Inwardly rectifying K+ (KIR) currents are present in some, but not all, vascular smooth muscles. We used patch-clamp methods to examine plasticity of this current by comparing contractile and proliferative phenotypes of a clonal human vascular smooth muscle cell line. Hyperpolarization of cells under voltage clamp elicited a large inward current that was selective for K+ and blocked by Ba2+. Current density was greater in proliferative compared with contractile cells (−4.5 ± 0.9 and −1.4 ± 0.3 pA/pF, respectively; P < 0.001). RT-PCR of mRNA from proliferative cells identified transcripts for Kir2.1 and Kir2.2 but not Kir2.3 potassium channels. Western blot analysis demonstrated greater expression of Kir2.1 protein in proliferative cells, consistent with the higher current density. Proliferative cells displayed a more negative membrane potential than contractile cells (−71 ± 2 and −35 ± 4 mV, respectively; P < 0.001). Ba2+ depolarized all cells, whereas small increases in extracellular K+ concentration elicited hyperpolarization only in contractile cells. Ba2+ inhibited [3H]thymidine incorporation, indicating a possible role for KIR channels in the regulation of proliferation. The phenotype-dependent plasticity of KIR channels may have relevance to vascular remodeling.
- ion channels
- inward rectifier
it has long been established that the resting membrane potential (RMP) of vascular smooth muscle cells (SMCs) depends primarily on the gradient for potassium ions across the plasma membrane (13, 14). An inwardly rectifying K+ (KIR) conductance in smooth muscle was first demonstrated by Edwards and coworkers (9), who demonstrated a K+-dependent inward current that rectified at potentials positive to the potassium equilibrium potential (E K) (8,9). However, because of the syncytial nature of smooth muscle tissue, recording of the KIR current was not possible until single-cell isolation techniques were adopted. The first demonstration of KIR currents in smooth muscle with the patch-clamp technique was by Quayle and coworkers (28), who characterized these currents in rat cerebral arteries. KIR currents have since been identified in rat hepatic, coronary, and mesenteric vascular SMCs (2, 10, 17,29).
Although KIR currents are present in many vascular SMCs, they are not as prevalent as other K+ current types, like the large conductance Ca2+-activated K+channel. Indeed, early studies demonstrated variability in smooth muscle of different vascular regions (8, 9) and thereby served to orient investigators searching for KIR channels to SMCs of specific vessels when patch-clamp techniques were established (28). Subsequent investigations revealed that KIR current is present in higher density in small-diameter arteries compared with larger, conduit vessels (27). This distribution suggests a fundamental diversity of KIRchannel expression in smooth muscle and is believed to contribute to functional differences between these types of vessels. However, it is uncertain whether KIR expression can be dynamically regulated in a given population of SMCs.
To determine whether KIR expression in vascular smooth muscle is related to cell differentiation, we characterized KIR current in HITB5 SMCs, a human internal thoracic artery SMC clone. The HITB5 SMCs have the ability to convert between the contractile and proliferative phenotypes in culture (19,20) and have allowed us to demonstrate (15) critical differences between these phenotypes, in terms of both contractile ability and ion channel complement. Here, we demonstrate Kir2.1 and Kir2.2 gene transcripts in human vascular SMCs as well as evidence that KIR channels are upregulated in proliferative compared with contractile cells. The phenotype-dependent plasticity of KIR expression revealed here indicates that these channels are subject to dynamic physiological regulation and may contribute to vascular remodeling.
MATERIALS AND METHODS
HITB5 SMCs and electrophysiology.
All studies were carried out with HITB5 SMCs, which can convert between a motile, proliferative phenotype and a contractile phenotype on withdrawal of serum from the medium (20). Cells studied were from subcultures 15–20 after cloning. The proliferative phenotype was attained by culturing cells in M199 with 10% FBS. The contractile phenotype was generated by culturing cells in M199 with no added serum. The electrophysiological properties of these cells were studied 72 h after serum withdrawal, a time established to yield contractile cells with increased expression of contractile-specific muscle proteins (20). For electrophysiological studies, cells were lifted from culture dishes with 0.25% trypsin-0.1 mM EDTA (GIBCO) and transferred to a 1-ml bath perfusion chamber. Cells were allowed to settle and adhere before being perfused with bath solution at a rate of 1–3 ml/min. The chamber was mounted on the stage of a Nikon inverted microscope. For whole cell recordings, the nystatin perforated-patch technique was used, with the electrode solution containing 250 μg/ml nystatin, as previously described (15).
