The effects of temperature change on lung liquid production by in vitro lungs from fetal guinea pigs

Lungs from fetal guinea pigs of 58-65 days of gestation were supported in vitro for 3 h, and lung liquid production rates were measured by a dye dilution technique. In 36 control preparations, incubated continuously at 37 degrees C, the average production rate in the first hour was 1.46 +/- 0.23 ml/h per kg body weight; there was no significant change over the following two h. In 36 further preparations the temperature was changed during the middle hour (ABA), with the following % reductions in production rates: at -1 degrees C (relative to 37 degrees C), 68.2 +/- 17.1%; -2 degrees C, 125.5 +/- 30.1% (reabsorption); -3 degrees C, 103.8 +/- 32.8% (reabsorption); -5 degrees C, 82.7 +/- 16.6%, -8 degrees C, 94.7 +/- 1.8 %; +2 degrees C, 100.7 +/- 12.6% (all significant, P less than 0.025-0.005). Slow recoveries followed a return to starting conditions, except after the increase in temperature, 10(-6) M amiloride abolished reabsorption, but not depression, during the maximal effects of temperature reduction (at -2 degrees C, n = 6); amiloride had no effect on control preparations (n = 6). These results suggest that: (a) reductions of 2-3 degrees C, as seen in the delivery room, abolish secretion, but not reabsorption of lung fluid; larger reductions stop both processes; (b) the reabsorptions seen after a fall in temperature depend on Na(+)-transport mechanisms; (c) lung liquid production was sensitive to a rise in temperature, so that fevers might adversely affect lung development, and (d) the fall in temperature at birth may be an important factor in the early reabsorption of lung liquid.     

Effects of norepinephrine on lung liquid production by in vitro lungs from fetal guinea pigs

Lungs from near-term fetal guinea pigs (61 +/- 2 days of gestation) were supported in vitro for 3 h; lung liquid production was monitored by a dye dilution method. Untreated control preparations produced fluid at 1.38 +/- 0.30 mL x kg(-1) body weight x h(-1), with no significant change (ANOVA; regression analysis); those given 1.24 x 10(-9) or 1.24 x 10(-8) M norepinephrine during the middle hour showed no significant change, but those given concentrations between 5.24 x 10(-8) and 1.24 x 10(-5) M all showed significant reductions or fluid reabsorption (based on 42 fetuses). The responses showed a linear relationship with the log concentration (r = 0.97). They appeared to involve alpha-adrenoreceptors, since responses to 10(-7) M norepinephrine were unaffected by 10(-6) M propranolol, but those to 10(-7) and 1.24 x 10(-6) M norepinephrine were abolished by 10(-6) and 1.78 x 10(-5) M phentolamine, respectively (based on 48 fetuses). Activation was through alpha2-adrenoreceptors, since responses to 10(-7) and 10(-5) M norepinephrine were abolished by 10(-4) M yohimbine, but not by 10(-5) M prazosin (based on 60 fetuses). The results show that norepinephrine is able to reduce lung liquid production when at plasma levels present at birth, and that it can produce reabsorption; unlike epinephrine, there was no reduction in responses at high concentrations. This work reintroduces a neglected factor, norepinephrine, into possible controls of lung liquid reabsorption, and opens up the potential for neural controls.     

