©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Factor-induced Phosphorylation and Activation of Aortic Smooth Muscle Na/CaExchanger (*)

Takahiro Iwamoto , Shigeo Wakabayashi , Munekazu Shigekawa (§)

From the (1) Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Although the Na/Caexchanger is one of the major Caextrusion systems in excitable tissues, little is known about its regulation via protein phosphorylation. We now present evidence that the Na/Caexchanger is phosphorylated in quiescent and growth factor-stimulated cultured aortic smooth muscle cells. The Na/Caexchanger was isolated from P-labeled cells by immunoprecipitation with a specific polyclonal antibody. Phosphorylation of the exchanger was increased by up to 1.7-fold in response to platelet-derived growth factor-BB (PDGF-BB), -thrombin, or phorbol 12-myristate 13-acetate (PMA). However, angiotensin II did not enhance the phosphorylation significantly. The extent of phosphorylation appeared to correlate with the growth factor-induced increase in cell 1,2-diacylglycerol. At least four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide map analysis of the phosphorylated exchanger, suggesting that phosphorylation occurred at multiple sites. PDGF-BB and PMA increased phosphorylation of the same phosphopeptides (in particular P1). Phosphorylated amino acids were exclusively serine residues in both quiescent and stimulated cells. We found that growth factors enhanced Na/Caexchange activity and that there was a good correlation between the growth factor-induced stimulations of phosphorylation and exchange activity. PDGF-BB-induced activation of the exchanger was abolished by prior long treatment of cells with PMA. These results suggest that the Na/Caexchanger is activated by protein kinase C-dependent phosphorylation in response to growth factors in vascular smooth muscle cells.


INTRODUCTION

The Na/Caexchanger of the plasma membrane is an electrogenic transporter with a 3Na/1Castoichiometry that plays a key role in regulating [Ca]() in many mammalian cells. The physiological importance of Na/Caexchange has been studied extensively in excitable tissues such as cardiac muscle, vascular smooth muscle, and nerve fiber (for review, see Ref. 1). The exchanger is primarily responsible for the removal of excess Cafrom cells during agonist or electrical stimulation.

The Na/Caexchanger (NCX1) has been cloned from heart (2) , aorta (3) , kidney (4) , and brain (5, 6) . By hydropathy analysis, the cardiac clone is modeled to have an amino-terminal cleaved signal sequence, 11 transmembrane segments, and a large hydrophilic cytoplasmic domain between the fifth and sixth transmembrane segments (2) . The clones isolated from different tissues are highly homologous to the cardiac clone, except for the diversity found in a small region near the carboxyl end of the intracellular domain, which is generated by alternative splicing (3, 6) . Recently, a new Na/Caexchanger isoform (NCX2) has been cloned that is a product of a different gene and abundantly expressed only in brain and skeletal muscle (7) . NCX1 and NCX2 have amino acid sequence identity of 65%, but their functional properties are rather similar (7) .

The regulatory properties of the cardiac Na/Caexchanger have most extensively been characterized using giant excised membrane patches from cardiomyocytes or Xenopus laevis oocytes expressing the cardiac exchanger. The cardiac exchanger activity is modulated positively by cytoplasmic Caand ATP (8) and negatively by cytoplasmic Na(9) and exchanger inhibitory peptide (10) . Interestingly, most of these regulatory properties can be abolished by treating the exchanger with chymotrypsin from the cytoplasmic side (8) or deletion mutagenesis of the cytoplasmic domain (11) . Therefore, the cytoplasmic domain is thought to be involved in the regulation of the exchanger.

For the cardiac Na/Caexchanger, there is currently no strong evidence that protein phosphorylation is involved in the regulation of its activity. In squid giant axons, however, there is much evidence suggesting that protein kinase-dependent phosphorylation is responsible for stimulation of the exchanger activity induced by MgATP (12, 13, 14) . On the other hand, the Na/Caexchanger in vascular smooth muscle cells is inhibited by depleting cellular ATP (15) . Furthermore, phorbol esters (16) , 8-bromo-cGMP (17) , norepinephrine and high K(18) , and PDGF-BB (19) were reported to stimulate the Na/Caexchange activity in smooth muscle cells and tissues. These findings suggest that phosphorylation of the exchanger itself or associated ancillary proteins may be involved in the exchanger activation in nerve and vascular smooth muscle cells.

