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(Circulation Research. 1995;76:522-529.)
© 1995 American Heart Association, Inc.

Articles

Calcium Signaling in Endothelial Cells Involves Activation of Tyrosine Kinases and Leads to Activation of Mitogen-Activated Protein Kinases

Ingrid Fleming, Beate Fisslthaler, Rudi Busse

From Zentrum der Physiologie, Klinikum der JWG-Universität, Frankfurt/Main, Germany.

Correspondence to Dr Ingrid Fleming, Zentrum der Physiologie, Klinikum der JWG-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany.

	Abstract

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Abstract The activation of endothelial cells following exposure to a variety of receptor-dependent and -independent stimuli is associated with the release of Ca²⁺ from intracellular stores as well as the influx of Ca²⁺ from the extracellular space. In the present study, we investigated the interaction between Ca²⁺ signaling in cultured human umbilical vein endothelial cells and tyrosine phosphorylation. Stimulation of endothelial cells with either bradykinin (100 nmol/L), histamine (1 µmol/L), or the Ca²⁺-ATPase inhibitor thapsigargin (30 nmol/L) resulted in a slightly delayed but prolonged tyrosine phosphorylation of two low molecular weight proteins (42 and 44 kD). These proteins were identified by immunoprecipitation as the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase). The agonist-induced tyrosine phosphorylation of the 42-/44-kD doublet was sensitive to the tyrosine kinase inhibitors genistein (100 µmol/L) and piceatannol (10 µmol/L) and was inhibited by the removal of Ca²⁺ from the extracellular medium. In fura 2–loaded endothelial cells, inhibition of tyrosine kinases attenuated Ca²⁺ signaling after stimulation with either bradykinin (30 nmol/L) or thapsigargin (30 nmol/L). Since inhibition of tyrosine kinases specifically attenuates the plateau phase of the Ca²⁺ response after stimulation, the effect of tyrosine kinase inhibition appeared to be mostly associated with the influx of Ca²⁺ from the extracellular space. These data demonstrate that the signal transduction cascade initiated by receptor-dependent and -independent stimulation of endothelial cells includes the following: a tyrosine kinase inhibitor–sensitive transmembranous influx of Ca²⁺ and the tyrosine phosphorylation of two cytosolic protein substrates identified as MAP kinases. Furthermore, on one hand, an increase in [Ca²⁺]_i was essential for tyrosine phosphorylation; on the other, the Ca²⁺ influx was modulated by tyrosine phosphorylation. This finding documents the mutual dependence of these two crucial signaling pathways in endothelial cells.

Key Words: endothelial cells • mitogen-activated protein kinase • intracellular Ca²⁺ • genistein

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is generally accepted that the signal transduction cascade initiated after the activation of endothelial cells by a variety of receptor-dependent and -independent stimuli is associated with an elevation in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i), characterized by an initial transient peak followed by a steady or oscillating plateau phase.^{1 2} The initial transient component reflects, at least in part, the inositol 1,4,5-trisphosphate (IP₃)–mediated release of Ca²⁺ from intracellular stores, whereas the second phase is characterized by a more prolonged transmembranous Ca²⁺ influx.^{2 3} The Ca²⁺ influx pathway that replenishes intracellular Ca²⁺ stores is thought to be activated after store emptying by the subsequent release and/or activation of one or more diffusible signal molecules.^{4 5} However, the means by which this cross talk between the Ca²⁺ store and the plasma membrane is achieved in endothelial cells remains a mystery. Recently, the activation of tyrosine kinases has been described in different cell types after agonist-induced Ca²⁺ mobilization.^{6 7 8 9} Therefore, we investigated the possible implication of tyrosine kinases in Ca²⁺-dependent signaling in endothelial cells. We report in the present study that stimulation of endothelial cells with receptor-dependent and -independent agonists leads to a Ca²⁺-dependent tyrosine phosphorylation of two cytosolic proteins identified as the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase). Inhibition of protein tyrosine phosphorylation, using the specific inhibitors genistein and piceatannol, not only prevented phosphorylation of the 42-/44-kD doublet but also drastically reduced agonist-stimulated Ca²⁺ influx into human endothelial cells. These findings document for the first time the interdependence of these two signal transduction pathways in endothelial cells.

	Materials and Methods

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Materials
Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) was purchased from Boehringer Mannheim GmbH; pluronic F-127, from Molecular Probes; genistein, ionomycin, and fura 2-AM, from Calbiochem-Novabiochem GmbH; thapsigargin, from Research Biochemicals International; N-nitro-L-arginine and HEPES, from Serva; medium 119 (M-119), from GIBCO BRL; and penicillin, streptomycin, L-glutamine, glutathione, and L(+)-ascorbic acid (Biotect protection medium), from Biochrom. Bradykinin and all other substances were obtained from Sigma Chemical Co.

