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Mechanisms of angiotensin II-induced expression of B₂ kinin receptors

¹Divisions of Endocrinology-Diabetes-Medical Genetics and Nephrology, Department of Medicine, Medical University of South Carolina, and ²Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina 29425

Submitted 6 August 2003 ; accepted in final form 3 November 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B₂ kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10^—9-10^—6 M)- and time (0–24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10^—7 M. ANG II (10^—7 M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT₁ receptor antagonist) and/or PD-123319 (AT₂ receptor antagonist) at 10 µM for 30 min, followed by ANG II (10^—7 M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT_1A receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44^mapk (PD-98059) and/or an inhibitor of p38^mapk (SB-202190), followed by ANG II (10^—7 M) for 24 h. Selective inhibition of the p42/p44^mapk pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.

AT₁ receptors; mitogen-activated protein kinase; vascular smooth muscle cells

ANG II, an octapeptide hormone, is the principal effector of the RAS. The biological effects observed in response to systemic or local production of ANG II are quite diverse and appear to play a critical role in the pathological adaptation to disease states involving the cardiovascular and renal systems. The cellular effects of ANG II are mediated through at least two high-affinity receptors subtyped AT₁ and AT₂, and both receptor subtypes belong to the seven-transmembrane G protein-coupled receptor superfamily (26, 27). The AT₁ receptor subtype mediates most of the physiological actions of ANG II and is widely expressed in adult tissues; in the vasculature AT₁ receptors are mainly expressed in vascular smooth muscle cells (VSMC) (48). Two other AT₁ receptor subtypes have been identified in rodents, designated AT_1A and AT_1B (12). In renal tissue, the expression of AT_1A and AT_1B receptors has been localized to the entire renal vasculature (42). The AT_1A and AT_1B receptor subtypes are pharmacologically indistinguishable, making it difficult to discern the function of each receptor with pharmacological tools. The functions of AT_1A and AT_1B receptors are not yet determined, but AT_1A receptor-knockout mice have been shown to have reduced blood pressure and reduced renal vascular resistance (11). The other major subtype of ANG II receptors, AT₂, is normally expressed at high levels in fetal tissues and decreases rapidly after birth but is expressed at low levels in adult tissues such as the vasculature and the kidney (36, 48).

The vasoactive nonapeptide BK is the principal effector of the KKS and has been implicated in the regulation of renal and cardiovascular function and vascular tone (23, 25). BK can be generated both systemically and locally within the vascular wall and can thus act in a paracrine and autocrine manner to influence vascular function (29, 30, 32). Most of the physiological actions of BK are attributed to activation of the B₂ kinin receptor (B2KR) subtype, which is a member of the seven-transmembrane G protein-coupled receptor superfamily (24). The physiological effects of BK are mediated via generation of second messengers such as nitric oxide and eicosanoids (16, 37). BK causes relaxation of the VSMC through the synthesis and release of nitric oxide from the endothelium (44). In contrast, injury to the integrity of the endothelium enables BK to directly increase intracellular calcium levels and induce VSMC contraction in a manner similar to ANG II (3).

We previously demonstrated (43) that the generation of BK in the interstitial fluid of the heart is significantly increased in response to ANG I infusion. The cross talk between BK and ANG II is still poorly defined. Therefore, the present study was designed to explore the potential role of ANG II in modulating the expression of B2KR, to identify the receptor subtype responsible for the actions of ANG II, and to delineate the cellular signals through which this regulation may occur.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture. Rat aortic VSMC from 75- to 150-g male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were prepared by a modification of the method of Majack and Clowes (22). A 2-cm segment of artery cleaned of fat and adventitia was incubated in 1 mg/ml collagenase for 3 h at room temperature. The artery was then cut into small sections; fixed to a culture flask for explantation in minimal essential medium containing 10% FCS, 100 mU/ml of penicillin, and 100 µg/ml of streptomycin; and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium was changed every 3–4 days, and cells were passaged every 6–8 days by harvesting with trypsin-EDTA. Semiconfluent (60–80%) cells were used in all studies by growing them in serum-free medium for 24–48 h before agonist stimulation. VSMC were identified by the following criteria: they stained positive for intracellular cytoskeletal fibrils of actin and smooth muscle cell-specific myosin (indicative of contractile cell) and negative for factor VIII antigens. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85–23, revised 1996).

