Serotonin Inhibits Voltage-Gated K+ Currents in Pulmonary Artery Smooth Muscle Cells
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Angel Cogolludo, Laura Moreno, Federica
参见附件。
the Department of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spain.
Abstract
Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K+ (KV) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of KV channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on KV channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hKV1.5 channels stably expressed in Ltk– cells. 5-HT reduced native KV and hKV1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on KV currents and contraction were markedly prevented by the 5-HT2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (G6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent -cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and this was increased on stimulation with 5-HT. Moreover, KV1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT2A receptors inhibits native KV and hKV1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. KV channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.
Key Words: potassium ion channels hypertension pulmonary arteries receptors
Introduction
Pulmonary hypertension (PH) is a heterogeneous group of disorders characterized by a sustained increase in pulmonary artery (PA) pressure leading to progressive right ventricular failure and death. Several lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) plays a central role in the pathogenesis of PH.1,2 First, 5-HT is an effective pulmonary vasoconstrictor and induces vascular smooth muscle hyperplasia.2–5 Moreover, plasma levels of 5-HT are increased in patients with primary PH.6 Conversely, mild pulmonary hypertension has been reported in some series of patients with carcinoid syndrome, a tumor of enterochromaffin cells releasing large amounts of 5-HT.7 Patients treated with fenfluramine or dexfenfluramine, anorectic drugs that induce platelet 5-HT release, inhibit the 5-HT transporter (5-HTT) and stimulate 5-HT receptors, also have a 23-fold increased risk of PH.8 In addition, 5-HTT overexpression or polymorphisms in the gene encoding the 5-HTT are associated with PH.9,10 Furthermore, mice lacking 5-HTT or 5-HT receptors (eg, 5-HT1B or 5-HT2B) show attenuated PH induced by hypoxia.3,11,12 Finally, specific pharmacological inhibition of 5-HT1B or 5-HT2A receptors or 5-HTT attenuates and/or reverses the development of PH and prolongs survival in animal models of PH.3,12–15
K+ channels play an essential role in regulating resting membrane potential, intracellular calcium concentration ([Ca2+]i), and contraction of vascular smooth muscle.16–18 Activation of K+ channels leads to hyperpolarization, whereas their inhibition causes membrane depolarization, activation of voltage-gated L-type Ca2+ channels, increase in [Ca2+]i, and vasoconstriction. Voltage-gated K+ (KV) channels present in pulmonary artery smooth muscle cells (PASMC) are inhibited by hypoxia, endothelin-1, thromboxane A2, and anorectic drugs.16,19–21 Moreover, decreased expression or function of KV channels in PASMC has been involved in the pathogenesis of primary and anorexigen-induced PH.21,22 From the variety of KV channels expressed in PASMC, special interest has been paid to KV1.5, because decreased expression or activity and mutations of KV1.5 occurs in primary PH22,23 and in vivo gene transfer of KV1.5 reduces PH and restores hypoxic pulmonary vasoconstriction.24 Furthermore, decreased expression and function of KV channels in PASMC leads to inhibition of apoptosis and promotes pulmonary vascular medial hypertrophy,25 whereas upregulation of KV1.5 correlates with an increase in apoptosis/proliferation ratio and prevents and reverses PH.26
As stated above, both 5-HT and KV channels may have an etiological role in PH. Unfortunately, the effects of 5-HT on KV channels in PASMC have not been analyzed yet. We hypothesized that 5-HT might inhibit KV channels, thereby establishing a link between these 2 pathogenic pathways in PH. Therefore, in the present study we have analyzed the effects of 5-HT on the current flowing through KV channels (IK(V)) recorded in rat PASMC and through cloned hKV1.5 channels (IKV1.5) stably expressed in Ltk– cells. The role of 5-HT receptors and 5-HTT in 5-HT-induced effects has also been studied.
Materials and Methods
All experiments were performed in accordance with the European Animals Act 1986 (Scientific Procedures) and approved by our institutional review board. A detailed description of the experimental methods is available in the online data supplement at http://circres.ahajournals.org.
Second- to third-order branches of the PA isolated from male Wistar rats were dissected and denuded of endothelium and PASMC were enzymatically isolated. Membrane currents were recorded in PASMC or in Ltk– cells stably expressing hKV1.5 channels using the whole-cell configuration of the patch-clamp technique and membrane potential was measured under current-clamp configuration.19,27 Coimmunoprecipitation, confocal immunostaining, and contractile tension studies are described in the online data supplement.
Results
Effects of 5-HT on IK(V) and Membrane Potential in PASMC
Addition of 5-HT (10 μmol/L) markedly inhibited IK(V) at all potentials tested (Figure 1A and 1B) and depolarized PASMC (Figure 1D) (P<0.01). The concentration-response curve for the inhibition of IK(V) by 5-HT at test potentials of –30 and +60 mV was fitted to a Hill equation leading to Emax values of 41.2±3.4% and 50.2±0.5% and EC50 of 1.23±0.42 and 2.33±0.09 μmol/L, respectively (Figure 1C), indicating a voltage-independent inhibition. KV currents recorded before and after treatment with 5-HT showed similar activation kinetics and voltage-activation curves (supplemental Figure I). After washing with 5-HT-free solution for 10 to 15 minutes, the effects of 5-HT on KV currents (n=5; not shown) or on membrane potential (Figure 1D; n=3) were only weakly or not reversed. In contrast, the effects of the KV channel blocker 4-aminopyridine (4-AP) (3 mmol/L) on KV currents (not shown; n=3) or on membrane potential were reversed by 80% following a 5 minutes washing period (Figure 1E; n=3).