Cell Culture
Human umbilical vein endothelial cells, isolated from umbilical cords as previously described,¹⁰ were seeded either on glass coverslips (Bachofer) or in culture dishes (35 mm, Falcon) containing M-119 and 20% heat-inactivated fetal calf serum (Vitromex) supplemented with penicillin (50 U/mL), streptomycin (50 µg/mL), L-glutamine (1 mmol/L), glutathione (5 mg/mL), and L(+)-ascorbic acid (5 mg/mL). [Ca²⁺]_i was estimated in cells grown on coverslips for 24 hours.

Immunoblotting
Confluent primary cultures of human umbilical vein endothelial cells were washed twice in HEPES-Tyrode solution and incubated at 37°C with or without various receptor-dependent and -independent stimuli as described in "Results." Thereafter, cells were washed with ice-cold HEPES-Tyrode solution containing NaF (100 mmol/L), Na₄P₂O₇ (15 mmol/L), Na₃VO₄ (2 mmol/L), leupeptin (2 µg/mL), pepstatin A (2 µg/mL), trypsin inhibitor (10 µg/mL), and phenylmethylsulfonyl fluoride (PMSF, 44 µg/mL) and harvested by scraping. The cell suspension was centrifuged at 13 000g for 60 seconds. Cells contained in the pellet were then lysed in buffer containing 1% (vol/vol) Triton X-100, left on ice for 5 minutes, and then centrifuged at 10 000g for 10 minutes. Approximately 70 µg protein from the resulting supernatant was separated by either 8% or 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) (Minigel Apparatus, Bio-Rad) and transblotted onto nitrocellulose paper. After washing for 1 hour at room temperature in a buffer containing Tris (50 mmol/L, pH 7.5), NaCl (200 mmol/L), 0.2% Triton X-100, 3% bovine serum albumin, and 10% horse serum, blots were incubated with either a mouse monoclonal anti-phosphotyrosine antibody (1 µg/mL) (UBI) or a rabbit polyclonal anti–MAP kinase (erk-CT) antibody (1 µg/mL) (UBI) at 4°C for 18 hours in a buffer containing Tris (500 mmol/L, pH 7.5), NaCl (200 mmol/L), and 0.2% Triton X-100. Blots were subsequently washed and incubated with a peroxidase-conjugated anti-mouse or anti-rabbit antibody (Amersham) at room temperature for 1 hour, washed again, and stained by the addition of diaminobenzidine (0.5 mg/mL) and H₂O₂ (5 µL of a 30% stock solution) as previously described.¹¹ Prestained molecular weight marker proteins (Bio-Rad) and a positive-control protein preparation from A431 cells containing the phosphorylated epidermal growth factor receptor (UBI) were used as standards for SDS-PAGE and phosphotyrosine immunodetection, respectively. All of the bands recognized by the anti-phosphotyrosine antibody could be competitively displaced by phosphotyrosine (not shown).

Immunoprecipitation of Tyrosine-Phosphorylated Proteins
The Triton-soluble 10 000g supernatant from control and bradykinin-stimulated confluent primary cultures of human umbilical vein endothelial cells was prepared as described, and 100 µg protein was diluted in 500 µL RIPA buffer containing Tris/HCl at pH 7.5 (50 mmol/L), NaCl (150 mmol/L), EGTA (1 mmol/L), sodium desoxycholate (0.25%), Nonidet P-40 (1%), Na₃VO₄ (1 mmol/L), NaF (100 mmol/L), Na₄P₂O₇ (15 mmol/L), leupeptin (2 µg/mL), pepstatin A (2 µg/mL), trypsin inhibitor (10 µg/mL), and PMSF (44 µg/mL). Tyrosine-phosphorylated proteins were precipitated by shaking gently overnight at 4°C with 30 µL of an anti-phosphotyrosine antibody covalently linked to agarose (UBI). The agarose was then recovered by centrifugation, washed five times with RIPA buffer, resuspended in SDS–sample buffer, and boiled for 10 minutes. The resulting supernatant was analyzed by SDS-PAGE and Western blotting.

