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(Hypertension. 2002;40:700.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Angiotensin II Triggers EGFR Tyrosine Kinase-Dependent Ca²⁺ Influx in Afferent Arterioles

From the Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Neb.

Correspondence to Pamela K. Carmines, PhD, Department of Physiology and Biophysics, University of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198–4575. E-mail pcarmines{at}unmc.edu

	Abstract

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We previously reported that inhibition of epidermal growth factor receptor tyrosine kinase activity attenuates renal arteriolar contractile responses to angiotensin II. We performed the present experiments to determine if epidermal growth factor receptor tyrosine kinase activity contributes to the afferent arteriolar intracellular [Ca²⁺] response to angiotensin II. Afferent arterioles were dissected from rat kidney and intracellular [Ca²⁺] was monitored with the use of fura-2. In normal Ringer’s bath containing 1.5 mmol/L Ca²⁺, basal intracellular [Ca²⁺] averaged 95±7 nmol/L and 100 nmol/L angiotensin II caused a rapid rise (peak =75±10 nmol/L) that waned to a plateau averaging 24±5 nmol/L above baseline. Pretreatment with 100 nmol/L AG1478 (epidermal growth factor receptor tyrosine kinase inhibitor) reduced both the peak and the plateau stages of the angiotensin II response (peak =42±7 nmol/L; plateau =8±4 nmol/L). A structurally unrelated epidermal growth factor receptor tyrosine kinase inhibitor also suppressed the peak response to angiotensin II, whereas tyrosine phosphatase inhibition enhanced the plateau phase of the response. In the presence of 100 nmol/L extracellular Ca²⁺, the angiotensin II response was characterized by a peak of diminished magnitude (=49±10 nmol/L; P<0.05 versus the response in normal Ringer’s bath) with no plateau, and this response was unaffected by AG1478. Moreover, angiotensin II stimulation of divalent cation influx (Mn²⁺ quench of fura-2 fluorescence) was decreased significantly by AG1478. We conclude that epidermal growth factor receptor tyrosine kinase activity contributes to the afferent arteriolar intracellular [Ca²⁺] response to angiotensin II and that this process involves promotion of Ca²⁺ influx.

Key Words: angiotensin • calcium • epidermal growth factor • renal circulation • protein kinases • muscle, smooth vascular

	Introduction

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In recent years, cross-talk between G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) has been well documented.¹ The resulting combinatorial signaling events allow GPCRs to take advantage of pathways downstream of RTKs to influence cell function. For example, GPCR signaling systems require epidermal growth factor receptor (EGFR) tyrosine kinase activation to trigger mitogen-activated protein kinase activation and transmission of mitogenic signals to the nucleus.² Cross-talk between the EGFR and the angiotensin II (Ang II) type 1 receptor (AT₁R, a GPCR) has been demonstrated in mesangial and vascular smooth muscle (VSM) cells. EGFR transactivation is evident in VSM as rapidly as 1 to 2 minutes after Ang II exposure,^1,3 raising the possibility that this process may contribute to the vasoconstrictor response to Ang II. Tyrosine kinases modulate Ang II effects on intracellular free Ca²⁺ concentration ([Ca²⁺]_i), intracellular pH, contraction, and growth in VSM, and tyrosine kinase inhibitors attenuate these Ang II-induced effects.^4–6 Virtually all studies concerning the role of tyrosine kinases in Ang II-mediated vascular responses have used large arteries or cultured VSM cells from these vessels, leaving uncertain the role of the EGFR in Ang II regulation of microvascular function. The recent report that aortic myocytes and renal preglomerular VSM cells display differing growth characteristics in culture⁷ lends credence to the contention that a direct parallel should not be drawn between the function of VSM cells residing in conduit arteries and the renal microvasculature.