RNA isolation and RT-PCR.
Confluent SMCs were washed twice in cold PBS, and total RNA was extracted with TRIzol reagent (BRL/Life Technologies). One microgram of the total RNA was reverse transcribed into cDNA with Superscript II transcriptase (BRL/Life Technologies) and oligo d(T) primers. The cDNA was then subjected to PCR amplification. Primers for Kir2.1 were designed based on published human cardiac inward rectifier potassium channel Kir2.1 sequence (GenBank accession no. U16861) and spanned a 600-bp cDNA fragment. For Kir2.1, primers were forward 5′-AAGTCCACACCCGACAACAGTG-3′ and reverse 5′-TTCTGGATTTGAGGAGCTGTGC-3′; for Kir2.2 (GenBank accession no. XM015523), forward 5′-CGCACTTCCACAAGACCTATG-3′ and reverse 5′-AAAACCAGCAAGGAGCACCG-3′, expected product size of 648 bp; and for Kir2.3 (GenBank accession no. XM009984), forward 5′-TTTGAGCCTGTGGTCTTCG-3′ and reverse 5′-TCCCCCAGTCTTTGAAACC-3′, expected product size of 531 bp. PCR was carried out for 35 cycles with denaturation at 94°C for 45 s, annealing at 54°C for 45 s, and extension at 72°C for 1.5 min. PCR product was resolved on 2% agarose gel and visualized by ethidium bromide staining. Single bands of the appropriate sizes were excised from gel, purified with a QIAquick gel extraction kit (Qiagen), and sequenced to confirm identity of the amplicons.
Western blot analysis for Kir2.1.
HITB5 SMCs cultured in M199 supplemented with 10% FBS or serum starved in M199 for 3 days were used for Western blot analysis for Kir2.1 protein as described previously (20). Total cellular protein content was quantified by the Lowry assay (Bio-Rad). Equal amounts of protein (20 μg) were loaded and run on 10% SDS-PAGE gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk overnight at 4°C and then incubated with rabbit anti-human Kir2.1 antibody (1:1,000 dilution; Alomone Labs) for 1 h at room temperature. Bound primary antibody was detected with horseradish peroxidase-conjugated sheep anti-rabbit IgG F(ab′)2(1:5,000 dilution; Amersham Pharmacia Biotech) for 1 h at room temperature, and blots were developed with SuperSignal ECL detection reagents (Pierce) before quantitation on a Bio-Rad GS-700 imaging densitometer.
To assess DNA synthesis rates, SMCs were pulse labeled with [3H]thymidine (20). Briefly, HITB5 cells were seeded in 96-well plates at 5 × 103cells/cm2. Cells were incubated with [3H]thymidine for 8 h (10 μCi/ml, 71 Ci/mmol; ICN) before being transferred onto glass fiber filters with an automated cell harvester (Tomtec). Incorporated [3H]thymidine was measured with a Trilax 1450 MicroBeta counter (Wallac). We monitored cell viability on the basis of inspection of cell morphology and only studied concentrations of Ba2+ at which there was no evidence for a toxic effect, as shown by rounding of the cell borders, increased spacing between cells, granularity of the cytoplasm, debris in the media, or any decline in cell confluence.
The Na+-HEPES bath solution used for perforated-patch recording contained (in mM) 130 NaCl, 5 KCl, 20 HEPES, 10d-glucose, 1 CaCl2, and 1 MgCl2 (pH set to 7.4 with NaOH). The electrode solution used for perforated-patch recording contained (in mM) 140 KCl, 0.4 CaCl2, 1 MgCl2, 20 HEPES, and 1 EGTA (pH set to 7.2 with KOH). The concentration of K+ in the bathing solution was raised by substitution of KCl for NaCl. For cell-attached patch recording, the bath and electrode solution was composed of (in mM) 135 KCl, 20 HEPES, 10 d-glucose, 1 MgCl2, and 1 CaCl2(pH set to 7.4 with KOH).