In the present study, we provide the first evidence that the Na/Caexchanger in rat aortic smooth muscle cells is phosphorylated and concomitantly activated in response to PDGF-BB, -thrombin, or phorbol ester. The regulation of this exchanger by growth factors is very interesting, since elevation of [Ca]has been suggested to be important for cell proliferation (20, 21) . Some of these results have been presented in abstract form (22) .


EXPERIMENTAL PROCEDURES

Cell Culture

Vascular smooth muscle cells were isolated from the thoracic aorta of male Wistar rats (200-300 g) by enzymatic dispersion as described by Chamley et al. (23) . The resulting cells were grown for 4-5 days in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO. After reaching confluence, cells were cultured in serum-free medium for an additional 24-48 h to enhance redifferentiation.

Antibody Production

A polyclonal anti-Na/Caexchanger antibody was raised by immunizing rabbits with a maltose-binding protein fusion protein containing a portion of the cytoplasmic domain (amino acids 273-769) of the dog cardiac Na/Caexchanger (2) . Preparation of the fusion protein and affinity purification of the antibody were described previously (24) .

Immunoprecipitation and Immunoblotting

Confluent smooth muscle cells in 100-mm dishes were labeled for 5 h at 37 °C in a phosphate-free, serum-free medium containing [P]orthophosphate (10 MBq/ml). The cells were stimulated with growth factors for 2-20 min and washed twice with ice-cold phosphate-buffered saline. Cell monolayers were scraped, suspended in 800 µl of ice-cold buffer A (50 m M Hepes/Tris (pH 7.4), 150 m M NaCl, 3 m M KCl, 25 m M sodium pyrophosphate, 10 m M ATP, 5 m M EDTA, 1 m M phenylmethylsulfonyl fluoride, 1 m M benzamidine hydrochloride, 10 units/ml aprotinin, 0.5 µg/ml leupeptin, and 0.5 µg/ml pepstatin A), and then centrifuged for 15 min at 100,000 g. The pellet was solubilized in 800 µl of ice-cold buffer A containing 1% CEfor 15 min, and the lysate was centrifuged for 30 min at 100,000 g. The solubilized sample was pretreated with protein A-Sepharose beads at 4 °C for 1 h and then incubated with an anti-Na/Caexchanger antibody at 4 °C for 1 h. To this mixture, 50 µl of a 50% slurry of protein A-Sepharose beads previously blocked for at least 1 h with a CEextract of Escherichia coli was added and the resultant mixture was incubated for 1 h at 4 °C in a rotating shaker. The beads were then washed eight times with buffer A containing 1% CE. Labeled proteins solubilized from beads by boiling in Laemmli's buffer were subjected to SDS-PAGE on a 7.5% gel and visualized by Bioimage analyzer (BAS2000, Fuji Film Co.).

SDS-PAGE and immunoblotting were performed essentially as described (25) . The immunoblots were visualized using the ECL detection system (Amersham Corp.).

Phosphopeptide Mapping

Phosphopeptide mapping analysis was carried out as described previously (26) , except that P-labeled Na/Caexchanger protein, which was recovered from SDS-PAGE gel slices and resuspended in 500 µl of 50 m M NHHCO(pH 8.0), was digested with 20 µg of TPCK-treated trypsin for 16 h, instead of 24 h, at 37 °C.

Phosphoamino Acid Analysis

Phosphoamino acid analysis was performed by the method of Cooper et al. (27) with modification. Briefly, the P-labeled Na/Caexchanger protein was first digested with TPCK-treated trypsin as described above and then hydrolyzed with 7 M HCl at 105 °C for 1 h. The hydrolysate was dried, washed four times with 500 µl of water, and then suspended in 10 µl of water containing internal standards (10 µg each of phosphoserine, phosphothreonine, and phosphotyrosine). Aliquots of the samples were applied onto thin-layer cellulose plates and amino acids were separated by electrophoresis for 1 h at 800 V in pyridine:acetic acid:water (5:45:950, v/v/v) (pH 3.6). Amino acids were reacted with ninhydrin and visualized using Bioimage analyzer.

