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Am J Physiol Heart Circ Physiol 277: H1061-H1068, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 3, H1061-H1068, September 1999

Calcium-calmodulin mediates bradykinin-induced MAPK phosphorylation and c-fos induction in vascular cells

Padma S. Naidu1, Victoria Velarde1, Christiana S. Kappler1, Roger C. Young2, Ronald K. Mayfield1, and Ayad A. Jaffa1,3

1 Departments of Medicine, 2 Obstetrics, and Gynecology, and 3 Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina and Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29425


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The vasoactive peptide bradykinin (BK) has been implicated in the pathophysiology of a number of vascular wall abnormalities, but the cellular mechanisms by which BK generates second messengers that alter vascular function are as yet undefined. Exposure of vascular smooth muscle cells (VSMC) to BK (10-7 M) produced a rapid and transient rise in intracellular calcium, which preceded an increase in tyrosine phosphorylation of mitogen-activated protein kinase (MAPK). MAPK activation by BK was observed as early as 1 min, peaked at 5 min, and returned to baseline by 20 min. Treatment of cells with the intracellular calcium chelator EGTA-acetoxymethyl ester inhibited BK-stimulated MAPK activation, suggesting that intracellular calcium mobilization contributes to the activation of MAPK. The calmodulin inhibitor W-7 also markedly inhibited BK-induced MAPK phosphorylation in the cytoplasm as well as in the nucleus. Moreover, the BK-induced increase in c-fos mRNA levels was significantly inhibited by the calmodulin inhibitor, indicating that calmodulin is required for BK signaling leading to c-fos induction. These results implicate the calcium-calmodulin pathway in the mechanisms for regulating MAPK activity and the resultant c-fos expression induced by BK in VSMC.

B2-kinin receptor; signal transduction; extracellular regulated kinases


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASOACTIVE NONAPEPTIDE bradykinin (BK) is the principal effector of the kallikrein-kinin system and has been implicated in the regulation of renal and cardiovascular function and vascular tone (25, 27). BK can be generated both systemically and locally within the vascular wall by the action of kallikrein on its kininogen substrate (29, 37, 38). Thus kinins could act in a paracrine or autocrine manner to influence vascular function. The physiological effects of BK are mediated via generation of second messengers such as nitric oxide and eicosanoids (19, 41). BK causes relaxation of vascular smooth muscle cells (VSMC) through synthesis and release of nitric oxide from the endothelium, but in states of vascular injury in which endothelial integrity is compromised, BK can act directly on VSMC to increase intracellular calcium and cause contraction (5).

Evidence from pharmacological and molecular cloning studies indicate that the BK B2 receptor is a member of the seven transmembrane G protein-coupled receptor superfamily (26). In VSMC activation of the B2-kinin receptor by BK stimulates phospholipase C activity via a heterotrimeric GTP-binding protein, leading to the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol, both of which are involved in intracellular calcium mobilization and activation of protein kinase C (8). Through a B2 receptor, BK has also been shown to stimulate VSMC proliferation and matrix formation, stimulate mitogen-activated protein kinase (MAPK) activation and nuclear translocation, induce expression of protooncogenes c-fos and c-jun and formation of the AP-1 complex (16, 44).

MAPKs also known as extracellular signal-regulated kinases belong to the group of serine-threonine kinases that are rapidly activated in response to growth-factor stimulation. They integrate multiple signals from various second messengers leading to cellular proliferation or differentiation (33, 42). The activated MAPK can regulate the expression of transcription factors such as c-fos through the phosphorylation of the transcription factor p62TCF (12). Induction of the protooncogene c-fos results in a complex formation with c-jun, which binds the AP-1 site, thus regulating the transcription of genes containing this element (18). Rises in intracellular calcium have also been shown to regulate the activity of MAPK and to induce the expression of c-fos in neuronal cells (34, 40). Some of the mechanisms through which elevated intracellular calcium modulates cellular events are through its association with calmodulin, forming a calcium-calmodulin complex that binds to target proteins and regulate their function (17).

