Contribution of Kv Channels to Phenotypic Remodeling of Human Uterine Artery Smooth Muscle Cells
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循环研究杂志 2005年第12期
the Departamento de Bioqueica y Biologe Molecular y Fisiologe e Instituto de Biologe y Geneetica Molecular (E.M.-V., A.M.-D., O.C., P.C., M.T.P.-G., J.R.L.-L.), Universidad de Valladolid y Consejo Superior de Investigaciones Ciente猣icas, Facultad de Medicina, Valladolid, Spain
Institut de Malalties Cardiovasculars (M.H.), Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer, Universidad de Barcelona, Barcelona, Spain.
Abstract
Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kv subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling.
Key Words: Kv channel expression vascular smooth muscle cells cell proliferation K+ currents
Introduction
The vascular smooth muscle cells (VSMCs) of mature animals are highly specialized cells whose main function is contraction. Although during vasculogenesis, the principal role of VSMCs is proliferation and synthesis of the matrix components of the vessel wall, differentiated VSMCs proliferate at an extremely low rate and express a unique repertoire of contractile proteins, ion channels, and signaling molecules required for contraction. However, VSMCs can undergo relatively rapid and reversible phenotypic changes in response to local environmental conditions. Accelerated proliferation of VSMCs is known to play a key role in atherosclerotic plaque formation and postangioplasty restenosis and is a common feature in hypertensive arteries.1
VSMCs express a large repertoire of ion channels and membrane receptors that vary widely among different vascular beds and are key determinants of the electrical and contractile responses of the cells.2eC5 Although release of intracellular Ca2+ is necessary for effective contraction, Ca2+ influx through voltage-activated Ca2+ (Cav) channels is responsible for initiating contraction, making resting membrane potential (Em) a primary determinant of vascular smooth muscle tone.4 Membrane depolarization opens Cav and raises [Ca2+]i, and cytosolic-free Ca2+ serves, in turn, as a critical signal transduction element in a variety of cell functions, such as contraction, migration, proliferation, and gene expression.6eC8
There is increasing evidence showing that K+ channels may have an important role in dedifferentiation and proliferation of VSMCs. Modulating Em, K+ channels can affect not only Ca2+ influx, a well-established factor influencing cell proliferation, but also the driving force for Na+-dependent transport, the intracellular pH, and the regulation of cell volume, all factors that also participate in proliferation and apoptotic processes.9,10 The role of K+ channels on cell proliferation is a complex modulatory activity that only certain K+ channels at specific times and locations can perform. This fact, together with the broad diversity of functional K+ currents among different vascular beds, has ravelled the analysis of the participation of K+ channels on VSMCs proliferation. At least 4 different types of K+ channels have been identified in VSMCs: inward rectifier, voltage-gated (Kv), ATP-gated, and Ca2+-gated K+ (BKCa) channels.2,4 The expression of these 4 types has been reported to vary among vascular beds as well as with vessel size.5,11 However, Kv and BKCa channels are present in virtually all vascular myocytes and strongly influence contractile responses.2 Moreover, several Kv channels have been described to participate in the proliferation process in pulmonary artery smooth muscles cells (SMCs).12,13
The goals of the present study were to characterize the expression profile of Kv channels in VSMCs from human uterine artery in contractile and proliferating phenotype and to evaluate their possible contribution to phenotypic modulation. Enzymatic dispersion of small pieces of uterine arteries provide VSMCs in contractile phenotype, which were studied within the same day, whereas proliferating VSMCs were obtained from explants of uterine arteries kept in culture for several passages. The molecular identification of the functional Kv subunits in both preparations shows that whereas Kv1 members are the main contributors to the Kv currents in contractile VSMCs, Kv3.4 expression is upregulated under proliferating conditions and represents the largest proportion of the Kv current in dedifferentiated VSMCs. Furthermore, selective blockade of Kv3.4 channels decreased proliferation rate, suggesting a direct relationship between channel function and uterine artery SMCs proliferation.
Materials and Methods
Uterine arteries were obtained from patients undergoing hysterectomy at the Clinic Hospital of Barcelona, with protocols approved by the Human Investigation Ethics Committee of the Hospital. Details of the materials and methods used in this study are in the online data supplement available at http://circres.ahajournals.org.
Results
Kv Currents in Freshly Dissociated and Cultured VSMCs
K+ currents were studied in acutely dissociated cells and in cultured cells obtained from human uterine arteries. After establishing the whole-cell configuration, currenteCvoltage (I/V) relationships for K+ currents were obtained every 2 minutes. After several records of I/V curves in control conditions, BKCa currents were subtracted by using the selective blocker paxilline (500 nmol/L).14,15 Figure 1 shows average I/V curves and sample records (insets) obtained in acutely dispersed cells (Figure 1A) and in cultured VSMCs (Figure 1B) before and after paxilline application. As previously reported,16 cultured cells exhibited tetrodotoxin-sensitive inward Na+ currents, which were absent in acutely dispersed cells (Figure 1B, inset). Depolarization elicited outward currents with an apparent threshold for activation positive to eC40 mV, and paxilline decreased current amplitudes at potentials positive to +30 mV. This effect was more evident in acutely dispersed cells. Cell capacitance was 40.21±2.45 pF in fresh VSMCs (n=28) and 44.58±6.17 pF in cultured cells (n=18). Current density was not significantly different between the 2 groups (19.04±2.76 versus 15.11±2.72 pA/pF at +80 mV), but BKCa current represents more than 70% of the outward K+ current in the freshly dissociated VSMCs and only 20% in the cultured cells. Furthermore, we consistently found a kinetic change with a larger proportion of inactivating K+ currents in cultured VSMCs (see traces in Figure 1). Resting Em measurements under perforated-patch conditions showed that contractile VSMCs had, on average, more hyperpolarized potentials than proliferating VSMCs (eC45.0±1.9 mV versus eC38.4±2.4 mV respectively, P<0.05; Figure 1C). Resting Em was not modified by paxilline in either of the 2 groups (data not shown). These observations indicate that the proliferation of human uterine artery VSMCs is associated with a decrease in the expression of BKCa currents and an increase in the contribution of Kv currents.
Pharmacological Characterization of Kv Currents
Whereas downregulation of BKCa currents with the phenotypic switch has been described previously,3,17 changes in Kv currents are not well characterized. Therefore, we characterized pharmacological Kv currents in both phenotypes after blocking BKCa currents with paxilline. Correolide was used to selectively block Kv1 currents,18 tetraethylammonium (TEA) sensitivity allowed the identification of Kv2 and Kv3 currents, and TEA- and correolide-insensitive current was attributed to Kv4 currents.19 The latter were also identified by their sensitivity to block by toxins such as heteropodatoxin20 and phrixotoxin.21
In acutely dispersed VSMCs (Figure 2), Kv1 currents were the predominant component of the K+ currents (Figure 2A). Correolide reduced Kv current amplitude by 21% or 70% at +80 mV when applied at 1 or 10 eol/L, respectively. At 10 eol/L, the effect was statistically significant at voltage values above eC30 mV. TEA was applied at low (micromolar) and high (millimolar) concentrations to dissect the contribution of high- and low-TEAeCsensitive Kv channels. There was a small reduction in the current amplitude in the presence of 100 eol/L TEA (15% inhibition at +80 mV; Figure 2B), which did not significantly increase after raising TEA up to 20 mmol/L. The onset of TEA inhibition appeared at more positive potentials than that of correolide (compare Figure 2A and 2B). The effect of TEA was the same on the total Kv current as on the correolide-insensitive fraction of the current, suggesting that Kv1 channels in acutely dispersed VSMCs are not sensitive to TEA (data not shown). The contribution of Kv4 channels to Kv currents was studied with specific blockers. Figure 2C shows an example in which 70 nmol/L phrixotoxin was used. The effect of the drug was voltage dependent (see inset). On average, phrixotoxin induced a 15% inhibition on the current amplitude at +80 mV, similar to the estimated proportion of TEA- and correolide-insensitive current (12.0±3.5%).
