Localization and accumulation of fibronectin in rabbit fetal lung tissue

An interaction between mesenchyme and epithelium is required for the normal differentiation of fetal lung tissue. This morphogenic interaction may be mediated, in part, by changes in the composition and/or structure of the extracellular matrix. Therefore, we characterized the localization and accumulation of fibronectin, an extracellular-matrix component, during several stages of lung development in the rabbit fetus in vivo as well as in day-21 rabbit fetal lung explants maintained in vitro. Fibronectin was detected immunocytochemically in the basement-membrane zone beneath the epithelial ducts in lung tissue obtained from rabbit fetuses at 19 and 21 days of gestation. In fetal lung tissue obtained at these early stages of lung development, mesenchymal cells were stained only at their periphery. Immunostaining for connective-tissue fibronectin increased greatly between days 24 and 31 of gestation. A similar increase in the intensity of immunostaining for connective-tissue fibronectin was observed in rabbit fetal lung explants that had been maintained in vitro for 7 days. The concentration of fibronectin in fetal lung tissue obtained at different days of gestation was determined using a specific enzyme-linked immunoadsorbent assay (ELISA) and was found to increase from 1.7 ng/micrograms protein in fetal lung tissue obtained at day 19 of gestation to 7.3 ng/micrograms protein in fetal lung tissue obtained at day 24 of gestation. The levels of fetal lung fibronectin then remained relatively constant through to day 31 of gestation. A similar increase in fibronectin concentration was observed in day-21 fetal lung explants maintained in vitro for 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysophosphatidylcholine uptake and metabolism in the adult rabbit lung

Tracer quantities of 3H-labeled lysoPC and 32P-labeled natural rabbit surfactant were given intratracheally via a bronchoscope and [14C]palmitate was given intravenously to 25 rabbits with labeled PC and lysoPC measured in the alveolar wash, lung homogenate, lamellar bodies and microsomes at five times from 10 min to 6 h after tracheal injection. Surprisingly, only 31% of the administered lysoPC remained in its original form in the total lungs (alveolar wash + lung homogenate) by 10 min, of which 77% was in the alveolar wash. Meanwhile, by 10 min an additional 37% was already converted to PC, of which more than 98% was in the lung homogenate. LysoPC continued to be rapidly and efficiently converted to PC, with 62% conversion measured at 3 h. The converted lysoPC initially appeared with high specific activity in microsomes, then in lamellar bodies, and finally in the alveolar wash. The intravascular palmitate labeled lung PC had similar specific activity-time profiles in the subcellular fractions, while intratracheally administered natural rabbit surfactant had a constantly low specific activity in microsomes and much higher specific activities in lamellar bodies and alveolar wash. Another 25 rabbits received intratracheal lysoPC labeled in both the choline and palmitate moieties and then were studied from 1 to 24 h after tracheal injection. The ratio of the palmitate to choline labels indicated uptake and conversion to PC primarily by direct acylation rather than transacylation and by intact reuptake and conversion rather than breakdown and resynthesis. LysoPC is an attractive 'metabolic probe' of surfactant metabolism which undergoes very rapid and efficient intracellular conversion to PC via a subcellular pathway that parallels the remodeling and de novo synthetic pathways.     

Uptake,efflux and metabolism of the polyamine putrescine in rabbit lung slices

The herbicide paraquat is a selective pulmonary toxin in many mammals, including man, and its pulmonary toxicity has been attributed to selective uptake by a polyamine transport system in lung. In the present study, we investigated the characteristics of this transport process in rabbit lung slices. [¹⁴C]Putrescine was accumulated by both saturable and non-saturable processes and the accumulated putrescine was non-effluxable over 60 min. The saturable component was inhibited by spermine and paraquat. Moreover, uptake studies in Na⁺-deficient medium indicated that the lack of Na⁺ may selectively enhance uptake via the non-saturable process. The two components also differed in the metabolic fate of accumulated substrate. At 0.6 μM putrescine, where the saturable process predominated, 98% of the ¹⁴C in the perchloric acid-soluble fraction of tissue homogenates was present as putrescine, whilst 3% of the accumulated substrate was found in the acid-insoluble fraction. With 500 μM putrescine, where the non-saturable process predominated, 82% of the ¹⁴C in the acid-soluble fraction was present as putrescine and 15% of accumulated putrescine was found in the acid-insoluble fraction. The acid-insoluble ¹⁴C was localised mainly in the 700 g and 4500 g pellets obtained after homogenising the tissue. We conclude that there are two components to putrescine uptake in rabbit lung slices, both of an apparently irreversible nature. We suggest that the components represent compartmentalisation of putrescine in selective pulmonary cell-types or separate subcellular organelles. The observed metabolism and covalent binding of putrescine appeared to be associated with the non-saturable component only.     

Glucocorticoid receptors in lung. Mechanism of specific glucocorticoid uptake by fetal rabbit lung nuclei.

