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(Hypertension. 1996;27:1097-1103.)
© 1996 American Heart Association, Inc.

Articles

Tyrosine Kinase Signaling Pathways Modulate Angiotensin II–Induced Calcium ([Ca²⁺]_i) Transients in Vascular Smooth Muscle Cells

R.M. Touyz; E.L. Schiffrin

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

	Abstract

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Abstract Tyrosine kinases have been implicated in vascular smooth muscle cell proliferation and contraction. Underlying mechanisms may involve Ca²⁺-dependent pathways. This study assesses relationships between angiotensin II (Ang II)–stimulated phospholipase C–mediated Ca²⁺ transients and tyrosine kinase–dependent pathways in vascular smooth muscle cells. Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was measured in primary cultured unpassaged vascular smooth muscle cells derived from mesenteric resistance vessels of Wistar-Kyoto rats with the use of fura 2 methodology. [Ca²⁺]_i effects of Ang II (1 nmol/L) were determined in vascular smooth muscle cells in which tyrosine kinase pathways were stimulated by insulin (70 µU/mL; 0.5 nmol/L), insulin-like growth factor-I (1 ng/mL; 0.13 nmol/L), or platelet-derived growth factor-BB (1 ng/mL; 0.04 nmol/L) and in cells in which tyrosine kinase was inhibited by specific inhibitors (1 µmol/L tyrphostin A-23 and genistein). Ang II elicited a rapid and transient [Ca²⁺]_i response (from 94±8 to 239±5.8 nmol/L). Activation of the receptor tyrosine kinase by insulin, platelet-derived growth factor, and insulin-like growth factor-I significantly reduced (P<.01) Ang II–induced [Ca²⁺]_i to 161±7, 189±3.7, and 183±5 nmol/L, respectively. In the presence of tyrphostin A-23 and genistein, Ang II–stimulated [Ca²⁺]_i remained persistently elevated and failed to return to basal levels. Tyrphostin A-1, the inactive tyrphostin analogue, had no significant effect on Ang II–induced [Ca²⁺]_i. This study demonstrates that activation of tyrosine kinase pathways reduces Ang II–elicited [Ca²⁺]_i responses, whereas tyrosine kinase inhibition prevents [Ca²⁺]_i recovery after agonist stimulation. Interaction between tyrosine kinase– and phospholipase C–dependent signaling pathways modulates vascular smooth muscle cell [Ca²⁺]_i responses to Ang II.

Key Words: angiotensin II • calcium • protein-tyrosine kinase • insulin • platelet-derived growth factor • muscle, smooth, vascular

	Introduction

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Contraction of vascular smooth muscle is a complex process involving multiple signal transduction regulatory pathways. Agonist-stimulated phospholipase C–mediated pathways have been well defined in VSMCs,^{1 2} and increasing evidence now suggests that tyrosine kinases, enzymes that phosphorylate proteins on tyrosine residues, also participate in VSMC function.^{3 4} The extent to which these enzymes contribute to smooth muscle signal transduction is unknown, but it may be possible that there is a cross-regulation between tyrosine kinase– and phospholipase C–linked pathways.

