Department of Oral Biology

• International Society of Tobacco Induced Diseases (ISPTID)
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• Canadian Association for Anatomy, Neurobiology and Cell Biology (CAANCB)
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• Biology of Breathing Group  more info >>

Respiratory Distress in the Premature Infant: Initiation of Surfactant Synthesis and Secretion

Research Program:
Neonatal Respiratory Distress Syndrome, the leading cause of death in premature infants, results from inability to expand the lungs properly. For some years it has been known that poor expansion characteristics are the result of insufficient production of a material called pulmonary surfactant. And in fact, the reason this substance is not produced in sufficient quantities is because the lungs of these infants are not mature enough. An interesting finding in the late 1960's suggested that glucocorticoids might be useful in accelerating the maturation of the lung. As a result this hormone was tested clinically for some years but generally its effects were not of a sufficiently uniform or predictable nature to make it useful. More recently investigations at the cellular level, particularly in cultures of isolated lung cells have shed light on the etiology of this disease as well as elucidated the characteristics and synthetic pathways for the pulmonary surfactant.

Surfactant is a complex mixture of phospholipids and proteins. Two phospholipids in particular, dipalmitoylphosphatidylcholine (DSPC) and phosphatidylglycerol (PG) are unique in form or abundance and as a result are used as markers for the pulmonary surfactant. In fact it is the amphipathic characteristics of these molecules, together with the surfactant proteins which allow the surfactant to adsorb to the air-liquid interface within the lung thereby reducing surface tension and facilitating proper expansion and deflation.

A scanning electron micrograph of mouse lung from the LUNGLAB showing a branching bronchiole with alveoli arranged around it.

Surfactant is produced by certain cells within the lungs called type II alveolar cells. Our research efforts are directed to examination of the method by which these cells differentiate and acquire the ability to synthesize and secrete the surfactant. Type II alveolar cells are isolated from fetal rabbit or rat lungs and grown in culture. With these cells we are examining the process of cellular specialization and differentiation, specifically, the dynamics of choline and phospholipid interactions in regulating the synthesis of DSPC in vitro.

Choline is the major precursor for synthesis of DSPC in these cells. Yet if the cells are incubated with 1-palmitoyl-2-oleoyl phosphatidylcholine or DSPC the rate of de novo synthesis of DSPC from [3H]choline is depressed. This suggests that the extracellular phospholipid environment affects cellular phospholipid synthesis. Indeed if the cells are exposed to radioactive phospholipid some of this material appears in the organic solvent soluble-extractable fraction. This indicates that these cells reutilize all or portions of surfactant material and this phospholipid re-incorporation pathway may contribute in a significant manner to alveolar surfactant levels and thus Type II cells stimulus-secretion coupling.

Many agents have been shown to stimulate secretion of the pulmonary surfactant. In particular we have found that isoproterenol or isoxsuprine induce the secretion of [3H]choline-labelled phospholipid. These compounds may effect this by several routes independent of accepted mechanisms involving cAMP. In fact evidence suggests that prostaglandins derived from the action of a phospholipase A2 on cell membrane diacylglycerol may regulate secretion. Alternatively inositol trisphosphate mobilization of intracellular calcium stores may be involved.

Both aspects are being examined by determining turnover of [14C]arachidonic acid or [3H]inositol into prostaglandins or inositolphosphates respectively. In addition we have also found that these cells bind [35S]GTP to several proteins and these proteins are affected by the differentiation state of the cells. Whether the GTP-binding proteins are associated with inositolphosphate turnover or cAMP activation is being investigated. Evidence from several recent studies indicates that protein kinase C activation is instrumental in activation of the surfactant secretion cycle. Studies from our lab have shown that exposure of type II cells to 4ß-phorbol ester which activates protein kinase C, stimulates phospholipid secretion.

Further studies are presently underway to examine the role and distribution of protein kinase C in subcellular fractions of whole lung as well as isolated fetal type II cells and fibroblasts. Developmental assessment of the enzyme activity in fetal rabbit lungs and isolated cells is being done.

In addition we have recently begun studies to define the role of a dynamic stretched environment in differentiation of the lung type II alveolar cells as a model for fetal breathing which occurs in utero. For these studies a FlexerCell apparatus is used to distend the monolayer of epithelial cells growing in vitro. Flexing of the cell monolayer has shown that this process increases the incorporation of [14C]glucose in cellular phospholipids and the incorporation of [3H]inositol into phosphatidylinositols and inositolphosphates. This suggests that the practice breathing that the fetus does prior to birth may play a role in differentiation of lung cells, especially the surfactant-producing type II alveolar cell.

