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Am J Physiol Lung Cell Mol Physiol 277: L282-L291, 1999;
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Vol. 277, Issue 2, L282-L291, August 1999

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

Sheu-Ling Lee, Wei-Wei Wang, Geraldine A. Finlay, and Barry L. Fanburg

Pulmonary and Critical Care Division, Department of Medicine, Tupper Research Institute, and New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous studies have shown that, through an active transport process, serotonin (5-HT) rapidly elevates O-2· 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 O-2· 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, O-2· 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 O-2· formation and may be initiated by either the 5-HT transporter or receptor depending on the cell type.

signal transduction; mitogen-activated protein kinase; superoxide; smooth muscle cell; fibroblast


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REACTIVE OXYGEN SPECIES (ROS) have now been recognized to be mediators of cellular proliferation for various types of nonphagocytic cells (4, 20, 32, 34, 35, 37, 42). The intracellular release of ROS in response to ligand stimulation acts as a second messenger in signal transduction, leading to cellular growth (11, 19, 43). However, the downstream targets of endogenous ROS are largely unknown. Mitogen-activated protein (MAP) kinases are known to provide an important intermediate signal transduction pathway in response to growth factors or other stimuli that lead to cellular proliferation or hypertrophy (7, 24). The best-characterized component of this pathway is the extracellular signal-regulated kinase (ERK) cascade (5, 13, 21). The regulation of the activation of MAP kinase signal by exogenous oxidants has been reported for neutrophils (10) and various other cell types (1, 15, 41, 42), including smooth muscle cells (SMCs; see Ref. 3). In addition to the effects of exogenous ROS, inhibition of intracellular ROS by either chemical or enzymatic antioxidants has been shown to inhibit MAP kinase signal transduction (22, 42).

Serotonin (5-HT) acts as a mitogen in both vascular and nonvascular cells through multiple intermediate mechanisms that have been proposed for this action (9). We have previously shown that superoxide mediates 5-HT-induced mitogenesis and that the superoxide formation is downstream to tyrosine phosphorylation of GTPase-activating protein (GAP) activated by 5-HT (31, 32). We have also found that initial signaling for SMCs from the bovine pulmonary artery occurs via active transport of 5-HT. Other investigators have reported that mitogenesis of other cell types produced by 5-HT is dependent upon action on 5-HT receptors (9). To try to sort out this possible discrepancy and to better identify downstream signaling pathways of 5-HT in this study, we have compared 5-HT-related properties of bovine pulmonary artery SMCs with those of Chinese hamster lung fibroblasts (CCL-39 cells), which have been previously identified by other investigators to respond to 5-HT to produce mitogenesis through a 5-HT receptor(s) (39, 40).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Ginkgo biloba extract (GK) 501 (batch no. 91196) was a gift from Dr. Fabio Soldati (Pharmaton, Lugano, Switzerland). Diphenyliodonium (DPI) was from ICN Pharmaceuticals (Costa Mesa, CA). Phospho-specific p44/42 MAP kinase (Thr202/Tyr204) antibody was from New England BioLabs (Beverly, MA). ERK1 polyclonal antibody was from Santa Crutz Biotechnology (Santa Cruz, CA). CGS-120668 maleate, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI HCl), alpha -methylserotonin maleate (m-5-HT), ketanserin tartrate, pirenperone, propranolol hydrochloride, and clomipramine were from RBI (Natick, MA). PD-98059 (2'-amino-3'-methoxyflavone) was from Calbiochem-Novabiochem International (San Diego, CA). Human recombinant basic fibroblast growth factor (FGF2) was from Promega (Madison, WI). All other reagents were from Sigma Chemical (St. Louis, MO).

Cell culture. SMCs from bovine pulmonary artery were isolated and cultured by a modification of the method of Ross as previously described (30). In these experiments, third- to fifth-passage SMCs were used. Chinese hamster lung fibroblasts CCL-39 cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in McCoy's 5A medium with 10% FBS.