Stimulation of VSMCs with vasoconstrictors such as Ang II, arginine vasopressin, norepinephrine, and endothelin-1 causes a rapid phospholipase Cß–mediated hydrolysis of inositol phospholipids, resulting in the production of two second messengers, 1,2-diacylglycerol and inositol triphosphate.⁵ Diacylglycerol activates protein kinase C, which influences Na⁺-H⁺ exchange, and inositol triphosphate mobilizes Ca²⁺ from intracellular Ca²⁺ stores, resulting in increased [Ca²⁺]_i and subsequent vascular smooth muscle contraction. Besides this classic pathway, it may be possible that protein tyrosine phosphorylation is also involved in the intracellular signaling of vasoconstrictor agonists in VSMCs. Several vasoactive peptides, including Ang II, endothelin-1, vasopressin, norepinephrine, and serotonin, induce tyrosine phosphorylation of many proteins in cultured cells.^{6 7 8 9} In intact vessels, tyrosine kinase inhibitors reversibly inhibit agonist-induced phasic and tonic contractions, suggesting that tyrosine kinase activity significantly participates in the regulation of signal transduction that is associated with smooth muscle contraction.^{10 11} Although the mechanisms underlying tyrosine kinase involvement in vasoconstriction are not well understood, Ca²⁺ probably plays a role. In cultured VSMCs, the Ca²⁺ ionophore ionomycin induces tyrosine phosphorylation, suggesting that Ca²⁺ can stimulate tyrosine kinase activity.⁴ Recent studies have demonstrated that various mitogenic factors, including PDGF, epidermal growth factor, fibroblast growth factor, and insulin, can induce a transient increase in [Ca²⁺]_i.^{12 13 14 15} These factors bind to receptor tyrosine kinases, which activate phospholipase C to convert phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol triphosphate, which in turn stimulates sarcoplasmic reticular release of Ca²⁺ and a consequent elevation in [Ca²⁺]_i.¹⁵ Studies investigating effects of tyrosine kinase inhibitors have also implicated a role of protein tyrosine phosphorylation in [Ca²⁺]_i regulation. In rabbit ear artery smooth muscle cells, the Ca²⁺ current is inhibited by the tyrosine kinase inhibitors genistein and tyrphostin A-23,¹⁶ and in rat mesenteric arteries, genistein decreases [Ca²⁺]_i.¹⁷ Interestingly, inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis but not intracellular free Ca²⁺ release in rat vascular smooth muscle.¹⁸

Stimulation of tyrosine kinase receptors, specifically insulin and IGF and epidermal growth factor receptors, modulate - and ß-adrenergic receptor–mediated events,^{19 20} and endothelin-1 receptor–linked responses are suppressed by interleukin-1ß.²¹ It is unknown whether other G protein–coupled receptors such as Ang II receptors are also influenced by tyrosine kinases and whether there is functional interaction between receptor tyrosine kinase and G protein–coupled receptor pathways.

In the present study, we investigated the possible involvement of tyrosine kinases in the intracellular signaling pathway of Ang II by determining effects of tyrosine kinase modulation on Ang II–stimulated [Ca²⁺]_i responses in primary cultured VSMCs.

	Methods

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Materials
All chemicals were of the highest reagent grade available. Ang II (rat) was from Peninsula Laboratories Inc. Genistein, tyrphostin A-1, and tyrphostin A-23 were from Calbiochem. Fura 2-acetoxymethyl ester (fura 2-AM) and pluronic F-127 were obtained from Molecular Probes Inc. DMSO was from Anachemia Canada Inc. Dulbecco's modified Eagle's medium was from GIBCO Canada, and Ham's F-12 medium was from Flow Laboratories Inc. IGF-I and PDGF-BB were from Boehringer Mannheim Biochemica. The other chemicals were obtained from Sigma Chemical Co, Fischer Scientific Co, and BDH Inc.

Rats
Male Wistar-Kyoto rats aged 17 weeks were purchased from Taconic Farms Inc (Germantown, NY). The rats were housed in the animal unit at the Clinical Research Institute of Montreal and were exposed to a 12-hour light/dark cycle at constant temperature (22°C) and relative humidity (60%). The study was approved by the Animal Ethics Committee of the Clinical Research Institute of Montreal and carried out according to the recommendations of the Canadian Council for Animal Care.

Cell Culture
The rats were killed by decapitation. Primary unpassaged cells derived from the mesenteric arteries of the rats were prepared according to previously described techniques.²² Briefly, mesenteric arteries (the main mesenteric vessel as well as first- and second-order branches) were cleaned of all adipose and connective tissue. Smooth muscle cells were dissociated by digestion of vascular arcades with 0.12 mg/mL elastase, 2 mg/mL collagenase (type 1), 0.36 mg/mL soybean trypsin inhibitor, 2 mg/mL bovine serum albumin, and 100 µg/mL gentamycin in Ham's F-12 medium for 60 minutes at 37°C. The tissue was filtered through a 100-µm nylon mesh, and the cell suspension was centrifuged at 200g and resuspended in Dulbecco's modified Eagle's medium containing 10% heat-inactivated calf serum, 2 mmol/L L-glutamine, 20 mmol/L HEPES (pH 7.4), 100 U/mL penicillin, and 100 µg/mL streptomycin. 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. The number of cells plated was approximately 3x10⁴ cells per milliliter. At confluence, the cells had the typical "hill-and-valley" pattern and tested positive for smooth muscle cell -actin, indicating the absence of contamination by other vascular cells such as fibroblasts or endothelial cells. Confluent cell cultures were rendered quiescent by serum deprivation and were maintained in serum-free medium for 36 hours before experimentation.