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Most of the work described above has been supported by the MRC of Canada and Manitoba Health Research Council.

We have recently begun work with other scientists at the National Research Council, Institute for Biodiagnostics into analyses of environmental pollutant effects on the pulmonary system. This approach uses fractions from lungs or lung cells of animals exposed to smoke, trichloroethylene, mercury and other pollutants. In addition isolated surfactant subfractions are also analyzed. The uniqueness of this approach is associated with our ability to analyze these samples by traditional biochemical analyses such as DNA and protein levels, and phospholipid analyses but further to determine molecular changes by Fourier-transform infrared spectroscopy with investigators at NRC.

Recent funding from NSERC has also provided us with both a Captive Bubble Surfactometer and Capillary Surfactometer which enables us to measure surface tension reducing abilities of the pulmonary surfactant. Ongoing collaborations with NRC as well as scientists at Dalhousie University have proved very useful. This work is supported by the National Science and Engineering Research Council of Canada (NSERC).

With Dr. T. Tsubai (Osaka Dental University) we are examining the regulation of differentiation and replication in mandibular condyle fibroblasts and the expression of IEG's (immediate early genes) cFOS and CJUN, in a model of arthritis centered in the temporomandibular joint. This work has shown that the mandibular condyle cells may be regulated by cytokines that hasten their progression through S phase and therefore contribute to the cell pool involved in joint healing. This work is supported by Manitoba Medical Services Foundation.

With Dr. T. Rand (St. Mary's University, Halifax) and Dr. M. Oulton (Dalhousie University Halifax) we are examining the effects of environmental pollutants such as mold toxin, mold spores (Stachybotry chartarum (atra)) and tobacco smoke components (nicotine and cotinine, a nicotine metabolite) on surfactant processing in adult lung and surfactant production in fetal and neonatal lung cells. Our studies suggest that these agents may significantly reduce production of surfactant and depress the replication rate of neonatal and fetal lungs cells.

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Present laboratory personnel:

• Mr. X. Xie, B.Sc. (Dent.) program
• Mr. J. Merluza, M.Sc. program, Oral Biology
• Ms. B. Weltman, B.Sc. (Dent.) program
• Ms. K. McCrae, Ph.D. program, Oral Biology
• Mr. R. Santos, B.Sc. (Dent.) program
• Dr. T. Tsubai, Adjunct Professor, University of Manitoba
• Ms. P. Irani, NSERC Undergraduate Fellowship
• Mr. C. Hillier, M.Sc. Program, Oral Biology
• Mr. J. Bilyk, M.Sc. Program, Anatomy