Incorporation of [3H]thymidine. The protocol used has been described in detail (30). In brief, plated SMCs were cultured for 72 h in RPMI medium containing 10% FBS followed by 72 h of growth arrest in medium containing 0.1% FBS. SMCs were then incubated at a density of 0.1 × 106 cells/35-mm petri dish with and without 1 µM 5-HT in the same medium for 20 h before being labeled with [methyl-3H]thymidine (0.1 mCi/ml, specific activity 20 Ci/mmol; New England Nuclear, Boston, MA) for 4 h. Iproniazid (an inhibitor of degradation of 5-HT by monoamine oxidase) or other inhibitors were added 30 min before the 5-HT. These agents alone at the concentrations reported did not alter the incorporation of [3H]thymidine by SMCs. After labeling, experiments were terminated by aspiration of medium and washing the cellular monolayer first with ice-cold PBS and then with cold 6% TCA. Cells were then dissolved in 0.2 N NaOH, and radioactivity was counted. The only modification to this protocol used for CCL-39 cells was that cells were growth arrested in McCoy's medium without FBS for 24 h followed by incubation with or without 5-HT in the presence or absence of inhibitors for 24 h and labeled with [3H]thymidine (0.5 mCi/ml) for the final 4 h.

Measurement of superoxide anion production in intact cells by a lucigenin-enhanced chemiluminescence assay. SMCs and CCL-39 cells were cultured in 100-mm petri dishes and growth arrested in medium containing 0.1% FBS. The assay was done as previously described (16, 32). 5-HT and other reagents were first added directly to the cellular monolayer. Cells were then trypsinized, pelleted by centrifugation, and resuspended in PBS containing 10 mM glucose and 1 mg/ml BSA. 5-HT and/or other reagents were then again added to the cellular suspensions in the cuvette. Cellular suspensions were loaded into a luminometer, and lucigenin (final concentration 500 µM) was automatically injected to start the reaction. A 15-s dark-adaptive period was carried out before each sample reading in the luminometer. Photoemission was recorded with 60-s integration for 5-10 min with a Lumac Biocounter M2010 (Lumac System, Titusville, FL). Buffer blank, lucigenin, or other reagents used alone in these studies produce negligible chemiluminescence. Data are expressed as degree of stimulation of chemiluminescence relative to control.

Preparation of whole cell extracts for electrophoresis. Cells were grown in 100-mm petri dishes to confluency and were growth arrested in medium containing 0.1% FBS. The cells were preincubated with inhibitors for 1-2 h before the addition of 1 µM 5-HT or other stimuli for periods indicated in RESULTS. Cellular monolayers were then washed two times with ice-cold PBS. Cell lysates were obtained by incubating the cellular monolayer in 1 ml of cell lysis buffer (47) containing 50 mM Tris · HCl, pH 7.5, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 40 µg/ml phenylmethylsulfonyl fluoride (PMSF), 0.07 µg/ml pepstatin, 1% Nonidet P-40, 150 mM NaCl, and 5 mM EDTA for 10 min at 4°C. The insoluble material was removed by centrifugation (14,000 g, 2 min), and the supernatant fraction was used for analysis. Twenty to fifty micrograms of protein of the whole cell lysate were subjected to SDS-PAGE on a 10% slab gel.

Immunoblotting with anti-phospho-specific p44/p42 MAP kinase antibody. After electrophoresis, gel proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Tropifluor, Tropix, Bedford, MA, or Immobilon-P, Millipore, Bedford, MA). After transfer, nonspecific PVDF binding sites were blocked with 5% HiPure liquid gelatin (Norland, New Brunswick, NJ) in buffer, pH 7.4, containing 75 mM sodium phosphate, 70 mM NaCl, 0.02% sodium azide, and 0.1% Tween 20. Blocking was done for 1 h at ambient temperature. The membrane was then treated overnight with a 1:1,000 dilution of alkaline phosphatase-conjugated anti-phospho-specific p44/42 MAP kinase antibody in blocking buffer at 4°C. Nitroblock (Tropix) was used according to the manufacturer's instructions. Subsequent washing, detection with the chemiluminescent substrate CSPD (disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'(5'-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate; Tropix), and film exposure were done as described previously (31). Densitometry was performed with a Millipore densitometer with Visage v4.6p software (Millipore). Data are expressed as degree of stimulation compared with control sample.

