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Circulation. 1996;93:2170-2177

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(Circulation. 1996;93:2170-2177.)
© 1996 American Heart Association, Inc.

Articles

Serotonin Stimulates the Expression of Thrombin Receptors in Cultured Vascular Smooth Muscle Cells

Role of Protein Kinase C and Protein Tyrosine Kinases

Valérie B. Schini-Kerth, PhD; Beate Fisslthaler, PhD; Ellen Van Obberghen-Schilling, PhD; Rudi Busse, MD, PhD

From the Center of Physiology, University Clinic of Frankfurt (Germany), and the Centre de Biochimie, Université de Nice (France) (E.V.O.-S.).


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background Thrombin has been implicated in the development of intimal thickening after balloon angioplasty. The action of thrombin on vascular cells involves the proteolytic activation of G protein–coupled receptors that are subjected to rapid and irreversible homologous desensitization. Hence, the amount and availability of thrombin-activatable receptors play a determinant role in thrombin responsiveness. The possibility that the platelet-derived product serotonin (5-HT) regulates expression of the thrombin receptor was examined in cultured rat aortic vascular smooth muscle cells.

Methods and Results Thrombin receptor expression was assessed at the mRNA level by Northern blot analysis and functionally by measurement of the release of 6-ketoprostaglandin F1{alpha}. 5-HT significantly enhanced thrombin receptor mRNA levels in a time- and concentration-dependent manner, an effect that was abolished by 5-HT2 receptor antagonists and by inhibition of protein kinase C but only slightly affected by inhibitors of protein tyrosine kinases. Enhanced thrombin receptor mRNA levels after exposure to 5-HT were associated with an increase in the thrombin-induced release of 6-ketoprostaglandin F1{alpha}.

Conclusions 5-HT stimulates the expression of thrombin receptors in vascular smooth muscle cells, probably via activation of 5-HT2 receptors and the subsequent activation of protein kinase C and possibly also protein tyrosine kinases. The upregulation of the synthesis of plasma membrane thrombin receptors by 5-HT released from aggregating platelets at sites of vascular injury may potentiate the mitogenic and constrictor actions of thrombin in the vascular wall.


Key Words: serotonin • arteriosclerosis • muscle, smooth • receptors, thrombin


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Intimal thickening, resulting mostly from the accumulation of VSMCs within the intima, is a major outcome of atherosclerosis and is especially prominent in restenosis after balloon angioplasty, endarterectomy, and coronary bypass graft surgery. Investigations using animal models have indicated that the pathophysiological mechanisms of restenosis are controlled, in large part, by vasoactive factors derived from different sources within the site of injury.1 2 3 4 After balloon catheterization of arteries, subendothelial surfaces are exposed to formed elements of the blood, resulting in the rapid activation of platelets and the coagulation cascade and in thrombus formation.5 6 Platelet aggregates release potent vasoactive factors and mitogens, including PDGF, epidermal growth factor, transforming growth factor-ß, and 5-HT.7 In addition to the platelet response, the clotting cascade also generates vasoactive factors, including thrombin and factor Xa.8 9 10 Moreover, several hours after the balloon injury, the blood vessel wall generates PDGF and bFGF, the synthesis of which is possibly stimulated by hemostatic and thrombotic factors.4 9 11 The platelet- and plasma-derived factors are likely to act synergistically with those released by the vascular wall to activate and maintain the smooth muscle cells in a proliferative state throughout the development of intimal thickening. However, because the proliferative response to balloon injury persists for several weeks, long after the platelet response has subsided,6 platelet-derived factors are unlikely to contribute to late restenosis. In contrast to the transient platelet response, substantial amounts of proteolytically active thrombin are generated at the luminal surface of balloon-injured arteries for several weeks, indicating that the serine protease may provide a sustained stimulus for cell proliferation.5 Indeed, several findings suggest that thrombin plays a major role in the development of intimal thickening. First, thrombin is a potent mitogen for VSMCs in culture.8 12 Second, the pattern of PDGF receptors and PDGF A-chain gene expression induced in vivo after balloon injury can be reproduced by thrombin, but not by other injury-induced factors, in cultured VSMCs.9 Finally, inhibition of thrombin with either hirudin or a combination of hirudin and D-Phe-Pro-Arg chloromethyl ketone can reduce intimal thickening in rabbit arteries.3 13