Relevant publications

• McCrae, K, Shaw, A., Mantsch, H.H., Thliveris, J.A., Das, R.M., Ahmed, M.K. and Scott, J.E. (1999). Lung cancer. Occurrence and new possibilities for detection. International Journal of Health Care Quality Assurance 12, I - XIII. 2000 Literati Club (London England) MCB University Press Award for Excellence, Most Outstanding paper.
• Tsubai, T., Higashi, Y. and Scott J.E. (2000) Effect of exogenous epidermal growth factor on the fetal rabbit mandibular condyle and isolated condylar fibroblasts. Arch. Oral Biol. 45, 507-15.
• Scott, J.E. (2000) The mandibular condyle:regulation of development and cell function. MDA Bulletin.
• McCrae, K., T. Rand, C. Mason, M.R. Oulton, C. Hastings, T. Cherlet, J.A. Thliveris, A. Shaw, H.H. Mantsch, J. MacDonald and J.E. Scott. (2001). Analysis of Stachybotrys chartarum (atra) spores and pulmonary surfactant from spore-exposed mice by Fourier transform infrared spectroscopy. Chem. Physics of Lipids 110, 1 Ð 10.
• McCrae, K.C., H.H. Mantsch, J.A. Thliveris, J.E. Scott, and R.A. Shaw (2002). Analysis of neoplastic changes in mouse lung using Fourier-transform infrared microspectroscopy. Vibrational Spectroscopy 28, 189-197.
• McCrae, K.C., Unruh, H., Shaw, A. Mantsch, H.H., Thliveris, J.A. and Scott J.E. (2001) Characterization of human lung neoplasms using Fourier transform infrared spectroscopy. Appl. Spectroscopy (submitted)
• Cherlet, T. and Scott, J.E. (2002) Tetrahydrocannabinol (THC) alters synthesis and release of surfactant-related material in isolated fetal rabbit type II cells. Drug Chem Tox. 25(2), 171-190.
• McCrae, K.C., Shaw, A., Mantsch, H.H., Thliveris, J.A. and Scott J.E. (2002) Nursing perspectives on the pulmonary surfactant. International Journal of Health Care Quality Assurance (in manuscript).
• Bergen H.T., Harrower, T.C., Manuel, P. and Scott, J.E. (2002) Identification of leptin receptors in lung and isolated fetal type II cells. Am. J. Respir. Cell Molec. Biol.27, 71-77.
• Anderson, J., Wozniak, A., Pilipowicz, O.,Yablonka-Reuveni, Z., Greenway, S., Craven, S., and Scott, E. (2003). C-met expression and mechanical activation of satellite cells on cultured muscle fibers. J. Histochem. Cytochem. 51 (11), 1437-1445.
• Scott, J.E. (2003) Pulmonary Biology Research in the Faculty of Dentistry MDA Bulletin.
• Wang, L., Liu, H.W., McNeill, K., Stelmack, G., Scott, J.E. and Halayko, A.J. (2004). Mechanical strain inhibits airway smooth muscle-specific gene transcription via protein kinase (PKC)-dependent signaling. Am. J. Respir. Cell Molec. Biol. 31, 54-61.
• McCrae, K.C., A. Shaw, J. Thliveris, and J.E. Scott (2004) Effect of a high cholesterol diet on pulmonary surfactant function in mice with pre-existing lung adenocarcinoma. Chem. Physics of Lipids (submitted)
• McCrae, K.C., Unruh, H., Shaw, R.A. Mantsch, H.H., Thliveris, J.A. & Scott J.E. (2004) Characterization of human lung neoplasms using Fourier transform infrared spectroscopy. (in manuscript)
• Scott, J.E. (2004) The Pulmonary Surfactant: Impact of Tobacco Smoke and Related Compounds on Surfactant and Lung Development (invited review). International Journal of Tobacco Induced Diseases 2(1), 3-25. Available online at: TOBACCO INDUCED DISEASES Volume 2, Issue 1, March 2004 (see Article 2.)
• McCrae, K.C., Rand, T.G., Shaw, R.A., Mantsch, H.H., Sowa, M.G., Thliveris, J.A. & Scott J.E. (2004) DNA fragmentation in developing lung fibroblasts exposed to Stachybotrys chartarum (atra) spores . Am. J. Respir. Cell Molec. Biol. (submitted)
• McCrae, K.C., Rand, T.G., Shaw, R.A., Mantsch, H.H., Sowa, M.G., Thliveris, J.A., & Scott J.E. (2004) The effects of a high cholesterol diet on pulmonary surfactant function. (in manuscript)
• McCrae, K.C., Shaw, R. A., Unruh, H. Mantsch, H.H., Thliveris, J.A., & Scott, J.E. (2004) The effects of lung adenocarcinoma on pulmonary lung surfactant function. (in manuscript)
• McCrae, K.C., Shaw, R. A., Unruh, H. Mantsch, H.H., Thliveris, J.A., & Scott, J.E. (2004) Biochemical characterization of pulmonary surfactant. (in manuscript)
• McCrae*, K.C., Shaw, A., Mantsch, H.H., Thliveris, J.A., Rand, T.G. and Scott, J.E. (2004) Correlation of DNA-specific alterations detected by the alkaline strand unwinding technique with infrared spectroscopic profile in isolated lung cells exposed to S. chartarum. Appl. Spectroscopy (submitted)
• Hastings, C., Rand, T., Bergen, H.T., Thliveris, J.A., Shaw, A., Mantsch, H.H. and Scott, J.E. (2004). Stachybotrys chartarum alters synthesis of disaturated phosphatidylcholine and activation of CTP:cholinephosphate cytidylyltransferase in isolated fetal rabbit type II cells. Toxicology Sci. (accepted - in revision)
• McCrae, K.C., Shaw, R. A., Unruh, H. Mantsch, H.H., Thliveris, J.A., & Scott, J.E. (2004) Pulmonary surfactant function in terminal airways: the effects of lung disease and nutrition status. (in manuscript)
• Funk, D.J., Graham, M.R., Girling, L.G., Thliveris, J., McManus, B.M., Walker, E.K-Y., Rector, E.S., Hillier, C., Scott, J.E. and Mutch W.A.C. (2004). A Comparison of Biologically Variable Ventilation to Recruitment in a Porcine Model of ARDS. Respiratory Res. Available online at: http://respiratory-research.com/content/5/1/22

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