We recently reported that renal arteriolar contractile responses to Ang II are suppressed by inhibition of EGFR tyrosine kinase activity⁸; however, the mechanism underlying the EGFR involvement in this critical microvascular contractile response to Ang II remains unclear. Two possible mechanisms might be involved in this process. First, binding of Ang II to the AT₁R in VSM triggers phospholipase Cß-dependent production of 1,4,5-inositol trisphosphate (IP₃), which binds to its receptor in sarcoplasmic reticulum to initiate Ca²⁺ release from this intracellular store. The resulting increase in [Ca²⁺]_i may provoke EGFR transactivation,¹ with subsequent tyrosine phosphorylation events promoting contraction by a mechanism such as increasing the Ca²⁺ sensitivity of the contractile apparatus.⁹ Alternatively, Ang II binding to the AT₁R may initiate Ca²⁺-independent transactivation of the EGFR, which then contributes to the Ang II-induced increase in [Ca²⁺]_i as might occur as a result of phospholipase C activation.¹⁰ An important difference between these two scenarios is that one involves EGFR-mediated events that favor contraction without altering intracellular Ca²⁺ homeostasis, whereas the other requires EGFR tyrosine kinase activity to achieve the full [Ca²⁺]_i response to Ang II. To distinguish between these scenarios in the renal microvasculature, we designed experiments to address the hypothesis that EGFR tyrosine kinase activity contributes to Ang II-induced [Ca²⁺]_i responses in afferent arterioles.

	Methods

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Animals
The procedures used in this study were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (SAS:VAF strain) weighing 250 to 300 g were purchased from Charles River Laboratories and provided free access to food and water before the study.

Isolation of Renal Afferent Arterioles
Renal afferent arterioles were isolated as previously described.¹¹ Briefly, after anesthetization with sodium pentobarbital (100 mg/kg IP), the kidneys were removed, immersed in cold Ringer’s solution, decapsulated, and cut into 2-mm slices. A medial slice was selected and placed in cold fura-2 loading solution (7 µmol/L fura-2 acetoxymethyl ester, 0.09% DMSO, and 0.018% Pluronic F-127 in normal Ringer’s solution). Segments of interlobular artery with single attached afferent arterioles (but devoid of glomeruli or tubular segments) were isolated by microdissection, taking care not to touch or stretch the afferent arterioles. After 1-hour incubation at room temperature in the fura-2 loading solution, the specimen was transferred to a chamber mounted on the stage of an inverted microscope (Nikon Diaphot model 300) and stabilized on the coverslip floor of the chamber with Cell-Tac adhesive (BD Biosciences) and/or a holding pipette.

[Ca²⁺]_i Measurement
Dual excitation wavelength fluorescence microscopy was used to monitor [Ca²⁺]_i in individual fura-2-loaded afferent arterioles as previously described.^11,12 Briefly, the arteriole was illuminated alternately with light at 340 and 380 nm wavelengths by means of a Deltascan dual monochromator system (Photon Technology International). An adjustable optical sampling window was positioned over the afferent arteriole, and emission fluorescence (510 nm) was collected with a photometer assembly. Arteriolar [Ca²⁺]_i was calculated with the use of the FeliX software package (Photon Technology International) according to the standard equation with a 0.85 viscosity correction factor.^13,14 Calibration of the fura-2 signal was performed daily.

Mn²⁺ Quench
Divalent cation influx was assessed on the basis of Mn²⁺-induced quenching of fura-2 fluorescence. Because the addition of Mn²⁺ generated a precipitate in Ringer’s solution, these experiments used a physiological saline solution (PSS) bath in which this phenomenon was absent. Individual arterioles were bathed in PSS containing 100 µmol/L Mn²⁺ and illuminated at an excitation wavelength of 360 nm (the isosbestic point of fura-2, insensitive to [Ca²⁺]_i); the emitted fluorescence (510 nm) was recorded with the use of the Deltascan system. To minimize the influence of other factors (eg, cell volume, fura-2 loading efficiency) on the results, 10 µmol/L ionomycin was applied at the end of each experiment to achieve maximal fluorescence quenching by Mn²⁺. This allowed normalization of the Mn²⁺ quench rate to the highest (pre-Mn²⁺) and lowest (Mn²⁺ plus ionomycin) fluorescence levels encountered within each individual experiment. Thus, the Mn²⁺ quench data are expressed as the percent decrease in fluorescence emission per second.