Values are provided as means ± SE, with sample sizes (n) indicating the number of cells studied. For patch-clamp experiments, only one patch was obtained per cell. Statistical comparisons were made with ANOVA or Student's t-test, withP < 0.05 considered to indicate significance.
Inward current in human vascular SMCs.
Whole cell currents were recorded from human SMCs with the perforated-patch technique. Hyperpolarizing the membrane from a holding potential of −60 mV elicited an inward current that peaked and declined slightly with prolonged depolarization (Fig.1). The inward current demonstrated strong rectification, with little outward current when the membrane was stepped to more positive potentials (Fig. 1 A). As this inward current represented the predominant current evident over the range of resting potentials, we sought to characterize it in detail.
When studied in other SMCs, KIR currents are inhibited by extracellular cations such as Ba2+ and Cs+(2, 28, 29). As the inward current in the HITB5 SMCs shared numerous characteristics with a strongly rectifying KIR current, we investigated its Ba2+sensitivity. Current elicited by step hyperpolarization from −60 to −120 mV was progressively blocked by increasing concentrations of Ba2+ (Fig. 1 B), with inhibition showing characteristic time dependence. When investigated with a ramp protocol, inhibition of KIR current by Ba2+ was recorded at all voltages, with small outward currents positive toE K abolished as well. Ba2+ was selective for the KIR current and had no effect on outward currents positive to 0 mV (Fig. 1 C). The Ba2+-sensitive current, determined as the difference between control current and that inhibited by 50 μM Ba2+, is shown expanded in the inset of Fig. 1 C. Here, the physiologically relevant outward current is evident at potentials positive to E K [−84 mV at 5 mM extracellular K+ concentration ([K+]o)], a feature only rarely evident in recordings from smooth muscles (29). To quantify blockade by Ba2+, current amplitude at −120 mV between 600 and 700 ms after hyperpolarization was plotted as a fraction of the control current level, revealing half-maximal block of the KIR current at 10 μM Ba2+ (Fig. 1 D).
To confirm the ionic selectivity of this current, we increased [K+]o, which resulted in the current-voltage relationship shifting toward more positive potentials (Fig.2 A). Under these conditions, the reversal potential of the inward current shifted from −79 ± 2 mV (n = 6) at 5 mM [K+]o to −6 ± 2 mV (n = 5) at 135 mM [K+]o. The current reversal potential was determined by extrapolation of the linear inward current at potentials negative to E K. The solid line in Fig.2 A represents the best fit of the reversal potentials of six cells over a range of [K+]o and demonstrates a 53-mV/10-fold change of [K+]o. The observed shift in reversal potential is consistent with the predicted values ofE K determined by the Nernst equation (Fig.2 B). Furthermore, the rectification of the outward current positive to E K also shifted with increasing [K+]o. This is a characteristic unique to strong inwardly rectifying K+ channels. The chord conductance was calculated from currents recorded in various [K+]o and increased with increasing [K+]o (Fig. 2 C). These results demonstrate that current reversal potential, rectification, and maximal conductance are all dependent on [K+]o, indicating a current selective for potassium.
Because of its limited expression and low levels in smooth muscle, the single-channel characteristics of the endogenously expressed KIR current have yet to be elucidated in native vascular SMCs. As the proliferative SMCs used here demonstrated large KIR currents, we investigated the single-channel properties with the cell-attached patch configuration. For these studies, cells were bathed in 135 mM K+ to clamp the membrane potential at 0 mV. On patch hyperpolarization to −120 mV, single-channel events of 3 pA were recorded (Fig. 3 A). Channel activity was robust, making it difficult to determine the number of channels in each patch. Assuming a reversal potential of 0 mV, the unitary conductance was estimated to be 25–30 pS. To confirm that the single-channel currents resulted from the same Ba2+-sensitive current, 50 μM Ba2+ was added to the patch pipette. In contrast to the robust channel currents observed in control traces, only brief, irregular inward currents were recorded in the presence of Ba2+ (Fig. 3 A,right). Current rectification at the single-channel level was investigated with a ramp protocol whereby the patch potential was commanded from 50 to −120 mV over 700 ms. Current amplitude decreased as the patch potential neared 0 mV, with clear rectification at positive potentials (Fig. 3 B). To estimate the single-channel conductance, currents were measured between −60 and −120 mV (Fig. 3 C). When unitary current amplitudes from multiple patches were averaged, the best-fit slope conductance was 31 pS.