Measurement of [Ca²⁺]_i
The cells grown on glass coverslips were washed three times with 2 mL modified Hanks' buffered saline solution containing (mmol/L) NaCl 137, KCl 5.4, NaHCO₃ 4.2, Na₂HPO₄ 3, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, glucose 10, and HEPES 5 (pH 7.4). The washed cells were loaded with fura 2-AM (4 µmol/L), which was dissolved in DMSO with 0.02% pluronic acid. The final DMSO concentration was less than 0.1% and had no effect on basal [Ca²⁺]_i. The cells were incubated for 30 minutes at 37°C in a humidified incubator (95% air/5% CO₂). The loaded cells were then washed three times with warmed (37°C) buffer and incubated for a further 15 minutes to ensure complete deesterification. Cells were finally washed once with fresh buffer. Under these loading conditions, the ratiometric (343/380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentation of fura 2. The coverslip containing cells was placed in a stainless steel chamber and mounted on the stage of an inverted microscope (Axiovert 135, Zeiss). Four glass rings (4 to 5 mm diameter) were placed on the coverslip according to previously described methods.²³ This allowed for four separate experiments for each coverslip.

[Ca²⁺]_i was measured in multiple cells simultaneously by fluorescent digital imaging with the Axiovert 135 inverted microscope and Attofluor Digital Fluorescence system (Attofluor Ratiovision, Zeiss). An emission wavelength of 520 nm and alternating excitatory wavelengths of 343 and 380 nm were used for measurement of fura 2 fluorescence. The Attofluor system was calibrated according to previously described methods²⁴ with the following equation: [Ca²⁺]_i (nmol/L)=K_d[(R-R_min)/(R_max-R)]xß, where R is the ratio of fluorescence at 343 and 380 nm; R_max and R_min are the ratios for fura-free acid at 343 and 380 nm in the presence of saturating calcium and zero calcium, respectively; and ß is the ratio of fluorescence of fura 2 at 380 nm in zero and saturating calcium.²⁵ K_d is the dissociation constant of fura 2 for Ca²⁺, assumed to be 224 nmol/L. Video images of fluorescence at 520 nm emission were obtained by an intensified charge-coupled device (CCD) 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 after background subtraction by the 380-nm image after background subtraction.

Protocols for Reagent Applications
The effects of various concentrations of Ang II (10^-11 to 10^-6 mol/L) on VSMC [Ca²⁺]_i were examined. In subsequent experiments, [Ca²⁺]_i responses elicited by a fixed concentration of 1 nmol/L Ang II were assessed in the presence of agents that activate a tyrosine kinase receptor–linked pathway (1 ng/mL [0.13 nmol/L] IGF-I, 1 ng/mL [0.04 nmol/L] PDGF, or 70 µU/mL [0.5 nmol/L] insulin) and in the presence of agents that inhibit tyrosine kinases (1 µmol/L tyrphostin A-23 or genistein). The effect of tyrphostin A-1 (1 µmol/L), an inactive tyrphostin analogue, was also assessed. For these experiments, cells were preincubated for 10 minutes with either tyrosine kinase modulator before Ang II addition (1 nmol/L final concentration). Cells were used for single applications, and repetitive determinations were not performed. The tyrphostins exhibited some intrinsic fluorescence that was corrected for in the final measurements.

Ang II, IGF-I, PDGF, and insulin were dissolved in the physiological buffer. Genistein and the tyrphostins were dissolved in DMSO, with the maximal DMSO concentration being less than 1%. Control experiments in which the vehicle was added were made before and after application of the agents.

Statistical Analysis
Mean values were calculated for multiple cells in each experiment, and then the mean of the experiments was determined and used for analysis. Ten to 15 cells were examined in each experimental field, and experiments were repeated four to eight times. Values are expressed as mean±SE. Comparison of mean values was performed by Student's t test where appropriate or by ANOVA followed by the Tukey-Kramer correction for multiple testing. A value of P<.05 was considered statistically significant. Concentration-response curves were fitted by nonlinear regression, and the concentration giving 50% of the maximal response (EC₅₀) was determined and pD₂ calculated as -log EC₅₀ (moles per liter).