Cell extracts. VSMC were washed twice, scraped in PBS containing 2 mM sodium vanadate, and centrifuged at 3,000 g for 5 min. Pellets were resuspended in 100 µl of lysis buffer (in mM: 20 Tris, 130 NaCl, 10 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 1 PMSF, and 2 Na vanadate with 10% glycerol, 100 mU/ml aprotinin, and 0.156 mg/ml benzamidine, pH 8.0), sonicated for 5 s, incubated on ice for 30 min, and centrifuged at maximum speed for 5 min. The supernatant was used as the protein source, and its concentration was determined by the method of Lowry et al. (19).

Western blotting of B2KR. To measure the protein levels of B2KR, 20–25 µg of soluble protein obtained as described in Cell extracts was separated by SDS-PAGE (12%) under reducing conditions and transferred to polyvinylidene difluoride membranes at 300 mA for 2 h. The membranes were blocked for 30 min in 1% BSA-Tris-buffered saline (in mM: 50 Tris pH 7.5 and 150 NaCl) at 37°C and immunoblotted with an anti-B₂-specific primary antibody (1:4,000 dilution) overnight at 4°C, followed by incubation of the membranes in a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were visualized with the chemiluminescence reagent Renaissance (NEN Life Science Products, Boston, MA), according to the procedure described by the supplier. Membranes were exposed to Kodak LS film, and bands were measured by densitometry and quantified by the NIH Image program. The B2KR antibody was raised against the second extracellular loop (amino acids 169–179) of the B₂ receptor and has the following amino acid sequence: RT-MKEYSDEGH (Zymed Labs, San Francisco, CA).

RT-PCR. RNA was extracted from cells with Tri Reagent (Molecular Research Center, Cincinnati, OH). RNA was then converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) according to the manufacturer's protocol at 37°C for 1 h. The PCR reaction was carried out in a 25-µl total volume containing 1x PCR buffer, 200 µM dNTPs, 2 ng/µl of each primer, 5 µl of first-strand cDNA, and 1 unit of Taq (Qiagen, Valencia, CA). The primers used for amplification of the rat B2KR were 5'-AAGACAGCAGTCACCATC-3' and 5'-GACAAACACCAGATCGGA-3'. The cycling conditions were an initial denaturation at 95°C for 5 min, followed by 40 cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 2 min. PCR reactions were visualized on a 1% agarose gel, photographs were taken, and densitometric analysis was performed with the NIH Image program.

AT_1A receptor-knockout mice. Breeding pairs of knockout mice in which the AT_1A receptor gene was disrupted by gene targeting were originally provided by Dr. Thomas Coffman (Duke University, Durham, NC). These animals were developed by using the 129J mouse germ cell line in the black 9 mouse strain (11). The knockout mice were repeatedly backcrossed to C57BL/6J wild-type mice for >20 generations to generate a stable breeding colony. The homozygous mice used in this study were generated by mating pairs of mice heterozygous (AT_1A^+/—) for the mutated gene. The body weights of AT_1A^—/— mice (23.9 ± 0.57 g; n = 10) were not significantly different from those of AT_1A^+/+ mice (19 ± 0.93 g; n = 6).

Statistical analysis. All data are expressed as means ± SE and were analyzed by ANOVA or Student's t-test for unpaired analysis. Values were considered significant if P < 0.05.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Regulation of B2KR by ANG II. To investigate whether ANG II increases the expression of B2KR, we performed both dose- and time-response experiments in VSMC. To determine the concentration of ANG II that will elicit maximal response, quiescent VSMC were treated with varying concentrations (10^—7-10^—9 M) of ANG II for 24 h. After treatment, proteins were isolated and B2KR protein levels were measured by Western blot analysis using an anti-B₂ receptor-specific antibody. Probing of VSMC lysate with the B₂ antibody showed a single immunoreactive band at 60 kDa. The results shown in Fig. 1A demonstrate that ANG II stimulated the expression of B2KR protein levels in a concentration-dependent manner, with maximal stimulation at 10^—7 M. The fold increase in B2KR protein levels was 3.1 ± 0.7, 2.8 ± 0.4, and 2.8 ± 0.4 in response to 10^—7, 10^—8, and 10^—9 M ANG II, respectively (P < 0.01; n = 6).

