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• W2138889204 abstract "Interactions between endothelial cells and extracellular matrix proteins are important determinants of endothelial cell signaling. Endothelial adhesion to fibronectin through αvβ3 integrins or the engagement and aggregation of luminal αvβ3 receptors by vitronectin triggers Ca2+ influx. However, the underlying signaling mechanisms are unknown. The electrophysiological basis of αvβ3 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 αvβ3 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 αvβ3 integrin. Interactions between endothelial cells and extracellular matrix proteins are important determinants of endothelial cell signaling. Endothelial adhesion to fibronectin through αvβ3 integrins or the engagement and aggregation of luminal αvβ3 receptors by vitronectin triggers Ca2+ influx. However, the underlying signaling mechanisms are unknown. The electrophysiological basis of αvβ3 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 αvβ3 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 αvβ3 integrin. Integrins are a large family of membrane-spanning, cell adhesion proteins composed of α and β subunits, with over 18 α and 8 β subunits combining to form more than 24 different heterodimers (1Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3739) Google Scholar, 2van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (789) Google Scholar). In the vascular system, integrins play various roles in coordinating cell function, such as adhesion, spreading, and migration (3Luscinskas F.W. Lawler J. FASEB J. 1994; 8: 929-938Crossref PubMed Scopus (264) Google Scholar, 4Eliceiri B.P. Circ. Res. 2001; 89: 1104-1110Crossref PubMed Scopus (308) Google Scholar). The vitronectin (VN) 1The 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. 1The 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. receptor, αvβ3 integrin, is expressed both luminally and abluminally on endothelium (5Conforti G. Dominguez-Jimenez C. Zanetti A. Gimbrone Jr., M.A. Cremona O. Marchisio P.C. Dejana E. Blood. 1992; 80: 437-446Crossref PubMed Google Scholar, 6Singh B. Fu C. Bhattacharya J. Am. J. Physiol. 2000; 278: L217-L226PubMed Google Scholar) and is thought to play an important role in several vascular pathologies (4Eliceiri B.P. Circ. Res. 2001; 89: 1104-1110Crossref PubMed Scopus (308) Google Scholar, 7Clemetson K.J. Cell. Mol. Life Sci. 1998; 54: 499-501Crossref Scopus (5) Google Scholar, 8Byzova T.V. Rabbani R. D'Souza S.E. Plow E.F. Thromb. Haemostasis. 1998; 80: 726-734Crossref PubMed Scopus (158) Google Scholar, 9Intengan H.D. Schiffrin E.L. Hypertension. 2000; 36: 312-318Crossref PubMed Scopus (351) Google Scholar). Soluble αvβ3 integrin ligands are also capable of acutely regulating vascular tone. For example, synthetic peptides containing the arginine-glycine-aspartic acid (RGD) sequence that binds αvβ3 integrin have been shown to block flow-induced, endothelium-dependent vasodilation in coronary arterioles (10Muller J.M. Chilian W.M. Davis M.J. Circ. Res. 1997; 80: 320-326Crossref PubMed Scopus (136) Google Scholar). It has therefore been suggested that soluble αvβ3 integrin ligands may acutely modulate blood flow by interacting with endothelial cell (EC) integrins (11Madden J.A. Christman N.J.T. Am. J. Physiol. 1999; 277: H2264-H2271PubMed Google Scholar, 12Bryan Jr., R.M. Marrelli S.P. Steenberg M.L. Schildmeyer L.A. Johnson T.D. Am. J. Physiol. 2001; 280: H2011-H2022Google Scholar). VN is a plasma glycoprotein first identified as a ligand of αvβ3 integrin and circulates at a concentration of 200-400 μg/ml in normal human plasma (13Germer M. Kanse S.M. Kirkegaard T. Kjoller L. Felding-Habermann B. Goodman S. Preissner K.T. Eur. J. Biochem. 1998; 253: 669-674Crossref PubMed Scopus (40) Google Scholar). VN can potentially interact with unbound αvβ3 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) (14Leavesley D.I. Schwartz M.A. Rosenfeld M. Cheresh D.A. J. Cell Biol. 1993; 121: 163-170Crossref PubMed Scopus (343) Google Scholar). Acute application of VN or cross-linking of αvβ3 integrin with the αvβ3 antibody, LM609, also increases EC [Ca2+]i (15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar). The [Ca2+]i increase occurs as a consequence of tyrosine phosphorylation of phospholipase C-γ1 following αvβ3 activation. It has been proposed that stimulation of αvβ3 integrin activates an unidentified Ca2+ influx pathway (14Leavesley D.I. Schwartz M.A. Rosenfeld M. Cheresh D.A. J. Cell Biol. 1993; 121: 163-170Crossref PubMed Scopus (343) Google Scholar, 15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar). Although integrins have been shown to regulate ion channels, including Ca2+ and K+ channels in other tissues (16Wildering W.C. Hermann P.M. Bulloch A.G.M. J. Neurosci. 