Fluid production by in vitro lungs from fetal guinea pigs

Lungs from fetal guinea pigs (54-67 days of gestation) were supported in vitro, and lung liquid secretion rates were measured by a dye-dilution technique. The average secretion rate in the first hour was 2.14 +/- 0.08 (SE) mL x kg-1 body weight.h-1 (0.21 +/- 0.01 mL/h) (n = 450); this was comparable to intact preparations. In an independent study of 30 lungs, secretion continued unchanged for 3 h, with no significant change in fluid composition. Between 54 days and term, production appeared to fall in terms of millilitres per kilogram per hour. The following agents were placed in the supporting saline during the middle hour of incubation. (i) Sodium iodoacetate: at 10(-4) M this produced a fall in secretion (fall, succeeding hours; 55.4 +/- 23.0 and 64.9 +/- 17.5%; n = 6); at 10(-3) M it stopped secretion (fall, succeeding hours; 87.2 +/- 10.3 and 100%, n = 6). (ii) Ouabain: at 10(-5) M there was no change in production (n = 6); at 10(-4) M, four preparations were unaffected, two reduced production. (iii) Epinephrine (10(-7) M) produced a significant fall in production in all cases (n = 6); in four preparations secretion reduced (average fall, 64.4 +/- 10.8%); in two preparations there was reabsorption (average rate, -1.03 mL.kg-1.h-1). This extends the effect of epinephrine to the guinea pig, and suggests that the in vitro preparation is a useful model for studies of the fetal lung.     

The effects of bumetanide and furosemide on lung liquid secretion in fetal sheep

Thirty-four experiments were carried out on the effects of loop diuretics on lung liquid secretion in 20 fetal sheep (128-145 days gestation) with indwelling catheters. Bumetanide placed in the lung liquid at 2.19 +/- 0.52 X 10(-4) M produced immediate reabsorption of fluid, and effects lasted 3 hr (n = 6). Bumetanide at 1.1 +/- 0.17 X 10(-5) M reduced secretion significantly for 2 hr (n = 4), but at 1.07 +/- 0.06 X 10(-6) M there was no clear effect (n = 6). Controls showed no significant change (n = 6). Furosemide was less effective. At 3.1 +/- 0.07 X 10(-3) M it produced an immediate reabsorption, which lasted 3 hr, but at 1.0 +/- 0.04 X 10(-4) M it increased secretion slightly (n = 4); controls showed no significant change (n = 6). The results are consistent with the presence of a chloride transport system, perhaps with sodium cotransport, as the major factor in fetal lung liquid secretion.     

Lung liquid production by in vitro lungs from fetal guinea pigs: studies with metabolic inhibitors.

Lungs from fetal guinea pigs (62 +/- 2 days of gestation) were supported in vitro for 3 h, and lung liquid production was measured by dye dilution. Eighteen untreated preparations produced fluid at 1.76 +/- 0.30 mL.kg-1 body weight.h-1 during the first hour, with no significant changes in later hours. When inhibitors of respiratory processes were placed in the outer saline during the middle hour, production changed significantly, as follows: (a) sodium iodoacetate at 10(-3) M stopped production (87.2 +/- 10.3 and 100% reductions, successive hours; n = 6), at 10(-4) M it reduced production (60.0 +/- 10.3 and 63.4 +/- 9.3% reduction, successive hours; n = 12); (b) sodium fluoride, 10(-3) M, almost stopped production (93.2 +/- 12.1 and 89.5 +/- 9.3% reductions, successive hours; n = 6); (c) sodium cyanide at high concentration (10(-3) M) reduced production slowly (35.5 +/- 12.3 and 73.1 +/- 22.4%; successive hours; n = 6); (d) sodium azide, 10(-3) M, also reduced production (67.6 +/- 14.2 and 59.7 +/- 14.0%, successive hours; n = 6); total lactate lost rose 1.8 +/- 0.5 fold; (e) dinitrophenol produced strong reabsorptions; at 10(-3) M, production fell 115.4 +/- 15.9 and 113.1 +/- 47.3%, successive hours (n = 4), and at 2 x 10(-4) M it fell 143.8 +/- 33.8 and 153.4 +/- 26.7%, successive hours (n = 6); total lactate lost rose 2- to 3-fold. Control preparations showed no significant changes. The results suggest that lung liquid production requires glycolysis and aerobic metabolism. However, reabsorption appears to continue on glycolysis alone, a particularly useful situation for neonates suffering respiratory distress.     

Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production.

