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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 1, July 1997 78-83

Inhibitory Effect of Heparin on Serotonin-induced Hyperplasia and Hypertrophy of Smooth Muscle Cells

Sheu-Ling Lee, Wei-Wei Wang, Patricia M. Joseph, Charles A. Hales, and Barry L. Fanburg

Pulmonary and Critical Care Divisions, Department of Medicine, New England Medical Center/Tufts University School of Medicine; and Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Serotonin (5-HT) produces both hyperplastic and hypertrophic effects on smooth muscle cell (SMC) in culture. Heparin is known to inhibit serum-induced hyperplasia of SMC but has not been previously tested on the stimulatory effect of 5-HT on SMC. Our present data show that at 24 h heparin inhibited by 50% the stimulation of 3H-thymidine incorporation into bovine pulmonary artery SMC and at 7 days totally reversed both cellular proliferation and enlargement of SMC produced by 1 µM 5-HT. Heparin failed to alter 5-HT uptake by SMC, but inhibited the stimulation of tyrosine phosphorylation of GTPase-activating protein, a proposed intermediate in the 5-HT stimulatory process. Thus heparin inhibits both hyperplastic and hypertrophic effects of 5-HT on SMC, perhaps through the inhibition of a phosphorylated intermediate protein.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Inhibition of of smooth muscle cell (SMC) proliferation by heparin that occurs both in vivo and in vitro is now a well recognized phenomenon (1). The antiproliferative action of heparin has been observed in SMC, mesangial cells, fibroblasts, and epithelial cells (5). Blockade of the cell cycle by heparin occurs either at the G0/G1 transition point (5) or at mid- to late-G1 progression (10). A variety of cellular intermediate processes such as protein kinase C (PKC) activation (9, 14), c-fos and c-myc gene expression, activator protein-1 (AP-1)/fos-jun binding activity (15), and AP-1 binding by post-translational modification of jun B (16) have been noted to be blocked by heparin. Recently, Dahlberg and colleagues (17) reported that antiproliferation of heparin correlated well with the inhibition of the rise of intracellular pH induced by recombinant human platelet-derived growth factor (PDGF) activated via a Na+/H+ antiporter. However, the precise mechanism of heparin's antiproliferative effect is still uncertain. The antiproliferative action of heparin on SMC has been demonstrated previously, primarily in serum-stimulated cells. The inhibition by heparin of cellular hypertrophy produced by angiotensin II was reported only recently in cardiomyocytes (18). We have found that serotonin (5-HT) is mitogenic for bovine pulmonary artery SMC producing both hyperplasia and hypertrophy through its action on a 5-HT membrane transporter (19), with a rapid elevation in tyrosine phosphorylation (Tyr-P) of GTPase-activating protein (GAP) (20) and early inductions of c-myc and alpha - and beta -actins (19). In this report we have evaluated whether the hyperplasia and hypertrophy of SMC induced by 5-HT might be reversed by heparin and, if so, the possible mechanism by which this may occur.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

Heparins were obtained from Sigma Chemical Co. (lot 64H0474; St. Louis, MO), Upjohn Co. (U-1394, lot 1438F; Kalamazoo, MI), Elkins-Sinn, Inc. (lot 26390; Cherry Hill, NJ) and Choay Inst. (lot IC-86172; Paris, France). RPMI 1640 medium, fetal bovine serum (FBS), and other reagents were from Sigma Chemical Co. PDGF-BB was from Pepro Tech, Rocky Hill, NJ. [Methyl-3H]-thymidine (specific activity 20 Ci/mmol) and [hydroxy-3H]-tryptamine creatine sulfate (specific activity 26.6 Ci/mmol) were from New England Nuclear Corp., Boston, MA. Recombinant anti-phosphotyrosine RC20 alkaline phosphatase conjugate was from Transduction Laboratories, Lexington, KY.

Cell Culture

SMC from bovine pulmonary artery (PA) were isolated and cultured by a modification of the method of Ross as previously described (21).