One of the earliest BK postreceptor signals is elevation in intracellular calcium. The relationship of the rise in intracellular calcium elicited by BK on cell signaling mechanisms leading to MAPK activation and c-fos induction is not defined. Therefore, in the present study, we investigated the role of intracellular calcium and calmodulin in BK stimulation of MAPK activation and c-fos mRNA expression in VSMC. We observed that BK stimulates p42mapk and p44mapk activation in the cytoplasm and nucleus via a calcium-calmodulin-dependent mechanism. Induction of c-fos mRNA expression by BK was also mediated via calmodulin. These results point to an important function of the calcium-calmodulin complex on the cellular responses initiated by BK in VSMC.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Rat aortic VSMC from male Sprague-Dawley rats (Charles-River Laboratories, Wilmington, MA) were prepared by a modification of the method of Majeck and Clowes (24). A 2-cm segment of artery cleaned of fat and adventitia was incubated in 1 mg/ml collagenase for 3 h at room temperature. The artery was then cut into small sections and fixed to a culture flask for explantation in minimal essential media containing 10% fetal calf serum (FCS), 1% nonessential amino acids, 100 mU/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2. Medium was changed every 3-4 days, and cells were passaged every 6-8 days by harvesting with trypsin-EDTA. Cell viability was assessed by standard dye exclusion techniques using 1% trypan blue. VSMC were identified by the following criteria. They stained positive for intracellular cytoskeletal fibrils of actin and smooth muscle cell specific myosin (indicative of contractile cell) and negative for factor VIII antigens. VSMC isolated by this procedure were homogeneous and were used in all studies between passage 2 and 4.

Measurement of intracellular calcium concentration by fura 2. VSMC grown to confluency onto round glass coverslips were rendered quiescent by growing them in serum-free media for 2 days. For fura 2 loading, cells were incubated at 37°C for 30 min in DMEM containing 5 µM of fura 2-acetoxymethyl ester (AM; Molecular Probes, Eugene, OR). The cells were then washed in bathing solution 1 containing the following composition (in mM): 140 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES-Na, pH 7.4. In some experiments, EGTA and fura 2 were simultaneously loaded into the cells by coincubation of 50 µM EGTA-AM and 5 µM fura 2-AM at 37°C for 30 min. The coverslips were then mounted in the flow chamber, and the intracellular calcium concentration ([Ca2+]i) was determined fluorometrically by using an Attofluor Ratio Vision fluorescence microscope with a ×40 objective (Atto Instruments, Rockville, MD). Emission intensities at wavelengths greater than 520 nm were obtained by alternatively exciting the fura 2 at 360 and 380 nm at 1 Hz. For each experimental run, the ratio of intensities was obtained in 10-20 regions of interest. Relative concentrations of intracellular calcium were calculated from the ratio of emission intensity using the 360 and 380 nm excitation wavelength. Reported responses were normalized to baseline fluorescence for each region of interest.

Cytosolic and nuclear extraction of proteins. Cytosolic and nuclear proteins were extracted from VSMC by the technique of Andrews and Faller (2). Quiescent VSMC grown in 10-cm dishes were stimulated with BK (10-7 M) for various times. The cells were suspended in 250 µl of lysis buffer {20 mM Tris, 130 mM NaCl, 10% glycerol, 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM Na vanadate, 100 mU/ml aprotinin, 0.15 mg/ml benzamidine, pH 8.0}, sonicated for 10 s and centrifuged at 13,000 g for 10 min. The supernatant was harvested as the cytosolic fraction. The pellet fraction was suspended in 500 µl of cold nuclear lysis buffer (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF), incubated on ice for 20 min, and centrifuged for 15 min at 4°C. The protein concentration in the nuclear and cytosolic fractions were determined by the Lowry method (22).

Western blotting of MAP kinases. To measure MAPK activity in cytosol and nuclear fractions, 20-25 µg of soluble protein obtained as previously described was subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins in the gel were transferred to polyvinylidene difluoride membrane and immunoblotted with rabbit polyclonal phospho-specific MAPK antibodies (1:1,000 dilution, New England Biolabs, Beverly, CA) that detect p42mapk and p44mapk. Total MAPK was measured in the same membranes by stripping the membrane and immunoblotting with anti-MAPK antibodies (1:6,000 × dilution). The immunoreactive tyrosine phosphorylated MAPK and total MAPK were detected by the CDP-Star chemiluminescent system (New England Biolabs).

RNA extraction and Northern blotting. Quiescent VSMC grown in 15-cm plates were stimulated with BK (10-7 M) for 30 min. Total RNA from the cells was extracted by the chloroform-phenol method (7). RNA yield was determined spectrophotometrically (Ultrospec III, Pharmacia) by absorbance at 260 nm.

