Lipopolysaccharide (LPS)-binding protein (LBP) binds with high affinity to LPS, and the LBP-LPS complex enhances cellular inflammatory responses to LPS. Although it is present in normal serum, LBP is also induced as part of the acute phase response. Synthesis of LBP is though to be limited to the liver, but we have recently reported significant extrahepatic (including pulmonary) LBP mRNA expression in in vivo rat models of sepsis and inflammation. In the present study, we tested the hypothesis that a cellular source of pulmonary LBP in the rat may be vascular smooth muscle, by exposing cultured rat pulmonary artery smooth muscle cells (RPASMC) to cytokines and LPS. Treatment of RPASMC for 4 and 24 h with a combination of tumor necrosis factor alpha, interleukin 1 beta (IL-1 beta), interferon gamma, and LPS resulted in significant LBP mRNA expression. Of this mixture, IL-1 beta alone was sufficient to induce LBP mRNA expression in both a time- and dose-dependent manner. The effects of IL-beta on LBP mRNA expression were significantly antagonized by IL-1 receptor antagonist protein. Furthermore, supernatants from RPASMC treated with IL-1 beta enhanced the binding of [125I]ASD-LPS by the macrophage cell line RAW 264.7, indicative of LBP bioactivity. We conclude that pulmonary artery smooth muscle cells stimulated with IL-1 beta produce a transcript for LBP or a homologous product in vitro. Local production of LBP could play an important role in the pulmonary response to inflammation and sepsis.

Cultured smooth muscle cells obtained from rat lung periphery (RPC) and proximal pulmonary artery (RSMC) expressed mRNA for serotonin (5-HT) type 2 receptor (5-HT2) and 5-HT transporter (by Northern blot analysis). Functional expression of these genes was evident since both cell types 1) bound 125I-labeled lysergic acid diethylamide (LSD; 5-HT2 receptor antagonist) that was equally effectively displaced by either ketanserin or mianserin; and 2) transported 5-[3H]HT in an imipramine-sensitive manner. Serotonin (10(-9)-10(-5) M) stimulated DNA synthesis (as measured by [3H]thymidine uptake) in RPC and RSMC. The 5-HT-induced increase in DNA synthesis was significantly inhibited in both cell types by the 5-HT2 receptor antagonist, ketanserin (10(-7)-10(-6) M), and by fluoxetine (10(-6) M), a putative 5-HT transport inhibitor. Acute exposure to 5-HT (1-100 microM) caused an abrupt rise in intracellular calcium ([Ca2+]i) in single pulmonary vascular smooth muscle cells as microspectrofluorometrically determined using the calcium-sensitive dye, fura 2. The 5-HT-induced change in [Ca2+]i was completely abolished in the presence of 10(-6) M ketanserin as well as imipramine or fluoxetine (10(-6) M). The calcium transients due to 5-HT persisted in a Na(+)-free condition (in which the transporter activity was completely abolished) and imipramine and fluoxetine (and ketanserin) were effective inhibitors of 5-HT under these conditions. Therefore, the 5-HT2 receptor, but not the transporter, is responsible for initiating the acute effects (e.g., calcium transients) of 5-HT in cultured rat pulmonary vascular smooth muscle cells and fluoxetine (1 microM) may have 5-HT2-receptor antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Metallothioneins (MT) are low molecular weight proteins that are important in providing protection against heavy metals such as cadmium. Other precise physiologic roles for this family of proteins are less clear, but fetal hepatic cell proliferation and differentiation may be regulated through changes in MT levels and attendant MT-mediated regulation of zinc levels. The role of MT in other developing tissue, most notably lung, is far less clear. Although expression of MT has been reported to be extremely low in early postnatal and mature lung, we hypothesized that MT has a more ubiquitous role in organ development and that pulmonary MT levels may be elevated during periods of rapid lung growth. Thus, we studied expression of MT in late-gestation fetal lambs. Sheep are particularly useful because alveolarization of lung parenchyma occurs before birth (by d 120 of a normal 147-d gestation). Immunoreactive MT was localized to bronchial epithelium of fetal, newborn, and mature sheep. The intensity of staining was greatest in the 130-d gestational age (saccular) lung, where positive reaction product was noted in the cytoplasm and nucleus of alveolar epithelial and interstitial cells. We next evaluated MT expression in developing lung tissue using Northern blot analysis and 32P-cDNA probes against the 3'-untranslated regions of mRNA encoding each of four known functional sheep MT (sMT) isoforms. Expression of sMT-II, sMT-Ia, and sMT-Ib was restricted to the saccular stage (120-132 d gestational age), and sMT-Ic mRNA was not detected in pulmonary samples from any stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-alpha (TGF-alpha) is a structural homologue of epidermal growth factor and competes for binding on a common transmembrane protein receptor/kinase. Although TGF-alpha appears to be more important than epidermal growth factor in embryogenesis and mammalian organogenesis, there is little information regarding its expression in developing lung. Accordingly, we measured levels of immunoreactive TGF-alpha and its gene expression in late-term fetal rat lung during the transition from the canalicular (19-20 d) to the saccular (21 d) stage. We report that at 19 d gestation intrapulmonary levels of TGF-alpha were 1.4 +/- 0.3 pmol/mg protein as determined by RIA, but had decreased by 50% at 21 d. To determine if the TGF-alpha gene is expressed in lung, RNA isolated from fetal rat lung was reverse transcribed, and a 302-bp fragment corresponding to a portion of the TGF-alpha gene was amplified by polymerase chain reaction. Southern blot hybridization with a 32P-labeled 2.3-kb EcoRI fragment of rat TGF-alpha cDNA clone showed a pattern of declining expression during late gestation. Therefore, fetal rat lung expresses TGF-alpha, as is evidenced by the synthesis of both the message and the protein. Because levels of protein were highest in the period of canalicular lung development when the respiratory acinus is formed and vascularized, a potential role for this intrapulmonary growth factor in pulmonary remodeling is suggested.