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Originally published In Press as doi:10.1074/jbc.M313791200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 13, 12959-12966, March 26, 2004
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Regulation of Ca2+-dependent K+ Current by {alpha}v{beta}3 Integrin Engagement in Vascular Endothelium*

Junya Kawasaki{ddagger}, George E. Davis§, and Michael J. Davis{ddagger}

From the Departments of {ddagger}Medical Physiology and §Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114

Received for publication, December 17, 2003 , and in revised form, January 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Interactions between endothelial cells and extracellular matrix proteins are important determinants of endothelial cell signaling. Endothelial adhesion to fibronectin through {alpha}v{beta}3 integrins or the engagement and aggregation of luminal {alpha}v{beta}3 receptors by vitronectin triggers Ca2+ influx. However, the underlying signaling mechanisms are unknown. The electrophysiological basis of {alpha}v{beta}3 integrin-mediated changes in endothelial cell Ca2+ signaling was studied using whole cell patch clamp and microfluorimetry. The resting membrane potential of bovine pulmonary artery endothelial cells averaged -60 ± 3 mV. In the absence of intracellular Ca2+ buffering, the application of soluble vitronectin (200 µg/ml) resulted in activation of an outwardly rectifying K+ current at holding potentials from -50 to +50 mV. Neither a significant shift in reversal potential (in voltage clamp mode) nor a change in membrane potential (in current clamp mode) occurred in response to vitronectin. Vitronectin-activated current was significantly inhibited by pretreatment with the {alpha}v{beta}3 integrin antibody LM609 by exchanging extracellular K+ with Cs+ or by the application of iberiotoxin, a selective inhibitor of large-conductance, Ca2+-activated K+ channels. With intracellular Ca2+ buffered by EGTA in the recording pipette, vitronectin-activated K+ current was abolished. Fura-2 microfluorimetry revealed that vitronectin induced a significant and sustained increase in intracellular Ca2+ concentration, although vitronectin-induced Ca2+ current could not be detected. This is the first report to show that an endothelial cell ion channel is regulated by integrin activation, and this K+ current likely plays a crucial role in maintaining membrane potential and a Ca2+ driving force during engagement and activation of endothelial cell {alpha}v{beta}3 integrin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are a large family of membrane-spanning, cell adhesion proteins composed of {alpha} and {beta} subunits, with over 18 {alpha} and 8 {beta} subunits combining to form more than 24 different heterodimers (1, 2). In the vascular system, integrins play various roles in coordinating cell function, such as adhesion, spreading, and migration (3, 4). The vitronectin (VN)1 receptor, {alpha}v{beta}3 integrin, is expressed both luminally and abluminally on endothelium (5, 6) and is thought to play an important role in several vascular pathologies (4, 7-9). Soluble {alpha}v{beta}3 integrin ligands are also capable of acutely regulating vascular tone. For example, synthetic peptides containing the arginine-glycine-aspartic acid (RGD) sequence that binds {alpha}v{beta}3 integrin have been shown to block flow-induced, endothelium-dependent vasodilation in coronary arterioles (10). It has therefore been suggested that soluble {alpha}v{beta}3 integrin ligands may acutely modulate blood flow by interacting with endothelial cell (EC) integrins (11, 12).

VN is a plasma glycoprotein first identified as a ligand of {alpha}v{beta}3 integrin and circulates at a concentration of 200-400 µg/ml in normal human plasma (13). VN can potentially interact with unbound {alpha}v{beta}3 integrin, and plasma levels of VN-containing complement complexes increase after complement activation. Adhesion to VN-covered substrates leads to an increase in EC intracellular Ca2+ concentration ([Ca2+]i) (14). Acute application of VN or cross-linking of {alpha}v{beta}3 integrin with the {alpha}v{beta}3 antibody, LM609, also increases EC [Ca2+]i (15). The [Ca2+]i increase occurs as a consequence of tyrosine phosphorylation of phospholipase C-{gamma}1 following {alpha}v{beta}3 activation. It has been proposed that stimulation of {alpha}v{beta}3 integrin activates an unidentified Ca2+ influx pathway (14, 15).