Thrombin exerts most, if not all, of its actions, including mitogenesis, on vascular cells via activation of the seven-transmembrane-domain thrombin receptor by a unique proteolytic mechanism. The serine protease binds to and cleaves the long extracellular amino-terminal extension of its receptor, unmasking a new amino-terminal that serves as a tethered ligand to effect receptor activation.14 15 Activation of the thrombin receptor can be elicited by proteolytically active forms of thrombin and by peptides that mimic the new amino-terminal of the thrombin receptor.14 16 Although exposure to thrombin results in irreversible proteolytic activation of the thrombin receptors, its action on cells is limited by the rapid desensitization of these receptors by mechanisms that involve phosphorylation and intracellular proteolysis.17 18 19 Recovery of thrombin responsiveness is due, in large part, to the replenishment of the cell surface with newly synthesized thrombin receptors, but recent evidence suggests that the mobilization of a limited intracellular pool of intact thrombin receptors may also be involved.17 18 19 Expression of the thrombin receptor is increased in balloon-injured arteries from the rat and in advanced atherosclerotic lesions in human arteries.20 21 Thus, these findings, in conjunction with those showing that balloon-injured arteries are exposed to active thrombin for several weeks,5 imply that thrombin may continue to exert a mitogenic effect throughout the development of intimal thickening and hence further support a pivotal role for thrombin in the vascular response to injury.

Although the stimulus responsible for enhanced in vivo expression of the thrombin receptor at sites of balloon injury remains unknown, factors released by aggregating platelets during the initial thrombotic and hemostatic events might be involved. Consistent with this hypothesis are the findings that PDGF causes a modest increase (about twofold) in the relative level of thrombin receptor mRNA in cultured VSMCs.21 Within platelets, 5-HT is a major nonpeptidergic substance that is stored at high concentrations in the dense granules along with other vasoactive factors, including ADP and ATP.7 Although little free 5-HT circulates in blood, its concentration increases markedly at sites of balloon injury for several days, presumably because of its release by aggregating platelets.22 The aim of the present study was to examine whether or not 5-HT affects the expression of the thrombin receptor in cultured VSMCs and, if so, to identify the underlying mechanisms.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
5-HT hydrochloride, calcium ionophore A23187, PMA, bovine fraction V albumin, TES, and HEPES were obtained from Sigma Chemical Co. Ketanserin tartrate, MDL-72222, and methiothepin mesylate were obtained from Research Biochemicals Inc. Erbstatin A and genistein were obtained from Biomol; PDGFAB and staurosporine from Boehringer Mannheim; MEM containing Earle's salts, trypsin, and FCS from PAN Systems; penicillin and streptomycin from Gibco BRL; and Ro-318220 from Roche Products Ltd. All plasticware was obtained from Greiner GmbH. [{alpha}-32P]dCTP (3000 Ci/mmol) was obtained from Hartmann Analytic. {alpha}-Thrombin (specific clotting activity, 3488.6 U/mg) was kindly provided by Dr JW Fenton II, Albany, NY. Male Wistar rats were obtained from Charles River Wiga Deutschland GmbH.

Cell Culture
Smooth muscle cells were isolated by elastase and collagenase digestion of thoracic aortas from male Wistar rats.23 Cells obtained from three different isolates were cultured serially in MEM containing Earle's salts, 2 mmol/L L-glutamine, 20 mmol/L TES, 20 mmol/L HEPES (both at pH 7.3), 100 U/mL of both penicillin and streptomycin, and 10% FBS. All experiments were performed on cells between passages 7 and 19. At confluence, the culture medium was replaced with serum-free medium containing 0.1% fatty acid–free BSA every 24-hour period for 2 days before treatment.