To test the involvement of 5-HT2A receptor on the IK(V) blocking properties of 5-HT, we performed experiments in the presence of the selective 5-HT2A receptor antagonist ketanserin (0.1 μmol/L). This drug had negligible effects on IK(V) but inhibited the effects of 5-HT on IK(V) (Figure 2) and on membrane potential (–46.3±2.1 and –45.2±2.1 before and after 5-HT, respectively; n=4). Similarly, the inhibitory effect of 5-HT on IK(V) was markedly attenuated in the presence of the 5-HTT inhibitor fluoxetine (0.1 μmol/L, Figure 2C; current-voltage [I-V] relationship in supplemental Figure II). However, other specific 5-HTT inhibitors such as fluvoxamine or citalopram or the 5-HT1B antagonist SB224289 (3 μmol/L) had no effects on the IK(V) blocking properties of 5-HT (Figure 2C; I-V relationships in supplemental Figure II).
Inhibition of hKV1.5 Currents by 5-HT
Figure 3 shows hKV1.5 current traces recorded in Ltk– cells stably expressing hKV1.5 channels. 5-HT (10 μmol/L) markedly inhibited hKV1.5 currents to a similar extent at all potentials tested, this effect being essentially prevented by ketanserin (0.1 μmol/L).
Role of Phospholipase C and Protein Kinases
To further assess the mechanisms involved in 5-HT-induced inhibition of IK(V), PASMC were perfused with different inhibitors before and during the addition of the agonist (summarized in Figure 4; I-V relationships in supplemental Figure III). The phospholipase C (PLC) inhibitor U73122 (3 μmol/L) and the classic protein kinase C (PKC) inhibitor G6976 (0.1 μmol/L) prevented the inhibitory effects of 5-HT on KV currents, whereas the PKC pseudosubstrate inhibitor (PKC-PI) (0.1 μmol/L, added in the internal solution) had no effect. The tyrosine kinases inhibitor genistein (10 μmol/L) per se caused a 25% inhibition of IK(V) and prevented the effects of 5-HT on the current. The inhibitory effect of genistein on IK(V) has been previously reported and seems to be independent of its tyrosine kinase inhibition activity.28 In the presence of the more selective tyrosine kinase inhibitor tyrphostin 23 (30 μmol/L), which had no effect per se, the effects of 5-HT on IK(V) were inhibited (Figure 4B and 4C). Likewise, tyrphostin 23 prevented the inhibitory effects of 5-HT on hKV1.5 recorded in Ltk– cells (Figure 4D). Furthermore, the incubation with the tyrosine phosphatase inhibitor vanadate (100 μmol/L) increased the inhibitory effect of a low concentration of 5-HT (0.1 μmol/L) on IK(V) in PASMC (Figure 4E). Taken together, these results indicated the involvement of tyrosine kinases in the electrophysiological effects of 5-HT. However, we did not find changes in the KV1.5 protein phosphorylation in tyrosine or serine residues, whereas at least 2 bands with a molecular mass of 20 to 24 kDa that coimmunoprecipitated with KV1.5 proteins showed increased tyrosine phosphorylation after 5 minutes of treatment with 5-HT (Figure 4F). Their nature is presently unknown.
KV1.5 Association With 5-HT2A Receptors and Caveolin-1
Homogenates from PA were immunoprecipitated with anti-KV1.5 antibodies and analyzed for 5-HT2A receptors and caveolin-1 content via Western blot analysis. Figure 5A shows that both proteins coimmunoprecipitated with KV1.5. Furthermore, the interaction among KV1.5, caveolin-1, and 5-HT2A augmented after stimulation with 5-HT. To analyze the potential functional role of caveolae, PASMC were incubated with -cyclodextrin (2% for 2 hours), a cholesterol-modifying agent that disrupts membrane lipid rafts. Interestingly, 5-HT failed to inhibit IK(V) in these lipid raft-disrupted myocytes (Figure 5B and 5C). In addition, concanavalin A, a widely used inhibitor of endocytotic processes, had no effect on IK(V) but prevented the effects of 5-HT on IK(V) (Figure 5D). Finally, confocal microscopy revealed that KV1.5 channels were localized preferentially in the plasma membrane of PASMC but were partly internalized on 5-HT stimulation, and this effect was prevented with concanavalin A (Figure 5E).
Contractile Responses to 5-HT
Stimulation of endothelium-denuded PA rings with 5-HT (10 μmol/L) induced a sustained contractile response of 113±6 mg (n=47). Under control conditions, the contraction elicited by 5-HT was suitably reproduced after a 30 minute washout (99±6% of the first contraction, P>0.05). Pretreatment with 0.1 μmol/L ketanserin or 3 μmol/L SB224289 before the second addition of 5-HT inhibited the vasoconstriction (Figure 6A). Fluoxetine, but not the other 5-HTT inhibitors fluvoxamine and citalopram, also caused a marked inhibition of the vasoconstriction induced by 5-HT. Furthermore, the contraction induced by 5-HT was markedly inhibited by the L-type Ca2+ channel blocker nifedipine (0.1 μmol/L), the classic PKC inhibitor G6976 (0.1 μmol/L), the tyrosine kinases inhibitors genistein (10 μmol/L) or tyrphostin 23 (30 μmol/L), or the endocytosis inhibitor concanavalin A (250 μg/mL, Figure 6B). In PA contracted with 5-HT (10 μmol/L), addition of tyrphostin 23 (10 μmol/L) induced a relaxant response of 38±8% (n=5; Figure 6C). However, when arteries were stimulated by 5-HT plus the KV channel inhibitor 4-AP (10 mmol/L), tyrphostin 23 had no relaxant effect (1±1%, n=4; P<0.05).
