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(Hypertension. 1997;30:222-229.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Regulates Vascular Smooth Muscle Cell pH, Contraction, and Growth Via Tyrosine Kinase–Dependent Signaling Pathways

Rhian M. Touyz; ; Ernesto L. Schiffrin

From the Medical Research Council (MRC) Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal (Quebec, Canada).

Correspondence to R.M. Touyz, MD, PhD, 110 Pine Ave W, Montreal, Quebec, Canada H2W 1R7.

	Abstract

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Abstract Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and pH (pH_i). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II–stimulated pH_i, and in simultaneously measured contractile and [Ca²⁺]_i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pH_i was determined by fluorescent digital imaging using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca²⁺]_i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [³H]thymidine and [³H]leucine incorporation, respectively. The Ang II receptor subtypes (AT₁ or AT₂) through which Ang II mediates effects were assessed with [Sar¹,Ile⁸]Ang II (a nonselective subtype antagonist), losartan (a selective AT₁ antagonist), and PD 123319 (a selective AT₂ antagonist). To determine whether tyrosine kinases influence Ang II–stimulated responses, cells were pretreated with 10^-5 mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pH_i in a dose-dependent manner (pD₂, 9.2±0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca²⁺]_i (pD₂, 7.4±0.1). Ang II (10^-7 mol/L) increased DNA ([³H]thymidine incorporation) and protein synthesis ([³H]leucine incorporation). [Sar¹,Ile⁸]Ang II and losartan but not PD 123319 abolished Ang II–elicited responses. Tyrphostin A-23 significantly attenuated Ang II–stimulated pH_i responses; it also inhibited [Ca²⁺]_i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II–stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II–mediated pH_i responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT₁ receptor subtype–mediated contraction and growth.

Key Words: calcium • signal transduction • muscle, smooth, vascular • hydrogen-ion concentration

	Introduction

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Angiotensin II, the final effector peptide of the renin-angiotensin system, mediates diverse physiological responses, including arterial vasoconstriction, renal sodium reabsorption, and aldosterone secretion.¹ It is also a growth-promoting factor for various cell types, including mesangial cells, endothelial cells, and VSMCs.^{2 3} In cultured VSMCs, Ang II induces hypertrophy and under certain conditions hyperplasia as well as increased extracellular matrix formation.⁴ Because of its contractile and growth-promoting functions, Ang II may play a pivotal physiological role in cardiovascular homeostasis and a pathogenic role in hypertension. Ang II mediates its functional responses in vascular smooth muscle via many intracellular pathways. Signal transduction is initiated when Ang II binds to specific cell membrane receptors, of which two subtypes, AT₁ and AT₂, have been characterized.⁵ The subclassification of receptors is based on their affinity for highly selective nonpeptide Ang II receptor antagonists.^{5 6} Losartan, SK&F 108566, and L-158,809 are some of the many potent, highly selective antagonists of both the binding of Ang II to AT₁ receptors and of AT₁-mediated functions. PD 123177 and its analogues and CGP 42112A totally inhibit Ang II binding to the AT₂ receptor at concentrations that have no effect on the AT₁ receptor. In VSMCs, Ang II activates phospholipase C (PLC), leading to the generation of inositol triphosphate and diacylglycerol, which are involved in intracellular Ca²⁺ mobilization and PKC activation, respectively.⁷ Intracellular Ca²⁺ release results in increased [Ca²⁺]_i, which mediates myosin-actin interaction, crossbridge cycling, and vascular smooth muscle contraction.^{7 8} PKC activation by 1,2-diacylglycerol regulates Na⁺-H⁺ exchange, which is one of the major controlling factors underlying the regulation of pH_i in VSMCs.⁹ Activation of the antiport results in intracellular alkalinization, which is an early signal in the process that initiates mitogenesis.¹⁰ Also, changes in pH_i influence actin-myosin crossbridge formation, thereby modulating the contractile properties of vascular smooth muscle.¹¹

