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(Hypertension. 1997;30:1440-1447.)
© 1997 American Heart Association, Inc.

Articles

Growth Factors Mediate Intracellular Signaling in Vascular Smooth Muscle Cells Through Protein Kinase C–Linked Pathways

Rhian M. Touyz; ; Ernesto L. Schiffrin

From the Experimental Hypertension Laboratory, MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal and Université de Montréal, Montreal, Quebec, Canada.

Correspondence to Rhian M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal (Quebec) Canada H2W 1R7. E-mail touyz{at}ircm.umontreal.ca

	Abstract

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Abstract Intracellular Ca²⁺ and pH are potent modulators of growth factor–induced mitogenesis and contraction. This study examined platelet-derived growth factor–(PDGF-BB) and insulin-like growth factor (IGF-1)–mediated signal transduction in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and intracellular pH (pH_i) were measured by fluorescence digital imaging using fura-2 AM and 2'7'-bis(2-carboxyethyl)-5⁶-carboxyfluorescein, respectively. Characteristics of [Ca²⁺]_i transients were determined by pre-exposing cells to Ca²⁺-free buffer, and involvement of the Na⁺/Ca²⁺ exchanger was assessed by withdrawal of extracellular Na⁺ and by exposure to dimethylbenzamil (Na⁺/Ca²⁺ exchange blocker). To determine whether pH_i responses were mediated via the Na⁺/H⁺ exchanger, cells were preincubated with 10^-5 mol/L 5-(N-ethyl-N-isopropyl)amiloride (a selective Na⁺/H⁺ exchange blocker). The role of protein kinase C (PKC) and tyrosine kinases in growth factor signaling was assessed by pre-exposing cells to calphostin C and chelerythrine chloride (selective PKC inhibitors; 10^-5 mol/L) and tyrphostin A23 (a selective tyrosine kinase inhibitor; 10^-5 mol/L). PDGF-BB and IGF-1 (1 to 10 ng/mL) increased [Ca²⁺]_i and pH_i in a dose-dependent manner. At concentrations greater than 1 ng/mL both growth factors induced a biphasic [Ca²⁺]_i response with an initial transient peak followed by a sustained elevation. At 5 ng/mL PDGF-BB and IGF-1 significantly increased [Ca²⁺]_i from 95±3 nmol/L to 328±28 and 251±18 nmol/L, respectively. Ca²⁺ withdrawal abolished the second phase of [Ca²⁺]_i elevation. Agonist-induced [Ca²⁺]_i responses were similarly altered by Na⁺ withdrawal, by Na⁺/Ca²⁺ exchange blockade, and by PKC inhibition; latency, the period from stimulus application to the first [Ca²⁺]_i peak, was increased, the initial [Ca²⁺]_i peak was attenuated, and the sustained phase was prolonged. PDGF-BB and IGF-1 (10 ng/mL) significantly increased pH_i from 6.89±0.04 nmol/L to 7.11±0.01 and 7.09±0.02 nmol/L, respectively. EIPA and calphostin C completely inhibited agonist-elicited alkalinization. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB and IGF-1, whose receptors have tyrosine kinase activity. In conclusion, PDGF-BB and IGF-1 elicit significant [Ca²⁺]_i and pH_i responses in VSMC. The underlying pathways that mediate these responses are partially dependent on Na⁺/Ca²⁺ transporters and the Na⁺/H⁺ exchanger, both of which are linked to PKC activation.

Key Words: growth factors • calcium • pH, intracellular • calphostin C • chelerythrine chloride • cultured cells

	Introduction

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A variety of polypeptide growth factors have been shown to be important in the physiological regulation of vascular smooth muscle cell contraction, migration, and growth^{1 2} and in pathological states such as hypertension and atherosclerosis.^{1 3} These include competence growth factors such as PDGF and progression factors such as IGF-1. Competence factors stimulate entry of cells into the G₁ phase of the cell cycle, whereas progression through the S phase and subsequent cellular division requires the presence of progression factors, of which IGF-1 is the most significant.⁴ PDGF is a potent mitogen for vascular smooth muscle cells. It exists as a dimeric disulfide–bonded molecule made up of combinations of A and B chains. PDGF is released by aggregated platelets in vivo, and in vitro both endothelial and smooth muscle cells have been shown to synthesize PDGF.⁵ IGF-1, which is closely related to insulin, has also been shown to be mitogenic to cultured vascular smooth muscle cells.^{1 4} The secretion of IGF-1 by smooth muscle cells is stimulated by various agonists, including PDGF and angiotensin II.⁶ This may have pathophysiological significance, since IGF-1 is a progression factor for PDGF.⁶ Thus, PDGF can induce the production of its own progression factor.

