Sheu-Ling Lee

Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture.

We have previously reported that serotonin (5-hydroxytryptamine [5HT]) alters cultured bovine pulmonary artery smooth muscle cell (SMC) configuration through two different regulatory mechanisms. We now report that 5HT also regulates SMC growth through these same two mechanisms--a stimulatory event initiated intracellularly and inhibition of growth resulting from a cell surface action. 5HT (1 microM) plus 0.1 mM iproniazid (a 5HT metabolic inhibitor) produced a severalfold stimulation of DNA synthesis (as measured by [3H]thymidine incorporation) of SMCs after a 17-24-hour incubation with only a slight elevation of cellular cAMP. This stimulatory effect responded synergistically with other growth factors including platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor and was effectively reversed by 5HT uptake inhibition. It was not produced by 5-hydroxyindoleacetic acid, a metabolite of 5HT. In the presence of 1 microM 5HT plus 0.1 mM isobutylmethylxanthine (IBMX), cAMP was elevated eightfold, dendritic formation occurred, and [3H]thymidine labeling of SMCs was inhibited. Inhibition of labeling by [3H]thymidine was mimicked by other agents that elevated cellular cAMP (10 microM histamine, 1 microM isoproterenol plus 0.1 mM IBMX, and 10 microM forskolin) and by 1 mM dibutyryl cAMP. This inhibitory effect was not blocked by either inhibition of 5HT uptake or 5HT-receptor antagonists ketanserin (5HT2); methiothepin, spiperone, and mianserin (5HT1/5HT2); and 3-tropanyl-indole-3-carboxylate and 3-tropanyl-3,5-dichlorobenzoate (5HT3). However, similar to 5HT, the 5HT1A agonist, (+/-)-8-hydroxy-(+/-)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalenehydrobromide, in association with IBMX, produced an elevation in cAMP and inhibition of labeling by [3H]thymidine. 5HT, in the presence of either iproniazid or IBMX, did not alter [Ca2+]i, indicating that [Ca2+]i was not a signal for either of these actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion

Our previous studies have shown that, through an active transport process, serotonin (5-HT) rapidly elevates[Formula: see text] formation, stimulates protein phosphorylation, and enhances proliferation of bovine pulmonary artery smooth muscle cells (SMCs). We presently show that 1 μM 5-HT also rapidly elevates phosphorylation and activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) 1 and ERK2 of SMCs, and the enhanced phosphorylation is blocked by the antioxidants Tiron, N-acetyl-l-cysteine (NAC), and Ginkgo biloba extract. Inhibition of MAP kinase with PD-98059 failed to block enhanced[Formula: see text] formation by 5-HT. Chinese hamster lung fibroblasts (CCL-39 cells), which demonstrate both 5-HT transporter and receptor activity, showed a similar response to 5-HT (i.e., enhanced mitogenesis, [Formula: see text]formation, and ERK1 and ERK2 phosphorylation and activation). Unlike SMCs, they also responded to 5-HT receptor agonists. We conclude that downstream signaling of MAP kinase is a generalized cellular response to 5-HT that occurs secondary to[Formula: see text] formation and may be initiated by either the 5-HT transporter or receptor depending on the cell type.

SUPEROXIDE AS AN INTERMEDIATE SIGNAL FOR SEROTONIN-INDUCED MITOGENESIS

Serotonin (5-HT) stimulates tyrosine phosphorylation and proliferation of bovine pulmonary artery smooth muscle cells (SMC) through its active transport (Lee et al, 1991). The present studies show that 5-HT also rapidly elevates O2.- formation by these cells within 10 minutes as measured by a lucigenin-enhanced chemiluminescence assay. The O2.- free radical quencher, Tiron, and N-acetyl-cysteine, a substrate for glutathione, block both the 5-HT-induced formation of O2.- and cellular proliferation. Similarly, inhibition of 5-HT transport with imipramine or treatment of cells with diphenyliodonium, a NAD(P)H oxidase inhibitor, block both 5-HT-induced elevation of O2.- and cellular proliferation. Alpha-hydroxyfarnesylphosphonic acid, an inhibitor of p21ras, also blocks 5-HT-induced proliferation. Endothelial cells from the same vessel show neither 5-HT-induced proliferation nor stimulation of O2.- formation. We conclude that 5-HT induced cellular proliferation of SMC through signaling pathways that utilize its transport system and O2.- formation.

Glycolytic activity and enhancement of serotonin uptake by endothelial cells exposed to hypoxia/anoxia.