When the same pharmacological study was performed in cultured VSMCs, a clearly different pattern was apparent (Figure 3). First, correolide (Figure 3A) had only a very modest effect (11.5±6.3% inhibition at +40 mV). Second, the predominant fast-inactivating current observed in these cells was almost completely abolished by low TEA (up to 1 mmol/L; Figure 3B). Finally, blockers of Kv4 channels had almost no effect (Figure 3C). Therefore, the predominant Kv current in cultured VSMCs is a transient current with high-TEA sensitivity, pointing to some members of the Kv3 subfamily, particularly Kv3.4. This suggestion was confirmed by exploring the effect of BDS-1, a selective blocker of Kv3.4 channels. BDS-1 (2.5 eol/L) decreased the current amplitude by &40% to 50% at potentials of eC20 mV or greater (Figure 3D). Taken together, these functional data suggest an extensive remodeling of Kv channels associated with the phenotypic modulation, with a predominant role of Kv1 in acutely dispersed and of Kv3 in cultured VSMCs, as indicated by the summary data of Figure 3E.
Expression Profile of Kv mRNA
To study the molecular correlates of the Kv currents, real-time PCR was performed in both contractile and proliferating VSMCs. The results, normalized to RPL18 mRNA amount and corrected for the amplification efficiency of each reaction (see online data supplement), are plotted in Figure 4A. Unexpectedly, Kv4 channels represent the largest amount in both preparations, accounting for 67% and 80% of the total Kv mRNA (see online data supplement). We did not detect expression of Kv3.1, Kv3.2, or Kv1.1 in any preparation. With the exception of Kv4.2 and Kv3.4 subunits, which undergo an increased expression in cultured VSMCs, proliferation induced a downregulation of mRNA of all Kv subunits present in fresh tissue. Figure 4B shows the magnitude of the changes in the expression of Kv subunit mRNA from proliferating VSMC, taking the levels of mRNA in contractile VSMCs as the calibrator. In general, there is a correlation between changes in mRNA levels and changes in functional expression when comparing contractile and proliferating VSMCs (compare Figures 4B and 3E): whereas Kv1 channels are downregulated, Kv3.4 channels upregulate their expression and their functional contribution in cultured VSMCs.
Expression and Subcellular Location of Kv Proteins
The above data show, nevertheless, some discrepancies between the mRNA levels of a given Kv subunit and its contribution to the total Kv current, which could be attributable to the absence of correlation between mRNA and protein levels or, alternatively, to altered membrane trafficking of the protein. To distinguish between these 2 possibilities, we determined protein expression by immunoblot and cellular location with immunocytochemistry. Representative Western blots both in freshly isolated tissues and in protein extracts from cultured cells are shown in Figure 5. Kv1 subunit proteins were detected in fresh tissues, being absent or expressed at much lower levels in cultured VSMCs, with the only exception of Kv1.3, which appears to be more abundant in protein extracts from cultured VSMCs, contrary to what we will predict according to mRNA levels. A similar inconsistency was found in the case of Kv3.4 protein, which seems to be more abundant in fresh VSMCs than in cultured cells. Finally, with regard to Kv4 subunit proteins, bands for Kv4.2 and Kv4.3 were observed in both tissue and cell cultures, with substantially higher levels of expression in protein extracts from tissues, revealing the presence in both preparations of Kv4 subunit proteins, whose functional contribution has been found to be very small or almost absent.
Next, we explored the subcellular location of some Kv subunit proteins, which had levels of expression (as determined by Western blot) that did not match their estimated levels of activity (as determined by functional studies), namely Kv1.3, Kv3.4, and Kv4 subfamily members. Kv1.3 was expressed in patches of membrane in acutely dissociated VSMCs (Figure 6A), whereas Kv4.3 proteins were found to colocalize with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) in the membranes of intracellular compartments both in cultured (Figure 6E) and freshly dissociated (data not shown) VSMCs. Finally, Kv3.4 immunoreactivity was detected in both preparations but in different cellular locations. Whereas, in freshly dissociated VSMCs, Kv3.4 colocalized with calreticulin and was absent from the plasma membrane (Figure 6B), in cultured VSMCs, Kv3.4 was almost exclusively found in a plasma membrane location (Figure 6C and D). Interestingly, whereas Kv3.4 mRNA increased in proliferating cells, total Kv3.4 subunit protein decreased, although it seemed to be more efficiently located in the plasma membrane.
Role of Kv3.4 in VSMCs Proliferation
The upregulation of Kv3.4 subunit mRNA during VSMC dedifferentiation and its transposition from a cytoplasmic to a membrane location suggest that the activity of Kv3.4 channels may be related to proliferation. To explore this hypothesis, we studied the proliferation rate of cultured VSMCs when the activity of the channels was blocked by low TEA concentrations (100 eol/L) or BDS-I toxin (Figure 7A). TEA application led to a decreased proliferation rate that was already significant after 24 hours of treatment. The difference increased with time and reached a plateau around 96 hours after TEA treatment. The same results were obtained when Kv3.4 channels were blocked with 2.5 eol/L BDS-I, whereas blockade of Kv4 channels with 70 nmol/L phrixotoxin did not affect proliferation rate (inset in Figure 7A). These experiments demonstrate a specific link between Kv3.4 (but not Kv4) channels and VSMC proliferation, although the contribution of other Kv channels to this process (ie, some Kv1 family members, as previously found in other preparations22,23) cannot be excluded.
The decreased number of cells in the presence of Kv3.4 channel blockers could reflect a decreased proliferation rate or an increase in the number of cells undergoing apoptosis. Because among the hallmarks of late-stage apoptosis is the fragmentation of nuclear chromatin, we performed a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay to detect apoptosis-induced DNA fragmentation both in control cells and in 100 eol/L TEAeCtreated cells (Figure 7B). There was no significant cell death in either of the 2 samples (2.1±0.9% in control versus 2.0±0.7% in TEA treated, n=10 fields from 2 independent experiments), indicating that TEA block of Kv3.4 channels does not induce apoptosis.
Discussion
In this work, we have characterized the Kv subunit profile underlying Kv currents of VSMCs from human uterine arteries and its changes when VSMCs switch from a contractile to a proliferating phenotype.