	Results

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Effects of IGF-I, PDGF, and Insulin on Ang II–Stimulated [Ca²⁺]_i
The growth factors IGF-I, PDGF, and insulin all induced a small but significant (P<.05) increase in [Ca²⁺]_i (Fig 1). The [Ca²⁺]_i response was rapid and transient and returned to baseline within 200 seconds after agonist application (Fig 2). Ang II increased [Ca²⁺]_i in a dose-dependent manner, with a pD₂ value of 7.9±0.3. At a concentration of 1 nmol/L, which is a low pharmacological dose that elicits significant responses, Ang II increased [Ca²⁺]_i to 239±5.8 nmol/L (Fig 1). Ang II–elicited [Ca²⁺]_i elevation was biphasic, with an initial peak followed by a plateau phase in which [Ca²⁺]_i was sustained at 118 nmol/L, which was significantly higher than basal [Ca²⁺]_i (90±2.2 nmol/L, P<.05; Fig 2). Within 8 minutes after stimulation, [Ca²⁺]_i levels had returned to baseline. When cells were preincubated for 10 minutes with IGF-I, PDGF, or insulin, all of which mediate effects through the tyrosine kinase pathway, Ang II–induced [Ca²⁺]_i responses were significantly reduced (P<.001, Ang II [Ca²⁺]_i versus growth factor plus Ang II [Ca²⁺]_i) (Fig 1). In addition, [Ca²⁺]_i recovery to baseline was facilitated in the presence of the growth factors.

View larger version (16K): [in this window] [in a new window]   Figure 1. Bar graph shows basal and Ang II (1 nmol/L)–stimulated [Ca2+]i responses in the absence and presence of insulin (70 µU/mL; 0.5 nmol/L), PDGF (1 ng/mL; 0.04 nmol/L), and IGF-I (IGF 1 in figure) (1 ng/mL; 0.13 nmol/L). *P<.05, **P<.01 vs basal; ***P<.001 vs other groups. Numbers in parentheses indicate number of experiments, with each experimental field comprising 10 to 15 cells.

View larger version (9K): [in this window] [in a new window]   Figure 2. Representative tracings of [Ca2+]i responses to 1 nmol/L Ang II. A represents the [Ca2+]i response without tyrosine kinase manipulation; B is the Ang II–induced response in cells preexposed for 10 minutes to 70 µU/mL (0.5 nmol/L) insulin; and C is the response in cells preexposed for 10 minutes to 1 µmol/L tyrphostin A-23. Arrow indicates time of Ang II application.

Effects of Tyrosine Kinase Inhibitors, Genistein, and Tyrphostins on Ang II–Stimulated [Ca²⁺]_i
For determination of the effects of tyrosine kinase inhibition on agonist-stimulated [Ca²⁺]_i transients, cells were preincubated for 10 minutes with genistein, tyrphostin A-23, or the inactive tyrphostin analogue A-21 before addition of IGF-I, PDGF, insulin, and Ang II. Both genistein (a selective tyrosine kinase inhibitor) and tyrphostin A-23 (a highly selective tyrosine kinase inhibitor) completely blocked the [Ca²⁺]_i response induced by IGF-I, PDGF, and insulin (Figs 3 and 4). Genistein had no significant effect on Ang II–stimulated peak [Ca²⁺]_i (207±14 nmol/L), whereas tyrphostin A-23 significantly reduced peak Ang II–induced [Ca²⁺]_i from 239±5.8 nmol/L (absence of tyrphostin A-23) to 195±6 nmol/L (presence of tyrphostin A-23) (P<.05; Figs 3 and 4). When cells were preincubated with the tyrosine kinase inhibitors plus IGF-I, PDGF, or insulin, the Ang II–induced peak [Ca²⁺]_i response was significantly reduced compared with Ang II–stimulated [Ca²⁺]_i in the absence of tyrosine kinase modulators (Figs 3 and 4).