2002; 22: 2419-2426Crossref PubMed Google Scholar, 17Lin B. Arai A.C. Lynch G. Gall C.M. J. Neurophysiol. 2003; 89: 2874-2878Crossref PubMed Scopus (80) Google Scholar, 18Kramar E.A. Bernard J.A. Gall C.M. Lynch G. J. Biol. Chem. 2003; 278: 10722-10730Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19Wu X. Mogford J.E. Platts S.H. Davis G.E. Meininger G.A. Davis M.J. J. Cell Biol. 1998; 143: 241-252Crossref PubMed Scopus (157) Google Scholar, 20Hofmann G. Bernabei P.A. Crociani O. Cherubini A. Guasti L. Pillozzi S. Lastraioli E. Polvani S. Bartolozzi B. Solazzo V. Gragnani L. Defilippi P. Rosati B. Wanke E. Olivotto M. Arcangeli A. J. Biol. Chem. 2001; 276: 4923-4931Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), 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. 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 (21Hamill O.P. Marty A. Neher E. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15035) Google Scholar) 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.Table IComposition of solutionsIntracellularExtracellularIIIIIIIVABCDNaCl136136140TEA aspartate140KCl40401405.95.9CsCl5.9NMDG140Potassium aspartate100100CaCl20.41.81.810MgCl211111.21.21.2EGTA51Glucose101010 Open table in a new tab Monoclonal antibodies against αvβ3 integrin (LM609; catalog number MAB1976Z) and α5β1 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). Fura-2 Microfluorimetry—Measurements of Fura-2 fluorescence were performed as described previously (22Sharma N.R. Davis M.J. Am. J. Physiol. 1996; 270: H267-H274PubMed Google Scholar). 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 (23Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). 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 αvβ3 or α5β1 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 (24Zar J.H. Biostatistical Analysis. 2nd Ed. Prentice-Hall, Inc., Englewood Cliffs, NJ1984: 196-198Google Scholar) 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. Localization of αvβ3 and α5β1 Integrin on BPAECs—Consistent with previous reports (5Conforti G. Dominguez-Jimenez C. Zanetti A. Gimbrone Jr., M.A. Cremona O. Marchisio P.C. Dejana E. Blood. 1992; 80: 437-446Crossref PubMed Google Scholar, 6Singh B. Fu C. Bhattacharya J. Am. J. Physiol. 2000; 278: L217-L226PubMed Google Scholar), both αvβ3 and α5β1 integrins were expressed on BPAECs (Fig. 1). αvβ3 integrin was preferentially localized to focal adhesions at the tips of actin filaments (Fig. 1A), whereas α5β1 integrin tended to be distributed around the cell center (Fig. 1B), possibly at fibrillar adhesions (25Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1785) Google Scholar). Both types of integrins were abundant on the surface of BPAECs under the conditions used in our experiments. 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. 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 αvβ3 Integrin Ab—To confirm that VN-activated current was mediated by αvβ3 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 α5β1 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 αvβ3 integrin. 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 αvβ3 integrin was required to initiate Ca2+ influx and [Ca2+]i increases (15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar). To test if aggregation of αvβ3 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 (26Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Crossref PubMed Google Scholar). 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). 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 (27Himmel H.M. Rasmusson R.L. Strauss H.C. Am. J. Physiol. 1994; 267: C1338-C1350Crossref PubMed Google Scholar), 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. 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 (28Kelly J.J. Moore T.M. Babal P. Diwan A.H. Stevens T. Thompson W.J. Am. J. Physiol. 1998; 274: L810-L819Crossref PubMed Google Scholar). Using solutions IV-D, in which Ca2+ was the primary charge carrier (28Kelly J.J. Moore T.M. Babal P. Diwan A.H. Stevens T. Thompson W.J. Am. J. Physiol. 1998; 274: L810-L819Crossref PubMed Google Scholar, 29Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Crossref PubMed Scopus (151) Google Scholar), 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 (30Schilling W.P. Rajan L. Strobl-Jager E. J. Biol. Chem. 1989; 264: 12838-12848Abstract Full Text PDF PubMed Google Scholar, 31Himmel H.M. Whorton A.R. Strauss H.C. Hypertension. 