Inward rectifier current density increases in proliferative phenotype.
In our previous investigation of the currents in HITB5 SMCs (15), we documented differences in current activation between contractile and proliferative cells. Ca2+-activated Cl− currents were found only in contractile cells, indicating plasticity of channel expression. A trend toward greater KIR current in the proliferative phenotype was also observed, which we have examined here in detail. Indeed, KIR current was generally larger in proliferative compared with contractile cells (Fig.4 A). To determine whether the amplitude was a reflection of cell size, we measured cell capacitance by off-line digital integration of capacitative currents. Both cell size and KIR current amplitude (as measured at −120 mV) demonstrated greater variability in proliferative compared with contractile cells (Fig. 4 B), with mean values of current amplitude at −120 mV of −999 ± 200 pA in proliferative cells compared with −129 ± 26 pA in the contractile phenotype. Cell size showed a similar trend, with proliferative cells demonstrating greater variability and an average capacitance of 231 ± 46 pF, whereas contractile cells were smaller with 124 ± 25 pF. When current was corrected for cell size, the proliferative phenotype demonstrated significantly larger KIR current density (−4.5 ± 0.9 pA/pF compared with −1.4 ± 0.3 pA/pF in contractile cells, P < 0.001; n = 25 for each). These findings therefore identify differences in KIR current activity between SMC phenotypes.
Identification of Kir2.1 and Kir2.2 mRNA in human vascular SMCs.
Whole cell and single-channel experiments provided evidence for a strongly rectifying K+-selective channel with characteristics of channels of the Kir2 family, of which Kir2.1 has been previously identified in smooth muscle (2, 5). To investigate the molecular identity of the channels contributing to the inward rectifier currents in human vascular SMCs, we isolated and reverse transcribed total RNA from proliferative HITB5 SMCs. This clonal cell line provided a tremendous opportunity to identify mRNA transcripts without contamination from other cell types. Amplifying smooth muscle cDNA with primers specific for Kir2.1 and Kir2.2 revealed single bands of the expected size (Fig.5 A). We also carried out positive controls for each set of primers with human brain cDNA. In contrast, there was no evidence of Kir2.3 in smooth muscle, although a band was present in the brain control (Fig. 5 A,right). Similar results were obtained in three independent experiments. Sequencing of the amplicons confirmed homology to the published gene sequences. RNA that had not undergone reverse transcription was also subjected to the amplification procedure, and no bands were evident (data not shown).
Although electrophysiological data established greater KIRcurrent density in proliferative compared with contractile cells, whether this increased activity was due to differences in channel expression or regulation was unknown. Therefore, we performed Western blot analysis for Kir2.1 protein on proliferative and contractile cells, detecting a band at ∼48 kDa in both phenotypes, similar to that previously reported for Kir2.1 (31). As seen in Fig.5, expression of Kir2.1 was greater in proliferative cells, with densitometric analysis of three replicate experiments revealing significantly greater Kir2.1 expression in proliferative compared with contractile cells (P < 0.05; n = 3).
KIR contributes to regulation of membrane potential.