View larger version (17K): [in this window] [in a new window]   Figure 3. Bar graph shows Ang II (1 nmol/L)–stimulated responses in cells preexposed to tyrphostin A-23 (Tyr) (1 µmol/L) alone or in combination with insulin (Ins) (70 µU/mL; 0.5 nmol/L), PDGF (1 ng/mL; 0.04 nmol/L), or IGF-I (IGF 1 in figure) (1 ng/mL; 0.13 nmol/L). **P<.01 vs Ang II; ***P<.001 vs Ang II; +P<.001 vs basal; P<.001. Numbers in parentheses indicate number of experiments, with each experimental field comprising 10 to 15 cells.

View larger version (18K): [in this window] [in a new window]   Figure 4. Bar graph shows Ang II (1 nmol/L)–stimulated [Ca2+]i responses in cells preincubated with genistein (Gen) (1 µmol/L) alone or in combination with insulin (Ins) (70 µU/mL; 0.5 nmol/L), PDGF (1 ng/mL; 0.04 nmol/L), or IGF-I (IGF 1 in figure) (1 ng/mL; 0.13 nmol/L). *P<.05 vs Ang II; ***P<.001 vs Ang II; +P<.001 vs basal; P<.001. Numbers in parentheses indicate number of experiments, with each experimental field comprising 10 to 15 cells.

[Ca²⁺]_i Recovery After Ang II Stimulation in the Presence of Tyrosine Kinase Inhibitors
In the presence of tyrosine kinase inhibitors, [Ca²⁺]_i recovery to basal levels was significantly blocked, and even 15 minutes after Ang II application [Ca²⁺]_i was still significantly elevated (Figs 2 and 5). Although [Ca²⁺]_i recovery was inhibited by both genistein and tyrphostin A-23, Ang II–elicited [Ca²⁺]_i was significantly higher in the presence of genistein compared with [Ca²⁺]_i in the presence of tyrphostin A-23 (P<.001, Fig 3). Similar patterns of Ang II–stimulated [Ca²⁺]_i occurred in cells that had been exposed to the tyrosine kinase inhibitors plus IGF-I, PDGF, or insulin (Fig 6).

View larger version (14K): [in this window] [in a new window]   Figure 5. Time course of [Ca2+]i recovery after Ang II (1 nmol/L) stimulation in the absence and presence of 70 µU/mL (0.5 nmol/L) insulin, 1 ng/mL (0.04 nmol/L) PDGF, and 1 ng/mL (0.13 nmol/L) IGF-I (IGF 1 in figure). Maximal stimulated [Ca2+]i was taken at 0 minutes, and recovery to basal values was measured thereafter. *P<.05 vs growth factor plus Ang II counterpart; **P<.01 vs growth factor plus Ang II–stimulated peak [Ca2+]i.

View larger version (17K): [in this window] [in a new window]   Figure 6. Time course of [Ca2+]i recovery after Ang II (1 nmol/L) stimulation in the absence and presence of tyrphostin A-23 (Tyr, 1 µmol/L) and genistein (Gen, 1 µmol/L). a, Results in the absence of growth factors; b, time course of [Ca2+]i recovery when cells were preincubated with tyrphostin A-23 plus PDGF or IGF-I. Maximal stimulated [Ca2+]i was taken at 0 minutes, and recovery to basal values was measured thereafter. **P<.01 vs Ang II–stimulated peak [Ca2+]i; +P<.001 vs Ang II counterpart; *P<.01 vs tyrphostin A-23.