In target cells, PDGF and IGF-1 bind to specific cell-surface receptors that have intrinsic tyrosine kinase activity, inducing various intracellular signal transduction pathways. PDGF, IGF-1, and other growth factors have been shown to influence cellular Ca²⁺ metabolism.^{7 8 9 10 11} They activate phospholipase C leading to the generation of inositol trisphosphate and diacylglycerol, which are involved in intracellular Ca²⁺ release and protein kinase C activation, respectively.^{9 10} In addition, PDGF stimulates Ca²⁺ influx.^{10 11} Intracellular Ca²⁺ mobilization and Ca²⁺ entry result in increased [Ca²⁺]_i, which mediates myosin-actin interaction, crossbridge formation, and vascular smooth muscle contraction.¹² Elevated [Ca²⁺]_i may also play a fundamental role in cell growth. Although there has been some controversy regarding the role of PDGF-induced [Ca²⁺]_i increases in mitogenesis, recent studies have demonstrated that the induction of replicative competence by PDGF is dependent on the maintenance of a sustained increase in [Ca²⁺]_i due to Ca²⁺ entry.^{11 13} Furthermore, PDGF-stimulated [Ca²⁺]_i elevation has been shown to be associated with tumor growth, which is inhibited in the presence of Ca²⁺ channel blockers.¹⁴

In addition to influencing [Ca²⁺]_i, PDGF activates the Na⁺/H⁺ exchanger by both protein kinase C–dependent and –independent pathways.¹⁵ In vascular smooth muscle cells, the Na⁺/H⁺ exchanger is one of the major controlling mechanisms underlying the regulation of pH_i.¹⁶ Activation of the antiport results in intracellular alkalinization, which is an early signal in the process that initiates mitogenesis.¹⁷ Also, changes in pH_i influence actin-myosin crossbridge formation, thereby modulating the contractile properties of vascular smooth muscle.^{17 18} Growth factor–generated [Ca²⁺]_i and pH_i signals may thus play an important role in vascular smooth muscle function.

The aims of the present study were to characterize [Ca²⁺]_i and pH_i transients induced by two potent growth factors, PDGF and IGF-1, and to elucidate some of the underlying mechanisms that regulate these agonist-stimulated second messengers in vascular smooth muscle cells.

	Methods

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Materials
PDGF-BB, IGF-1, dimethylbenzamil, and chelerythrine chloride were from Calbiochem. Fura-2-acetoxymethyl ester (fura-2 AM), 2'7'-bis(2-carboxyethyl)-5⁶-carboxyfluorescein (BCECF-AM), and pluronic F-127 were obtained from Molecular Probes Inc. Dimethyl sulfoxide was from Anachemia Canada Inc. Dulbecco's modified Eagle's medium (DMEM) was from GIBCO Canada and Ham's F-12 medium was from Flow Laboratories Inc. All other chemicals were from Sigma Chemical Co, Fisher Scientific Co, and BDH Inc.

Cell Culture
Vascular smooth muscle cells were isolated from mesenteric arteries of adult male Sprague-Dawley rats as previously described.^{19 20} Briefly, mesenteric arteries were cleaned of adipose and connective tissues, smooth muscle cells were dissociated by digestion of vascular arcades, the tissue was filtered, and the cell suspension centrifuged and resuspended in DMEM containing heat-inactivated calf serum, L-glutamine, HEPES, penicillin, and streptomycin. Vascular smooth muscle cells were grown on round glass coverslips (25 mm diameter) in plastic six-well dishes and maintained at 37°C in a humidified incubator in an atmosphere of 95% air/5% carbon dioxide. Cells were studied at confluence (7 to 10 days postplating). Before experimentation, confluent cultures of vascular smooth muscle cells were rendered quiescent by serum deprivation and maintenance in a serum-free medium for 36 hours.

