Mukul K. Basu

pH, temperature, humidity and the dynamic force-area curve of dipalmitoyl lecithin

Both high pH at 25 degrees C and humidity at 37 degrees C prevent DPL films from attaining zero surface tension, whereas humidity at 25 degrees C and high pH at 37 degrees C do not. At 37 degrees C DPL lowered surface tension to zero when spread from organic solvent or when absorbed from aqueous 0.15 M NaCl in the surface balance in which the surface film was exposed to the room air (dry film). Upon saturation of the atmosphere with water vapor in equilibrium with the aqueous phase at 37 degrees C in a closed chamber, DPL lost the ability to produce zero surface tension, and the gamma min of the DPL film increased from zero to 22 dyne/cm. Addition of DPL in chloroform to distilled water before dispersion by sonication did not prevent the effect of the humidity. However, when the chloroform solution of DPL was added to 0.15 M NaCl before sonication, the adsorbed film produced immediately a stable gamma min of zero in a saturated atmosphere, 37 degrees C. In the absence of chloroform, with DPL adsorbed from either distilled water or 0.15 M NaCl, the effect of humidity was reversed either by removing the chamber and returning the wet film to room air or by introducing small quantities of dispersing agents such as cholesteryl palmitate. However, whereas the effect of humidifying the air was reversible indefinitely, the effect of cholesteryl palmitate (zero surface tension, wet or dry film) was irreversible. This means that there are substances or conditions that can assist DPL films in maintaining zero surface tension when such films are exposed to humidity-saturated air at 37 degrees C.

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37 degrees C and incubated for five hours with 3H-choline and 14C-palmitate in the presence of various concentrations of prostaglandins (PGS) E2 and F2alpha. In the control (no PG) the level of % palmitate incorporation was 13.5 x as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 muM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 x better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 muM, PGE2 was much more effective than PGF2alpha in stimulating the incorporation of both precursors.

Effect of serum albumin on dynamic force-area curve of dipalmitoyl lecithin.

In line with previous findings at 25 degrees C, solutions of serum albumin in the subphase stabilized the surface activity of DPL spread films at 25 degrees C as well as 37 degrees C. In contrast, films adsorbed from mixtures of DPL and albumin exhibitied a marked inhibitory action of the albumin on DPL activity. The inhibitory effect increased with the relative protein concentration but, with albumin/DPL ratios smaller than 2, the DPL activity was regained gradually with cycling. With larger albumin/DPE negative effect of albumin was counteracted by higher temperatures (37 degrees C vs 25 degress C) and modest cholesterol concentrations; with greater cholesterol concentrations the known inhibitory effect of cholesterol prevailed. The inhibitory effect of albumin was potentiated by humidity; saturation of the atmosphere with water vapor at 37 degrees C abolished the DPL character of DPL-RSA mixtures and prevented its return (zero surface tension) upon reversal of the atmosphere from saturated water vapor to dry air. The data are important in the interpretation of the surface activity of pulmonary washings and other pulmonary extracts.

Surface potentials and ion sorption of dimyristoyl-, dipalmitoyl- and distearoyl phosphatidylcholine films on NaCl and CaCl2 solutions.

Abstract When either dimyristoyl-, dipalmitoyl-, or distearoyl phosphatidylcholine was spread on the aqueous phase, the electrical potential at collapse pressure on 75 mM CaCl2 was about 70 mV higher than on 150 mM NaCl; the difference between Ca2+ and Na+ fell gradually to 15–25 mV at the lower pressures. When, however, the electrolyte was injected under the dipalmitoyl phosphatidylcholine film preformed on distilled water, the surface potential at any film pressure was the same on water, NaCl or CaCl2 in the first 4 min; but, the presence of ⩾1 mol% acidic phospholipid (dicetyl phosphate or dipalmitoyl phosphatidic acid), together with dipalmitoyl phosphatidylcholine in mixed films, caused the surface potential to increase rapidly in the order CaCl2 > NaCl > water, by a quantity that was proportional to the concentration of the acidic lipid in the film and Na+ or Ca2+ in the aqueous phase. It is suggested that, in the absence of acidic phospholipids (contaminants), the modest rise in surface potential is due to penetration of the electrolytes, as well as waters, ion pairs. Measurements of surface radioactivity (45Ca2+) and infrared spectra of the interfacial material led to the discovery of a new species, Ca(OH)2, at the airwater interface of CaCl2 solutions. A direct correlation exists between adsorption of Ca2+ and Ca(OH)2 formation, both of which are enhanced by the presence of dipalmitoyl phosphatidylcholine at the interface; no specific correlation, however, can be established between either surface potential and ion binding of the neutral phospholipid or surface potential and ion pair penetration, since it is not yet possible to identify the electrically significant ion pair in the ionic maze of the interfacial lattice.

Effects of cholesterol and cholesteryl ester on dynamic surface tension of dipalmitoyl lecithin

The lipid composition of cell membranes, serum lipoproteins, and various subcellular particles determines the lipid-lipid interactions that are in part responsiblefor the architecture, and the physical properties (such as plasticity, viscosity, thermal transitions) of the resulting structures as well as for the biochemical function of the related aqueous surfaces. Cholesterol exists free in cell membranes (1), is carried also free and in the form of cholesteryl esters by serum lipoproteins (2), and is found in both forms in pulmonary lavage, which is obtained by irrigation of the lung with 0.15 M NaCI (3, 4) and is known to affect the surface activity of pulmonary surfactant (5, 6). Also known are the binding of cholesterol to phosphatidyl cholines (lecithins) (7-9) and the influence of cholesteryl esters on the equilibrium force-area curves of lecithins monomolecular films (10). In this communication, we wish to describe the influence of cholesterol and cholesteryl palmitate on the dynamic force-area curve of dipalmitoyl lecithin at 25 and 37°C in a modified Langmuir trough. A similar study, using adsorbed DPL-cholesterol mixed films by the bubble method has been published recently (6). In our approach we study both adsorbed and spread films, whereby we are able to consider the effect of the physical state of the lipid and of the mode in which the lipid is applied to the interface (3, 5). MATERIALS AND METHODS