In addition to activation of traditional phosphoinositide-PLC–mediated Ca²⁺ signaling pathways commonly associated with Ang II, it has recently been suggested that tyrosine kinase–dependent pathways are associated with Ang II responses in vascular smooth muscle.^{12 13} Protein tyrosine kinases are phosphotransferases that are involved in the transduction pathways leading to both normal and aberrant cell growth and differentiation. In cultured VSMCs, Ang II phosphorylates multiple substrates, leading to activation of many growth-associated pathways, namely, PLC-, Jak-STAT (signal transducers and activators of transcription), and Ras-Raf-MAP (mitogen-activated protein) kinases.^{14 15 16 17} Activation of these pathways leads to the initiation of a growth response. Ang II has been shown to induce an increase in expression of the growth-associated proto-oncogenes c-fos, c-jun, and c-myc in many cell types, which further supports the trophic role of this peptide.¹⁸ Tyrosine kinases have also been implicated as mediators of gastric and vascular smooth muscle contraction and may play a role in Ang II–stimulated vascular contractile responses.¹⁹ A recent study demonstrated that in vivo administration of Ang II in normotensive WKY induced an increase in overall tyrosine phosphorylation in aorta and that this was coupled to contractile activity and an increase in blood pressure.²⁰ Furthermore, the selective tyrosine kinase inhibitor tyrphostin-25 when given in vivo inhibited all the Ang II–mediated effects.²⁰ Since the initiation of cell contraction involves the modulation of myofilaments within the cytoskeleton, whereas cell growth requires transmission of a nuclear message, Ang II must have at least two distinct vascular functions, contraction and mitogenesis. The exact signaling pathways that underlie these effects have not been well defined, but it is possible that there is cross talk between the phosphoinositide pathway and tyrosine ki- nase–dependent signaling pathways. Ang II–generated PKC, pH_i, and Ca²⁺ signals may play a role in phosphorylation and activation of protein tyrosine kinases, whereas tyrosine kinases in turn may retroactively influence [Ca²⁺]_i and pH_i. We recently demonstrated that G protein–coupled receptor-linked [Ca²⁺]_i transients are modulated by tyrosine kinase–dependent pathways.²¹ It is unclear whether these pathways also influence agonist-induced pH_i responses in VSMCs.

The aims of the present study were (1) to investigate the role of tyrosine kinases in Ang II–stimulated pH_i responses in VSMCs; (2) to determine whether the functional effectors of agonist-mediated second messengers, ie, contraction and growth, are stimulated by Ang II and whether tyrosine kinases modulate these effects; and (3) to characterize the membrane receptor subtype through which Ang II elicits its cellular actions. Unlike our previous studies in which we assessed only [Ca²⁺]_i responses, in the present study we measured Ang II–stimulated [Ca²⁺]_i transients simultaneously with VSMC contraction. This allows for the determination of the temporal relationship between [Ca²⁺]_i signaling and contraction at the single-cell level. VSMCs from mesenteric arteries of WKY were studied. For the pH_i, [Ca²⁺]_i, and contractile studies, we used only primary cultured unpassaged cells, which exhibit a contractile phenotype and have undergone little phenotypic change relative to the original smooth muscle cells in blood vessels. Growth studies were performed in low-passaged cultured cells, which have a proliferative phenotype.

	Methods

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Materials
All chemicals were of the highest reagent grade available. Ang II was from Peninsula Laboratories Inc. Fura 2–acetoxymethyl ester (fura 2-AM), 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), and pluronic F-127 were obtained from Molecular Probes Inc. Dimethyl sulfoxide was from Anachemia Canada Inc. Tyrphostins were from Calbiochem. DMEM was from GIBCO Canada, and Ham's F-12 medium was from Flow Laboratories Inc. All other chemicals were from Fisher Scientific Co, BDH Inc, and Sigma Chemical Co.