Incorporation of [3H]thymidine

The protocol used has been previously described (21). In brief, plated SMC were cultured for 72 h in medium containing 10% FBS followed by 72-96 h growth arrest in medium containing 0.1% FBS and antibiotics. Cells which were at a density of approximately 0.1 × 106 cells/35-mm Petri dish were then incubated with 5-HT in the same medium containing 0.6 mM ascorbic acid for 20 h prior to being labeled with [methyl-3H]-thymidine (0.1 µCi/ml) for 4 h. Heparin was added 20 min before the 5-HT. Heparin alone at the concentrations reported did not alter the incorporation of 3H-thymidine by quiescent SMC that were not treated with 5-HT. After labeling with thymidine, experiments were terminated by aspiration of medium and then washing of the cellular monolayer, first with ice-cold phosphate-buffered saline (PBS) and then with cold 6% trichloroacetic acid. Cells were then dissolved in 0.2 N NaOH and radioactivity was counted.

Cell Number and Size Analysis

At the end of the incubation period, control and treated cells were trypsinized and diluted in Isoton. Cell number and size distribution measurements were carried out using a Coulter counter equipped with a Coulter Channelyzer and an x-y Recorder 4 (Coulter, Hialeah, FL) as previously described (19). Changes in size were determined and expressed as percent change from the control.

Measurement of 5-HT Uptake

5-HT uptake was measured as previously described (22). The monolayer of cells cultured in a 35-mm Petri dish was rinsed twice 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. 3H-5-HT (8.3 µCi/ml) was added from a stock solution containing ascorbic acid (10 µg/ml) and EDTA (10 µg/ml) as antioxidants. 5-HT concentration was 15 nM. The uptake of 5-HT by PA SMC was saturated in 30 min at this concentration of 5-HT; therefore, incubations were carried out for 10 min. After the incubation, medium was removed and monolayers were washed 3 times with ice-cold PBS plus 15 mM dextrose. Cells were then dissolved in 0.2 N NaOH and radioactivity was counted.

Preparation of Whole Cell Extracts and Electrophoresis

Cells were grown in 100-mm Petri dishes to confluency and were growth-arrested for 72-96 h. The cells were exposed to 1 µM 5-HT for 10 min and then were washed twice with ice-cold PBS. Cell lysates were obtained by incubating the cellular monolayer in 0.75 ml cell lysis buffer (23) containing 50 ml Tris HCl (pH 7.5), 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 10 µg/ml aprotine, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 40 µg/ml phenylmethylsulfonyl fluoride, 0.07 µg/ml pepstatin, 1% NP-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 protein measurement and electrophoresis.

Thirty micrograms protein of whole cell lysates was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on an 8.5% slab gel in a model SE-600 apparatus (Hoefer Scientific, San Francisco, CA) according to Laemmli (24).

Immunoblotting with Antiphosphotyrosine Antibody

After electrophoresis, gel proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Tropifluor; Tropix, Bedford, MA or Immobilon-P; Millipore Inc., 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 sodium chloride, 0.02% sodium azide, and 0.1% Tween-20. Blocking was done for 1 h at ambient temperature. The membrane was then treated with a 1/25,000 dilution of alkaline phosphatase (AP)-conjugated anti-phosphotyrosine antibody in blocking buffer for 90 min at ambient temperature with gentle agitation. Nitro-block (Tropix) was used according to the manufacturer's instructions. Subsequent washing, detection with the chemiluminescent substrate AMPPD (Tropix), and film exposure were done as described previously (25). Densitometry was determined with a Millipore densitometer with Visage V4.6p software (Millipore).

Statistics

Each treatment was carried out at least in triplicate experiments, and a representative experiment is shown. Unpaired Student's t test was used for statistical comparisons.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Heparin on Cellular Hyperplasia and Hypertrophy