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was measured with the ratiometric fluorescent dye fura-2 AM according to previously described methods.²⁰ On the day of the study, the culture medium was replaced 30 minutes before loading with warmed (37°C) modified Hanks' buffered saline containing (in mmol/L) 137 NaCl, 4.2 NaHCO₃, 3 NaHPO₄, 5.4 KCl, 0.4 KH₂PO₄, 1.3 CaCl₂, 0.5 MgCl₂, 0.8 MgSO₄, 10 glucose, and 5 HEPES (pH=7.4). The cells, attached to the glass coverslips were washed three times with 2 mL modified Hanks' buffer. The washed cells were loaded with fura-2 AM (4 µmol/L) that was dissolved in dimethyl sulfoxide containing 0.02% pluronic F-127 and incubated for 30 minutes at 37°C in a humidified incubator (5% CO₂/95% air). Under these loading conditions, the ratiometric (343/380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentalization of fura-2. The loaded cells were then washed three times with Hanks' buffer and used after a 5-minute stabilization period. All washing procedures and experiments were performed at room temperature, thereby minimizing compartmentalization and cell extrusion of the dye. Four glass rings (diameter 4 to 5 mm) were placed on the coverslip containing cells, and a seal was formed between the ring and coverslip using vacuum grease (Dow Corning). Each ring was filled with 50 µL warmed Hanks' buffer. This method allowed for four separate experiments per coverslip.

[Ca²⁺]_i was measured in single cells in cell clusters by fluorescent digital imaging. The advantages of this system are that multiple cells can be examined simultaneously and that the cells under investigation can be imaged throughout the experiment. Cells were investigated using an Axiovert 135 inverted microscope (40x oil immersion objective) and Attofluor Digital Fluorescence System (Zeiss, Germany) using alternating excitatory wavelengths of 343 and 380 nm. Video images of fluorescence at 520 nm emission were obtained using an intensified charge-coupled device camera system (Zeiss) with the output digitized to a resolution of 512x480 pixels. Images of fluorescence ratios were then obtained by dividing, pixel-by-pixel, the 343-nm image by the 380-nm image after background subtraction. [Ca²⁺]_i was calculated by in situ calibration techniques using the formula of Grynkiewicz et al,²¹ [Ca²⁺]_i=K_dxß(R-R_min)/(R_max-R), where K_d is the dissociation constant for fura-2-Ca²⁺ and taken to be 224 nmol/L,²¹ ß is defined as the ratio of fluorescence at 380 nm and zero Ca²⁺ (F_{380 min}) to saturating Ca²⁺ (F_{380 max}) conditions, and R is the ratio of fluorescence obtained with excitation at 343 and 380 nm with min and max subscripts denoting the ratios obtained under Ca²⁺-free and Ca²⁺-saturating conditions, respectively. Maximum (F_max) and minimum (F_min) fluorescence intensities were obtained for each experiment by exposure to 10 µmol/L ionomycin and 3 mmol/L ethylene glycol-bis (ß-aminoethyl ether)-N, N,N¹-N¹-tetraacetic acid, respectively.

Measurement of pH_i
pH_i was measured with the pH-sensitive dye BCECF-AM according to previously described methods.^{22 23} On the day of the study, the culture medium was replaced 30 minutes before loading with warmed (37°C) modified Hanks' buffered saline. The cells attached to the glass coverslips were washed three times with 2 mL modified Hanks' buffer. The washed cells were loaded with BCECF-AM (2 µmol/L) that was dissolved in dimethyl sulfoxide containing 0.02% pluronic F-127 and incubated for 30 minutes at 37°C in a humidified incubator (5% CO₂/95% air). The loaded cells were then washed three times with Hanks' buffer and used after a 10-minute stabilization period. The coverslip was prepared as described above for [Ca²⁺]_i.

pH_i was measured in single cells in cell clusters by fluorescent digital imaging. The Attofluor Digital Fluorescence System with alternating excitatory wavelengths of 488 and 460 nm, and an emission wavelength of 520 nm was used to measure pH_i. pH_i was calculated from a calibration curve obtained for each experiment by determining the fluorescence ratios at pH_i values of 7.4, 7.2, 7.0, and 6.8. pH_i was set by incubating the coverslip in K⁺-rich buffer in the presence of 10 µmol/L nigericin (an exogenous K⁺/H⁺ exchange ionophore).²⁴