Although integrins have been shown to regulate ion channels, including Ca2+ and K+ channels in other tissues (16-20), it is not known if integrins regulate EC ion channels. The rapid effects of integrin activation on EC [Ca2+]i suggest that ion channels are involved in this response, and the purpose of the present study was to directly test this idea.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Bovine pulmonary artery ECs (BPAECs) were obtained at passage 1 (BioWhittaker, Walkersville, MD) and cultured on 35-mm plastic 6-well dishes in minimum essential medium containing 10% fetal bovine serum, human epithelial growth factor (10 µg/liter), bovine brain extract, hydrocortisone (1 mg/liter), penicillin (100 units/ml), streptomycin (100 units/ml), and heparin (25 units/ml). Cells between passages 4 and 9 were used for both patch clamp and microfluorimetry after trypsinization (0.25 mg/ml with EDTA) and plating on gelatin-coated coverslips 24-48 h prior to an experiment. Only single cells were studied in all protocols.

Electrophysiology—Conventional whole cell current recordings were performed (21) using an EPC-9 patch clamp system (HEKA Elektronik, Lambrecht, Germany). Continuous recordings of membrane potential (Em), membrane current (Im), cell capacitance, and/or seal resistance were simultaneously monitored using Pulse and X-Chart software (HEKA). Patch pipettes were pulled from borosilicate glass (catalog number 7040, Sutter Instruments, Novato, CA) with resistances of 3-8 megaohms. To determine current-voltage (I-V) relationships, 1-s voltage ramps from -120 to +60 mV or voltage steps between -120 and +20 mV in 20-mV increments (200 ms duration) were applied. Data were fitted using IGOR analysis routines (Wavemetrics, Lake Oswego, OR).

Solutions and Reagents—The compositions of all solutions are listed in Table I. All experiments were performed at 35 °C using a bath temperature controller (HCC-100, Dagan Corp., Minneapolis, MN). The calculated equilibrium potentials for the standard experimental conditions, solutions I-A (solution I in the pipette and solution A in the bath), were -84.1 mV for K+ (EK) and -32.8 mV for Cl- (ECl) at 35 °C.


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  		TABLE I
Composition of solutions

Values are in mmol/liter. All solutions contained 10 mmol/liter HEPES. All intracellular solutions contained 2 mmol/liter MgATP. pH was adjusted to 7.2 by KOH (I, II, and III) or methanesulfonic acid (IV) and to 7.4 by NaOH (A, B, and C) or 20% TEA-OH (D). TEA, tetraethylammonium; NMDG, N-methyl-D-glucamine.

 
Monoclonal antibodies against {alpha}v{beta}3 integrin (LM609; catalog number MAB1976Z) and {alpha}5{beta}1 integrin (catalog number MAB1999) were obtained from Chemicon (Temecula, CA). Donkey anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Fura-2 and Fura-2 AM were obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma-Aldrich.

Bradykinin, thapsigargin, and La3+ were added to the appropriate solutions at the concentrations indicated in the Fig. 6 legend and applied to individual cells from wide-tipped glass micropipettes connected to a Picospritzer (Parker-Hannifin, Fairfield, NJ). The low shear stress associated with local solution exchange did not in itself induce changes in current (n = 4).



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  		FIG. 6.
TG, but not VN or BK, induces detectable Ca2+ current (ICa). Voltage pulses (from -120 to +20 mV in 20-mV increments from VH = 0 mV) were performed both at rest (panel A) and during stimulation with either VN (200 µmol/liter; panel B), BK (1 µmol/liter; panel C), or TG (1 µmol/liter; panel D) in solutions IV-D. The calibration bar in A also applies to panels B and C. E, typical time course of whole cell current observed at VH = -120 mV when TG (1 µmol/liter) was present in the pipette. Within 30 s after whole cell current recording was initiated, the response to TG reached its peak (§). La3+ (50 µmol/liter) was applied when the amplitude of the current stabilized. F, data obtained from voltage pulses shown in panels A-D are taken at the times indicted by squares (160-180 ms into the pulse) and summarized and plotted as the Im-Em relationship. Symbols shown in panel F represent rest (closed circles; n = 12); VN (200 µmol/liter; open squares; n = 6); BK (1 µmol/liter; open triangles; n = 6); and TG (1 µmol/liter; closed triangles; n = 5). Note the different scale from that used for the other figures. Data are mean ± S.E. *, p < 0.05 compared with rest.