We have previously reported that 5-HT acts as a mitogen on SMC by stimulating DNA synthesis after 24 h incubation, and producing cellular hyperplasia and hypertrophy after 7 days' incubation (19). Heparin (Sigma) at 100 and 200 µg/ml inhibited by approximately 50% or more the [3H]thymidine incorporation stimulated by 10% FBS, 20 ng/ml PDGF and 1 µM 5-HT (Figure 1). There was no inhibition by Sigma heparin at 10 µg/ml. These results are unlike those of Johnson and associates (26), who reported that heparin failed to affect the stimulation of DNA synthesis of human airway SMC by PDGF. The increase in cell number at day 7 produced by 5-HT in our studies was also inhibited by heparin (Sigma), as shown in Figure 2. Cellular hypertrophy produced by 5-HT was also reversed by heparin (Figures 3 and 4). Heparin from sources other than Sigma (Elkins-Sinn, Upjohn, and Choay) were tested for their antihyperplastic and antihypertrophic effects. Figure 5 shows that heparin from these three sources at 10 µg/ ml concentration inhibited 5-HT-stimulated [3H]thymidine incorporation by approximately 50% and abolished the 5-HT-induced hyperplasia (Figure 6). Heparin from Elkins-Sinn and Upjohn at a concentration of 10 µg/ml also blocked the hypertrophic effect of 5-HT (Figure 7). Thus heparin from Sigma was less potent than the heparin from the other sources and was ineffective against the hypertrophic effect at 10 µg/ml.


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  		Figure 1.   Stimulation of [3H]thymidine incorporation into SMC by 10% FBS, 20 ng/ml PDGF, and 1 µM 5-HT were inhibited by pre-incubation with 100 and 200 µg/ml heparin (Sigma). Mean ± SD (n = 4 dishes).


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  		Figure 2.   The increase in cell number produced by 7 days' incubation with 5-HT was reversed by co-incubation with 100 and 200 µg/ml heparin from Sigma. * Significantly different from control, P < 0.05; ** significantly different from 5-HT, P < 0.05. Mean ± SD (n = 4 dishes).


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  		Figure 3.   The cellular hypertrophy produced by 7 days' incubation with 5-HT was reversed by 100 and 200 µg/ml heparin (Sigma). * Significantly different from control, P < 0.05; ** significantly different from 5-HT, P < 0.05. Mean ± SD (n = 4 dishes).


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  		Figure 4.   The shift of a cell-size distribution curve produced by incubation with 1 µM 5-HT for 7 days was reversed to that of quiescent cells by co-incubation with 200 µg/ml heparin (Sigma).


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  		Figure 5.   Stimulation of [3H]thymidine incorporation by 1 µM 5-HT was inhibited by 10 µg/ml heparin from Choay, Elkins-Sinn, and Upjohn. * Significantly different from control, P < 0.05; ** significantly different from 5-HT, P < 0.05. Mean ± SD (n = 4 dishes).


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  		Figure 6.   The increase in cell number after 7 days' incubation with 5-HT was abolished by co-incubation with 10 µg/ml heparin from Choay, Elkins-Sinn, and Upjohn. These heparins alone at this concentration did not affect the cell number of control cells. * Significantly different from control, P < 0.05; ** significantly different from 5-HT alone, P < 0.05. Mean ± SD (n = 4 dishes).


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  		Figure 7.   Antihypertrophic effect of heparins from Choay, Elkins-Sinn, and Upjohn at a concentration of 10 µg/ml on SMC after 7 days' incubation with 5-HT. Heparin from Upjohn and Elkins-Sinn exhibited higher antihypertrophic effect than heparin from Choay. * Significantly different from control, P < 0.05; ** significantly different from 5-HT, P < 0.05. Mean ± SD (n = 4 dishes).

Effect of Heparin on 5-HT Transport

We have reported that 5-HT-induced mitogenesis is mediated through the action of a 5-HT membrane transporter and that this mitogenic effect is blocked by 5-HT uptake inhibitors such as imipramine and fluoxetine (27). Thus one possible mechanism by which heparin might influence the mitogenic effect of 5-HT would be through an action on the transport process. However, heparin from all the sources we tested failed to alter the 5-HT uptake of SMC, as shown in Figure 8.


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  		Figure 8.   Heparin from different sources at various concentrations failed to alter 5-HT uptake by SMC. Mean ± SD; n = 4 dishes.

Effect of Heparin on Protein Tyr-P Induced by 5-HT

In previous studies we have found that 5-HT-induced mitogenesis is reversed by actions of tyrosine kinase inhibitors (27). We later identified the association of Tyr-P of p120, identified as GAP and an unidentified p65, which co-precipitates with p120 (28), with this mitogenic process (20). Therefore, we set out to determine whether heparin might block Tyr-P of intermediate cellular proteins, a mechanism that has been associated with cellular proliferation induced by many growth factors (29). Both 5-HT and 10% serum stimulated Tyr-P of p120 and p65 after 10 min incubation. Pre-incubation of cells with heparin (Sigma) prior to 10 min stimulation with 5-HT or 10% serum markedly inhibited the enhanced Tyr-P of p120 and p65, as shown in Figure 9.