Somatostatin-like immunoreactivity was measured by radioimmunoassay with a monoclonal antibody in lungs from perinatal guinea pigs (62 +/- 2 days of gestation). Fetuses delivered by Caesarean section and dissected before breathing showed 4748 +/- 758 pg/lung (n = 25). Fetuses allowed to breathe (neonates) showed marked increases in activity: 7629 +/- 1355 pg/lung (n = 12) after breathing 30 seconds, and 10729 +/- 1064 pg/lung (n = 6) after breathing 3 minutes (2.3-fold increase, P < 0.005). Values then declined (5203 +/- 1050 pg/lung (n = 9) at 30 minutes; 1458 +/- 105 pg/lung (n = 4) at 60 minutes). Changes were similar in pg/g wet tissue. HPLC characterized the immunoreactive peptides as somatostatin-14 (SS-14) and somatostatin-28 (SS-28) in both fetuses and neonates (n = 11). SS-28 made up only 13.7 +/- 1.7% of the activity; this percentage did not change with breathing. The effects of synthetic SS-14 on lung liquid production were investigated in in vitro lungs from 42 fetal guinea pigs. All 21 preparations immersed in 10(-5)-10(-7) M SS-14 during the middle hour of 3 h incubations reduced production, often approaching zero after treatment (rates, ml/kg body weight per h, succeeding hours: 10(-5) M (n = 9), 3.09 +/- 0.68, 0.93 +/- 0.39, -0.05 +/- 0.60 (fall significant during and after treatment, P < 0.025-0.005); 10(-6) M (n = 6), 3.06 +/- 0.68, 1.29 +/- 0.58, 0.36 +/- 0.38 (P < 0.05-0.005); 10(-7) M (n = 6), 1.96 +/- 0.66, 1.11 +/- 0.34, 0.64 +/- 0.28 (P < 0.05-0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cAMP, its analogues, and forskolin on lung fluid production by in vitro lung preparations from fetal guinea pigs.

Lungs from fetal guinea pigs (62 +/- 1 days of gestation) were supported in vitro for 3 h and fluid production was determined by a dye dilution method, based on Blue Dextran 2000. Twenty untreated lungs produced fluid at 1.41 +/- 0.22 mL.kg-1 body weight.h-1, with no significant changes during later hours. Treatments with analogues of cAMP, cAMP, or forskolin during the middle hour reduced production significantly. Dibutyryl cAMP at 10(-3) M produced reabsorption (117.8 +/- 13.6% reduction, p less than 0.001, n = 10); at 10(-4) M it reduced production (77.3 +/- 11.0% fall, p less than 0.001, n = 10). 8-Bromo-cAMP appeared more effective; at 10(-4) M it caused slight reabsorption (109.0 +/- 8.9% reduction, p less than 0.001, n = 6) and at lower concentrations it decreased production (at 10(-6) M, 67.6 +/- 9.6% fall, p less than 0.001, n = 6; at 10(-7) M, 40.0 +/- 14.3% fall, p less than 0.001, n = 6). At high doses, cAMP itself produced similar effects (at 5 x 10(-3) M, 141.6 +/- 22.8% reduction, p less than 0.001, n = 6); at 10(-4) it was ineffective (n = 3). Forskolin at 10(-6) M induced the strongest reabsorptions seen (159.1 +/- 10.9% reduction, p less than 0.001, n = 6); at lower concentrations it reduced production (at 10(-8) M, 73.8 +/- 5.5% fall, p less than 0.001, n = 6; at 10(-9) M, 29.2 +/- 9.2% fall, p less than 0.05, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of arginine vasopressin and epinephrine on lung liquid production in fetal goats