Total RNA (20 µg), obtained as previously described, was denatured at 65°C for 15 min and ran on a 1.5% agarose gel. The gel was stained with ethidium bromide to determine the position in each lane of the 28S and 18S ribosomal RNA and to demonstrate that similar amounts of intact RNA were used for each sample. Total RNA was transferred from the gel to Nytran membrane filters by a Possiblot Pressure Transducer (Stratagene, La Jolla, CA) and hybridized at 60°C for 18-24 h, with nick translated cDNA probes labeled with 32P using a nick-translation kit (Bethesda Research Laboratories, Bethesda, MD). The hybridized membranes were washed and exposed to film. Autoradiographs (Kodak XAR-5 film, Eastman Kodak, Rochester, NY) of the membranes were obtained and scanned, and the intensities of the bands were quantified by a Joyce Loebl microdensitometer.

Statistical analysis. Data are expressed as means ± SE and were analyzed by analysis of variance for repeated measures and by using the Student's t-test for unpaired analysis. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of MAPK by BK. Treatment of VSMC with 10-7 M BK resulted in a time-dependent increase in tyrosine phosphorylation of p42mapk and p44mapk, compared with unstimulated cells (Fig. 1). MAPK phosphorylation was detectable as early as 1 min, peaked at 5 min, and declined to basal values by 20 min. BK treatment produced a seven- to eightfold increase in MAPK phosphorylation relative to basal activity. In subsequent experiments, MAPK measurements were carried out with BK stimulation for 5 min.


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  		Fig. 1.   Bradykinin (BK) stimulates mitogen-activated protein kinase (MAPK) phosphorylation in vascular smooth muscle cells (VSMC). VSMC were treated with BK (10-7 M) for indicated times. MAPK phosphorylation in cell lysate was measured by immunoblots using anti-phosphotyrosine-MAPK antibodies (PY-MAPK). Total MAPK was measured in same immunoblot by stripping membrane and reincubating with anti-total MAPK antibodies. Blots shown are representative of 5 separate experiments. Bar graph represents intensities of both p42mapk and p44mapk bands measured in a densitometer relative to total MAPK and expressed as percent phosphorylation relative to control. * P < 0.001 vs. control.

Calcium and calmodulin role in BK-induced MAPK activation. VSMC preloaded with fura 2 were monitored for changes in intracellular calcium after exposure to BK (10-7 M) in normal and calcium-free medium. BK evoked a rapid and transient rise in intracellular calcium that peaked at 1-30 s and gradually returned to baseline within 1 min (Fig. 2). This rise in intracellular calcium by BK occurred despite the removal of extracellular calcium from the media, suggesting that transmembrane calcium influx is not necessary for the rise in intracellular calcium. In support of this finding, preincubation of VSMC with the intracellular calcium chelator EGTA-AM (50 µM) for 30 min completely prevented the rise in intracellular calcium by BK.


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  		Fig. 2.   Effect of EGTA-acetoxymethyl ester (AM) on BK-induced rise in intracellular calcium concentration ([Ca2+]i). Fura 2-loaded VSMC were stimulated with BK (10-7 M) in presence (dotted line) and absence (solid line) of EGTA-AM (50 µM). BK was added at 60 s. Fluorescence ratio is ratio of emission intensities obtained using excitation wavelengths of 360 and 380 nm, normalized for each cell. Each tracing represents an average obtained from 10 cells within a single experiment. Graph is representative of at least 4 separate experiments.