Discussion
This is the first study demonstrating regulation of the native PA KV channels and cloned human hKV1.5 channels by 5-HT. The main findings of the study can be summarized as follows. 5-HT depolarized PASMC and inhibited the KV current in PASMC and in Ltk– cells expressing cloned hKV1.5 but did not modify the activation curve and the kinetics of the currents. These effects were inhibited by antagonists/inhibitors of 5-HT2A receptors, PLC, classic PKCs, and tyrosine kinases and by cyclodextrin and concanavalin A. In addition, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and KV1.5 channels internalized when cells were stimulated with 5-HT. Finally, the contraction induced by 5-HT in isolated rat PA was inhibited by the same drugs preventing KV channels inhibition.
Decreased KV channel activity leads to depolarization, opening of L-type voltage-dependent Ca2+ channels, increased [Ca2+]i, and vasoconstriction.16–18 Thus, KV channel blockers such as 4-AP induce depolarization and pulmonary vasoconstriction. In the present report, 5-HT caused KV current inhibition with a similar potency (EC50 values of 2 μmol/L) than that previously reported for pulmonary vasoconstriction (EC50 values of 1 and 3 μmol/L in fawn-hooded and Sprague-Dawley rats, respectively29) and with a similar or higher efficacy (50% inhibition) than other vasoactive factors known to inhibit KV channels such as endothelin-1 (29%20), the thromboxane A2 analog U46619 (56%19), or hypoxia (49%30). In addition, 5-HT caused membrane depolarization; its vasoconstrictor response was inhibited by the L-type Ca2+ channel blocker nifedipine and by the same drugs preventing KV channel inhibition. Furthermore, in 5-HT-contracted PA, the relaxation induced by the tyrosine kinase inhibitor tyrphostin 23 was abolished by 4-AP. All of these results are consistent with the view that 5-HT-induced vasoconstriction is, at least in part, mediated by KV channel inhibition. Accordingly, 5-HT has been previously reported to block KV channels in carotid body type I cells31 and in choroid plexus epithelial cells.32
As outlined in the introduction, multiple evidences indicate that 5-HT plays a key role in the pathogenesis of PH. However, the molecular target of 5-HT is not clearly defined, and it seems likely that several G protein-coupled receptors, as well as the 5-HTT, are involved in PH. 5-HT-induced pulmonary vasoconstriction seems to be mainly attributable to the activation of 5-HT2A and 5-HT1B/1D receptors.4,29 In the present study, 5-HT-induced vasoconstriction was inhibited by the selective antagonists of 5-HT2A and 5-HT1B receptors ketanserin and SB224289, respectively, whereas the inhibition of KV currents was prevented only by ketanserin. Therefore, 5-HT-induced vasoconstriction was mediated by at least 2 types of receptors, 5-HT2A and 5-HT1B, but only the former signaled via KV channels. The 5-HTT inhibitor fluoxetine also inhibited the effects of 5-HT on KV currents and vasoconstriction. In contrast, other selective 5-HTT inhibitors such as fluvoxamine and citalopram at concentrations sufficient to fully inhibit the transporter had no effect. The different behavior of fluoxetine suggests that this drug is acting via a 5-HTT-independent mechanism. In fact, fluoxetine inhibited (+)-norfenfluramine- and 5-HT-induced vasoconstriction in mice lacking 5-HTT.33 One possibility is that fluoxetine antagonized 5-HT2A receptors, because this drug, in contrast to the other 5-HTT inhibitors tested, has a relatively high affinity against the 5-HT2A receptor (Ki140 nmol/L).34 This drug, 1 of the most widely prescribed antidepressant, has been recently proposed as a novel treatment for PH.13 Its inhibitory effect on 5-HT-induced KV current attenuation described herein might contribute to its ability to prevent and reverse PH.
KV channels exist as tetramers formed by 4 transmembrane KV subunits35 combined with modulatory cytosolic KV subunits. In human PA, 22 transcripts of KV (KV1.1 to -1.7, KV1.10, KV2.1, KV3.1, KV3.3, KV3.4, KV4.1, KV4.2, KV5.1, KV6.1 to -6.3, KV9.1, KV9.3, KV10.1, and KV11.1), and 3 of KV subunits (KV1 to -3) have been identified by RT-PCR.36 Further diversity can be found in native channels because heterotetramers can be formed by the combination of distinct KV subunits. Because KV1.5 subunits are believed to be major contributors of the native KV currents in PA,16 we analyzed the effects of 5-HT on the KV current carried by human cloned KV1.5 channels expressed in Ltk– cells. 5-HT induced an inhibitory effect on this current of similar characteristics of that in native PA myocytes. This effect was also prevented by ketanserin and tyrphostin 23, suggesting a similar signaling pathway in both types of cells. In addition, we found a strong expression of 5-HT2A receptors by Western blot in Ltk– cells (not shown). Thus, KV1.5 channels are likely candidates to underlie 5-HT-sensitive currents in native cells, even when we cannot rule out that other KV channel subunits might also contribute to the effects of 5-HT.
