Control of pulmonary surfactant secretion in adult California sea lions

Marine mammals have a spectacular suite of respiratory adaptations to deal with the extreme pressures associated with deep diving. In particular, maintaining a functional pulmonary surfactant system at depth is critical for marine mammals to ensure that inspiration is possible upon re-emergence. Pulmonary surfactant is secreted from alveolar type II (ATII) cells and is crucial for normal lung function. It is not known whether ATII cells have the ability to continue to secrete pulmonary surfactant under pressure, or how secretion is maintained and controlled. We show here that surfactant secretion in California sea lions (Zalophus californianus) was increased after high pressures (25 and 50 atm) of short duration (30 min), but was unaffected by high pressures of long duration (2 h). This is in contrast to a similar sized terrestrial mammal (sheep), where surfactant secretion was increased after high pressures of both long and short duration. Z. californianus and terrestrial mammals also show similar responses to stimulatory hormones and autonomic neurotransmitters. It therefore seems that an increase in the quantity of surfactant in seal lungs after diving is most likely caused by mechanostimulation induced by pressure and volume changes, and that seals are adapted to maintain constant levels of surfactant under long periods of high pressure.     

Positive Selection in the N-Terminal Extramembrane Domain of Lung Surfactant Protein C (SP-C) in Marine Mammals

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (ω) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Richard Kliman     

Surfactant from diving aquatic mammals.

Diving mammals that descend to depths of 50-70 m or greater fully collapse the gas exchanging portions of their lungs and then reexpand these areas with ascent. To investigate whether these animals may have evolved a uniquely developed surfactant system to facilitate repetitive alveolar collapse and expansion, we have analyzed surfactant in bronchoalveolar lavage fluid (BAL) obtained from nine pinnipeds and from pigs and humans. In contrast to BAL from terrestrial mammals, BAL from pinnipeds has a higher concentration of phospholipid and relatively more fluidic phosphatidylcholine molecular species, perhaps to facilitate rapid spreading during alveolar reexpansion. Normalized concentrations of hydrophobic surfactant proteins B and C were not significantly different among pinnipeds and terrestrial mammals by immunologic assay, but separation of proteins by gel electrophoresis indicated a greater content of surfactant protein B in elephant seal surfactant than in human surfactant. Remarkably, surfactant from the deepest diving pinnipeds produced moderately elevated in vitro minimum surface tension measurements, a finding not explained by the presence of protein or neutral lipid inhibitors. Further study of the composition and function of pinniped surfactants may contribute to the design of optimized therapeutic surfactants.     

Phosphatidylinositol and not phosphatidylglycerol is the important minor phospholipid in rhesus-monkey surfactant

Pulmonary surfactant was isolated from lung tissue and alveolar washes of lungs of adult rhesus monkeys (Macaca mulatta). The phospholipid composition was determined and compared to the composition of human surfactant fractions. Contrary to human surfactant, phosphatidylinositol is the major acidic phospholipid, whereas phosphatidylglycerol is only a minor component in rhesus-monkey surfactant. These differences are not caused by a difference in plasma myo-inositol concentrations between the two species.     

The fecal viral flora of California sea lions

California sea lions are one of the major marine mammal species along the Pacific coast of North America. Sea lions are susceptible to a wide variety of viruses, some of which can be transmitted to or from terrestrial mammals. Using an unbiased viral metagenomic approach, we surveyed the fecal virome in California sea lions of different ages and health statuses. Averages of 1.6 and 2.5 distinct mammalian viral species were shed by pups and juvenile sea lions, respectively. Previously undescribed mammalian viruses from four RNA virus families (Astroviridae, Picornaviridae, Caliciviridae, and Reoviridae) and one DNA virus family (Parvoviridae) were characterized. The first complete or partial genomes of sapeloviruses, sapoviruses, noroviruses, and bocavirus in marine mammals are reported. Astroviruses and bocaviruses showed the highest prevalence and abundance in California sea lion feces. The diversity of bacteriophages was higher in unweaned sea lion pups than in juveniles and animals in rehabilitation, where the phage community consisted largely of phages related to the family Microviridae. This study increases our understanding of the viral diversity in marine mammals, highlights the high rate of enteric viral infections in these highly social carnivores, and may be used as a baseline viral survey for comparison with samples from California sea lions during unexplained disease outbreaks.     

Eustachian tube surfactant is different from alveolar surfactant: determination of phospholipid composition of porcine eustachian tube lavage fluid.