Tyrphostin A-1, the inactive tyrphostin analogue, had no significant effect on Ang II–induced [Ca²⁺]_i responses (229±18 nmol/L in the presence of tyrphostin A-1) or on [Ca²⁺]_i recovery.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we assessed the effects of tyrosine kinase modulators on Ang II–induced [Ca²⁺]_i responses in cultured VSMCs. Since Ang II mediates its effects through G protein–coupled membrane receptors that activate phospholipase C, this study indirectly investigates the interaction between tyrosine kinase– and phospholipase C–linked pathways. Results from the present study demonstrate that (1) IGF-I, PDGF, and insulin, at physiological or low pharmacological concentrations, induce a transient rise in [Ca²⁺]_i; (2) activation of tyrosine kinase signaling pathways by IGF-I, PDGF, and insulin attenuates Ang II–stimulated [Ca²⁺]_i and promotes [Ca²⁺]_i recovery; and (3) tyrosine kinase inhibition reduces peak [Ca²⁺]_i and prevents the return of [Ca²⁺]_i to basal levels after Ang II stimulation. Although the inhibitory effects of genistein and tyrphostin A-23 on tyrosine phosphorylation were not directly assessed in the present study, this property of these agents at the concentrations used has been previously well documented.^{26 27} Furthermore, in this study we have demonstrated that these tyrosine kinase inhibitors completely blocked the [Ca²⁺]_i rise induced by IGF-I, PDGF, and insulin. In addition, tyrphostin A-1, the inactive tyrphostin analogue, had no significant effect on agonist-induced responses. These data indicate that in our cell preparation, genistein and tyrphostin A-23, at a concentration of 1 µmol/L, inhibit receptor tyrosine kinase activity.

The [Ca²⁺]_i-elevating effects of growth factors in VSMCs have been previously demonstrated.^{12 13 14 15 28} Mogami and Kojima²⁹ reported that PDGF induces a rapid increase in [Ca²⁺]_i followed by a sustained plateau phase that is mediated by a calcium entry mechanism distinct from L-type voltage-dependent calcium channels. In the present study, IGF-I, PDGF, and insulin induced a transient [Ca²⁺]_i peak that returned rapidly to basal levels. At the concentrations we used, we did not observe a [Ca²⁺]_i plateau phase. However, PDGF at higher concentrations (>5 ng/mL, data not shown) induced a biphasic [Ca²⁺]_i response similar to that reported by others.²⁹ Epidermal growth factor, which also acts through receptor tyrosine kinases, has been shown to cause rapid increases in intracellular Ca²⁺ fluxes and to modulate acute regulation of blood flow. In sheep, parenteral administration of epidermal growth factor results in acute reduction of peripheral resistance and an increase in cardiac output.³⁰ In dogs, intra-arterial injection of epidermal growth factor induces vasodilation,³¹ whereas in intact rat vessel strips, it elicits a contractile response.³⁰ These data suggest that vascular smooth muscle is a potential target for growth factors and that tyrosine kinase pathways are involved in the regulation of vascular smooth muscle contraction.

The major finding of the present study is that Ang II–elicited [Ca²⁺]_i responses are modulated by tyrosine kinases. Ang II has been shown to stimulate tyrosine phosphorylation, and although interaction between tyrosine kinase–stimulated pathways and vasoconstrictor-mediated actions have been previously investigated, little is known about the direct effects of receptor tyrosine kinase activation on Ang II–mediated responses. Ang II is a potent vasoconstrictor and mitogen that elicits its contractile (and possibly mitogenic) effects by increasing VSMC [Ca²⁺]_i. As demonstrated here as well as in our previous studies^{14 23 32} and those of others,^{33 34} Ang II increases [Ca²⁺]_i in a dose-dependent manner. When cells were prestimulated with IGF-I, PDGF, or insulin, the Ang II–induced [Ca²⁺]_i responses were significantly attenuated. These effects are not due to tachyphylaxis, as we have demonstrated that repeated stimulation of cells with various agents that raise [Ca²⁺]_i do not alter Ang II–elicited [Ca²⁺]_i responses.¹⁴ In previous studies, we and others reported that insulin (at pharmacological and physiological concentrations) reduces agonist-induced contractile and [Ca²⁺]_i responses in cultured VSMCs and in intact vessels,^{14 35 36 37} possibly by decreasing calcium influx via voltage-dependent channels or by reduced inositol triphosphate–releasable calcium stores.^{38 39} Those data support our present results, which suggest that activation of tyrosine kinase signaling pathways may function as a negative modulator of Ang II–induced [Ca²⁺]_i and contractile responses.

