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Endocrinology Vol. 140, No. 3 1385-1391
Copyright © 1999 by The Endocrine Society

ARTICLES

Raf-1 Kinase Activation by Angiotensin II in Adrenal Glomerulosa Cells: Roles of Gi, Phosphatidylinositol 3-Kinase, and Ca2+ Influx

Roger D. Smith1, Albert J. Baukal, Paul Dent2 and Kevin J. Catt

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510; and Molecular Radiology Section, Virginia Commonwealth University (P.D.), Richmond, Virginia 23298-0058

Address all correspondence and requests for reprints to: Dr. K. J. Catt, ERRB, NICHD, National Institutes of Health, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: catt{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Little is known of the mechanisms leading to mitogen-activated protein kinase (MAPK) activation via Gq-coupled receptors. We therefore examined the pathways by which angiotensin II (Ang II) activates Raf-1 kinase, an upstream intermediate in the pathway to MAPK, via the Gq-coupled AT1 angiotensin receptor in bovine adrenal glomerulosa (BAG) cells. Ang II caused a rapid and transient activation of Raf-1 that reached a peak at 5–10 min. Ang II was a potent stimulus of Raf-1 activation with an ED50 of 10 pM and a maximal response at 1 nM, although higher Ang II concentrations elicited a submaximal response. Ang II-stimulated Raf-1 activity was unaffected by down-regulation of protein kinase C and intracellular Ca2+ chelation (using BAPTA) but was partially inhibited by pertussis toxin, and was abolished by manumycin A. Removal of extracellular Ca2+ (by EGTA) or blockade of L type Ca2+ channels (by nifedipine), as well as inhibition of MEK-1 kinase (by PD98059), enhanced Raf-1 activity, whereas wortmannin (100 nM) inhibited approximately one half of Ang II-stimulated Raf-1 activity. Hence, Raf-1 kinase activation by Ang II in BAG cells is dependent on Ras, is mediated in part via Gi and phosphatidylinositol 3-kinase, and is negatively regulated via Ca2+ influx and a downstream signaling element(s).


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
INVESTIGATION of the signaling pathways that mediate the proliferative responses of cells to mitogenic stimuli has been the subject of intensive study in recent years. It is now well established that phosphorylation of the ubiquitous signaling intermediate, mitogen-activated protein kinase (MAPK), plays a critical role in mitogenesis. Upon activation by phosphorylation, MAPK translocates to the nucleus, where it phosphorylates (and thereby activates) several targets including transcription factors critical to the control of cellular proliferation. The best understood pathway to MAPK activation is that activated by growth factor receptor tyrosine kinases such as the EGF receptor (EGF-R) (reviewed in Refs. 1, 2, 3). In its active autophosphorylated state, the EGF-R recruits to the plasma membrane (via adapter proteins such as Grb2 and Shc) the guanine nucleotide exchange factor, m-Sos, which mediates the exchange of GDP for GTP on plasma membrane-anchored Ras. The GTP-bound Ras recruits to the plasma membrane and activates (via a poorly understood mechanism) Raf-1 kinase. Activated Raf-1 then phosphorylates and activates the MAPK kinase, MEK-1, which, in turn, phosphorylates and activates MAPK.

In addition to this well characterized pathway to MAPK activation, it is now increasingly apparent that many members of the expanding superfamily of G protein-coupled receptors (GPCRs), which lack intrinsic kinase activity, are also able to activate MAPK (reviewed in Refs. 4, 5, 6). Furthermore, inherited mutations of certain GPCRs that result in constitutive receptor activation are associated with various disease states in man characterized by proliferative disregulation (7). Individual GPCRs activate one or more of the principle G protein subtypes that include Gs (which activates adenylate cyclase), Gi (which inhibits adenylate cyclase), and Gq/11 which activates phospholipase C-ß [and thence, protein kinase C (PKC)]. Although the role of Gs in mitogenesis is complex, and its ability to mediate cell proliferation appears to be restricted to a limited number of cell types, many of the Gi-coupled GPCRs have been reported to mediate mitogenesis in a variety of cell types (4, 5, 6). The pathway from Gi-coupled GPCRs appears to be mediated by the release of Gß{gamma} complexes from pertussis toxin (PTX)-sensitive G proteins because MAPK activation is inhibited by expression of Gß{gamma} sequestrants (such as ßARKct or {alpha}-transducin) or pretreatment of cells with PTX (8, 9). However, the pathways leading to MAPK activation via Gq-coupled GPCRs are largely insensitive to Gß{gamma} sequestrants and PTX, indicating that these pathways are mainly activated either by the {alpha} subunit of Gq/11 or by some other mechanism(s).