Northern Blot Analysis
Total cellular RNA was prepared by acid guanidinium thiocyanate extraction and quantified by absorbance at 260 nm. Total RNA was size-fractionated by electrophoresis on 1% agarose gels containing 2.2 mol/L formaldehyde in a buffer of 20 mmol/L MOPS, 5 mmol/L sodium acetate, and 1 mmol/L EDTA, pH 7.0. RNA was transferred to nylon membranes (Hybond-N, Amersham-Buchler) and fixed by baking at 80°C for 2 hours. The membranes were prehybridized at 60°C for 4 to 6 hours in a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 5xDenhardt's solution, 4xSSC, 0.02% SDS, and 250 µg/mL denatured salmon sperm DNA. A 2000-bp Pst I restriction fragment from a Chinese hamster thrombin receptor cDNA clone was labeled with 30 µCi of [{alpha}-32P]dCTP (3000 Ci/mmol) by use of a labeling kit from Pharmacia. The [32P]DNA was purified by gel filtration (Nick columns, Pharmacia). The Northern blots were hybridized in the same buffer as for prehybridization at 60°C for 16 to 20 hours with the labeled DNA. After hybridization, the blots were washed twice at 42°C in 2xSSC and 0.1% SDS for 30 minutes and then twice in 0.2xSSC and 0.1% SDS at 55°C for 30 minutes. After hybridization with the thrombin receptor probe, filters were hybridized with a 32P-labeled probe for 18S ribosomal RNA. Autoradiography was performed with Fuji RX film with intensifying screens (DuPont de Nemours) at -70°C. The autoradiographs were analyzed by scanning densitometry. Thrombin receptor mRNA levels were normalized to their respective 18S ribosomal RNA levels and expressed in arbitrary units as a fold increase of the signal obtained with untreated cells.

Release of 6-Ketoprostaglandin F1{alpha}
Cells were incubated at 37°C in fresh serum-free culture medium in the absence or presence of 5-HT for 6 hours. The conditioned medium was then removed, and the cells were washed twice with HEPES–Tyrode's solution. After an additional 30-minute equilibration period, cells were either untreated or stimulated with thrombin or PDGFAB. Thereafter, aliquots were removed from the incubation medium, and the amount of 6-ketoprostaglandin F1{alpha} present in each sample was determined by radioimmunoassay.

Statistical Analyses
Results are shown as mean±SEM. Statistical analyses were performed with Student's paired t test (two-tailed) or an ANOVA followed by Fisher's protected least significant difference test to identify differences between two treatments. A value of P<.05 was considered to be statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of 5-HT on Expression of Thrombin Receptor mRNA
Expression of the thrombin receptor was assessed in serum-deprived quiescent rat aortic smooth muscle cells by Northern blot analysis. As shown in Figs 1Down and 3Down, a low level of thrombin receptor mRNA could be detected in untreated smooth muscle cells. The transcript level was markedly increased after exposure of the cells to 5-HT (1 µmol/L, Fig 1Down). As early as 1 hour after treatment, a 2.2-fold (mean of two different experiments) increase in the level of thrombin receptor mRNA was found. Thereafter, the signal increased steadily to reach a peak value of 4.8±1.0-fold (21 different experiments) at 4 hours and then returned to baseline (0.9-fold, mean of two different experiments) over the next 11 hours (Fig 1Down). Therefore, a 4-hour treatment period was chosen for further characterization of the action of 5-HT. The response to 5-HT was concentration dependent, with a threshold concentration of {approx}10 nmol/L (Fig 2Down).