To determine whether the rise in intracellular calcium elicited by BK plays a role in MAPK activation, VSMC were pretreated with 50 µM EGTA-AM for 30 min followed by BK stimulation for 5 min. In the absence of EGTA-AM, BK produced a sevenfold increase in MAPK phosphorylation compared with unstimulated cells. However, pretreatment of VSMC with EGTA-AM inhibited the BK-induced MAPK phosphorylation by about 50% (Fig. 3A). EGTA-AM alone did not effect basal MAPK phosphorylation, nor did it influence BK-induced total tyrosine phosphorylation. To rule out a role for influx of calcium through L-type-mediated calcium channels in BK-induced MAPK activation, we treated VSMC with 1 µM nifedipine, followed by BK stimulation. Blockade of L-type calcium channels with nifedipine failed to inhibit BK-induced p42mapk and p44mapk phosphorylation (Fig. 3B). We also carried out experiments to study the effects of BK on MAPK activation in calcium-free medium to determine whether extracellular calcium is required for MAPK activation. The results show similar activation of MAPK by BK in calcium-containing as well as calcium-free media (Fig. 3C). Furthermore, addition of EGTA (3 mM) to the extracellular media did not alter MAPK activation in response to BK (Fig. 3C). Taken together, these data indicate that the rise in intracellular calcium, rather than calcium influx, contributes significantly to BK-induced MAPK activation in VSMC. The failure to completely block BK-induced MAPK phosphorylation by EGTA-AM, in the face of complete elimination of the rise in intracellular calcium, indicates that BK promotes MAPK activation via calcium-dependent as well as calcium-independent mechanisms.


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  		Fig. 3.   Role of intracellular calcium in BK-induced MAPK activation. VSMC were pretreated with EGTA-AM (50 µM) for 30 min (A), nifedipine (1 µM) for 1 min (B), and/or in extracellular calcium-free medium ± EGTA, followed by BK (10-7 M) stimulation for 5 min (C). MAPK phosphorylation in the cell lysate was measured by immunoblots using anti-phosphotyrosine-MAPK antibodies. Total MAPK was measured in same immunoblot by stripping membrane and reincubating with anti-total MAPK antibodies. Blots are representative of 6 separate experiments. Bar graphs represent intensities of both p42mapk and p44mapk bands measured in densitometer relative to total MAPK and expressed as percent phosphorylation relative to control. * P < 0.01 vs. control. # P < 0.01 vs. BK.

In a separate experiment we investigated the effects of EGTA-AM on MAPK phosphorylation by agents that do not elicit release of intracellular calcium. VSMC were treated with phorbol 12-myristate 13-acetate (PMA 5 µM) for 5 min, in the presence and absence of EGTA-AM. PMA resulted in a 3.54 ± 0.35-fold increase in MAPK activation compared with unstimulated controls. A similar increase (3.71 ± 0.30) in MAPK activation was observed in response to PMA in the presence of EGTA-AM. This finding suggests that the inhibitory effect of EGTA-AM on MAPK activation is specific for agonists that stimulate rise in intracellular calcium such as BK.

Rises in intracellular free calcium concentration result in the reversible formation of a calcium-calmodulin complex. A major mechanism for calcium signal transduction is via activation of calmodulin and calmodulin-dependent protein kinase II. To evaluate the requirement for calmodulin in the activation of MAPK in response to BK, VSMC were pretreated for 45 min with a calmodulin inhibitor W-7 (30 µM). The inhibitor was used at concentrations that achieved half-maximal inhibition of calmodulin (13). Once again, BK produced a marked increase in tyrosine phosphorylation of MAPK. This was inhibited by W-7 to a level not significantly different from unstimulated control cells (Fig. 4A). Another study was also carried out to assess the effects of a second calmodulin inhibitor, calmidazolium, on BK-induced MAPK phosphorylation. Treatment of VSMC with BK (10-8 M) resulted in a 2.45 ± 0.16-fold increase in MAPK phosphorylation compared with unstimulated controls (P < 0.006). However, in the presence of calmidazolium (10 µg), the increase in MAPK activation in response to BK was reduced to 1.81 ± 0.13 (P < 0.03 BK vs. BK-calmidazolium, respectively, n = 3).


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  		Fig. 4.   Role of calmodulin in BK-induced MAPK activation. VSMC were pretreated for 45 min with calmodulin inhibitor W-7 (30 µM, A) and/or calmodulin kinase II inhibitor KN-93 (30 µM, B), followed by either BK or angiotensin II (ANG II) stimulation (10-7 M) for 5 min. MAPK phosphorylation in the cell lysate was measured by immunoblots using anti-phosphotyrosine-MAPK antibodies. Total MAPK was measured in same immunoblot by stripping the membrane and reincubating with anti-total MAPK antibodies. Blots shown are representative of 5 separate experiments. Bar graph represents intensities of both p42mapk and p44mapk bands measured in a densitometer relative to total MAPK and expressed as percent phosphorylation relative to control. * P < 0.005 vs. control. # P < 0.005 vs. BK. dagger  P < 0.005 vs. ANG II.