 
Fura-2 Microfluorimetry—Measurements of Fura-2 fluorescence were performed as described previously (22). Briefly, BPAECs on coverslips were loaded with 10 µmol/liter Fura-2/AM in solution A for 30 min at 25 °C, followed by washing for 10 min at 35 °C. Fluorescence emission at 510 nm associated with alternating 340/380-nm excitation (at ~15 Hz) was used as an index of [Ca2+]i (23). Calibrations were performed using Fura-2 (pentapotassium salt) and the Molecular Probes calibration kit. Solution A was used for all Fura-2 experiments on cells, except for Ca2+-free bath protocols (solution C).

Immunofluorescence Staining—BPAECs plated on gelatin-coated coverslips were fixed with phosphate-buffered saline (PBS) containing (in mmol/liter) 2.7 KCl, 1.5 KH2PO4, 157 NaCl, and 8 NaH2PO4 with 4% paraformaldehyde. Fixation was followed by five washes in PBS containing 0.1 mmol/liter glycine. The cells were permeabilized in PBS with 0.1% Triton X-100, rinsed five times, and incubated with or without {alpha}v{beta}3 or {alpha}5{beta}1 integrin Ab (1:100 dilution), in PBS containing 0.1% Triton X-100, 0.9% sodium citrate, and 0.025% NaN3 for 60 min, followed by five rinses. Cells were then incubated in goat anti-mouse secondary IgG conjugated to Alexa 488 (1:300 dilution) for 60 min and rinsed five times. This was followed by phalloidin-rhodamine (1:50 dilution) treatment for 40 min to stain actin filaments. Images were collected using an Orca cooled-CCD camera (Hamamatsu Photonics K. K., Hamamatsu, Japan) and processed with Metamorph (Universal Imaging Corp., Downington, PA).

Data Analysis—Analysis of variance was used to determine statistically significant differences between I-V curves. Scheffe's post hoc tests (24) were used to compare differences between different groups at the same holding potentials. Paired and unpaired t tests were used to test differences between groups for Em or the reversal potential for whole cell current (Erev). Values of p < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of {alpha}v{beta}3 and {alpha}5{beta}1 Integrin on BPAECs—Consistent with previous reports (5, 6), both {alpha}v{beta}3 and {alpha}5{beta}1 integrins were expressed on BPAECs (Fig. 1). {alpha}v{beta}3 integrin was preferentially localized to focal adhesions at the tips of actin filaments (Fig. 1A), whereas {alpha}5{beta}1 integrin tended to be distributed around the cell center (Fig. 1B), possibly at fibrillar adhesions (25). Both types of integrins were abundant on the surface of BPAECs under the conditions used in our experiments.



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  		FIG. 1.
Both {alpha}v{beta}3 and {alpha}5{beta}1 integrins are present in BPAECs. Immunofluorescence staining revealed the presence of both {alpha}v{beta}3 (A) and {alpha}5{beta}1 (B) integrins (both shown in green) on the surface of BPAECs. Actin filaments were stained with phalloidin-rhodamine (red). A control in which no primary Ab was added is shown in panel C. Bar, 5 µm.

 
Vitronectin Activates a Whole Cell Current in BPAECs—The basic characteristics of unstimulated BPAECs in solutions I-A were as follows: cell capacitance = 29.8 ± 1 pF (n = 56); Em = -60.1 ± 3 mV (n = 42); Erev = -57.5 ± 3 mV (n = 52). In solutions I-A, BPAECs (n = 52) exhibited an inward rectifying K+ current as the predominant resting current with mean = -12.6 ± 1 pA/pF at holding potential (VH) = -120 mV. At VH = -50 mV, VN (200 µg/ml) activated a significant outward current (from 0.2 ± 0.1 to 5.0 ± 1 pA/pF) in 16 of 22 (73%) cells (Fig. 2A). The I-V relationships for the cells at rest and after activation of current by VN, as determined using voltage ramps, are shown in Fig. 2B. Whole cell currents in the presence of VN were outwardly rectifying and relatively large at voltages from -50 to +50 mV.