Measurement of [Ca²⁺]_i
For the measurement of [Ca²⁺]_i, endothelial cells were loaded with the fluorescent Ca²⁺-sensitive dye fura 2 by incubation with 3 µmol/L fura 2-AM and 0.025% (wt/vol) pluronic F-127 at 37°C for 90 minutes. Thereafter, the coverslips were washed in HEPES-modified Tyrode's solution of the following composition (mmol/L): NaCl 132, KCl 4, CaCl₂ 1, MgCl₂ 0.5, HEPES 9.5, and glucose 5. Coverslips were then mounted in a flow chamber, superfused with HEPES-Tyrode solution containing 0.3% bovine serum albumin, and placed on the stage of an inverted microscope (Diaphot-TMB, Nikon). [Ca²⁺]_i was determined fluorometrically by using continuous rapid alternating excitation from dual monochromators set at 340 and 380 nm (Deltascan, Photon Technology). Incident light passed through a filter block (BA 480, Nikon) and was focused onto the sample by means of an objective (Fluor 40, Nikon). Emitted light was collected by the objective, and fluorescence >480 nm was detected by a photon-counting photomultiplier (model D-104, Photon Technology). Photomultiplier digital output was collected by an IBM-compatible computer. At the end of each experiment, cells were superfused with a buffer containing Ca²⁺ (1.5 mmol/L) and ionomycin (1 µmol/L). After a stable 340/380 ratio was achieved (maximum, R_max), the buffer was changed to one containing EGTA (20 mmol/L) and ionomycin (1 µmol/L; minimum, R_min). The background signal was determined after the addition of MnCl₂ (10 mmol/L) to the perfusate, and [Ca²⁺]_i was calculated by assuming a dissociation constant for fura 2 at 37°C of 220 nmol/L and using the following relation as described by Grynkiewicz et al¹² :

Statistical Analysis
Unless otherwise indicated, data are expressed as mean±SEM. Statistical evaluation was performed by using Student's t test for unpaired data, one-way ANOVA followed by a Bonferroni t test, or ANOVA for repeated measures, where appropriate. Values of P<.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Bradykinin, Histamine, and Thapsigargin on Protein Tyrosine Phosphorylation in Human Endothelial Cells
Immunoblotting of protein from confluent primary cultures of human endothelial cells using a monoclonal anti-phosphotyrosine antibody revealed a relatively high basal level of apparently constitutively tyrosine-phosphorylated proteins (Fig 1).

View larger version (45K): [in this window] [in a new window]   Figure 1. Immunoblots (top) and bar graph (bottom) showing the time course of the bradykinin-stimulated increase in tyrosine phosphorylation of specific proteins in human umbilical vein endothelial cells. Cells were incubated in the presence and absence of bradykinin (100 nmol/L) at 37°C for the indicated times and then analyzed for tyrosine phosphorylation by immunoblotting as described. The quantification of tyrosine phosphorylation was performed by densitometric scanning of the blots, and the density of the 42-kD bands (open columns) and 44-kD bands (hatched columns) was normalized by comparison with the density of the 58-kD band, which was unaffected by the addition of bradykinin. The results are representative of four separate experiments.

Stimulation of endothelial cells with bradykinin (100 nmol/L) resulted in the time-dependent phosphorylation of two low molecular weight proteins (Fig 1). The smaller of the two proteins (42 kD) could be detected in unstimulated cells, but in bradykinin-stimulated cells no increase in tyrosine phosphorylation was evident after 30 seconds or 1 minute (not shown). However, a significant increase in tyrosine phosphorylation was detected 2 minutes after the addition of bradykinin. The slightly larger protein (44 kD), although not apparent in control cells, was clearly tyrosine-phosphorylated 5 minutes after the addition of bradykinin. Maximal phosphorylation of both proteins was detected after 5 minutes, was decreased slightly by 10 minutes, but was still detectable as long as the cells remained in contact with the agonist. Bradykinin-stimulated tyrosine phosphorylation was found to be reversible, since a time-dependent decrease of both bands was observed after agonist removal, with no phosphorylation being detected after 10 minutes (not shown).

Stimulation of endothelial cells with histamine (1 µmol/L, 5 minutes) and thapsigargin (30 nmol/L, 15 minutes) resulted in the tyrosine phosphorylation of two low molecular weight proteins (42- and 44-kD), which were identical to those phosphorylated after stimulation with bradykinin (Fig 2). Similar results were also obtained with ionomycin (0.5 µmol/L, 5 minutes; not shown).

View larger version (46K): [in this window] [in a new window]   Figure 2. Tyrosine phosphorylation of specific proteins by bradykinin, histamine, and thapsigargin in human umbilical vein endothelial cells. Cells were incubated in the presence and absence of bradykinin (100 nmol/L, 5 minutes), histamine (1µmol/L, 5 minutes), or thapsigargin (30 nmol/L, 15 minutes) at 37°C and then analyzed for tyrosine phosphoproteins by immunoblotting. The results are representative of three experiments.

Effect of Ca²⁺ Removal on Bradykinin-Stimulated Tyrosine Phosphorylation
Protein tyrosine phosphorylation of solvent-treated endothelial cells was unaltered by incubation for up to 15 minutes in Ca²⁺-free HEPES-Tyrode solution containing EGTA (0.2 mmol/L) (Fig 3). The bradykinin-stimulated tyrosine phosphorylation of the 42- and 44-kD proteins, on the other hand, was found to be transient and was markedly attenuated by the removal of extracellular Ca²⁺. Indeed the 42- and the 44-kD proteins could only barely be detected 5 minutes after the addition of bradykinin (Fig 3).