Measurement of 5-HT uptake. 5-HT uptake was measured as previously described (27, 28). The effect of hypothermia was tested by carrying out uptake experiments in the cold room at 4°C. Media used were equilibrated at 4°C before measuring uptake. For assessment of uptake in Na2+-free medium, NaCl in PBS was replaced by LiCl, and Na2HPO4 · 7H2O was replaced by tris(hydroxymethyl)aminomethane. The pH of the final solution was adjusted to 7.4 by adding 1 N KOH. The CCL-39 cell monolayer was rinsed two times with PBS containing 15 mM dextrose (pH 7.4) and was incubated for 30 min in this solution containing 0.1 mM iproniazid to block monoamine oxidase activity. 5-[3H]HT (5-[1,2-3H(N)]hydroxytryptamine creatinine sulfate, specific activity 30 Ci/mmol; New England Nuclear) was added from a stock solution containing ascorbic acid (10 µg/ml) and EDTA (10 µg/ml) as antioxidants. The 5-HT concentration used in this study was 15 nM. The uptake of 5-HT by CCL-39 cells was saturated in 20 min; therefore, incubations were carried out for 10 min. Under these conditions, radioactivity taken up by cells was no more than 1% of that in the medium. After the incubation, media were removed, and monolayers were washed three times with ice-cold PBS plus 0.1 mM imipramine (5-HT uptake inhibitor). Cells were dissolved in 0.2 N NaOH, and the radioactivity was counted.

MAP kinase activity assay. MAP kinase activity was measured in immune complexes using myelin basic protein as the substrate (44). The whole cell lysates obtained as described in Preparation of whole cell extracts for electrophoresis were used for immunoprecipitation (IP) for kinase assay. IP was performed by binding ERK1 polyclonal antibody to protein A-Sepharose beads (Repligen, Cambridge, MA), and 50 µg protein of cell lysates were added. This mixture was gently rocked at 4°C for 1 h. The precipitated immune complex was then washed one time with Triton lysis buffer (20 mM Tris buffer, pH 7.4, 137 mM NaCl, 2 mM EDTA, pH 7.4, 1% Triton X-100, 25 mM beta -glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10% glycerol, 0.2 mM PMSF with 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and one time with kinase assay buffer (125 mM HEPES, pH 7.5, 100 mM beta -glycerophosphate, 12.5 mM MgCl2, 100 mM p-nitrophenyl phosphate, and 0.5 mM sodium orthovanadate) plus protease inhibitors. The pellet was then resuspended in 20 µl of kinase assay buffer supplemented with 0.1 mg/ml of myelin basic protein (Sigma), 25 µM ATP, and 10 µCi [gamma -32P]ATP (New England Nuclear) and incubated at 30°C for 20 min. The reaction was terminated by addition of 7 µl of 4× Laemmli sample buffer. Samples were then boiled for 5-10 min, centrifuged, and resolved by 12.5% SDS-PAGE. Gels were dried and quantitated by ImageQuant software on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Data are expressed as degree of kinase activity relative to quiescent control.

Quantitation. Each treatment was carried out in at least triplicate experiments, and a representative experiment is shown in Figs. 1-10. Values are means ± SD (n = 4). Unpaired Student's t-test was used for determination of the significance of the results.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously reported that 5-HT elevates superoxide formation, stimulates protein phosphorylation, and enhances proliferation of bovine pulmonary artery SMCs in culture (30-32). Exposure of these SMCs to 1 µM 5-HT also rapidly elevated phosphorylation of both p44 and p42 (ERK1 and ERK2) MAP kinases by 7- to 10-fold within 5 min (Fig. 1A). This elevation returned to baseline by 1 h. Omission of Na+ from the cellular medium prevented the stimulation of the MAP kinases by 5-HT (Fig. 1B). PD-98059, a specific inhibitor of MAP kinase kinase (2, 8), dose dependently inhibited stimulation of DNA synthesis by 5-HT (Fig. 2). Thus 5-HT-induced mitogenesis of SMCs appears to occur via signaling through a MAP kinase-dependent pathway. Antioxidants such as Tiron [4,5-dihydroxy-(1,3-benzene disulfonic acid)], NAC, and GK dose dependently inhibited 5-HT-stimulated DNA synthesis of SMCs (23, 32, 33) and also blocked the 5-HT-elevated phosphorylation of ERK1 and ERK2, as shown in Fig. 3. However, the MAP kinase inhibitor PD-98059, which had no quenching effect on O-2· generated from interaction of xanthine (60 µM) with xanthine oxidase (2 mU/ml; unpublished data), also failed to show any effect on O-2· released from SMCs incubated with 5-HT.


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  		Fig. 1.   A: time course of serotonin (5-HT)-induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase of smooth muscle cells (SMCs) [extracellular signal-regulated kinase (ERK) 1 (open bars)/ERK2 (filled bars), respectively]. Immunoblotting and densitometry were done as described in MATERIALS AND METHODS. Data are expressed as degree of stimulation compared with control (Contl) sample. B: lack of influence of 5-HT on phosphorylation of p44/p42 when SMCs are incubated in Na+-free medium.