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  		FIG. 2.
VN activates whole cell current. A, change in whole cell current in response to VN (200 µg/ml) was monitored at VH = -50 mV in solutions I-A. Voltage ramps (-120 mV to +60 mV) under resting conditions (trace 1) and during (trace 2) application of VN are shown in panel B. The numbers shown in panel A correspond to the Im-Em relationships shown in panel B. In panel B, the net VN-activated whole cell current (trace 2-1) was obtained by subtracting trace 1 from trace 2. C, summaries of results from 16 different cells at rest (open triangles), during stimulation with VN (closed circles), and the difference currents (open squares). Data are mean ± S.E.

 
The net current evoked by VN, after subtracting the resting inward rectifying current, is shown in Fig. 2, B and C. VN significantly increased the amplitude of the whole cell difference current from 1.7 ± 1 to 30.1 ± 5 pA/pF at VH = +60 mV (n = 16). This was associated with a slight shift in Erev to more negative potentials (-66.4 ± 2 to -70.6 ± 1 mV; n = 16). In a different series of experiments, Em was measured under current clamp conditions (n = 11). There was no significant difference between the average Em at rest, -64.4 ± 5 mV, and during VN stimulation, -65.3 ± 5 mV. These data suggest that the resting Em of BPAECs is determined by a K+ current and that VN activates a whole cell current whose Erev is close to EK.

Vitronectin-activated Current Is Inhibited by {alpha}v{beta}3 Integrin Ab—To confirm that VN-activated current was mediated by {alpha}v{beta}3 integrin engagement, BPAECs were pretreated with integrin-specific antibodies prior to VN application. In the presence of LM609 (200 µg/ml), VN-induced current was completely inhibited (Fig. 3A; n = 5). However, pretreatment with {alpha}5{beta}1 integrin Ab did not have a significant effect on VN-induced current (Fig. 3B; n = 4). These results suggest that VN-activated current results from the specific activation of {alpha}v{beta}3 integrin.



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  		FIG. 3.
Pretreatment with {alpha}v{beta}3 integrin Ab inhibits VN-induced whole cell current. Voltage ramps (-120 mV to +60 mV) were used to obtain Im-Em relations in response to VN with and without pretreatment with integrin antibody. Panel A shows the effect of pretreatment with LM609 (200 µg/ml), a monoclonal Ab against {alpha}v{beta}3 (200 µg/ml; squares; n = 5), and panel B shows the effect of pretreatment with a monoclonal Ab against {alpha}5{beta}1 integrin (200 µg/ml; triangles; n = 4) for 30 min at 25 °C prior to the stimulation with VN. For both data sets, circles represent the control response to VN without pretreatment, which are the same data as in Fig. 2C (circles). In panel B, four of six cells (67%) responded to VN. Solutions are I-A. Data are mean ± S.E. *, p < 0.05 compared with the value without pretreatment.

 
Acute application of soluble LM609 alone failed to elicit any change in current (n = 5; data not shown). In a previous study of BPAECs, aggregation of {alpha}v{beta}3 integrin was required to initiate Ca2+ influx and [Ca2+]i increases (15). To test if aggregation of {alpha}v{beta}3 integrin by LM609 would stimulate current, BPAECs were pretreated with LM609 in solution A for 30 min at 25 °C, followed by acute application of secondary Ab (IgG, 200 µg/ml). However, this procedure failed to significantly activate current (n = 5, data not shown).

Vitronectin-activated Current Is Carried by K+—The negative Erev of VN-induced current suggested that the current was carried primarily by K+. To test this assumption further, extracellular K+ was substituted with solutions I-B containing Cs+, an inhibitor of multiple K+ channels (26). Cs+ bath solution completely blocked both the inward rectifying current at rest and the VN-activated current (Fig. 4A; n = 8). In addition, VN did not significantly shift Erev from its more depolarized initial value (-46.3 ± 4 mV in Cs+ bath; -43.3 ± 5 mV during VN). The effect of iberiotoxin (IBTX), a selective inhibitor of large conductance Ca2+-activated K+ (KCa) channels, was subsequently tested. In standard bath solution (solution A), IBTX (100 nmol/liter) completely inhibited the VN-activated outward current without significantly affecting the inward rectifying component of current (Fig. 4B).



