Fred Possmayer

Bovine pulmonary surfactant: Chemical composition and physical properties

Bovine pulmonary surfactant was obtained by endotracheal lavage of lungs from newly slaughtered cows followed by differential centrifugation. Lipid extracts of bovine surfactant contained 3% neutral lipid, mainly as cholesterol and diacylglycerol and 97% phospholipid. Phosphatidylcholine (79%) and phosphatidylglycerol (11%) accounted for most of the phospholipids with smaller amounts of phosphatidyl-ethanolamine, phosphatidylinositol, lyso-bis-phosphatidic, acid and sphingomyelin. Fatty acid analysis revealed high levels of palmitate in phosphatidylcholine and to a lesser extent phosphatidylglycerol, but not in the other diacylphospholipids. Phosphatidylcholine was 53% disaturated and phosphatidylglycerol was 23% disaturated. Monoenoic species accounted for the major proportion of the remaining, lipid. The protein content was 10% as estimated by the Lowry procedure and 5% when determined by amino acid analysis. Extraction with chloroform/methanol removed ca. 90% of the protein but had no effect on the surfactant properties as evaluated by a pulsating bubble technique.

POSITIONAL SPECIFICITY OF SATURATED AND UNSATURATED FATTY ACIDS IN PHOSPHATIDIC ACID FROM RAT LIVER

Abstract 1. 1. The relative incorporation of a number of radioactive fatty acids into the different glycerolipids of rat liver microsomes has been investigated. 2. 2. Studies on the distribution of the radioactivity incorporated into phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid showed that in all three lipids, the majority of the saturated fatty acids were at the 1-position while the polyunsaturated fatty acids were largely confined to the 2-position. 3. 3. The phosphatidic acid fraction of rat liver was isolated in a pure form, and its fatty acid distribution agreed with the results of the incorporation studies. 4. 4. The incorporation of radioactive fatty acids into different molecular species of phosphatidic acid, separated as dimethyl phosphatidates was investigated.

P2X7 Receptors on Osteoblasts Couple to Production of Lysophosphatidic Acid: A Signaling Axis Promoting Osteogenesis

Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures.

The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.

Deleterious Effect of Tracheal Obstruction on Type II Pneumocytes in Fetal Sheep

It was previously shown that tracheal obstruction accelerated fetal lung growth and eventually reversed the pulmonary hypoplasia in experimental diaphragmatic hernia. We have successfully developed a reversible tracheal obstruction technique in fetal sheep using balloon occlusion and showed that 3 wk of obstruction induced significant lung growth of the same magnitude as the tracheal ligation. The purpose of this study was to examine the effects of 1 and 3 wk of tracheal occlusion on the alveolar cell population with specific attention to the type II pneumocytes. We first showed that 1 wk of occlusion induced a significant increase in lung weight and in alveolar surface area. We then used the surfactant protein C (SP-C) mRNA as a specific marker of differentiated type II pneumocytes. Total RNA was isolated from fetal sheep lung with or without tracheal occlusion, and Northern blots were hybridized with a cDNA probe specific for the sheep SP-C. The results show a dramatic decrease in SP-C mRNA expression (8.8-fold, p < 0.01). In situ hybridization showed a marked decrease in the density of cells expressing SP-C, as well as the amount of SP-C mRNA expressed by the cells. The effect was present as early as 1 wk of occlusion. The sparseness of type II pneumocytes was further confirmed by electron microscopy. We thus conclude that tracheal obstruction causes a profound decrease in the number of type II pneumocytes in the lungs. Given the crucial role of type II pneumocytes in surfactant production, we could speculate that, if tracheal occlusion is able to accelerate lung growth, the final product is probably surfactant-deficient.