Experimental Protocols
[Ca²⁺]_i and pH_i were measured in unstimulated cells and in cells exposed to 1 to 10 ng/mL PDGF-BB or IGF-1. To elucidate whether extracellular calcium contributes to growth factor–elicited [Ca²⁺]_i responses, cells were exposed to Ca²⁺-free buffer, which was prepared by omitting CaCl₂ from the Hanks' buffer and by adding 1.5 mmol/L EGTA. For these experiments, cells were pre-exposed to Ca²⁺-free buffer for 10 minutes before the addition of PDGF-BB or IGF-1. The role of Na⁺/Ca²⁺-dependent transport was determined by repeating the experiments in Na⁺-free buffer that was prepared by replacing NaCl with choline chloride. To further investigate the role of the Na⁺/Ca²⁺ exchanger, cells were pre-exposed to the Na⁺/Ca²⁺ exchange blocker dimethylbenzamil (50 µmol/L).²⁵ A highly selective Na⁺/H⁺ exchange blocker EIPA, was used to assess the role of the Na⁺/H⁺ exchanger in pH_i responses to PDGF-BB and IGF-1. Cells were preincubated for 10 minutes with 10^-5 mol/L EIPA before the addition of the agonists. To determine whether protein kinase C contributes to growth factor–elicited [Ca²⁺]_i and pH_i, cells were pretreated for 10 minutes to two different highly selective protein kinase C inhibitors, calphostin C and chelerythrine chloride. These inhibitors were used at a final concentration of 10^-5 mol/L, which has been shown to completely inhibit protein kinase C activity. To assess whether lower concentrations of calphostin C and chelerythrine chloride also influence agonist-induced [Ca²⁺]_i responses, full dose-response curves were obtained for these agents.

Cells were used for single experiments and repetitive determinations on single cells were not performed. The maximum peak ratio recorded was considered to be the maximal response to the agonist. Cell response rate was greater than 90%. At various intervals throughout the experiment, [Ca²⁺]_i and pH_i effects of vehicle (Hanks' buffer) were determined. Hanks' buffer had no effect on [Ca²⁺]_i or pH_i.

Statistical Analysis
Each experiment was repeated at least four times using different cell preparations. Data obtained from fluorescent digital imaging studies, where multiple cells (8 to 20 cells) were examined in each experimental field, were calculated as the mean [Ca²⁺]_i or pH_i per experiment and then as the mean of multiple experiments. Results are presented as mean±SEM and compared by Student's t test or by ANOVA where appropriate. Tukey-Kramers' correction was used to compensate for multiple testing procedures. P<.05 was considered significant. Concentration-response curves were fitted by nonlinear regression. The concentration (in mol/L) giving 50% inhibition of response (IC₅₀) was determined, and pI₂ calculated as -logIC₅₀.

	Results

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Effects of PDGF-BB and IGF-1 on [Ca²⁺]_i
Basal [Ca²⁺]_i in vascular smooth muscle cells was 95±3 nmol/L. Both PDGF-BB and IGF-1 increased [Ca²⁺]_i with responses for PDGF-BB being greater than those for IGF-1. As shown in Fig 1a, PDGF-BB (5 ng/mL) induced with a delay of about 60 seconds, an initial transient (first phase), and a subsequent lower steady state (second-phase) elevation of [Ca²⁺]_i. The peak levels of the first and the steady state levels of the second phase are shown in Fig 2. These responses were dose-dependent.

View larger version (11K): [in this window] [in a new window]   Figure 1. Representative fluorescent tracings of [Ca2+]i responses to 5 ng/mL PDGF-BB in cells exposed to normal buffer (tracing a), in cells exposed to Ca2+-free buffer (tracing b), in cells exposed to Na+-free buffer (tracing c), in cells pre-exposed to the Na+/Ca2+ exchange blocker dimethylbenzamil (50 µmol/L) (tracing d), and in cells pretreated with the protein kinase C inhibitor calphostin C (10-5 mol/L) (tracing e). Cells were pre-exposed to the various experimental conditions for 10 minutes prior to PDGF-BB stimulation. The arrow indicates time of PDGF-BB addition. [Ca2+]i is expressed as nmol/L.

View larger version (19K): [in this window] [in a new window]   Figure 2. Line graphs demonstrate effects of increasing concentrations of PDGF-BB and IGF-1 on [Ca2+]i responses in vascular smooth muscle cells. The first [Ca2+]i phase refers to the initial [Ca2+]i peak and the second [Ca2+]i phase refers to the second component of [Ca2+]i elevation. Each data point is the mean±SEM of 6 to 8 different experiments with each experiment comprising 10 to 20 cells.