Phosphatidylcholine (PC) is the main phospholipid in lung surfactant and, more specifically, dipalmitoyl PC (PC16:0/16:0) is the major surface-active component. Several studies have tentatively shown that eustachian tube lavage fluid (ETLF) contains surface-active material. The aim of the present study was to determine, using electrospray ionization mass spectrometry, whether the phospholipid molecular species composition of ETLF is similar to that of lung surfactant. PC was the main component of both ETLF and bronchoalveolar lavage fluid (BALF). The concentration of phosphatidylethanolamine was higher and phosphatidylglycerol was undetectable in ETLF compared with BALF. The molecular species composition of PC in ETLF was notably different from that of BALF, palmitoyloleoyl PC being the major component. Importantly, given its predominance in BALF PC, the concentration of PC16:0/16:0 was low in ETLF. As expected on the basis of this molecular species composition of PC, ETLF did not generate low surface tension values under dynamic compression in a pulsating bubble surfactometer.We conclude that the surfactant in ET is different from lung surfactant, and that low surface tension is not a major determinant of ETLF function.     

The composition and function of the pulmonary surfactant system during metamorphosis in the tiger salamander Ambystoma tigrinum

Mammalian lungs secrete a mixture of surface-active lipids (surfactant), which greatly reduces the surface tension of the fluid coating the inner lung surface, thereby reducing the risk of collapse upon deflation and increasing compliance upon inflation. During foetal lung maturation, these lipids become enriched in the primary and active ingredient, a disaturated phopholipid. However, disaturated phospholipids exist in their inactive gellike form at temperatures below 37°C and thus are inappropriate for controlling surface tension in the lungs of many ectotherms. We examined the development of the composition and function of the surfactant system of the tiger salamander (Ambystoma tigrinum) during metamorphosis from the fully aquatic larva (termed stage I) through an intermediate air-breathing larval form (stage IV) to the terrestrial adult (stage VII). Biochemical analysis of lung washings from these three life stages revealed a decrease in the percentage of disaturated phospholipid per total phospholipid (23.03 versus 15.92%) with lung maturity. The relative cholesterol content remained constant. The increased level of phospholipid saturation in the fully aquatic larvae may reflect their generally higher body temperature and the higher external hydrostatic compression forces exerted on the lungs, compared to the terrestrial adults. Opening pressure (pressure required for initial lung opening) prior to lavage decreased from larval to adult salamanders (7.96 versus 4.69 cm H₂O), indicating a decrease in resistance to opening with lung development. Opening pressure increased after lavage in older aquatic (stage IV) larvae (5.36 versus 9.80 cm H₂O) and in the adults (4.69 versus 7.65 cm H₂O), indicating that the surfactant system in salamanders may have an antiglue function which prevents apposing epithelial surfaces from adhering together.Abbreviations bm body mass - Chol cholesterol - DSP disaturated phospholipid - PC phosphatidylcholine - PL phospholipid - postlav postlavage - prelav prelavage - P-V pressure-volume - RH relative humidity - T_b body temperature - USP unsaturated phospholipid - WL wet lung mass     

The energetic consequences of behavioral variation in a marine carnivore

Intraspecific variability in foraging behavior has been documented across a range of taxonomic groups, yet the energetic consequences of this variation are not well understood for many species. Understanding the effect of behavioral variation on energy expenditure and acquisition is particularly crucial for mammalian carnivores because they have high energy requirements that place considerable pressure on prey populations. To determine the influence of behavior on energy expenditure and balance, we combined simultaneous measurements of at‐sea field metabolic rate (FMR) and foraging behavior in a marine carnivore that exhibits intraspecific behavioral variation, the California sea lion (Zalophus californianus). Sea lions exhibited variability in at‐sea FMR, with some individuals expending energy at a maximum of twice the rate of others. This variation was in part attributable to differences in diving behavior that may have been reflective of diet; however, this was only true for sea lions using a foraging strategy consisting of epipelagic (<200 m within the water column) and benthic dives. In contrast, sea lions that used a deep‐diving foraging strategy all had similar values of at‐sea FMR that were unrelated to diving behavior. Energy intake did not differ between foraging strategies and was unrelated to energy expenditure. Our findings suggest that energy expenditure in California sea lions may be influenced by interactions between diet and oxygen conservation strategies. There were no apparent energetic trade‐offs between foraging strategies, although there was preliminary evidence that foraging strategies may differ in their variability in energy balance. The energetic consequences of behavioral variation may influence the reproductive success of female sea lions and result in differential impacts of individuals on prey populations. These findings highlight the importance of quantifying the relationships between energy expenditure and foraging behavior in other carnivores for studies addressing fundamental and applied physiological and ecological questions.     

