Terence E. Nicholas

The pulmonary consequences of a deep breath

We used the isolated rat lung perfused with Krebs bicarbonate and 4.5% albumin, to examine the effect of a transient increase in peak inspired pressure (PIP). The lung was ventilated with 5% CO2 in O2 at a Vr of 2.5 ml, an f of 60 min-1 and an end expired pressure of 2 cm H2O. After 30 min we increased the PIP from 9 to 18 cm H2O for one breath; following a further 30 sec of normal ventilation we lavaged the lung. The large breath increased the amount of alveolar surfactant phospholipids (PLalv) (control: 7.0 +/- 0.73 (11); large breath: 8.3 +/- 1.33 (14), mean +/- SD in mg . g dry lung-1), and decreased the percentage of PLalv associated with tubular myelin (control: 30.2 +/- 3.49% (9); deep breath: 25.4 +/- 2.99% (9). In rats that had received 20 microCi . kg-1 of [methyl-3H]choline chloride 3 h previously, there was also an increase in the tritium in PLalv expressed as a percent of that in tissue (control: 4.4 +/- 0.77% (5); deep breath: 5.7 +/- 1.0% (7). The deep breath also resulted in an increase in oxygen diffusing capacity. We conclude, that a single deep breath results in the opening of atelectatic alveoli, the release of pulmonary surfactant and possibly also the transfer of PLalv from the tubular myelin to the monomolecular phase.

Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells.

We examined the effects of short‐term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol‐induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Body Temperature Alters the Lipid Composition of Pulmonary Surfactant in the Lizard Ctenophorus nuchalis

In any 24-h period the body temperature (Tb) of the central Australian agamid lizard, Ctenophorus nuchalis, may vary from 13 to 45 degrees C; the mean preferred Tb is 37 degrees C. We have analyzed surfactant-type lipids in lizards that underwent rapid changes in Tb from 37 degrees C to 14, 19, 27, or 44 degrees C. Lipids were extracted from lung lavage and lamellar body fractions, and phospholipids and cholesterol components were measured. There was no change in either the total amount or relative proportions of the different classes of phospholipids, but cooling increased the cholesterol content of lavage. An increase in the cholesterol: phospholipid ratio was evident within 2 h of cooling to 19 degrees C and was maintained for at least 48 h. The ratio increased from 8% at 37 degrees C, to 15% after 4 h at 19 degrees C, and 18% after 4 h at 14 degrees C. Possibly the increase in cholesterol promotes fluidity and absorption of surfactant within the alveoli of lizards with low Tb. Cold lizards collapse their lungs during prolonged periods of apnea and the surfactant may prevent the epithelial walls from adhering.

Partitioning lung and plasma proteins: circulating surfactant proteins as biomarkers of alveolocapillary permeability.

1. The alveolocapillary membrane faces an extraordinary task in partitioning the plasma and lung hypophase proteins, with a surface area approximately 50‐fold that of the body and only 0.1–0.2 μm thick.

Pulmonary-type surfactants in the lungs of terrestrial and aquatic amphibians.

We examined the composition and function of pulmonary surfactants in amphibians inhabiting aquatic and terrestrial habitats with particular regard to the influences of (1) variations in body temperature, (2) external hydrostatic pressure and (3) breathing pattern. Two fully aquatic salamanders, and the completely terrestrial cane toad Bufo marinus (all maintained at 21-23 degrees C) were selected. Whereas one of the salamanders (Siren intermedia) possessed gills and lungs, Amphiuma tridactylum only possessed lungs. We determined the amounts of cholesterol (Chol), disaturated phospholipids (DSP) and total phospholipid (PL) in lavage of all three species, and also determined the types of phospholipids of B. marinus and A. tridactylum. DSP lowers surface tension at the air-water interface in the lung, while Chol and unsaturated phospholipids assist spreading and maintain the DSP in its disordered, liquid-crystalline state at high lung volumes. All three species had significant amounts of pulmonary-type surfactant. The two aquatic salamanders had identical ratios of both Chol/PL and DSP/PL both of which in turn were nearly twice those of B. marinus. All three species had similar Chol/DSP ratios. Aquatic salamanders sustain high external hydrostatic pressures exerted by the aquatic environment and tend to collapse their lungs during expiration. We hypothesize that these salamanders might require a DSP-rich surfactant to prevent the epithelial surfaces from adhering and large amounts of Chol to keep the DSP fluid. The terrestrial B. marinus has less DSP, suggesting a surfactant which is fluid over a large range of temperatures. Possibly, cane toads do not require a DSP rich surfactant as they neither collapse their lungs on deflation, nor experience external hydrostatic pressures promoting lung collapse. The PL profile of B. marinus lavage was similar to that of other frogs and mammals, containing phosphatidylcholine (PC) as the predominant phospholipid together with substantial amounts of phosphatidylglycerol (PG). On the other hand, although A. tridactylum exhibited high levels of PC, it contained phosphatidylinositol (PI) in place of PG, a pattern typical of reptiles and birds.

Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells

The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12-O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (alpha, betaII, delta, and eta) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12- O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (α, βII, δ, and η) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.

IMMUNOHISTOCHEMICAL LOCALIZATION AND CHARACTERIZATION OF A RAT CLARA CELL 26-kDa PROTEIN (CC26) WITH SIMILARITIES TO GLUTATHIONE PEROXIDASE AND PHOSPHOLIPASE A2

We have purified and partially sequenced a 26-kDa protein isolated from rat lung lavage. Two-dimensional electrophoresis and Western blotting using an antibody we have raised to this protein indicated that CC26 has 3 isoforms with pIs between 4.9 and 5.5 and is neither a component of surfactant nor present in plasma. The first 10 amino acids of the N-terminal of all 3 isoforms were identical. The first 25 amino acids of the N-terminal sequence were identical to a rat acidic calcium-independent phospholipase A2 and one amino acid different from a mouse nonselenium glutathione peroxidase. Light immunohistochemistry showed a strong reaction with the airway hypophase from the trachea down to the terminal bronchioles, but not in the alveolus. Immunohistochemistry at the electron microscopy level showed that CC26 was localized to the dense secretory bodies and endoplasmic reticulum of the Clara cell and secretory granules of tracheal nonciliated serous and goblet cells. Heavily labeled Clara cell dense secretory bodies were observed in the process of being exocytosed. Biochemical and further sequence analysis will be required to determine if this protein is either a nonselenium glutathione peroxidase or a calcium-independent phospholipase A2.

Analysis of Pulmonary Phospholipid Compartments in the Unanesthetized Rat During Prolonged Periods of Hyperpnea

We exposed rats to 4% CO2-10% O2-86% N2 for 24 h before infusing with 20 microCi kg-1 (methyl-3H)choline chloride. They were then exposed for periods up to 48 h, at which times their lungs were degassed and lavaged. The lavage fluid was divided into a tubular myelin-rich (PLalv-1) and a tubular myelin-poor (PLalv-2) fraction. Lamellar body (lb) and microsomal (m) fractions were prepared from the lung tissue and the amount of phospholipid (PL) was determined in each of the 4 fractions. Specific activity (sp.act.) curves were constructed for both control and hyperpneic groups. Exposure to the gas doubled both tidal volume and frequency of breathing. Total PLalv, PLlb and PLm were all markedly elevated, which, when taken in conjunction with the changes in sp.act. in these fractions, suggests that the rate of surfactant PL synthesis was increased within 24 h. The shapes of the sp.act. curves suggest precursor-product relationships between PLlb and PLalv-1 and between PLalv-1 and PLalv-2. However, when we applied analysis based on the Zilversmit steady-state equation, instead of the expected straight line, we found a marked clockwise hysteresis that did not return to the origin. Whereas this may reflect PLalv being supplied from 2 tissue pools, we argue that, in fact, we are not dealing with classic compartmental precursor-product relationships.

The physiological significance of 11β-hydroxysteroid dehydrogenase in the rat lung

We have examined the interconversion of cortisone (E) and cortisol (F) in rat lung homogenate and microsomal fraction and in the isolated rat lung perfused with Krebs bicarbonate solution containing 4.5% albumin. In the perfused lung the apparent Km was 5.1 microM E and the Vmax was 9 nmol . g(-1) . min-1. The ability of the lung to reduce E to F was enhanced both by 7 days prior exposure of the rat to an ambient temperature of 2 degrees C and by starvation of the rat for 3 days. The activity was inhibited by adrenalectomy and castration of 7 days duration. Whereas little steroid oxidation occurred in the perfused lung, preparations of lung homogenates and microsomal fraction readily reduced or oxidised the 11-position of the corticoid molecule depending on the preponderance of either NADPH or NADP, respectively. We conclude, that the predominance of the reductive reaction in the whole rat lung under physiological conditions reflects the very active pentose-phosphate shunt in the lung, which produces NADPH. We suggest that this ability of the lung to activate E to F may exert a fine control over the arterial concentration of unbound, physiologically active, 11-hydroxylated steroid.

A comparison of the surfactant associated lipids derived from reptilian and mammalian lungs

The lungs of the central netted dragon Ctenophorus nuchalis are bag-like, with most of the gas exchange region located in the anterior third. Although the faveoli are much larger than the mammalian alveoli, the lizard at 37 degrees C has approximately 70 times more surfactant phospholipid per unit area of respiratory surface than does a similar sized mammal. However, when expressed as per wet lung weight, lizards and rats possessed similar amounts of phospholipids. Dipalmitoylphosphatidylcholine was the principal phospholipid in both species. However, major differences existed in the phospholipid, neutral lipid and fatty acid profiles. Whereas the lizard contained neither phosphatidylglycerol nor phosphatidylethanolamine it had more cholesterol, esterified cholesterol, acylglycerides and unsaturated fatty acids. Although the ratio of saturated:unsaturated fatty acids was similar in rats and lizards, palmitic acid predominated in the former. The composition of lizard surfactant suggests that it would adsorb rapidly at reduced body temperature.