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  		FIG. 4.
VN-activated whole cell current is carried by K+. Voltage ramps (-120 mV to +60 mV) were used to obtain Im-Em relations in response to VN using solutions I-A (circles), solutions I-B (extracellular [Cs+] = 5.9 mmol/liter; [K+] = 0; squares in panel A; n = 8) or solutions I-A with IBTX (100 nmol/liter; triangles in panel B; n = 11). Control data (circles) are the same as in Fig. 2C (circles). In panel C, the solid line represents Erev for VN-induced whole cell current during the stimulation with VN in varying extracellular [K+] at 5.9, 30, or 120 mmol/liter. Data plotted in triangles represent the response to VN in solutions III-A, where intracellular [Cl-] = 142 mmol/liter. The dotted line represents the predicted equilibrium potential for K+ (EK) at 35 °C. For solutions I-A, EK and ECl were -84 and -32.8 mV, respectively, at 35 °C. For solutions III-A, ECl was -1.1 mV. Data are mean ± S.E. *, p < 0.05 compared with the value in solutions I-A without IBTX.

 
To further test if the VN-induced current was carried by K+, the net VN-induced current was measured at various extracellular K+ concentrations ([K+]o) equalling 5.9, 30, or 120 mmol/liter, and Erev was plotted as a function of [K+]o (Fig. 4C). As determined from the slope of the graph, Erev changed by 50.5 mV per 10-fold increase in [K+]o (Fig. 4C; solid line). The predicted slope for a pure K+ current at 35 °C is 61.1 mV per 10-fold increase in [K+]o (Fig. 4C; dotted line). In contrast, changing [Cl-]i by switching from solutions I-A (ECl = -32.8 mV) to III-A (ECl = -1.1 mV), with [K+]o = 5.9 mmol/liter in both cases, did not significantly shift Erev (Fig. 4C). Collectively, these results suggest that VN-evoked current is almost exclusively carried by K+ through a large conductance KCa channel.

Vitronectin-activated Current Requires an Increase in Intra-cellular Ca2+—Agonist-induced increases in [Ca2+]i are known to activate K+ current in ECs (27), so the role of intracellular Ca2+ in mediating the effects of VN was tested. When intracellular Ca2+ was strongly buffered with 5 mmol/liter EGTA in the pipette (solutions II-A), VN failed to activate any significant current at VH = -50 mV (Fig. 5A). Periodic voltage ramps from -120 mV to +60 mV confirmed that there was no activation of current by VN at other potentials (Fig. 5B). The average I-V relationships with and without EGTA in the pipette solution are shown in Fig. 5C. Thus, VN-evoked current was completely abolished when intracellular Ca2+ was strongly buffered by EGTA. These results suggest that an increase in [Ca2+]i is required for activation of K+ current by VN.



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  		FIG. 5.
Intracellular Ca2+ buffering inhibits VN-induced whole cell current. A, the change in whole cell current by VN (200 µg/ml) was monitored at VH = -50 mV during strong buffering of intracellular Ca2+ by 5 mmol/liter EGTA (solutions II-A). B, voltage ramps (-120 mV to +60 mV) before (trace 1) and during (trace 2) VN application. The numbers in panel A correspond to the Im-Em relationship curves shown in panel B. In panel B, VN-activated whole cell current data (trace 2-1) was obtained by subtracting trace 1 from trace 2. C, summary of six experiments in response to VN in solutions II-A ([EGTA]i = 5 mmol/liter; squares). The data plotted as circles [EGTA]i = 0) are the same as in Fig. 2C (circles). Data are mean ± S.E. *, p < 0.05 compared with the value in solutions I-A.

 
Mechanism of the VN-induced [Ca2+]i Increase—To test if VN would activate a non-voltage-gated Ca2+ current (ICa) that might contribute to secondary activation of a KCa channel, protocols were performed using solutions previously used to record store-operated cation currents (28). Using solutions IV-D, in which Ca2+ was the primary charge carrier (28, 29), ICa averaged -0.09 ± 0.01 pA/pF (VH = -120 mV) and Erev averaged -86 ± 6 mV in unstimulated cells (Fig. 6, A and F). One minute after application of VN (200 µg/ml), ICa was -0.07 ± 0.02 pA/pF (VH = -120 mV; Fig. 6, B and F) and Erev was -89 ± 11 mV. Neither value was significantly different from its corresponding value in unstimulated cells.