Measurement of Na -dependent CaUptake

For Naloading, confluent cells in 24-well dishes were incubated in 1 ml of BSS (10 m M Hepes/Tris (pH 7.4), 146 m M NaCl, 4 m M KCl, 2 m M MgCl, 1 m M CaCl, 10 m M glucose, and 0.1% BSA) containing 1 m M ouabain, 10 µ M monensin, 10 m M NaHCO, and 3 m M NaHPOat 37 °C for 20 min. Cells were then incubated for 3 min with a Ca-free Naloading solution to reduce elevated [Ca]to a basal level. Cauptake was initiated by switching the medium to a Na-free BSS (replacing NaCl with equimolar KCl) or normal BSS, both of which contained 370 KBq/ml Ca, 1 m M ouabain, and 10 µ M verapamil. After a 30-s incubation, Cauptake was stopped by washing cells five times with an ice-cold solution containing 10 m M Hepes/Tris (pH 7.4), 120 m M choline chloride, and 10 m M LaCl. For the growth factor treatment, the solutions contained growth factors from 10 min before the start of Cauptake measurement. Cells were solubilized in 1% SDS and 10 m M NaBO, and aliquots were taken for determination of radioactivity and protein. Na/Caexchange activity was estimated by subtracting Cauptake activity in the normal BSS from that in Na-free BSS. Protein was measured by a modified Lowry method (28) with BSA as a standard. Measurement of [Ca]-[Ca]in monolayered cultured cells was measured using fura-2 as a fluorescent Caindicator as described previously (29) . Measurement of [Na]-Cell Nacontent was measured by equilibrating cells with BSS containing Na(740 KBq/ml), and [Na]was calculated by adopting 4.5 µl/mg protein as the total intracellular water space (30) .

Measurement of 1,2-Diacylglycerol

Confluent cells in 12-well dishes were stimulated with PDGF-BB in 1 ml of BSS. After appropriate intervals, incubation was terminated by adding 1 ml of ice-cold methanol. After extraction of lipids, 1,2-diacylglycerol was measured with the sn-1,2-diacylglycerol assay reagent system (Amersham) using a thin layer chromatography to separate phosphatidic acid converted from 1,2-diacylglycerol.

Statistical Analysis

Experimental results are expressed as means ± S.D. Significant differences were assessed with a one-way analysis of variance followed by Dunnett's test. A p value of <0.05 was considered significant.

Materials

CaCland NaCl were obtained from DuPont NEN, and [P]orthophosphate and sn-1,2-diacylglycerol assay reagent system were obtained from Amersham. BSA (fatty acid-free), -thrombin, cyclopiazonic acid, monensin, ouabain, PMA, and verapamil were from Sigma. PDGF-BB was purchased from Becton Dickson Laboratories. Ionomycin was from Calbiochem. Fura-2/acetoxymetylester was from Dojindo Laboratories. Angiotensin II was from the Peptide Institute. CEwas purchased from Nikkol Chemical Co. Ltd. (Tokyo, Japan). All other chemicals were of analytical grade.