After exposure of fetal rabbit lungs to glucocorticoid in vivo or in vitro, the hormone binds to specific receptors localized in the cytoplasm and in the nuclei. The present studies are compatible with a mechanism by which the nuclear receptor originates from the cytoplasm and arises from a hormone-, temperature-, and ionic strength-dependent transfer of the cytoplasmic receptor into the nucleus. This conclusion is reached from the following observations. Specific binding of glucocorticoid to nuclei from lungs not previously exposed to the hormone is not observed unless the cytosol is also present. In the presence of cytosol, nuclear uptake of the hormone is very slow at 0 degrees but is highly enhanced with increasing temperature. Concomitantly with the increased nuclear uptake there is an equiivalent loss of glucocortoid-receptor complex from the cytosol, indicating that the complex is transferred to the nuclei by a temperature-dependent process. Although the nuclei do not bind the cytoplasmic complex at 0 degrees, they do so provided that the cytosol is briefly heated in the presence of hormone prior to mixing with the nuclei. Thus the cytoplasmic complex must first be activated before it can bind to nuclei..     

Mechanism of polyamine spermidine uptake by Xenopus laevis oocytes

In this study, the mechanisms of polyamine spermidine (Spd) uptake were investigated in Xenopus laevis oocytes. Spd uptake followed a sigmoidal kinetics with [S]90/[S]10 = 3 microM and Hill interaction coefficient (n) = 2. The order of magnitude of uptake and efflux was similar (t1/2 = 45 min). The equilibrium potential for Spd, calculated by Nenrst equation, was 90.78 mV. Free energy change for the uptake (delta G) was found to be 2.31 Kcal/mole of Spd. During efflux, Spd was not converted into putrescine or spermine. It seems that there are two types of Spd uptake pathways: Na(+)-dependent and Na(+)-independent since replacement of Na+ from incubation medium did not completely abolish the Spd uptake. The Na(+)-dependent component of Spd uptake was shared neither by system A nor by system ASC amino acids.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

Localization of an uptake hydrogenase in anabaena

Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H₂ uptake was detected (0.60-1.44 μmoles/[mg of chlorophyll a per hour]) in all three strains when grown with N₂ as the sole source of nitrogen. H₂ uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH₄⁺ and lacking heterocysts. H₂ uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N₂/CO₂, vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase.     

Use of liposomes in probing the uptake of liposomal phosphatidylcholine by rabbit lung in vitro.

The purpose of this study was to characterize the uptake of liposomal phosphatidylcholine by lung tissue and its subcellular organelles. Multilamellar liposomes were prepared from egg yolk phosphatidylcholine, dicetyl phosphate, and cholesterol (molar ratio 7 : 2 : 1). Liposomal phosphatidylcholine labeled with [1-14C]dipalmitoyl phosphatidylcholine was taken up by lung slices and incorporated into subcellular organelles including lamellar bodies, mitochondria, and microsomes. In addition, when liposomes were incubated with lamellar bodies, mitochondria, or microsomes, the transfer of liposomal phosphatidylcholine to these subcellular fractions was facilitated by the cytosolic fraction. In tissue slice experiments after 1 h of incubation, about 86% of the total radioactivity absorbed by lung slices and subcellular organelles was recovered in phosphatidylcholine. The ratio of the radioactivity of fatty acids at 1- and 2-positions of dipalmitoyl phosphatidylcholine recovered from all fractions was nearly 1 : 1. This suggests that most phosphatidylcholine molecules were taken up intact. In conclusion, this study provides a method using liposomes as a tool for probing the phosphatidylcholine transfer mechanism in lung.     

Swelling and ion uptake in cat cerebrocortical slices:

Cat cerebrocortical slices incubating in medium containing normal K+ concentrations were exposed to a number of different transmitters. Norepinephrine, histamine and adenosine or 2-chloroadenosine caused increased swelling of the slices associated with an increased Na+ and Cl- content. These effects were seen only when both Cl- and HCO3- were present in the medium, and were inhibited by a number of anion transport inhibitors. These characteristics were identical to those of the HCO3(-)-dependent component of the swelling induced by high K+ levels in the medium. Other transmitters, namely 5-hydroxytryptamine, dopamine, and gamma-amino butyric acid, were ineffective. The effects of norepinephrine, histamine and 2-chloroadenosine were antagonised by propranolol and phentolamine, chlorpheniramine and diphenhydramine, and theophylline respectively. These antagonists also inhibited HCO3(-)-dependent, K+-stimulated swelling. The transmitters which induced swelling also stimulated the carbonic anhydrase activity of cerebrocortical slices. We conclude from these data that the HCO3(-)-dependent component of K+-stimulated swelling may be due to K+-stimulated release of transmitters. Furthermore, the fact that the transmitters which induce swelling have also been reported to be most effective in increasing cAMP content in both brain slices or cultured astrocytes is consistent with the swelling response being mediated via cAMP-induced changes and being predominantly localized to astrocytes.     

Substrate specificity of uptake of diamines in mouse brain slices

Brain slices upon incubation accumulate diamines (cadaverine and putrescine) from the medium against a concentration gradient up to an intracellular-to-medium ratio of 8. The transport system is different from the various systems for amino acids, among which is the transport system for basic amino acids. Diamine uptake, in contrast to amino acid uptake, is independent of Na⁺ and is increased at higher pH. There is some overlap among these transport classes—basic amino acids have a low affinity to the diamine system and some heteroexchange (stimulation of uptake) can be observed at very high concentrations between diamines and some amino acids (glycine, β alanine, γ-aminobutyrate, possibly also with proline and taurine). The diamine system seems also to be separate from the monoamine uptake systems. The results indicate the presence of numerous systems for metabolite transport in the brain with some overlap between systems.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.