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  		Fig. 2.   Inhibition by PD-98059 of 5-HT-induced DNA synthesis of SMCs. Experiments were done as described in MATERIALS AND METHODS. Values are means ± SD (n = 4 dishes), cpm, Counts/min; TdR, thymidine. Significantly different (P < 0.05) from: * quiescent control; ** 5-HT alone.



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  		Fig. 3.   Inhibitory effect of PD-98059 (PD; 10 µM), Tiron (2 mM), N-acetyl-L-cysteine (NAC; 10 mM), and Ginkgo biloba (GK; 200 µg/ml) on 5-HT-induced phosphorylation of p44/p42 of SMCs. Experiments were done as described in MATERIALS AND METHODS.

With the use of various inhibitors of 5-HT uptake, we have previously concluded that 5-HT produces its mitogenic effect on vascular SMCs through action on a 5-HT transporter (30). On the other hand, various other studies have reported that 5-HT produces a mitogenic response through action on 5-HT receptors (9). One such report is that of Seuwen and colleagues (39, 40), which showed that 5-HT stimulates mitogenesis of CCL-39 fibroblasts by action on a 5-HT1B receptor. We have confirmed the mitogenic effect of 5-HT on CCL-39 cells (Fig. 4) and have also found that this effect is blocked by a number of inhibitors of 5-HT transport, including imipramine, clomipramine, and fluoxetine (Fig. 4). This observation prompted us to study the 5-HT transport activity of CCL-39 cells. As shown in Fig. 5A, CCL-39 cells, like SMCs, rapidly accumulate 5-HT, and the uptake is blocked by a variety of agents and conditions that are known to inhibit 5-HT transport (28, 29), including the use of Na2+-free medium, cold temperature, and agents such as imipramine, propranolol, ketanserin, and verapamil (Fig. 5B).


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  		Fig. 4.   Effect of 5-HT transporter inhibitors [imipramine (IMP), clomipramine (Clom), and fluoxetine (Flu)] and 5-HT2 receptor antagonists [ketanserin (Ket) and pirenperone (Pir)] on 5-HT-induced DNA synthesis of CCL-39 cells. Experiments were done as noted in MATERIALS AND METHODS. Concentrations of inhibitors as follows: 0.1 mM for imipramine and 10 µM for the others. Values are means ± SD (n = 4 dishes). Significantly different (P < 0.05) from: * quiescent control; ** 5-HT alone.




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  		Fig. 5.   5-HT uptake by CCL-39 cells. A: time course of 5-HT uptake of CCL-39 cells at 15 nM 5-HT. B: 5-HT transport by CCL-39 cells is inhibited by Na+-free medium, cold temperature, and uptake inhibitors such as imipramine (0.1 mM), propanolol (Prop), ketanserin, and verapamil (Verp) at 10 µM concentration. Values are means ± SD (n = 4 dishes). * Significantly different from control, P < 0.05.

DOI and m-5-HT are 5-HT analogs that are thought to act through stimulation of a 5-HT2 receptor. Contrary to their failure to stimulate proliferation of SMCs (Fig. 6), these agents produced a two- to threefold stimulation of proliferation of CCL-39 cells (Fig. 7). The CCL-39 cells also showed a mitogenic response to CGS-12066B dimaleate, a specific 5-HT1B-receptor agonist (Fig. 7).


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  		Fig. 6.   Failure of stimulation of mitogenic response of SMCs by 5-HT receptor agonists [CGS (5-HT1B receptor), (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and alpha -methylserotonin maleate (m-5-HT; 5-HT2 receptor)] in contrast to a 6- to 7-fold stimulation of DNA synthesis induced by 5-HT. Values are means ± SD (n = 4 dishes). * Significantly different from quiescent control, P < 0.05.



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  		Fig. 7.   Inhibitory effect of PD-98059 on the mitogenic response of CCL-39 cells to 5-HT, CGS-120668 (CGS), DOI, and m-5-HT. Experiments were done as noted in MATERIALS AND METHODS. Values are means ± SD (n = 4 dishes). Significantly different (P < 0.05) from: * quiescent control; ** stimulated control.

