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5-Hydroxytryptamine Stimulates Phosphorylation of p44/p42 Mitogen-Activated Protein Kinase Activation in Bovine Aortic Endothelial Cell Cultures

McDuffie, J. Eric; Motley, Evangeline D.*; Limbird, Lee E.; Maleque, Mohammed A.

Journal of Cardiovascular Pharmacology: March 2000 - Volume 35 - Issue 3 - p 398-402

5-Hydroxytryptamine (5-HT) is sequestered and released by endothelial cells, acts as an endothelial cell mitogen, promotes the release of nitric oxide (NO), and has been associated with the p44/p42 mitogen-activated protein kinase (MAPK) cascade. NO also acts as a cell mitogen and promotes signals that culminate in the phosphorylation of MAPK. The aim of this study was to test whether endothelial 5-HT receptors stimulate dual (tyrosyl- and threonyl-) phosphorylation of MAPK through a mitogen-activated protein kinase kinase-1 (MEK-1) and eNOS-dependent pathway in bovine aortic endothelial cells (BAECs). As shown by Western blot analysis, 5-HT and the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) stimulate time- and concentration-dependent (0.001-10 μM) phosphorylation of MAPK in these cells. The agonist-stimulated phosphorylation of MAPK was blocked by the 5-HT1B-receptor antagonist isamoltane (0.01-10 μM) and the MEK-1 inhibitor PD 098059 {[2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one]; 0.01-10 μM}. The eNOS inhibitor L-Nω-iminoethyl-L-ornithine (L-NIO; 0.01-10 μM) failed to block the 1 μM 5-NOT-stimulated responses. Our findings suggest that the 5-HT receptors (specifically 5-HT1B) mediate signals to MEK-1 and subsequently to MAPK through an eNOS-independent pathway in BAECs.

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan; *Department of Anatomy and Physiology, Meharry Medical College, †Department of Pharmacology, Vanderbilt University, and ‡Department of Pharmacology, Meharry Medical College, Nashville, Tennessee, U.S.A.

Received December 4, 1998; revision accepted October 28, 1999.

Address correspondence and reprint requests to Dr. J. E. McDuffie at University of Michigan Medical School, Department of Pathology, MSRB I Room 7520, Ann Arbor, MI 48109, U.S.A. E-mail:

5-Hydroxytryptamine (5-HT) is highly involved in the regulation of vascular reactivity (1). Serotonergic receptors expressed in endothelial cells include 5-HT1B, 5-HT2B, and 5-HT4(2). The 5-HT1B receptor (formally classified as rat 5-HT1D and human 5-HT1Dβ) is negatively coupled to adenylyl cyclase (2) and mediates increases in intracellular calcium (3). More recently the 5-HT1B receptor has been linked to the activation of the mitogen-activated protein kinase (MAPK) in a stably transfected Chinese hamster ovary (CHO) cell line expressing 5-HT1B receptors (4). Previously we reported evidence that 5-HT evokes endothelial nitric oxide synthase (eNOS) activation after activation of 5-HT1B receptors in bovine aortic endothelial cells (BAECs) (5). The activation of eNOS precedes the release of nitric oxide (NO), which mediates endothelium-dependent relaxation of blood vessels (6). Impairment of endothelium-dependent relaxation responses has been associated with MAPK (7). NO and NO-related species (NOx) act as endothelial cell mitogens and stimulate the activation of MAPK (8). The stimulation of MAPK by 5-HT has been associated with the Gi-coupled 5-HT1A receptor (9) and the Gq-coupled 5-HT2A(10) and 5-HT2B receptors (11). MAPK plays an important role in the 5-HT2A receptor-mediated contraction of arteries (10). Neither the putative role of MAPK in the endothelial 5-HT receptor-mediated MAPK signal-trandsuction pathways nor the biologic relevance of NO in the 5-HT receptor-mediated phosphorylation of MAPK have been previously defined. Although the mitogenic signals mediated by endothelial 5-HT receptors have been extensively investigated (12), further investigation is needed to determine whether a link exists between 5-HT receptor-mediated phosphorylation of MAPK and NO signaling in endothelial cells.

In this study, we examined the role of endothelial 5-HT receptors in the dual (tyrosyl- and threonyl-) phosphorylation of p44/p42 MAPK in BAECs. Specifically we tested whether 5-HT- and the 5-HT1B-receptor agonist 5-nonyloxytryptamine (5-NOT) stimulate phosphorylation of MAPK in BAECs using Western blot analysis. We found that the two agonists stimulate dual phosphorylation of MAPK. Isamoltane (5-HT1B-receptor antagonist) and PD 098059 (MEK-1 antagonist) effectively blocked the agonist-stimulated phosphorylation of MAPK, whereas L-NIO (eNOS selective antagonist) did not alter the agonist-evoked responses. These findings suggest that 5-HT1B receptors signal to MEK-1 and subsequently to MAPK through an eNOS-independent pathway, accounting in part for the molecular signals involved in 5-HT-stimulated mitogenesis in endothelial cells.