We next investigated the contribution of the inwardly rectifying K+ current to the membrane potential of both contractile and proliferative cells. Figure6 A shows a representative trace from a proliferative cell recorded in current-clamp configuration. Current was injected at 5-s intervals to evaluate membrane conductance. External Ba2+ caused rapid and reversible depolarization, indicating that KIR contributed to setting the RMP. Furthermore, increases in [K+]o from 5 to 135 mM resulted in progressive depolarization, close to that predicted by the Nernst equation. Voltage deflections recorded during current injection increased in amplitude during Ba2+ application, as expected during channel inhibition. In contrast, conductance increased with higher [K+]o, resulting in smaller voltage deflections (Fig. 6). On average, proliferative cells demonstrated a more negative resting potential compared with contractile cells (−71 ± 2 mV for 9 proliferative cells, −35 ± 4 mV for 5 contractile cells; P < 0.001; Fig. 6 B). Ba2+ (50 or 100 μM) depolarized cells of both phenotypes, with proliferative cells dropping to −50 ± 6 mV (P < 0.005; n = 6) and contractile cells to −25 ± 3 mV (P < 0.05;n = 3). These data demonstrate that the Ba2+-sensitive current contributes to setting the RMP of each phenotype. Moreover, the more hyperpolarized RMP of the proliferative cells may reflect the increased Kir2.1 channel expression shown above.
Because contractile cells demonstrated a less polarized RMP than proliferative cells, and one that was more positive thanE K, we hypothesized that these cells would demonstrate membrane hyperpolarization in response to small increases in extracellular K+. Indeed, when 12 contractile cells were exposed to an increase in [K+]o from 5 to 12 mM, a small hyperpolarization of the membrane potential was detected in 5 cells (−4 ± 1 mV). Voltage-clamp analysis on the same cells confirmed that these five cells demonstrated KIR current compared with the seven cells that did not hyperpolarize in response to increased [K+]o. A representative voltage trace can be seen in Fig. 7 A. Increasing [K+]o from 5 to 12 mM caused a prompt hyperpolarization. As the increased K+ was washed out, the membrane potential quickly returned to resting levels. Voltage-clamp analysis of the same cell as in Fig. 7 Ademonstrated an inwardly rectifying current that was shifted by the increased [K+]o (Fig. 7 B). Moreover, the reversal potential of the current in 12 mM [K+]o was clearly shifted negatively compared with that in 5 mM [K+]o (−46 mV in 12 mM [K+]o, −37 mV in 5 mM [K+]o; Fig. 7 C). Thus cell hyperpolarization in response to small increases in [K+]o is directly demonstrated to be due to a shift of the KIR current-voltage relationship.
Ba2+ inhibits [3H]thymidine incorporation in proliferative vascular SMCs.
Proliferative HITB5 cells demonstrated greater Kir2.1 expression than contractile cells. Furthermore, KIR activity contributed to the more hyperpolarized RMP of the proliferative compared with contractile cells. Therefore, we chose to investigate the contribution of KIR current to cell proliferation. Proliferative SMCs were pulse labeled with [3H]thymidine, and incorporation was assessed. The addition of Ba2+ decreased [3H]thymidine incorporation in a concentration-dependent manner (Fig. 8) suggestive of a decrease in cell proliferation. DNA synthesis was reduced to 50% in the presence of 1 mM Ba2+, indicating a decrease in potency compared with channel block at −120 mV, which is in keeping with the steep voltage dependence of Ba2+ blockade (29).
In the present study, we demonstrated phenotype-dependent plasticity of KIR current and channel expression in human vascular SMCs. Patch-clamp recording provided evidence for differential activity of this current in the HITB5 clone, with proliferative cells exhibiting greater current than contractile cells. RT-PCR revealed transcripts for Kir2.1 and Kir2.2. Western blot analysis for Kir2.1 revealed increased Kir2.1 protein in proliferative cells, consistent with the regulated expression of this channel. KIR current also contributed to setting the RMP, with proliferative cells showing a more hyperpolarized resting potential compared with contractile cells. KIR current may participate in the regulation of vascular SMC proliferation, as extracellular Ba2+ inhibits [3H]thymidine incorporation in proliferative cells.