Serotonin (5-HT) uptake by bovine pulmonary artery endothelial cells in culture was stimulated several fold by exposure of cells for 24 hours to 0–3% O2. The cells appeared normal morphologically, excluded trypan blue dye, showed normal levels of release of lactate dehydrogenase, and contained normal amounts of adenosine triphosphate (ATP). Incubation of cells with iodoacetate (3–5 μM), an inhibitor of glycolytic metabolism, prevented the stimulation of 5-HT uptake but did not impair cellular proliferation. A similar, but less pronounced, effect was produced by 1 mM deoxyglucose. On the other hand, the mitochondrial inhibitors antimycin A, sodium azide, potassium cyanide, and 2,4-dinitrophenol inhibited cellular replication similar to the effect of anoxia but did not alter the stimulation of 5-HT uptake. 5-HT uptake correlated strongly with lactate production by the cells exposed to anoxia (r = 0.98). We conclude that stimulation of 5-HT uptake by prolonged exposure of the endothelial cell to hypoxia/anoxia causes alteration of a membrane function (serotonin transport) of the endothelial cell. This alteration is associated with enhanced glycolytic activity of the cell during exposure to hypoxia. The mechanism for translation of enhanced glycolysis to later stimulation of 5-HT uptake remains to be determined. Endothelial cellular replication is dependent on aerobic metabolism.

Dexfenfluramine as a mitogen signal via the formation of superoxide anion

Fenfluramine and its isomer, dexfenfluramine (d‐Fen), have been used extensively as appetite suppressants and have been associated with pulmonary hypertension and cardiac valvular insufficiency. The findings have been linked to alteration in levels of serotonin (5‐HT), but these agents also directly influence K+ channels and intracellular Ca2+. Our study shows for the first time that d‐Fen also produces a mitogenic effect on fibroblasts in culture. The mitogenic effect occurs at 10–100‐µM concentrations; includes both cellular hyperplasia (assayed by [3H]‐thymidine incorporation and cell counts) and hypertrophy (assayed by [3H]‐leucine incorporation and Coulter counter analysis); and is initiated by cell signaling through a rapid elevation of superoxide, protein tyrosine phosphorylation, and ERK1/ERK2 MAP kinase activation, similar to the cellular effects produced by 5‐HT. The enhanced [3H]‐thymidine incorporation and ERK1/ERK2 MAP kinase activation are blocked by an antioxidant (Tiron), a NADPH oxidase inhibitor (DPI), a tyrosine phosphorylation inhibitor (tyrphostin), and a MAP kinase kinase inhibitor (PD 98059), which suggests that reactive oxygen species and the MAP kinase pathway play critical roles in the d‐Fen‐induced mitogenesis. Blockage of the effect by 5‐HT transporter inhibitors and 5‐HT receptor antagonists suggests that d‐Fen shares a common cellular ligand and signaling pathway with 5‐HT.

A role for the serotonin transporter in hypoxia-induced pulmonary hypertension

Serotonin (5-hydroxytryptamine; 5-HT) is one of many vasoactive substances postulated to participate in the development of hypoxia-induced pulmonary hypertension. Pulmonary vasoactive responses to hypoxia are intensified by 5-HT (1), but attempts to block hypoxia-induced pulmonary hypertension with 5-HT receptor antagonists have met with mixed success. Furthermore, it has been difficult to establish a causal relationship between 5-HT and the physiological response to hypoxia. Lacking in much of this work is a clear distinction between two classes of molecules, 5-HT transporters and 5-HT receptors, either or both of which may participate in the response of pulmonary vascular smooth muscle cells to 5-HT. High levels of 5-HT have been associated with pulmonary hypertension in several systems. Herve et al. (2) described a patient with pulmonary hypertension who had high levels of circulating 5-HT due to a platelet storage disease, and other individuals have been described with primary pulmonary hypertension associated with elevated serum 5-HT levels (3). In the fawn-hooded rat, an animal model for platelet storage disease that exhibits high circulating levels of 5-HT, mild hypoxia also leads to pulmonary hypertension (4, 5). Finally, pulmonary hypertension is also associated with appetite suppressants, such as fenfluramine or dexfenfluramine, which block reuptake of 5-HT.