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Chemicals and reagents

L-Nω-iminoethyl-L-ornithine (L-NIO) and PD 098059 {[2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one]} were purchased from Calbiochem. 5-Nonyloxytryptamine (5-NOT) and isamoltane were purchased from Tocris. All other chemicals and reagents were purchased from Sigma.

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Cell culture and Western blot analysis

Bovine aortic endothelial cells (BAECs) were purchased from Clonetics and cultured (≤15 passages) according to the supplier's reference protocol. Cell cultures were grown on six-well plates and made quiescent by incubation with serum-free Dulbecco's modified Eagle's Medium (DMEM) for 24 h before use. The cells were stimulated with either vehicle (0.1% dimethyl sulfoxide), agonist, and/or antagonists at 37°C in serum-free DMEM for specified durations. The reactions were terminated by the replacement of medium with 100 μl of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer [62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 1% β-mercaptoethanol, and 0.1% bromophenol blue, pH 6.8 (13)]. After brief sonication (10 s), samples were boiled for 5 min at 95°C and centrifuged (14,000 g, 5 min) at 4°C. The resulting supernatants (25 μl) were subjected to SDS-PAGE. Proteins in the gel were transferred to Hybond-C extra nitrocellulose membrane (Amersham). After blocking with 5% milk, the membrane was treated with primary anti-rabbit polyclonal immunoglobulin G (IgG) phosphospecific p44/42 MAPK antibody (1:2,000; New England Biolabs) and subsequently incubated with secondary anti-rabbit Ig, peroxidase-linked species-specific whole antibody (1:2,000, Amersham). Blots were reprobed using a primary anti-rabbit ERK2-specific antibody (New England Biolabs) to demonstrate that the autoradiographic densities at 42 and 44 kDa were not due to unequivalent protein loading. The quantity of MAPK protein expressed remained unchanged after the addition of ligand(s). Immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) detection system (Amersham) and quantified using an Adobe Photo Imaging System.

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Endothelial 5-HT receptor-mediated MAPK phosphorylation

We examined the phosphorylation of p44/p42 MAPK in BAECs by Western blot analysis with a phosphospecific anti-MAPK antibody after stimulation with 5-HT and the 5-HT1B-receptor agonist 5-NOT. Phosphorylation of MAPK was maximal by 10 min after 5-HT and 5-NOT stimulation (Fig. 1A). A concentration-dependent (1 nM-10 μM) increase in phosphorylation of MAPK was observed with both 5-HT and 5-NOT (Fig. 1B). 5-NOT "maximally" stimulated MAPK phosphorylation to a higher degree when compared with 5-HT. The marked increase in the phosphorylation of p44/p42 MAPK by 5-NOT, when compared with 5-HT, is similar to its effects on the inhibition of adenylyl cyclase (14). The 5-HT- and 5-NOT-stimulated responses were attenuated by the 5-HT1B antagonist isamoltane in a concentration-dependent manner (Fig. 1C). The high concentration(s) of isamoltane used to block the agonist-stimulated MAPK phosphorylation are in agreement with the concentration (10 μM) needed to displace 5-HT from 5-HT1B receptors (∼50% displacement) in radioligand-binding assays (15). Therefore we concluded that the observed agonist-evoked responses occurred after the activation of 5-HT1B receptors.

FIG. 1

FIG. 1

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Effects of PD 098059 on 5-HT receptor-mediated phosphorylation of MAPK

MAPK phosphorylation occurs after the activation of MEK-1 (16), which plays a role in the regulation of receptor-mediated vascular reactivity (10). The MEK-1 inhibitor PD 098059 is capable of inhibiting both tyrosyl- and threonyl-phosphorylation of p42 and p44 MAPK (10). PD 098059 (1 μM) effectively inhibited the 5-HT- and 5-NOT-stimulated phosphorylation of MAPK (Fig. 2). These data suggest that 5-HT and 5-HT1B agonists stimulate MAPK phosphorylation through a MEK-1-dependent pathway.

FIG. 2

FIG. 2

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Effects of L-NIO on 5-HT receptor-mediated phosphorylation of MAPK

It has been shown that 5-HT promotes the release of NO (6), and the 5-HT1B receptor mediates activation of eNOS in BAECs (5). To examine the sensitivity of 5-HT-stimulated phosphorylation of MAPK to an eNOS-selective antagonist, BAECs were preincubated with L-NIO. L-NIO (0.01-10 μM) pretreatment failed to block the 5-HT- and 5-NOT-evoked responses (Fig. 3). Whereas the 5-HT1B receptor mediates NO release in endothelial cells, the failure of L-NIO to block the agonist-stimulated responses clearly suggests that these receptors signal to MAPK independent of their ability to promote intracellular NO accumulation.