With the exception of pulmonary arteries, little is known regarding the expression profile of Kv channels in human resistance arteries.24eC27 However, it is well established that Kv channels play an important role in regulating contraction through their effects on Em and by integrating a variety of vasoactive signals.2 Moreover, abnormal expression profiles of Kv channels have been involved in the pathogenesis of arterial hypertension and vascular proliferation, leading to new opportunities for developing drugs for targeting disease-specific changes in ion channel expression.28,29 Identification of the contributing Kv genes is crucial to pursuit this approach.
The large diversity of Kv channel genes, together with their possibilities of heteromultimerization, association with accessory subunits, and alternative splicing, leads to an enormous diversity in the molecular composition and properties of Kv channel complexes.19 Often, the number of expressed Kv subunits appears to be much larger than the number of apparent Kv current components.30 The oligomeric composition of Kv channels and the factors regulating their folding and trafficking to the cell membrane31 could contribute to explain these discrepancies.
We have tried to correlate the electrophysiological and pharmacological properties of Kv currents with the expression of Kv subunits, both at mRNA and protein levels. Although the role of BKCa currents in resistance arteries in vivo cannot be evaluated with this study, the apparent activation thresholds of BKCa and Kv currents (see Figure 1) suggests a small contribution of BKCa current to the resting Em in relaxed cells, which is supported by the measured resting Em values (Figure 1C) and its insensitivity to paxilline. Our observations echo the results obtained in other VSMC preparations, where Kv currents activated at potentials 20 to 30 mV more negative than BKCa currents.15,32
Our results showing that Kv1 members are the predominant components of the Kv currents are in good agreement with previous reports from other resistance vessels.33eC37 The pharmacological data from acute VSMCs also indicate the contribution of Kv2, Kv3, and Kv4 channels. Kv2.1 currents have been described in rat mesenteric artery VSMCs38 and have been found to be the major contributor to the Kv current in VSMCs from conduit arteries such as rat aorta.14,15,17 mRNA and even protein for Kv3.4 and Kv4 channels have been previously described in rat mesenteric SMCs, but their functional correlates were not found.38
Real-time PCR confirmed the presence of all the Kv studied but Kv1.1, Kv3.1 and Kv3.2. It also showed an inconsistency with the functional data, as the more represented Kv subunit mRNAs (Kv4 subunits), represent only around 15% of the total Kv current. Western blot and immunocytochemistry data indicate that although Kv4 protein (mainly Kv4.3) is present in VSMCs, it is located mostly intracellularly and, therefore, cannot contribute to Kv currents.
On the other hand, the results from cultured VSMCs show remarkable differences, with relevant implications. First, our data demonstrate that VSMC proliferation leads to an important Kv channel remodeling; therefore, caution must be taken when interpreting data on ion channel expression or function obtained from cultured VSMCs, a common situation when studying human tissues. Also, the remodeling of Kv currents correlates well with the changes in mRNA, again with the notable exception of Kv4 subunits (see the online data supplement). Specific localization and trafficking mechanisms are relevant to Kv4 channel function in most cells where these channels have been studied. In addition, a surprisingly large number of ancillary subunits and scaffolding proteins that can interact with the primary subunits altering channel trafficking have been described (for a review, see Birnbaum et al39). The study of the expression pattern of these auxiliary subunits may contribute to understanding the lack of functional expression of Kv4 channels in fresh and cultured VSMCs. For the other Kv subunits, we observed an overall reduction in the expression of Kv1 and Kv2 mRNA and an increase in the amount of Kv3.4 mRNA. These changes are congruous with the electrophysiological and pharmacological profile of cultured VSMCs (Figure 3), where the predominant Kv current is an inactivating current, highly sensitive to micromolar doses of TEA.
Since the first description by DeCoursey et al40 of the possible role of K+ channels in T-cell mitogenesis, many other K+ channels have been implicated in normal and/or pathological cell proliferation in different preparations.10 Proliferation associates with either changes in the expression of channels already present (ie, the switch between Kv1.3 and intermediate-conductance Ca2+-activated K+ [IKCa] channels in T cells,40 between Kv1.5 and Kv1.3 in proliferating microglia,22 or between BKCa and IKCa in rat aortic SMCs41,42) or with the concomitant appearance of channels absent in the native tissues, such as Kv10.1 channels in several tumor cell lines.10,43 We have also explored the expression of Kv10.1 and IKCa1 in acute and cultured VSMCs, finding a significant upregulation of Kv10.1 mRNA in cultured VSMCs and no changes in IKCa mRNA (see the online data supplement).
Regulation during VSMC proliferation has been described for L- and T-type Cav channels44eC46 and for IKCa channels.3,41,42 For Kv channels, a role in VSMC proliferation through their effects on Em is well established in the pulmonary circulation, where the decreased activity of Kv1.5 (and also Kv2.1) in hypoxic pulmonary hypertension has been shown to contribute to vascular hypertrophy by decreasing apoptosis.23,24
Our results provide data in concordance with some of these previous reports and also some novel findings that increase the number of players contributing to VSMCs proliferation. (1) We demonstrate a marked decrease in the expression of all Kv1 channels. Downregulation of Kv1.5 is in agreement with the previous findings in pulmonary arteries, although its participation in the phenotypic change of human uterine artery SMCs was not further explored. (2) We found a decrease in Kv2 channel expression in proliferating VSMCs, an observation previously reported in immature rat aortic myocytes.17 Interestingly, some of the functional changes observed in cultured VSMCs, such as the decreased in BKCa currents and the increase in the inactivating component of the Kv current, have also been reported in neonatal myocytes, suggesting a relationship between the proliferating phenotype and the immature VSMCs. (3) We show, for the first time, the presence and functional upregulation of Kv3.4 channels with the phenotypic change, most likely associated with their translocation from an intracellular location to the plasma membrane and their contribution to cell proliferation. This translocation of Kv3.4 protein could be associated with the stimulation of mitogen-activated protein kinase pathways or growth factors that are probably involved in VSMC proliferation and that have been shown to contribute to the membrane trafficking of BK channels in neurons.47 Finally, the proliferation assays demonstrate that Kv3.4 channels are necessary for VSMCs to proliferate, as selective blockade of these channels decreases the number of viable cells without increasing apoptosis.
In summary, the present work provides a thorough functional and molecular characterization of the Kv subunits expressed in VSMCs from human uterine artery and its modification by cell proliferation. We report Kv channel remodeling associated with the phenotypic change, and we found that Kv3.4 activity is related to cell proliferation; therefore, pharmacological modulation of this channel could become relevant for the regulation of VSMC proliferation associated with both physiological and pathological processes.
Acknowledgments
This work was supported by Instituto de Salud Carlos III grants G03/011 (Red Respira) and G03/045 (Red Heracles) and PI041044 (to J.R.L.-L.) and Ministerio de Educacie y Ciencia grant BFU2004-05551 (to M.T.P.-G.). We thank Esperanza Alonso and Miriam Castro for excellent technical assistance and Merck Co for the gift of correolide.
References
Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995; 75: 487eC517.
Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol. 1995; 268: C799eCC822.
Neylon CB. Potassium channels and vascular proliferation. Vascul Pharmacol. 2002; 38: 35eC41.
Jackson WF. Ion channels and vascular tone. Hypertension. 2000; 35: 173.