The major actions of the octapeptide hormone, angiotensin II (Ang II), in its regulation of fluid and electrolyte balance, and in cardiovascular homeostasis, are mediated via the Gq-coupled AT1 receptor (AT1-R) (reviewed in Ref. 10). Hypertrophic and hyperplastic actions of Ang II (acting through the AT1-R) have been observed in a variety of target cells. For example, the hormone stimulates hypertrophy of aortic smooth muscle cells (11, 12) and is mitogenic toward renal arteriolar smooth muscle cells (13), cardiac fibroblasts (14), and rat intestinal epithelial cells (15). A major trophic target of Ang II is the adrenal cortex, where the peptide not only stimulates aldosterone release but also plays an important role in the glomerulosa cell hyperplasia that results from dietary sodium restriction (16).

We have previously reported that Ang II is mitogenic in primary cultures of bovine adrenal glomerulosa (BAG) cells (17). Acting via the AT1-R, Ang II increased thymidine incorporation into DNA, increased the proportion of cells in S phase, and stimulated the proliferation of BAG cells. An analysis of the growth-promoting pathways in BAG cells revealed that Ang II stimulated multiple PKC-dependent and -independent pathways to MAPK (18). To further clarify the pathways by which Gq-coupled GPCRs activate the upstream signaling elements in the MAPK cascade, we have investigated the mechanisms by which Ang II activates Raf-1 kinase in BAG cells.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Materials
DMEM, Medium 199 (M199), donor horse serum (DHS), FBS, and antibiotic/antimycotic solutions were from Biofluids (Rockville, MD). Angiotensin II was from Peninsula Laboratories, Inc. (Belmont, CA). {gamma}[32P]ATP was from Amersham (Arlington Heights, IL). Protein A-Sepharose and acidic fibroblast growth factor were from Oncogene Research Products (Cambridge, MA). (His)6-MEK-1 was prepared as previously described (19). Rabbit polyclonal anti-Raf-1 (sc133) was from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA) and PTX was from List Biologicals (Campbell, CA). Nifedipine, calmidazolium, BAPTA-AM, manumycin A, genistein and herbimycin A were from Calbiochem(La Jolla, CA). PD98059 was from Alexis Biochemicals (San Diego, CA). Wortmannin, LY294002, lysophosphatidic acid and all other fine chemicals, which were of analytical grade or higher, were from Sigma Chemical Co. (St. Louis, MO).

Cell culture
Primary cultures of glomerulosa cells were prepared from bovine adrenal glands as previously described (20). Cells were plated at 107 per 10 cm plastic culture dish (Becton Dickinson and Co., Lincoln, NJ) in DMEM containing 10% (vol/vol) DHS, 2% (vol/vol) FBS, 100 µg/ml streptomycin, 100 IU/ml penicillin, 5 µg/ml fungizone, 25 µg/ml gentamicin, 8 µg/ml trimethoprim and 40 µg/ml sulfamethoxazole. Cells were cultured in a humidified atmosphere of 5% CO2 in air at 37 C and formed confluent monolayers after 3 days.