To determine possible underlying mechanisms for growth factor–induced [Ca²⁺]_i responses, experiments were performed in the absence of extracellular Ca²⁺. Ca²⁺ withdrawal reduced the first [Ca²⁺]_i phase and inhibited the second phase of [Ca²⁺]_i elevation (Figs 1b and 3), suggesting that the first phase of the [Ca²⁺]_i transient is mediated mainly by intracellular Ca²⁺ mobilization and partially by Ca²⁺ influx, whereas the second phase is mediated primarily by the influx of extracellular Ca²⁺.

View larger version (25K): [in this window] [in a new window]   Figure 3. Bar graphs demonstrate effects of PDGF-BB (upper panel) and IGF-1 (lower panel) on [Ca2+]i responses in cells exposed to buffer with and without Ca2+. Definitions are the same as in Fig 2. Results are mean±SEM. Numbers in parentheses indicate number of experiments. *P<.05 vs counterpart in buffer with Ca2+; **P<.01 vs basal; ***P<.001 vs basal; P<.001 vs first [Ca2+]i phase.

To determine the role of Na⁺/Ca²⁺-dependent transport on growth factor–elicited [Ca²⁺]_i responses, experiments were performed in Na⁺-free medium in which choline chloride replaced NaCl. In the absence of extracellular Na⁺, basal [Ca²⁺]_i was unchanged but the second phase of agonist-induced [Ca²⁺]_i elevation was increased and sustained and failed to return to basal levels (Figs 1c and 4). To further evaluate the role of the Na⁺/Ca²⁺ exchanger, effects of PDGF-BB were assessed in the presence of dimethylbenzamil, an amiloride analogue that selectively blocks Na⁺/Ca²⁺ exchange.²⁵ Dimethylbenzamil had no effect on basal [Ca²⁺]_i (Fig 5) but increased the latency phase, the period from stimulus application to the initial first [Ca²⁺]_i peak, and significantly reduced the first phase of PDGF-BB–induced [Ca²⁺]_i (Table 1, Figs 1d and 5). The second phase of PDGF-BB–stimulated [Ca²⁺]_i was significantly elevated (P<.01), and the response was sustained compared with PDGF-BB–induced responses in the absence of dimethylbenzamil (Fig 5).

View larger version (27K): [in this window] [in a new window]   Figure 4. Bar graphs demonstrate effects of 5 ng/mL PDGF-BB (upper panel) and IGF-1 (lower panel) on [Ca2+]i responses in cells exposed to buffer with and without Na+. Definitions for [Ca2+]i phases are the same as in Fig 2. Results are mean±SEM. Numbers in parentheses indicate number of experiments. *P<.05 vs counterpart in buffer containing Na+; **P<.01 vs basal; ***P<.001 vs basal; P<.001 vs first [Ca2+]i phase.

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graphs demonstrate effects of 5 ng/mL PDGF-BB in the absence and presence of the Na+/Ca2+ exchange blocker dimethylbenzamil (benz.) (50 µmol/L). Cells were pre-exposed to dimethylbenzamil for 10 minutes before the addition of PDGF-BB. Results are mean±SEM. Numbers in parentheses indicate number of experiments, with each experiment comprising 5 to 10 cells. *P<.05 vs PDGF-BB in the absence of dimethylbenzamil; P<.001 vs first Ca2+ phase; ***P<.001 vs basal and dimethylbenzamil groups.

View this table: [in this window] [in a new window]   Table 1. Latency Phase of [Ca2+]i Response in Vascular Smooth Muscle Cells Exposed to 5 ng/mL PDGF-BB or IGF-1 in Various Experimental Conditions

To assess whether these agonist-induced changes may be associated with protein kinase C, [Ca²⁺]_i effects of PDGF-BB and IGF-1 were determined in cells that had been pre-exposed to 10^-5 mol/L calphostin C and chelerythrine chloride. Chelerythrine exhibited some intrinsic fluorescence that was accounted for in the final calculations. Protein kinase C blockade significantly altered growth factor–elicited [Ca²⁺]_i transients (Fig 1e). The latency phase was increased (Table 1), the first phase [Ca²⁺]_i peak was attenuated and the second phase of [Ca²⁺]_i elevation was increased and sustained (Figs 1e and 6). To determine whether lower concentrations of calphostin C and chelerythrine chloride also influence agonist-induced [Ca²⁺]_i responses, PDGF-BB [Ca²⁺]_i effects were assessed in the presence of various concentrations of protein kinase C inhibitors. Both calphostin C and chelerythrine reduced peak [Ca²⁺]_i in a dose-dependent manner (pI₂=8.5±0.31, calphostin C, pI₂=7.3±0.14, chelerythrine chloride; Fig 7). At concentrations greater than 10^-9 mol/L and 10^-10 mol/L for calphostin C and chelerythrine, respectively, the second [Ca²⁺]_i phase was increased and remained persistently elevated (Fig 7). This [Ca²⁺]_i profile was similar to that when extracellular Na⁺ was withdrawn or when Na⁺/Ca²⁺ exchange was blocked. Preincubation of cells with the selective tyrosine kinase inhibitor tyrphostin A-23 completely abolished PDGF-BB–and IGF-1–mediated [Ca²⁺]_i responses (Table 2).