View larger version (64K): [in this window] [in a new window]   Figure 3. Effect of removal of extracellular Ca2+ on the time course of the bradykinin-stimulated increase in tyrosine phosphorylation of specific proteins in human umbilical vein endothelial cells. Cells were washed with a Ca2+-free HEPES-Tyrode solution containing EGTA (0.2 mmol/L), incubated in the presence and absence of bradykinin (100 nmol/L) at 37°C for the indicated times, and then analyzed for tyrosine phosphorylation by immunoblotting as described. The results are representative of three experiments.

Effect of Genistein and Piceatannol on Bradykinin-Stimulated Tyrosine Phosphorylation
Preincubation of endothelial cells with the tyrosine kinase inhibitor genistein (100 µmol/L, 15 minutes) was without effect on protein tyrosine phosphorylation in unstimulated cells (Fig 4). However, addition of bradykinin (100 nmol/L, 5 minutes) to genistein-treated cells did not result in tyrosine phosphorylation of either the 42- or 44-kD proteins. Similarly, piceatannol (10 µmol/L), a tyrosine kinase inhibitor that acts via a mechanism distinct from that of genistein, also prevented the bradykinin-stimulated tyrosine phosphorylation of the two proteins. However, incubation with piceatannol alone resulted in the appearance of at least two proteins of between 53 and 57 kD (Fig 4).

View larger version (73K): [in this window] [in a new window]   Figure 4. Effect of tyrosine kinase inhibitors on the bradykinin-stimulated increase in tyrosine phosphorylation in human endothelial cells. Cells were incubated in the presence and absence of bradykinin (100 nmol/L, 5 minutes) after pretreatment with either solvent (dimethyl sulfoxide, 2 hours), genistein (100 µmol/L, 15 minutes), or piceatannol (10 µmol/L, 2 hours). Tyrosine phosphorylation was assayed by immunoblotting as described. The results are representative of three separate experiments.

Effect of Bradykinin on MAP Kinase Phosphorylation in Human Endothelial Cells
Immunoblotting of protein from confluent primary cultures of human endothelial cells using a polyclonal anti–MAP kinase antibody revealed the presence of two MAP kinases in endothelial cells corresponding to molecular weights of 42 and 44 kD. Within 5 minutes of stimulation with bradykinin (100 nmol/L), a slight but distinct shift in the position of approximately one half of the 42-kD MAP kinase toward a lower mobility could be detected (Fig 5, left). This decrease in the mobility of the 42-kD protein presumably reflects the phosphorylation of the MAP kinase at threonine and tyrosine residues, which is required for enzyme activity.¹³

View larger version (58K): [in this window] [in a new window]   Figure 5. Identification of the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase) in control and bradykinin-stimulated human umbilical vein endothelial cells. Cells were incubated in the presence and absence of bradykinin (100 nmol/L, 5 minutes) at 37°C, and MAP kinase was detected by immunoblotting of cell extracts (left) or immunoprecipitates (right) of tyrosine-phosphorylated proteins. The results are representative of three experiments.

To substantiate the hypothesis that stimulation of endothelial cells with bradykinin results in the tyrosine phosphorylation of the 42- and 44-kD MAP kinases, tyrosine-phosphorylated proteins present in the cell extract were immunoprecipitated by using an anti-phosphotyrosine antibody. When immunoprecipitated protein from control cells was blotted with anti–MAP kinase antibody, only one band (44 kD) was visible, an observation that is consistent with a basal level of tyrosine phosphorylation under control conditions (Fig 5, right). After cell stimulation with bradykinin, the density of the 44-kD band increased at least fourfold, and a less densely stained band of 42 kD became clearly visible. Although the 44-kD MAP kinase was more readily immunoprecipitated than the 42-kD isoform, these results indicate that bradykinin does indeed stimulate the tyrosine phosphorylation of both the 42- and 44-kD MAP kinases.

Effect of Tyrosine Kinase Inhibition on Bradykinin-Stimulated Increases in [Ca²⁺]_i
The effect of tyrosine kinase inhibition on Ca²⁺ signaling was investigated in endothelial cells loaded with the Ca²⁺-sensitive dye fura 2. Superfusion of endothelial cells with bradykinin (30 nmol/L) resulted in a rapid and transient peak increase in [Ca²⁺]_i followed by a maintained elevation of [Ca²⁺]_i above resting values (Fig 6A). These observations are in accordance with previously reported data.^{1 2 14}

View larger version (20K): [in this window] [in a new window]   Figure 6. Graphs showing the effect of tyrosine kinase inhibition on the bradykinin-stimulated Ca2+ response in human endothelial cells. Fura 2–loaded cells were incubated with either solvent (dimethyl sulfoxide, 2 hours, ), genistein (100 µmol/L, 15 minutes and present throughout the experiment, ), or piceatannol (10 µmol/L, 2 hours, ). The increase in [Ca2+]i in response to bradykinin (100 nmol/L) was assayed in the presence of extracellular Ca2+ (A) and in Ca2+-free buffer containing EGTA (0.2 mmol/L) with the subsequent addition of 1 mmol/L Ca2+ (B). Results are presented as the mean±SEM of data obtained in five separate experiments.

