Heather A. Barr

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.

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.

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.

INTRACELLULAR STORAGE OF SURFACTANT AND PROINFLAMMATORY CYTOKINES IN CO-CULTURED ALVEOLAR EPITHELIUM AND MACROPHAGES IN RESPONSE TO INCREASING CO2 AND CYCLIC CELL STRETCH

Cell stretch stimulates both surfactant and cytokine production. The authors proposed that stretch, through these effects, modifies the pathogenesis of lipopolysaccharide-induced acute lung injury (ALI), and that this is CO2 dependent. Rat alveolar type II cells and macrophages were co-cultured with lipopolysaccharide in 5%, 10%, or 20% CO2 ± stretch (30%, 60 cycles/min) for 6 hours. Intracellular TNF-α and IL-6 increased whereas secreted cytokine and surfactant decreased with increasing CO2. Stretch independently increased intracellular TNF-α and decreased IL-6 secretion. Elevated CO2 may therefore diminish secretion of proinflammatory cytokines by alveolar cells, contributing to an explanation for protective hypercapnia in ALI.

Turnover of cholesterol and dipalmitoyl phosphatidylcholine in surfactant of adult rat lung.

In order to compare the turnover of two major surfactant components, [1α,2α(n)-3H]cholesterol and [methyl14C choline] dipalmitoyl phosphatidylcholine (DPPC) were introduced as lamellar bodiesvia the trachea into lightly anesthetized rats which were then allowed to recover. The radiotracers were assumed to have entered the alveolar surfactant pool and to have subsequently recycled in part into the lamellar bodies of alveolar type II cells. For DPPC, the specific activityvs. time curves of tubular myelin rich (alv-1) and tubular myelin poor (alv-2) alveolar lavage fractions were similar, and there was a plausible precursor-product relationship between lamellar bodies and either (or both) of these compartments. In contrast, however, the specific activities of alv-1 and alv-2 for cholesterol were quite different, allowing us to reject the hypothesis of a precursor-product relationship between classical lamellar bodies and alv-2. The estimated turover time for DPPC in alv-1 was 240 or 206 min, depending on which subfraction of lamellar bodies one takes to be the precursor. For cholesterol it was 583 or 624 min. These longer turnover times for cholesterol should lead to a greater than twofold increase in the relative concentration of cholesterol in the putative product compartment. Such an increase was not found. We interpret this as reflecting either noncompartmental behavior of the alveolar surfactant pool, or multiple pools of lamellar bodies with different turnover times. We conclude that two major components of pulmonary surfactant, cholesterol and DPPC, are handled differently, and that for at least one of these substances, the widely accepted scenario of a compartmental precursor-product relationship between lamellar bodies and alveolar surfactant must be rejected.

Composition of alveolar surfactant changes with training in humans.

We test the hypothesis that the changes we observed previously in the relative amounts of disaturated phospholipids (DSP), cholesterol (CHOL), and surfactant protein‐A (SP‐A) in human alveolar surfactant in response to acute exercise, and which were related to fitness, can be induced by training.

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO2/13%O2/82% N2 for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-14C] choline chloride into phosphocholine, [methyl-14C]phosphocholine into CDPcholine, and [14C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.

Control of alveolar surfactant in rats at rest and during prolonged hyperpnoea: pharmacological evidence for two tissue pools of surfactant.

1 Propranolol, atropine and indomethacin (i.p.) affect neither the amount (PLalv), nor the specific activity (PLalvsp.act.) of alveolar surfactant‐type phospholipids lavaged from the lungs of unanaesthetized rats, either at rest or made hyperpnoeic for 24 h with 5%CO2/13%O2/82%N2. 2 Whereas salbutamol (280 μg kg−1 body weight, i.p.) consistently increased PLalv and PLalvsp.act., pilocarpine (1.5, 3, 10 and 50 mg kg−1, i.p.) and labetalol (1 and 5 mg kg−1, i.p.) had no effect. The dose of pilocarpine reported by others to release surfactant (150 mg kg−1) induced marked salivation, diarrhoea, chromodacryorrhoea and a three‐fold increase in tidal volume. 3 In the isolated perfused lung of the rat, salbutamol (1.5 μm) consistently increased PLalvsp.act, whereas pilocarpine (0.1 and 1 μM) had no effect on these variables. 4 In the isolated perfused lung, the maximum amount of surfactant that could be released by salbutamol (0.5 mM) was smaller than that which could be released in response to an increase in tidal volume (peak inflation pressure 28 cmH2O). 5 When the concentration of salbutamol in the isolated perfused lung was adjusted to produce the same increase in PLalv as did a single simulated deep breath, the PLalvsp.act was significantly increased by salbutamol, but not by the simulated deep breath. 6 We conclude, that neither the autonomic nervous system nor the prostaglandin system is essential for the release of surfactant at rest or during hyperpnoea. Furthermore, we suggest that two pools of surfactant, with different release mechanisms, exist in lung tissue.

Determination of time constants in kinetic studies involving radiolabeled tracer molecules

In tracer kinetic studies, two homogeneous well-mixed compartments may be related as precursor and product. The time constant for the product compartment can then be estimated from the two specific activity vs. time curves. We examine ten methods of estimating this time constant for data with several different error patterns, and conclude that the present practice of transforming the data to a straight line yields an inefficient but unbiased estimate of the time constant. A least squares estimate, appropriately weighted and taking into account that both precursor and product specific activities are subject to error is expected theoretically and found in practice, to be most efficient. It is however, mathematically complicated. A modified approach using a minimum chi 2 estimate in which precursor specific activities are treated as error-free, is almost as efficient. It is mathematically simple, and outperforms the currently used estimators.