In previous studies,19,27 we reported that KV channel inhibition induced by the thromboxane A2 analog U46619 in both rat and porcine PA, involved the activation of PKC, an atypical PKC that is insensitive to diacylglycerol. However, 5-HT-induced inhibition of KV current was not significantly affected by the specific PKC inhibitor. 5-HT2A receptors signal primarily through heterotrimeric proteins of the Gq/11 subfamily, activation of PLC, the subsequent formation of diacylglycerol, and activation of classic diacylglycerol-sensitive PKC.37 Likewise, the effects of 5-HT on KV currents were prevented by U73122, a PLC inhibitor, and by G6976, an inhibitor of classic diacylglycerol-sensitive PKCs, that does not inhibit PKC and did not modify the effects of U46619 in rat PA.19 Accordingly, KV channel inhibition by 5-HT in carotid body type I cells and rat choroid plexus epithelial cells was sensitive to PKC inhibitors.31,32 In addition, 5-HT-induced (but not U46619-induced) inhibition was prevented by genistein, a widely used tyrosine kinase inhibitor, and by tyrphostin 23, a selective tyrosine kinase inhibitor. Furthermore, PA contracted by 5-HT attained a relaxant response on treatment with tyrphostin 23, suggesting that tyrosine kinase(s) is a significant player in the response to 5-HT. Incubation with the KV channel blocker 4-AP before application of tyrphostin 23 completely blocked its relaxing effect, strongly supporting the idea that KV channels are coupled to tyrosine kinase activation after 5-HT receptor stimulation. Because the degree of phosphorylation of tyrosine residues results from the balance of the phosphorylating activity of tyrosine kinases and the dephosphorylating activity of tyrosine phosphatases, it is expected that inhibition of the latter may result in an enhancement of tyrosine kinase mediated effects. In fact, the tyrosine phosphatase inhibitor vanadate potentiated the inhibitory effects of low concentrations of 5-HT on KV currents. We found no changes at the level of tyrosine or serine phosphorylation in KV1.5 channel protein from cells incubated with 5-HT, ruling out a direct phosphorylation of KV1.5 subunits. However, we found that 5-HT induced tyrosine phosphorylation in at least 2 unknown proteins (molecular mass of 20 to 24 kDa), which coimmunoprecipitated with KV1.5. Taken together, these results suggest that activation of PLC, classic PKC, and tyrosine kinases are involved in the signaling pathway of 5-HT. Likewise, 5-HT2A receptors have been coupled with activation of tyrosine kinases including Src and JAK2 via PKC-dependent or -independent pathways.38,39
Caveolae are specialized microdomains in the plasma membrane composed of integral membrane proteins, including the protein caveolin-1, that are involved in endocytotic and signal-transduction processes.40 Caveolae serve to compartmentalize and integrate numerous signaling events, such as PKC, tyrosine kinases, mitogen-activated protein kinase, ion channels, G proteins, and G protein-coupled receptors.40 Caveolin has been reported to colocalize with KV1.5 channels on the membrane of transfected Ltk– cells41 and with 5-HT2A receptors on the membrane of transfected HEK-293 cells and C6 glioma cells.42 We found that KV1.5 coimmunoprecipitated with 5-HT2A receptors and caveolin-1 in native PA. Therefore, this association could serve to cluster the 5-HT2A receptor and signaling proteins with the K+ channel, allowing for specific modulation while preventing crosstalk with other pathways. Depletion of membrane cholesterol disrupts caveolar lipid rafts without changing the amplitude of KV1.5 currents.41 We found that lipid raft disruption with cyclodextrin prevented the inhibitory effects of 5-HT on KV currents, indicating the functional importance of this compartmentalization. Concanavalin A binds to cell surface glycoproteins and impairs their mobility within the membrane bilayer and is widely used to block endocytotic processes,43 including 5-HT2A receptor endocytosis in HEK-293 cells.44 In the present study, concanavalin A prevented the inhibitory effects on KV currents and the vasoconstriction induced by 5-HT. Likewise, endocytosis of KV1.2 and KV1.5 channels has been reported to be a key mechanism in the control of KV channel activity, which is regulated by tyrosine kinases.45,46 Confocal images of PASMC also showed that KV1.5 immunofluorescence changed from a preferential membrane to cytosolic localization on stimulation with 5-HT, and this effect was prevented by concanavalin A. Taken together, these results suggest that the 5-HT-induced inhibition of KV currents and the subsequent vasoconstriction of PA result from the interaction of 5-HT2A receptors with KV channels within caveolar membrane microdomains and involve tyrosine phosphorylation and endocytotic processes. This is consistent with a reduction in KV current amplitude without modification of its biophysical features.
PH has a multifactorial origin2,47 characterized by sustained elevation of pulmonary arterial pressure associated with vasoconstriction and PA smooth muscle proliferation. Both 5-HT and KV channels have been implicated in the pathogenesis of PH. In the present study, we demonstrate that there is a link between these 2 key pathogenetic factors. Thus, 5-HT, via activation of 5-HT2A receptors, inhibited the activity of KV channels, resulting in pulmonary vasoconstriction. Decreased KV channel activity not only causes pulmonary vasoconstriction but also contributes to pulmonary vascular medial hypertrophy by inhibiting apoptotic cell shrinkage and apoptosis,25 whereas overexpression of the KV1.5 gene (KCNA5) induces apoptosis in PASMC.48 Thus, KV channel inhibition might also be involved in the medial hypertrophy of PA induced by 5-HT.5,49 Additionally, it must be noted that KV channel blockade has also been involved in the vasoconstriction and pulmonary hypertension induced by anorexigens and the antiparkinsonian drug pergolide.21,50 Interestingly, dexfenfluramine and pergolide activate 5-HT2A receptors,34,51 which suggests that KV channel blockade induced by these drugs might be secondary to 5-HT2A receptor activation.
In conclusion, 5-HT inhibits KV currents in PA myocytes and in Ltk– cells stably transfected with human KV1.5 channels through the activation of 5-HT2A receptors, PLC, classic PKCs, tyrosine kinases, and endocytotic processes. This effect is involved, at least partly, in PA vasoconstriction and might contribute to the development of human PH.
Acknowledgments
This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF2005-03770, SAF2005-04609, and AGL2004-06685-C04-1) and from Comunidad Autónoma de Madrid (GR/SAL/0594/2004). A.C, L.M., and F.L. are supported by RECAVA (Red Temática de Investigación Cardiovascular), Ministerio de Educación y Ciencia (FPU), and CSIC (I3P grant), respectively.
Footnotes
Original received October 19, 2005; revision received February 22, 2006; accepted February 28, 2006.