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  		Figure 1. Northern blot showing the time course of thrombin receptor mRNA expression induced by 5-HT (1 µmol/L). Quiescent rat aortic smooth muscle cells were treated as indicated in "Methods." Hybridization was performed either with the thrombin receptor cDNA probe (top) or with the probe for 18S ribosomal RNA (center). Thrombin receptor mRNA levels and 18S ribosomal RNA levels were quantified by densitometry, and changes in thrombin receptor mRNA levels relative to 18S ribosomal RNA levels are shown (bottom). Similar observations were obtained in two independent experiments.



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  		Figure 3. Northern blot showing the effect of various 5-HT receptor antagonists on the induction of thrombin receptor mRNA expression evoked by 5-HT. Quiescent rat aortic smooth muscle cells were incubated with either methiothepin (5-HT1/5-HT2 receptor antagonist), ketanserin (5-HT2 receptor antagonist), or MDL-72222 (5-HT3 receptor antagonist) for 15 minutes before the addition of 5-HT or vehicle for 4 hours. Hybridization was performed either with the thrombin receptor cDNA probe (top) or with the probe for 18S ribosomal RNA (center). Thrombin receptor mRNA levels and 18S ribosomal RNA levels were quantified by densitometry, and changes in thrombin receptor mRNA levels relative to 18S ribosomal RNA levels are shown (bottom). Similar observations were obtained in three independent experiments.



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  		Figure 2. Concentration-dependent effect of 5-HT on the expression of the thrombin receptor mRNA. Quiescent rat aortic smooth muscle cells were exposed to 5-HT for 4 hours. Results are shown as mean±SEM values obtained from three different experiments.

Effect of 5-HT Receptor Antagonists on 5-HT–Induced Increase in Thrombin Receptor mRNA Expression
To determine the type of 5-HT receptor(s) involved in mediating the enhanced expression of thrombin receptor mRNA in response to 5-HT, the effects of various 5-HT receptor antagonists were investigated. Preincubation of cells for 15 minutes with either ketanserin (1 µmol/L, 5-HT2 receptor antagonist) or methiothepin (1 µmol/L, 5-HT1/5-HT2 receptor antagonist) before the addition of 5-HT (1 µmol/L) abolished the stimulatory effect of 5-HT (the inhibitory effects were 101.9±13.2% and 91.4±12.5%, respectively, three different experiments, Fig 3Up). The stimulatory action of 5-HT was also inhibited by ritanserin (5-HT2 receptor antagonist; data not shown). In contrast, preincubation of cells with MDL-72222 (0.1 µmol/L, 5-HT3–type receptor antagonist) affected the response to 5-HT only minimally (an inhibition of 15.4±26.2% was found, three different experiments, Fig 3Up). None of these 5-HT receptor antagonists alone increased the expression of thrombin receptor mRNA (Fig 3Up).

Putative Role of Protein Kinases and [Ca2+]i in the Expression of Thrombin Receptor mRNA
The interaction of 5-HT with serotonergic receptors on vascular smooth muscle activates several intracellular signal transduction pathways, which results in increases in [Ca2+]i and activation of protein kinase C and protein tyrosine kinases.24 25 26 Therefore, the role of these signal transduction pathways in modulating expression of the thrombin receptor mRNA was examined. The results shown in Fig 4Down indicate that increasing [Ca2+]i by exposing the cells to the calcium ionophore A23187 (1 µmol/L) had no effect on thrombin receptor mRNA levels. Direct activation of protein kinase C obtained by short-term exposure of the cells to PMA (0.3 µmol/L) was also without effect, as was a combination of the two treatments (A23187 plus PMA). Next, involvement of protein kinase C in 5-HT (1 µmol/L)–stimulated expression of thrombin receptor mRNA was assessed both by use of two different protein kinase C inhibitors, Ro-318220 (0.1 µmol/L) and staurosporine (10 nmol/L),27 and by downregulation of the enzyme after prolonged exposure of cells to PMA (0.3 µmol/L).28 As the results in Fig 5Down indicate, both treatments abolished the response to 5-HT, indicating that protein kinase C is involved in this effect. Finally, the role of protein tyrosine kinases in mediating the cellular effects of 5-HT was assessed with erbstatin A (10 µmol/L) and genistein (10 µmol/L), two different inhibitors of protein tyrosine kinases.29 Although erbstatin A and genistein reduced the 5-HT (1 µmol/L)–stimulated level of thrombin receptor mRNA by about 41% and 38%, respectively, these decreases were not statistically significant (Fig 6Down). However, when the data obtained with the two protein tyrosine kinase inhibitors were combined and analyzed, a statistically significant inhibition of the 5-HT effect was found (the relative thrombin receptor mRNA level was reduced from 3.4±0.5 to 2.4±0.4, 10 different experiments, P<.05). Exposure of cells to either Ro-318220, staurosporine, PMA, erbstatin A, or genistein alone affected the level of the thrombin receptor mRNA only minimally (Figs 5Down and 6Down).