Cell Culture
The study was approved by the Animal Ethics Committee of the Clinical Research Institute of Montreal (IRCM) and carried out according to the recommendations of the Canadian Council for Animal Care. Male WKY (n=20) (Taconic Farms Inc, Germantown, NY) were used. The rats were housed under standardized conditions (12-hour light/dark cycle at a constant temperature of 22°C and relative humidity of 60%) in the animal unit at the IRCM. The rats were killed by decapitation. VSMCs derived from mesenteric arteries were isolated, phenotypically characterized, and propagated as described in detail previously.^{22 23} Briefly, mesenteric arteries were cleaned of adipose and connective tissue, VSMCs were dissociated by digestion of vascular arcades, the tissue was filtered, and the cell suspension was centrifuged and resuspended in DMEM containing heat-inactivated calf serum, L-glutamine, HEPES, penicillin, and streptomycin. For pH_i, [Ca²⁺]_i, and contraction studies, VSMCs 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% CO₂. For pH_i studies, cells were studied at confluence (typically 7 to 10 days after plating). Before experimentation, confluent cultures of VSMCs were rendered quiescent by serum deprivation and maintenance in a serum-free medium for 36 hours. For [Ca²⁺]_i and contraction studies, cells were plated onto round glass coverslips that were coated with rat tail tendon collagen gels that were prepared as follows²⁴ : WKY tail tendons were sterilized in 70% ethanol for 4 hours, minced, and extracted with 0.1% acetic acid for 48 hours at 4°C. The protein concentration of the supernatant was adjusted to 0.15 mg/mL and titrated to pH 8.0 with NaOH at 4°C. One milliliter was placed on glass coverslips in six-well plates at room temperature. The gels formed within 30 minutes and were incubated with DMEM overnight before seeding with isolated VSMCs. After seeding with cells, the multiwell dishes containing glass coverslips were incubated in a humidified incubator maintained at 37°C and equilibrated with 5% CO₂ and 95% air. After 48 hours and every 48 hours thereafter, the medium was replaced with 1 mL DMEM containing 0.5% fetal calf serum.

Measurement of pH_i
pH_i was measured with the pH-sensitive dye BCECF-AM according to previously described methods.²⁵ On the day of the study, the culture medium was replaced 30 minutes before loading with warmed (37°C) modified Hanks' buffered saline containing (mmol/L) NaCl 137, NaHCO₃ 4.2, NaHPO₄ 3, KCl 5.4, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, glucose 10, and HEPES 5. 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). Under these loading conditions, the ratiometric fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentalization of dye. The loaded cells were then washed three times with Hanks' buffer and used after a 5- to 10-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.

pH_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 with an Axiovert 135 inverted microscope (x40 oil immersion objective) and an Attofluor Digital Fluorescence System (Zeiss) using alternating excitatory wavelengths of 488 and 460 nm. Video images of fluorescence at the emission wavelength of 520 nm were obtained with an intensified charge-coupled device (CCD) camera system (Zeiss), with the output digitized to a resolution of 512x480 pixels. 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).²⁶

Simultaneous Measurement of VSMC [Ca²⁺]_i and Contraction
After 7 days, Ang II–induced [Ca²⁺]_i and contractile responses of VSMCs were measured, as previously described.^{27 28} The gel-coated coverslips with attached fura 2–loaded cells were placed on the stage of the Axiovert microscope. After a 10-minute stabilization period, a field of cells was photographed to obtain baseline images. Ang II was then added, and serial images were taken of the same field of cells at 10- to 15-second intervals after Ang II addition. The lengths of the longest axes of cells were measured in the first image, and lengths of the same cells were measured in the subsequent photographs. For each cell, the percent contraction from the baseline length was calculated, and these values were averaged for all cells. The average baseline cell lengths were consistent between preparations. [Ca²⁺]_i was measured at the same time that images were captured. Cells grown on the rat tail collagen gels were loaded with the fluorescent dye fura 2-AM (4 µmol/L) as described previously.^{29 30} Excitation wavelengths of 343 and 380 nm and an emission wavelength of 520 nm were used. [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al³¹ : [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/L³¹ ; 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 EGTA, respectively.

Experimental Protocols
pH_i, [Ca²⁺]_i, and contraction were measured in unstimulated cells and in cells exposed to increasing concentrations (10^-10 to 10^-5 mol/L) of Ang II in the absence and presence of the selective tyrosine kinase inhibitor tyrphostin A-23 and its inactive analogue tyrphostin A-1 (10^-5 mol/L). To determine whether tyrphostin A-23 had a dose-dependent effect on Ang II–stimulated [Ca²⁺]_i, we pretreated cells for 10 minutes with increasing concentrations (10^-5 to 10^-10 mol/L) of tyrphostin A-23 before adding Ang II (10^-9 mol/L). Ang II was used at a concentration of 10^-9 mol/L because we previously demonstrated that this concentration elicits significant [Ca²⁺]_i responses and corresponds approximately to the EC₃₀ value (concentration giving 30% of maximal response). Furthermore, 10^-9 mol/L Ang II is a high physiological concentration and should induce responses that are probably the maximal ones occurring in vivo. To determine the receptor subtype through which Ang II mediates [Ca²⁺]_i and pH_i responses, we preexposed cells to 10^-6 mol/L [Sar¹,Ile⁸]Ang II (a nonselective angiotensin receptor antagonist), losartan (a selective AT₁ antagonist), and PD 123319 (an AT₂-specific ligand) for 10 minutes before addition of 10^-9 mol/L Ang II. To verify at least to some extent that the tyrosine kinase inhibitor used in the present study did not inhibit non–tyrosine kinase–mediated pathways, we assessed the effects of tyrphostins on ionomycin (10^-5 mol/L)–induced [Ca²⁺]_i responses. Ionomycin is a Ca²⁺ ionophore that increases [Ca²⁺]_i through non–tyrosine kinase–dependent pathways.