Chemicals and Reagents
Fura-2 and fura-2 acetoxymethyl ester were purchased from Molecular Probes. AG1478, AG9, and phenylarsine oxide (PAO) were from Calbiochem. All other chemicals were purchased from Sigma Chemical. None of the pharmacological agents exhibited autofluorescence at the excitation and emission wavelengths used for fura-2. For measurement of arteriolar [Ca²⁺]_i responses, all agents were administered with the normal Ringer’s bathing solution (in mmol/L: 148 NaCl, 5 KCl, 1 MgSO₄, 1.6 Na₂HPO₄, 0.4 NaH₂PO₄, 1.5 CaCl₂, and 5 d-glucose). A low Ca²⁺ (100 nmol/L) modification of the Ringer’s bath was used in some experiments. A PSS bath (in mmol/L: 145 NaCl, 4 KCl, 1.5 CaCl₂, 5.5 d-glucose, 10 HEPES) was used in the Mn²⁺ quench experiments.

Experiment Protocols
Effect of EGFR Tyrosine Kinase or Tyrosine Phosphatase Inhibition on [Ca²⁺]_i Responses to Ang II
After a stabilization period, baseline afferent arteriolar [Ca²⁺]_i was determined during exposure to normal Ringer’s bath. The vessel was then subjected to one of the following 10 minute-treatments: (a) untreated (no drugs), (b) EGFR tyrosine kinase inhibition by exposure to 100 nmol/L AG1478 or 10 µmol/L 4,5-dianilinophthalamide (DAPH), (c) exposure to the inactive analog of AG1478 (100 µmol/L AG9), or (d) phosphotyrosine phosphatase inhibition by exposure to 10 µmol/L PAO. Finally, 100 nmol/L Ang II was applied in the continued presence of the pretreatment drug for 5 minutes.

Effect of Low Ca²⁺ Bath and EGFR Tyrosine Kinase Inhibition on [Ca²⁺]_i Responses to Ang II
After a stabilization period, baseline arteriolar [Ca²⁺]_i was measured in normal Ringer’s bath, followed by exposure to a low Ca²⁺ Ringer’s bath (100 nmol/L Ca²⁺). Subsequently, 100 nmol/L Ang II was added to the low Ca²⁺ bathing solution. Other arterioles were subjected to the identical manipulations, except that the baseline period was followed by 10-minute exposure to 100 nmol/L AG1478, which was also included in the low Ca²⁺ and low Ca²⁺ plus Ang II solutions for these experiments.

Effect of EGFR Tyrosine Kinase Inhibition on Mn²⁺ Quench Response to Ang II
Afferent arterioles were randomly assigned to untreated and EGFR tyrosine inhibition groups. Each arteriole was initially bathed in PSS during a stabilization period lasting at least 5 minutes. In the untreated group, the arteriole was then exposed to PSS containing 100 µmol/L Mn²⁺ for 120 seconds to establish the baseline quench rate. Subsequently, the arteriole was exposed to Mn²⁺-containing PSS containing 100 nmol/L Ang II for 5 minutes. Finally, 10 µmol/L ionomycin was applied at the end of each experiment. In the EGFR tyrosine kinase inhibition group, the initial stabilization period was followed by 8-minute exposure to PSS containing 100 nmol/L AG1478. Then, the bathing solution was changed to PSS containing both AG1478 and 100 µmol/L Mn²⁺, and 120 seconds was allowed to establish the baseline quench rate. The subsequent Ang II and ionomycin treatments were imposed as described above, except that AG1478 was included in all bathing solutions.

Statistical Analyses
Statistical analysis was performed by ANOVA or repeated-measures ANOVA followed by the Newman-Keuls multiple range test. Statistical computations were performed with the SigmaStat 2.03 software package (SPSS Inc), with statistical significance defined as P<0.05. All data are reported as mean±SEM (n=number of arterioles).

	Results

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Basal afferent arteriolar [Ca²⁺]_i averaged 95±7 nmol/L (n=42) and did not differ between treatment groups. Moreover, baseline [Ca²⁺]_i was unaltered by treatment with either AG9 (=-5±2 nmol/L, n=10), AG1478 (=3±2 nmol/L, n=8), or DAPH (=-10±10 nmol/L, n=7). Thus, EGFR tyrosine kinase inhibition failed to alter basal [Ca²⁺]_i under these experimental conditions.