Michelakis ED, Reeve HL, Huang JM, Tolarova S, Nelson DP, Weir EK, Archer SL. Potassium channel diversity in vascular smooth muscle cells. Can J Physiol Pharmacol. 1997; 75: 889eC897.
Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation. 1995; 91: 1107eC1115.
Landsberg JW, Yuan JXJ. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci. 2004; 19: 44eC50.
Pulver RA, Rose-Curtis P, Roe MW, Wellman GC, Lounsbury KM. Store-operated Ca2+ entry activates the CREB transcription factor in vascular smooth muscle. Circ Res. 2004; 94: 1351eC1358.
Wonderlin WF, Strobl JS. Potassium channels, proliferation and G1 progression. J Membr Biol. 1996; 154: 91eC107.
Pardo LA. Voltage-gated potassium channels in cell proliferation. Physiology. 2004; 19: 285eC292.
Archer SL. Diversity of phenotype and function of vascular smooth muscle cells. J Lab Clin Med. 1996; 127: 524eC529.
Mandegar M, Yuan JX. Role of K+ channels in pulmonary hypertension. Vascul Pharmacol. 2002; 38: 25eC33.
Archer S, Rich S. Primary pulmonary hypertension: a vascular biology and translational research "work in progress". Circulation. 2000; 102: 2781eC2791.
Li G, Cheung DW. Effects of paxilline on K+ channels in rat mesenteric arterial cells. Eur J Pharmacol. 1999; 372: 103eC107.
Tammaro P, Smith AL, Hutchings SR, Smirnov SV. Pharmacological evidence for a key role of voltage-gated K+ channels in the function of rat aortic smooth muscle cells. Br J Pharmacol. 2004; 143: 303eC317.
Cox RH, Zhou Z, Tulenko TN. Voltage-gated sodium channels in human aortic smooth muscle cells. J Vasc Res. 1998; 35: 310eC317.
Belevych AE, Beck R, Tammaro P, Poston L, Smirnov SV. Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes. Cardiovasc Res. 2002; 54: 152eC161.
Hanner M, Schmalhofer WA, Green B, Bordallo C, Liu J, Slaughter RS, Kaczorowski GJ, Garcia ML. Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction. J Biol Chem. 1999; 274: 25237eC25244.
Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann N Y Acad Sci. 1999; 868: 233eC285.
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491eC498.
Diochot S, Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol. 1999; 126: 251eC263.
Kotecha SA, Schlichter LC. A Kv1.5 to Kv1.3 switch in endogenous hippocampal microglia and a role in proliferation. J Neurosci. 1999; 19: 10680eC10693.
Brevnova EE, Platoshyn O, Zhang S, Yuan JXJ. Overexpression of human KCNA5 increases IK(V) and enhances apoptosis. Am J Physiol Cell Physiol. 2004; 287: C715eCC722.
Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin L, Yuan JXJ. Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes. Am J Physiol. 2001; 280: L801eCL812.
Sweeney M, Yuan J. Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels. Respir Res. 2000; 1: 40eC48.
Michelakis ED, Weir EK. The pathobiology of pulmonary hypertension. Smooth muscle cells and ion channels. Clin Chest Med. 2001; 22: 419eC432.
Thebaud B, Michelakis ED, Wu XC, Moudgil R, Kuzyk M, Dyck JR, Harry G, Hashimoto K, Haromy A, Rebeyka I, Archer SL. Oxygen-sensitive Kv channel gene transfer confers oxygen responsiveness to preterm rabbit and remodeled human ductus arteriosus: implications for infants with patent ductus arteriosus. Circulation. 2004; 110: 1372eC1379.
Cox RH. Changes in the expression and function of arterial potassium channels during hypertension. Vascul Pharmacol. 2002; 38: 13eC23.
Cox RH, Rusch NJ. New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure. Microcirculation. 2002; 9: 243eC257.
Cox RH. Molecular determinants of voltage-gated potassium currents in vascular smooth muscle. Cell Biochem Biophys. 2005; 42: 167eC196.
Heusser K, Schwappach B. Trafficking of potassium channels. Curr Opin Neurobiol. 2005; 15: 364eC369.
Cheong A, Quinn K, Dedman AM, Beech DJ. Activation thresholds of K-v, BK and Cl-Ca channels in smooth muscle cells in pial precapillary arterioles. J Vasc Res. 2002; 39: 122eC130.
Fountain SJ, Cheong A, Flemming R, Mair L, Sivaprasadarao A, Beech DJ. Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle. J Physiol. 2004; 556: 29eC42.
Albarwani S, Nemetz LT, Madden JA, Tobin AA, England SK, Pratt PF, Rusch NJ. Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels. J Physiol. 2003; 551: 751eC763.
Fergus DJ, Martens JR, England SK. Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle. Pflugers Arch. 2003; 445: 697eC704.
Plane F, Johnson R, Kerr P, Wiehler W, Thorneloe K, Ishii K, Chen T, Cole W. Heteromultimeric Kv1 channels contribute to myogenic control of arterial diameter. Circ Res. 2005; 96: 216eC224.
Cheong A, Dedman AM, Xu SZ, Beech DJ. K-v alpha 1 channels in murine arterioles: differential cellular expression and regulation of diameter. Am J Physiol Heart Circ Physiol. 2001; 281: H1057eCH1065.
Xu C, Lu Y, Tang G, Wang R. Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 1999; 277: G1055eCG1063.
Birnbaum SG, Varga AW, Yuan LL, Anderson AE, Sweatt JD, Schrader LA. Structure and function of Kv4-family transient potassium channels. Physiol Rev. 2004; 84: 803eC833.
DeCoursey TE, Chandy KG, Gupta S, Cahalan MD. Voltage-gated K+ channels in human lymphocyte-T:a role in mitogenesis Nature. 1984; 307: 465eC468.
Neylon CB, Lang RJ, Fu Y, Bobik A, Reinhart PH. Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function. Circ Res. 1999; 85: e33eCe43.
Kohler R, Wulff H, Eichler I, Kneifel M, Neumann D, Knorr A, Grgic I, Kampfe D, Si H, Wibawa J, Real R, Borner K, Brakemeier S, Orzechowski HD, Reusch HP, Paul M, Chandy KG, Hoyer J. Blockade of the intermediate-conductance calcium-activated potassium channel as a new therapeutic strategy for restenosis. Circulation. 2003; 108: 1119eC1125.
Pardo LA, del Camino D, Sanchez A, Alves F, Bruggemann A, Beckh S, Stuhmer W. Oncogenic potential of EAG K(+) channels. EMBO J. 1999; 18: 5540eC5547.
Kuga T, Kobayashi S, Hirakawa Y, Kanaide H, Takeshita A. Cell cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture. Circ Res. 1996; 79: 14eC19.
Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 1998; 12: 593eC601.
Rodman DM, Reese K, Harral J, Fouty B, Wu S, West J, Hoedt-Miller M, Tada Y, Li KX, Cool C, Fagan K, Cribbs L. Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes. Circ Res. 2005; 96: 864eC872.
Chae KS, Dryer SE. The p38 mitogen-activated protein kinase pathway negatively regulates Ca2+-activated K+ channel trafficking in developing parasympathetic neurons. J Neurochem. 2005; 94: 367eC379.(Eduardo Miguel-Velado, Al)
Institut de Malalties Cardiovasculars (M.H.), Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer, Universidad de Barcelona, Barcelona, Spain.