View larger version (17K): [in this window] [in a new window]   Fig. 1. ANG II stimulates B2 kinin receptor (B2KR) protein levels. A: dose response: quiescent vascular smooth muscle cells (VSMC) were treated with varying doses of ANG II (10—7 M-10—9 M) for 24 h. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with specific anti-B2 receptor antibody (1:4,000). Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 6 experiments. C, control. B: time response: quiescent VSMC were treated with ANG II (10—7 M) for 4, 6, and 24 h. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with specific anti-B2 receptor antibody (1:4,000). Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 13 experiments. *P < 0.01 vs. control.

To determine the optimal time for increased expression of B2KR, quiescent VSMC were treated with 10^—7 M ANG II for various times (4, 6, and 24 h). This concentration of ANG II was selected because it evoked the maximal response in B2KR expression. The results shown in Fig. 1B demonstrate that B2KR protein levels were increased by 4 h, peaked at 6 h, and remained elevated at 24 h (1.6 ± 0.3-, 2.8 ± 0.4-, and 2.7 ± 0.4-fold increase above control at 4, 6, and 24 h, respectively, P < 0.01; n = 13).

Furthermore, to determine whether ANG II was also affecting the mRNA expression of B2KR, quiescent VSMC were stimulated with ANG II (10^—7 M) for various times (4, 6, and 24 h) to determine optimal stimulation. The results in Fig. 2 show a time-dependent increase in the expression of the B2KR. B2KR mRNA levels were increased at 4 h, peaked at 6 h, and returned to control levels after 24 h of treatment with ANG II (P < 0.05; n = 4).

View larger version (22K): [in this window] [in a new window]   Fig. 2. ANG II induces the expression of B2KR mRNA levels. Quiescent cells were treated with ANG II (10—7 M) for 4, 6, and 24 h. RNA was isolated, and RT-PCR was performed with rat B2KR-specific primers. Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 4 experiments. *P < 0.05 vs. time 0 (control).

The above time-response study was repeated with real-time PCR. B2KR mRNA levels were expressed relative to -actin mRNA levels. The results demonstrate that ANG II stimulation resulted in a significant increase in B2KR mRNA levels at 4 h compared with unstimulated cells (2.45 ± 0.1- vs. 1.25 ± 0.03-fold increase, ANG II 4 h vs. control, respectively, P < 0.006; n = 5). This increase in B2KR mRNA levels in response to ANG II was maintained at 6 h (1.84 ± 0.02- vs. 1.25 ± 0.03-fold increase, ANG II 6 h vs. control, respectively, P < 0.01; n = 5) and returned to baseline levels by 24 h. Thus the data generated from real-time PCR support our data generated with RT-PCR. These findings are consistent with our protein data in that the ANG II-induced increase in B2KR expression is seen first at the mRNA level and later at the protein level.

Effect of actinomycin D on ANG II-induced B2KR expression. To explore whether ANG II modulates the expression of B2KR at the transcriptional or posttranscriptional level, quiescent VSMC were pretreated with actinomycin D (1 µM) for 2 h, followed by ANG II (10^—7 M) stimulation for 6 h for measurements of B2KR mRNA levels and for 24 h for measurements of B2KR protein levels. The results shown in Fig. 3A demonstrate that ANG II produced a 1.7 ± 0.2-fold increase in B2KR mRNA levels compared with unstimulated controls (P < 0.01; n = 6). However, in the presence of actinomycin D, the increase in B2KR mRNA levels induced by ANG II was completely abolished (1.7 ± 0.2- vs. 0.7 ± 0.04-fold increase, ANG II vs. actinomycin D + ANG II, P < 0.002; n = 5). -Actin mRNA levels measured in the same cells were not altered by any of the treatments (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effect of actinomycin D on ANG II-induced regulation of B2KR. A: quiescent VSMC were pretreated with actinomycin (1 µM) for 2 h, followed by stimulation with ANG II (10—7 M) for 6 h. RNA was isolated, and RT-PCR was performed with rat B2KR-specific primers and/or -actin-specific primers. Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 6 experiments. B: VSMC were pretreated with actinomycin D (1 µM) for 2 h, followed by stimulation with ANG II (10—7 M) for 24 h. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with specific anti-B2 receptor antibody (1:4,000). Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 6 experiments. *P < 0.01 vs. control; P < 0.002 vs. ANG II.