Figure 1 illustrates the impact of EGFR tyrosine kinase inhibition on afferent arteriolar [Ca²⁺]_i responses to Ang II. In untreated arterioles (n=17), 100 nmol/L Ang II caused a typical rapid [Ca²⁺]_i increase (=75±10 nmol/L above baseline, referred to as the peak response) followed by decline in [Ca²⁺]_i until it reached a plateau (3 minutes after the peak; =24±5 nmol/L above baseline). The inactive tyrphostin compound (100 µmol/L AG9) did not change the amplitude or the shape of the response to Ang II (=83±13 nmol/L peak and 17±6 nmol/L plateau; n=10). In contrast, the peak [Ca²⁺]_i response to Ang II was blunted by both EGFR tyrosine kinase inhibitors (AG1478: =42±7 nmol/L above baseline, n=8; DAPH: =30±6 nmol/L, n=8; both P<0.05 versus untreated). The time required for the peak [Ca²⁺]_i response to wane by 50% (t_0.5) was also reduced by EGFR tyrosine kinase inhibition, averaging 152±22 seconds in untreated arterioles and 101±14 seconds in AG1478-treated arterioles (P<0.05). DAPH tended to diminish the plateau response (=12±3 nmol/L), although this effect did not achieve statistical significance; however, AG1478 significantly decreased the magnitude of the plateau [Ca²⁺]_i response to Ang II (=8±4 nmol/L above baseline; P<0.05 versus untreated). Thus, EGFR tyrosine kinase inhibition attenuated the afferent arteriolar [Ca²⁺] response to Ang II.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of EGFR tyrosine kinase inhibition (100 nmol/L AG1478 or 10 µmol/L DAPH) on renal arteriolar [Ca2+]i responses to 100 nmol/L Ang II. AG9 (100 µmol/L) is an inactive analog of AG1478. A, Representative responses. B, Mean peak and plateau responses. *P<0.05 vs untreated peak; P<0.05 vs untreated plateau.

The phosphotyrosine phosphatase inhibitor PAO (10 µmol/L) evoked a slow rise in afferent arteriolar [Ca²⁺]_i from the initial baseline value of 100±10 nmol/L (n=7) to achieve a stable value that averaged 205±13 nmol/L 10 minutes after PAO exposure. From this PAO baseline value, 100 nmol/L Ang II elicited an [Ca²⁺]_i response that differed markedly from that observed in untreated vessels (Figure 2). PAO enhanced the plateau phase of the [Ca ²⁺]_i response (=71±14 nmol/L; P<0.05 versus untreated), whereas it did not alter the peak [Ca²⁺]_i response (=68±18 nmol/L; P>0.05 versus untreated). Thus, phosphotyrosine phosphatase inhibition increased baseline [Ca²⁺]_i in afferent arterioles and augmented the plateau phase of the [Ca²⁺]_i response to Ang II.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of phosphotyrosine phosphatase inhibition (10 µmol/L PAO) on renal arteriolar [Ca2+]i response to 100 nmol/L Ang II. A, Representative responses. B, Mean peak and plateau responses. P<0.05 vs untreated plateau.