Abstract
Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kv subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling.
Key Words: Kv channel expression vascular smooth muscle cells cell proliferation K+ currents
Introduction
The vascular smooth muscle cells (VSMCs) of mature animals are highly specialized cells whose main function is contraction. Although during vasculogenesis, the principal role of VSMCs is proliferation and synthesis of the matrix components of the vessel wall, differentiated VSMCs proliferate at an extremely low rate and express a unique repertoire of contractile proteins, ion channels, and signaling molecules required for contraction. However, VSMCs can undergo relatively rapid and reversible phenotypic changes in response to local environmental conditions. Accelerated proliferation of VSMCs is known to play a key role in atherosclerotic plaque formation and postangioplasty restenosis and is a common feature in hypertensive arteries.1
VSMCs express a large repertoire of ion channels and membrane receptors that vary widely among different vascular beds and are key determinants of the electrical and contractile responses of the cells.2eC5 Although release of intracellular Ca2+ is necessary for effective contraction, Ca2+ influx through voltage-activated Ca2+ (Cav) channels is responsible for initiating contraction, making resting membrane potential (Em) a primary determinant of vascular smooth muscle tone.4 Membrane depolarization opens Cav and raises [Ca2+]i, and cytosolic-free Ca2+ serves, in turn, as a critical signal transduction element in a variety of cell functions, such as contraction, migration, proliferation, and gene expression.6eC8
There is increasing evidence showing that K+ channels may have an important role in dedifferentiation and proliferation of VSMCs. Modulating Em, K+ channels can affect not only Ca2+ influx, a well-established factor influencing cell proliferation, but also the driving force for Na+-dependent transport, the intracellular pH, and the regulation of cell volume, all factors that also participate in proliferation and apoptotic processes.9,10 The role of K+ channels on cell proliferation is a complex modulatory activity that only certain K+ channels at specific times and locations can perform. This fact, together with the broad diversity of functional K+ currents among different vascular beds, has ravelled the analysis of the participation of K+ channels on VSMCs proliferation. At least 4 different types of K+ channels have been identified in VSMCs: inward rectifier, voltage-gated (Kv), ATP-gated, and Ca2+-gated K+ (BKCa) channels.2,4 The expression of these 4 types has been reported to vary among vascular beds as well as with vessel size.5,11 However, Kv and BKCa channels are present in virtually all vascular myocytes and strongly influence contractile responses.2 Moreover, several Kv channels have been described to participate in the proliferation process in pulmonary artery smooth muscles cells (SMCs).12,13
The goals of the present study were to characterize the expression profile of Kv channels in VSMCs from human uterine artery in contractile and proliferating phenotype and to evaluate their possible contribution to phenotypic modulation. Enzymatic dispersion of small pieces of uterine arteries provide VSMCs in contractile phenotype, which were studied within the same day, whereas proliferating VSMCs were obtained from explants of uterine arteries kept in culture for several passages. The molecular identification of the functional Kv subunits in both preparations shows that whereas Kv1 members are the main contributors to the Kv currents in contractile VSMCs, Kv3.4 expression is upregulated under proliferating conditions and represents the largest proportion of the Kv current in dedifferentiated VSMCs. Furthermore, selective blockade of Kv3.4 channels decreased proliferation rate, suggesting a direct relationship between channel function and uterine artery SMCs proliferation.
Materials and Methods
Uterine arteries were obtained from patients undergoing hysterectomy at the Clinic Hospital of Barcelona, with protocols approved by the Human Investigation Ethics Committee of the Hospital. Details of the materials and methods used in this study are in the online data supplement available at http://circres.ahajournals.org.
Results
Kv Currents in Freshly Dissociated and Cultured VSMCs
K+ currents were studied in acutely dissociated cells and in cultured cells obtained from human uterine arteries. After establishing the whole-cell configuration, currenteCvoltage (I/V) relationships for K+ currents were obtained every 2 minutes. After several records of I/V curves in control conditions, BKCa currents were subtracted by using the selective blocker paxilline (500 nmol/L).14,15 Figure 1 shows average I/V curves and sample records (insets) obtained in acutely dispersed cells (Figure 1A) and in cultured VSMCs (Figure 1B) before and after paxilline application. As previously reported,16 cultured cells exhibited tetrodotoxin-sensitive inward Na+ currents, which were absent in acutely dispersed cells (Figure 1B, inset). Depolarization elicited outward currents with an apparent threshold for activation positive to eC40 mV, and paxilline decreased current amplitudes at potentials positive to +30 mV. This effect was more evident in acutely dispersed cells. Cell capacitance was 40.21±2.45 pF in fresh VSMCs (n=28) and 44.58±6.17 pF in cultured cells (n=18). Current density was not significantly different between the 2 groups (19.04±2.76 versus 15.11±2.72 pA/pF at +80 mV), but BKCa current represents more than 70% of the outward K+ current in the freshly dissociated VSMCs and only 20% in the cultured cells. Furthermore, we consistently found a kinetic change with a larger proportion of inactivating K+ currents in cultured VSMCs (see traces in Figure 1). Resting Em measurements under perforated-patch conditions showed that contractile VSMCs had, on average, more hyperpolarized potentials than proliferating VSMCs (eC45.0±1.9 mV versus eC38.4±2.4 mV respectively, P<0.05; Figure 1C). Resting Em was not modified by paxilline in either of the 2 groups (data not shown). These observations indicate that the proliferation of human uterine artery VSMCs is associated with a decrease in the expression of BKCa currents and an increase in the contribution of Kv currents.
Pharmacological Characterization of Kv Currents
Whereas downregulation of BKCa currents with the phenotypic switch has been described previously,3,17 changes in Kv currents are not well characterized. Therefore, we characterized pharmacological Kv currents in both phenotypes after blocking BKCa currents with paxilline. Correolide was used to selectively block Kv1 currents,18 tetraethylammonium (TEA) sensitivity allowed the identification of Kv2 and Kv3 currents, and TEA- and correolide-insensitive current was attributed to Kv4 currents.19 The latter were also identified by their sensitivity to block by toxins such as heteropodatoxin20 and phrixotoxin.21
In acutely dispersed VSMCs (Figure 2), Kv1 currents were the predominant component of the K+ currents (Figure 2A). Correolide reduced Kv current amplitude by 21% or 70% at +80 mV when applied at 1 or 10 eol/L, respectively. At 10 eol/L, the effect was statistically significant at voltage values above eC30 mV. TEA was applied at low (micromolar) and high (millimolar) concentrations to dissect the contribution of high- and low-TEAeCsensitive Kv channels. There was a small reduction in the current amplitude in the presence of 100 eol/L TEA (15% inhibition at +80 mV; Figure 2B), which did not significantly increase after raising TEA up to 20 mmol/L. The onset of TEA inhibition appeared at more positive potentials than that of correolide (compare Figure 2A and 2B). The effect of TEA was the same on the total Kv current as on the correolide-insensitive fraction of the current, suggesting that Kv1 channels in acutely dispersed VSMCs are not sensitive to TEA (data not shown). The contribution of Kv4 channels to Kv currents was studied with specific blockers. Figure 2C shows an example in which 70 nmol/L phrixotoxin was used. The effect of the drug was voltage dependent (see inset). On average, phrixotoxin induced a 15% inhibition on the current amplitude at +80 mV, similar to the estimated proportion of TEA- and correolide-insensitive current (12.0±3.5%).