The effects of arginine vasopressin (AVP) and epinephrine on lung liquid secretion were investigated in 67 acute fetal goats (116 days of gestation to term) with intact umbilical cords after caesarean section. Secretion was measured by an impermeant tracer technique. AVP was infused intravenously (1.6-39.2 mU/(kg.min); 2 h) into 26 fetuses. All fetuses below 130 days of gestation, except one, showed no response. All above 133 days reduced secretion, or turned to reabsorption, except at the lowest infusion rate. The effect persisted, and usually increased postinfusion. Expansion of the lungs with saline did not change the response. The percentage reductions were linearly related to the logarithm of the infusion rate (threshold, 1.42 mU/(kg.min]. The absolute reductions were linearly related to fetal weight. Epinephrine was infused intravenously (0.30-6.72 micrograms/(kg.min); 1-2 h) into 12 fetuses. All fetuses (118 days to term) reduced secretion or reabsorbed by the second hour. At the highest infusion rate, reabsorption was immediate; at the lowest, secretion increased slightly, then fell significantly in the second hour. Epinephrine acted at levels considered physiological at delivery in the sheep. AVP appears to act at plasma levels found in most vaginal deliveries; it may augment epinephrine-induced reabsorption during stress, and help long-term removal of lung fluid.     

The rate of production of lung liquid in fetal goats, and the effect of expansion of the lungs

Fetal goats (95-155 days of gestation), delivered by Caesarian section, under chloralose (50 mg/kg), with intact umbilical cords, were monitored for lung fluid production by an impermeant tracer method. The average rate of production was 13.4 +/- 2.3 ml/h, or 6.0 +/- 0.9 ml/h per kg. Production rose exponentially towards birth, by both parameters, significant P less than 0.0001 (ml/h), or P less than 0.0005 (ml/h per kg). This indicated an increase in lung liquid production due to both growth and increased activity of the tissues. However, considerable individual variability, even between twins, suggested that this general pattern was modulated by physiological requirements. In 14 fetuses (plus 12 controls), expansion of the lungs with volumes of saline similar to those of the first inspirations (saline, 15-40 ml, matched to the optical density of the lung fluid; inspiration by spirometer, 20-25 ml), caused reduction of secretion or reabsorption of fluid. Secretion changed to reabsorption at about 50% expansion, and the probable physiological limit was estimated at 62-68% expansion. The logarithm of the % fall in production was linearly related to the % expansion (r = 0.88; P less than 0.0001); therefore, small expansions (equivalent to intrauterine breathing) would have little effect, but larger changes such as first breaths, would produce rapidly escalating effects. Controls showed no similar activity. Changes in Na+ and Cl- ions are in parallel to those of water. However, K+ ions moved into the lungs after expansion, in the opposite direction to Na+ ions, and against their concentration gradient. It is suggested that expansion of the lungs activates a Na+/K+ ATP-ase pump to aid reabsorption of salt and water at birth.     

Oxytocin-induced labor augments IL-1beta-stimulated lung fluid absorption in fetal guinea pig lungs

We tested the hypothesis that oxytocin-induced labor augmented IL-1beta-induced/-stimulated lung fluid absorption in preterm guinea pig fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. At day 3, oxytocin was administered, and fetuses were delivered by abdominal hysterotomy at 61 and by oxytocin-induced birth at 68 days gestation. Delivered fetuses were instilled with isosmolar 5% albumin into the lungs, and lung fluid movement was measured over 1 h by mass balance. Lung fluid absorption was induced in 61-day and stimulated in 68-day gestation lungs by IL-1beta. Labor induction by oxytocin augmented IL-1beta-induced/-stimulated lung fluid absorption. Metyrapone pretreatment did not affect oxytocin-induced/-stimulated lung fluid absorption, while completely blocking IL-1beta-induced/-stimulated fluid absorption. Fetal lung fluid absorption, when present, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive sodium channels were critical for fluid absorption. Epithelial sodium channel and Na-K-ATPase subunit expressions were both increased by IL-1beta, but not further by oxytocin. Our results indicate that IL-1beta release into the maternal blood circulation positively affects lung maturation due to the IL-1beta-induced release of cortisol and thus prepares the lungs for the epinephrine surge associated with labor.     