RESULTS

Phosphorylation of the Na/CaExchanger by PDGF-BB and Other Agents

We investigated growth factor-induced phosphorylation of the Na/Caexchanger by immunoprecipitation from serum-depleted rat aortic smooth muscle cells metabolically labeled with [P]orthophosphate. The antibody used, which was raised against a portion of the cytoplasmic domain of the dog cardiac Na/Caexchanger and affinity-purified, recognized a broad band covering the expected molecular mass for the Na/Caexchanger (125 kDa), when the immunoprecipitated material was analyzed by SDS-PAGE and protein staining (Fig. 1 A). This broad band may be caused by glycosylation of the exchanger protein (1) or the presence of alternatively spliced exchanger isoforms (3, 6) . We found that this protein band was phosphorylated in serum-depleted, unstimulated cells (Fig. 1 B). When these cells were treated with 10 or 20 ng/ml PDGF-BB for 10 min, the extent of phosphorylation increased to 146 ± 9% or 166 ± 15% ( n = 3) of that for the untreated cells (Fig. 1 B). Treatment of cells with 10 n M -thrombin or 100 n M PMA for 10 min also increased the phosphorylation to 140 ± 9% or 145 ± 11% ( n = 3), respectively (Fig. 1, B and C). However, 100 n M angiotensin II did not increase the phosphorylation significantly (118 ± 6%, n = 3) under the equivalent experimental conditions (Fig. 1 C). Thus PDGF-BB, -thrombin, and PMA induced Na/Caexchanger phosphorylation in serum-depleted aortic smooth muscle cells. We found that removal of extracellular Cadecreased the basal and PDGF-BB-induced exchanger phosphorylation slightly; in 4 m M EGTA, the basal phosphorylation was 83% ( n = 2) of the control value, whereas PDGF-BB (10 ng/ml, 10 min) increased the exchanger phosphorylation to 132% ( n = 2) under the same conditions.


Figure 1: Immunological detection of phosphorylated Na/Ca exchanger in unstimulated and growth factor-stimulated smooth muscle cells. Rat aortic smooth muscle cells labeled with ( panels B and C) or without ( panel A) [P]orthophosphate (10 MBq/ml) were treated with growth factors for 10 min and then solubilized with 1% CElysis buffer. Cell lysates were incubated with a polyclonal antibody against the dog cardiac Na/Caexchanger (1/100 dilution), and the resultant immunoprecipitates were subjected to SDS-PAGE. In panel A, a nitrocellulose transfer of gel was probed with the polyclonal antibody and visualized by using ECL detection system. The upper band is the Na/Caexchanger, whereas the lower band is immunoglobulin derived from the immunoprecipitate. In panels B and C, phosphorylated proteins in the gels were visualized by Bioimage analyzer. In each panel, C, unstimulated cells; PDGF 10 and 20, cells stimulated with 10 or 20 ng/ml PDGF-BB; T, cells stimulated with 10 n M -thrombin; AII, cells stimulated with 100 n M angiotensin II; PMA, cells stimulated with 100 n M PMA. Molecular mass markers (in kDa) are shown on the left.



A time course of PDGF-BB-induced phosphorylation of the Na/Caexchanger is shown in Fig. 2, and the results of three similar experiments analyzed densitometrically are summarized in Fig. 3. In the same latter figure, we also show the time courses of PDGF-BB-induced changes in [Ca]and a 1,2-diacylglycerol level. The PDGF-BB-induced phosphorylation increased with time, reaching 169 ± 17% ( n = 3) of the control level at 20 min. Under the same conditions, [Ca]reached a peak (450 ± 40 n M, n = 4) within 2 min, followed by a slow decline. The formation of 1,2-diacylglycerol, on the other hand, increased from a basal level of 91 ± 8 pmol/10cells ( n = 4) to a high level of 191 ± 20 pmol/10cells at 20 min. In contrast to PDGF-BB, 100 n M angiotensin II, which did not enhance the exchanger phosphorylation significantly (Fig. 1 C), increased the formation of 1,2-diacylglycerol to a lower level (126 ± 16 pmol/10cells ( n = 4) at 10 min), as compared with PDGF-BB (189 ± 30 pmol/10cells ( n = 4) at 10 min).

Phosphopeptide Mapping and Phosphoamino Acid Analysis of the Na/CaExchanger

To characterize sites for the basal and stimuli-enhanced phosphorylation in the Na/Caexchanger, we performed two-dimensional tryptic phosphopeptide mapping as described under ``Experimental Procedures'' (Fig. 4). Tryptic digestion of the P-labeled Na/Caexchanger in unstimulated cells generated four major phosphopeptides (P1, P2, P3, and P4). Occasionally one minor spot (P5), which may represent a partially digested phosphopeptide, was observed. Stimulation of cells with PDGF-BB (20 ng/ml) and PMA (100 n M) for 20 min resulted in a significant increase in the amounts of P label incorporated into P1 and, to a lesser extent, into P2. In unstimulated cells, P1 was weakly phosphorylated, representing less than 15% of the total P label incorporated. In contrast, it accounted for 40-50% of the total P label in PDGF-BB- and PMA-stimulated cells. These results indicate that phosphorylation of the Na/Caexchanger occurs at multiple sites and that PDGF-BB and PMA enhance phosphorylation of the same phosphopeptides.