When the same pharmacological study was performed in cultured VSMCs, a clearly different pattern was apparent (Figure 3). First, correolide (Figure 3A) had only a very modest effect (11.5±6.3% inhibition at +40 mV). Second, the predominant fast-inactivating current observed in these cells was almost completely abolished by low TEA (up to 1 mmol/L; Figure 3B). Finally, blockers of Kv4 channels had almost no effect (Figure 3C). Therefore, the predominant Kv current in cultured VSMCs is a transient current with high-TEA sensitivity, pointing to some members of the Kv3 subfamily, particularly Kv3.4. This suggestion was confirmed by exploring the effect of BDS-1, a selective blocker of Kv3.4 channels. BDS-1 (2.5 eol/L) decreased the current amplitude by &40% to 50% at potentials of eC20 mV or greater (Figure 3D). Taken together, these functional data suggest an extensive remodeling of Kv channels associated with the phenotypic modulation, with a predominant role of Kv1 in acutely dispersed and of Kv3 in cultured VSMCs, as indicated by the summary data of Figure 3E.
Expression Profile of Kv mRNA
To study the molecular correlates of the Kv currents, real-time PCR was performed in both contractile and proliferating VSMCs. The results, normalized to RPL18 mRNA amount and corrected for the amplification efficiency of each reaction (see online data supplement), are plotted in Figure 4A. Unexpectedly, Kv4 channels represent the largest amount in both preparations, accounting for 67% and 80% of the total Kv mRNA (see online data supplement). We did not detect expression of Kv3.1, Kv3.2, or Kv1.1 in any preparation. With the exception of Kv4.2 and Kv3.4 subunits, which undergo an increased expression in cultured VSMCs, proliferation induced a downregulation of mRNA of all Kv subunits present in fresh tissue. Figure 4B shows the magnitude of the changes in the expression of Kv subunit mRNA from proliferating VSMC, taking the levels of mRNA in contractile VSMCs as the calibrator. In general, there is a correlation between changes in mRNA levels and changes in functional expression when comparing contractile and proliferating VSMCs (compare Figures 4B and 3E): whereas Kv1 channels are downregulated, Kv3.4 channels upregulate their expression and their functional contribution in cultured VSMCs.
Expression and Subcellular Location of Kv Proteins
The above data show, nevertheless, some discrepancies between the mRNA levels of a given Kv subunit and its contribution to the total Kv current, which could be attributable to the absence of correlation between mRNA and protein levels or, alternatively, to altered membrane trafficking of the protein. To distinguish between these 2 possibilities, we determined protein expression by immunoblot and cellular location with immunocytochemistry. Representative Western blots both in freshly isolated tissues and in protein extracts from cultured cells are shown in Figure 5. Kv1 subunit proteins were detected in fresh tissues, being absent or expressed at much lower levels in cultured VSMCs, with the only exception of Kv1.3, which appears to be more abundant in protein extracts from cultured VSMCs, contrary to what we will predict according to mRNA levels. A similar inconsistency was found in the case of Kv3.4 protein, which seems to be more abundant in fresh VSMCs than in cultured cells. Finally, with regard to Kv4 subunit proteins, bands for Kv4.2 and Kv4.3 were observed in both tissue and cell cultures, with substantially higher levels of expression in protein extracts from tissues, revealing the presence in both preparations of Kv4 subunit proteins, whose functional contribution has been found to be very small or almost absent.
Next, we explored the subcellular location of some Kv subunit proteins, which had levels of expression (as determined by Western blot) that did not match their estimated levels of activity (as determined by functional studies), namely Kv1.3, Kv3.4, and Kv4 subfamily members. Kv1.3 was expressed in patches of membrane in acutely dissociated VSMCs (Figure 6A), whereas Kv4.3 proteins were found to colocalize with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) in the membranes of intracellular compartments both in cultured (Figure 6E) and freshly dissociated (data not shown) VSMCs. Finally, Kv3.4 immunoreactivity was detected in both preparations but in different cellular locations. Whereas, in freshly dissociated VSMCs, Kv3.4 colocalized with calreticulin and was absent from the plasma membrane (Figure 6B), in cultured VSMCs, Kv3.4 was almost exclusively found in a plasma membrane location (Figure 6C and D). Interestingly, whereas Kv3.4 mRNA increased in proliferating cells, total Kv3.4 subunit protein decreased, although it seemed to be more efficiently located in the plasma membrane.
Role of Kv3.4 in VSMCs Proliferation
The upregulation of Kv3.4 subunit mRNA during VSMC dedifferentiation and its transposition from a cytoplasmic to a membrane location suggest that the activity of Kv3.4 channels may be related to proliferation. To explore this hypothesis, we studied the proliferation rate of cultured VSMCs when the activity of the channels was blocked by low TEA concentrations (100 eol/L) or BDS-I toxin (Figure 7A). TEA application led to a decreased proliferation rate that was already significant after 24 hours of treatment. The difference increased with time and reached a plateau around 96 hours after TEA treatment. The same results were obtained when Kv3.4 channels were blocked with 2.5 eol/L BDS-I, whereas blockade of Kv4 channels with 70 nmol/L phrixotoxin did not affect proliferation rate (inset in Figure 7A). These experiments demonstrate a specific link between Kv3.4 (but not Kv4) channels and VSMC proliferation, although the contribution of other Kv channels to this process (ie, some Kv1 family members, as previously found in other preparations22,23) cannot be excluded.
The decreased number of cells in the presence of Kv3.4 channel blockers could reflect a decreased proliferation rate or an increase in the number of cells undergoing apoptosis. Because among the hallmarks of late-stage apoptosis is the fragmentation of nuclear chromatin, we performed a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay to detect apoptosis-induced DNA fragmentation both in control cells and in 100 eol/L TEAeCtreated cells (Figure 7B). There was no significant cell death in either of the 2 samples (2.1±0.9% in control versus 2.0±0.7% in TEA treated, n=10 fields from 2 independent experiments), indicating that TEA block of Kv3.4 channels does not induce apoptosis.
Discussion
In this work, we have characterized the Kv subunit profile underlying Kv currents of VSMCs from human uterine arteries and its changes when VSMCs switch from a contractile to a proliferating phenotype.
With the exception of pulmonary arteries, little is known regarding the expression profile of Kv channels in human resistance arteries.24eC27 However, it is well established that Kv channels play an important role in regulating contraction through their effects on Em and by integrating a variety of vasoactive signals.2 Moreover, abnormal expression profiles of Kv channels have been involved in the pathogenesis of arterial hypertension and vascular proliferation, leading to new opportunities for developing drugs for targeting disease-specific changes in ion channel expression.28,29 Identification of the contributing Kv genes is crucial to pursuit this approach.
