig.1 C; n= 6). In contrast, preparations treated with epinephrine above physiological levels (10−6 M and 10−5 M) showed no significant change (same tests; Fig. 1,D andE). This establishes the disappearance of responses at high concentrations of epinephrine.
Effects of General α- and β-Adrenoreceptor Antagonists on Maximal Responses to Epinephrine (10−7 M)
The significant reductions in fluid production by 10−7 M epinephrine (Fig.2 A; n= 6; 2 reabsorptions) were not abolished by incubation with the β-antagonist propranolol (10−6 M; Fig.2 B; n= 6; 1 reabsorption). ANOVA showed no significant differences in responses with or without propranolol. Preparations treated with 10−6 M propranolol alone and untreated controls showed no significant changes (ANOVA, regression analysis; Fig. 2, C andD; n= 6). There was no evidence for β-receptor activation. In contrast, the effects of 10−7 M epinephrine (Fig. 3 A;n = 6) were abolished by the α-antagonist phentolamine (10−6 M; Fig.3 B; n= 6). Preparations treated with 10−6 M phentolamine alone and untreated controls showed no significant changes (ANOVA, regression analysis; Fig. 3, C andD; n= 6). Therefore, α-receptor blockade appeared to eliminate responses to epinephrine.
The data suggest that epinephrine reduces lung liquid production in fetal guinea pigs by activation of α-, not β-, receptors.
Effects of α1- and α2-Adrenoreceptor Antagonists on Maximal Responses to 10−7 M Epinephrine
These studies tested more specific α-receptor antagonists.
α1-Receptor blockade by prazosin.
Studies were carried out on 24 fetuses of 60 ± 2 days of gestation and 79.4 ± 18.4 (SD) g body wt. Results are shown in Fig.4. The significant reductions in fluid production produced by 10−7M epinephrine (Fig. 4 A;n = 6; 2 reabsorptions) were not blocked by the α1-antagonist prazosin (10−5 M; Fig.4 B; n= 6; 1 reabsorption). ANOVA showed no significant differences in responses with or without prazosin. Preparations treated with 10−5 M prazosin alone and untreated controls showed no significant changes (ANOVA, regression analysis; Fig. 4, C andD; n= 6). Therefore, there was no evidence for α1-receptor activation by 10−7 M epinephrine.
α2-Receptor blockade by yohimbine.
Studies were carried out on 24 fetuses of 60 ± 2 days of gestation and 80.5 ± 13.5 (SD) g body wt. Results are shown in Fig.5. The marked reductions in fluid production produced by 10−7M epinephrine (Fig. 5 A;n = 6) were completely abolished by the α2-antagonist yohimbine (10−4 M; Fig.5 B, n= 6); in fact, a small but nonsignificant increase in production was seen (see below). Preparations treated with 10−4 M yohimbine alone and untreated controls showed no significant changes (ANOVA, regression analysis; Fig. 5, C andD; n= 6). Yohimbine appeared to be an effective antagonist of the effects of epinephrine; therefore, activation appeared to be through the α2-adrenoreceptor in the guinea pig.
Effects of α1- and α2-Adrenoreceptor Antagonists on Responses to High Concentrations of Epinephrine (10−5 M)
These studies repeated the above experiments, but at concentrations of epinephrine that appeared to be ineffective.
α1-Blockade by prazosin.
Studies were based on 24 fetuses of 60 ± 1 day of gestation and 75.4 ± 11.3 (SD) g body wt, and the results are given in Fig.6. As shown earlier, 10−5 M epinephrine was without effect (Fig. 6 A;n = 6), but in the presence of the α1-antagonist prazosin (10−5 M) there were strong and significant declines in production (P < 0.005–0.0005; ANOVA, regression analysis), and two preparations showed reabsorption (Fig.6 B; n= 6). Preparations treated with 10−5 M prazosin alone and untreated controls showed no significant changes (same tests; Fig. 6,C andD; n= 6). Therefore, blockade of α1-receptors appeared to allow high concentrations of epinephrine to reduce fluid production or cause reabsorption, as at lower concentrations of the hormone.