Age-related changes in hematocrit in the Galápagos sea lion (Zalophus wollebaeki) and the Weddell seal (Leptonychotes weddellii)

In mammals, hematocrit (Hct) is optimized between the divergent requirements of blood flow characteristics and oxygen transport and storage capacity. This trade‐off plays a particularly major role in marine mammals, in which oxygen demand during sustained diving requires high Hct levels. Galápagos sea lions (Zalophus wollebaeki) need a long time after birth to develop from terrestrial life to the state of an independent forager at sea. We here show that pups were born with high Hct of 45%, then reduced Hct during the first 40 d of life to 31% while they remain constantly ashore, and increased Hct again until the adult level (57%) is reached at 1 yr of age when they begin to dive for foraging at sea. A similar, but much more rapidly changing pattern is seen in Weddell seal pups, but not in northern elephant seals, where no reduction in Hct is seen after birth. These and similarly changing patterns in terrestrial mammals likely reflect species specific functional adjustments during development due to a trade‐off between the costs of circulation and the changing need to store and transport oxygen.     

Limb movements and locomotor function in the California sea lion (Zalophus californianus)

The peculiar amphibious mode of life of California sea lions suggests that their locomotor systems may contain adaptations both to life on land and in the water. Previous studies of their locomotor behaviour have been either superficial or based on inferences which were derived from limb structure. Limb movements associated with locomotor behaviour in California sea lions are described on the basis of frame-by-frame analysis of slow motion cinematography of typical aquatic and terrestrial locomotor sequences. Results are compared to reports of terrestrial and aquatic locomotor behaviour in fissiped carnivores, whose locomotor behaviour is presumed to reflect the framework from which the locomotor behaviour of sea lions was derived. The major distinction between sea lions and fissipeds in terms of aquatic locomotor behaviour involves the use of the forelimb in sea lions. Propulsive thrust is generated by medial rotation, adduction and retraction of the forelimbs in sea lions, in contrast to nearly pure limb retraction in fissipeds. The major features which distinguish terrestrial locomotor behaviour in sea lions from that of fissipeds are use of the manus as a transverse rather than sagittal propulsive lever and extensive use of posterior axial and head and neck movements rather than hindlimb movements. The biomechanical implications of these movements are used to elucidate their potentially adaptive features.     

Changes in quantity, composition, and surface activity of alveolar surfactant at birth

We hypothesized that when the lung makes the transition from the fluid- to the air-filled state at birth, there are changes in physical and functional properties of the alveolar surfactant. To test this hypothesis, newborn rabbits were killed at different times in the first 24 h of life, their lungs lavaged with ice-cold saline, and the lavage fluid subfractionated by differential centrifugation. The phospholipid and protein content and composition and the kinetics of surface tension lowering of the subfractions were examined. We found that with the onset of breathing, shifts occur in the distribution of surfactant subfractions as a surfactant apoprotein-free phospholipid fraction is generated. The ratio of rapidly sedimentable apoprotein-rich to slowly sedimentable, apoprotein-free fractions decreases from 31 at birth to 4 at 24 h of life. Concurrently, rates of surface tension lowering by the subfractions increase with time. The results suggest that the adult pattern of pool sizes and surface activity of alveolar surfactant is not present at birth but evolves slowly over the 1st day of life.     

Reversibility of lung inflammation caused by SP-B deficiency

Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1beta, and macrophage inflammatory protein-2 concentrations were increased after removal of doxycycline, indicating pulmonary inflammation. Restoration of SP-B expression following administration of doxycycline rapidly reversed SP-B-dependent abnormalities in lung mechanics and inflammation. SP-B deficiency is sufficient to cause lung dysfunction and inflammation in adult mice. SP-B reversed inflammation and maintained lung function in vivo, indicating its potential utility for the prevention and treatment of pulmonary injury and surfactant deficiency.     