KIR current was identified in HITB5 SMCs by its inward rectification, sensitivity to external Ba2+, and K+ selectivity. These characteristics resemble those previously demonstrated for KIR currents in SMCs from cerebral and coronary arteries (2, 27-29) and are indicative of a channel from the Kir2 family (23). Although the inward component of the KIR current has been used for identification and characterization in most studies on vascular myocytes, the physiologically significant outward component of the current has until now only been clearly shown in cell types other than smooth muscle (16) and oocytes expressing Kir2.1 (18). The robust expression of this channel in the proliferative phenotype allowed us to demonstrate the outward current for the first time in human SMCs.
The K+ selectivity of the KIR current in the present study was evaluated by increasing [K+]o, and the reversal potential was shifted by a 53-mV/10-fold change in [K+]o, similar to previous reports (16, 28, 29) and consistent with the value predicted by the Nernst equation for a K+-selective channel. Furthermore, the conductance increased with a rise in [K+]o, further implicating a channel of the Kir2 family. These channels are also characterized by their sensitivity to external Ba2+ in the low micromolar range, similar to previous reports in vascular SMCs (2, 28, 29). Many earlier studies of KIR in smooth muscle were carried out with elevated [K+]o to enhance the inwardly rectifying current and increase the driving force. When the blocker data from Bradley and coworkers (2) are considered at −40 mV (approximately the driving force used in the present study), the half-blockade by Ba2+ occurred at 7.2 μM, close to what we observed. The single-channel properties of the KIRcurrent were also investigated. The KIR current activity in the proliferative phenotype was robust, allowing us to demonstrate current rectification at the single-channel level as well as blockade by Ba2+. The unitary conductance of 25–30 pS we found is in the range reported for Kir2.1 (22 pS) and Kir2.2 (34 pS) channels in other cell types (23). This is the first description of single-channel KIR currents in vascular SMCs, and it further supports the conclusion that this channel is of the Kir2 family.
RT-PCR analysis of total cell mRNA revealed mRNA transcripts for Kir2.1 and Kir2.2, but not Kir2.3, in human vascular myocytes. Electrophysiological reports suggested that the current derived from a member of the Kir2 family, and indeed Kir2.1 mRNA has been identified in the rat basilar artery (2, 5) as well as in the coronary and mesenteric artery (2). It is of interest that immunocytochemical studies indicate the presence of all three Kir channel types in rat cerebral and basilar arteries, although expression of Kir2.3 was reported to be weak compared with Kir2.1 and Kir2.2 (31).
Interestingly, smooth muscle KIR channels have so far only been investigated in the contractile phenotype. Here we compare the expression and activity of Kir2.1 in both the contractile and proliferative phenotypes of a vascular SMC cell clone (20). These cells bidirectionally switch phenotype, allowing for the comparison of phenotypes while avoiding complications arising from extended time in culture (12, 24). This enabled us to demonstrate that loss of the contractile phenotype need not be accompanied by a concurrent loss of large conductance Ca2+-activated K+ channels in SMCs (15), as suggested earlier (22). However, Ca2+-activated Cl− current activity was observed exclusively in contractile cells, indicating plasticity of Cl− channel expression in vascular SMCs (15). Here we have identified another example of phenotype-dependent expression of ion channels in human SMCs. Patch-clamp electrophysiology and Western blot analysis demonstrated greater KIR current density and protein in the proliferative compared with the contractile phenotype, indicating that the expression of this channel can be dynamically regulated within a defined population of vascular SMCs.
The involvement of KIR channels in setting the membrane potential of contractile SMCs was investigated, as the resting potential is a key determinant of myogenic tone (5, 9). KIR channels have also been implicated in the phenomenon of K+-induced hyperpolarization whereby small increases in [K+]o cause vasodilation in vitro (3,4, 10, 17, 21, 27) and in vivo (5). A study of Kir2.1 knockout mice confirmed this role for KIR, as these animals no longer demonstrate vasodilation in response to increases in [K+]o (33). In the present study, we demonstrate for the first time K+-induced hyperpolarization at the level of the individual vascular SMC cell with the current-clamp technique. Contractile cells that had an average membrane potential of approximately −35 mV hyperpolarized in response to a small elevation in [K+]o. Moreover, those cells that did not show inward rectifier current in voltage clamp also did not hyperpolarize, implicating KIR in this process.