Raf-1 kinase assay
Confluent monolayers of bovine adrenal glomerulosa cells in 10 cm dishes were rendered quiescent by overnight incubation in serum-free M199 containing antibiotics as detailed above. After treatment with test agents as required, cells were washed three times with ice-cold PBS, drained, and scraped into ice-cold lysis buffer (LB, 10 mM Na phosphate, pH 7.0, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% (wt/vol) Na deoxycholate, 1% (vol/vol) NP40, 0.1% (wt/vol) SDS) containing freshly added 1 µg/ml aprotinin, 200 µM Na3VO4, 14 mM ß-mercapto-ethanol and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. After clarification at 14,000 x g, the supernatant was incubated with 2% (vol/vol) protein A-Sepharose for 2–4 h at 4 C. Raf-1 was immunoprecipitated from the precleared supernatant by the addition of 1 µg of rabbit anti-Raf-1 antibody and 2% (vol/vol) protein A-Sepharose overnight with tumbling at 4 C. After three washes in ice-cold LB lacking protease inhibitors, the immune complexes were washed twice in kinase assay buffer (KAB, 15 mM MgCl2, 10 µM Na3VO4, 25 mM Tris, pH 7.5), and resuspended in 40 µl KAB containing 0.5 µg (His)6-MEK-1 as substrate. After preincubation for 10 min at 30 C, the kinase reaction was initiated by the addition of 20 µCi {gamma}[32P]ATP (50 µM final concentration) for a further 30 min at 30 C with frequent mixing. The reaction was terminated on ice and, after sedimentation of the Sepharose beads, the supernatants were transferred to fresh tubes. One half volume of 3 x Laemmli sample buffer (21) containing 2 mM ATP was added and the samples were boiled for 10 min. The (His)6-MEK-1 was resolved by SDS-PAGE, fixed for 20 min in 50% (vol/vol) methanol/7.5% (vol/vol) acetic acid, washed extensively with frequent changes in 5% (vol/vol) methanol/7.5% (vol/vol) acetic acid, and dried using Gel-Dry (Novex, San Diego, CA). Radioactivity incorporated into (His)6-MEK-1 was visualized and quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Ang II activates Raf-1 kinase in bovine adrenal glomerulosa cells
The in vitro Raf-1 kinase assay using (His)6-MEK-1 as substrate detected low levels of Raf-1 activity in unstimulated BAG cells. Addition of Ang II (1 nM) elicited a transient increase in the activity of Raf-1 that was apparent after 1 min, reached a peak value at 5–10 min, and declined thereafter (Fig. 1Go). Because the level of measured Raf-1 activity in both unstimulated and Ang II-stimulated cells varied between experiments, and the level of Raf-1 activity in unstimulated cells was low, Raf-1 activity values were subsequently expressed as a percentage of Ang II-stimulated activity in each experiment. No Ang II-stimulated Raf-1 activity was detected when the anti-Raf-1 antibody was omitted from the immunoprecipitation reaction, or when it was preincubated with an excess of immunizing peptide (data not shown), indicating that the stimulated Raf-1 activity detected in the assay is specific to the antibody. Inclusion of the specific PKC inhibitor, bisindolylmaleimide (1 µM), in the kinase assay buffer had no effect on Ang II-stimulated Raf-1 activity (data not shown), indicating that the phosphorylation of (His)6-MEK-1 was not due to coprecipitation of PKC.



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  		Figure 1. Time-course of Ang II-stimulated Raf-1 activation. In A, serum-deprived glomerulosa cells were exposed to 1 nM Ang II for the indicated times. In this and subsequent experiments, cell lysates were treated with the anti-Raf-1 antibody and the immunoprecipitates were incubated with {gamma}[32P]ATP and (His)6-MEK-1 substrate for 30 min at 30 C. Following resolution by SDS-PAGE, radioactivity incorporated into (His)6-MEK-1 was visualized and quantitated in a PhosphorImager. In B, data represent the average (± range) Raf-1 activity from two independent experiments.

