Summary
Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 50∶1 for 1 h at 37° C. A 1-mm3 pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-μm cryosections were cut at -100° C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus and cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p<0.001) whereas Cl was double (p<0.001), K was 29% lower (p<0.001), and Ca was 4 times higher (p<0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrate elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.
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References
Becker EL, Talley JV, Showell HJ, Naccache PH, Sha'afi RI (1978) Activation of the rabbit polymorphonuclear leukocyte membrane “Na+,K+” ATPase by chemotactic factor. J Cell Biol 77:329–333
Bell SW, Masters SK, Ingram P, Waters M, Shelburne JD (1979) Ultrastructure and x-ray microanalysis of macrophages exposed to cadmium chloride. Scan Electron Microsc III:111–121
Bianchi CP (1968) Cell calcium. Butterworths, London
Cameron IL, Smith NKR, Pool TB, Sparks RL (1980) Intracellular concentration of sodium and other elements as related to mitogenesis and oncogenesis in vivo. Cancer Res 40:1493–1500
Castranova V, Bowman L, Miles PR (1979) Transmembrane potential and ionic content of rat alveolar macrophages. J Cell Physiol 101:471–480
deVries CR, Ingram P, Walker SR, Linton RW, Gutknecht WF, Shelburne JD (1983) Acute toxicity of lead particulates on pulmonary alveolar macrophages. Ultrastructural and microanalytical studies. Lab Invest 48:35–44
Gullvag BM, Nilsen A, Mylius E (1980) X-ray micro-analytical studies of alveolar macrophages (AM) from expectorate samples as part of a test of effect of occupational particulate air pollution. Scan Electron Microsc 111:339–348
Hagler HK, Burton KP, Greico CA, Lopez LE, Buja LM (1980) Techniques for cryosectioning and X-ray microanalysis in the study of normal and injured mycardium. Scan Electron Microsc II:493–498
Hall TA (1979) Biological X-ray microanalysis. J Microsc 117:145–163
Hinkle DE, Wiersma W, Jurs SG (1979) Applied statistics for the behavioral sciences. Houghton Mifflin Co., Boston
Kavet RI, Brain JD (1977) Phagocytosis: quantification of rates and intercellular heterogeneity. J Appl Physiol: Respir Environ Exercise Physiol 42:432–437
Lindsey JR, Baker HJ, Overcash RG, Cassell GH, Hunt CE (1971) Murine chronic respiratory disease. Am J Pathol 64:675–708
Mackaness GB (1970) The monocyte in cellular immunity. Semin Hematol 7:172
Masters SK, Bell SW, Ingram P, Adams DO, Shelburne JD (1979) Preparative techniques for freezing and freeze-sectioning macrophages for energy dispersive X-ray microanalysis. Scan Electron Microsc III:97–110, 122
Myrvik QN, Leake ES, Fariss B (1961) Lysozyme content of alveolar and peritoneal macrophages from the rabbit. J Immunol 86:133–136
Naum Y (1975) Growth of pulmonary alveolar macrophages in vitro: responses to media conditioned by lung cell lines. Cytobios 14:211–220
Parod RJ, Brain JD (1983) Uptake of latex particles by pulmonary macrophages: role of calcium. Am J Physiol 245:C227-C234
Putney JW (1978) Stimulus-permeability coupling: role of calcium in the receptor regulation of membrane permeability. Pharm Rev 30:209–245
Robin ED, Smith JD, Tanser AR, Adamson JS, Millen JE, Packer B (1971) Ion and macromolecular transport in the alveolar macrophage. Biochem Biophys Acta 241:117–128
Rubin RP (1970) The role of calcium in the release of neurotransmitter substances and hormones. Pharmacol Rev 22:389–428
Rubin RP (1982) Calcium and Cellular Secretion. Plenum Press, New York
Sandow A (1965) Excitation-contraction coupling in skeletal muscle. Pharmacol Rev 17:265–320
Showell HJ, Becker EL (1976) The effects of external K+ and Na+ on the chemotaxis of rabbit peritoneal neutrophils. J Immunol 116:99–105
Smith NR, Sparks RL, Pool TB, Cameron IL (1978) Differences in the intracellular concentration of elements in normal and cancerous liver cells as determined by X-ray microanalysis. Cancer Res 38:1952–1959
Smith NKR, Stabler SB, Cameron IL, Medina D (1981) X-ray microanalysis of electrolyte content of normal, preneoplastic and neoplastic mouse mammary tissue. Cancer Res 41:3877–3880
Smith NKR, Morris SS, Richter MR, Cameron IL (1983a) Intracellular elemental content of cardiac and skeletal muscle of normal and dystrophic hamsters. Muscle Nerve 6:481–489
Smith NKR, Lewinski A, Bartke A, Stabler-Morris S (1983b) X-ray microanalysis of cardiocytes in the Snell dwarf mouse. Biochem Biophys Res Comm 114:234–239
Soderland SC, Naum Y (1973) Growth of pulmonary macrophages in vitro. Nature 245:150–152
Somlyo AV, Shuman H, Somlyo AP (1977) Elemental distribution in striated muscle and the effects of hypertonicity. Electron probe analysis of cryo sections. J Cell Biol 74:828–857
Stossel TP (1973) Quantitative studies of phagocytosis. Kinetic effects of cations and heat-labile opsonin. J Cell Biol 58:346–356
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Smith, N.K.R., Lewinski, A.K., Mangos, J.A. et al. Stimulus-permeability coupling in rat pulmonary macrophages challenged by Pseudomonas aeruginosa . Cell Tissue Res. 240, 461–465 (1985). https://doi.org/10.1007/BF00222360
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DOI: https://doi.org/10.1007/BF00222360