Phosphoamino acid analysis was performed on the phosphorylated Na/Caexchanger (Fig. 5). The Na/Caexchanger phosphorylation in the basal, and PDGF-BB- and PMA-stimulated cells occurred exclusively on serine residues, not on threonine or tyrosine residues. The result indicates the involvement of serine kinase(s) in the Na/Caexchanger phosphorylation.

Effect of PDGF-BB and Other Agents on Na/CaExchanger Activity

We examined the effect of treatment with PDGF-BB (10 or 20 ng/ml), -thrombin (10 n M), angiotensin II (100 n M), or PMA (100 n M) for 10 min on Na-dependent Cauptake (reverse mode of the Na/Caexchanger) in Na-loaded rat aortic smooth muscle cells, as described under ``Experimental Procedures'' (Fig. 6). Because these agents except for PMA increased [Ca]from basal levels of 100-150 n M to peak levels of 400-550 n M and because Na/Caexchange depends on [Ca], cells had been exposed to a Ca-free solution containing these agents during the last 3 min of the 10-min treatment in order to reduce [Ca]to a basal level.

As shown in Fig. 6, treatment of cells with PDGF-BB or -thrombin caused a significant increase (120-130% of the control value) in Na-dependent Cauptake. The extent of such PDGF-BB-induced activation was similar, when cells had been incubated with 30 to 146 m M Naand loaded with different levels of Na(data not shown). It should be mentioned that cell Naloading itself was not affected by prior treatment with PDGF-BB; when cells were equilibrated with 146 m M Nain the presence and absence of 20 ng/ml PDGF-BB, [Na]immediately after the transfer of cells to the Cauptake medium were 97 ± 4 and 99 ± 5 m M ( n = 4) in control and PDGF-BB-treated cells, respectively. On the other hand, angiotensin II did not affect Na-dependent Cauptake (Fig. 6). However, PMA enhanced the Cauptake significantly. It is important to note that there is apparent parallelism between these growth factor-induced increases in Na/Caexchange activity and the phosphorylation of the exchanger (Fig. 6).


Figure 6: Correlation between phosphorylation and activity of Na/Ca exchanger. Cells cultured in 24-well dish were stimulated with no agent ( C), 10 or 20 ng/ml PDGF-BB ( PDGF 10 or 20), 10 n M -thrombin ( T), 100 n M angiotensin II ( AII), or 100 n M PMA for 10 min. Na-dependent Cauptake was determined as described under ``Experimental Procedures.'' P incorporation into the immunoprecipitated Na/Caexchanger (see Fig. 1) was quantified by densitometry using Bioimage analyzer. Results are presented as the mean ± S.D. ( n = 3 or 4). *, significant difference from unstimulated cells.



We also examined whether PDGF-BB was able to enhance Na-dependent decline in [Ca]via the forward mode of Na/Caexchanger in aortic smooth muscle cells (Fig. 7). We treated cells with 50 µ M cyclopiazonic acid in Ca- and Na-free, high pH (pH 8.8) BSS containing 20 m M Mg, which inhibits Caextrusion via the Na/Caexchanger and the ATP-dependent Capump (31) . After [Ca]reached a plateau level of about 300 n M (about 2 min later), Nawas added extracellularly to a final concentration of 50 m M. In unstimulated cells, Naevoked a decline in [Ca](initial rate of decline, 33 ± 6 n M/10 s ( n = 3)) (Fig. 7 A), although addition of choline chloride, in place of NaCl, did not affect [Ca](data not shown). When cells were stimulated with 10 ng/ml PDGF-BB for 20 min, the initial rate of Na-induced [Ca]decline significantly increased to 89 ± 12 n M/10 s ( n = 3), and [Ca]reached a lower steady state level (Fig. 7 A). Interestingly, the PDGF-BB-induced stimulation of Na-dependent decline in [Ca]was abolished (initial rate of decline, 36 ± 8 n M/10 s ( n = 3)) by pretreatment of cells with 100 n M PMA for 24 h, suggesting that protein kinase C is involved in such activation of Na/Caexchanger (Fig. 7 B).


