Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

Hormonal regulation of beta-adrenergic receptors in fetal rabbit lung in organ culture

Explants of fetal rabbit lung were established on the 25th day of gestation. These were maintained in serum-free medium for periods up to 10 days. During this time, the cultures exhibited morphological changes typical of terminal lung differentiation. Morphological evidence was also obtained for synthesis and secretion of pulmonary surfactant in these explants. beta-Adrenergic receptors were identified in these lung explants. Exposure of the explants to 10(-7)M dexamethasone on the third day of culture resulted in a significant increase in the number of beta-adrenergic receptors in the tissue without a change in receptor affinity. The effect of dexamethasone in organ culture was dose-dependent, a maximum increase in receptor number being observed within 48 hours of incubation with a hormone concentration of 1 x 10(-7)M. Exposure of the explant tissue to 1 x 10(-7)M triiodothyronine resulted in no significant increase in the concentration of beta-adrenergic receptors and no change in receptor affinity. These results suggest that glucocorticoids may potentiate the effects of beta-adrenergic agents in the fetal lung by increasing the numbers of their receptors. The effects of triiodothyronine upon the fetal lung do not appear to be mediated by this mechanism.     

Dissociation of beta-adrenergic stimulation of renin secretion and prostacyclin synthesis in the rabbit kidney

The effect of inhibition of prostaglandin (PG) synthesis with indomethacin on basal and isoproterenol-stimulated renin secretion was examined in the isolated perfused rabbit kidney. 6-keto PGF1 alpha' the stable metabolite of prostacyclin, was measured in urine by radioimmunoassay using 125I labelled histamine coupled to 6-keto PGF1 alpha as ligand. The level in urine, prior to isolation and perfusion of the kidney, was 10.7 +/- 5.6 ng/min, and this was reduced to 0.32 +/- 0.25 ng/min (P less than 0.05) in rabbits treated with 2.0 mg/kg of indomethacin. Renin release was markedly stimulated by intrarenal infusion of isoproterenol (0.1 microgram/min) but urinary 6-keto PGF1 alpha did not change. These responses were not affected by indomethacin treatment. Renal perfusion pressure, perfusate flow rate and consequently renal vascular resistance, remained relatively constant during the course of perfusion and were unaltered by indomethacin treatment. These results therefore do not support a role for PGs, and in particular prostacyclin, in the renin response to beta-adrenergic stimulation with isoproterenol.     

Inhibition by colchicine of phospholipid secretion induced by lung distension

The effect of colchicine, a microtubule disruptor, on phospholipid secretion stimulated by distension of fetal rabbit lungs was investigated. After colchicine injection and breathing for 45 min, pups were killed and their lungs were lavaged with colchicine. Controls were injected and lavaged with saline. All lungs were given static air inflation and a final lavage, and the returns were analyzed for phospholipid DNA, and lactate dehydrogenase. The first lavage after breathing yielded 33% less phospholipid with colchicine, 3.83 compared with 5.72 mg/g dry lung wt (P less than 0.05). The postinflation phospholipid yield was also significantly reduced with colchicine from 1.04 to 0.70 mg/g dry lung wt (P less than 0.05). The postinflation DNA was significantly reduced with colchicine, from 1.26 to 0.44 micrograms (P less than 0.01), suggesting reduced alveolar macrophages. Colchicine did not change the recovery by lavage of exogenous radioactive phospholipid. As reflected by ATP and lactate levels, tissue metabolism was well maintained. The results are interpreted to mean that colchicine reduced simultaneously lavage-associated phospholipid secretion, inflation-produced phospholipid secretion, and macrophage migration.     

Surfactant secretion in a newborn rabbit lung slice model

We describe a slice model for the study of pulmonary surfactant secretion in newborn rabbits. Full term rabbits were delivered by cesarean section and injected intraperitoneally with [Me-3H]choline. Four hours later they were killed, the lungs were perfused to remove blood, slices (0.5 mm thick) were prepared and incubated in buffer at 37 degrees C. The composition of the lipids initially released into the medium resembled those of lung tissue rather than surfactant. Following 3 changes of medium, however, the composition of the lipids released was very similar to that of lung lavage. Phosphatidylcholine accounted for over 70% of the total while phosphatidylethanolamine and sphingomyelin accounted for only 7% and 4%, respectively. 52% of the phosphatidylcholine was disaturated. Less than 5% of the tissue lactate dehydrogenase was released into the medium. The rate of phosphatidyl[Me-3H]choline release during this period was, therefore, measured. Release of phosphatidyl[Me-3H]choline was linear with time and was temperature-dependent. Prostaglandin E2 stimulated its rate of release by 20% while indomethacin and flufenamic acid, inhibitors of prostaglandin synthesis, inhibited it by 52% and 37%, respectively. The calcium ionophore A23187 in the presence of Ca2+ stimulated release by 40% while colchicine an cytochalasin B inhibited it by 36% and 32%, respectively. These data suggest that both prostaglandins and Ca2+ are involved in surfactant release and that intact microtubular and microfilament systems may also be necessary.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Effects of betamethasone on phospholipid content,composition and biosynthesis in the fetal rabbit lung

Administration of betamethasone (0.2 mg/kg, intramuscularly) to pregnant rabbits had the following effects on the fetal lung at 26–27 days gestation. It increased the amount of phosphatidylcholine in lung lavage by 70% and almost doubled the phosphatidylcholine/sphingomyelin ratio, it increased the rate of incorporation of choline into phosphatidylcholine in fetal lung slices by up to 90%, it increased the activities of pulmonary cholinephosphate cytidylyltransferase and phosphatidate phosphatase by 50% and it reduced the amount of lung glycogen to 60% of the amount in the controls. Betamethasone had no effect on the activities of pulmonary cholinephosphotransferase or lysolecithin: lysolecithin acyltransferase but it sligthly decreased the activity of choline kinase.Betamethasone administration to the doe did not increase the amount of surfactant phospholipid in fetal lung lavage to as great an extent as did direct administration of cortisol to the fetuses. Neither did betamethasone stimulate the activity of pulmonary cholinephosphotransferase. These data suggest that agents other than glucocorticoids mediate the stress-induced acceleration of fetal lung maturation and surfactant production.     