Isolation and characterization of plamsa membrane from monolayer cultures of epithelial type II lung cells

Abstract Pneumocyte type II produces a phospholipid, dipalmitoyl lecithin, which is stored in and secreted from the cells inclusion bodies and is indispensable for alveolar stability. Cloned rat lung type II cells were harvested at monolayer confluence and homogenized in swelling buffer. After sequential differential centrifugations, the crude membrane fraction was subjected to discontinuous sucrose density gradient centrifugation at 65,000 × g . Quality of the relevant fractions was monitored by enzyme activities and phase contrast and electron microscopy of two major bands at densities 1.16 and 1.18, respectively. The less dense band contained only small quantities of organelles, little cytochrome c oxidase, and some glucose 6-phosphatase, but had a significant (Na + , K + )-ATPase activity; this and ultrastructural evidence certified the product as a suitable plasma membrane preparation. Upon sodium dodecyl sulfate-poly acrylamide gel electrophoresis, the protein pattern consisted of 11 major protein bands between 13,000 and 68,000 M r ranges, and several minor ones. The lipid pattern was studied by two-dimensional thin layer chromatograpy, followed by various group reactions (e.g., amine, unsaturation, phosphorus, sugars). In the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, palmitic acid was the least abundant of four major fatty acids, accounting for 14.20% in phosphatidyl choline and 5.70% in phosphatidyl ethanolamine, whereas the most abundant were stearic and palmitoleic with about 28% each in phosphatidyl choline, and palmitoleic (29.90%) and oleic (23.05%) in the ethanolamine phosphatide. Apparently, the palmitic acid containing phosphatidyl choline must be in the lamellar inclusion bodies of type II cells and not in their plasma membranes.

Cultured lung cells: effects of isotopic dilution and some undefined stimulation on the incorporation of radiolabeled palmitate and choline into phosphatidyl choline (lecithin).

Abstract Using radiolabels, we studied the effect of certain experimental conditions on the incorporation of choline and palmitate into phosphatidyl choline (lecithin) of cloned type II rat lung cells. When the label was changed from methyl-3H to 1,2-14C the incorporation of choline was reduced to 1/3; in contrast, when the label was moved from 1-14C to 9,10-3H, the incorporation of free palmitate was more than doubled. Removal of choline from the culture medium caused trebling of choline incorporation and appreciable decrease in palmitate uptake, indicating respectively an expected effect of increased choline label concentration in the absence of carrier, and a marked dependence of palmitate on choline incorporation. Removal of fetal calf serum produced more than 2/3 decrease in palmitate incorporation (instead of an expected increase because of palmitate isotope concentration), whereas choline uptake was not affected, meaning respectively that either serum hormones or serum lipids, or both simultaneously, are important for palmitate but not for choline incorporation. This is only the beginning of a host of studies required to clarify the role of fetal calf serum constituents onto lecithin biosynthesis in cultured lung cells and finally gain a full appreciation of the biosynthetic pathways of phosphatidyl choline (“surfactant”) in type II cells of alveolar epithelium.

Cultured Lung Cells: Interplay Effects of Beta-Mimetics, Prostaglandins and Corticosteroids in the Biosynthesis of Dipalmitoyl Lecithin

Abstract Cell lines derived from type II lung cells were used to study interplays of substances affecting incorporation of labeled precursors [1-14C] palmitate and [methyl-3H] choline into phosphatidyl choline. Ethanol stimulated markedly biosynthesis of dipalmitoyl phosphatidyl choline in cloned rabbit lung cells; the stimulating action of ethanol was reduced very much by cortisol and less by ritodrine. In the presence of 0.1 μᴍ isoproterenol, two prostaglandins, E2 and F2a, caused marked depressions in the incorporation of both precursors by cell line A 549 derived from human lung adenocarcinoma. One concluded that among the agents studied, ethanol and cortisol are potent antagonists, and so were also the prostaglandins and isoproterenol.

Surface behavior of prostaglandins (PGs)

Abstract This study establishes some correlations between molecular structure and surface function of six prostaglandins in a model membrane system. Using spread films at the air/water interface, we determined surface pressure and surface potential of PGs A1, A2, E1, E2, F1α and F2α. All the PGs formed films with low pressure (0 to 9 dyne/cm) and relatively low surface potentials (ΔV = 10 to 250 mV). On 0.15 M NaCl, the π and ΔV values were in the order E1 > F1α > A1 > F1α > A1 > F2α = A2 > E2 and F1α > E1 > A1 > A2 > F2α > E2 respectively. Clearly, the cis unsaturation in the carboxylic chain of the PG2 series conferred greater instability to the films, as indicated by the lowest π and ΔV values. Also, members of the PG1 series penetrated films of dipalmitoyl phosphatidyl choline (DPPC) better than PG2 did, the ablest being E1 (Δπ = 12 dyne/cm) and the poorest F2α (Δπ = 2 dyne/cm); penetration of E1 and F2α was independent of the initial pressure (πi) of the DPPC film, whereas with A1 and F1α Δπ decreased as πi increased. The PGs expressed marked discriminating capacities for the electrolyte, as indicated by differences in their π and ΔV responses to Na+ and Ca++ as well as for the lipid, as indicated by different penetration (Δπ values) into DPPC films.