 
Ang II was a potent stimulus of Raf-1 activation in BAG cells, with half-maximal stimulation at 10 pM and maximal activation by a peptide concentration of 1 nM (Fig. 2Go). In a total of 29 experiments, the mean (± SEM) Ang II-induced increase in Raf-1 activity was 3.6 (± 0.2)-fold over control. Ang II concentrations greater than 1 nM elicited a submaximal increase in Raf-1 activity, with 100 nM Ang II stimulating approximately 65% of the activity measured in response to 1 nM Ang II (Fig. 2Go).



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  		Figure 2. Concentration-dependent activation of Raf-1 by Ang II. In A, glomerulosa cells were exposed to the indicated concentrations of Ang II for 5 min. In B, data represent the mean (± SEM) Raf-1 activity from three independent experiments.

 
The role of second messengers in Raf-1 activation
Because two of the well characterized signaling events consequent upon Ang II stimulation of BAG cells are activation of PKC and elevation of intracellular [Ca2+] ([Ca2+]i) (10), we evaluated the role of each of these events in the Ang II stimulation of Raf-1 activity. Short-term (5 min) treatment of BAG cells with the phorbol ester, tetradecanoylphorbol-13-acetate (TPA), was as effective as Ang II in activating Raf-1 (Fig. 3Go), indicating that PKC activation is sufficient to activate Raf-1. However, whereas down-regulation of PKC by prolonged (48 h) exposure of BAG cells to 2 µM TPA abolished the TPA stimulation of Raf-1, the Ang II stimulation of Raf-1 was unaffected, indicating that this activation pathway is independent of PKC (Fig. 3Go). Consistent with this result, pretreatment of BAG cells with the specific PKC inhibitor, bisindolylmaleimide (1 µM), also abolished the TPA stimulation of Raf-1 but did not inhibit Ang II-stimulated Raf-1 activation (data not shown).



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  		Figure 3. Ang II activates Raf-1 independently of PKC. In A, glomerulosa cells were pretreated with vehicle (Con) or 2 µM TPA for 48 h. Cells were then exposed to vehicle (C), 1 nM Ang II (A) or 200 nM TPA (T) for a further 5 min as indicated. In B, data from control (unshaded) or TPA-pretreated (shaded) cells represent the mean (± SEM) Raf-1 activity from three independent experiments.

 
Treatment of BAG cells with the Ca2+ ionophore, ionomycin, also activated Raf-1 but was a weak stimulus compared with Ang II (data not shown). However, chelation of intracellular Ca2+ with BAPTA did not inhibit Ang II-induced Raf-1 activation (Fig. 4AGo), indicating that Raf-1 activation is independent of changes in [Ca2+]i. Consistent with this finding, treatment of the cells with the calmodulin inhibitor, calmidazolium, also had no effect on Ang II-stimulated Raf-1 activity (Fig. 4AGo). However, incubation of BAG cells in either Ca2+-free medium containing 0.1 mM EGTA, or in the presence of the L type Ca2+ channel antagonist, nifedipine (3 µM), potentiated the Raf-1 activation induced by Ang II (Fig. 4Go, B and C), indicating that Ca2+ influx exerts a negative regulatory influence in this response.



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  		Figure 4. The role of Ca2+ in Ang II-stimulated Raf-1 activity. In A, glomerulosa cells were pretreated with vehicle (Con), 1 µM BAPTA (for 40 min) or 10 µM calmidazolium (CZM) (for 10 min) as indicated. Cells were then exposed to vehicle (C) or 1 nM Ang II (A) for a further 5 min. In B, cells were exposed to vehicle (Con), 0.1 mM EGTA in Ca2+-free medium (EGTA) or 3 µM nifedipine (Nif) for 10 min. Cells were then exposed to vehicle (C) or 1 nM Ang II (A) for a further 5 min as indicated. In C, data from control (unshaded) or Ang II-stimulated (shaded) cells represent the mean (± SEM) Raf-1 activity from four independent experiments.