We next explored the role of the calmodulin kinase II inhibitor KN-93 (30 µM) on MAPK activation in response to BK (Fig. 4B). VSMC pretreated with the calmodulin kinase II inhibitor KN-93 for 30 min were followed by stimulation with either BK (10-7 M) or angiotensin II (ANG II, 10-7 M) for 5 min. MAPK activation in response to ANG II has been shown to involve activation of calcium-calmodulin protein kinase II pathway (1). The results shown in Fig. 4 demonstrate that both BK and ANG II produced a significant increase in MAPK phosphorylation compared with unstimulated cells (P < 0.001, n = 6). This increase in MAPK phosphorylation in response to either BK and/or ANG II was completely abolished in the presence of the calmodulin kinase II inhibitor KN-93 (Fig. 4). KN-93 had no significant effect on basal phosphorylation of MAPK. These findings suggest that MAPK activation by BK seems to occur via a calmodulin-calmodulin kinase II-dependent mechanism.

Nuclear phosphorylation of MAPK by BK is calmodulin dependent. The nuclear targets for MAPK include the phosphorylation of Elk-1/TCF, which in turn leads to transcriptional activation at the serum response element and induction of c-fos mRNA levels (12). For MAPK to influence c-fos expression, it requires translocation to the nucleus. To assess the role of calmodulin in BK-induced nuclear phosphorylation of MAPK, we measured the tyrosine phosphorylation of MAPK in the cytosol and nuclear extracts of VSMC treated with 10-7 M BK, in the presence and absence of calmodulin inhibitor W-7. Treatment of VSMC with BK for 5 min resulted in a marked increase in tyrosine phosphorylation of MAPK in both cytosolic and nuclear fractions (Fig. 5). Pretreatment of VSMC with W-7 significantly decreased both cytosolic and nuclear MAPK phosphorylation in response to BK. Phosphorylated p42mapk and p44mapk in the cytosol of W-7-treated cells were reduced by approximately 50% compared with BK-stimulated cells. Nuclear phosphorylation of p42mapk and p44mapk in response to BK was also significantly reduced by the calmodulin inhibitor.


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  		Fig. 5.   BK-induced activation of cytosolic and nuclear MAPK is blocked by calmodulin inhibitor. VSMC were pretreated for 45 min with calmodulin inhibitor W-7 (30 µM) followed by BK stimulation (10-7 M) for 5 min. MAPK phosphorylation in cytoplasm and nuclear extracts was measured by immunoblots using anti-phosphotyrosine-MAPK antibodies. Total MAPK was measured in same immunoblot by stripping membrane and reincubating with anti-total MAPK antibodies. Blot is representative of 4 separate experiments. Bar graph represents intensities of both p42mapk and p44mapk bands in cytoplasm and nucleus measured in densitometer relative to total MAPK and expressed as percent phosphorylation relative to control. * P < 0.006 vs. control. # P < 0.006 vs. BK.

BK-induced c-fos mRNA expression is regulated by calmodulin. To evaluate whether the induction in c-fos mRNA levels by BK are calmodulin dependent, we measured c-fos mRNA levels in VSMC pretreated with W-7 for 45 min, followed by BK (10-7 M) stimulation for 30 min. As shown in Fig. 6, c-fos mRNA levels were undetectable at 0 min but were markedly induced within 30 min of BK stimulation. However, in the presence of the calmodulin inhibitor W-7, the induction of c-fos mRNA by BK was significantly suppressed (6,996 ± 470 vs. 4,235 ± 452 densitometric units; BK vs. BK-W-7, respectively, P < 0.002). beta -actin mRNA levels measured in the same cells were not altered by any of the treatments (7,515 ± 1,330, 10,013 ± 288, 6,869 ± 444, 7,490 ± 181, densitometric units, control vs. BK vs. W-7 vs. W-7-BK, respectively, Fig. 6).


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  		Fig. 6.   Effect of calmodulin inhibitor on BK-induced c-fos expression. VSMC were pretreated for 45 min with calmodulin inhibitor W-7 (30 µM) followed by BK stimulation (10-7 M) for 30 min. RNA was extracted from cells, and c-fos and beta -actin mRNA levels were measured by Northern blot analysis. Bar graph represents relative intensities of c-fos mRNA levels/beta -actin mRNA levels. Blot shown is representative of at least 4 separate experiments. * P < 0.05 vs. control. dagger  P < 0.05 vs. BK.

