Published as doi: 10.1096/fj.05-3923fje.
(The FASEB Journal. 2006;20:991-993.)
© 2006 FASEB
Signaling pathways mediating VEGF165-induced calcium transients and membrane depolarization in human endothelial cells
Nancy S. Dawson*,
David C. Zawieja*,1,
Mack H. Wu
and
Harris J. Granger*
* Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas, USA; and
Department of Surgery, U.C. Davis Medical Center, M.I.N.D. Institute, Sacramento, California, USA
1Correspondence: Lymphatic Biology Division, Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114 USA. E-mail: dcz{at}tamu.edu
SPECIFIC AIMS
Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and microvascular permeability and accelerates tumor growth and metastases. The aims of the present study were 1) to simultaneously image changes in intracellular calcium concentration ([Ca2+]i) and membrane potential in VEGF165 –stimulated human umbilical endothelial cells (HUVEC), and 2) to determine how specific signal transduction molecules and ion channels may modulate these transients.
PRINCIPAL FINDINGS
1. VEGF165 induces rapid changes in cytosolic calcium concentration and membrane potential in HUVEC.
Changes in intracellular calcium and membrane potential were monitored simultaneously in HUVEC using the fluorescent indicators, indo-1 AM and DiSBAC2(3), respectively. Application of VEGF165 (100 ng/ml) induces a rapid increase in [Ca2+]i), followed by a sustained plateau phase where [Ca2+]i, remains slightly elevated above baseline (Fig. 1
A). Membrane potential also shows a biphasic response. Initially, as [Ca2+]i increases, a slight, transient membrane hyperpolarization occurs, but as the [Ca2+]i peaks and then decays toward control, a strong depolarization develops, which is sustained (Fig. 1A
). Increases in [Ca2+]i may reflect release from internal calcium stores and/or influx from the extracellular space. We determined the relative contribution from each calcium compartment and evaluated the concomitant effect on membrane polarization. Pretreatment with thapsigargin (TG) depletes calcium stores so that no calcium transients are elicited by VEGF165, and depolarization is diminished to 
50%. Blockade of nonselective calcium entry channels (NSCC) with LaCl3 results in 40% inhibition of the calcium peak and abolishes the plateau. To further elucidate the role of specific calcium entry channels, we utilized the organic channel blockers, LOE-908 (which inhibits NSCC1 and NSCC2) and SKF96365 (which inhibits both store-operated calcium channels (SOC) and NSCC2). LOE-908 reduces the calcium peak to 35–45% of the control, abolishes the calcium plateau, and blocks depolarization by 50–60% (Fig. 1C
); SKF shows a similar calcium profile and blocks depolarization by 80% (Fig. 1D
).
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Figure 1. A) Simultaneous responses of cytosolic calcium and membrane potential in HUVEC following application of 100 ng/ml human recombinant VEGF165. The time of addition of VEGF165 is indicated by the vertical line. For comparison, control responses to VEGF165 obtained from Figure 1
A are represented by a dotted line without error bars in panels B–D. B) Effect of VEGFR-2 inhibition on calcium transients and membrane depolarization after application of 100 ng/ml VEGF165. C) Effect of pretreatment of HUVEC with LOE-908, the nonselective cation channel blocker (NSCC1 and NSCC2) on calcium and membrane potential responses to VEGF165. D) Effect of pretreatment of HUVEC with SKF96365, the store-operated and nonselective cation channel blocker (SOC and NSCC2), on calcium and membrane potential responses to VEGF165.
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2. Inhibitors of VEGFR2-receptor tyrosine kinase, src kinase, and inositol-triphosphate (IP3) eliminate both the calcium and membrane potential transients
Because many proangiogenic characteristics of VEGF are mediated through VEGFR2, specific inhibitors of four known molecules in the VEGF165-VEGFR2 signal transduction cascade [ras, phospholipase C
l (PLC l)-inositol phosphate (IP3), src kinase and phosphatidylinositol-3-kinase (PI3K)] were used to probe their involvement. We found that calcium and membrane potential transients are both eliminated with inhibitors to VEGFR2 receptor tyrosine kinase (Fig. 1B
), src kinase (PP2), and the IP3 receptor (IP3R) (2-APB). These data strongly suggest the sufficiency and necessity of VEGFR2 and the downstream requirement for src kinase and IP3-operated calcium channels to elicit the calcium and membrane potential transients. However, in the 10-min period of VEGF stimulation studied, we did not see evidence of immediate downstream ras signaling, since a farnesyltransferase inhibitor (FTI-277) (that blocks ras translocation to the plasma membrane), did not alter [Ca2+]i or membrane potential.