Due to the absence of a clear response to VN, bradykinin (BK), an agonist known to induce Ca2+ release and influx (30, 31), and thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump (28, 32), were used as positive controls. One minute after application of BK (1 µM), ICa averaged -0.13 ± 0.04 pA/pF (VH = -120 mV; Fig. 6, C and F) and Erev averaged -76 ± 10 mV. Neither value was significantly different from its corresponding value in unstimulated cells. In contrast, when TG (1 µM) was preloaded into the patch pipette, there was a rapid activation of inward current, which then partially inactivated and stabilized after ~3 min (Fig. 6E). ICa peaked at -0.87 ± 0.08 pA/pF, and Erev shifted to +3 ± 1 mV (VH = -120 mV; Fig. 6, D and F) within 30 s after cell membrane rupture by pipette suction (Fig. 6E). The addition of 50 µM La3+ to the bath solution inhibited current activation (Fig. 6E) from -0.87 ± 0.08 to -0.09 ± 0.01 pA/pF (VH = -120 mV) and shifted Erev from +2.8 ± 1 back to -86.8 ± 2 mV (n = 5).

To test if VN would elevate [Ca2+]i, even though VN-induced ICa activation could not be detected, VN was applied to non-voltage clamped BPAECs loaded with Fura-2/AM. Indeed, VN (200 µg/ml) induced a significant [Ca2+]i increase in BPAECs (Fig. 7A), as reported previously (15). LM609 alone did not significantly alter [Ca2+]i, as reported in the same study (15), nor was a significant [Ca2+]i increase detected in LM609-pretreated cells when a secondary Ab (anti-mouse IgG, 200 µg/ml) was applied to aggregate {alpha}v{beta}3 integrin (although several different durations and temperatures for LM609 pretreatment were tested). Because the magnitude of the VN-induced [Ca2+]i increase was rather modest, BK and TG were used as positive controls known to elevate EC [Ca2+]i (31, 33). Both 1 µmol/liter BK (Fig. 7B) and 1 µmol/liter TG (Fig. 7C) induced significant and sustained [Ca2+]i increases, with the TG response (210 ± 22%, n = 6) being substantially higher than that for VN (118 ± 4%, n = 8) or BK (138 ± 12%, n = 5) (Fig. 7D). These sustained [Ca2+]i increases required Ca2+ influx, because they were prevented when [Ca2+]o was absent from the bath solution (solution C; Fig. 7C).



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  		FIG. 7.
VN, BK and TG elevate [Ca2+]i. Single BPAECs were loaded with 10 µmol/liter Fura-2, and [Ca2+]i was measured during stimulation with VN (panel A; 200 µg/ml), BK (panel B; 1 µmol/liter), or TG (panel C; 1 µmol/liter) in 1.8 mmol/liter Ca2+ bath (solution A). When VN was applied, two of eight cells had a positive response (>30% increase over rest). D, sustained phase of the [Ca2+]i increase in 1.8 mmol/liter Ca2+ bath (solid bars; n = 5-8) or Ca2+-free bath (open bars; solution C; n = 4-6) was sampled ~3 min after application of each agonist. [Ca2+]i values are normalized as the percentage increase above resting F340/F380 ratios. Data are mean ± S.E. *, p < 0.05 versus VN; #, p < 0.05 versus BK.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A novel finding of this study is that the interaction between {alpha}v{beta}3 integrin and the extracellular matrix protein VN results in rapid activation of at least one type of endothelial cell ion channel. The following observations suggest that the major VN-activated current is a KCa current. (i) Neither Erev nor Em significantly shifted from EK (-84.1 mV) during VN application. (ii) Erev for VN-activated current shifted with changes in [K+]o close to what would be predicted for a pure K+ current. (iii) VN-activated K+ current was completely inhibited by buffering intracellular Ca2+. (iv) VN-activated K+ current was blocked by Cs+ and IBTX. Activation of the KCa current by VN did not appear to substantially hyperpolarize cultured BPAECs due to their already negative resting membrane potentials (-64 mV), but it would be predicted to hyperpolarize other ECs with more positive resting potentials (34-36). Although a Ca2+ current directly activated by VN could not be detected, our collective electrophysiological and microfluorimetric data suggest that a Ca2+-permeable channel is also activated by VN in these cells and that its activation underlies the plateau phase of the VN-stimulated [Ca2+]i increase. These results show, for the first time, that ion channels in ECs are rapidly activated in response to EC integrin interaction with an extracellular matrix protein. This points to a new mechanism for the control of vascular resistance under physiological conditions and during adaptive responses to vascular injury.