View larger version (30K): [in this window] [in a new window]   Figure 6. Bar graphs demonstrate effects of 5 ng/mL PDGF-BB (upper panel) and IGF-1 (lower panel) on [Ca2+]i responses in the absence and presence of 10-5 mol/L calphostin C (calC) or chelerythrine chloride (chel). Cells were preincubated with either inhibitor for 10 minutes before stimulation with PDGF-BB or IGF-1. Results are mean±SEM. Numbers in parentheses indicate number of experiments, with each experiment comprising many cells. *P<.05 vs IGF-1 counterpart in the absence of protein kinase C inhibition; **P<.01 vs basal; ***P<.001 vs basal; P<.001 vs first [Ca2+]i phase.

View larger version (21K): [in this window] [in a new window]   Figure 7. Line graphs demonstrate effects of increasing concentrations of protein kinase C inhibitors calphostin C and chelerythrine chloride on PDGF-BB (5 ng/mL) –induced [Ca2+]i responses. Each data point is the mean±SEM of 3 to 5 experiments.

View this table: [in this window] [in a new window]   Table 2. Effects of Selective Tyrosine Kinase Inhibitor Tyrphostin A-23 (10-5 mol/L) on [Ca2+]i and pHi Responses Induced by 5 ng/mL PDGF-BB and IGF-1

Effects of PDGF-BB and IGF-1 on pH_i
Basal pH_i in vascular smooth muscle cells was 6.89±0.04. Both growth factors induced intracellular alkalinization in a dose-dependent manner (Fig 8). Unlike Ca²⁺ transients, the pH_i response was sustained (Fig 9). To determine whether growth factors mediate pH_i effects through the Na⁺/H⁺ exchanger, cells were pre-exposed to EIPA, a selective Na⁺/H⁺ exchange blocker. EIPA did not alter basal pH_i but inhibited PDGF-BB–and IGF-1–induced alkalinization (Figs 9 and 10). Similar results were obtained when cells were exposed to the protein kinase C inhibitor calphostin C (Figs 9 and 10). Pre-exposure of cells to tyrphostin A23 completely abolished PDGF-BB and IGF-1 pH_i effects (Table 2).

View larger version (14K): [in this window] [in a new window]   Figure 8. Line graphs demonstrate effects of increasing concentrations of PDGF-BB and IGF-1 on pHi responses in vascular smooth muscle cells. Each data point is the mean±SEM of 4 to 6 experiments with each experiment comprising 8 to 20 cells.

View larger version (4K): [in this window] [in a new window]   Figure 9. Representative fluorescent tracings of pHi responses to 10 ng/mL PDGF-BB in vascular smooth muscle cells in control conditions (tracing a), in cells pre-exposed to 10-5 mol/L EIPA, a selective Na+/H+ exchange blocker (tracing b), and in cells pretreated with 10-5 mol/L calphostin C, a selective protein kinase C inhibitor (tracing c). Cells were preincubated with either inhibitor for 10 minutes before PDGF-BB stimulation. The arrow indicates time of agonist addition.

View larger version (15K): [in this window] [in a new window]   Figure 10. Bar graphs demonstrate effects of 10 ng/mL PDGF-BB (upper panel) and IGF-1 (lower panel) on pHi responses in the absence and presence of 10-5 mol/L EIPA or calphostin C (calC). Cells were pretreated with either inhibitor for 10 minutes before agonist stimulation. Results are mean±SEM. Numbers in parentheses indicate number of experiments. *P<.05 vs other groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study identifies some of the intracellular signaling events mediated by two important mitogens, PDGF-BB and IGF-1, in vascular smooth muscle cells. We demonstrate that these two growth factors dose-dependently increase [Ca²⁺]_i and pH_i through protein kinase C–dependent pathways that are linked to Na⁺/Ca²⁺ transporters and the Na⁺/H⁺ exchanger.