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  		Figure 4. Either increasing [Ca2+]i by treating VSMCs with the calcium ionophore A23187 (1 µmol/L), activation of protein kinase C by treatment of VSMCs with PMA (0.3 µmol/L), or a combination of the two treatments for 4 hours did not cause the induction of thrombin receptor mRNA expression. The effect of 5-HT (1 µmol/L) is also shown. *Significant stimulatory effect of 5-HT. Results are shown as mean±SEM values resulting from three to five different experiments.



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  		Figure 5. Effects of protein kinase C inhibition on 5-HT–stimulated expression of thrombin receptor mRNA. Quiescent rat aortic smooth muscle cells were incubated in the absence or presence of either Ro-318220 (0.1 µmol/L, Ro) or staurosporine (10 nmol/L, Stauro) for 15 minutes, or with PMA (0.3 µmol/L) for 24 hours before the addition of 5-HT (1 µmol/L) or vehicle for 4 hours. Results are shown as mean±SEM values obtained from four to six different experiments. *Significant stimulatory effect of 5-HT; {dagger}significant inhibitory effect of either Ro-318220, staurosporine, or PMA.



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  		Figure 6. Effects of inhibition of protein tyrosine kinases on 5-HT–stimulated expression of thrombin receptor mRNA. Quiescent rat aortic smooth muscle cells were incubated in the absence or presence of (A) genistein (10 µmol/L) and (B) erbstatin A (10 µmol/L) for 15 minutes before the addition of 5-HT (1 µmol/L) or vehicle for 4 hours. Results are shown as mean±SEM values obtained from four to six different experiments. *Significant stimulatory effect of 5-HT.

Release of 6-Ketoprostaglandin F1{alpha}
In light of the results showing that 5-HT can positively regulate thrombin receptor mRNA expression, experiments were performed to determine the effect of 5-HT on the functional expression of the receptor. However, thrombin receptor protein could not be detected either in membrane preparations or in glycoprotein-enriched fractions from control cells and from 5-HT–treated cells (1 µmol/L for either 6 or 15 hours) by Western blot analysis using two different polyclonal antibodies raised against the rodent tethered ligand peptide. Since these antibodies were able to detect the receptor in membranes from cells that express a high level of the thrombin receptor, such as CCL39 fibroblasts (data not shown), the lack of detection of the thrombin receptor in smooth muscle cells is most likely a result of a low level of receptor expression. Therefore, the level of thrombin receptors in smooth muscle cells was assessed functionally by measurement of the release of 6-ketoprostaglandin F1{alpha} evoked by a submaximally effective concentration of thrombin.30 Thrombin caused a time-dependent accumulation of 6-ketoprostaglandin F1{alpha} in the incubation medium (Fig 7Down). Consistent with a 5-HT–induced increase in the levels of thrombin receptor expression at the cell surface, this response to thrombin was enhanced significantly in 5-HT–pretreated cells (Fig 7Down). Negligible amounts of 6-ketoprostaglandin F1{alpha} were released in the absence of thrombin from control or 5-HT–pretreated cells (Fig 7Down). The possibility that the greater responsiveness of the 5-HT–treated cells to thrombin reflects a generalized change in cell metabolism was ruled out by the observation that similar amounts of 6-ketoprostaglandin F1{alpha} were released from control and 5-HT–treated cells by PDGFAB (TableDown).