 
To determine whether Raf-1 is also subject to negative regulation by downstream signaling element(s) in the MAPK cascade, we examined the effect of the selective MEK-1 inhibitor, PD98059 (10 µM) (22), in BAG cells. PD98059 had no apparent effect on the Raf-1 activity stimulated by fibroblast growth factor (FGF) but significantly increased (by approximately 50%) the Raf-1 activity stimulated by Ang II (Fig. 5Go). These findings indicate that MEK-1 (and/or an element(s) downstream of MEK-1) engages in negative feedback regulation of the Raf-1 activation induced by Ang II, but not that of FGF, in BAG cells. It is also apparent that the magnitude of Raf-1 activation elicited by FGF was appreciably greater than that stimulated by Ang II.



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  		Figure 5. MEK inhibition enhances Ang II-stimulated Raf-1 activity. In A, glomerulosa cells were pretreated with vehicle (Con) or 10 µM PD98059 for 10 min. Cells were then exposed to vehicle (C), 1 nM Ang II (A) or 50 ng/ml acidic FGF (F) for a further 5 min as indicated. In B, data from control (unshaded) or PD98059-pretreated (shaded) cells represent the mean (± SEM) Raf-1 activity from four independent experiments. Note the difference in scale between the ordinate axes for the Ang II and FGF data.

 
Raf-1 activation requires Ras
The current paradigm of Raf-1 activation via receptor tyrosine kinases entails a translocation of Raf-1 from the cytosol to the plasma membrane, where it is activated (via a poorly understood mechanism) by membrane-anchored Ras (1, 2, 3). We therefore determined the subcellular location of Ang II-stimulated Raf-1 activity, and compared its location to that stimulated by FGF, in BAG cells. Raf-1 activity stimulated by either Ang II or FGF was largely confined to membranes (prepared by differential centrifugation), and very little activity was present in the cytosol (data not shown).

We have previously demonstrated that Ang II activates Ras in BAG cells (18). To determine whether the Ang II-induced activation of Raf-1 is dependent on Ras, BAG cells were pretreated for 1 h with manumycin A (30 µM) before stimulation with either Ang II or FGF. This inhibitor of Ras farnesyl transferase prevents Ras lipidation and impairs its membrane association and activation (23). In control cells, the magnitude of FGF-stimulated Raf-1 activity was approximately 7-fold greater than that of Ang II (Fig. 6Go), and after manumycin A treatment both the FGF and Ang II stimulation of Raf-1 was abolished. Taken together, these results indicate that the activation of Raf-1 by both agonists requires that Ras is localized to the plasma membrane.



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  		Figure 6. Effect of manumycin A on Ang II-stimulated Raf-1 activation. In A, glomerulosa cells were pretreated with vehicle (Con) or 30 µM manumycin A (Man A) for 1 h. Cells were then exposed to vehicle (C), 1 nM Ang II (A) or 50 ng/ml acidic FGF (F) for a further 5 min as indicated. In B, data from control (unshaded) or manumycin A-pretreated (shaded) cells represent the mean (± SEM) Raf-1 activity from four independent experiments. Note the difference in scale between the ordinate axes for the Ang II and FGF data.

 
Upstream elements in Raf-1 activation
The activation of mitogenic signaling pathways via Gi-coupled receptors is mediated by the release of ß{gamma} complexes from PTX-sensitive G proteins (8, 9). Because the AT1-R is known to couple to Gi in certain cell types (10), we evaluated the involvement of PTX-sensitive G proteins in the activation of Raf-1 by Ang II in BAG cells. Although the majority (~70%) of Raf-1 activity stimulated by lysophosphatidic acid (LPA: which acts via a GPCR that couples to both Gi and Gq (24)) was inhibited by overnight treatment of the cells with PTX (100 ng/ml), a significant component (~40%) of Ang II-stimulated Raf-1 activity was also inhibited by the toxin (Fig. 7Go). This result suggests that Ang II activates a Gi-dependent pathway to Raf-1 in BAG cells, although the major pathway appears to be independent of Gi (and presumably operates through Gq). Conversely, the major pathway to Raf-1 activated by LPA in BAG cells is through Gi, although LPA also appears to activate a Gi-independent pathway (presumably through Gq) in these cells.