Superoxide scavenging effect of Ginkgo biloba extract on serotonin-induced mitogenesis

We have reported previously that serotonin (5-HT) stimulates the mitogenesis of bovine pulmonary artery smooth muscle cells (SMCs) through active transport of 5-HT and cellular signaling that includes elevation of superoxide (O2.-) and enhancement of protein tyrosine phosphorylation. Ginkgo biloba extract 501 (EGb 501), which has been demonstrated to act as an antioxidant, was found to block both the elevated O2.- and the proliferative and hypertrophic influences of 5-HT on SMCs, but not to directly inhibit the associated activation of NAD(P)H oxidase or the stimulation of phosphorylation of GTPase-activating protein (GAP). A similar effect of Ginkgo biloba extract 501 occurred on Chinese hamster lung fibroblasts (CCL-39), where 5-HT receptor, as opposed to transporter, action has been associated with mitogenesis. We conclude from these studies that Ginkgo biloba extract 501 quenches O2.- formation by 5-HT, thereby blocking its mitogenic effect. Stimulation of protein tyrosine phosphorylation of GAP by 5-HT appears to precede the elevation of O2.-.

Reduction of Endothelial Cell Related TGFβ Activity by Thiols

Transforming growth factor β (TGFβ) may play an important role in diseases characterized by pulmonary fibrosis. We have previously demonstrated that thiols inhibit the pro-oxidant effects of TGFβ1 in bovine pulmonary artery endothelial cells (BPAEC). To help define the mechanism of this observation we have examined the effect of reduced (GSH) and oxidized (GSSG) glutathione, N-acetyl cysteine (NAC) and cysteine (CYS) on the biological activity of a) TGFβ released by bovine pulmonary artery endothelial cells (BPAEC) into culture medium, and b) commercially available porcine platelet TGFβ1. The biological activity of TGFβ (following activation) released into the medium from cultured BPAEC was significantly reduced when the cells were cultured in the presence of 10 mM GSH or 10 mM NAC for 24 h (10 mM GSH: 85.7 ± 50 pg/ml/106 cells and 10 mM NAC: 127.3 ± 35 pg/ml/106 cells, compared with control: 541 ± 8.9 pg/ml/106 cells; p < 0.05). Thiols (10 mM GSH, 10 mM NAC and 5 mM cysteine), added directly to cell-free...

Ultrastructural changes of bovine pulmonary artery endothelial cells irradiated in vitro with a 137Cs source

Bovine pulmonary artery endothelial cells in culture were evaluated by phase-contrast and electron microscopy at various times after being irradiated with 137Cs in vitro. Cells irradiated prior to reaching confluency showed vacuolization aand increased numbers of lysosomes beginning at 48 hr after irradiation with 300-500 rad and at 24 hr after irradiation with 1500-5000 rad. After 7 days the morphological changes appeared to be reversible for cells receiving the lower doses, but were progressive for higher doses of radiation. The same qualitative changes, with a delayed onset, were observed for cells irradiated at confluency. An observed decrease in the endoplasmic reticulum and polysomes occurred only late in the course of radiation injury. There was no observable structural alteration of mitochondria even when there was evidence of otherwise marked cytoplasmic injury. We conclude that structural changes of the lysosomes constitute an early phase of injury by irradiation of the endothelial cell in culture, while decreases in endoplasmic reticulum and polysomes occur relatively late. The mitochondrial structure of the endothelial cell appears to be relatively resistant to radiation. All morphological changes occur subsequent to impaired transport of alpha-aminoisobutyric acid, which is observed within 6 hr as previously reported.

Ultrastructural changes in bovine pulmonary artery endothelial cells exposed to 80% O2 in vitro.

SummaryBovine pulmonary artery endothelial cells in culture were exposed for up to 7 d to a gas mixture containing 80% O2, 5% CO2, and 15% N2 (hyperoxia) and were compared by phase contrast and electron microscopy to cells exposed to a gas mixture containing 20% O2, 5% CO2, and 75% N2. Cells exposed to hyperoxia became enlarged and showed vacuolization and increased lysosomes within 24 to 48 h. These changes were progressive over the 7 d period of exposure. Between 3 and 7 d of exposure to hyperoxia the cells showed reductions in polysomes and endoplasmic reticulum. Despite the other marked cytoplasmic changes, the appearance of mitochondria of oxygen-exposed cells remained unchanged from those of air-exposed cells throughout the 7 d period. Preconfluent and confluent cells responded qualitatively similarly to hyperoxia, but morphological evidence of injury occurred more rapidly for preconfluent cells. We conclude that the initial early structural injury of the endothelial cell exposed to hyperoxia occurs in lysosomes and that the mitochondrial structure is relatively resistant to injury.