The tyrosine kinase inhibitor genistein (100 µmol/L) was without effect on basal levels of [Ca²⁺]_i, with resting [Ca²⁺]_i being 87±15 nmol/L in control cells and 82±34 nmol/L in genistein-treated cells (P=NS, n=5). However, genistein (100 µmol/L) attenuated the peak increase in [Ca²⁺]_i after the addition of bradykinin (inhibition was 54±6%; P<.01, n=5) as well as the plateau phase of the Ca²⁺ response (inhibition was 62±3%; P<.01, n=5) as measured 12 minutes after the addition of bradykinin (Fig 6A).

In the absence of extracellular Ca²⁺, the bradykinin-stimulated increase in [Ca²⁺]_i was similar in solvent- and genistein-treated cells ([Ca²⁺]_i being 265.8±49.9 nmol/L and 262±15 nmol/L, respectively; P=NS, n=5) (Fig 6B). Addition of extracellular Ca²⁺ (1 mmol/L) to these cells caused a characteristic rapid increase in [Ca²⁺]_i, which was significantly attenuated in genistein-treated cells. After the inhibition of tyrosine kinases, the peak increase in [Ca²⁺]_i was inhibited by 71.3±4.6% (P<.001, n=5), and the plateau level of [Ca²⁺]_i 6 minutes after the addition of extracellular Ca²⁺ was attenuated by 60.6±12% (P<.05, n=5) (Fig 6B).

Pretreatment of endothelial cells with the tyrosine kinase inhibitor piceatannol (10 µmol/L) resulted in qualitatively the same effects as genistein on the bradykinin-stimulated Ca²⁺ response (Fig 6). Although the initial Ca²⁺ increase in response to bradykinin tended to be decreased in piceatannol-treated cells, this effect failed to attain statistical significance.

Effect of Tyrosine Kinase Inhibition on Thapsigargin-Stimulated Increases in [Ca²⁺]_i
Thapsigargin (30 nmol/L), which (independent of an increase in inositol phosphates) increases [Ca²⁺]_i by inhibiting the microsomal Ca²⁺-ATPase,^{15 16} induced a slightly delayed but long-lasting increase in endothelial [Ca²⁺]_i (Fig 7A). This response was significantly attenuated in genistein-treated cells, with [Ca²⁺]_i being 450±25 nmol/L in solvent-pretreated cells compared with 161±28 nmol/L in genistein-pretreated cells (P<.0001, n=5), as observed 12 minutes after the addition of thapsigargin (Fig 7A).

View larger version (19K): [in this window] [in a new window]   Figure 7. Graphs showing the effect of tyrosine kinase inhibition on the thapsigargin-stimulated Ca2+ response in human endothelial cells. Fura 2–loaded cells were incubated with either solvent (dimethyl sulfoxide, 15 minutes, ), genistein (100 µmol/L, 15 minutes and present throughout the experiment, ), or piceatannol (10 µmol/L, 2 hours, ). The increase in [Ca2+]i in response to thapsigargin (30 nmol/L) was assayed in the presence of extracellular Ca2+ (A) and in Ca2+-free buffer containing EGTA (0.2 mmol/L) with the subsequent addition of 1 mmol/L Ca2+ (B). Results are presented as the mean±SEM of data obtained in four separate experiments.

In the absence of extracellular Ca²⁺, the thapsigargin-stimulated increase in [Ca²⁺]_i was similar in solvent- and genistein-treated cells, with [Ca²⁺]_i being 81±12 and 80±4 nmol/L, respectively (P=NS, n=5) (Fig 7B). Addition of extracellular Ca²⁺ (1 mmol/L) caused a rapid increase in [Ca²⁺]_i, which was significantly attenuated by the inhibition of tyrosine kinases. In genistein-treated cells, the increase in [Ca²⁺]_i observed 8 minutes after the addition of extracellular Ca²⁺ was only 30.0±2.0% (P<.0001, n=5) of that detected in solvent-treated cells (Fig 7B).