FIG. 3

FIG. 3

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Several stimuli (i.e, 5-HT and NO) generate signals leading to the phosphorylation of MAPK, which is clearly implicated in the regulation of gene transcription and protein synthesis (16-18). Previous reports suggested that the mitogenic effects of 5-HT on endothelial cells may involve Gq-coupled 5-HT2 receptors; however, it has been shown that pertussis toxin (PTX) attenuates these effects, which suggests the involvement of Gi-coupled 5-HT receptors (19). The Gi-coupled 5-HT1B receptor mediates eNOS activation and the subsequent release of NO and endothelium-dependent vasodilation (5). In this study, we showed that dual phosphorylation of MAPK after 5-HT is mediated by Gi-coupled 5-HT1B receptors through subsequent activation of MEK-1, but not by intracellular NO accumulation in BAECs.

The data from this study show that 5-NOT elicits time-(maximal within 10 min, Fig. 1A) and concentration-dependent (Fig. 1B) increases in phosphorylation of MAPK. 5-NOT acts as a full agonist for 5-HT1B receptors at concentrations ≤1 μM(14). The 1 μM 5-NOT-evoked response was qualitatively identical to that observed in 1 μM 5-HT-stimulated cells after pretreatment with the 5-HT1B inhibitor isamoltane (Fig. 1C), which further suggests the involvement of 5-HT1B receptors. These observations are consistent with other findings in this cell type, in which both isamoltane and the Gi-coupled protein inhibitor, PTX, effectively blocked 5-HT- and 5-NOT-stimulated eNOS activation (5). These data are also in agreement with findings in transfected CHO-1B cells, which suggests that the 5-HT1B receptor signals to the activation of MAPK through Gi proteins (4). The failure of isamoltane to attenuate the 1 μM 5-HT-stimulated response completely (p42 MAPK phosphorylation) may be associated with the decrease in receptor selectivity exhibited by isamoltane at higher concentrations (4,15). However, we were able to demonstrate inhibition of maximal 5-NOT-stimulated p44 MAPK phosphorylation with 10 μM isamoltane. The p42 MAPK phosphorylation evoked by 5-NOT, as opposed to that by 5-HT, also was blocked by 10 μM isamoltane. This suggests that p42 MAPK phosphorylation may result after the activation of other endogenous 5-HT receptors.

The phosphorylation of MAPK by 5-HT and 5-NOT was sensitive to the MEK-1 antagonist PD 098059 (Fig. 2). These findings are consistent with earlier reports that suggest that PD 098059 blocks 5-HT-stimulated tyrosylphosphorylation of MAPK in endothelium-denuded rat aortic ring preparations (10). PD 098059 elicited similar inhibitory effects on 5-NOT-stimulated MAPK phosphorylation (Fig. 2). In parallel experiments in which eNOS activity was measured, 10 μM PD 098059 (concentration producing 100% reduction of the 5-HT-stimulated phosphorylation of MAPK) did not block the 5-HT-stimulated eNOS activity (data not shown). In addition, a similar study suggested that vascular endothelium-derived growth factor (VEGF)-stimulated NOS activity is blocked by the nonselective NOS inhibitor L-Nω-monomethyl-L-arginine (L-NMMA), but not by PD 098059 in coronary venular endothelial cells (18). Therefore we ruled out the possibility that PD 098059 could affect other transducing pathways upstream of the activation of MAPK in our system.

Pretreatment of BAECs with the eNOS-selective inhibitor L-NIO did not alter the 5-NOT-stimulated phosphorylation of MAPK (Fig. 3). These findings suggest that NO does not serve as a putative signal-transduction molecule between the endothelial 5-HT receptors and MAPK. It may be concluded that because 5-HT plays a significant role in the regulation of endothelium-dependent vessel relaxation (2,5,6), agents such as PD 098059 could be used therapeutically to mitigate pathophysiologic conditions (i.e., hypertension and atherosclerosis) that may arise after 5-HT-receptor activation.

In conclusion, the 5-HT1B receptor-mediated phosphorylation of MAPK in BAECs appears to be MEK-1 dependent and eNOS independent. Although it is yet speculative whether NO plays a significant role in the regulation of endothelial cell growth by other mitogens (18), our studies with BAECs lend novel insights into the molecular mechanisms underlying 5-HT1B receptor-mediated mitogenic signals in endothelial cells.

Acknowledgment: We to thank Gerald Frank, Tadashi Yamakawa, M.D., Ph.D., and Edward Yun for their technical assistance. This work was supported by grants from the National Science Foundation and National Institutes of Health, Heart and Lung Institute: NSF 9550699, HL07809, and HL03320.

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5-Hydroxytryptamine; Protein kinase; Nitric oxide; Endothelium

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