On the basis of in vivo animal studies and on experiments of nature, it has been suggested that fetal breathing movements are essential for normal lung growth in utero. To study this hypothesis in vitro, we examined the effect of mechanical stretch on proliferation of fetal rat lung cells maintained in organotypic culture to provide a three-dimensional matrix. Initial studies demonstrated that stretch-mediated effects on cell division and DNA synthesis in such cultures were influenced by cell inoculation density, fetal calf serum concentration, and by the amplitude, frequency, periodicity, and duration of the applied stretch. After a 48-h exposure to an intermittent stretch pattern (5% elongation, 60 stretches/min for 15 min of each hour), cell number increased 10% (P less than 0.05), cell doubling time was reduced from 71 to 55 h (P less than 0.05), [3H]thymidine incorporation into DNA increased 61% (P less than 0.01), and the [3H]thymidine-labeling index increased 2.8-fold (P less than 0.001) compared with nonstretched controls. This effect did not appear to be mediated by prostaglandins or leukotrienes because the prostaglandin synthase inhibitors ibuprofen (2.5-50 microM) or BW 755C (5 microM), leukotriene biosynthesis inhibitors BW 755C (5 microM) or MK-886 (0.3 microM), and leukotriene D4 receptor antagonist MK-571 (0.3 microM) did not block stretch-mediated effects. We conclude that mechanical forces act directly to stimulate fetal rat lung cell growth and that these results are compatible with a significant role for fetal breathing in normal fetal lung growth.(ABSTRACT TRUNCATED AT 250 WORDS)

The signaling pathways by which intermittent strain (60 cycles/min, 15 min/h) regulates proliferation of mixed fetal rat lung cell in vitro have been investigated. Adenosine 3',5'-cyclic monophosphate (cAMP) content and cAMP-dependent protein kinase (PKA) activity were not affected by strain. The stimulatory effect of strain on DNA synthesis was also not influenced by the cyclic nucleotide-dependent protein kinase inhibitors H-8 or HA-1004, the adenylate cyclase inhibitor SQ-22536, or a PKA inhibitor and cAMP antagonist, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). In contrast, intracellular concentrations of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), were dramatically increased after a short period of strain. This increase in second messengers was accompanied by an increased tyrosine phosphorylation of phospholipase C-gamma 1. Phospholipase D activity was also increased by strain. Mechanical strain elicited a shift in the subcellular distribution of PKC activity from cytosol to membranes shortly after the onset of strain. The specific activity of PKC in the membranes increased 6- to 10-fold within 5-15 min and remained increased throughout a 48-h period of intermittent strain. Strain-induced PKC activation and DNA synthesis were blocked by the PKC inhibitors H-7, staurosporine, and calphostin C, as well as by the phospholipase C inhibitor U-73,122. We conclude that mechanical strain of mixed fetal rat lung cells activates phospholipid turnover via phospholipases, followed by PKC activation, which then triggers the downstream events that lead to cell proliferation.

An intermittent mechanical strain regimen, which simulates fetal breathing movements, has been shown to enhance DNA synthesis and cell division of fetal rat lung cells. The signaling mechanism through which the physical stimulus is transduced is unknown. Herein, we report that mechanical strain (5% elongation, 60 cycles/min) of fetal lung cells, cultured in a three-dimensional environment provided by Gelfoam sponges, increased the mRNA levels of platelet-derived growth factor B (PDGF-B) and beta-receptor (PDGF-beta-R) within 5 min of the onset of strain. Both PDGF-BB and PDGF-beta-R proteins were increased after a 24-h intermittent strain (15 min/h). Phosphorothioate antisense PDGF-B oligonucleotides (ON) at 15 microM abolished the strain-enhanced DNA synthesis and cell growth. Scrambled PDGF-B ON had no such effect. A neutralizing PDGF-BB antibody (10 micrograms/ml) also attenuated strain-induced DNA synthesis. Furthermore, the strain-induced stimulatory effect on DNA synthesis of fetal lung cells was blocked by tyrphostin 9 (1 microM), a PDGF receptor-associated tyrosine kinase inhibitor, but not by its inactive structural analogue tyrphostin 1. Antisense but not sense PDGF-beta-R ON (10 microM) also abrogated the strain-enhanced DNA synthesis. These results suggest that physical forces such as fetal breathing movements regulate fetal lung cell growth by controlling PDGF-B and PDGF-beta-R gene expression.

Because of their high degree of unsaturation, polyunsaturated fatty acids (PUFA) in mammals, with mainly 18, 20 and 22 carbons, can easily be autooxidized, and converted into many oxidized derivatives and degradation products. This short review reports on some of those relevant to the evaluation of oxidative stress in situ. In addition, the enzyme-dependent oxygenation by both dioxygenases and monooxygenases is briefly reviewed by functional and/or metabolic categories, pointing out the structure variety and the analytical approaches.

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