The effects of actinomycin D on B2KR protein levels were also examined, and the results are shown in Fig. 3B. Once again, treatment of VSMC with ANG II produced a 6.3 ± 1.5-fold increase in B2KR protein levels compared with unstimulated cells (P < 0.014; n = 6). However, in the presence of actinomycin D, the increase in B2KR protein levels induced by ANG II was completely abolished (6.3 ± 1.5- vs. 0.2 ± 0.04-fold increase, ANG II vs. actinomycin D + ANG II, P < 0.0001; n = 6). Together, these findings indicate that ANG II may regulate the expression of B2KR at the transcriptional level.

Role of ANG II type 1 receptor subtype in regulation of B2KR expression. Two receptor subtypes can be distinguished pharmacologically through which ANG II can mediate its response, AT₁ and AT₂ (26, 27). To determine which of these receptor subtypes is involved in the induction of B2KR, we used the specific receptor antagonist for each receptor subtype. Losartan is a specific receptor antagonist for the AT₁ receptor subtype, and PD-123319 is a specific receptor antagonist for the AT₂ receptor subtype (6, 21). Quiescent VSMC were pretreated with either PD-123319 or losartan at 10 µM for 30 min, followed by stimulation with ANG II (10^—7 M) for 24 h. The results shown in Fig. 4A demonstrate that selective inhibition of the AT₂ receptor subtype by PD-123319 does not significantly alter the increased expression of B2KR in response to ANG II (3.3 ± 0.9- vs. 5.0 ± 2.0-fold increase, ANG II vs. PD-123319 + ANG II, P > 0.05; n = 3). On the other hand, when the AT₁ receptor subtype was selectively blocked with losartan, the increase in B2KR protein levels induced by ANG II stimulation was significantly reduced (3.9 ± 1.3- vs. 2.0 ± 1.0-fold increase, ANG II vs. losartan + ANG II, P < 0.05; n = 8; Fig. 4B). These findings provide evidence that ANG II modulates the expression of B2KR via activation of the type 1 receptor subtype.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of ANG II receptor blockers on B2KR expression. Quiescent VSMC were pretreated with the AT2 receptor blocker PD-123319 (10 µM; A) and/or the AT1 receptor blocker losartan (10 µM; B) for 30 min, followed by stimulation with ANG II (10—7 M) for 24 h. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with specific anti-B2 receptor antibody (1:4,000). Bar graphs represent the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 3–8 experiments. *P < 0.01 vs. control; P < 0.05 vs. ANG II.

To further explore the role of AT₁ receptors in regulating the levels of B2KR, we measured the expression of B2KR in AT_1A receptor-knockout mice (AT_1A^—/—) and in the wild type (AT_1A^+/+). The results shown in Fig. 5 indicate that the mRNA levels of B2KR measured in renal tissue of AT_1A^—/— were significantly lower than those measured in AT_1A^+/+. The B2KR mRNA level in AT_1A^—/— was 11.1 ± 3 densitometric units (n = 10), and in AT_1A^+/+ it was 15.5 ± 0.9 densitometric units (n = 6; P < 0.0004). -Actin mRNA levels measured in the same samples were not significantly different between AT_1A^—/— and AT_1A^+/+ (17.5 ± 5.3 vs. 16.8 ± 1.1 densitometric units, AT_1A^—/— vs. AT_1A⁺/⁺, P > 0.05). These findings demonstrate for the first time that targeted disruption of AT_1A^—/— results in decreased expression of B2KR.

View larger version (17K): [in this window] [in a new window]   Fig. 5. B2KR expression in AT1A receptor-knockout mice. A: B2KR mRNA levels were measured in AT1A receptor-knockout mice (AT1A—/—; n = 10) and in the wild type (AT1A+/+; n = 6). Bar graph represents the means ± SE of the intensities of the bands expressed as fold increase above control. B: -actin mRNA levels were measured at the same time in the same samples. *P < 0.004 vs. AT1A+/+.