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  		Figure 9.   (Top panel ) The enhancement of Tyr-P of p120 and p65 by 10 min incubation with 10% FBS or 1 µM 5-HT was markedly inhibited by pre-incubation of cells with heparin (Sigma) at both 100 and 200 µg/ml concentrations. A moderate inhibition of Tyr-P of an unidentified protein between 34 and 56 KD was noted in heparin-treated cells, which was not stimulated by serum and 5-HT. (Bottom panel) Densitometric measurements of stimulation of phosphorylation of SMC. Treatments 1-7 are as indicated in top panel .

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pulmonary vascular remodeling is known to occur following a variety of insults, including lung injury (32) and exposure to hypoxia (33, 34). An unknown triggering event causes smooth muscle cellular proliferation and hypertrophy which may result in pulmonary hypertension. In addition to conventional growth factors, there is increasing evidence to suggest that vasoactive substances such as arginine vasopressin, endothelin-1, angiotensin II, and 5-HT may participate in the mitogenic response (35).

It has been postulated that platelets adhering to the vascular wall may be the source of 5-HT that produces SMC mitogenesis (38). Also, plasma levels of 5-HT may be elevated in certain platelet storage diseases (39) and primary pulmonary hypertension (40). There is a similar elevation of plasma 5-HT in the fawn-hooded rat (41, 42), an experimental model of pulmonary hypertension. Weight-reduction drugs that inhibit re-uptake of 5-HT by platelets have been associated with pulmonary hypertension in humans (43). Thus there are now several pieces of information that suggest at least a partial role for 5-HT in vascular remodeling.

We have previously demonstrated that 5-HT uptake by PA SMC is stimulated by exposures to hypoxia (22) and that 5-HT in concentrations as low as 0.1-1 µM stimulates both proliferation and hypertrophy of SMC in culture (19). Furthermore, the mitogenic action of 5-HT is synergistic with that of conventional peptide growth factors (19, 21).

Heparin, a well known inhibitor of SMC proliferation (5), is often used in the treatment of pulmonary hypertension. It has been demonstrated to inhibit cellular proliferation induced by serum and PDGF, but has not previously been tested for its influence on hyperplasia and hypertrophy produced by 5-HT or for its influence on cellular hypertrophy, in general, except for a report showing that it prevents hypertrophy produced by angiotensin II in cardiomyocytes (18). As shown in our studies, heparin inhibits both cellular proliferation and hypertrophy of pulmonary vascular SMC in culture. These effects may be related to heparin's inhibition of hypoxia-induced experimental pulmonary hypertension in vivo (4) and inhibition of the Na+/ H+ exchanger and PDGF-induced cellular proliferation in vitro (17). Most of our studies have been done with heparin obtained from Sigma, because this was the source most readily available to us. Furthermore, we were careful to identify the lot number of the heparin used because substantial variations in the antiproliferative actions of heparin exist for different lot numbers and suppliers (44).

The mechanism by which heparin inhibits SMC proliferation has not been clearly identified. It is known that cellular binding and internalization of heparin correlates well with its antiproliferative action (18, 45, 46). Whether this represents a competitive binding of a receptor for a growth factor or an interference with an intracellular signaling mechanism is not known. It has been variously proposed that the antihyperplastic action of heparin occurs through inhibition of PKC, AP-1 binding, c-fos/c-myc gene expression, or other intracellular signaling processes (9, 14 -16, 47).