Mechanisms of Activation of K+ Current by {alpha}v{beta}3 Integrin—The mechanism underlying VN-activated K+ current may be similar to those of other K+ currents activated by agonists. By measuring whole cell current and [Ca2+]i simultaneously in bovine aortic EC (BAEC), Himmel et al. (27) found that several agonists, including BK, induced [Ca2+]i increases and subsequently activated K+ current. BK-induced current was completely abolished after intracellular Ca2+ chelation with EGTA, similar to our results for VN. There are some differences, however, between the characteristics of the current activated by BK and VN. In BAEC, BK not only activated a K+ current but also Cl- and non-selective cation currents that were dependent on the [Ca2+]i increase. Thus, BK shifted Erev from -79 to -63 mV, away from EK (-83 mV) (27). In our study of BPAECs, VN tended to shift Erev toward a more negative potential (-66 mV to -71 mV), which is consistent with more selective activation of a K+ current. Indeed, significant activation of another current during K+ channel inhibition by Cs+ could not be detected, and IBTX, a selective blocker of large conductance KCa channels, inhibited the VN-activated current.

Recent studies have revealed that integrin ligands acutely regulate several types of ion channels (16-20). For example, integrins have been found to modulate KCa current in at least two different types of cells. Attachment of fibronectin-coated beads to leukemia cells led to hyperpolarization (37), an effect most likely mediated by activation of KCa current. However, this effect was not completely abolished by strong intracellular Ca2+ buffering (38); therefore, the KCa current was probably not activated by an increase in [Ca2+]i but by another mechanism such as channel phosphorylation. Mechanical stimulation of human articular chondrocytes through {beta}1 integrins was shown to induce membrane hyperpolarization by autocrine production of interleukin-4 (39, 40). The hyperpolarization in that case was due to activation of apamin-sensitive, small conductance KCa channels and was blocked by pretreatment with {beta}1 integrin Ab (41). It is not clear whether an increase in [Ca2+]i was critical to that response, because [Ca2+]i changes were not measured or prevented in those experiments. Collectively, these studies suggest that integrin engagement and activation can result in KCa channel activation by several different pathways.

Intracellular signal transduction by integrins usually involves tyrosine kinase activation (1). Indeed, several lines of evidence suggest that non-receptor tyrosine kinases directly regulate KCa channels. In Chinese hamster ovary cells co-transfected with prolactin receptors and KCa channels, the addition of prolactin led to channel activation that persisted after patch excision and was inhibited by an antibody to Janus tyrosine kinase 2 (JAK2) (42). These results suggest that the channels were directly regulated by JAK2 or a downstream kinase. In human embryonic kidney 293 cells, co-expression of KCa channels with c-Src, another non-receptor tyrosine kinase, led to Ca2+-sensitive enhancement of K+ current (43). This enhancement was mediated by phosphorylation of residue Tyr-766 on the C-terminus of the channel's {alpha}-subunit. Interestingly, both phosphorylation of the channel and potentiation of current were more pronounced at high levels of intracellular Ca2+ (43). It is possible that the KCa current activated by VN-{alpha}v{beta}3 interaction in BPAECs may also involve a Ca2+-sensitive, tyrosine kinase-dependent pathway. Whether this mechanism involves phosphorylation of a specific C-terminal tyrosine residue on the BKCa channel remains to be investigated. Our observation that VN-activated K+ current is blocked by EGTA does not preclude direct regulation of the channel by tyrosine phosphorylation if that process also requires a relatively high [Ca2+]i (43).