Cell Growth Studies
We used VSMCs passaged between three and six times for cell growth studies. Cells were seeded into 24-well plates and cultured for 24 hours in DMEM as described above. After 24 hours, the culture medium was replaced with serum-free medium containing insulin (5x10^-7 mol/L) and transferrin (5 µg/mL) for 48 hours to render the cells quiescent. DNA synthesis was evaluated by measuring [³H]thymidine incorporation into DNA, and protein synthesis was determined by measuring [³H]leucine incorporation.^{32 33} Quiescent cells were stimulated for 24 hours with Ang II (10^-7 mol/L) in the absence and presence of 10^-6 mol/L [Sar¹,Ile⁸]Ang II, losartan, PD 123319, or tyrphostins (10^-5 mol/L). The cells were then incubated with 5 µmol/L [³H]thymidine (40 Ci/mmol) and cultured for a further hour. For protein synthesis studies, 1 µmol/L [³H]leucine was added at the same time Ang II was added. Radioactive medium was removed, and the cells were washed five times with ice-cold Hanks' buffer. Cells exposed to [³H]thymidine were then incubated with trichloroacetic acid (0.75 mol/L) for 2 hours at 70°C. Cells exposed to [³H]leucine were washed with 5% ice-cold trichloroacetic acid and then incubated with 1 mol/L NaOH for 60 minutes at room temperature. Relative [³H]thymidine and [³H]leucine incorporation was determined by liquid scintillation counting.

Data Analysis
Each experiment was repeated at least four times. Data obtained from imaging studies, in which multiple cells were examined in each experimental field, were calculated as the mean [Ca²⁺]_i and pH_i per experiment and then as the mean of multiple experiments. Results are presented as mean±SEM and compared by ANOVA for repeated measures. Tukey-Kramer's correction was used to compensate for multiple testing. Concentration-response curves were fitted by nonlinear regression, and the concentration, in moles per liter, giving 50% response (EC₅₀) (or 50% inhibition, IC₅₀) was determined and pD₂ (or pI₂) calculated as -log EC₅₀ (or IC₅₀). A value of P<.05 was considered significant.

	Results

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Effects of Tyrosine Kinase Inhibition on Ang II–Induced pH_i
Ang II elicited a biphasic effect on pH_i, with an initial and brief acidification followed by a prolonged alkalinization (Fig 1). The acidification period was transient and not related to the Ang II concentration, whereas the alkalinization phase was dose-dependent. Ang II increased pH_i in a concentration-dependent fashion (pD₂=9.2±0.1) (Fig 2). When cells were pretreated with the selective tyrosine kinase inhibitor tyrphostin A-23 (10^-5 mol/L), the pH_i response induced by Ang II was significantly reduced (Fig 2). Tyrphostin A-23 alone did not alter basal pH_i. The inactive analogue tyrphostin A-1 had no effect on Ang II–stimulated pH_i (Fig 2).

View larger version (6K): [in this window] [in a new window]   Figure 1. VSMC pHi response to Ang II (10-8 mol/L). Arrow indicates time of Ang II addition.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of increasing Ang II concentration in the absence and presence of tyrphostin A-23 (selective tyrosine kinase inhibitor) and tyrphostin A-1 (inactive analogue) on pHi responses in VSMCs. Cells were preexposed to 10-5 mol/L of either tyrphostin for 10 minutes before Ang II application. Each data point is mean±SEM of four to six different experiments, with each experimental field comprising 10 to 20 cells. *P<.05, **P<.01 vs other groups.