Role of MAPK pathway in ANG II-induced B2KR expression. It was shown previously that ANG II can activate both the p44/p42 and p38 MAPK pathways in VSMC (40). To determine whether either of these MAPK pathways would mediate the effects through which ANG II regulates the expression of B2KR, we examined the effects of specific cell-permeant inhibitors such as PD-98059 (New England BioLabs), which specifically inhibits the p42/p44^mapk activator MAPK kinase, and SB-202190 (Calbiochem, San Diego, CA), which specifically inhibits the activity of p38^mapk (5, 18). Quiescent VSMC were pretreated with PD-98059 (40 µM) and/or SB-202190 (10 µM) for 30 min, followed by stimulation with ANG II (10^—7 M) for 24 h. The results are shown in Fig. 6. Once again, ANG II produced an 8.7 ± 2.6-fold increase in B2KR protein levels compared with unstimulated controls (P < 0.02; n = 8; Fig. 6A). This increase in B2KR protein levels was significantly reduced by the p42/p44^mapk inhibitor PD-98059 (8.86 ± 2.64- vs. 4.8 ± 1.9-fold increase, ANG II vs. PD-98059 + ANG II, P < 0.003; n = 8). On the other hand, selective inhibition of p38^mapk by SB-202190 did not significantly alter the increased expression of B2KR in response to ANG II (2.4 ± 0.5- vs. 2.8 ± 0.8-fold increase, ANG II vs. SB-202190 + ANG II, P > 0.05; n = 4; Fig. 6B). These findings demonstrate that ANG II utilizes the p42/44^mapk pathway to modulate the expression of B2KR in VSMC.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Role of MAPK in ANG II-induced regulation of B2KR. Quiescent VSMC were pretreated with a MAPK kinase inhibitor (PD-98059, 40 µM; A) or a p38mapk inhibitor (SB-202190, 10 µM; B) for 30 min, followed by stimulation with ANG II (10—7 M) for 24 h. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with specific anti-B2 receptor antibody (1:4,000). Bar graphs represent the means ± SE of the intensities of the bands expressed as fold increase above control. Blots are representative of 4–8 experiments. *P < 0.01 vs. control; P < 0.003 vs. ANG II.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
In the present study we have demonstrated that ANG II exerts a significant effect on the expression of B2KR in VSMC. We have shown that ANG II increases the mRNA and protein levels of B2KR in vascular tissue. This effect of ANG II was mediated via activation of the type 1 receptor subtype. In addition, the upregulation of B2KR protein levels in response to ANG II is mediated via activation of the MAPK pathway.

Many studies have demonstrated a pivotal role for ANG II in modulating the functional and structural integrity of the arterial wall under physiological and pathological conditions. The majority of ANG II actions on vascular wall have been attributed to its binding to two high-affinity receptors, AT₁ and AT₂. Both receptor subtypes are differentially expressed in the vasculature. The AT₁ receptor is predominantly expressed in VSMC, whereas AT₂ receptor expression predominates in the adventitia and is undetectable in VSMC (15, 48). Thus the majority of actions of ANG II on VSMC are mediated via activation of AT₁ receptors. In this regard, in the present study we found that ANG II increases the expression of B2KR in VSMC. This effect of ANG II was inhibited by losartan, a specific receptor antagonist for the AT₁ receptor subtype, but not by PD-123319, a specific receptor antagonist for the AT₂ receptor subtype, indicating that ANG II regulates the expression of B2KR via activation of the type 1 receptor subtype.

This finding is consistent with an earlier report by Gavras and colleagues (17) demonstrating that infusion of ANG II results in the upregulation of B2KR mRNA levels in cardiac myocytes and VSMC. Furthermore, our results also demonstrate that targeted disruption of AT_1A^—/— results in decreased expression of B2KR, thus implicating for the first time a role for the AT_1A receptor subtype in modulating the expression of B2KR.

The cellular mechanisms by which ANG II alters vascular function are under intense investigation, and recent studies have shown that ANG II can influence VSMC function in a variety of ways. Stimulation of VSMC with ANG II leads to an increase in intracellular calcium, activation of the MAPK (p42, p44, p38) pathway, PKC, and generation of reactive oxygen species (ROS). These second messengers have been implicated in mediating the vascular complications associated with ANG II such as cellular proliferation, hypertrophy, hyperplasia, and matrix deposition (40).