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  		Figure 7. Effect of 5-HT pretreatment on the thrombin-stimulated 6-ketoprostaglandin F1{alpha} release. Quiescent VSMCs were incubated in serum-free medium in either the presence or absence of 5-HT for 6 hours. Thereafter, the medium was replaced three times to wash out the indolamine. Control and 5-HT–treated cells were then incubated with thrombin or vehicle (control). At indicated times, an aliquot was taken from the incubation medium, and the amount of 6-ketoprostaglandin F1{alpha} present in each sample was determined by radioimmunoassay. Results are shown as mean±SEM of two different experiments performed in quadruplicate. *Significantly greater release of 6-ketoprostaglandin F1{alpha}.


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  		Table 1. Effect of 5-HT Pretreatment on the Release of 6-Ketoprostaglandin F1{alpha} Evoked by Thrombin and PDGFAB in VSMCs


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In situ hybridization and immunohistochemical studies have shown that the expression of the thrombin receptor is upregulated in diseased blood vessels from both humans and animals.20 21 In carotid arteries removed from healthy baboons and rats, the thrombin receptor is expressed at low levels in both endothelial cells and VSMCs.21 Within hours of balloon angioplasty of rat carotid arteries, there is an increased expression of the thrombin receptor mRNA in medial smooth muscle cells. Thereafter, the signal remains elevated in medial and intimal smooth muscle cells throughout the development of intimal thickening.21 Abundant thrombin receptor mRNA has also been detected in neointimal smooth muscle cells after endarterectomy in the baboon carotid artery as well as in early atherosclerotic lesions of human arteries.20 21 In addition, advanced human atherosclerotic lesions have elevated thrombin receptor mRNA levels, which are concentrated predominantly in areas rich in macrophages and VSMCs.20 These findings suggest that the enhanced expression of the thrombin receptor is a consequence of the vascular injury. Moreover, the association of the increased thrombin receptor mRNA levels within VSMCs, especially the proliferating neointimal cells, suggests that this event may be of importance for the proliferative response to injury. As a possible consequence of the increased thrombin receptor mRNA level, the plasma membrane would be constantly supplied with newly synthesized thrombin receptors, and hence thrombin may continue to exert its mitogenic effect during the development of intimal thickening.

The mechanisms regulating the in vivo expression of the thrombin receptor at sites of vascular injury remain to be identified. The rapid induction (within 6 hours) of thrombin receptor mRNA expression in the blood vessel wall after balloon injury21 suggests that hemostatic and thrombotic events occurring during the early phase of the vascular response to injury might be involved. Consistent with a role of platelets are the present findings indicating that the platelet-derived product 5-HT upregulated the expression of the thrombin receptor mRNA in cultured VSMCs. The observed fivefold increase in the relative steady-state level of the thrombin receptor mRNA may result from increased transcription of the thrombin receptor gene and/or stabilization of the thrombin receptor mRNA. However, Western blot analysis using polyclonal antibodies raised against the rodent thrombin receptor was not sensitive enough to detect the level of thrombin receptor protein in both control and 5-HT–treated smooth muscle cells. Thus, it remains to be demonstrated that the increases in thrombin receptor mRNA levels occur at the protein level as well. Nevertheless, the present findings that the thrombin-induced release of 6-ketoprostaglandin F1{alpha} was selectively increased in smooth muscle cells that had been exposed to 5-HT for several hours would suggest that 5-HT also increases the level of functional thrombin receptors in VSMCs. 5-HT upregulated the expression of the thrombin receptor mRNA in a transient manner, with a maximal effect occurring at 4 hours. This relatively short-term action may be explained, at least in part, by a continuous decrease in the amount of 5-HT in the incubation medium over time, because the indolamine breaks down rapidly in solution at neutral pH.31 In addition, the action of 5-HT may be limited by its uptake and subsequent intracellular metabolism by monoamine oxidases in VSMCs.32 The importance of this mechanism is indicated by the findings that inhibitors of monoamine oxidases potentiated the mitogenic effect of 5-HT in cultured pulmonary artery smooth muscle cells33 and reuptake blockers increased the formation of inositol phosphate induced by 5-HT in VSMCs.34 Despite these potential mechanisms limiting the action of 5-HT, a consistent upregulation of the thrombin receptor mRNA level was found at 10 nmol/L. This threshold value is in close agreement with the ability of 5-HT to contract isolated canine coronary and pulmonary arteries,35 36 rat jugular vein,37 and human mesenteric, cerebral, coronary, and internal mammary arteries38 39 and to stimulate mitogenesis in cultured bovine aortic smooth muscle cells.40