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

Parasympathetic control of secretion of pancreatic juice in the rabbit (author's transl)]

The secretion of pancreatic juice in response to stimulation of the vagus nerves in the neck, and to injection of parasympathomimetic drugs, was studied in anesthetized rabbits. Vagal stimulation caused a marked increase of protein content in pancreatic juice, and also a small increase in flow of juice, highly variable from one amimal to another, preceded by a short period of inhibition. Atropine supressed vagal effect on protein concentration, but failed to abolish the flow response. Injection of parasympathomimetic drugs closely imitated the results of vagal stimulation, with the difference that secretory action was fully abolished by atropine. The possibility that effects the parasympathetic stimulation on the flow of juice were secondary to vasodilatation, was discussed. The obtained data were compared with those described for other mammalian species, and the conclusion was reached that rabbit behaves like carnivora rather than herbivora.     

Adenosine and leukotrienes have a regulatory role in lung surfactant secretion in the newborn rabbit

Previous studies have shown that secretion of phosphatidylcholine in cultured adult rat type II pneumocytes is stimulated by purinoceptor agonists and leukotrienes. The objective of the present study was to determine if such agents have a physiological role in the regulation of surfactant secretion. We chose the newborn rabbit as the experimental model, since in this system there is a marked increase in surfactant secretion immediately after birth. We examined the effects of an inhibitor of leukotriene biosynthesis, nordihydroguaiaretic acid, two leukotriene antagonists, FPL-55712 and FPL-57231, and a P1 purinoceptor antagonist, 8-phenyltheophylline, on this increase. Newborn rabbits were delivered by Cesarean section at 30 days gestation. Some animals in each litter were killed immediately, while others were injected with test agents or solvent vehicle while still in the amniotic sacs. After breathing for 3 h in an incubator, these animals were also killed. The lungs were lavaged with saline and the phospholipid content and composition of the lung lavage liquid was measured. In control animals, there was a greater than 2-fold increase in the amounts of total phospholipid and phosphatidylcholine and in the phosphatidylcholine/sphingomyelin ratio during the 3 h period of breathing. The increases in total phospholipid and phosphatidylcholine were decreased 38-62% by the antagonists, while the increase in the phosphatidylcholine/sphingomyelin ratio was decreased 61-77%. These data show that the ventilation-induced increase in secretion of lung surfactant in the newborn rabbit is inhibited by leukotriene and P1 receptor antagonists and by an inhibitor of leukotriene biosynthesis and, when taken together with the data from the tissue culture system, support a role for leukotrienes and adenosine in the physiological regulation of surfactant secretion.     

Electrostatic control of phospholipid polymorphism

A regular progression of polymorphic phase behavior was observed for mixtures of the anionic phospholipid, cardiolipin, and the cationic phospholipid derivative, 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine. As revealed by freeze-fracture electron microscopy and small-angle x-ray diffraction, whereas the two lipids separately assume only lamellar phases, their mixtures exhibit a symmetrical (depending on charge ratio and not polarity) sequence of nonlamellar phases. The inverted hexagonal phase, H(II,) formed from equimolar mixtures of the two lipids, i.e., at net charge neutrality (charge ratio (CR((+/-))) = 1:1). When one type of lipid was in significant excess (CR((+/-)) = 2:1 or CR((+/-)) = 1:2), a bicontinuous cubic structure was observed. These cubic phases were very similar to those sometimes present in cellular organelles that contain cardiolipin. Increasing the excess of cationic or anionic charge to CR((+/-)) = 4:1 or CR((+/-)) = 1:4 led to the appearance of membrane bilayers with numerous interlamellar contacts, i.e., sponge structures. It is evident that interactions between cationic and anionic moieties can influence the packing of polar heads and hence control polymorphic phase transitions. The facile isothermal, polymorphic interconversion of these lipids may have important biological and technical implications.     

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Characterization of the beta-adrenergic receptor mediating secretion of parathyroid hormone

In vitro incubation studies with bovine parathyroid gland slices compared the relative responsiveness of parathyroid hormone (PTH) secretion to isoprotherenol, epinephrine or norepinephrine. Isoproterenol was the most potent and norepinephrine the least potent of the three stimuli, suggesting a beta 2 type of an adrenergic response. However in this in vitro system, tazalol, a selective beta 1 adrenergic agonist significantly stimulated PTH secretion, whereas terbutaline, a selective beta 2 agonist had no effect. In addition, practolol, a selective beta 1 adrenergic antagonist blocked isoproterenol- or tazolol-stimulated PTH secretion. In vivo studies in normal human subjects showed that injection of te nonselective beta agonist, isoproterenol, (0.15 mg s.c.) significantly increased, whereas injection of the selective beta 2 agonist, terbulatine (0.3 mg s.c.) had no effect on serum PTH levels. These latter studies with putative selective beta adrenergic agents suggest that the beta adrenergic receptor mediating PTH secretion is of the beta 1 type (in contrast to the studies above with nonselective agents). The studies suggest that the beta adrenergic receptor mediating PTH secretion apparently differs from the classical beta 1 receptor described in th myocardium or the classical beta 2 receptor described in the bronchial smooth muscle.