The localization of kinin receptors within the vascular wall and their activation by BK imply that this system has a role in the regulation of vascular function (25). The vasoactive non-apeptide BK, which is the principal effector of the KKS, can be generated both systemically and locally within the vascular wall (16). Recent studies in our laboratory and by others provide some insights as to how a change in kinin receptors could alter VSMC function. We showed previously (28, 41) that activation of B2KR in VSMC by BK elicits a rise in intracellular calcium, increases proliferation, stimulates MAPK activation and nuclear translocation, and induces expression of c-fos and c-jun genes and the formation of the activator protein-1 complex. The cellular mechanism through which BK stimulates MAPK activation and c-fos mRNA expression in VSMC involves the activation of the calcium/calmodulin pathway, src kinase, PKC, and MAPK kinase and generation of ROS (8, 28, 41). More recently, we showed (4) that BK, like ANG II, is capable of stimulating the expression of extracellular matrix proteins via autocrine activation of transforming growth factor-. Furthermore, BK B2KR antagonists have been shown to attenuate neointimal proliferation after angioplasty, with a concomitant decrease in the activation of the MAPK pathway (46). Together, our findings demonstrate that BK and ANG II share common signaling pathways leading to changes in vascular tone and architecture. Thus it is conceivable that the upregulation of B₂ receptors by ANG II may provide a mechanistic pathway through which ANG II could propagate or amplify its intracellular signals, ultimately promoting changes in VSMC function. In this regard, a recent study by AbdAlla et al. (1) demonstrated that the AT₁ receptor communicates with the B2KR receptor and forms heterodimers with enhanced function in response to ANG II stimulation.

It is widely accepted that ANG II stimulation results in the activation of members of the MAPK family, and activation of this pathway is known to be important in regulating gene expression and vascular cell growth and function (40). To elucidate which member of the MAPK family may be responsible for the ANG II-induced increase in B2KR, we elected to study the role of p42/p44^mapk and p38^mapk. Our findings indicate that inhibition of p42/p44^mapk suppressed the expression of B2KR levels in response to ANG II stimulation whereas inhibition of the p38^mapk pathway did not alter the expression of B2KR in response to ANG II. This finding demonstrates that the regulation of B₂ receptors by ANG II is mediated in part via activation of the p42/p44^mapk pathway.

Although the cellular mechanism by which ANG II regulates the expression of B2KR is not fully elucidated, several possibilities may exist. ANG II has been shown to activate transcription factors such as NF-B and to induce phosphorylation of cAMP response element binding protein (7, 39). Interestingly, two putative cAMP response element sites have been identified on the B2KR gene promoter (20). Thus one possible mechanism through which ANG II could regulate the expression of B₂ receptors is by enhancing the activity of these transcription factors, which in turn may induce transcription of the B2KR gene. The recent identification of a p53 binding site on the B2KR gene by El-Dahr and colleagues (31) may offer another possible target through which ANG II may promote transactivation of the B2KR gene.

Another possible mechanism through which ANG II can modulate the expression of B2KR is via generation of ROS. This production of free radicals that is caused by ANG II may function as second messenger upstream of the MAPK pathway to stimulate signal transduction pathways leading to transcriptional regulation of the B2KR gene (2, 9).

In summary, we have shown that the expression of B2KR in VSMC is upregulated by ANG II and that this regulation occurs at the transcriptional level of the B2KR gene. The results also implicate p42/p44^mapk as a key player in modulating the expression of B2KR in VSMC in response to ANG II challenge. These data provide further rationale for studying the cross talk between ANG II and BK receptors in the progression and pathogenesis of vascular dysfunction in disease states such as diabetes and hypertension.

	ACKNOWLEDGMENTS

 
GRANTS

This work was supported by National Institutes of Health Grants DK-46543, HL-71255, and HL-55782, a Research Grant from the American Society of Nephrology (A. A. Jaffa), and a Merit Review Grant from the Research Service of the Department of Veterans Affairs (F. N. Hutchison).

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. A. Jaffa, Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical Univ. of South Carolina, 114 Doughty St., PO Box 250776, Charleston, SC 29425 (E-mail: jaffaa{at}musc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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