The effects of mechanical stretching on fetal rat lung cell prostacyclin production

A model system was used to determine the effect of stretch on prostacyclin (PGI) production by organotypic fetal rat lung cultures grown on gelatin foam in vitro, measured by RIA of 6-keto-PGF1_α (6KF) in the culture medium. The stretching apparatus was programmable for stretch of varying frequency and duration. The effective stimuli for PGI production were: continuous pulsatile stretch> intermittent pulsatile stretch> permanent stretch (p<0.05). The rate of PGI production was greatest in the first 15min of pulsatile stretch and was associated with a 70% increase in cAMP production (p < 0.05). When the effect of magnitude of stretch was compared (15% vs 28% extension), there was a significant increase with a maximum in the 28% stretch group double that of the 15% stretch group (p<0.01). PGI production in response to pulsatile stretching was inhibited by indomethacin but not by pretreatment with cortisol. These results suggest that the production of PGI by lung cells may be significantly affected by the frequency and magnitude of pulsatile stretching.     

The effects of mechanical stretching on fetal rat lung cell prostacyclin production.

A model system was used to determine the effect of stretch on prostacyclin (PGI) production by organotypic fetal rat lung cultures grown on gelatin foam in vitro, measured by RIA of 6-keto-PGF1 alpha (6KF) in the culture medium. The stretching apparatus was programmable for stretch of varying frequency and duration. The effective stimuli for PGI production were: continuous pulsatile stretch greater than intermittent pulsatile stretch greater than permanent stretch (p less than 0.05). The rate of PGI production was greatest in the first 15 min of pulsatile stretch and was associated with a 70% increase in cAMP production (p less than 0.05). When the effect of magnitude of stretch was compared (15% vs 28% extension), there was a significant increase with a maximum in the 28% stretch group double that of the 15% stretch group (p less than 0.01). PGI production in response to pulsatile stretching was inhibited by indomethacin but not by pretreatment with cortisol. These results suggest that the production of PGI by lung cells may be significantly affected by the frequency and magnitude of pulsatile stretching.     

Acetaminophen metabolism in vivo by pregnant, fetal, and neonatal guinea pigs

Acetaminophen (50 mg/kg body weight) was administered by iv injection to pregnant guinea pigs (60-65 days of gestation) and by ip injection to cesarean-derived term (67 days of gestation) pups. At suitable time intervals after treatment, the concentrations of drug, glucuronide (GLU), and sulfate (SO4) in blood plasma, urine, and bile were measured by high-performance liquid chromatography (HPLC). At 60-65 days of gestation, guinea pig fetuses formed both GLU and SO4, an approximate ratio of 2:1 being observed with mean concentrations of the order of 43 and 27 micrograms/mL being measured for GLU and SO4, respectively at 180 min post-treatment. At the same time interval, the major detoxification product found in the blood plasma of the pregnant dams was GLU (104 micrograms/mL) with only minute amounts (4.2 micrograms/mL) of SO4 being detected. In cesarean-derived and acetaminophen-treated pups, euthanized at 2 or 4 hr post-treatment, plasma levels of GLU were approximately twofold higher relative to the concentration of SO4 at both time intervals. Significant differences were not observed in either bile or urine at 2 hr post-treatment but by 4 hr after treatment the levels of GLU found in the bile and urine were two- or threefold higher than those of SO4. In contrast to the adult guinea pig where GLU forms some 90% of the urinary excretory product and SO4 accounts for only 7%, the SO4 pathway of detoxification appears to be of significant importance to the fetal and neonatal animal.     

Met-enkephalin effects on histamine-induced bronchoconstriction in guinea pigs

We investigated the effects of the neuropeptide met-enkephalin on histamine-induced bronhoconstriction in an experimental model of asthma. Classic Konzett and R?ssler's method of whole body plethysmography modified by Gjuris, was applied in the study. This method represents a standard experimental model of bronchoconstriction, suitable for the evaluation of peptide effects on the histamine-induced bronchoconstriction. The results of the measurements implicate a dose-related modulatory effect of met-enkephalin on the bronchoconstrictor action of histamine. Met-enkephalin doses of 1 mg/kg and 10 mg/kg, respectively, caused statistically significant reduction of the histamine-induced bronchoconstriction. Estimated ED50 dose was 0.235 mg/kg. Further studies are needed to define practical and therapeutical use of the presented observations in respiratory pharmacology.     