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the Department of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spain.
Abstract
Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) and voltage-gated K+ (KV) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of KV channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on KV channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hKV1.5 channels stably expressed in Ltk– cells. 5-HT reduced native KV and hKV1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on KV currents and contraction were markedly prevented by the 5-HT2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (G6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent -cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and this was increased on stimulation with 5-HT. Moreover, KV1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT2A receptors inhibits native KV and hKV1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. KV channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH.
Key Words: potassium ion channels hypertension pulmonary arteries receptors
Introduction
Pulmonary hypertension (PH) is a heterogeneous group of disorders characterized by a sustained increase in pulmonary artery (PA) pressure leading to progressive right ventricular failure and death. Several lines of evidence indicate that serotonin (5-hydroxytryptamine [5-HT]) plays a central role in the pathogenesis of PH.1,2 First, 5-HT is an effective pulmonary vasoconstrictor and induces vascular smooth muscle hyperplasia.2–5 Moreover, plasma levels of 5-HT are increased in patients with primary PH.6 Conversely, mild pulmonary hypertension has been reported in some series of patients with carcinoid syndrome, a tumor of enterochromaffin cells releasing large amounts of 5-HT.7 Patients treated with fenfluramine or dexfenfluramine, anorectic drugs that induce platelet 5-HT release, inhibit the 5-HT transporter (5-HTT) and stimulate 5-HT receptors, also have a 23-fold increased risk of PH.8 In addition, 5-HTT overexpression or polymorphisms in the gene encoding the 5-HTT are associated with PH.9,10 Furthermore, mice lacking 5-HTT or 5-HT receptors (eg, 5-HT1B or 5-HT2B) show attenuated PH induced by hypoxia.3,11,12 Finally, specific pharmacological inhibition of 5-HT1B or 5-HT2A receptors or 5-HTT attenuates and/or reverses the development of PH and prolongs survival in animal models of PH.3,12–15
K+ channels play an essential role in regulating resting membrane potential, intracellular calcium concentration ([Ca2+]i), and contraction of vascular smooth muscle.16–18 Activation of K+ channels leads to hyperpolarization, whereas their inhibition causes membrane depolarization, activation of voltage-gated L-type Ca2+ channels, increase in [Ca2+]i, and vasoconstriction. Voltage-gated K+ (KV) channels present in pulmonary artery smooth muscle cells (PASMC) are inhibited by hypoxia, endothelin-1, thromboxane A2, and anorectic drugs.16,19–21 Moreover, decreased expression or function of KV channels in PASMC has been involved in the pathogenesis of primary and anorexigen-induced PH.21,22 From the variety of KV channels expressed in PASMC, special interest has been paid to KV1.5, because decreased expression or activity and mutations of KV1.5 occurs in primary PH22,23 and in vivo gene transfer of KV1.5 reduces PH and restores hypoxic pulmonary vasoconstriction.24 Furthermore, decreased expression and function of KV channels in PASMC leads to inhibition of apoptosis and promotes pulmonary vascular medial hypertrophy,25 whereas upregulation of KV1.5 correlates with an increase in apoptosis/proliferation ratio and prevents and reverses PH.26
As stated above, both 5-HT and KV channels may have an etiological role in PH. Unfortunately, the effects of 5-HT on KV channels in PASMC have not been analyzed yet. We hypothesized that 5-HT might inhibit KV channels, thereby establishing a link between these 2 pathogenic pathways in PH. Therefore, in the present study we have analyzed the effects of 5-HT on the current flowing through KV channels (IK(V)) recorded in rat PASMC and through cloned hKV1.5 channels (IKV1.5) stably expressed in Ltk– cells. The role of 5-HT receptors and 5-HTT in 5-HT-induced effects has also been studied.
Materials and Methods
All experiments were performed in accordance with the European Animals Act 1986 (Scientific Procedures) and approved by our institutional review board. A detailed description of the experimental methods is available in the online data supplement at http://circres.ahajournals.org.
Second- to third-order branches of the PA isolated from male Wistar rats were dissected and denuded of endothelium and PASMC were enzymatically isolated. Membrane currents were recorded in PASMC or in Ltk– cells stably expressing hKV1.5 channels using the whole-cell configuration of the patch-clamp technique and membrane potential was measured under current-clamp configuration.19,27 Coimmunoprecipitation, confocal immunostaining, and contractile tension studies are described in the online data supplement.
Results
Effects of 5-HT on IK(V) and Membrane Potential in PASMC
Addition of 5-HT (10 μmol/L) markedly inhibited IK(V) at all potentials tested (Figure 1A and 1B) and depolarized PASMC (Figure 1D) (P<0.01). The concentration-response curve for the inhibition of IK(V) by 5-HT at test potentials of –30 and +60 mV was fitted to a Hill equation leading to Emax values of 41.2±3.4% and 50.2±0.5% and EC50 of 1.23±0.42 and 2.33±0.09 μmol/L, respectively (Figure 1C), indicating a voltage-independent inhibition. KV currents recorded before and after treatment with 5-HT showed similar activation kinetics and voltage-activation curves (supplemental Figure I). After washing with 5-HT-free solution for 10 to 15 minutes, the effects of 5-HT on KV currents (n=5; not shown) or on membrane potential (Figure 1D; n=3) were only weakly or not reversed. In contrast, the effects of the KV channel blocker 4-aminopyridine (4-AP) (3 mmol/L) on KV currents (not shown; n=3) or on membrane potential were reversed by 80% following a 5 minutes washing period (Figure 1E; n=3).