Tyr-P of protein appears to play a significant intermediary role in the cellular proliferative and hypertrophic responses to 5-HT that we have demonstrated (20). Inhibition of tyrosine kinase, but not a number of other signaling pathways, clearly blocks the stimulation of both processes by 5-HT (20). PKC does not participate in the 5-HT stimulatory process we have observed, and intracellular Ca2+ is not elevated by 5-HT in the cells we have studied. Direct assessment of stimulation of phosphorylated proteins by 5-HT shows that enhanced phosphorylation of p120 and p65 predominate and that the p120 component consists largely of GAP which plays an important role in the cellular ras pathway (20). We have demonstrated in these studies that, like proliferation and hypertrophy, stimulation of phosphorylation of p120 and p65 is inhibited by heparin. Stimulation of these cells by 5-HT is coupled to the action of a 5-HT transporter, but not a cell-surface receptor (27). Heparin failed to inhibit the active transport of 5-HT by our cells, thereby excluding an influence on this process as the cause for inhibition of hyperplasia and hypertrophy. It is likely that the action of heparin is through an intracellular event. Whether heparin might have a direct effect on tyrosine kinase activity needs further evaluation.

    Footnotes

Address correspondence to: Sheu-Ling Lee, Ph.D., New England Medical Center, Pulmonary and Critical Care Div., 750 Washington St., NEMC #265, Boston, MA 02111.

(Received in original form July 30, 1996 and in revised form December 30, 1996).

Acknowledgments: The authors thank Deborah LaPerche for her assistance in the preparation of this manuscript. This study was supported by the National Heart, Lung, and Blood Institute, Grants HL32723 and HL39150.

Abbreviations AP-1, activator protein-1; FBS, fetal bovine serum; 5-HT, serotonin; GAP, GTPase-activating protein; PA, pulmonary artery; PBS, phosphate-buffered saline; PDGF, recombinant human platelet-derived growth factor; SMC, smooth muscle cell; Tyr-P, tyrosine phosphorylation.

    References
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Clowes, A. W., and M. J. Karnovsky. 1977. Supression by heparin of smooth muscle cell proliferation in injured arteries. Nature 265: 625-626 [Medline].

2. Guyton, J. R., R. D. Rosenberg, A. W. Clowes, and M. J. Karnovsky. 1980. Inhibition of rat arterial smooth muscle cell proliferation by heparin. Circ. Res. 46: 625-634 [Free Full Text].

3. Hoover, R. L., R. Rosenberg, W. Hearing, and M. J. Karnovsky. 1980. Inhibition of rat arterial smooth muscle cell proliferation by heparin. II. In vitro studies. Circ. Res. 47: 578-583 [Abstract/Free Full Text].

4. Thompson, B. T., C. R. Spence, S. P. Janssens, P. M. Joseph, and C. A. Hales. 1994. Inhibition of hypoxic pulmonary hypertension by heparins of differing in vitro antiproliferative potency. Am. J. Respir. Crit. Care Med. 149: 1512-1517 [Abstract].

5. Castellot, J. J. Jr., L. V. Favreau, M. J. Karnovsky, and R. D. Rosenberg. 1982. Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin: possible role of a platelet endoglycosidase. J. Biol. Chem. 257: 11256-11260 [Abstract/Free Full Text].

6. Groggel, G. C., G. N. Marinides, P. Hovingh, E. Hammond, and A. Linker. 1990. Inhibition of rat mesangial cell growth by heparan sulfate. Am. J. Physiol. 258: F259-F265 [Abstract/Free Full Text].

7. Floege, J., E. Eng, B. A. Young, W. G. Couser, and R. J. Johnson. 1993. Heparin suppresses mesangial cell proliferation and matrix expansion in experimental mesangioproliferative glomerulonephritis. Kidney Int. 43: 369-380 [Medline].

8. Wright, T. C. Jr., T. V. Johnstone, J. J. Castellot Jr., and M. J. Karnovsky. 1985. Inhibition of rat cervical epithelial cell growth by heparin and its reversal by EGF. J. Cell. Physiol. 125: 499-506 [Medline].

9. Wright, T. C. Jr., L. A. Pukac, J. J. Castellot Jr., M. J. Karnovsky, R. A. Levine, H. Y. Kim-Park, and J. Campisi. 1989. Heparin suppresses the induction of c-fos and c-myc mRNA in murine fibroblasts by selective inhibition of a protein kinase C-dependent pathway. Proc. Natl. Acad. Sci. USA 86: 3199-3203 [Abstract/Free Full Text].