Pretreatment of endothelial cells with the tyrosine kinase inhibitor piceatannol (10 mmol/L) also significantly attenuated the portion of the Ca²⁺ response to thapsigargin that is normally attributed to Ca²⁺ influx (Fig 7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study demonstrate that a common part of the signal transduction cascade initiated by receptor-dependent and -independent stimulation of endothelial cells includes a tyrosine kinase inhibitor–sensitive transmembranous influx of Ca²⁺ and the phosphorylation of two cytosolic protein substrates with apparent molecular weights of 42 and 44 kD. Tyrosine phosphorylation of the 42-/44-kD doublet would appear to be a phenomenon dependent on the presence of extracellular Ca²⁺, since phosphorylation was barely detectable after Ca²⁺ removal. By use of immunoprecipitation techniques, the 42- and 44-kD tyrosine-phosphorylated proteins were identified as MAP kinases. In addition, a bradykinin-induced shift in the electrophoretic mobility of at least the 42-kD MAP kinase was demonstrated.

The occurrence of phosphorylated tyrosine as a stable yet reversible modification of proteins in endothelial cells implies the existence of one or more protein tyrosine kinases and also of protein tyrosine phosphatases. The last 5 years has seen a vast increase in the number of systems in which tyrosine phosphorylation has been implicated (for review see Reference 17¹⁷ ). Similarly, the list of known tyrosine-phosphorylated substrate proteins has also expanded and now includes many proteins with known enzymatic activities or assumed functions, such as the 1 and 2 isoforms of phospholipase C (PLC), phosphatidylinositol 3-kinase, MAP kinase, the Ras GTPase–activating protein, and the putative nucleotide exchange factor Vav.¹⁸ Although endothelial cells have been reported to contain a modest level of tyrosine kinase activity,¹⁹ their involvement in the intracellular signal transduction cascade was thought to be restricted mainly to events instigated after the occupancy of growth factor receptors known to have inherent tyrosine kinase activity.¹⁹ This concept would now appear to be an underestimation of the role of tyrosine phosphorylation in cell signaling. Indeed, there is an increasing amount of evidence suggesting that after occupancy, G protein–coupled receptors, which are devoid of inherent protein tyrosine kinase activity, can in fact rapidly induce tyrosine phosphorylation of cellular substrate proteins and of the receptors themselves. For example, the mitogenic actions of endothelin involve a protein tyrosine kinase–based mechanism,²⁰ and angiotensin II and vasopressin are able to activate tyrosine kinase pathways to cause contraction in certain intact smooth muscle preparations.²¹ More recently, a fusion protein comprising the intracellular tail of the AT₁ receptor was found to be an excellent substrate for the src family of protein tyrosine kinases.²² This phenomenon, at least in the case of the AT₁ receptor, has been shown to be linked to the tyrosine phosphorylation of PLC-1 and to a subsequent increase in the formation of IP₃.²³ Since G protein–coupled receptors share a similar molecular architecture, this situation could also apply to the kinin and histamine receptors. Indeed, the C-terminal tail of the B₂-kinin receptor, which contains tyrosine residues susceptible to modification by phosphorylation,^{24 25} has recently been reported to be tyrosine-phosphorylated after occupancy.²⁶ The recruitment of a nonreceptor tyrosine kinase into the signal transduction pathway for a variety of agonists that act via receptors lacking intrinsic tyrosine kinase activity would therefore appear to be a more general phenomenon.

However, tyrosine phosphorylation of the kinin or histamine receptor in human endothelial cells is unlikely to be able to account for the effects observed in the present study, since the same two cytosolic proteins were also tyrosine-phosphorylated after cell stimulation with the receptor-independent agonist ionomycin and the Ca²⁺-ATPase inhibitor thapsigargin, both of which enhance Ca²⁺ influx by stimulating the store-regulated Ca²⁺ influx pathway.^{27 28} From the experiments using an antibody that recognizes the 43-, 42-, and 44-kD MAP kinases, encoded by the erk1, mapk, and mpk genes, we were able to show that the 42-/44-kD doublet corresponds exactly to the MAP kinases present in endothelial cells. Moreover, phosphotyrosine immunoprecipitation experiments revealed that bradykinin stimulation is indeed associated with an increased tyrosine phosphorylation of the 42- and 44-kD MAP kinases when compared with solvent-treated cells.

The tyrosine phosphorylation and activation of MAP kinase is, on the other hand, unlikely to be involved in the very early steps of signal transduction following cell activation with bradykinin, histamine, and thapsigargin. Rather, because of the dependence of tyrosine phosphorylation of the 42- and 44-kD proteins on extracellular Ca²⁺, it would appear that their activation occurs at a point downstream to the increase in [Ca²⁺]_i. This would fit with the observations that no increase in MAP kinase tyrosine phosphorylation was observed until 2 minutes after the addition of bradykinin and that maximum tyrosine phosphorylation was relatively delayed, being observed 5 minutes after cell stimulation. However, the increase in [Ca²⁺]_i was relatively rapid, with the peak increase being recorded 1 minute after bradykinin administration. Although our finding that an increase in [Ca²⁺]_i could indeed be sufficient to activate MAP kinase in endothelial cells was at first sight unexpected, it is supported by the observation that both the Ca²⁺ ionophore and thapsigargin have been shown to stimulate the 44- and 42-kD MAP kinase isoenzymes in human fibroblasts and epidermal carcinoma cells.²⁹ In the latter cell types, however, extracellular Ca²⁺ was not required to activate the MAP kinases, thus suggesting that activation required only the release of intracellular Ca²⁺. However, the Ca²⁺ dependency of MAP kinase activation is not a generally observed phenomenon, since an increase in [Ca²⁺]_i failed to activate MAP kinase in COS cells.³⁰