3. Inhibitors of PI3 kinase and chloride channels modulate membrane depolarization, in a calcium-independent manner
PI3K is known to directly associate with ligated VEGFR2. To test its involvement in the rise of [Ca2+]i and the shift in membrane potential, we used a PI3K-specific inhibitor, wortmannin, and found that it reduces membrane depolarization by 60% but does not significantly alter the calcium transient.
Because calcium currents are believed to be too small to explain the significant depolarization that we observed in VEGF165-activated cells (see Fig. 1B
), we explored the involvement of specific chloride channels. Pretreatment of HUVEC with the voltage-independent, chloride channel blocker, tamoxifen gave a similar profile as that observed with wortmannin.
CONCLUSIONS AND SIGNIFICANCE
To our knowledge, this is the first study to use calcium- and membrane-sensitive fluorescent dyes to investigate their dynamic and concomitant relationship in VEGF165-stimulated endothelial cells, and to use inhibitors to identify key signaling molecules that modulate these transients. Binding of VEGF165 to VEGFR2 initiates a complex signaling pathway, known to involve dimerization and phosphorylation of the tyrosine kinase receptor, recruitment of src-homology (SH)-2 domain-containing proteins, (PI3K and PLC1), elevation of [Ca2+]i, and calcium influx.
Calcium response
We show that VEGFR-2 activation significantly increases [Ca2+]i and shifts the resting membrane potential in HUVEC. A rise in [Ca2+]i can reflect release of Ca2+ from intracellular stores, entry of Ca2+ from the extracellular space, and the concerted effect of several calcium-sequestration mechanisms. We show that VEGF165 induces a biphasic increase in [Ca2+]i, consisting of an initial strong calcium peak that lasts for 2 min, followed by a sustained plateau phase that remains above baseline for the 10-min period of observation (Fig. 1A
). By using various inhibitors of calcium influx pathways, we show that the strong calcium peak represents a combination of both internal calcium stores release and calcium influx, since blockade with LaCl3, LOE-908, and SKF96365 all produce a reduction in the initial calcium peak. Depletion of calcium stores with TG or inhibition of stores release with 2-APB, completely blocks both the initial peak and the plateau phase of the calcium transient.
Calcium influx is believed to occur through capacitative calcium channels (CCE)/SOC and/or receptor-activated NSCC channels. Our experiments with LaCl3, LOE-908, SKF 96365 and 2-APB substantiate this entry pathway for calcium influx, and implicate SOC-like channels as the primary calcium influx pathway for refilling stores in VEGF165-stimulated cells.
Inhibitors of VEGFR-2 and the IP3R abolish stores release and calcium influx (Fig. 1B
), an inhibitor of src kinase abolishes calcium stores release, whereas, inhibitors of PI3K and chloride channels do not significantly alter stores release or calcium influx. VEGF165-stimulation of VEGFR2 tyrosine kinase activity is necessary for calcium signaling (Fig. 1B
). The studies using 2-APB show that stores release and calcium influx require IP3/IP3R signaling presumably through IP3R-gated channels in the endoplasmic reticulum, near the plasma membrane. The studies with PP2 suggest a dominant role for src kinase, a SH-2 domain-containing protein, in regulating internal calcium release, possibly through activating the PLC1-IP3-IP3R cascade.
The calcium transients implicate calcium entry, activated by stores-depletion in a manner that may open chloride channels, such as calcium-activated chloride channels (CaCC) and perhaps vol-regulated anion channels (VRAC). Such channels are thought to be involved in maintaining a polarized membrane and stabilizing the driving force for calcium influx. We show that inhibitors of chloride channels and PI3K do not directly alter the calcium transients but strongly influence the membrane potential.