10. Castellot, J. J. Jr., K. Wong, B. Herman, R. L. Hoover, D. F. Albertini, T. C. Wright, B. L. Caleb, and M. J. Karnovsky. 1985. Binding and internalization of heparin by vascular smooth muscle cells. J. Cell. Physiol. 124: 13-20 [Medline].

11. Majesky, M. W., S. M. Schwartz, M. M. Clowes, and A. W. Clowes. 1987. Heparin regulates smooth muscle S phase entry in the injured rat carotid artery. Circ. Res. 61: 296-300 [Abstract/Free Full Text].

12. Castellot, J. J. Jr., L. A. Pukac, B. L. Caleb, T. C. Wright, and M. J. Karnovsky. 1989. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J. Cell Biol. 109: 3147-3155 [Abstract/Free Full Text].

13. Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E. Sonenshein. 1989. Heparin prevents vascular smooth muscle cell progession through the G1 phase of the cell cycle. J. Biol. Chem. 264: 6990-6995 [Abstract/Free Full Text].

14. Pukac, L. A., M. E. Ottlinger, and M. J. Karnovsky. 1992. Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J. Biol. Chem. 267: 3707-3711 [Abstract/Free Full Text].

15. Busch, S. J., G. A. Martin, R. L. Barnhart, M. Mano, A. D. Cardin, and R. L. Jackson. 1992. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription. J. Cell Biol. 116: 31-42 [Abstract/Free Full Text].

16. Au, Y. P. T., G. Dobrowolska, D. R. Morris, and A. W. Clowes. 1994. Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B.  Circ. Res. 75: 15-22 [Abstract/Free Full Text].

17. Dahlberg, C. G. W., B. T. Thompson, P. M. Joseph, H. G. Garg, C. R. Spence, D. A. Quinn, J. V. Bonventre, and C. A. Hales. 1996. Differential effect of three commercial heparins on Na+/H+ exchange and growth of PASMC. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14):L260-L265.

18. Akimoto, H., H. Ito, M. Tanaka, S. Adachi, M. Hata, M. Lin, H. Fujisaka, F. Marumo, and M. Hiroe. 1996. Heparin and heparin sulfate block angiotensin II-induced hypertrophy in cultured neonatal rat cardiomyocytes. Circulation 93: 810-816 [Abstract/Free Full Text].

19. Lee, S. L., W. W. Wang, J. J. Lanzillo, and B. L. Fanburg. 1994. Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am. J. Physiol. 266(Lung Cell. Mol. Physiol. 10):L46-L52.

20. Lee, S. L., W. W. Wang, and B. L. Fanburg. 1997. Association of tyrosine phosphorylation of GTPase-activating protein with mitogenic action of serotonin. Am. J. Physiol. 272(Cell Physiol. 41):C223-C230.

21. Lee, S. L., W. W. Wang, B. J. Moore, and B. L. Fanburg. 1991. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ. Res. 68: 1362-1368 [Abstract/Free Full Text].

22. Lee, S. L., J. Dunn, F. S. Yu, and B. L. Fanburg. 1989. Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture. J. Cell. Physiol. 138: 145-153 [Medline].

23. Welham, M. J., and J. W. Schrader. 1992. Steel factor-induced tyrosine phosphorylation in murine mast cells. J. Immunol. 149: 2772-2783 [Abstract].

24. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227: 680-685 [Medline].

25. Lee, S. L., J. Stevens, W. W. Wang, and J. J. Lanzillo. 1994. A liquid gelatin blocking reagent for Western blotting with chemiluminescent detection. BioTechniques 17: 60-62 [Medline].

26. Johnson, P. R. A., C. L. Armour, D. Carey, and J. L. Black. 1995. Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture. Am. J. Physiol. 269(Lung Cell. Mol. Physiol. 13):L514-L519.

27. Lee, S. L., W. W. Wang, J. J. Lanzillo, and B. L. Fanburg. 1994. Regulation of serotonin-induced DNA synthesis of bovine pulmonary artery smooth muscle cells. Am. J. Physiol. 266(Lung Cell. Mol. Physiol. 10):L53-L60.

28. Ellis, C., M. Moran, F. McCormick, and T. Pawson. 1990. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature 343: 377-381 [Medline].

29. Leeb-Lundberg, L. M. F., and X. H. Song. 1991. Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-KDa group of proteins in Swiss 3T3 cells. J. Biol. Chem. 266: 7746-7749 [Abstract/Free Full Text].