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  		Figure 7. Effect of PTX on Ang II-stimulated Raf-1 activity. In A, glomerulosa cells were pretreated overnight with vehicle (Con) or 100 ng/ml PTX. Cells were then exposed to vehicle (C), 1 nM Ang II (A) or 10 µM lysophosphatidic acid (L) for a further 5 min as indicated. In B, data from control (unshaded) or PTX-pretreated (shaded) cells represent the mean (± SEM) Raf-1 activity from three independent experiments.

 
Several reports have suggested that Ang II activates Ras (and, thence, the MAPK pathway) via a tyrosine kinase(s) (25, 26, 27, 28). To evaluate the possible involvement of a tyrosine kinase(s) in the Ang II activation of Raf-1, BAG cells were pretreated with the tyrosine kinase inhibitor, genistein (500 µM), before stimulation with Ang II. However, genistein only inhibited approximately 40% of the Raf-1 activity stimulated by Ang II, indicating that the major pathway to Raf-1 in BAG cells does not involve a genistein-sensitive tyrosine kinase (data not shown). In addition, pretreatment of BAG cells with another tyrosine kinase inhibitor, herbimycin A (3 µM), had very little effect on the Ang II stimulation of Raf-1 (data not shown). These findings indicate that tyrosine kinases do not play a major role in the Ang II-induced activation of Raf-1 in BAG cells.

Because phosphatidylinositol-3-kinase (PtdIns-3-K) has been implicated in signaling to MAPK via Gi-coupled GPCRs (29), we evaluated its possible role in the Ang II stimulation of Raf-1 in BAG cells using the potent inhibitor of PtdIns-3-K, wortmannin (WT) (30). Pretreatment of the cells with low (100 pM-1 nM) WT concentrations caused a small (35% at 1 nM) but significant enhancement of Ang II-induced Raf-1 activation (Fig. 8Go, A and B). However, WT concentrations > 1 nM inhibited Ang II-stimulated Raf-1 activity in a dose-dependent manner such that approximately 50% of the Ang II-stimulated Raf-1 activity observed in the absence of WT, and approximately 60% of the Ang II-stimulated Raf-1 activity observed in the presence of 1 nM WT, was inhibited by 100 nM WT. In other experiments, WT (up to 100 nM) had no effect on basal Raf-1 activity (data not shown).



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  		Figure 8. Effect of PtdIns-3-K inhibition on Ang II-stimulated Raf-1 activity. In A, glomerulosa cells were pretreated with vehicle (Con, 0) or the indicated concentrations of wortmannin (WT) for 10 min. Cells were then exposed to vehicle (Con) or 1 nM Ang II for a further 5 min as indicated. In B, data represent the mean (± SEM) Raf-1 activity from three independent experiments. The concentration-dependent effects of LY294002 on Ang II-stimulated Raf-1 activation are shown in panel C.