Mitogenic peptides such as PDGF, a competence growth factor, and IGF-1, a progression growth factor, are important mitogens for vascular smooth muscle cells. In addition, these agents have been shown to modulate smooth muscle contraction as well as to stimulate extracellular matrix biosynthesis.^{2 13 26 27} Thus, growth factors contribute significantly to vascular smooth muscle cell function and may be important in pathological states of the vasculature such as hypertension, atherosclerosis, and diabetes or after intravascular injury. Both peptides mediate their effects through specific cell membrane receptors that possess an intrinsic tyrosine kinase activity that is stimulated on ligand binding. Many intracellular events occur rapidly after exposure of growth factors, including activation of several proteins, increased phosphatidylinositol turnover, activation of protein kinase C, induction of growth-related genes, and, as demonstrated here, increased intracellular [Ca²⁺]_i and alkalinization.

The significance of growth factor–induced [Ca²⁺]_i responses have been debated in the past, but it is becoming increasingly evident that these changes are important in contraction and mitogenesis. Mogami and Kojima²⁸ reported that Ca²⁺ influx is a prerequisite for DNA synthesis induced by PDGF in vascular smooth muscle cells, and Failli et al¹¹ demonstrated that PDGF-induced mitogenesis in human hepatic cells requires extracellular Ca.²⁺ Kobayashi et al²⁹ showed that Ca²⁺-dependent effects elicited by PDGF may be only mitogenic at pathologically high [Ca²⁺]_i. Because of the potential importance of growth factor–stimulated [Ca²⁺]_i in vascular smooth muscle function, we assessed some of the putative mechanisms that regulate [Ca²⁺]_i responses elicited by PDGF-BB and IGF-1. After a latency period of about 60 seconds, at concentrations greater than 1 ng/mL PDGF-BB and IGF-1 induced a biphasic Ca²⁺ response in primary cultured vascular smooth muscle cells. The lag phase may represent the kinetics of the peptides binding to their receptors and the intracellular pathways activated by the bound receptors. The time course of PDGF-induced phospholipase C–mediated hydrolysis of phosphoinositides is slow compared with that of vasoconstrictors such as angiotensin II,³⁰ and this may be reflected as the delayed [Ca²⁺]_i response. The initial [Ca²⁺]_i transient was followed by a subsequent lower steady state elevation of [Ca²⁺]_i. At all tested concentrations, PDGF-BB effects were greater than those of IGF-1. This may be related to differences in receptor density. Extracellular Ca²⁺ withdrawal inhibited the second phase but not the first phase of the [Ca²⁺]_i response, indicating that the first and second phases may be mediated primarily through intracellular Ca²⁺ mobilization and Ca²⁺ influx, respectively. PDGF-induced Ca²⁺ influx may occur through mechanisms distinct from L-type voltage-dependent channels, as previous studies demonstrated that nickel (which may block L-type and T-type channels) blocks PDGF-stimulated Ca²⁺ influx, whereas L-type Ca²⁺ channel antagonists do not.^{28 31}