Both 5-HT and m-5-HT produced a rapid increase in phosphorylation of ERK1 and ERK2 by 10- to 20-fold in CCL-39 cells (Fig. 8A). All of these stimulations of ERK1 and ERK2 were blocked by both 5-HT uptake inhibitors (imipramine, clomipramine, and fluoxetine) and 5-HT2-receptor antagonists (ketanserin and pirenperone) as shown in Fig. 8, Aa and Ab, respectively. MAP kinase activity of CCL-39 cells was elevated by 5-HT and m-5-HT 5.5- and 3.4-fold, respectively; and this stimulation was inhibited by 5-HT transporter or receptor inhibitors (Fig. 8B).



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  		Fig. 8.   Inhibitory effect of 5-HT transporter inhibitors (imipramine, clomipramine, and fluoxetine) and 5-HT2-receptor antagonists (ketanserin and pireperone) on phosphorylation of p44/p42 (A) and induced MAP kinase activity (B) by 5-HT (lanes 2 and 4-6) and m-5-HT (lanes 3, 7, and 8) vs. control (lane 1) in CCL-39 cells. Treatments in the immunoblot of B correspond with those in the densitometric results. Experiments were done as noted in MATERIALS AND METHODS. Concentrations of inhibitors as follows: 0.1 mM imipramine and 10 µM for the others. Densitometric results in A are phosphorylation of 5-HT ± inhibitors (a) and m-5-HT ± receptor antagonists (b). Open bars, p44; hatched bars, p42.

We assessed the importance of MAP kinase in the proliferative process of CCL-39 cells stimulated by 5-HT and 5-HT agonists. As shown in Fig. 7, [3H]thymidine incorporation of these cells induced by 5-HT and receptor agonists was inhibited by PD-98059, a known inhibitor of MAP kinase.

We have previously reported that O-2· is important in 5-HT signaling of mitogenesis in SMCs (32). Therefore, we tested a variety of antioxidants and an inhibitor of NAD(P)H oxidase (DPI) on the responses of CCL-39 cells to 5-HT. DPI alone stimulated [3H]thymidine incorporation. Tiron, DPI, GK, and NAC dose dependently inhibited both [3H]thymidine incorporation and phosphorylation of ERK1 and ERK2 induced by 5-HT in CCL-39 cells (Fig. 9, A-D). Similar to our observations with SMCs (30), 5-HT also acted synergistically with 10 ng/ml FGF2 in stimulating DNA synthesis and activating ERK1 and ERK2 of CCL-39 cells (Fig. 10, A and B). Tiron, NAC, DPI, and GK inhibited O-2· generation from this cell type when it was incubated with 5-HT plus FGF2 (Fig. 10C). These antioxidants also inhibited both DNA synthesis (Fig. 10A) and the activation of ERK1 and ERK2 (Fig. 10B). PD-98059, however, showed no effect on release of O-2· from CCL-39 cells produced by treatments with either 5-HT (data not shown) or with 5-HT plus FGF2 (Fig. 10C) but highly inhibited DNA synthesis induced by these agents.





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  		Fig. 9.   Inhibitory effect of antioxidants (Tiron, NAC, and GK) and diphenyliodonium (DPI) on 5-HT-induced DNA synthesis (A and B) and phosphorylation of p44/p42 (C) of CCL-39 cells induced by either 5-HT (a) or m-5-HT (b). Open bars, p44; hatched bars, p42. Concentrations of inhibitors in C as follows: 10 µM PD-98059, 150 µg/ml GK, 2 mM Tiron, and 10 mM NAC. DPI (0.1 mM) inhibits phosphorylation of p44/42 as shown in D. Experiments were done as noted in MATERIALS AND METHODS. Values are means ± SD (n = 4 dishes). Significantly different (P < 0.05) from: * quiescent control; ** 5-HT alone.





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  		Fig. 10.   Effect of 1 µM 5-HT plus 10 ng/ml fibroblast growth factor (FGF2) on DNA synthesis (A) and phosphorylation of p44/p42 of CCL-39 cells (B). Antioxidants (2 mM Tiron, 10 mM NAC, and 150 µg/ml GK) and 0.1 mM DPI inhibit stimulated DNA synthesis, phosphorylation of p44/p42, and O-2· release from CCL-39 cells incubated with 5-HT plus FGF2. However, PD-98059 (10 µM) fails to show any inhibitory effect on O-2· release (C). Experiments were done as noted in MATERIALS AND METHODS. Values are means ± SD (n = 4 dishes). Significantly different (P < 0.05) from: * quiescent control; ** 5-HT plus FGF2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

From the studies presented and other data we have published, some general statements can be made about at least part of the sequence of events whereby 5-HT signals cells to proliferate. Depending upon the cell type, the data indicate that the initial signal occurs either through internalization of 5-HT via the 5-HT transporter or by way of a 5-HT receptor on the cell surface. GAP is phosphorylated early in the series of subsequent events (31), then O-2· is formed intracellularly (32), possibly through activation of p21ras (38). In response to the O-2· formation, MAP kinases, specifically ERK1 and ERK2, are phosphorylated and activated.