Integrin-mediated Ca2+ Signaling in ECs—Cheresh and coworkers (14) first reported VN-induced [Ca2+]i increases in human umbilical vein ECs as they adhered to a VN-covered substrate. Based on the use of pharmacological inhibitors, the [Ca2+]i increase was attributed to activation of a non-voltage-dependent Ca2+ channel. Bhattacharya et al. (15) demonstrated that both VN and cross-linking of {alpha}v{beta}3 integrin by LM609 increased [Ca2+]i in BPAECs. The [Ca2+]i increase occurred as a consequence of tyrosine phosphorylation of phospholipase C-{gamma}1 and an increase in inositol 1,4,5-trisphosphate levels that would induce Ca2+ release from Ca2+ stores. However, Ca2+ influx was required for sustained elevation in [Ca2+]i. The mechanism for Ca2+ entry was not shown, because neither study examined changes in the electrophysiological mechanisms underlying the [Ca2+]i increase.

Conceptually, integrin activation could increase [Ca2+]i by increasing the driving force for passive calcium entry or by activating an ion channel or transporter to alter Ca2+ conductance. In other types of non-excitable cells, multiple lines of evidence suggest that integrin activation increases Ca2+ conductance by activating a Ca2+-permeable ion channel. For example, thrombin application in human platelets leads to increased Ca2+ channel activity via activation of {alpha}IIb{beta}3 integrin (44). In Madin-Darby canine kidney cells, the application of beads coated with the RGD peptide elicits an [Ca2+]i increase that correlates with bead adhesion, suggesting the involvement of integrins in regulating Ca2+ homeostasis (45, 46).

Our observation that the Erev of VN-activated current did not shift exactly as predicted for a pure K+ current (Fig. 4C) suggests that another ion channel is also activated by VN. In non-excitable cells, including ECs, a store-operated Ca2+ current (ICa) is considered to play a predominant role in controlling Ca2+ influx (47-49). Some laboratories have been able to detect this current (28, 50, 51), whereas others have not (52, 53). In ECs, the amplitude of ICa is typically very small (~ -0.1 pA/pF at VH = 0 mV), even in response to a non-physiological stimulus such as inositol 1,4,5-trisphosphate inclusion in the pipette (50). To detect ICa following {alpha}v{beta}3 integrin activation, intracellular solutions containing weakly buffered free Ca2+ and extracellular solutions containing high Ca2+ in the absence of any other membrane-permeable cations (solutions IV-D) were used. Under the same conditions, supra-physiological stimulation with TG activated an easily detectable ICa of -0.87 pA/pF (VH = -120 mV). However, no significant ICa could be detected in response to VN or BK, even though both agents produced small but sustained [Ca2+] increases. The VN and BK responses could reflect intracellular Ca2+ release only and/or weak activation of ICa below the detection threshold of whole cell current recording.

In conclusion, we report for the first time that engagement of {alpha}v{beta}3 integrin by an extracellular matrix protein leads to activation of an endothelial cell K+ current. The current has the characteristics of a KCa channel that is activated secondary to Ca2+ influx and/or release. Activation of whole cell KCa current following integrin engagement would hyperpolarize the endothelium, particularly in electrically coupled ECs in vivo, where the resting Em is relatively depolarized (54). This would sustain the plateau phase of the [Ca2+]i transient by enhancing the electrochemical driving force for Ca2+ (34-36). Thus, {alpha}v{beta}3 engagement by extracellular matrix proteins is predicted to regulate production of Ca2+-dependent EC vasoactive substances such as endothelium-derived hyperpolarizing factor (EDHF), nitric oxide, prostacyclin, and endothelin and thereby acutely modulate vascular tone (10-12). In this way, VN and other {alpha}v{beta}3 integrin ligands have the potential to acutely regulate the local circulation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL-60180 (to M. J. D.) and HL-59971 to (to G. E. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Medical Physiology, Rm. 346, Reynolds Medical Bldg., Texas A&M University College of Medicine, College Station, TX 77845. Tel.: 979-845-7819; Fax: 979-847-8635; E-mail: mjd{at}tamu.edu.

1 The abbreviations used are: VN, vitronectin; Ab, antibody; BK, bradykinin; BPAEC, bovine pulmonary artery endothelial cell; BAEC, bovine aortic endothelial cell; EC, endothelial cell; Erev, reversal potential; IBTX, iberiotoxin; KCa, Ca2+-activated K+; Im, membrane current; ICa, Ca2+ current; PBS, phosphate-buffered saline; TG, thapsigargin; VH, holding potential. Back


   ACKNOWLEDGMENTS
 
Judy Davidson gave extensive assistance in preparing and proofreading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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