Abstract
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 microg/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.
Highlights
Integrins are a large family of membrane-spanning, cell adhesion proteins composed of ␣ and  subunits, with over 18 ␣ and 8  subunits combining to form more than 24 different heterodimers [1, 2]
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 ␣v3 integrin
Localization of ␣v3 and ␣51 Integrin on BPAECs—Consistent with previous reports [5, 6], both ␣v3 and ␣51 integrins were expressed on BPAECs (Fig. 1). ␣v3 integrin was preferentially localized to focal adhesions at the tips of actin filaments (Fig. 1A), whereas ␣51 integrin tended to be distributed around the cell center (Fig. 1B), possibly at fibrillar adhesions
Summary
Integrins are a large family of membrane-spanning, cell adhesion proteins composed of ␣ and  subunits, with over 18 ␣ and 8  subunits combining to form more than 24 different heterodimers [1, 2]. Integrins play various roles in coordinating cell function, such as adhesion, spreading, and migration [3, 4]. ␣v3 integrin, is expressed both luminally and abluminally on endothelium [5, 6] and is thought to play an important role in several vascular pathologies [4, 7,8,9]. Soluble ␣v3 integrin ligands are capable of acutely regulating vascular tone. It has been suggested that soluble ␣v3 integrin ligands may acutely modulate blood flow by interacting with endothelial cell (EC) integrins [11, 12]. VN can potentially interact with unbound ␣v3 integrin, and plasma levels of VNcontaining complement complexes increase after complement activation. The rapid effects of integrin activation on EC [Ca2ϩ]i suggest that ion channels are involved in this response, and the purpose of the present study was to directly test this idea
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