Interannual variation in the at‐sea behavior of California sea lions (Zalophus californianus)

To be successful, marine predators must alter their foraging behavior in response to changes in their environment. To understand the impact and severity of environmental change on a population it is necessary to first describe typical foraging patterns and identify the underlying variability that exists in foraging behavior. Therefore, we characterized the at‐sea behavior of adult female California sea lions (n = 32) over three years (2003, 2004, and 2005) using satellite transmitters and time‐depth recorders and examined how foraging behavior varied among years. In all years, sea lions traveled on average 84.7 ± 11.1 km from the rookery during foraging trips that were 3.2 ± 0.3 d. Sea lions spent 42.7% ± 1.9% of their time at sea diving and displayed short (2.2 ± 0.2 min), shallow dives (58.5 ± 8.5 m). Among individuals, there was significant variation in both dive behavior and movement patterns, which was found in all years. Among years, differences were found in trip durations, distances traveled, and some dive variables (e.g., dive duration and bottom time) as sea lions faced moderate variability in their foraging habitat (increased sea‐surface temperatures, decreased upwelling, and potential decreased prey abundance). The flexibility we found in the foraging behavior of California sea lions may be a mechanism to cope with environmental variability among years and could be linked to the continuing growth of sea lion populations.     

Non-specificity of surfactant deficiency in neonatal respiratory disorders.

The phospholipid content of lung fluid taken from 77 babies during the first day of life was studied. Babies with hyaline membrane disease had low concentrations of the surfactant phospholipids phosphatidylcholine, phosphatidylinositol, and phosphatidylglycerol. The palmitic acid content in phosphatidylcholine was also lower than normal. Surfactant deficiency was not, however, specific for hyaline membrane disease, as similar phospholipid abnormalities were observed in babies with congenital pneumonia and transient tachypnoea of the newborn. These findings have important clinical implications. They are relevant to research into surfactant substitution and cast doubts on the value of the antenatal phospholipid lung profile of amniotic fluid in predicting the risk of hyaline membrane disease.     

The in vivo labeling with acetate and palmitate of lung phospholipids from developing and adult rabbits

The labeling with radiolabeled acetate and palmitate of lung, microsomes isolated from lung, and surfactant phospholipids from adult, 3-day-old, and newborn rabbits was studied. The half-life of phosphatidylcholine from lung and microsomal fractions was shorter when labeled with acetate than when labeled with palmitate. Half-time values similarly measured for phosphatidylglycerol, phosphatidylinositol or phosphatidylethanolamine were not different for the two labels. Acetate and palmitate-labeled phospholipids appeared in the surfactant fraction with similar accumulation curves. The relative specific activities of acetate-labeled phosphatidylcholine from adult, 3-day-old, and newborn rabbits, respectively, were 1.30, 1.86 and 1.77 times those measured for those measured for the palmitate label. Surfactant phosphatidylinositol and phosphatidylethanolamine from 3-day-old animals similarly were labeled preferentially with acetate. However, phosphatidylglycerol purified from the surfactant fraction contained equivalent relative amounts of the acetate and palmitate labels in 3-day-old and adult rabbits. These results suggest that the type II pneumocyte may use acetate preferentially for the synthesis of palmitic acid which then is incorporated into surfactant phospholipids.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.     

Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A(2)-mediated surfactant dysfunction

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A(2) (sPLA(2)) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA(2)s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA(2)s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA(2)s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA(2) is less clear and may be the combined result of both mechanisms.     

Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

Phosphatidylglycerol in lung surfactant. III. Possible modifier of surfactant function.

Lamellar bodies and alveolar lavage from adult mammalian lung contain unusually high concentrations of phosphatidylglycerol that could serve as a sensitive indicator of surfactant. Phosphatidylglycerol was absent and phosphatidylinositol was correspondingly prominent in surfactant from the preterm rabbit fetus. Phosphatidylglycerol rapidly appeared and phosphatidylinositol decreased following the delivery. Surfactant isolated from the prematurely born rabbit or from humans with respiratory distress syndrome never contained phosphatidylglycerol. Comparison between lamellar bodies from fetal and postnatal rabbits revealed remarkably similar composition except for the acidic phospholipids; however, the physico-chemical properties were different. The compressibility of the surface film (i.e. the ratio of the fractional decrease in surface area and the corresponding decrease in surface tension) at low surface tensions was higher with fetal than with postnatal surfactant, whereas the difference in minimum surface tensions was small. These data suggest that phosphatidylglycerol is not an essential component required for the formation of the complex, but it improves the properties of surfactant in stabilizing the alveoli.