In contrast, hyperpolarization in response to small increases in [K+]o was not observed in SMCs of the proliferative phenotype. This is consistent with the hyperpolarized RMP of these cells; on average, proliferative cells had a RMP of approximately −70 mV. Increases in [K+]oonly caused membrane depolarization due to the positive shift inE K. However, proliferative cells were dependent on KIR for the determination of the membrane potential, suggesting that the inward rectifier current, and the resulting hyperpolarization, serves an important functional role. Indeed, [3H]thymidine incorporation was decreased by addition of the KIR channel blocker Ba2+, suggesting that KIR may participate in the regulation of cell proliferation.
Membrane potential is believed to be a vital determinant of vascular SMC proliferation, as depolarized potentials will allow for greater Ca2+ influx, a known mitogenic signal (1), through voltage-dependent L-type Ca2+ channels (26). Evidence to support this idea comes from studies documenting the regulation of several classes of K+currents, including voltage-dependent delayed rectifier current (26) and ATP-sensitive K+ channels (6), where in both cases proliferating cells exhibit reduced K+ current density. These findings raise the possibility that different mechanisms may regulate the growth of smooth muscle cells originating in different tissues. However, Curtis and Scholfield (7) demonstrated the presence of a nifedipine-sensitive but non-L-type Ca2+ influx pathway in arteriolar SMCs and concluded that a hyperpolarized membrane potential would maximize Ca2+ influx by increasing the Ca2+ driving force, dogma generally reserved for nonexcitable cells. KIR currents play a critical role in proliferation of other cell types, possibly by affecting the RMP. Schlichter and colleagues (30) showed that KIRcurrents are necessary for colony stimulating factor-1-induced proliferation of microglial cells. Also, Vaur and associates (32) observed depolarization and decreased proliferation with blockade of KIR current in a pituitary cell line.
Further evidence for the role of Kir2.1 in cell proliferation and development comes from genetic disruption of Kir2.1. Zaritsky and coworkers (33) suggested that the lethal cleft palate observed in Kir2.1-deficient mice is due to a breakdown in cell signaling, proliferation, or differentiation of various cell types. These conclusions are supported by the phenotype of patients suffering from Andersen syndrome, which was recently attributed to mutations in Kir2.1 and is characterized by cardiac arrythmias, skeletal muscle myotonia, and dysmorphia (25). Plaster and coworkers (25) recognized that the cleft palate of the Kir2.1 knockout mouse corresponded to the dysmorphic features seen with Andersen syndrome and concluded that Kir2.1 plays a critical role in development. Furthermore, decreasing Kir2.1 expression has been shown to retard skeletal muscle development in vitro (11). These findings are consistent with our data and suggest that Kir2.1 expression may be a key factor in vascular SMC proliferation.
In conclusion, we demonstrated phenotype-dependent plasticity of KIR expression and activity in human vascular SMCs. These channels contribute to both the regulation of myogenic tone and the rate of SMC [3H]thymidine incorporation. The dynamically regulated expression of KIR, including Kir2.1, may play a critical role in physiological processes such as vascular remodeling.
We gratefully acknowledge the technical assistance of Caroline Van Den Diepstraten.
This work was supported by Canadian Institutes of Health Research (CIHR) Grants MT10019 and MT11715 and Heart and Stroke Foundation of Canada Grant T4458. T. Karkanis was supported by an Ontario Graduate Scholarship in Science and Technology and S. Li by a Premier's Research Excellence Award (to J. G. Pickering). J. G. Pickering is a Career Investigator of the Heart and Stroke Foundation of Canada. S. M. Sims was supported by a CIHR Scientist award.
Present address of S. Li: Dept. of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey, One Robert Wood Johnson Pl., PO Box 19, New Brunswick, NJ 08903-0019.
Address for reprint requests and other correspondence: S. M. Sims, Dept. of Physiology and Pharmacology, Faculty of Medicine and Dentistry, Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 21, 2003;10.1152/ajpheart.00559.2002
- Copyright © 2003 the American Physiological Society