 
A similar biphasic concentration-dependent effect on Ang II-stimulated Raf-1 kinase activity was also obtained when the specific PtdIns-3-K inhibitor, LY294002 (31), was employed in place of WT. Pretreatment of the cells with low (100 pM-100 nM) concentrations of LY294002 enhanced Ang II-induced Raf-1 activation, whereas LY294002 concentrations > 100 nM inhibited (in a dose-dependent manner) the Ang II-stimulated Raf-1 activity observed in the presence of 100 nM LY 294002 (Fig. 8CGo). Like WT, LY294002 also had no effect on basal Raf-1 activity (data not shown). Because WT concentrations in the 1–100 nM range (30), and LY294002 concentrations in the 100 nM-10 µM range (31), are believed to specifically inhibit PtdIns-3-K, it appears that a significant component of the Ang II-stimulated Raf-1 response is mediated by PtdIns-3-K.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Ang II was a potent stimulus of Raf-1 activation in BAG cells, with an ED50 (around 10 pM) that is similar to the ED50 (3–10 pM) at which Ang II activates MAPK in these cells (18). However, in contrast to the dose-response curve for MAPK activation, Ang II-induced Raf-1 activation reached a peak at 1 nM and declined thereafter to approximately 65% of the peak value at higher (> 1 nM) concentrations of the peptide. This result implies that, although Raf-1 activation is maximal at subsaturating (1 nM) Ang II concentrations, higher (> 1 nM) concentrations of the peptide also activate a pathway(s) that negatively regulates Raf-1 activity. Because removal of extracellular Ca2+ and blockade of L type Ca2+ channels each enhanced Ang II-induced Raf-1 activation, it is possible that this proposed negative regulatory pathway that operates at Ang II concentrations > 1 nM is mediated via Ca2+ influx. Interestingly, although higher (> 1 nM) Ang II concentrations submaximally activated Raf-1, the same peptide concentrations maximally activated MAPK (18). This apparent discrepancy between the actions of higher (> 1 nM) Ang II concentrations on Raf-1 and MAPK activity probably reflects amplification of the Raf-1 signal such that partial activation of Raf-1 elicits a maximal MAPK response. Consistent with this hypothesis, the potency with which Ang II activated MAPK (ED50 at 3–10 pM) was slightly higher than that with which it activated Raf-1 (ED50 at 10 pM) (18).

Inhibition of Ras farnesyl transferase with manumycin A abolished Raf-1 activation induced by either Ang II or FGF, indicating that both pathways upstream of Raf-1 require membrane-associated Ras. In contrast, MEK-1 inhibition by PD98059 enhanced the activation of Raf-1 by Ang II but had no apparent effect on FGF stimulation of Raf-1. Hence, in addition to the negative regulatory effect of higher (> 1 nM) Ang II concentrations, Raf-1 appears also to be negatively regulated either by MEK-1 itself or by an element downstream of MEK-1 such as MAPK. The site(s) at which such negative regulation is exerted is unknown. However, because PD98059 had no apparent effect on the FGF stimulation of Raf-1, it is may not occur at the level of Ras or of Raf-1 itself because both elements are common to the Ang II- and FGF-stimulated pathways. Negative regulation by the downstream signaling element(s) may therefore occur either at the level of the AT1-R itself or at an intermediate(s) between the receptor and Ras.

The stimulation of Raf-1 by Ang II was mediated via multiple G proteins because, although a major component of Raf-1 activation was insensitive to PTX (which inactivates Gi and Go), a significant component was PTX-sensitive. However, this PTX-sensitive component of Raf-1 activation does not appear to be required for the proliferative responses of BAG cells to Ang II because PTX had no major effect on Ang II-stimulated MAPK activation (18). Also, both the mitogenic and early gene responses of BAG cells to Ang II are mediated by PTX-insensitive pathways (17).

Ang II has previously been found to activate Ras and/or Raf-1 in several cell types. For example, Ang II-induced Raf-1 activation was reported in cardiac myocytes (32), vascular smooth muscle cells (VSMCs) (33), hypothalamic/brain stem neurones (34) and AT1-R-expressing CHO cells (35). In addition, Ang II-induced activation of Ras was observed in vascular smooth muscle cells (25, 36, 37) and cardiac myocytes (26). However, in contrast to the PKC independence of Raf-1 activation observed in BAG cells, Raf-1 activation was dependent on PKC in cardiac myocytes (32), vascular smooth muscle cells (33) and AT1-R-expressing CHO cells (35). The causal relationship between Ras and Raf-1 activation has been addressed either by the expression of dominant-negative Ras or by using the Ras farnesyl transferase inhibitor, manumycin A. Whereas Ras was not required for MAPK activation in VSMCs (36, 37) and AT1-R-expressing CHO cells (35), dominant-negative Ras inhibited c-fos transcription in H295R adrenal cells (38) and manumycin A inhibited MAPK activation in cardiac myocytes (32). The pathways by which Ang II activates Ras and Raf-1 therefore appear to vary considerably between different cell types.

