HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/7/1324 00-0431fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LEE, S.-L. Articles by FANBURG, B. L. Search for Related Content PubMed PubMed Citation Articles by LEE, S.-L. Articles by FANBURG, B. L.

Dexfenfluramine as a mitogen signal via the formation of superoxide anion ¹

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

²Correspondence: New England Medical Center, Pulmonary and Critical Care Division, 750 Washington St., #257, Boston, MA 02111, USA. E-mail:bfanburg{at}lifespan.org

SPECIFIC AIMS

Fenfluramine (l-Fen) and its dextro-rotary stereoisomer dexfenfluramine (d-Fen) have been used extensively as appetite suppressants and have been associated with the development of both pulmonary hypertension and fibrosis of valves of the heart. Mitogenesis of fibroblasts and smooth muscle cells in the resulting pathology has been postulated to occur by blockage of serotonin (5-HT) reuptake by fenfluramine. The present study was undertaken to determine whether d-Fen itself is a direct cellular mitogen and, if so, to identify the signaling pathways through which the mitogenesis occurs.

PRINCIPAL FINDINGS

1. d-Fen produces a mitogenic effect and superoxide anion (O₂^.-) release from fibroblasts (CCL-39 cells)
d-Fen, but not l-Fen, dose-dependently stimulates cellular [³H]-thymidine and [³H]-leucine incorporation and induces cellular hyperplasia and hypertrophy after 7 days. d-Fen rapidly induces a twofold and long-lasting elevation of superoxide anion (O₂^.-) release from these cells. Both O₂^.- stimulation and cellular mitogenesis are inhibited by the flavoprotein inhibitor DPI.

2. d-Fen activates phosphorylation of ERK1/ERK2 MAP kinase
d-Fen rapidly activates ERK1 and ERK2 (p44/p42) MAP kinases. Chemical antioxidants [NAC, Tiron, and Ginkgo biloba extract (GK)]; a NADPH oxidase inhibitor, DPI; a MAP kinase inhibitor, PD98059 (PD); and a tyrosine kinase inhibitor, tyrphostin (Tyr) inhibit both d-Fen- induced ERK1/ERK2 MAP kinase activation (Fig. 1 ) and [³H]-thymidine incorporation.

View larger version (26K): [in this window] [in a new window]   Figure 1. d-Fen induces a rapid phosphorylation of p44/p42 MAP kinase that is highly inhibited by 1 µM PD98059 (PD), 10 mM NAC, 100 µM Tiron, 10 µM DPI, 10 µM tyrphostin (Tyr), 200 µg/ml Ginkgo biloba extract (GK), and 10 µM of imipramine (Imp), fluoxetine (Flu), ketanserin (Ket), or pirenperone (Pir).

3. Inhibitory effect of 5-HT transporter inhibitors and 5-HT2 receptor antagonists on d-Fen-induced mitogenesis and ERK1/ERK2 MAP kinase activation
Preincubation of cells with 5-HT transporter inhibitors (imipramine, fluoxetine, clomipramine, and zimelidine) and 5-HT2 receptor antagonists (ketanserin and pirenperone) inhibit d-Fen-induced ERK1/ERK2 MAP kinase activation (Fig. 1) and [³H]-thymidine incorporation.

CONCLUSIONS

It has been speculated that pulmonary hypertension and heart valvular disease associated with appetite suppressants are caused by elevated serum levels of 5-HT produced by these agents. Although 5-HT has been reported to act as a mitogen for smooth muscle cells (SMCs), fibroblasts, and mesangial cells in culture, fenfluramine has never been studied for its direct cellular effect other than its inhibition of K⁺ channels and increase in intracellular Ca²⁺ of SMCs. The present study shows for the first time that d-Fen, but not l-Fen, produces a mitogenic effect on fibroblasts in culture and signals through pathways that mimic those induced by 5-HT. Inhibition of d-Fen-induced ERK1/ERK2 MAP kinase activation and [³H]-thymidine incorporation by antioxidants DPI, PD98059, and tyrphostin suggests that reactive oxygen species and ERK MAPK play a critical role in the d-Fen-induced mitogenesis. The cellular ligand for fenfluramine has not been identified. The response of CCL-39 cells to d-Fen with protein tyrosine phosphorylation, O₂^.- formation, and ERK MAP kinase phosphorylation is identical to the signaling pathway we have previously described for 5-HT. We tested whether 5-HT transporter inhibitors, 5-HT2 receptor antagonists, and 5-HT itself have any effect on d-Fen-induced mitogenesis. Our data show that 5-HT transporter inhibitors and 5-HT2 receptor antagonists reduce, whereas 5-HT itself additively stimulates, d-Fen-induced cellular mitogenesis, suggesting that the stimulatory effect of d-Fen is regulated through a ligand similar or identical to that activated by 5-HT (Fig. 2 ). Our findings are consistent with recent reports that d-Fen and other anorexic agents known or suspected to increase the risk of pulmonary hypertension are 5-HT transporter substrates and that 5-HT2B receptors might also be associated with fenfluramine-induced cardiopathy. Further, antidepressants with a high affinity for the 5-HT transporter have been predicted to reduce the risk of developing d-Fen-associated pulmonary hypertension.

View larger version (10K): [in this window] [in a new window]   Figure 2. Schematic diagram of cellular transduction pathway for d-Fen.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0431fje ; to cite this article, use FASEB J. (March 28, 2001) 10.1096/fj.00-0431fje

This article has been cited by other articles:

				Y. Dempsie, I. Morecroft, D. J. Welsh, N. A. MacRitchie, N. Herold, L. Loughlin, M. Nilsen, A. J. Peacock, A. Harmar, M. Bader, et al. Converging Evidence in Support of the Serotonin Hypothesis of Dexfenfluramine-Induced Pulmonary Hypertension With Novel Transgenic Mice Circulation, June 3, 2008; 117(22): 2928 - 2937. [Abstract] [Full Text] [PDF]

				A. E. Linder, W. Ni, T. Szasz, R. Burnett, J. Diaz, T. J. Geddes, D. M. Kuhn, and S. W. Watts A Serotonergic System in Veins: Serotonin Transporter-Independent Uptake J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 714 - 722. [Abstract] [Full Text] [PDF]

				E. K. Weir, M. Obreztchikova, and Z. Hong Fenfluramine: riddle or Rosetta stone? Eur. Respir. J., February 1, 2008; 31(2): 232 - 235. [Full Text] [PDF]

				E. D. Willers, J. H. Newman, J. E. Loyd, I. M. Robbins, L. A. Wheeler, M. A. Prince, K. C. Stanton, J. A. Cogan, J. R. Runo, D. Byrne, et al. Serotonin Transporter Polymorphisms in Familial and Idiopathic Pulmonary Arterial Hypertension Am. J. Respir. Crit. Care Med., April 1, 2006; 173(7): 798 - 802. [Abstract] [Full Text] [PDF]

				Y. J. Suzuki, R. M. Day, C. C. Tan, T. H. Sandven, Q. Liang, J. D. Molkentin, and B. L. Fanburg Activation of GATA-4 by Serotonin in Pulmonary Artery Smooth Muscle Cells J. Biol. Chem., May 2, 2003; 278(19): 17525 - 17531. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/7/1324 00-0431fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LEE, S.-L. Articles by FANBURG, B. L. Search for Related Content PubMed PubMed Citation Articles by LEE, S.-L. Articles by FANBURG, B. L.