To test the involvement of 5-HT2A receptor on the IK(V) blocking properties of 5-HT, we performed experiments in the presence of the selective 5-HT2A receptor antagonist ketanserin (0.1 μmol/L). This drug had negligible effects on IK(V) but inhibited the effects of 5-HT on IK(V) (Figure 2) and on membrane potential (–46.3±2.1 and –45.2±2.1 before and after 5-HT, respectively; n=4). Similarly, the inhibitory effect of 5-HT on IK(V) was markedly attenuated in the presence of the 5-HTT inhibitor fluoxetine (0.1 μmol/L, Figure 2C; current-voltage [I-V] relationship in supplemental Figure II). However, other specific 5-HTT inhibitors such as fluvoxamine or citalopram or the 5-HT1B antagonist SB224289 (3 μmol/L) had no effects on the IK(V) blocking properties of 5-HT (Figure 2C; I-V relationships in supplemental Figure II).
Inhibition of hKV1.5 Currents by 5-HT
Figure 3 shows hKV1.5 current traces recorded in Ltk– cells stably expressing hKV1.5 channels. 5-HT (10 μmol/L) markedly inhibited hKV1.5 currents to a similar extent at all potentials tested, this effect being essentially prevented by ketanserin (0.1 μmol/L).
Role of Phospholipase C and Protein Kinases
To further assess the mechanisms involved in 5-HT-induced inhibition of IK(V), PASMC were perfused with different inhibitors before and during the addition of the agonist (summarized in Figure 4; I-V relationships in supplemental Figure III). The phospholipase C (PLC) inhibitor U73122 (3 μmol/L) and the classic protein kinase C (PKC) inhibitor G6976 (0.1 μmol/L) prevented the inhibitory effects of 5-HT on KV currents, whereas the PKC pseudosubstrate inhibitor (PKC-PI) (0.1 μmol/L, added in the internal solution) had no effect. The tyrosine kinases inhibitor genistein (10 μmol/L) per se caused a 25% inhibition of IK(V) and prevented the effects of 5-HT on the current. The inhibitory effect of genistein on IK(V) has been previously reported and seems to be independent of its tyrosine kinase inhibition activity.28 In the presence of the more selective tyrosine kinase inhibitor tyrphostin 23 (30 μmol/L), which had no effect per se, the effects of 5-HT on IK(V) were inhibited (Figure 4B and 4C). Likewise, tyrphostin 23 prevented the inhibitory effects of 5-HT on hKV1.5 recorded in Ltk– cells (Figure 4D). Furthermore, the incubation with the tyrosine phosphatase inhibitor vanadate (100 μmol/L) increased the inhibitory effect of a low concentration of 5-HT (0.1 μmol/L) on IK(V) in PASMC (Figure 4E). Taken together, these results indicated the involvement of tyrosine kinases in the electrophysiological effects of 5-HT. However, we did not find changes in the KV1.5 protein phosphorylation in tyrosine or serine residues, whereas at least 2 bands with a molecular mass of 20 to 24 kDa that coimmunoprecipitated with KV1.5 proteins showed increased tyrosine phosphorylation after 5 minutes of treatment with 5-HT (Figure 4F). Their nature is presently unknown.
KV1.5 Association With 5-HT2A Receptors and Caveolin-1
Homogenates from PA were immunoprecipitated with anti-KV1.5 antibodies and analyzed for 5-HT2A receptors and caveolin-1 content via Western blot analysis. Figure 5A shows that both proteins coimmunoprecipitated with KV1.5. Furthermore, the interaction among KV1.5, caveolin-1, and 5-HT2A augmented after stimulation with 5-HT. To analyze the potential functional role of caveolae, PASMC were incubated with -cyclodextrin (2% for 2 hours), a cholesterol-modifying agent that disrupts membrane lipid rafts. Interestingly, 5-HT failed to inhibit IK(V) in these lipid raft-disrupted myocytes (Figure 5B and 5C). In addition, concanavalin A, a widely used inhibitor of endocytotic processes, had no effect on IK(V) but prevented the effects of 5-HT on IK(V) (Figure 5D). Finally, confocal microscopy revealed that KV1.5 channels were localized preferentially in the plasma membrane of PASMC but were partly internalized on 5-HT stimulation, and this effect was prevented with concanavalin A (Figure 5E).
Contractile Responses to 5-HT
Stimulation of endothelium-denuded PA rings with 5-HT (10 μmol/L) induced a sustained contractile response of 113±6 mg (n=47). Under control conditions, the contraction elicited by 5-HT was suitably reproduced after a 30 minute washout (99±6% of the first contraction, P>0.05). Pretreatment with 0.1 μmol/L ketanserin or 3 μmol/L SB224289 before the second addition of 5-HT inhibited the vasoconstriction (Figure 6A). Fluoxetine, but not the other 5-HTT inhibitors fluvoxamine and citalopram, also caused a marked inhibition of the vasoconstriction induced by 5-HT. Furthermore, the contraction induced by 5-HT was markedly inhibited by the L-type Ca2+ channel blocker nifedipine (0.1 μmol/L), the classic PKC inhibitor G6976 (0.1 μmol/L), the tyrosine kinases inhibitors genistein (10 μmol/L) or tyrphostin 23 (30 μmol/L), or the endocytosis inhibitor concanavalin A (250 μg/mL, Figure 6B). In PA contracted with 5-HT (10 μmol/L), addition of tyrphostin 23 (10 μmol/L) induced a relaxant response of 38±8% (n=5; Figure 6C). However, when arteries were stimulated by 5-HT plus the KV channel inhibitor 4-AP (10 mmol/L), tyrphostin 23 had no relaxant effect (1±1%, n=4; P<0.05).