Figure 7: Effect of PDGF-BB on Na-dependent [Ca] decline measured in PMA-pretreated cells in Na- and Ca-free high pH/high Mg medium. Cells were treated with or without 10 ng/ml PDGF-BB for 20 min in BSS after they had been incubated with ( panel B) or without ( panel A) 100 n M PMA for 24 h. Then they were transferred to a Ca- and Na-free, high pH medium (pH 8.8) containing 20 m M Mgand 50 µ M cyclopiazonic acid. After [Ca] reached a plateau level (about 300 n M), NaCl was applied to the medium to a final concentration of 50 m M to induce Na-dependent [Ca] decline.




DISCUSSION

In the present study, we demonstrated that the Na/Caexchanger was phosphorylated in quiescent smooth muscle cells and that the phosphorylation was enhanced by up to 1.7-fold in response to PDGF-BB, -thrombin, or PMA (Figs. 1, 3, and 6). This is the first demonstration of phosphorylation of the Na/Caexchanger in response to the physiological ligands.

PDGF-BB was the most potent stimulator of exchanger phosphorylation among the agents tested. Tryptic phosphopeptide map analysis revealed that the same four major phosphopeptides (P1, P2, P3, and P4) were generated in unstimulated as well as PDGF-BB- or PMA-stimulated cells, suggesting that the exchanger phosphorylation occurs at multiple sites (Fig. 4). Phosphorylation of P1 and, to a lesser extent, of P2 was enhanced in PDGF-BB- and PMA-treated cells, and the phosphopeptide maps were essentially the same for these cells. P1 contains major site(s) for phosphorylation by PDGF-BB and phorbol ester, because P label incorporation into P1, which accounted for less than 15% of the total exchanger phosphorylation, increased to 40-50% of the total phosphorylation after PDGF-BB and PMA treatment. Furthermore, phosphorylated amino acids in the exchanger were exclusively serine residues in both quiescent and PDGF-BB- or PMA-stimulated cells (Fig. 5), indicating the involvement of serine protein kinase(s). Taken together, these results suggest that the Na/Caexchanger in vascular smooth muscle cells is phosphorylated in response to PDGF-BB or -thrombin via a protein kinase C-dependent pathway. We do not know, however, whether protein kinase C directly phosphorylates the exchanger or whether it phosphorylates and activates a secondary serine/threonine kinase. The involvement of protein kinase C is consistent with the well known facts that signaling by PDGF-BB and -thrombin occurs through activation of phospholipase C-1 and phospholipase C-1, respectively, that produce inositol 1,4,5-triphosphate and 1,2-diacylglycerol from phosphatidylinositol 4,5-bisphosphate (32) . Indeed, we observed elevation of 1,2-diacylglycerol in PDGF-BB-stimulated cells (Fig. 3). At present, however, we cannot rule out the possibility that other protein kinases such as Ca/calmodulin kinase II were also involved in the exchanger phosphorylation, because PDGF-BB induced a prolonged increase in [Ca](Fig. 3) and because increased [Ca]seemed to be able to enhance the exchanger phosphorylation (see ``Results'').


Figure 4: Two-dimensional tryptic phosphopeptide maps of Na/Ca exchanger. Cells labeled with [P]orthophosphate were stimulated with no agent ( C), PDGF-BB (20 ng/ml), or PMA (100 n M) for 20 min. The phosphorylated Na/Caexchanger was isolated by immunoprecipitation and subsequent SDS-PAGE. The Na/Caexchanger protein eluted from gels was digested with TPCK-treated trypsin and subjected to two-dimensional phosphopeptide mapping as described under ``Experimental Procedures.'' Phosphopeptides were visualized by Bioimage analyzer. The right bottom panel schematically represents the location of phosphopeptides and direction of peptide migration ( arrow).