Antiradiation effects of leucynferon on dogs and guinea pigs

In experiments with two species of animals (dogs, guinea pigs) irradiated with sublethal and lethal doses of gamma-rays, it was observed, that leucynferon had antiradiation effect. Course of injections: dogs--8 injections subcutaneus: 2.0 ml (1, 3, 5, 8, 12, 14, 21, 34 days after irradiation); guinea pigs--14 injections subcutaneus, 0.2 ml (1-14 days after irradiation). Therapeutical effect was explained by capacity of the preparation to defend the hemopoietic organs from the radiation and to stimulate hemopoiesis. Leucynferon hindered the development of acute radiation sickness symptoms. Immunoreactivity of dogs and guinea pigs in experimental group was more complete and restored faster. The growth of the automicroflora on the skin was restrained. Production of interferon-gamma (which is a function of T-lymphocytes) was restored faster.     

Hyperpnea-induced production of TBHP-initiated chemiluminescence in guinea pigs

Antioxidants attenuate hyperpnea-induced airway constriction. It was hypothesized that this type of airway constriction is closely related to reactive oxygen species (ROS). However, there is no direct evidence of an increase in ROS during or right after the course of hyperpnea. To detect ROS production induced by hyperpnea, forty one guinea pigs were divided into four groups: control; control with 95% O2-5% CO2; hyperpnea with 95% air-5% CO2; and hyperpnea with 95% O2-5% CO2. Three minutes following hyperpnea or at the equivalent time, we obtained bronchoalveolar lavage (BAL) and measured its chemiluminescence (CL) counts. In addition, hyperpnea with 95% O2-5% CO2 gas mixture was carried out and BAL was collected 3 minutes after the hyperpnea in an additional forty animals. We measured CL counts in BAL samples before and after the treatments of the following ROS scavenger(s) or saline in vitro: control (saline); superoxide dismutase (SOD); catalase; dimethylthiourea (DMTU); and SOD+catalase+DMTU. Hyperpnea with 95% O2-5% CO2, but not with 95% air-5% CO2, gas mixture induced significant increase in t-butyl hydroperoxide-initiated CL counts, which were inhibited by DMTU, catalase, or SOD in vitro. Our data suggest that hyperpnea with a 95% O2-5% CO2, but not with 95% air-5% CO2, gas mixture induced an increase in ROS production.     

Propranolol antagonism to the effect of furosemide on the compostion of endolymph in guinea pigs.

Furosemide, administered intravenously (50 mg/kg) to guinea pigs, caused an increase in the sodium concentration and a decrease in the potassium concentration of endolymph, and a fall in the endolymphatic potential. The furosemide-induced electrolyte changes were prevented by pretreatment of five guinea pigs with propranolol given intravenously (2 mg/kg). The fall in the endolymphatic potential was not prevented by propranolol. Local administration of furosemide to the perilymphatic or endolymphatic space caused a fall in the endolymphatic potential, but had no effect upon the concentrations os sodium and potassium of endolymph. These studies provide additional information suggesting the mutual independence of the endolymphatic potential and sodium and potassium concentration gradients.     

Metabolism of testosterone in human,rat and rabbit fetal lungs and in rabbit neonatal lung in vitro

• 1.1. Metabolism of 4-¹⁴C-testosterone was investigated in human, rat and rabbit fetal lung subcellular fractions and also in rabbit neonatal lungs. Androst-4-ene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and both 5α- and 5β-androstane-3α,17β-diols were identified as metabolites of testosterone.
• 2.2. The microsomal fraction produced mainly 5α-reduced epimers while the cytosol incubations resulted in 5β-reduced metabolites.
• 3.3. No conjugation was found.
• 4.4. The amounts of polar metabolites in the microsomal incubations and the amounts of dihydroxy-lated metabolites in the soluble fraction incubations were statistically significantly greater in the neonatal rabbit lung incubations compared with those of fetal lungs.