α2-Blockade by yohimbine.
Studies were based on 24 fetuses of 61 ± 2 days of gestation and 81.9 ± 14.2 (SD) g body wt. Results are shown in Fig.7. As shown previously, 10−5 M epinephrine was without effect (Fig. 7 A;n = 6), but in the presence of the α2-receptor antagonist yohimbine (10−4 M) it produced significant increases in fluid production in every experiment (P < 0.05–0.025; ANOVA, regression analysis; Fig. 7 B,n = 6). Preparations treated with 10−4 M yohimbine alone and untreated controls showed no significant changes (same tests; Fig. 7,C andD; n= 6). It appeared that blockade of α2-receptors allowed high concentrations of epinephrine to activate α1-receptors and increase fluid production.
The data suggest that high, unphysiological concentrations of epinephrine activate α1- and α2-adrenoreceptors: stimulation of α1-receptors can increase fluid production, whereas activation of α2-receptors can inhibit it; the final result is no apparent change.
The results presented here show that 10−8 and 10−7 M epinephrine reduce lung liquid production or produce reabsorption in in vitro lungs from fetal guinea pigs (P < 0.001–0.0005) but are ineffective at high, unphysiological concentrations (10−6 and 10−5 M). Responses at the most effective concentration (10−7 M) appeared to be through α-adrenoreceptors, since the general α-receptor antagonist phentolamine abolished responses, but the general β-adrenoreceptor antagonist propranolol was without effect. This was confirmed by use of more specific blockers. The α2-antagonist yohimbine eliminated responses to 10−7M epinephrine, but the α1-antagonist prazosin was without effect; therefore, activation appeared to be through α2-adrenoreceptors. At high, ineffective concentrations of epinephrine (10−5 M), responses could be produced if these antagonists were used together with the hormone. In the presence of prazosin, the inhibition of fluid production seen at lower concentrations of epinephrine reappeared; in the presence of yohimbine, production of fluid was stimulated. This suggests that the disappearance of responses at high, unphysiological concentrations of epinephrine was the result of two opposing processes:1) reduction of production by activation of α2-adrenoreceptors (as at lower concentrations) and 2) simultaneous increases of production through activation of α1-adrenoreceptors.
The ability of 10−8 and 10−7 M epinephrine to reduce lung liquid production or produce reabsorption is in agreement with earlier work on fetal sheep and goats and with studies of catecholamine-related drugs in fetal rabbits (4, 17, 24, 31). The effect is generally considered physiological, since it occurs at plasma levels seen at birth, but it is unlikely to be long lasting, since it reverses readily after infusion (4). In general, the situation is similar in the in vitro preparations studied here.
The antagonist studies reported here utilized 10−7 M epinephrine, rather than 10−8 M epinephrine used in our pilot experiments. These two concentrations span the probable levels in fetal plasma during delivery. In sheep, plasma levels reach 3.8 × 10−8 M in the early neonate (4), and after normal vaginal deliveries in humans the average plasma level in the umbilical artery is 1.0 ± 2.0 × 10−8 M, with some values as high as 6.1 × 10−8 M (based on 13 independent studies, adapted from Refs. 20 and 22). Values during delivery are not known in fetal guinea pigs, but during asphyxia concentrations in these fetuses are ∼10−7 M, with some measurements as high as 1.4 × 10−7 M (14). Therefore, 10−7 M epinephrine probably represents the upper limit of physiological concentrations. Because 10−7 M epinephrine produced maximal effects on lung liquid production, this concentration seemed appropriate for this more extended study of antagonists. Nevertheless, the effects of propranolol and phentolamine were the same at 10−8 M epinephrine used in pilot studies and at 10−7 M epinephrine reported here.