Membrane depolarization
Potassium channels (including Ca2+-activated K+ channels, KCa, inward-rectifying K+ channels, Kir, and voltage-dependent K+ channels) are the major class of ion channels thought to determine membrane potential. Cultured, preconfluent HUVEC cells, like those used in our experiments, are reported to have a resting membrane potential of –27 to –52 mV. Additionally, chloride channels are also implicated as regulators of membrane potential in endothelial cells. Endothelial cells with more negative potentials are termed K+-type cells (expressing a more prominent hyperpolarization via potassium channels) and cells with less negative potentials are called Cl– type cells (expressing the involvement of chloride channels). Activation of VRAC and CaCC cause depolarization and are suggested to be modulators of the inward driving force for calcium, and NSCC are also proposed to "tune" the membrane potential in resting and activated cells.
VEGF165 stimulation clearly shifts the resting membrane potential of HUVEC (Fig. 1A
). Immediately after stimulation, a slight transient hyperpolarization develops, which is followed by a strong and sustained depolarization. The biphasic nature of the membrane potential profile is a "mirror-like image" of the concomitant, biphasic changes occurring in the calcium transients (Fig. 1A
) (i.e., where the slight hyperpolarization appears to be correlated with calcium release (peak) and the pronounced depolarization phase correlated with the sustained phase of calcium influx). We found that various inhibitors of calcium influx (Fig. 1C
and D) attenuate the membrane depolarization 50–80%, showing a definite calcium-influx-sensitive component to membrane depolarization. Inhibitors of the calcium stores release and influx pathway (e.g., VEGFR2/src kinase-/PLC1-IP3-IP3R) abolish the membrane depolarization completely, whereas inhibitors of PI3K and chloride channels attenuate the membrane depolarization by 50%. We propose that KCa are activated in response to the rise in [Ca2+]i, accounting for the initial membrane hyperpolarization, which provides a favorable electrochemical gradient for calcium entry and enhances the elevation of intracellular [Ca2+]i (peak). Depletion of calcium stores activates calcium entry through SOC channels, which shift the membrane potential toward depolarization, thereby stabilizing the driving force for calcium influx, and providing negative feedback for additional calcium entry. The precise mechanism for this is not understood; however, the experiments with LaCl3, SKF96365, PP2, 2-APB, and RTKI clearly show that a large rise in [Ca2+]i is strongly coupled to the depolarization. The prolonged depolarization may activate anion channels in the plasma membrane, such as CaCC and/or VRAC/ (I Cl, swell), or potentially by KCa.
Several experiments strongly suggest that some aspects of membrane depolarization are calcium-dependent and others are calcium-independent. Since calcium influx inhibitors (LOE 908 and SKF96365) attenuated the membrane depolarization, calcium influx may be responsible for the calcium-dependent portion of membrane depolarization. VEGF-mediated calcium influx is abolished, and the magnitude of depolarization is significantly reduced when internal calcium stores are depleted (TG or LaCl3). TG pretreatment (30 min) completely abolished the VEGF-induced calcium transients but only partially attenuated depolarization, suggesting a potential role for calcium-independent activation of ion channels such as NSCC, VRAC, or closure of Kir channels. The wortmannin and tamoxifen profiles for calcium and membrane potential also suggest that VEGF-induced membrane depolarization is regulated, in a similar manner. We propose that the PI3 kinase pathway may directly or indirectly activate VRAC, as we observed a partial depolarization from VEGF165 after pretreatment with wortmannin, without alteration of the VEGF-induced calcium transients.
In conclusion, we show that VEGF165 alters [Ca2+]i and membrane potential in HUVEC cells. VEGF induces a rapid peak in calcium followed by a sustained plateau above baseline, and these calcium changes are accompanied by a transient hyperpolarization and a sustained large depolarization, respectively. Figure 2
depicts our understanding of the integrated response of the endothelium to VEGF stimulation, and shows key signaling molecules that appear to regulate calcium and membrane potential transients.
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Figure 2. Schematic depiction of the integrated response of the endothelium to VEGF stimulation, showing key signaling molecules that appear to regulate calcium and membrane potential transients.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3923fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