Two subsequent series of experiments addressed the Ca²⁺ pool influenced by EGFR tyrosine kinase in the response to Ang II. The initial experiments examined the impact of a low Ca²⁺ (100 nmol/L) bath on the response. This extracellular [Ca²⁺] was chosen to prevent Ca²⁺ entry while avoiding depletion of the intracellular Ca²⁺ store (which might occur during exposure to a Ca²⁺-free, EGTA-containing bath). In these experiments (n=22), baseline arteriolar [Ca²⁺]_i averaged 87±7 nmol/L during exposure to normal Ringer’ bath and 73±6 nmol/L during subsequent exposure to the low Ca²⁺ bath (either in the absence or presence of AG1478). These values did not differ significantly by paired analysis. As shown in Figure 3, low Ca²⁺ bath decreased the peak Ang II-induced [Ca²⁺]_i response by 40% (=43±10 nmol/L above baseline; n=11) compared with the response observed in normal Ringer’s bath. Low Ca²⁺ bath also markedly reduced the time required for the peak [Ca²⁺]_i response to subside, as indicated by a decrease in t_0.5 from 152±22 seconds in normal Ringer’s bath to 48±6 seconds in low Ca²⁺ bath (P<0.05). Moreover, no plateau phase was evident in arterioles exposed to Ang II in low Ca²⁺ bath (=-5±3 nmol/L). The [Ca²⁺]_i response to Ang II that remained evident in low Ca²⁺ bath is considered to represent the contribution of Ca²⁺ release from intracellular stores. Inclusion of 100 nmol/L AG1478 in the low Ca²⁺ bath did not alter magnitude or time course (t_0.5=55±7 seconds) of the Ang II response, compared with the effect of low Ca²⁺ alone. Thus, AG1478 did not change the Ca²⁺ release-dependent component of the [Ca²⁺]_i response to Ang II, suggesting that EGFR tyrosine kinase inhibition attenuates the afferent arteriolar response to Ang II by an effect on Ca²⁺ influx.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of extracellular [Ca2+] on renal arteriolar responses to Ang II in the presence and absence of EGFR tyrosine kinase inhibition (100 nmol/L AG1478). A, Representative responses. B, Mean peak and plateau responses. *P<0.05 vs normal Ca2+ peak; P<0.05 vs normal Ca2+ plateau.

The Mn²⁺ quench technique was used to further evaluate the contention that EGFR tyrosine kinase activity contributes to Ang II-induced Ca²⁺ influx. The left panel of Figure 4A illustrates the time course of fura-2 fluorescence emission changes at the Ca²⁺-insensitive wavelength (360 nm) during a typical experiment performed in the absence of AG1478. Fluorescence emissions were constant (decreasing 0.01±0.01% per second) in the absence of Mn²⁺, indicating minimal leak of fura-2 from the cells under basal conditions. On exposure to 100 µmol/L Mn²⁺, fluorescence intensity began to decline at a rate of 0.11±0.02% per second, reflecting the basal rate of divalent cation influx and subsequent quenching of fura-2 fluorescence by Mn²⁺ binding. Subsequent exposure to 100 nmol/L Ang II (in the continued presence of extracellular Mn²⁺) accelerated Mn²⁺ quench to 0.40±0.05% per second during the first 60 seconds, followed by a slowed quench rate (0.06±0.01% per second measured 2.5 to 3.5 minutes after Ang II exposure; P>0.05 versus Mn²⁺ alone). Finally, addition of 10 µmol/L ionomycin allowed maximal Mn²⁺ quenching of fura-2 fluorescence. The right panel of Figure 4A illustrates the impact of AG1478 on Mn²⁺ quench in afferent arterioles and the response of this parameter to Ang II. AG1478 alone had no effect on fura-2 fluorescence emissions. In the presence of AG1478, Mn²⁺ quenching of fura-2 fluorescence was evident at a rate of 0.16±0.02% per second, a value comparable to that observed in the absence of AG1478 and indicating no effect of AG1478 on the basal rate of divalent cation influx. However, compared with the untreated group, Ang II-stimulated acceleration of Mn²⁺ quench was suppressed in AG1478-treated vessels during the first 60 seconds (Figure 4B), although the response evident after 2.5 to 3.5 minutes (0.06±0.01% per second) was comparable to that observed in untreated vessels. Thus, EGFR tyrosine kinase inhibition decreased the rapid divalent cation influx response to Ang II in isolated afferent arterioles.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of EGFR tyrosine kinase inhibition on Ang II-stimulated Mn2+ quenching of fura-2 fluorescence. A, Representative responses. B, Mean Ang II-induced acceleration of Mn2+ quench rate. AG1478, 100 nmol/L in PSS; Mn, 100 µmol/L MnCl2 in PSS; ionomycin, 10 µmol/L in PSS. *P<0.05 vs Ang II alone.