The activation of α-adrenoreceptors was entirely unexpected. Although only a limited number of species have been investigated, studies with epinephrine, propranolol, and various catecholamine-related drugs have suggested that fluid reabsorption depends on β-activation in sheep, rabbits, and rats (8, 11, 16, 31, 32). However, norepinephrine is known to be capable of reducing production through α-adrenoreceptors in fetal sheep (13), and failure of various β-receptor antagonists to prevent clearance of fluid at delivery in sheep and rabbits showed that mechanisms outside the β-receptor system must exist (8, 19). The work reported here suggests that species differences may be important.
The work with the more specific α-antagonists supported these conclusions. Although there is some overlap, prazosin is far more potent at inhibiting α1-receptors and yohimbine is more potent at blocking α2-receptors; in fact, this has been the basis for α-receptor classification (5). Therefore, the results given here suggested that the α2-adrenoreceptor was involved in fluid reabsorption, and the α1-receptor had no influence at physiological levels of epinephrine. α2-Receptors are known to exist in guinea pig lungs, at least in airways, where they predominate (30). In general, these receptors mediate their effects through Gi protein and the adenyl cyclase system (5), and this system is known to influence fluid reabsorption (15). However, there is a problem. Adenosine 3′,5′-cyclic monophosphate (cAMP) stimulates reabsorption (15), but the usual action of the α2-receptor is to inhibit, not stimulate, release of cAMP (5). Therefore, it is possible that the receptor is acting through its other effects, such as modulation of ion channels (5). However, there is a more interesting possibility. The α2-receptor might act through the usual adenyl cyclase system, but to stimulate its activity. Although not the general rule, this has been seen in a number of tissues from different species (23). Therefore, it may be particularly significant that epinephrine has been shown to generate cAMP through α-receptor activation in the lungs of adult guinea pigs, an action blocked by phentolamine (23). In addition, generation of cAMP through α2-receptor activation has been seen in isolated tracheal cells from rabbits, so such mechanisms are not unreasonable in the pulmonary system (18). The final response is also reasonable. Activation of α2-receptors has been shown to produce reabsorption of Na+ and Cl− in the intestine, and the lungs, like the intestines, are modifications of the alimentary tract (7, 10).
The work also suggested that α1-receptors could have effects, but to stimulate secretion, and only at high, unphysiological concentrations of epinephrine. Again, α1-receptors have been demonstrated in the lungs and trachea of guinea pigs, but in lower numbers than α2-receptors (1,30). In other tissues, α1-receptors act through the polyinositol system and elevate intracellular Ca2+ (5, 12). However, the mechanisms used here and the way in which fluid production is increased are not clear. Perhaps the most remarkable aspect was the consistency of the responses, since factors that consistently raise fluid production have been difficult to find (21). However, the opposing effects of α1-receptors, which stimulate fluid production, and α2-receptors, which inhibit it, can explain the unusual disappearance of effects of epinephrine at high unphysiological concentrations. At this time, stimulation of production must be regarded as an interesting pharmacological effect; however, there must be some reservation, since there was also an increase in production at physiological levels of epinephrine, although this increase could not be shown to be statistically significant. Nevertheless, this study shows more clearly than most that stimulation of production is a possibility.
It must be remembered that any in vitro method, whether by tissue culture or isolated organ, is never entirely physiological and needs to be extended to the intact animal. Nevertheless, this in vitro model gives a good basis for further investigation and has many assets. It allows elimination of external influences, such as reflexes, and a reduction in the variables in a complex situation, such as elimination of vascular and colloid osmotic effects. It allows the use of hormones and antagonists at precise concentrations, uninfluenced by placental destruction or loss, and the use of agents that are toxic or have widespread effects in the whole animal. These factors were an asset here. The results suggest that there can be species differences in the mechanisms that help drain the lungs at birth, so those that operate in the human may be more complex than we believe.
We thank the Dept. of Zoology for financial assistance and the National Research Council of Canada for Operating Grant NSERC-582584 (A. M. Perks).
Address for reprint requests: A. M. Perks, Dept. of Zoology, University of British Columbia, 6270 University Bl., Vancouver, BC, Canada V6T 1Z4.
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