Recent reports have suggested a role for src family tyrosine kinases, as well as elevation of intracellular [Ca2+]i, in the signaling pathway(s) from the AT1-R (25, 26, 27) and other Gq-coupled GPCRs (39) to Ras and the MAPK cascade. For example, electroporation of anti-src antibodies (27), or treatment with genistein (25) abolished Ang II-induced Ras activation in VSMCs. Furthermore, a src family kinase SH3 domain-specific peptide inhibited Ang II-induced guanine nucleotide exchange activity in cardiac myocytes (26), and both genistein and herbimycin A inhibited MAPK activation mediated by expressed (Gq-coupled) {alpha}1B adrenergic receptors ({alpha}1B-ARs) in HEK293 cells (28). In addition, Ang II-induced MAPK activation was inhibited by intracellular Ca2+ chelation in {alpha}1B-AR-expressing HEK293 cells (28), and by either intracellular Ca2+ chelation or treatment with the calmodulin inhibitor, calmidazolium, in VSMCs (25). In contrast to these reports, however, neither calmidazolium nor intracellular Ca2+ chelation with BAPTA had any effect, and inhibition of Ca2+ influx potentiated, the Ang II stimulation of Raf-1 in BAG cells. Furthermore, although a minor component (similar in magnitude to that which was sensitive to PTX) of Raf-1 activation was sensitive to genistein, the majority of Raf-1 activation in BAG cells was insensitive to tyrosine kinase inhibition. Similarly, although Ang II-induced tyrosine phosphorylation of the adaptor protein, shc, was reported in cardiac myocytes (26), we were unable to reproducibly demonstrate any Ang II-induced tyrosine phosphorylation of shc in BAG cells (data not shown).

The effects of the PtdIns-3-K inhibitors, wortmannin (WT) and LY294002, on Raf-1 activation were biphasic, with an initial increase in activity at lower concentrations of each inhibitor. However, WT concentrations in the nanomolar range, and LY294002 concentrations 100-fold higher, dose dependently inhibited the maximal Ang II-induced Raf-1 activation observed in the presence of 1 nM WT, or 100 nM LY294002, respectively. Thus, approximately one half of the Ang II-stimulated Raf-1 activity observed in the presence of 1 nM WT or 100 nM LY294002 was inhibited at WT and LY294002 concentrations of 100 nM or 10 µM, respectively. Because these concentrations of WT (30) and LY 294002 (31) are known to fully inhibit PtdIns-3-K, these findings are consistent with a role for PtdIns-3-K in the Ang II-induced activation of Raf-1. However, it is not yet clear why very low WT (and LY294002) concentrations enhanced Raf-1 activity.

In conclusion, we have demonstrated that Ang II potently and transiently activates multiple Ras-dependent pathways to Raf-1 kinase in BAG cells. Although a major pathway to Raf-1 operates independently of Gi (presumably via Gq), a significant component of Raf-1 activation was mediated via Gi. Raf-1 activity was subject to negative regulation at Ang II concentrations > 1 nM (possibly via Ca2+ influx), and also via a downstream signaling element(s) in the MAPK pathway. In contrast to the mechanisms of Raf-1 activation induced by Ang II in other cell types, Raf-1 activation in BAG cells was independent of PKC activation and elevation of [Ca2+]i, and was predominantly independent of tyrosine kinases. However, in common with the mechanism of Raf-1 activation via Gi-coupled GPCRs, Raf-1 activation via the Gq-coupled AT1-R in BAG cells was partially dependent on Gi and PtdIns-3-K.


   Acknowledgments
 
We thank Tamas Balla for many fruitful discussions and Xue Zhao for preparing bovine adrenal glomerulosa cells.


   Footnotes
 
1 Supported in part by an International Fellowship (FS-95018) from the British Heart Foundation. Back

2 Supported by PHS Grant R-01 DK-52825 and a fellowship from the V Foundation. Back

Received June 18, 1998.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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