	Discussion

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Ang II induces afferent arteriolar constriction through its interaction with AT₁ receptors,¹⁵ which mediate Ang II-induced Ca²⁺ signaling responses in VSM cells.¹⁶ Tyrosine kinases have been shown to influence vascular contraction in large arteries and cultured myocytes from these vessels.^17,18 Moreover, we recently reported that renal arteriolar contractile responses to Ang II are suppressed by inhibition of EGFR tyrosine kinase activity.⁸ The present study explored the mechanism of EGFR tyrosine kinase involvement in the Ang II-induced response of the renal preglomerular microvasculature. A microscope-based fluorescence photometry system was used to monitor [Ca²⁺]_i responses to Ang II in nonperfused, nonpressurized afferent arterioles. A similar approach has been used by other investigators to probe [Ca²⁺]_i signaling events in arterioles isolated from a variety of tissues.^19–22 Limitations of this approach have been considered in detail previously¹¹ and include the assumption that VSM cells provide the major source of fura-2 fluorescence emissions. Although endothelial cells probably load with fura-2 in this experimental setting, their fluorescence emissions should represent a relatively minor component of the overall signal because of the larger mass of VSM cells. Indeed, Praddaude et al²³ detected no significant influence of deendothelialization on rat afferent arteriolar [Ca²⁺]_i responses to Ang II in this same experimental setting. Thus, the afferent arteriolar [Ca²⁺]_i responses observed in the present study are assumed to provide a semiquantitative indication of events occurring chiefly in the resident VSM cells. In accord with this contention, our observations confirm that Ang II typically provokes a biphasic afferent arteriolar [Ca²⁺]_i response composed of a rapid initial transient and a sustained plateau phase, as reported to occur in many pure VSM cell populations.^4,24,25

The role of EGFR tyrosine kinase in Ang II-induced [Ca²⁺]_i signaling was assessed by using two different EGFR tyrosine kinase inhibitors. AG1478 is a synthetic tyrphostin compound that competes for the substrate-binding site to affect tyrosine kinase activity.²⁶ AG1478 acts as a very potent and selective EGFR tyrosine kinase inhibitor (IC₅₀=3 nmol/L), influencing other tyrosine kinases only at much higher concentrations.²⁷ We have shown that tyrphostin inhibition of tyrosine kinase activity does not alter K⁺-induced afferent arteriolar constriction,⁸ arguing against the possibility that these compounds exert a direct effect on L-type Ca²⁺ channels.²⁸ The other EGFR tyrosine kinase inhibitor used in the present study was DAPH, a nontyrphostin compound that interacts with the ATP binding site of the enzyme.²⁹ Although DAPH inhibits EGFR tyrosine kinase activity (IC₅₀=0.8 µmol/L), it also affects protein kinase C (IC₅₀=6.0 µmol/L) and Src kinase (IC₅₀=16 µmol/L).³⁰ Compared with DAPH, AG1478 is a more selective EGFR tyrosine kinase inhibitor (IC₅₀>100 µmol/L for platelet-derived growth receptor tyrosine kinase).²⁷ Both AG1478 and DAPH blunted the peak afferent arteriolar [Ca²⁺]_i response to Ang II. AG1478 also decreased the plateau phase of [Ca²⁺]_i response to Ang II, whereas the impact of DAPH on this parameter did not achieve statistical significance (perhaps because of nonspecific effects of this compound). AG9, an inactive tyrphostin analog (IC₅₀>1250 µmol/L for EGFR tyrosine kinase),²⁷ failed to alter the [Ca²⁺]_i response to Ang II in the present study or the contractile response to Ang II in our previous study.⁸ This observation is consistent with the idea that AG1478 suppresses the afferent arteriolar Ang II response through its ability to inhibit tyrosine kinase activity rather than through a nonspecific effect of tyrphostin compounds. Our observations extend to the renal microvascular level reports that tyrosine kinase inhibition attenuates Ang II-induced [Ca²⁺]_i and contraction responses in VSM cells from aorta or mesenteric artery.⁴

Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation reflects a balance between phosphatase and kinase activities. Altered regulation of phosphatase and/or kinase activity has been shown to lead to abnormal growth and differentiation.³¹ If tyrosine phosphorylation caused by EGFR tyrosine kinase activation contributes to the [Ca²⁺]_i response to Ang II, we reasoned that tyrosine phosphatase inhibition should exaggerate the response by impeding dephosphorylation. This postulate was explored through the use of the membrane-permeable phosphotyrosine phosphatase inhibitor PAO (IC₅₀=18 µmol/L). PAO had two striking effects on afferent arteriolar [Ca²⁺]_i. First, although neither EGFR tyrosine kinase inhibitor altered basal [Ca²⁺]_i, PAO caused a slow but marked increase in this parameter. These observations imply that net EGFR tyrosine kinase-dependent protein phosphorylation is maintained low in afferent arterioles by phosphotyrosine phosphatase activity under basal conditions and that interruption of the normal phosphorylation/dephosphorylation cycle by inhibiting dephosphorylation ultimately allows accumulation of Ca²⁺ in the cytosol. The mechanism underlying this effect was not addressed in the present study; however, the PAO-induced increase in [Ca²⁺]_i in endothelial cells has been attributed to Ca²⁺ influx.³² PAO also enhanced the plateau [Ca²⁺]_i response to Ang II in afferent arterioles, further suggesting that tyrosine phosphorylation events contribute to the magnitude of the sustained response to this agonist and that this event is normally limited by a dephosphorylation event. The plateau phase of the VSM [Ca²⁺]_i response to Ang II is involved in sustaining the contractile response³³ and generally results from Ca²⁺ influx (in accord with our observations; Figure 3). Thus, the ability of PAO to exaggerate the plateau phase of the Ang II-induced [Ca²⁺]_i response suggests that tyrosine phosphorylation promotes Ang II-induced Ca²⁺ influx, thereby sustaining the contractile response. It is not clear why PAO did not affect peak [Ca²⁺]_i response to Ang II, although it is possible that the influence of dephosphorylation on the Ang II response is slower in onset than activation of the EGFR tyrosine kinase. It is also possible that activation of phosphotyrosine phosphatase occurs secondary to EGFR tyrosine kinase activation in response to Ang II and that dephosphorylation events contribute to the waning of the [Ca²⁺]_i from the peak value to the sustained plateau. Further studies are required to explore this postulate. It is at least clear that inhibition of phosphotyrosine phosphatase activity allows an exaggerated [Ca²⁺]_i response to Ang II, consistent with a role of tyrosine phosphorylation in the Ca²⁺ signaling response.

The peak [Ca²⁺]_i response to Ang II was reduced in magnitude and the plateau response was abolished in arterioles bathed in low Ca²⁺ media, indicating involvement of Ca²⁺ influx in both phases of the response. It has been reported that the initial transient [Ca²⁺]_i response to Ang II is generated primarily by IP₃-induced mobilization of intracellular Ca²⁺ and to a lesser extent by Ca²⁺-induced Ca²⁺ release.⁴ However, we found that the low Ca²⁺ bath shortened peak [Ca²⁺]_i response duration by 33% and reduced its magnitude by 40%, similar to reports that nifedipine reduces the initial phasic afferent arteriolar [Ca²⁺]_i signal by 40% to 50%.²¹ These observations are consistent with involvement of both Ca²⁺ mobilization and Ca²⁺ influx through voltage-gated channels in producing the initial peak [Ca²⁺]_i response to Ang II in the afferent arteriole. The low Ca²⁺ bath also abolished the plateau phase of the response, in accord with the contention that this phase of [Ca²⁺]_i response to Ang II is dependent entirely on external Ca²⁺ and is the result of Ca²⁺ influx.^34,35 Involvement of EGFR tyrosine kinase activity in Ang II-induced Ca²⁺ influx is implied by the ability of AG1478 to reduce the magnitude and duration of the peak [Ca²⁺]_i response, as well as to diminish the magnitude of the plateau, similar to the effect of the low Ca²⁺ bath. Moreover, EGFR tyrosine kinase inhibition failed to alter the magnitude or time course of the Ang II response in the low Ca²⁺ bath, indicating that EGFR activation has no impact on the Ca²⁺ mobilization component of the response. Rather, these data imply that EGFR tyrosine kinase activity mainly contributes to the Ca²⁺ influx response to Ang II. Further support for this contention is provided by the ability of AG1478 to suppress the Ang II-stimulated acceleration of Mn²⁺ quench of fura-2 fluorescence (an indicator of Ca²⁺ influx). In our study, Ang II caused rapid Mn²⁺ quenching of fura-2 fluorescence, consistent with the observations of Loutzenhiser and Loutzenhiser.²¹ The Mn²⁺ quench rate then slowed to a value comparable to baseline in both untreated and AG1478-treated arterioles. It is possible that the sensitivity of this measure of divalent cation influx may be insufficient to resolve the rate of Ca²⁺ influx required to sustain the modest (25 nmol/L) plateau elevation in [Ca²⁺]_i evoked by Ang II in our experimental setting.