Effects of Ang II and Tyrphostin A-23 on Simultaneous Measurements of VSMC [Ca²⁺]_i and Contraction
We used VSMCs loaded with fura 2 and grown on rat tail tendon collagen gels to determine whether Ang II simultaneously increases [Ca²⁺]_i and contracts individual cells. The majority of cells (approximately 68%) that had attached and spread out on the collagen gel contracted after exposure to Ang II. Maximal contraction was obtained with 10^-6 mol/L Ang II (Fig 3) and occurred within 5 to 7 minutes (Figs 3 and 4). [Ca²⁺]_i effects induced by Ang II were rapid and transient, with the [Ca²⁺]_i peak occurring within 60 seconds of Ang II application (Fig 4). Thus, the peak [Ca²⁺]_i response preceded the maximum contractile response, indicating a temporal dissociation between the biochemical and functional responses. Ang II increased [Ca²⁺]_i in a dose-dependent manner (pD₂=7.4±0.1) (Fig 5). To determine whether tyrosine kinase pathways influence Ang II–stimulated [Ca²⁺]_i and contractile responses, we assessed the action of tyrphostin A-23 on Ang II–induced effects. Tyrphostin A-23 alone had no effect on VSMC length or shape but significantly reduced the contractile response elicited by Ang II (Fig 3). Tyrphostin A-23 dose-dependently decreased peak [Ca²⁺]_i (pI₂=7.3±0.36) (Fig 6), and latency, the period from stimulus application to the peak response, was increased. Ionomycin significantly increased [Ca²⁺]_i (913±23 nmol/L). Neither tyrphostin A-1 (962±23 nmol/L) nor tyrphostin A-23 (916±45 nmol/L) had any significant effect on ionomycin-induced [Ca²⁺]_i. We performed these studies as positive controls for tyrphostin A-23 to verify that non–tyrosine kinase–dependent pathways are not affected by the tyrosine kinase inhibitor.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effects of Ang II in the absence and presence of tyrphostins on VSMC contraction. Top, Ang II–stimulated contractile responses measured 5 minutes after agonist addition. n=3 experiments per data point. Bottom, Contractile effects of 10-7 mol/L Ang II in cells pretreated with 10-5 mol/L tyrphostin (Tyr) A-23 or A-1. *P<.05 vs other groups.

View larger version (16K): [in this window] [in a new window]   Figure 4. Time course of [Ca2+]i changes (top) and VSMC contractile responses (bottom) after Ang II (10-7 mol/L) stimulation. Ang II was added at 0 seconds. Each data point is mean±SEM of four experiments.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effects of increasing concentrations of Ang II on [Ca2+]i responses in VSMCs. Each data point is mean±SEM of four to six experiments, with each experimental field comprising many cells.

View larger version (15K): [in this window] [in a new window]   Figure 6. Effects of increasing concentrations of tyrphostin A-23 on Ang II–stimulated [Ca2+]i responses in VSMCs. Cells were exposed to tyrphostin A-23 for 10 minutes before Ang II (10-9 mol/L) was added. pI2=7.3±0.36. Each data point is mean of three to five experiments, with each experimental field comprising 10 to 18 cells.

Effects of Tyrphostin A-23 on Ang II–Stimulated [³H]Thymidine and [³H]Leucine Incorporation
Ang II stimulation for 24 hours significantly increased incorporation of [³H]thymidine and [³H]leucine into quiescent VSMCs. Maximal stimulation was observed with 10^-7 mol/L (Fig 7). Ang II–stimulated [³H]leucine incorporation was greater than [³H]thymidine incorporation (Fig 7), suggesting that Ang II may be a more potent hypertrophic than hyperplastic growth factor in rat mesenteric VSMCs. Exposure of cells to tyrphostin A-23 for 24 hours completely inhibited Ang II–stimulated incorporation of [³H]thymidine and [³H]leucine (Fig 7). Tyrphostin A-1 had no effect on Ang II–stimulated cell growth (Fig 7).

View larger version (18K): [in this window] [in a new window]   Figure 7. [3H]Thymidine and [3H]leucine incorporation in VSMCs stimulated with 10-7 mol/L Ang II in the absence and presence of 10-5 mol/L tyrphostins (Tyr). Quiescent cells were stimulated for 24 hours. Growth assays were performed in triplicate and repeated three times. **P<.01 vs other groups.