The most unexpected finding of the present study was the interdependence of Ca²⁺ and tyrosine phosphorylation. On one hand, an increase in [Ca²⁺]_i was essential for maintained tyrosine phosphorylation; on the other hand, the Ca²⁺ influx was modulated by tyrosine kinase inhibitors. In fura 2–loaded endothelial cells, the tyrosine kinase inhibitors genistein and piceatannol, which prevented the agonist-induced tyrosine phosphorylation, attenuated the Ca²⁺ response to bradykinin and thapsigargin. In the presence of extracellular Ca²⁺, both the peak and plateau phases of the Ca²⁺ response to these agonists were altered. However, the experiments performed in the absence of Ca²⁺ demonstrated that inhibition of tyrosine kinase activity selectively attenuated the plateau phase of the increase in [Ca²⁺]_i by 55%. The phenomenon of Ca²⁺ overshoot, which is observed after the addition of Ca²⁺ to cells stimulated in the absence of extracellular Ca²⁺, clearly shows that depletion of intracellular stores by agonists results in an increased permeability of the plasma membrane to Ca²⁺ and is consistent with previously published data.^{4 5} Since inhibition of tyrosine kinases specifically attenuates this portion of the Ca²⁺ response following stimulation of endothelial cells with either receptor-dependent or -independent agonists, the effect of tyrosine kinase inhibition appeared to be mostly associated with the influx of Ca²⁺ from the extracellular space. These findings suggest that not only are changes in [Ca²⁺]_i able to alter protein tyrosine phosphorylation but that tyrosine phosphorylation of specific proteins may be involved in the regulation of [Ca²⁺]_i. Since increases in Ca²⁺ lead to tyrosine phosphorylation and tyrosine phosphorylation leads to increases in [Ca²⁺]_i, it could be expected that a positive feedback would be rapidly established, resulting in a cellular Ca²⁺ overload. However, the level of phosphorylation of a protein reflects the ratio of its rates of phosphorylation and dephosphorylation, which are determined by the intrinsic activities of the kinase(s) and phosphatase(s) for which the protein is a substrate. Therefore, it is more than likely that a positive feedback on Ca²⁺ influx in endothelial cells is avoided by the concomitant bradykinin-induced activation of protein tyrosine phosphatase(s). Evidence to support the existence of such a control mechanism can be derived from experiments using a number of cell types, including cultured human endothelial cells (I. Fleming, unpublished observations), in which protein tyrosine phosphatase inhibitors have been found to instigate an increase in [Ca²⁺]_i.^{31 32}

Recently, there have been several reports linking agonist-induced Ca²⁺ mobilization with activation of tyrosine kinases in platelets,⁶ liver epithelial cells,⁹ and human foreskin fibroblasts.³³ In platelets, for example, tyrosine phosphorylation of a group of proteins was reported to be transiently elevated after a thrombin-stimulated increase in [Ca²⁺]_i,⁶ a phenomenon later shown to be closely correlated with the synthesis of phosphatidylinositol 3,4-bisphosphate.⁷ Thrombin-induced protein tyrosine phosphorylation was sensitive to the chelation of intracellular Ca²⁺ and could be mimicked by inhibiting the Ca²⁺-ATPase, either reversibly by decreasing temperature or irreversibly by the administration of thapsigargin.⁶ These observations hinted that, at least in platelets, the mobilization of intracellular Ca²⁺ stores favors tyrosine phosphorylation, whereas homeostatic levels of Ca²⁺ in storage compartments favor tyrosine dephosphorylation of specific proteins.⁶ In addition, protein tyrosine kinase inhibitors such as genistein have been reported to inhibit the thrombin-induced increase in platelet [Ca²⁺]_i and the subsequent aggregation.⁸ However, the involvement of tyrosine kinases in Ca²⁺ signaling is not necessarily a universal phenomenon, since tyrosine kinase activity is not required for the activation of Ca²⁺ influx pathways in all cell types.^{34 35 36}