1993; 21: 112-127Crossref PubMed Scopus (233) Google Scholar), and thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump (28Kelly J.J. Moore T.M. Babal P. Diwan A.H. Stevens T. Thompson W.J. Am. J. Physiol. 1998; 274: L810-L819Crossref PubMed Google Scholar, 32Inesi G. Sagara Y. Arch. Biochem. Biophys. 1992; 298: 313-317Crossref PubMed Scopus (159) Google Scholar), 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 (15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar). LM609 alone did not significantly alter [Ca2+]i, as reported in the same study (15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar), 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 αvβ3 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 (31Himmel H.M. Whorton A.R. Strauss H.C. Hypertension. 1993; 21: 112-127Crossref PubMed Scopus (233) Google Scholar, 33Kawasaki J. Hirano K. Hirano M. Nishimura J. Fujishima M. Kanaide H. Eur. J. Pharmacol. 1999; 373: 111-120Crossref PubMed Scopus (15) Google Scholar). 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). A novel finding of this study is that the interaction between αvβ3 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 (34Peppelenbosch M.P. Tertoolen L.G. de Laat S.W. J. Biol. Chem. 1991; 266: 19938-19944Abstract Full Text PDF PubMed Google Scholar, 35Sharma N.R. Davis M.J. Am. J. Physiol. 1994; 266: H156-H164PubMed Google Scholar, 36Vaca L. Licea A. Possani L.D. Am. J. Physiol. 1996; 270: C819-C824Crossref PubMed Google Scholar). 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 αvβ3 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. (27Himmel H.M. Rasmusson R.L. Strauss H.C. Am. J. Physiol. 1994; 267: C1338-C1350Crossref PubMed Google Scholar) 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) (27Himmel H.M. Rasmusson R.L. Strauss H.C. Am. J. Physiol. 1994; 267: C1338-C1350Crossref PubMed Google Scholar). 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 (16Wildering W.C. Hermann P.M. Bulloch A.G.M. J. Neurosci. 2002; 22: 2419-2426Crossref PubMed Google Scholar, 17Lin B. Arai A.C. Lynch G. Gall C.M. J. Neurophysiol. 2003; 89: 2874-2878Crossref PubMed Scopus (80) Google Scholar, 18Kramar E.A. Bernard J.A. Gall C.M. Lynch G. J. Biol. Chem. 2003; 278: 10722-10730Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19Wu X. Mogford J.E. Platts S.H. Davis G.E. Meininger G.A. Davis M.J. J. Cell Biol. 1998; 143: 241-252Crossref PubMed Scopus (157) Google Scholar, 20Hofmann G. Bernabei P.A. Crociani O. Cherubini A. Guasti L. Pillozzi S. Lastraioli E. Polvani S. Bartolozzi B. Solazzo V. Gragnani L. Defilippi P. Rosati B. Wanke E. Olivotto M. Arcangeli A. J. Biol. Chem. 2001; 276: 4923-4931Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 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 (37Arcangeli A. Becchetti A. Del Bene M.R. Wanke E. Olivotto M. Biochem. Biophys. Res. Commun. 1991; 177: 1266-1272Crossref PubMed Scopus (14) Google Scholar), an effect most likely mediated by activation of KCa current. However, this effect was not completely abolished by strong intracellular Ca2+ buffering (38Becchetti A. Arcangeli A. Del Bene M.R. Olivotto M. Wanke E. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1992; 248: 235-240Crossref PubMed Scopus (49) Google Scholar); 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 β1 integrins was shown to induce membrane hyperpolarization by autocrine production of interleukin-4 (39Salter D.M. Robb J.E. Wright M.O. J. Bone Miner. Res. 1997; 12: 1133-1141Crossref PubMed Scopus (125) Google Scholar, 40Battistella-Patterson A.S. Wang S. Wright G.L. Can. J. Physiol. Pharmacol. 1997; 75: 1287-1299Crossref PubMed Scopus (28) Google Scholar). The hyperpolarization in that case was due to activation of apamin-sensitive, small conductance KCa channels and was blocked by pretreatment with β1 integrin Ab (41Millward-Sadler S.J. Wright M.O. Lee H. Nishida K. Caldwell H. Nuki G. Salter D.M. J. Cell Biol. 1999; 145: 183-189Crossref PubMed Scopus (145) Google Scholar). 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 (1Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3739) Google Scholar). 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) (42Prevarskaya N.B. Skryma R.N. Vacher P. Daniel N. Djiane J. Dufy B. J. Biol. Chem. 1995; 270: 24292-24299Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). 