Characterization of VSMC Angiotensin Receptor Subtype
The angiotensin receptor subtypes involved in pH_i, [Ca²⁺]_i, and cellular growth responses were determined with the nonspecific angiotensin subtype receptor blocker [Sar¹,Ile⁸]Ang II and the AT₁- and AT₂-selective antagonists losartan and PD 123319, respectively. [Sar¹,Ile⁸]Ang II (10^-6 mol/L) and losartan (10^-6 mol/L) abolished Ang II–mediated responses, whereas PD 123319 did not alter Ang II–stimulated effects (Fig 8). Treatment of VSMCs with 10^-6 mol/L of any of the antagonists alone had no effect on basal pH_i, [Ca²⁺]_i, or [³H]thymidine or [³H]leucine incorporation.

View larger version (33K): [in this window] [in a new window]   Figure 8. Effects of [Sar1,Ile8]Ang II (Sar), losartan, and PD 123319 (PD) on Ang II–elicited pHi responses (top left), Ang II–induced [Ca2+]i responses (top right), and Ang II–stimulated [3H]thymidine (bottom left) and [3H]leucine (bottom right) incorporation in VSMCs. Cells were preincubated with either Ang II receptor antagonist (10-6 mol/L) for 10 minutes before Ang II (10-7 mol/L for [3H]thymidine and [3H]leucine incorporation and 10-9 mol/L for pHi and [Ca2+]i) was added. *P<.05, **P<.01 vs other groups.

	Discussion

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In the present study, we explored the intracellular second messengers underlying Ang II–mediated contraction and growth in VSMCs and evaluated whether tyrosine kinases play a role in Ang II–induced cellular responses. Our data demonstrate that Ang II increases pH_i and stimulates both the contraction and growth of VSMCs. These effects, which are mediated via the AT₁ receptor subtype, are inhibited to varying degrees by the selective tyrosine kinase inhibitor tyrphostin A-23, suggesting that Ang II effects are partially dependent on tyrosine kinase–linked signaling pathways, whereas the inactive analogue tyrphostin A-1 was ineffective.

VSMC pH_i is an important second messenger in the transduction of contractile and growth stimuli. Intracellular alkalinization stimulates DNA synthesis and cell growth and increases actin-myosin sensitivity to Ca²⁺, thereby increasing vascular contraction and tone. In the present study, Ang II increased VSMC pH_i in a dose-dependent manner, and it may be through these responses that Ang II mediates, at least in part, its contractile and growth effects. Mechanisms underlying Ang II–induced pH_i changes are linked to PLC-induced hydrolysis of inositol phospholipids and activation of the PKC-Na⁺-H⁺ pathway.^{7 34} Besides this classic pathway, it may be possible that protein tyrosine kinases are also involved. The importance of tyrosine phosphorylation in Ang II signal transduction has recently been reviewed.³⁵ Although there is increasing evidence implicating tyrosine kinases in [Ca²⁺]_i homeostasis, there are few data on the role of tyrosine kinase pathways in pH_i regulation. Our results demonstrate that tyrphostin A-23 attenuated Ang II–stimulated pH_i responses, suggesting a significant contribution of protein tyrosine kinases in the regulation of pH_i. This may be through PKC, the Na⁺-H⁺ antiport, or possibly through pathways that influence Ca²⁺, such as the Na⁺-Ca²⁺ exchanger. Tyrphostins, developed by Levitzki,³⁶ bind the substrate subsite of the protein tyrosine kinase domain, have reversible inhibitory effects, and are highly specific for tyrosine phosphorylation. Other tyrosine kinase inhibitors, such as genistein, are less specific for tyrosine kinases, and although we studied them previously,²¹ we did not use them here. In our present investigations, the inactive analogue tyrphostin A-1 did not alter Ang II–mediated responses, whereas tyrphostin A-23 significantly attenuated agonist-elicited responses, suggesting that effects were specifically linked to tyrosine kinases. Our results obtained with ionomycin and the inability of tyrphostin A-23 to inhibit ionomycin-elicited responses further support the tyrosine kinase–specific effects reported here.