The specific [Ca²⁺]_i profile of PDGF-BB and IGF-1 may be associated with mechanisms that regulate Ca²⁺ efflux. In vascular smooth muscle cells, Ca²⁺ is extruded from the cell by Ca²⁺-ATPase and by the Na/Ca²⁺ exchanger.³² Ca²⁺-ATPase rapidly and actively transports Ca²⁺ across the plasma membrane. The Na⁺/Ca²⁺ transporter is an electrogenic transporter with a 3 Na⁺/1 Ca²⁺ stoichiometry and plays a key role in maintaining [Ca²⁺]_i at constant levels.³³ The Na⁺/Ca²⁺ exchanger in vascular smooth muscle cells is modulated positively by [Ca²⁺]_i and negatively by cytoplasmic [Na⁺]_i, and has been shown to be activated by phorbol esters as well as by PDGF.^{34 35} To determine the role of Na⁺/Ca²⁺-dependent transporters in growth factor–stimulated [Ca²⁺]_i, experiments were performed in Na⁺-free medium as well as in the presence of dimethylbenzamil, an amiloride analogue that selectively blocks Na⁺/Ca²⁺ exchange. Under these conditions, basal [Ca²⁺]_i was unchanged, indicating that the Na⁺/Ca²⁺ exchanger is inactive in the basal state. However, growth factor–induced [Ca²⁺]_i responses were significantly altered. The latency period was increased, the first phase of [Ca²⁺]_i elevation was reduced, and the second phase was increased and sustained. [Ca²⁺]_i failed to return to basal levels. The prolonged lag phase may represent changes in kinetics of agonist-receptor binding and initiation of intracellular biochemical pathways. Thus, in the absence of extracellular Na⁺ or in the presence of Na⁺/Ca²⁺ exchanger blockade, agonist-stimulated Ca²⁺ accumulates in the cytoplasm, resulting in the sustained second phase of [Ca²⁺]_i elevation. Interestingly, in the presence of various concentrations of two different highly selective protein kinase C inhibitors, agonist-stimulated [Ca²⁺]_i responses were similar to those in Na⁺-free medium or when Na⁺/Ca²⁺ exchange was blocked. These results suggest that [Ca²⁺]_i transients elicited by PDGF-BB and IGF-1 are regulated by a Na⁺/Ca²⁺-dependent transporter that is linked to protein kinase C–dependent pathways. Iwamoto et al³⁶ recently reported that activity of the Na⁺/Ca²⁺ exchanger in rat aortic vascular smooth muscle cells is positively regulated by growth factors and that PDGF-BB, but not angiotensin II, –induced increase in Na⁺/Ca²⁺ exchange activity is mediated by protein kinase C. Growth factors may differ in their capacity to activate protein kinase C and the Na⁺-Ca²⁺ exchanger, which, in turn, may underlie the differential [Ca²⁺]_i responses and associated signaling effects. For example, the slower, more prolonged [Ca²⁺]_i responses of PDGF-BB and IGF-1 may be important for the mitogenic properties of these peptides, whereas the more acute [Ca²⁺]_i transient characteristic of angiotensin II and vasopressin may be important for contraction.

A novel finding in the present study is the alkalinizing effect of PDGF-BB and IGF-1. Vascular smooth muscle cell pH_i is an important second messenger in the transduction of contractile and growth stimuli. Intracellular alkalinization stimulates DNA synthesis and cell growth, but also increases actin-myosin sensitivity to [Ca²⁺]_i thereby increasing contraction and tone.^{17 18} PDGF-BB and IGF-1 increased pH_i in a dose-dependent manner, and it may be through these responses that growth factors mediate, at least in part, their contractile and mitogenic effects. Agonist-stimulated pH_i responses were completely abolished by EIPA, a selective Na⁺/H⁺ exchange blocker, and by the protein kinase C inhibitor calphostin C. Thus, growth factor–induced alkalinization in vascular smooth muscle cells may be mediated by the Na⁺/H⁺ exchanger and modulated by protein kinase C–dependent pathways. Previous studies have shown that PDGF-BB activates the Na⁺/H⁺ exchanger.¹⁵ In vascular smooth muscle cells, Ca²⁺ also appears to play a critical role in PDGF-induced activation of this antiport. Depletion of cell Ca²⁺ by removal of extracellular Ca²⁺ completely blocked PDGF activation of Na⁺/H⁺ exchange.¹⁵ Hence agonist-stimulated [Ca²⁺]_i changes may influence regulation of intracellular pH_i. Interactions between PDGF-AB–stimulated [Ca²⁺]_i and pH_i have also been shown in Syrian hamster embryo cells.³⁷ In these cells, however, Ca²⁺ influx was associated with intracellular acidification, which was a major determinant of cellular proliferation.

In conclusion, the present study demonstrates that PDGF-BB and IGF-1 increase [Ca²⁺]_i and pH_i in vascular smooth muscle cells. These effects are mediated through protein kinase C–dependent pathways, which modulate [Ca²⁺]_i and pH_i through a Na⁺/Ca²⁺-dependent transporter and the Na⁺/H⁺ exchanger, respectively. These intracellular signaling events may be required for the mitogenic and contractile actions of growth factors in vascular smooth muscle cells.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular free Ca2+ concentration EIPA = 5-(N-ethyl-N-isopropyl)amiloride IGF-1 = insulin-like growth factor PDGF = platelet-derived growth factor(s) pHi = intracellular pH

	Acknowledgments

 
This work was supported by a group grant to the Multidisciplinary Research Group on Hypertension and by grant MT-14080, both from the Medical Research Council of Canada, and by grants from the Heart and Stroke Foundation of Quebec. The authors thank Carole Tremblay for her secretarial help.

Received March 17, 1997; first decision April 14, 1997; accepted June 12, 1997.

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