In recent years, it has become evident that GPCRs (such as the AT₁R) transactivate receptor tyrosine kinases (particularly the EGFR^36,37), allowing GPCRs to take advantage of signaling pathways downstream of RTKs to exert their effects on the cells.¹ AT₁R-EGFR cross-talk has been studied primarily in the context of the mitogenic effect of Ang II on aortic myocytes. Results of our previous study⁸ and the present study suggest that EGFR tyrosine kinase activity is involved in Ang II-induced activation of afferent arterioles, which represent a pivotal microvascular target of Ang II. Tyrosine phosphorylation involvement in this process is indicated by the observations that EGFR tyrosine kinase inhibition attenuated by 50% both the contractile response to 10 nmol/L Ang II⁸ and the [Ca²⁺]_i response to 100 nmol/L Ang II and that tyrosine phosphatase inhibition markedly enhanced the plateau phase of the [Ca²⁺]_i response. The mechanism of AT₁R-induced EGFR transactivation is not well understood. Moreover, the mechanism through which EGFR tyrosine kinase influences Ca²⁺ influx to contribute to the contractile response of this critical microvascular segment remains speculative. It is possible that Ang II activation of the EGFR tyrosine kinase promotes Ca²⁺ influx by influencing voltage-gated Ca²⁺ channels,³⁸ which represent the primary Ca²⁺ influx pathway used by Ang II in the afferent arteriole.^21,35 Alternatively, tyrosine kinase activity may module Ca²⁺-activated Cl^- channels, which have been suggested to contribute to Ang II-induced afferent arteriolar depolarization.³⁹

In summary, EGFR tyrosine kinase inhibitors blunted the [Ca²⁺]_i response to Ang II in rat renal afferent arterioles, and phosphotyrosine phosphatase inhibition exaggerated the response. The [Ca²⁺]_i response to Ang II in the low Ca²⁺ bath, assumed to represent Ca²⁺ mobilization from intracellular stores, was not altered by EGFR tyrosine kinase inhibition. However, EGFR tyrosine kinase inhibition suppressed the rapid divalent cation influx response to Ang II. These accumulated observations indicate that Ang II-induced afferent arteriolar contraction involves participation of EGFR tyrosine kinase activity that acts, at least in part, to promote Ca²⁺ influx (Figure 5). Further studies are required to determine the processes linking the AT₁R and the EGFR, as well as the mechanism through which EGFR tyrosine kinase influences Ca²⁺ influx to promote the contractile response of this critical microvascular bed.

View larger version (20K): [in this window] [in a new window]   Figure 5. Simplified scheme illustrating the apparent role of EGFR in eliciting the afferent arteriolar contractile response to Ang II.

	Acknowledgments

 
These studies were supported by funding from the National Institutes of Health (DK39202). We thank Rachel W. Fallet for excellent technical support.

	Footnotes

 
Presented in part at Experimental Biology 2002, New Orleans, La, April 20–24, 2002, and at the ASN/ISN World Congress of Nephrology, San Francisco, Calif, October 13–17, 2001, and published in abstract form (J Am Soc Nephrol 2001;12:496A and FASEB J 2002;16:A417).

Received May 9, 2002; first decision June 11, 2002; accepted August 14, 2002.

	References

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