The large diversity of Kv channel genes, together with their possibilities of heteromultimerization, association with accessory subunits, and alternative splicing, leads to an enormous diversity in the molecular composition and properties of Kv channel complexes.19 Often, the number of expressed Kv subunits appears to be much larger than the number of apparent Kv current components.30 The oligomeric composition of Kv channels and the factors regulating their folding and trafficking to the cell membrane31 could contribute to explain these discrepancies.
We have tried to correlate the electrophysiological and pharmacological properties of Kv currents with the expression of Kv subunits, both at mRNA and protein levels. Although the role of BKCa currents in resistance arteries in vivo cannot be evaluated with this study, the apparent activation thresholds of BKCa and Kv currents (see Figure 1) suggests a small contribution of BKCa current to the resting Em in relaxed cells, which is supported by the measured resting Em values (Figure 1C) and its insensitivity to paxilline. Our observations echo the results obtained in other VSMC preparations, where Kv currents activated at potentials 20 to 30 mV more negative than BKCa currents.15,32
Our results showing that Kv1 members are the predominant components of the Kv currents are in good agreement with previous reports from other resistance vessels.33eC37 The pharmacological data from acute VSMCs also indicate the contribution of Kv2, Kv3, and Kv4 channels. Kv2.1 currents have been described in rat mesenteric artery VSMCs38 and have been found to be the major contributor to the Kv current in VSMCs from conduit arteries such as rat aorta.14,15,17 mRNA and even protein for Kv3.4 and Kv4 channels have been previously described in rat mesenteric SMCs, but their functional correlates were not found.38
Real-time PCR confirmed the presence of all the Kv studied but Kv1.1, Kv3.1 and Kv3.2. It also showed an inconsistency with the functional data, as the more represented Kv subunit mRNAs (Kv4 subunits), represent only around 15% of the total Kv current. Western blot and immunocytochemistry data indicate that although Kv4 protein (mainly Kv4.3) is present in VSMCs, it is located mostly intracellularly and, therefore, cannot contribute to Kv currents.
On the other hand, the results from cultured VSMCs show remarkable differences, with relevant implications. First, our data demonstrate that VSMC proliferation leads to an important Kv channel remodeling; therefore, caution must be taken when interpreting data on ion channel expression or function obtained from cultured VSMCs, a common situation when studying human tissues. Also, the remodeling of Kv currents correlates well with the changes in mRNA, again with the notable exception of Kv4 subunits (see the online data supplement). Specific localization and trafficking mechanisms are relevant to Kv4 channel function in most cells where these channels have been studied. In addition, a surprisingly large number of ancillary subunits and scaffolding proteins that can interact with the primary subunits altering channel trafficking have been described (for a review, see Birnbaum et al39). The study of the expression pattern of these auxiliary subunits may contribute to understanding the lack of functional expression of Kv4 channels in fresh and cultured VSMCs. For the other Kv subunits, we observed an overall reduction in the expression of Kv1 and Kv2 mRNA and an increase in the amount of Kv3.4 mRNA. These changes are congruous with the electrophysiological and pharmacological profile of cultured VSMCs (Figure 3), where the predominant Kv current is an inactivating current, highly sensitive to micromolar doses of TEA.
Since the first description by DeCoursey et al40 of the possible role of K+ channels in T-cell mitogenesis, many other K+ channels have been implicated in normal and/or pathological cell proliferation in different preparations.10 Proliferation associates with either changes in the expression of channels already present (ie, the switch between Kv1.3 and intermediate-conductance Ca2+-activated K+ [IKCa] channels in T cells,40 between Kv1.5 and Kv1.3 in proliferating microglia,22 or between BKCa and IKCa in rat aortic SMCs41,42) or with the concomitant appearance of channels absent in the native tissues, such as Kv10.1 channels in several tumor cell lines.10,43 We have also explored the expression of Kv10.1 and IKCa1 in acute and cultured VSMCs, finding a significant upregulation of Kv10.1 mRNA in cultured VSMCs and no changes in IKCa mRNA (see the online data supplement).
Regulation during VSMC proliferation has been described for L- and T-type Cav channels44eC46 and for IKCa channels.3,41,42 For Kv channels, a role in VSMC proliferation through their effects on Em is well established in the pulmonary circulation, where the decreased activity of Kv1.5 (and also Kv2.1) in hypoxic pulmonary hypertension has been shown to contribute to vascular hypertrophy by decreasing apoptosis.23,24
Our results provide data in concordance with some of these previous reports and also some novel findings that increase the number of players contributing to VSMCs proliferation. (1) We demonstrate a marked decrease in the expression of all Kv1 channels. Downregulation of Kv1.5 is in agreement with the previous findings in pulmonary arteries, although its participation in the phenotypic change of human uterine artery SMCs was not further explored. (2) We found a decrease in Kv2 channel expression in proliferating VSMCs, an observation previously reported in immature rat aortic myocytes.17 Interestingly, some of the functional changes observed in cultured VSMCs, such as the decreased in BKCa currents and the increase in the inactivating component of the Kv current, have also been reported in neonatal myocytes, suggesting a relationship between the proliferating phenotype and the immature VSMCs. (3) We show, for the first time, the presence and functional upregulation of Kv3.4 channels with the phenotypic change, most likely associated with their translocation from an intracellular location to the plasma membrane and their contribution to cell proliferation. This translocation of Kv3.4 protein could be associated with the stimulation of mitogen-activated protein kinase pathways or growth factors that are probably involved in VSMC proliferation and that have been shown to contribute to the membrane trafficking of BK channels in neurons.47 Finally, the proliferation assays demonstrate that Kv3.4 channels are necessary for VSMCs to proliferate, as selective blockade of these channels decreases the number of viable cells without increasing apoptosis.
In summary, the present work provides a thorough functional and molecular characterization of the Kv subunits expressed in VSMCs from human uterine artery and its modification by cell proliferation. We report Kv channel remodeling associated with the phenotypic change, and we found that Kv3.4 activity is related to cell proliferation; therefore, pharmacological modulation of this channel could become relevant for the regulation of VSMC proliferation associated with both physiological and pathological processes.
Acknowledgments
This work was supported by Instituto de Salud Carlos III grants G03/011 (Red Respira) and G03/045 (Red Heracles) and PI041044 (to J.R.L.-L.) and Ministerio de Educacie y Ciencia grant BFU2004-05551 (to M.T.P.-G.). We thank Esperanza Alonso and Miriam Castro for excellent technical assistance and Merck Co for the gift of correolide.
References
Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995; 75: 487eC517.
Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol. 1995; 268: C799eCC822.
Neylon CB. Potassium channels and vascular proliferation. Vascul Pharmacol. 2002; 38: 35eC41.
Jackson WF. Ion channels and vascular tone. Hypertension. 2000; 35: 173.
Michelakis ED, Reeve HL, Huang JM, Tolarova S, Nelson DP, Weir EK, Archer SL. Potassium channel diversity in vascular smooth muscle cells. Can J Physiol Pharmacol. 1997; 75: 889eC897.
Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation. 1995; 91: 1107eC1115.
Landsberg JW, Yuan JXJ. Calcium and TRP channels in pulmonary vascular smooth muscle cell proliferation. News Physiol Sci. 2004; 19: 44eC50.