Figure 5: Phosphoamino acid analysis of phosphorylated Na/Ca exchanger. Cells labeled with [P]orthophosphate were stimulated with no agents ( C), PDGF-BB (20 ng/ml), or PMA (100 n M) for 20 min. The phosphorylated Na/Caexchanger was isolated by immunoprecipitation and subsequent SDS-PAGE. After tryptic digestion and acid hydrolysis, phosphoamino acids were analyzed by thin-layer electrophoresis and then visualized by Bioimage analyzer. P-Ser, P-Thy, and P-Tyr represent the positions of phosphoserine, phosphothreonine, and phosphotyrosine, respectively.




Figure 3: Time courses of PDGF-BB-induced changes in Na/Ca exchanger phosphorylation, [Ca], and 1,2-diacylglycerol level. In cells stimulated with 10 ng/ml PDGF-BB for indicated periods of time, changes in the Na/Caexchanger phosphorylation, [Ca], and the1,2-diacylglycerol content were determined as described under ``Experimental Procedures.'' P incorporation into the Na/Caexchanger ( cf. Fig. 2) was quantified by densitometry using Bioimage analyzer. Results are presented as the mean ± S.D. ( n = 3 or 4). *, significant difference from cells at 0 min.



Primary structure of the Na/Caexchanger in rat aortic smooth muscle cells is highly homologous to the canine cardiac exchanger except for the NH-terminal portion, which is presumably a cleaved signal sequence, and part (amino acid residues 570-621) of the large cytoplasmic domain (3) . The cytoplasmic domain is considered to be involved in the regulation of the exchanger as briefly discussed in the introduction. The cytoplasmic domain of the smooth muscle Na/Caexchanger contains several candidate phosphorylation site sequences for serine/threonine kinases such as protein kinase C, Ca/calmodulin kinase II, and cAMP-dependent protein kinase (33) . One of such sequences Arg-Lys-Ala-Val-Ser, which is also conserved in the cardiac Na/Caexchanger (2) , is a good candidate for the phosphorylation site.

Regulation of the Na/Caexchanger in vascular smooth muscle cells is important to maintain cellular Cahomeostasis. Recently, phorbol esters (16) , cGMP (17) , norepinephrine and high K(18) , and PDGF-BB (19) have been reported to stimulate Na/Caexchange in A7r5 cells, rat aortic smooth muscle cells, and rabbit aortic rings. However, there has been no information about the mechanism by which these agents enhance the exchange activity, except that norepinephrine and high Kwere later shown to cause cytoplasmic Naretention that may partially explain the observed increase in Na/Caexchange (34) .

In this study, we determined the effects of PDGF-BB and other agents on Na-dependent Cauptake (the reverse mode of Na/Caexchange) in rat aortic smooth muscle cells. We found that PDGF-BB, -thrombin, and PMA significantly enhanced the Na-dependent Cauptake (Fig. 6). Furthermore, PDGF-BB stimulated Na-dependent [Ca]decline under conditions in which the plasma membrane and sarcoplasmic reticulum Capumps were inhibited (Fig. 7). It should be noted that PDGF-BB stimulated Na-dependent Cauptake only by about 1.3-fold, whereas it stimulated Na-dependent [Ca]decline to a greater extent (about 3-fold when the initial rate of [Ca]decline was measured) ( cf. Figs. 6 and 7). For this apparent discrepancy, we are currently unable to provide an explanation. It should also be noted that stimulation of Na-dependent [Ca]decline by PDGF-BB was abolished by prolonged pretreatment of cells with PMA, a procedure that down-regulates the protein kinase C content in the plasma membrane (35) . All these results suggest that activity of the Na/Caexchanger is positively regulated by growth factors and that the PDGF-BB-induced increase in Na/Caexchange activity, like the phosphorylation of the exchanger, is mediated via a protein kinase C-dependent pathway.