5-HT interacts with a family of receptors to affect diverse physiological responses in both the central nervous and vascular systems. Serotonergic receptors have been defined according to their interaction with various agonists and antagonists and the transduction signal they activate. From molecular cloning, these receptors have been subdivided into ligand-gated receptors and G-protein–coupled receptors.41 The latter class is divided into three subclasses according to the second messenger pathways to which the receptors are coupled. The 5-HT1 receptors interact negatively with adenylyl cyclase, the 5-HT2 receptors are coupled to the activation of phospholipase C, and the 5-HT4, 5-HT6, and 5-HT7 receptor subtypes interact positively with adenylyl cyclase. The direct contractile effect of 5-HT on the vascular smooth muscle is mediated predominantly by 5-HT2 receptors in most vascular beds.32 37 43 In human arteries, 5-HT contracted coronary and mesenteric arteries by activating a mixture of both 5-HT1–like and 5-HT2 receptors but contracted large cerebral arteries solely via 5-HT1–like receptors resembling the 5-HT1D subtype.39 44 Conversely, the mitogenic and chemotactic effect of 5-HT on cultured VSMCs, including those from rat aorta, is mediated predominantly by 5-HT2 receptors.40 45 46 47 48 A major role for 5-HT2 receptors is also suggested in the present study, because the expression of the thrombin receptor mRNA by 5-HT was inhibited by the 5-HT2 receptor antagonists ketanserin and ritanserin and by the nonselective 5-HT1/5-HT2 receptor antagonist methiothepin, whereas the 5-HT3 antagonist MDL-72222 was without effect.

Molecular cloning and characterization of the rat and hamster 5-HT2 receptors have indicated that the receptor is coupled to activation of phospholipase C via a Gq protein, resulting in the liberation of the second messengers diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which mobilizes calcium from intracellular stores.49 50 51 Both increases in [Ca2+]i and hydrolysis of phosphoinositide are induced by 5-HT in the vascular smooth muscle as well as in many other tissues.34 52 The present findings showing that inhibition of protein kinase C abolished the 5-HT–stimulated induction of thrombin receptor mRNA expression implicate the involvement of protein kinase C in the signal transduction cascade leading to thrombin receptor expression. Surprisingly, the direct activation of protein kinase C by PMA did not mimic the stimulatory effect of 5-HT. One explanation for this finding is that activation of serotonergic receptors may be coupled to an additional intracellular effector pathway that must be activated concomitantly with the protein kinase C pathway to stimulate the expression of the thrombin receptor. Because the calcium ionophore A23187 neither alone nor in combination with PMA stimulated thrombin receptor mRNA expression, increases in [Ca2+]i are unlikely to contribute to the signaling pathways leading to thrombin receptor mRNA expression in 5-HT–stimulated cells. In addition, the mitogenic action of 5-HT in cultured bovine pulmonary smooth muscle cells has been associated with enhanced tyrosine phosphorylation.26 A possible involvement of protein tyrosine kinases in the intracellular signaling pathway leading to the expression of the thrombin receptor mRNA by 5-HT is also suggested in the present study, because inhibitors of protein tyrosine kinases reduced the 5-HT effect, although only to a small extent. Altogether, the present observations support the concept that the 5-HT–stimulated expression of the thrombin receptor in VSMCs occurs via activation of the 5-HT2 subtype of serotonergic receptors and is coupled to a signaling pathway that involves protein kinase C activation and possibly also protein tyrosine kinases.