We also investigated VSMC functional responses induced by Ang II. In the present study, we used VSMCs grown on collagen gels in the presence of 0.5% fetal calf serum to study contraction. Cells do not proliferate significantly under these conditions and retain a contractile phenotype, as evidenced by their ability to contract and by expression of cytoskeletal markers.³⁰ Ang II contracted individual VSMCs and dose-dependently increased [Ca²⁺]_i. However, there was a temporal difference between the generation of the [Ca²⁺]_i transient and contraction. [Ca²⁺]_i increased before contraction, suggesting that elevated [Ca²⁺]_i may sensitize actin-myosin crossbridge formation before contraction is induced. It is also possible that the prolonged alkalinization phase elicited by Ang II stimulation plays a role. When cells were preincubated for 10 minutes with tyrphostin A-23, Ang II–induced [Ca²⁺]_i and contractile effects were reduced but not completely abolished, suggesting only partial dependency on tyrosine kinases. Our findings on isolated cultured cells support other studies which demonstrated that tyrosine kinase inhibitors reduced Ang II–stimulated vascular contraction in intact vessels.^{37 38} These data together with our results indicate that tyrosine kinase activity significantly participates in the regulation of signal transduction associated with Ang II–stimulated contraction of smooth muscle.

Besides its vasoconstrictor role, Ang II has also been suggested to act as a cellular growth factor and thus contribute to pathological conditions such as atherosclerosis and hypertension. However, the mode by which this vasoconstrictor peptide affects cell growth still remains controversial. In some studies, Ang II has been demonstrated to have proliferative effects on serum-deprived VSMCs, and in other reports, it has been shown either to have no proliferative effects or to have hypertrophic effects without hyperplasia.^{39 40 41} It has also been reported that Ang II promotes mitogenesis when combined with other growth promoters or with minimal concentrations of serum.^{3 42} In the present study, we determined the effect of Ang II on DNA and protein synthesis as indicators of cell hyperplasia and hypertrophy, respectively. Ang II at a concentration of 10^-7 mol/L significantly increased both DNA and protein synthesis in cultured VSMCs in serum-free medium containing selenium, iron, and transferrin. The effect on protein synthesis was greater than that on DNA synthesis, suggesting that Ang II may have a more potent hypertrophic than hyperplastic effect in cells cultured under conditions described in the present study. These results are in agreement with others in which it was reported that Ang II induces hypertrophy, not hyperplasia, of cultured rat aortic VSMCs.^{39 42} Almost all growth factors mediate their mitogenic effects through tyrosine kinase–linked signaling pathways.^{16 43} In the present study, tyrphostin A-23 attenuated both DNA and protein synthesis, whereas its inactive analogue tyrphostin A-1 did not alter Ang II–stimulated growth. Tyrosine kinase inhibition negatively influenced [Ca²⁺]_i and pH_i responses, which may be contributory factors in the attenuated growth effects of Ang II observed in the presence of tyrphostin A-23. These results indicate the importance of Ang II in VSMC growth and demonstrate that tyrosine kinases play a significant role in Ang II– stimulated mitogenesis.

In our study, we characterized the receptor subtypes mediating Ang II responses in WKY-derived VSMCs. [Sar¹,Ile⁸]Ang II and losartan completely blocked the cellular effects of Ang II, whereas PD 123319 had no effect. Similar results have been reported with cells derived from Sprague-Dawley rats.⁴⁴ Thus, Ang II appears to mediate contraction as well as hypertrophy and hyperplasia of VSMCs exclusively via the AT₁ receptor subtype.

In conclusion, our results provide new evidence for a role of tyrosine kinases in Ang II–mediated pH_i responses in VSMCs and indicate that tyrosine kinases participate in the regulation of signal transduction that is associated with AT₁ receptor subtype–mediated contraction and growth. These data further support a role of tyrosine kinase–dependent pathways in Ang II signaling in vascular smooth muscle.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1, AT2 = angiotensin type 1, type 2 (receptor) [Ca2+]i = intracellular free Ca2+ concentration DMEM = Dulbecco's modified Eagle's medium pHi = intracellular pH PKC = protein kinase C VSMC = vascular smooth muscle cell WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension. The authors thank Carole Tremblay for her secretarial help.

Received September 19, 1996; first decision October 21, 1996; accepted January 10, 1997.

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