P2X7 Nucleotide Receptors Mediate Blebbing in Osteoblasts through a Pathway Involving Lysophosphatidic Acid

Extracellular nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 receptors in many cell types, including osteoblasts. P2X7 receptors are ATP-gated cation channels that can induce formation of large membrane pores. Disruption of the gene encoding the P2X7 receptor leads to decreased periosteal bone formation and insensitivity of the skeleton to mechanical stimulation. Our purpose was to investigate signaling pathways coupled to P2X7 activation in osteoblasts. Live cell imaging showed that ATP or 2 ′,3 ′-O-(4-benzoylbenzoyl)-ATP (BzATP), but not UTP, UDP, or 2-methylthio-ADP, induced dynamic membrane blebbing in calvarial osteoblasts. Blebbing was observed in calvarial cells from wildtype but not P2X7 knock-out mice. P2X7 receptors coupled to activation of phospholipase D and A2, inhibition of which suppressed BzATP-induced blebbing. Activation of these phospholipases leads to production of lysophosphatidic acid (LPA). LPA caused dynamic blebbing in osteoblasts from both wild-type and P2X7 knock-out mice, similar to that induced by BzATP in wildtype cells. However, LPA-induced blebbing was more rapid in onset and was not affected by inhibition of phospholipase D or A2. Blockade or desensitization of LPA receptors suppressed blebbing in response to LPA and BzATP, without affecting P2X7-stimulated pore formation. Thus, LPA functions downstream of P2X7 receptors to induce membrane blebbing. Furthermore, inhibition of Rho-associated kinase abolished blebbing induced by both BzATP and LPA. In summary, we propose a novel signaling axis that links P2X7 receptors through phospholipases to production of LPA and activation of Rho-associated kinase. This pathway may contribute to P2X7-stimulated osteogenesis during skeletal development and mechanotransduction.

Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)

The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders

Gastrointestinal symptoms and altered blood phospholipid profiles have been reported in patients with autism spectrum disorders (ASD). Most of the phospholipid analyses have been conducted on the fatty acid composition of isolated phospholipid classes following hydrolysis. A paucity of information exists on how the intact phospholipid molecular species are altered in ASD. We applied ESI/MS to determine how brain and blood intact phospholipid species were altered during the induction of ASD-like behaviors in rats following intraventricular infusions with the enteric bacterial metabolite propionic acid. Animals were infused daily for 8 days, locomotor activity assessed, and animals killed during the induced behaviors. Propionic acid infusions increased locomotor activity. Lipid analysis revealed treatment altered 21 brain and 30 blood phospholipid molecular species. Notable alterations were observed in the composition of brain SM, diacyl mono and polyunsaturated PC, PI, PS, PE, and plasmalogen PC and PE molecular species. These alterations suggest that the propionic acid rat model is a useful tool to study aberrations in lipid metabolism known to affect membrane fluidity, peroxisomal function, gap junction coupling capacity, signaling, and neuroinflammation, all of which may be associated with the pathogenesis of ASD.

Surface activity in situ, in vivo, and in the captive bubble surfactometer

For studies of the mechanical effects of lung surfactants, the captive bubble surfactometer (CBS) combines the advantages of the continuous film of Pattles bubbles with the feasibility of the Langmuir-Wilhelmy balance to produce surface tension-area hysteresis loops. The CBS allows the compression of films to very low and stable surface tensions of 1-2 mN/m. Such low and stable surface tensions are in line with results obtained from pressure-volume studies on excised lungs. In addition, the CBS is useful to test other essential physical properties of the surfactant system, including: (1) rapid film formation (within seconds) through adsorption from the hypophase; (2) low film compressibility with a fall in surface tension to very low (<2 mN/m) values during surface compression; and (3) effective replenishment of the surface film on expansion by the incorporation of surfactant material from material associated with the surface (the surface associated surfactant reservoir). Morphological observations of films fixed in situ or in vitro reveal frequently their multilayered structure, which is consistent with the concept of the surface reservoir. The deviation of the bubbles from a Laplacian shape at very low surface tension and the morphological observations suggest that the surfactant film cannot be considered a simple monolayer.