Pulver RA, Rose-Curtis P, Roe MW, Wellman GC, Lounsbury KM. Store-operated Ca2+ entry activates the CREB transcription factor in vascular smooth muscle. Circ Res. 2004; 94: 1351eC1358.
Wonderlin WF, Strobl JS. Potassium channels, proliferation and G1 progression. J Membr Biol. 1996; 154: 91eC107.
Pardo LA. Voltage-gated potassium channels in cell proliferation. Physiology. 2004; 19: 285eC292.
Archer SL. Diversity of phenotype and function of vascular smooth muscle cells. J Lab Clin Med. 1996; 127: 524eC529.
Mandegar M, Yuan JX. Role of K+ channels in pulmonary hypertension. Vascul Pharmacol. 2002; 38: 25eC33.
Archer S, Rich S. Primary pulmonary hypertension: a vascular biology and translational research "work in progress". Circulation. 2000; 102: 2781eC2791.
Li G, Cheung DW. Effects of paxilline on K+ channels in rat mesenteric arterial cells. Eur J Pharmacol. 1999; 372: 103eC107.
Tammaro P, Smith AL, Hutchings SR, Smirnov SV. Pharmacological evidence for a key role of voltage-gated K+ channels in the function of rat aortic smooth muscle cells. Br J Pharmacol. 2004; 143: 303eC317.
Cox RH, Zhou Z, Tulenko TN. Voltage-gated sodium channels in human aortic smooth muscle cells. J Vasc Res. 1998; 35: 310eC317.
Belevych AE, Beck R, Tammaro P, Poston L, Smirnov SV. Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes. Cardiovasc Res. 2002; 54: 152eC161.
Hanner M, Schmalhofer WA, Green B, Bordallo C, Liu J, Slaughter RS, Kaczorowski GJ, Garcia ML. Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction. J Biol Chem. 1999; 274: 25237eC25244.
Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, Rudy B. Molecular diversity of K+ channels. Ann N Y Acad Sci. 1999; 868: 233eC285.
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491eC498.
Diochot S, Drici MD, Moinier D, Fink M, Lazdunski M. Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis. Br J Pharmacol. 1999; 126: 251eC263.
Kotecha SA, Schlichter LC. A Kv1.5 to Kv1.3 switch in endogenous hippocampal microglia and a role in proliferation. J Neurosci. 1999; 19: 10680eC10693.
Brevnova EE, Platoshyn O, Zhang S, Yuan JXJ. Overexpression of human KCNA5 increases IK(V) and enhances apoptosis. Am J Physiol Cell Physiol. 2004; 287: C715eCC722.
Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin L, Yuan JXJ. Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes. Am J Physiol. 2001; 280: L801eCL812.
Sweeney M, Yuan J. Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels. Respir Res. 2000; 1: 40eC48.
Michelakis ED, Weir EK. The pathobiology of pulmonary hypertension. Smooth muscle cells and ion channels. Clin Chest Med. 2001; 22: 419eC432.
Thebaud B, Michelakis ED, Wu XC, Moudgil R, Kuzyk M, Dyck JR, Harry G, Hashimoto K, Haromy A, Rebeyka I, Archer SL. Oxygen-sensitive Kv channel gene transfer confers oxygen responsiveness to preterm rabbit and remodeled human ductus arteriosus: implications for infants with patent ductus arteriosus. Circulation. 2004; 110: 1372eC1379.
Cox RH. Changes in the expression and function of arterial potassium channels during hypertension. Vascul Pharmacol. 2002; 38: 13eC23.
Cox RH, Rusch NJ. New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure. Microcirculation. 2002; 9: 243eC257.
Cox RH. Molecular determinants of voltage-gated potassium currents in vascular smooth muscle. Cell Biochem Biophys. 2005; 42: 167eC196.
Heusser K, Schwappach B. Trafficking of potassium channels. Curr Opin Neurobiol. 2005; 15: 364eC369.
Cheong A, Quinn K, Dedman AM, Beech DJ. Activation thresholds of K-v, BK and Cl-Ca channels in smooth muscle cells in pial precapillary arterioles. J Vasc Res. 2002; 39: 122eC130.
Fountain SJ, Cheong A, Flemming R, Mair L, Sivaprasadarao A, Beech DJ. Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle. J Physiol. 2004; 556: 29eC42.
Albarwani S, Nemetz LT, Madden JA, Tobin AA, England SK, Pratt PF, Rusch NJ. Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels. J Physiol. 2003; 551: 751eC763.
Fergus DJ, Martens JR, England SK. Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle. Pflugers Arch. 2003; 445: 697eC704.
Plane F, Johnson R, Kerr P, Wiehler W, Thorneloe K, Ishii K, Chen T, Cole W. Heteromultimeric Kv1 channels contribute to myogenic control of arterial diameter. Circ Res. 2005; 96: 216eC224.
Cheong A, Dedman AM, Xu SZ, Beech DJ. K-v alpha 1 channels in murine arterioles: differential cellular expression and regulation of diameter. Am J Physiol Heart Circ Physiol. 2001; 281: H1057eCH1065.
Xu C, Lu Y, Tang G, Wang R. Expression of voltage-dependent K+ channel genes in mesenteric artery smooth muscle cells. Am J Physiol Gastrointest Liver Physiol. 1999; 277: G1055eCG1063.
Birnbaum SG, Varga AW, Yuan LL, Anderson AE, Sweatt JD, Schrader LA. Structure and function of Kv4-family transient potassium channels. Physiol Rev. 2004; 84: 803eC833.
DeCoursey TE, Chandy KG, Gupta S, Cahalan MD. Voltage-gated K+ channels in human lymphocyte-T:a role in mitogenesis Nature. 1984; 307: 465eC468.
Neylon CB, Lang RJ, Fu Y, Bobik A, Reinhart PH. Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function. Circ Res. 1999; 85: e33eCe43.
Kohler R, Wulff H, Eichler I, Kneifel M, Neumann D, Knorr A, Grgic I, Kampfe D, Si H, Wibawa J, Real R, Borner K, Brakemeier S, Orzechowski HD, Reusch HP, Paul M, Chandy KG, Hoyer J. Blockade of the intermediate-conductance calcium-activated potassium channel as a new therapeutic strategy for restenosis. Circulation. 2003; 108: 1119eC1125.
Pardo LA, del Camino D, Sanchez A, Alves F, Bruggemann A, Beckh S, Stuhmer W. Oncogenic potential of EAG K(+) channels. EMBO J. 1999; 18: 5540eC5547.
Kuga T, Kobayashi S, Hirakawa Y, Kanaide H, Takeshita A. Cell cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture. Circ Res. 1996; 79: 14eC19.
Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 1998; 12: 593eC601.
Rodman DM, Reese K, Harral J, Fouty B, Wu S, West J, Hoedt-Miller M, Tada Y, Li KX, Cool C, Fagan K, Cribbs L. Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes. Circ Res. 2005; 96: 864eC872.
Chae KS, Dryer SE. The p38 mitogen-activated protein kinase pathway negatively regulates Ca2+-activated K+ channel trafficking in developing parasympathetic neurons. J Neurochem. 2005; 94: 367eC379.(Eduardo Miguel-Velado, Al)