Whether the initiating signal occurs through a 5-HT transporter or a 5-HT receptor has been controversial. The 5-HT transporter and the 5-HT receptor(s) are different molecules that have now been cloned separately (9). Their functional properties in cells have been largely defined with the use of well-characterized transport inhibitors or receptor antagonists along with use of other known features of an active transport process, such as dependence on Na+ and inhibition by cold. All of our data obtained with bovine pulmonary artery SMCs have clearly defined the participation of a 5-HT transporter, internalization of 5-HT, and a requirement for a pertussis toxin-insensitive G protein (9) in the initiation of a proliferation process for this cell type (30).

Because other cells, including Chinese hamster lung fibroblasts, have been described by other investigators to show a proliferative response to 5-HT through a more conventional cell membrane receptor action (as has been described for growth factors in general), we undertook in the present experiments to study for the first time the ability of the CCL-39 fibroblasts to actively transport 5-HT. These studies showed that the CCL-39 cells, indeed, actively transport 5-HT and that the uptake process for these cells is also, like that of the bovine pulmonary artery SMCs, a mechanism by which proliferation is initiated. A confusing factor for these cells is the observation that, unlike SMCs, they also show a mitogenic response to purported 5-HT receptor agonists, and the mitogenic response to 5-HT is inhibited by 5-HT receptor antagonists. The results of these pharmacological studies are somewhat difficult to interpret but suggest that more than one 5-HT receptor type may exist for CCL-39 cells and may be responsive to 5-HT. Why this cell type may react to 5-HT and show a proliferative response through either a 5-HT receptor or transporter is unclear. The transporter action appears to predominate, since all methods to inhibit transport of 5-HT, including exposure to cold and removal of Na+ from the medium, strongly block cellular proliferation caused by 5-HT.

Regardless of the initiating mechanism, both SMCs and the CCL-39 fibroblasts show stimulation of formation of O-2· by 5-HT, and uses of antioxidants such as Tiron, NAC, and GK all block the proliferative process. DPI, which is a nonspecific inhibitor of NAD(P)H oxidase, also blocks the process, suggesting that a NAD(P)H oxidase may participate in the signaling mechanism. Similarly, both cell types rapidly respond to 5-HT by showing enhanced phosphorylation of the MAP kinases, ERK1, ERK2, and MAP kinase activity, which are blocked by antioxidants. Activation of MAP kinase is generally believed to be regulated by Ras protein (6, 17). 5-HT activation of ERK1 and ERK2 has been reported to occur in bovine tracheal SMCs and CHO-K1 fibroblasts through actions on 5-HT1A, 5-HT2, and 5-HT2B receptors (14, 18, 26) and in 5-HT-induced vascular contraction through a 5-HT2A receptor (12, 45, 46). Our studies show that this action can also happen through a 5-HT transporter.

It was recently recognized that superoxide relays the oncogenic message of Ras protein for cellular growth (36). Lander et al. (25) proposed that p21ras is a common signaling target of ROS and cellular redox stress. Our data show that antioxidants have no effect on the 5-HT-induced elevation of tyrosine phosphorylation of GAP (33) but do inhibit MAP kinase activation. This finding is consistent with previous observations that NAC suppresses the phosphorylation of MAP kinase induced by platelet-derived growth factor (42) and nerve growth factor (22) in cell types different from ours. Inhibition of 5-HT-induced cellular proliferation by the MAP kinase inhibitor PD-98059 supports the role of MAP kinase in the proliferative process produced by 5-HT. Similarly, failure to block 5-HT-induced O-2· formation with PD-98059 indicates that O-2· formation is upstream to phosphorylation of ERK1 and ERK2. Thus these and our other previous data (31-33) strongly suggest that O-2· formation is downstream to tyrosine phosphorylation of GAP and upstream to MAP kinase activation. Superoxide formation appears to be a critical step in signaling 5-HT-induced cellular growth.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-32723.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S.-L. Lee, New England Medical Center, Pulmonary and Critical Care Division, 750 Washington St., NEMC #265, Boston, MA 02111.

Received 28 December 1998; accepted in final form 6 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(2):L282-L291
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