As far as we know, this study demonstrates for the first time that tyrosine kinase inhibitors modulate Ang II–stimulated [Ca²⁺]_i in VSMCs. The relationship between Ang II and tyrosine kinases has been investigated in contractile studies of muscle strips and isolated vessels but has not been previously studied in primary cultured unpassaged VSMCs. In gastric smooth muscle and porcine coronary artery, Ang II induced a contractile response similar to that elicited by epidermal growth factor.³⁰ In these preparations, the Ang II effect was blocked by tyrphostin and genistein at concentrations that selectively block epidermal growth factor contraction, implying that a tyrosine kinase pathway is involved in the contractile actions of Ang II. Similar pathways have been described for vasopressin and prostaglandin F₂.⁴⁰ In the present study, neither genistein nor tyrphostin A-23 blocked the Ang II–induced [Ca²⁺]_i peak, but they significantly inhibited [Ca²⁺]_i recovery to basal levels. These results suggest that tyrosine phosphorylation plays a role in the regulation of Ca²⁺ transients and in Ca²⁺ normalization after Ang II stimulation. Although the underlying mechanisms for this are unknown, it is possible that Ca²⁺ efflux pathways and/or Ca²⁺ refilling mechanisms are blocked. The major transporter for Ca²⁺ efflux in VSMCs is via the plasma membrane Ca²⁺-ATPase pump, which mobilizes Ca²⁺ out of the cell.⁴¹ Ca²⁺ reentry into reticular stores is facilitated by sarcoplasmic reticular Ca²⁺-ATPase.⁴² Since Ca²⁺-ATPase is stimulated by insulin,³⁹ which is linked to a tyrosine kinase receptor–mediated pathway, it is possible that inhibition of tyrosine kinases may inhibit Ca²⁺-ATPase activity, thereby preventing Ca²⁺ efflux and Ca²⁺ filling into Ca²⁺ stores. This would result in sustained [Ca²⁺]_i elevation and possibly in [Ca²⁺]_i overload. These proposals await clarification. Genistein and tyrphostin A-23 may also exert their effects via tyrosine kinase–independent pathways. Both agents influence Ca²⁺ and K⁺ channels,^{16 43} and genistein has been shown to inhibit postreceptor effects of insulin without inhibiting the insulin receptor tyrosine kinase.⁴⁴

Genistein failed to alter the Ang II–induced [Ca²⁺]_i peak, whereas tyrphostin A-23 significantly reduced maximum [Ca²⁺]_i. Also, sustained [Ca²⁺]_i after Ang II application was significantly higher in genistein-treated cells compared with that in tyrphostin A-23–treated cells. These differential responses may be attributable to the fact that genistein has some effect on other protein kinases and signal transduction systems besides tyrosine kinases, whereas tyrphostin A-23 selectively inhibits tyrosine kinases.⁴⁵ Yang et al⁴⁶ also demonstrated variable inhibitory effects of genistein and tyrphostin. In guinea pig gastric circular muscle, genistein partially inhibited prostaglandin F₂–induced contraction, whereas tyrphostin had no effect; and in Ang II–stimulated muscle, both genistein and tyrphostin inhibited contraction by only 43%. Unlike the present findings and those of Yang et al, Marrero et al⁴⁷ recently reported that Ang II–mediated inositol triphosphate production, and probably [Ca²⁺]_i, are inhibited by genistein. In our study, tyrphostin A-23 but not genistein inhibited peak [Ca²⁺]_i. These contradictory findings may be related to the fact that we studied primary cultured unpassaged mesenteric VSMCs that were stimulated with 10^-9 mol/L Ang II in the presence of 1 µmol/L genistein, whereas Marrero et al examined passaged aortic VSMCs that had been exposed to 10^-7 mol/L Ang II and 120 µmol/L genistein.

In conclusion, data from the present study raise the possibility that tyrosine kinases influence [Ca²⁺]_i responses elicited by Ang II in VSMCs. It is likely that there is cross-regulation between tyrosine kinase–linked receptors and G protein–coupled receptors that may play an important role in the modulation of [Ca²⁺]_i and vascular smooth muscle function.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular free Ca2+ concentration Ang II = angiotensin II DMSO = dimethyl sulfoxide IGF-I = insulin-like growth factor-I PDGF = platelet-derived growth factor-BB VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by a group grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension. Dr Touyz is the recipient of a fellowship from the Medical Research Council of Canada.

	Footnotes

 
Reprint requests to Dr E.L. Schiffrin, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, H2W 1R7, Canada.

Received September 21, 1995; first decision December 21, 1995; accepted January 8, 1996.

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