Discussion
This is the first study demonstrating regulation of the native PA KV channels and cloned human hKV1.5 channels by 5-HT. The main findings of the study can be summarized as follows. 5-HT depolarized PASMC and inhibited the KV current in PASMC and in Ltk– cells expressing cloned hKV1.5 but did not modify the activation curve and the kinetics of the currents. These effects were inhibited by antagonists/inhibitors of 5-HT2A receptors, PLC, classic PKCs, and tyrosine kinases and by cyclodextrin and concanavalin A. In addition, 5-HT2A receptors and caveolin-1 coimmunoprecipitated with KV1.5 channels, and KV1.5 channels internalized when cells were stimulated with 5-HT. Finally, the contraction induced by 5-HT in isolated rat PA was inhibited by the same drugs preventing KV channels inhibition.
Decreased KV channel activity leads to depolarization, opening of L-type voltage-dependent Ca2+ channels, increased [Ca2+]i, and vasoconstriction.16–18 Thus, KV channel blockers such as 4-AP induce depolarization and pulmonary vasoconstriction. In the present report, 5-HT caused KV current inhibition with a similar potency (EC50 values of 2 μmol/L) than that previously reported for pulmonary vasoconstriction (EC50 values of 1 and 3 μmol/L in fawn-hooded and Sprague-Dawley rats, respectively29) and with a similar or higher efficacy (50% inhibition) than other vasoactive factors known to inhibit KV channels such as endothelin-1 (29%20), the thromboxane A2 analog U46619 (56%19), or hypoxia (49%30). In addition, 5-HT caused membrane depolarization; its vasoconstrictor response was inhibited by the L-type Ca2+ channel blocker nifedipine and by the same drugs preventing KV channel inhibition. Furthermore, in 5-HT-contracted PA, the relaxation induced by the tyrosine kinase inhibitor tyrphostin 23 was abolished by 4-AP. All of these results are consistent with the view that 5-HT-induced vasoconstriction is, at least in part, mediated by KV channel inhibition. Accordingly, 5-HT has been previously reported to block KV channels in carotid body type I cells31 and in choroid plexus epithelial cells.32
As outlined in the introduction, multiple evidences indicate that 5-HT plays a key role in the pathogenesis of PH. However, the molecular target of 5-HT is not clearly defined, and it seems likely that several G protein-coupled receptors, as well as the 5-HTT, are involved in PH. 5-HT-induced pulmonary vasoconstriction seems to be mainly attributable to the activation of 5-HT2A and 5-HT1B/1D receptors.4,29 In the present study, 5-HT-induced vasoconstriction was inhibited by the selective antagonists of 5-HT2A and 5-HT1B receptors ketanserin and SB224289, respectively, whereas the inhibition of KV currents was prevented only by ketanserin. Therefore, 5-HT-induced vasoconstriction was mediated by at least 2 types of receptors, 5-HT2A and 5-HT1B, but only the former signaled via KV channels. The 5-HTT inhibitor fluoxetine also inhibited the effects of 5-HT on KV currents and vasoconstriction. In contrast, other selective 5-HTT inhibitors such as fluvoxamine and citalopram at concentrations sufficient to fully inhibit the transporter had no effect. The different behavior of fluoxetine suggests that this drug is acting via a 5-HTT-independent mechanism. In fact, fluoxetine inhibited (+)-norfenfluramine- and 5-HT-induced vasoconstriction in mice lacking 5-HTT.33 One possibility is that fluoxetine antagonized 5-HT2A receptors, because this drug, in contrast to the other 5-HTT inhibitors tested, has a relatively high affinity against the 5-HT2A receptor (Ki140 nmol/L).34 This drug, 1 of the most widely prescribed antidepressant, has been recently proposed as a novel treatment for PH.13 Its inhibitory effect on 5-HT-induced KV current attenuation described herein might contribute to its ability to prevent and reverse PH.
KV channels exist as tetramers formed by 4 transmembrane KV subunits35 combined with modulatory cytosolic KV subunits. In human PA, 22 transcripts of KV (KV1.1 to -1.7, KV1.10, KV2.1, KV3.1, KV3.3, KV3.4, KV4.1, KV4.2, KV5.1, KV6.1 to -6.3, KV9.1, KV9.3, KV10.1, and KV11.1), and 3 of KV subunits (KV1 to -3) have been identified by RT-PCR.36 Further diversity can be found in native channels because heterotetramers can be formed by the combination of distinct KV subunits. Because KV1.5 subunits are believed to be major contributors of the native KV currents in PA,16 we analyzed the effects of 5-HT on the KV current carried by human cloned KV1.5 channels expressed in Ltk– cells. 5-HT induced an inhibitory effect on this current of similar characteristics of that in native PA myocytes. This effect was also prevented by ketanserin and tyrphostin 23, suggesting a similar signaling pathway in both types of cells. In addition, we found a strong expression of 5-HT2A receptors by Western blot in Ltk– cells (not shown). Thus, KV1.5 channels are likely candidates to underlie 5-HT-sensitive currents in native cells, even when we cannot rule out that other KV channel subunits might also contribute to the effects of 5-HT.