In the present study, the two compounds that were used to inhibit protein tyrosine kinase activity have distinctly different mechanisms of action. While genistein inhibits protein tyrosine kinases by competing with ATP rather than the substrate, piceatannol specifically inhibits a number of tyrosine kinases, including p56lck, p60src, and the epidermal growth factor receptor by competing with the peptide or protein substrate binding site.³⁷ In unstimulated endothelial cells, genistein was without effect on the constitutively tyrosine-phosphorylated proteins, whereas incubation of the cells with piceatannol led to the detection of a band of proteins of 55 to 57 kD. This band is unlikely to represent a protein(s) that is activated by the compound per se but most probably represents an increase in the mobility of a larger cytosolic protein, as a consequence of its tyrosine dephosphorylation during the 2-hour incubation period with the tyrosine kinase inhibitor. There have been a number of concerns expressed regarding the specificity of genistein, which at concentrations >100 µmol/L, has been shown to inhibit protein kinase A. However, since the catalytic subunit of protein kinase A is apparently not affected by treatment with piceatannol³⁷ and since genistein and piceatannol produced essentially the same effects, it would appear that the effect of these compounds on agonist-stimulated Ca²⁺ influx is a consequence of their shared capacity to inhibit protein tyrosine kinase activity.

Comparison of the kinetics of agonist-induced increase in [Ca²⁺]_i with that of tyrosine phosphorylation suggests that the tyrosine-phosphorylated protein involved in regulating Ca²⁺ influx is in all probability not a MAP kinase. Rather, a more likely candidate would be a protein either attached to the membrane or whose presence is masked by the constitutively tyrosine-phosphorylated proteins detected in the cytosol of control cells. Moreover, the protein involved in regulating Ca²⁺ influx would be expected to be tyrosine-phosphorylated immediately after addition of the agonist, even in the absence of extracellular Ca²⁺, and to remain phosphorylated until store filling was complete. Such a tyrosine-phosphorylated protein has been described in platelets⁶ and has been tentatively identified as the microfilament-associated protein vinculin.³⁸ To date, however, there has been no description of such a tyrosine-phosphorylated protein in endothelial cells.

It is likely that the activation of tyrosine kinases represents only one link in the chain of events involved in regulating Ca²⁺ influx into endothelial cells. Indeed, a number of recent reports have suggested that the regulation of Ca²⁺ influx also involves serine-threonine kinases³⁹ and small G proteins.^{40 41} The involvement of such a kinase cascade in endothelial cells is suggested by our observation that okadaic acid and calyculin A, two potent serine-threonine phosphatase inhibitors,⁴² attenuated Ca²⁺ influx into human umbilical vein endothelial cells (I. Fleming, unpublished observation). Therefore, it would seem that the intracellular signal transmitted by empty Ca²⁺ stores to initiate a transmembranous Ca²⁺ influx involves a process in which the dephosphorylation of a serine-threonine–phosphorylated protein and the activation of a protein tyrosine kinase are both implicated. Although the kinase cascade involved has not been identified, there is a certain amount of circumstantial evidence to support the hypothesis that activation of the Ras-GTP/Raf pathway, which forms part of the kinase cascade upstream from the MAP kinase, is important in Ca²⁺ influx. For example, inhibition of GTP hydrolysis by introducing nonhydrolyzable guanine nucleotide analogues, such as GTPS,¹ into cells not only inhibits Ras but also inhibits thapsigargin-induced Ca²⁺ entry into mouse lacrimal acinar cells⁴⁰ and rat basophilic leukemia cells.⁴¹ In addition, it has been shown that GTP increased the size of the IP₃-sensitive Ca²⁺ store in digitonin-permeabilized hepatocytes.⁴³

In summary, we propose that the activation of tyrosine kinases forms part of the cascade of events linking the emptying of internal Ca²⁺ stores with the activation of the Ca²⁺ influx pathway in human endothelial cells. One hypothesis that could account for such findings would be that Ca²⁺ released from intracellular stores might activate a Ca²⁺-dependent tyrosine kinase(s), which could then in turn activate a Ca²⁺ influx pathway. In addition, our finding that the MAP kinase was tyrosine-phosphorylated, and presumably activated, in endothelial cells after an increase in [Ca²⁺]_i has wide-ranging implications for these cells, since MAP kinase recognizes many different substrates in the cell, including growth factor receptors, microtubule-associated proteins, specific serine-threonine protein kinases, phospholipase A₂, and transcription factors.⁴⁴ Although the involvement of the Ras-GTP/Raf/MAP kinase pathway in Ca²⁺ signaling is at the moment speculative, its activation after increases in endothelial [Ca²⁺]_i, such as that seen in response to shear stress,^{45 46} may open up a whole new field of research in endothelial cell physiology as a means of linking this mechanical signal to alterations in gene transcription.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Bu 436/4-3) and the Deutsche Gesellschaft für Herz- und Kreislaufforschung. The authors are indebted to Dr Markus Hecker for advice and discussion and to Isabel Winter and Annette Kirsch for expert technical assistance.

Received July 28, 1994; accepted December 13, 1994.

	References

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