In healthy humans and animals, the plasma and vascular tissue concentration of 5-HT is maintained at low levels as a consequence of uptake and storage in platelets as well as the degradation by monoamine oxidases in endothelial cells and VSMCs. However, injury to the blood vessel wall resulting in the disruption or dysfunction of the endothelium (eg, balloon angioplasty, atherosclerosis) disturbs these homeostatic mechanisms. Indeed, increased levels of 5-HT, similar to those used in the present study, have been found locally in canine coronary arteries at sites of endothelial injury and stenosis,53 in the canine coronary sinus after experimentally induced endothelial injury,54 and also within the coronary bed of patients with unstable angina.55 In humans, intracoronary infusions of 5-HT at concentrations as low as 0.25 µmol/L evoked a coronary dilation in healthy subjects but a paradoxical vasoconstriction in patients with a dysfunctional endothelium, such as in coronary artery disease.56 5-HT is most likely released by mural thrombosis at sites of injury, since a close correlation has been found between the level of 5-HT and platelet deposition.53 54 Furthermore, micromolar concentrations of 5-HT can be reached after activation of platelet suspensions of cell density equivalent to those found in vivo.35 57 Thus, at sites of injury, the blood vessel wall may be chronically exposed to concentrations of platelet-derived 5-HT within the range necessary to evoke biological responses. In conjunction with these previous findings, the present data suggest that an important role of 5-HT in vascular injury, in addition to its ability to promote platelet aggregation and stimulate vascular tone and growth, is the stimulation of thrombin receptor expression. 5-HT may not be the sole platelet product that is able to affect the expression of the thrombin receptor in the blood vessel wall, because PDGF is also known to increase thrombin receptor mRNA levels in cultured VSMCs.21 A similar upregulation of the thrombin receptor mRNA expression has also been found with bFGF, a mitogen that has also been implicated in the proliferative response to balloon injury,58 in cultured VSMCs.21 59 Since 5-HT synergizes with PDGF and bFGF to stimulate mitogenesis in VSMCs,33 40 it is likely that these factors may also act synergistically to stimulate the synthesis of the thrombin receptor in the blood vessel wall.


*    Selected Abbreviations and Acronyms
 
bFGF = basic fibroblast growth factor
[Ca2+]i = intracellular calcium
5-HT = serotonin
PDGF = platelet-derived growth factor
PMA = phorbol 12-myristate 13-acetate
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Bu 436/4-3 and Schi 389/1-1) and a grant from the Institut de Recherches Internationales Servier (Paris, France). The authors thank Dr P.M. Vanhoutte (Institut de Recherches Internationales Servier) for critical reading of the manuscript and Edeltraud Thielen and Gaby Hoch for technical assistance.


*    Footnotes
 
Reprint requests to V.B. Schini-Kerth, PhD, Zentrum der Physiologie, Klinikum der JWG-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.

Received October 3, 1995; revision received December 5, 1995; accepted December 10, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Friedman RJ, Stemerman MB, Wenz B, Moore S, Gauldie J, Gent M, Tiell ML, Spaet TH. The effect of thrombocytopenia on experimental arteriosclerotic lesion formation in rabbits: smooth muscle cell proliferation and re-endothelialization. J Clin Invest. 1977;60:1191-1201.
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