In previous studies,19,27 we reported that KV channel inhibition induced by the thromboxane A2 analog U46619 in both rat and porcine PA, involved the activation of PKC, an atypical PKC that is insensitive to diacylglycerol. However, 5-HT-induced inhibition of KV current was not significantly affected by the specific PKC inhibitor. 5-HT2A receptors signal primarily through heterotrimeric proteins of the Gq/11 subfamily, activation of PLC, the subsequent formation of diacylglycerol, and activation of classic diacylglycerol-sensitive PKC.37 Likewise, the effects of 5-HT on KV currents were prevented by U73122, a PLC inhibitor, and by G6976, an inhibitor of classic diacylglycerol-sensitive PKCs, that does not inhibit PKC and did not modify the effects of U46619 in rat PA.19 Accordingly, KV channel inhibition by 5-HT in carotid body type I cells and rat choroid plexus epithelial cells was sensitive to PKC inhibitors.31,32 In addition, 5-HT-induced (but not U46619-induced) inhibition was prevented by genistein, a widely used tyrosine kinase inhibitor, and by tyrphostin 23, a selective tyrosine kinase inhibitor. Furthermore, PA contracted by 5-HT attained a relaxant response on treatment with tyrphostin 23, suggesting that tyrosine kinase(s) is a significant player in the response to 5-HT. Incubation with the KV channel blocker 4-AP before application of tyrphostin 23 completely blocked its relaxing effect, strongly supporting the idea that KV channels are coupled to tyrosine kinase activation after 5-HT receptor stimulation. Because the degree of phosphorylation of tyrosine residues results from the balance of the phosphorylating activity of tyrosine kinases and the dephosphorylating activity of tyrosine phosphatases, it is expected that inhibition of the latter may result in an enhancement of tyrosine kinase mediated effects. In fact, the tyrosine phosphatase inhibitor vanadate potentiated the inhibitory effects of low concentrations of 5-HT on KV currents. We found no changes at the level of tyrosine or serine phosphorylation in KV1.5 channel protein from cells incubated with 5-HT, ruling out a direct phosphorylation of KV1.5 subunits. However, we found that 5-HT induced tyrosine phosphorylation in at least 2 unknown proteins (molecular mass of 20 to 24 kDa), which coimmunoprecipitated with KV1.5. Taken together, these results suggest that activation of PLC, classic PKC, and tyrosine kinases are involved in the signaling pathway of 5-HT. Likewise, 5-HT2A receptors have been coupled with activation of tyrosine kinases including Src and JAK2 via PKC-dependent or -independent pathways.38,39
Caveolae are specialized microdomains in the plasma membrane composed of integral membrane proteins, including the protein caveolin-1, that are involved in endocytotic and signal-transduction processes.40 Caveolae serve to compartmentalize and integrate numerous signaling events, such as PKC, tyrosine kinases, mitogen-activated protein kinase, ion channels, G proteins, and G protein-coupled receptors.40 Caveolin has been reported to colocalize with KV1.5 channels on the membrane of transfected Ltk– cells41 and with 5-HT2A receptors on the membrane of transfected HEK-293 cells and C6 glioma cells.42 We found that KV1.5 coimmunoprecipitated with 5-HT2A receptors and caveolin-1 in native PA. Therefore, this association could serve to cluster the 5-HT2A receptor and signaling proteins with the K+ channel, allowing for specific modulation while preventing crosstalk with other pathways. Depletion of membrane cholesterol disrupts caveolar lipid rafts without changing the amplitude of KV1.5 currents.41 We found that lipid raft disruption with cyclodextrin prevented the inhibitory effects of 5-HT on KV currents, indicating the functional importance of this compartmentalization. Concanavalin A binds to cell surface glycoproteins and impairs their mobility within the membrane bilayer and is widely used to block endocytotic processes,43 including 5-HT2A receptor endocytosis in HEK-293 cells.44 In the present study, concanavalin A prevented the inhibitory effects on KV currents and the vasoconstriction induced by 5-HT. Likewise, endocytosis of KV1.2 and KV1.5 channels has been reported to be a key mechanism in the control of KV channel activity, which is regulated by tyrosine kinases.45,46 Confocal images of PASMC also showed that KV1.5 immunofluorescence changed from a preferential membrane to cytosolic localization on stimulation with 5-HT, and this effect was prevented by concanavalin A. Taken together, these results suggest that the 5-HT-induced inhibition of KV currents and the subsequent vasoconstriction of PA result from the interaction of 5-HT2A receptors with KV channels within caveolar membrane microdomains and involve tyrosine phosphorylation and endocytotic processes. This is consistent with a reduction in KV current amplitude without modification of its biophysical features.
PH has a multifactorial origin2,47 characterized by sustained elevation of pulmonary arterial pressure associated with vasoconstriction and PA smooth muscle proliferation. Both 5-HT and KV channels have been implicated in the pathogenesis of PH. In the present study, we demonstrate that there is a link between these 2 key pathogenetic factors. Thus, 5-HT, via activation of 5-HT2A receptors, inhibited the activity of KV channels, resulting in pulmonary vasoconstriction. Decreased KV channel activity not only causes pulmonary vasoconstriction but also contributes to pulmonary vascular medial hypertrophy by inhibiting apoptotic cell shrinkage and apoptosis,25 whereas overexpression of the KV1.5 gene (KCNA5) induces apoptosis in PASMC.48 Thus, KV channel inhibition might also be involved in the medial hypertrophy of PA induced by 5-HT.5,49 Additionally, it must be noted that KV channel blockade has also been involved in the vasoconstriction and pulmonary hypertension induced by anorexigens and the antiparkinsonian drug pergolide.21,50 Interestingly, dexfenfluramine and pergolide activate 5-HT2A receptors,34,51 which suggests that KV channel blockade induced by these drugs might be secondary to 5-HT2A receptor activation.
In conclusion, 5-HT inhibits KV currents in PA myocytes and in Ltk– cells stably transfected with human KV1.5 channels through the activation of 5-HT2A receptors, PLC, classic PKCs, tyrosine kinases, and endocytotic processes. This effect is involved, at least partly, in PA vasoconstriction and might contribute to the development of human PH.
Acknowledgments
This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF2005-03770, SAF2005-04609, and AGL2004-06685-C04-1) and from Comunidad Autónoma de Madrid (GR/SAL/0594/2004). A.C, L.M., and F.L. are supported by RECAVA (Red Temática de Investigación Cardiovascular), Ministerio de Educación y Ciencia (FPU), and CSIC (I3P grant), respectively.
Footnotes
Original received October 19, 2005; revision received February 22, 2006; accepted February 28, 2006.
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