In contrast to other growth factors tested, angiotensin II did not significantly increase both Na/Caexchange and phosphorylation of the exchanger (Figs. 1 C and 6). -Thrombin and PMA induced intermediate levels of activation of these parameters (Fig. 6). Thus, there is apparently a good correlation between the extents of stimulation of Na/Caexchange and phosphorylation of the exchanger induced by different growth factors. We found that angiotensin II increased 1,2-diacylglycerol content to a low level under our experimental conditions when compared with PDGF-BB (see ``Results''). Therefore, the inability of angiotensin II to enhance Na/Caexchange and the exchanger phosphorylation seems to be due to a low level of 1,2-diacylglycerol produced. Production of different levels of 1,2-diacylglycerol in response to PDGF-BB and angiotensin II seems to indicate that these two growth factors differ in their capacity to activate protein kinase C in the primary cultured smooth muscle cells used in this study.

PDGF and -thrombin are powerful mitogens for cells of mesenchymal origin including vascular smooth muscle cells (36) . They increase [Ca]transiently and induce cascades of events such as activation of ion transporters, phosphorylation of proteins, and expression of a number of genes that are associated with cell proliferation. The increase in [Ca]may be required for mitogenic signaling at least in certain cell types, since Cablockers and chelators are able to inhibit growth factor-induced mitogenesis in those cells (20, 21) . On the other hand, transient exposure of growth-arrested vascular smooth muscle or other types of cells to PDGF, -thrombin, or phorbol esters result in activation of the Na/Hexchanger and Na/K/Clcotransporter (37, 38, 39) . In addition, phorbol esters are able to stimulate the plasma membrane Capump in vascular smooth muscle cells and other cell types (29, 40) . We presented evidence suggesting that protein kinase C directly phosphorylates and activates the plasma membrane Ca-ATPase (41, 42) . Our finding that PDGF-BB, -thrombin, and PMA stimulate the Na/Caexchanger in aortic smooth muscle cells indicate that this exchanger is also one of the targets involved in the growth factor-induced ionic changes that may be required for the cellular proliferative cycle to proceed properly.

Regulation of the Na/Caexchanger has been studied most extensively in canine cardiomyocytes and squid giant axons (1) . In squid giant axons, ATP and its slowly hydrolyzable analog ATPS have been shown to activate the Na/Caexchanger in the presence of Mgand micromolar cytoplasmic Ca(13) , and inorganic phosphate and vanadate have been shown to markedly enhance the ATP-dependent stimulation (12, 14) . These findings strongly support the view that the Na/Caexchanger is regulated by phosphorylation in squid axons. In contrast, ATP-dependent stimulation of Na/Caexchange in cardiomyocytes under patch clamp conditions does not require intracellular Ca, cannot be mimicked by ATPS, and is not enhanced by phosphatase inhibitors (43) . These results suggest no involvement of kinases or phosphatases in regulation of the cardiac exchanger, although it was once reported that the exchange activity of cardiac sarcolemma vesicles was modulated by a calmodulin-dependent kinase and phosphatase (44) . The stimulation of the cardiac exchanger by ATP under patch conditions is thus thought to be caused indirectly by phospholipid asymmetry in the sarcolemma generated by a lipid flipase (45) . Therefore regulation of the Na/Caexchanger by phosphorylation currently seems to be a property shared only by squid axon and smooth muscle exchangers, although we have no information regarding how similar are these mammalian and non-mammalian exchanger isoforms with respect to their structure and regulation. Regulation of the cardiac isoform by protein phosphorylation obviously requires further investigation.


FOOTNOTES

*
This work was supported by Grant-in-aid 321 for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan and by Special Coordination Funds Promoting Science and Technology (Encouragement System of COE) for the Science and Technology Agency of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5, Suita, Osaka 565, Japan. Tel.: 81-6-833-5012 (ext. 2519); Fax: 81-6-872-7485.

The abbreviations used are: [Ca], intracellular Caconcentration; PDGF, platelet-derived growth factor; CE, octaethylene glycol mono- n-dodecyl ether; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; TPCK, tosylamide-2-phenylethylchloromethyl ketone; Na, intracellular Na; Na, extracellular Na; BSS, balanced salt solution; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; ATPS, adenosine 5`- O-(thiotriphosphate).


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