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 (43Ling S. Woronuk G. Sy L. Lev S. Braun A.P. J. Biol. Chem. 2000; 275: 30683-30689Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This enhancement was mediated by phosphorylation of residue Tyr-766 on the C-terminus of the channel's α-subunit. Interestingly, both phosphorylation of the channel and potentiation of current were more pronounced at high levels of intracellular Ca2+ (43Ling S. Woronuk G. Sy L. Lev S. Braun A.P. J. Biol. Chem. 2000; 275: 30683-30689Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). It is possible that the KCa current activated by VN-αvβ3 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 (43Ling S. Woronuk G. Sy L. Lev S. Braun A.P. J. Biol. Chem. 2000; 275: 30683-30689Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Integrin-mediated Ca2+ Signaling in ECs—Cheresh and coworkers (14Leavesley D.I. Schwartz M.A. Rosenfeld M. Cheresh D.A. J. Cell Biol. 1993; 121: 163-170Crossref PubMed Scopus (343) Google Scholar) 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. (15Bhattacharya S. Ying X. Fu C. Patel R. Kuebler W. Greenberg S. Bhattacharya J. Circ. Res. 2000; 86: 456-462Crossref PubMed Scopus (34) Google Scholar) demonstrated that both VN and cross-linking of αvβ3 integrin by LM609 increased [Ca2+]i in BPAECs. The [Ca2+]i increase occurred as a consequence of tyrosine phosphorylation of phospholipase C-γ1 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 αIIbβ3 integrin (44Fujimoto T. Fujimura K. Kuramoto A. J. Biol. Chem. 1991; 266: 16370-16375Abstract Full Text PDF PubMed Google Scholar). 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 (45Sjaastad M.D. Angres B. Lewis R.S. Nelson W.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8214-8218Crossref PubMed Scopus (47) Google Scholar, 46Sjaastad M.D. Lewis R.S. Nelson W.J. Mol. Biol. Cell. 1996; 7: 1025-1041Crossref PubMed Scopus (58) Google Scholar). 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 (47Parekh A.B. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 48Rosado J.A. Graves D. Sage S.O. Biochem. J. 2000; 351: 429-437Crossref PubMed Scopus (86) Google Scholar, 49Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weissgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Crossref PubMed Scopus (504) Google Scholar). Some laboratories have been able to detect this current (28Kelly J.J. Moore T.M. Babal P. Diwan A.H. Stevens T. Thompson W.J. Am. J. Physiol. 1998; 274: L810-L819Crossref PubMed Google Scholar, 50Fasolato C. Nilius B. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 69-74Crossref PubMed Scopus (95) Google Scholar, 51Sharma N.R. Davis M.J. Am. J. Physiol. 1995; 268: H962-H973PubMed Google Scholar), whereas others have not (52De Smet P. Oike M. Droogmans G. Van Driessche W. Nilius B. Pfluegers Arch. Eur. J. Physiol. 1994; 428: 94-96Crossref PubMed Scopus (15) Google Scholar, 53Fierro L. Lund P.E. Parekh A.B. Pfluegers Arch. Eur. J. Physiol. 2000; 440: 580-587PubMed Google Scholar). 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 (50Fasolato C. Nilius B. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 69-74Crossref PubMed Scopus (95) Google Scholar). To detect ICa following αvβ3 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 αvβ3 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 (54Daut J. Mehrki G. Nees S. Newman W.H. J. Physiol. 1988; 402: 237-254Crossref PubMed Scopus (49) Google Scholar). This would sustain the plateau phase of the [Ca2+]i transient by enhancing the electrochemical driving force for Ca2+ (34Peppelenbosch M.P. Tertoolen L.G. de Laat S.W. J. Biol. Chem. 1991; 266: 19938-19944Abstract Full Text PDF PubMed Google Scholar, 35Sharma N.R. Davis M.J. Am. J. Physiol. 1994; 266: H156-H164PubMed Google Scholar, 36Vaca L. Licea A. Possani L.D. Am. J. Physiol. 1996; 270: C819-C824Crossref PubMed Google Scholar). Thus, αvβ3 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 (10Muller J.M. Chilian W.M. Davis M.J. Circ. Res. 1997; 80: 320-326Crossref PubMed Scopus (136) Google Scholar, 11Madden J.A. Christman N.J.T. Am. J. Physiol. 1999; 277: H2264-H2271PubMed Google Scholar, 12Bryan Jr., R.M. Marrelli S.P. Steenberg M.L. Schildmeyer L.A. Johnson T.D. Am. J. Physiol. 2001; 280: H2011-H2022Google Scholar). In this way, VN and other αvβ3 integrin ligands have the potential to acutely regulate the local circulation. Judy Davidson gave extensive assistance in preparing and proofreading the manuscript." @default.
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• W2138889204 title "Regulation of Ca2+-dependent K+ Current by αvβ3 Integrin Engagement in Vascular Endothelium" @default.
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