30. Zachary, I., J. Gil, W. Lehmann, J. Sinnett-Smith, and E. Rozengurt. 1991. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 88: 4577-4581 [Abstract/Free Full Text].

31. Schvartz, I., O. Ittoop, G. Davidai, and E. Hazum. 1992. Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells. Peptides 13: 159-163 [Medline].

32. Kanai, Y., S. Hori, T. Tanaka, M. Yasuoka, K. Watanabe, N. Aikawa, and Y. Hosoda. 1993. Role of 5-hydroxytryptamine in the progression of monocrotaline induced pulmonary artery in rats. Circ. Res. 27: 1619-1623 .

33. Hultgren, H. N., J. Kelly, and H. Miller. 1965. Pulmonary circulation in acclimatized man at high attitude. J. Appl. Physiol. 20: 233-238 [Abstract/Free Full Text].

34. Reid, L.. 1979. The pulmonary circulation: remodeling in growth and disease. Am. Rev. Respir. Dis. 119: 531-546 [Medline].

35. Geisterfer, A. A. T., M. J. Peach, and G. K. Owens. 1988. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ. Res. 62: 749-756 [Abstract/Free Full Text].

36. Geisterfer, A. A. T., and G. K. Owens. 1989. Arginine vasopressin-induced hypertrophy of cultured rat aortic smooth muscle cells. Hypertension 14: 413-420 [Abstract/Free Full Text].

37. Mitsuhashi, M., and D. G. Payan. 1987. The mitogenic effects of vasoactive neuropeptides on cultured smooth muscle cells. Life Sci. 40: 853-861 [Medline].

38. Vanhoutte, P. M.. 1983. 5-hydroxytryptamine and vascular disease. Federation Pro. 42: 233-237 .

39. Herve, P., L. Drouet, C. Dosquet, J. M. Launay, B. Rain, G. Simonneau, J. Caen, and P. Duroux. 1990. Primary pulmonary hypertension in a patient with a familial platelet storage pool disease: role of serotonin. Am. J. Med. 89: 117-120 [Medline].

40. Herve, P., J. M. Lunay, M. L. Scrobohaci, F. Brenot, G. Simonneau, P. Petitpretz, P. Poubeau, J. Cerrina, P. Duroux, and L. Drouet. 1995. Increased plasma serotonin in primary pulmonary hypertension. Am. J. Med. 99: 249-254 [Medline].

41. Tschopp, T. B., and M. B. Zucker. 1972. Hereditary defect in platelet function in rats. Blood 40: 217-226 [Abstract/Free Full Text].

42. Sato, K., S. Webb, A. Tucker, M. Rabinovitch, R. F. O'Brien, I. F. McMurtry, and T. J. Stelzner. 1992. Factors influencing the idiopathic development of pulmonary hypertension in the Fawn Hooded rat. Am. Rev. Respir. Dis. 145: 793-797 [Medline].

43. Brenot, F., P. Herve, P. Petitpretz, F. Parent, P. Duroux, and G. Simonneau. 1993. Primary pulmonary hypertension and fenfluramine use. Br. Heart J. 70: 537-541 [Abstract/Free Full Text].

44. Castellot, J. J. Jr., J. Choay, J. C. Lormeau, M. Petitou, E. Sache, and M. J. Karnovsky. 1986. Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-o sulfate group. J. Cell Biol. 102: 1979-1984 [Abstract/Free Full Text].

45. Letourneur, D., B. L. Caleb, and J. J. Castellot Jr.. 1995. Heparin binding, internalization, and metabolism in vascular smooth muscle cells. I. Upregulation of heparin binding correlates with antiproliferative activity. J. Cell. Physiol. 165: 676-686 [Medline].

46. Barzu, T., M. Pascal, M. Maman, C. Roque, F. Lafont, and A. Rousselet. 1996. Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures. J. Cell. Physiol. 167: 8-21 [Medline].

47. Reilly, C. F., L. M. S. Fritze, and R. D. Rosenberg. 1986. Heparin inhibition of smooth muscle cell proliferation: a cellular site of action. J. Cell. Physiol. 129: 11-19 [Medline].




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