Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-beta1 (TGF-beta1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 +/- 0.1; L2, 1.2 +/- 0.1 fmol/10(6) cells) and released (L1, 9.2 +/- 0.5; L2, 2.3 +/-0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC(50) 8 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-beta1 downregulated ET-1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone.

Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension. Participation Of arterial smooth muscle cells.

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

Kirsten sarcoma virus-immortalized mast cell lines. Reversible inhibition of growth by dexamethasone and evidence for the presence of an autocrine growth factor.

Expansion of mast cell numbers occurs in vivo during certain inflammatory reactions, including active fibrosis, parasite infestations, and immediate hypersensitivity reactions. T cell-produced cytokines, including IL-3 and IL-4, are thought to control this mast cell proliferation in part, and glucocorticoid regulation of T cell-produced cytokines is thought to account for diminished mast cell proliferation during administration of glucocorticoids in vivo. Here we show that glucocorticoids have a direct inhibitory effect on proliferation of Kirsten sarcoma virus-immortalized mast cells (KiSV-MC) in vitro, with an ID50 of 1.0 +/- 0.2 nM dexamethasone (mean +/- SD, n = 4). At 10 nM dexamethasone, KiSV-MC proliferation was inhibited by 83 +/- 5% (mean +/- SD, n = 4). As determined by trypan blue staining and [3H]TdR incorporation, the glucocorticoid-mediated growth inhibition was due to diminished mast cell proliferation rather than cell death and was completely reversible after 6 days of glucocorticoid treatment. By cell cycle analysis, glucocorticoids diminished the percentage of mast cells in S phase and increased the percentage in G0-G1 phase. Although we show that the KiSV-MC proliferate via an autocrine mechanism, glucocorticoid treatment of the KiSV-MC did not inhibit their production of the autocrine growth factor. During 6 days of treatment with 1 to 1000 nM dexamethasone, mast cell carboxypeptidase activity increased by a maximum of 3.5-fold. In contrast, total chymotryptic and tryptic esterase activities diminished by as much as 40% with dexamethasone treatment. We conclude that glucocorticoids directly affect mast cell growth and differentiation at levels equal to the reported Kd for glucocorticoid receptors on other immune cells.