The effects of EGTA-AM on BK-induced c-fos mRNA levels were also examined. VSMC were pretreated with 50 µM EGTA-AM for 30 min followed by BK (10-7 M) stimulation for 30 min. Once again, basal c-fos mRNA levels were undetectable in control unstimulated cells (0.19 ± 0.07, c-fos mRNA/beta -actin mRNA, n = 5) but were markedly induced in response to BK stimulation (1.51 ± 0.05, c-fos mRNA/beta -actin mRNA, P < 0.001, BK vs. control, respectively). However, in the presence of EGTA-AM, the increase in c-fos mRNA induced by BK was reduced by about 35% (1.12 ± 0.23, c-fos mRNA/beta -actin mRNA, n = 5). This reduction in c-fos mRNA by EGTA-AM tended to but was not statistically significant (P < 0.06, BK vs. BK-EGTA-AM). EGTA-AM had no significant effect on basal c-fos mRNA levels (0.18 ± 0.05, c-fos mRNA/beta -actin mRNA, n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The findings of the present study demonstrate that activation of MAPK and induction of c-fos in response to BK challenge in VSMC involve a rise in intracellular calcium and activation of the calcium-calmodulin complex. The peak activation of MAPK by BK was observed at 5 min, whereas the rise in intracellular calcium elicited by BK peaked within 30 s and declined to baseline by 60 s. EGTA-AM binding of calcium and the calmodulin inhibitor did not entirely block MAPK activation, indicating that intracellular calcium rise and calmodulin activation is only one of the upstream second messengers utilized by BK to activate MAPK in VSMC. In the present studies, we have also shown the induction of c-fos by BK involves activation of MAPK by a calcium-calmodulin-dependent pathway.

One of the earliest postreceptor events in BK transmembrane signaling is elevation in intracellular calcium. Our data suggest that this [Ca2+]i rise produced by BK in VSMC probably occurs through release of calcium from intracellular stores. From other available data, it is likely that the BK B2 receptor initiates calcium release following activation of phospholipase C-beta through pertussis toxin-insensitive Gq, which converts phosphatidylinositol [4-5]-disphosphate into Ins(1,4,5)P3 and diacylglycerol (20). Ins(1,4,5)P3 in turn can act as an intracellular second messenger by binding to specialized tetrameric Ins(1,4,5)P3 receptors that span the endoplasmic reticular membrane to trigger release of calcium from the endoplasmic reticulum (3). Support for this mechanism comes from our finding that removal of extracellular calcium did not abrogate the rise in [Ca2+]i in response to BK. Studies from other cells show that the peak for Ins(1,4,5)P3 formation following BK exposure occurs within 15 to 20 s, corresponds to or precedes the rapid and transient release of calcium (14, 31).

A role for calcium as an intracellular signal mediating proliferation induced by various growth factors in different cell types has been established (4, 30, 35). In VSMC, a rise in intracellular calcium has been implicated in proliferation and migration after endothelial injury; however, the mechanism through which calcium results in VSMC proliferation is as yet undefined (15, 28). In the present study we have shown that the increase in [Ca2+]i elicited by BK rather than an influx of calcium through L-type calcium channels is required for MAPK activation. Furthermore, removal of calcium from the extracellular medium and or addition of EGTA to the extracellular medium did not alter the response of BK to activate MAPK. Taken together, our data seem to suggest that mobilization of calcium from intracellular stores by BK is sufficient to activate MAPK. Our findings support previous published data demonstrating that the rise in intracellular calcium elicited by thapsigargin, ANG II, and ionomycin contributes to the activation of MAPK (1, 6, 9). Although the cellular mechanism(s) through which calcium activates MAPK is not clearly defined, the involvement of Ras, Raf and MEK has been suggested (34). We have preliminary evidence to suggest that MAPK activation by BK is inhibited by a specific inhibitor of MEK (unpublished observations).

The calcium-binding protein calmodulin is implicated in the regulation of many cellular signaling pathways triggered by calcium, including progression of cell cycle and regulation of cell proliferation (17, 23, 39). Calmodulin binds to and activates several cellular proteins in response to a rise in intracellular calcium. The results of the present study indicate that activation of cytosolic and nuclear MAPK in response to BK are mediated through a calmodulin-dependent pathway. Support for this pathway comes from the findings that inhibition of calmodulin with the cell-permeable inhibitors W-7 and/or calmidazolium significantly reduced MAPK activation in response to BK. The calmodulin kinase II inhibitor KN-93 also inhibited the response of BK to stimulate MAPK activation. Our data are in agreement with a recent study by Abraham et al. (1) that implicated a role for calmodulin-dependent kinase II in MAPK activation in response to ionomycin stimulation.

Our results indicate that one of the nuclear targets for calmodulin is the induction of the protooncogene c-fos, which binds with c-jun to form the AP-1 complex transcription factor, thereby regulating the expression of genes containing this element (18). The present data show that treatment of VSMC with the calmodulin inhibitor W-7 significantly reduced the increase in c-fos mRNA level observed in response to BK stimulation. This is the first indication that BK stimulates c-fos induction via a calmodulin-dependent pathway. Although the induction of c-fos transcription can be mediated by several cis-activating elements that are recognized by proteins that are activated in response to an external signal, the sequence of events leading to c-fos induction by BK has not been completely defined. Because calmodulin has been shown to be present in the nucleus, it is possible that calmodulin through its binding to nuclear calcium could influence c-fos expression directly or indirectly by activating calmodulin kinase II, which has been shown to contribute to c-fos expression (45).

Another pathway through which BK could induce c-fos expression is via activation of MAPK through a calmodulin-dependent pathway. Support for such a pathway comes from the finding that the calmodulin inhibitor blocks not only BK-induced p42mapk and p44mapk phosphorylation in the cytoplasm but also phosphorylation of p42mapk and p44mapk in the nucleus. Once nuclear p42mapk and p44mapk are activated, they result in the phosphorylation of p62TCF/Elk-1 proteins leading to enhanced c-fos transcription (12). In this regard, a recent study by El-Dahr et al. (10), showed that the tyrosyl phosphorylation of Elk-1 in response to BK is mediated via MAPK activation in mesangial cells. Furthermore, the calmodulin-kinase cascade has been shown to activate transcription through phosphorylation of Elk-1 (11). An alternative pathway through which BK can induce c-fos expression is through intracellular calcium mobilization and hence MAPK activation. In this regard, our findings indicate that the intracellular calcium chelator EGTA-AM decreased (35%) c-fos mRNA expression in response to BK. Other studies have reported that the increase in c-fos expression in response to endothelin or ionomycin can be attenuated in the presence of 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid, an intracellular calcium chelator (32, 43).

In addition to calcium binding, phosphorylation of calmodulin may regulate its mode of action. Tyrosine phosphorylation of calmodulin by Src kinases and/or the insulin receptor kinase was shown to enhance biological activity, whereas serine and threonine phosphorylation decreased calmodulin activity (36). In this regard, BK could activate calmodulin by two distinct regulatory pathways, one through a rise in intracellular calcium and the other via activation of cytoplasmic tyrosine kinases such as Src, PYK2, and or focal adhesion kinase 125 (21).

In summary, our results suggest that activation of the B2 receptor by BK initiates multiple signals and subsequent cross-talk between second messengers leading to the release of intracellular calcium, which binds to calmodulin and results in the activation and nuclear translocation of MAPK. Activated nuclear MAPK can phosphorylate TCF/Elk-1, resulting in induction of the transcription factor c-fos, leading to further downstream signaling events that can regulate VSMC function. A better understanding of the signal transduction pathways by which BK modulates vascular function provides the basis for investigating the paracrine or autocrine effects of BK on vascular smooth muscle cell proliferation in states of vascular injury and disease.


    ACKNOWLEDGEMENTS

We thank Kim Sutton and Rory Hession for technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health (NIH) Grants DK-46543 and HL-55782, a Research Award from the American Diabetes Association (A. A. Jaffa), a Merit Review Grant from the Research Service of the Department of Veterans Affairs (R. K. Mayfield), a Postdoctoral Fellowship Award from the Juvenile Diabetes Foundation (V. Velarde), and a Training Fellowship from the NIH HL-07260 (P. S. Naidu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. A. Jaffa, Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425 (E-mail: jaffaa{at}musc.edu).

Received 20 August 1998; accepted in final form 29 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 277(3):H1061-H1068
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