Departments of 1 Pharmacology and 2 Medicine, University of California, San Diego, School of Medicine, La Jolla, California 92093-0636
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ABSTRACT |
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We have studied
Gq-linked ANG II signaling [inositol phosphate (IP)
accumulation, Ca2+ mobilization] in primary cultures of
rat cardiac fibroblasts (CFs) and have found that ANG II initiates a
protein kinase C (PKC)-mediated negative feedback loop that rapidly
terminates the ANG II response. Pharmacological inhibition of PKC by
staurosporine and GF-109203X doubled IP production over that achieved
in response to ANG II alone. Inhibition of PKC also led to larger
Ca2+ transients in response to ANG II, suggesting that
Ca2+ mobilization was proportional to
Gq-phospholipase C-IP3 activity under
the conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMA
halved IP formation, with an EC501 nM; 4
-PMA was
inactive. Time course data demonstrated that ANG II-mediated IP
production fully desensitized within 30 s; PKC inhibition reduced
the rate and extent of this desensitization. In cells desensitized to
ANG II, a purinergic agonist still mobilized intracellular
Ca2+, indicating that desensitization was homologous. The
ANG II-induced Ca2+ signal was fully resensitized within 30 min. The data demonstrate that a large portion of the
IP-Ca2+ responses of rat CFs to ANG II are short-lived
because of rapid, PKC-mediated desensitization.
AT1 receptor; angiotensin II; purinergic receptor; inositol phosphates; intracellular calcium
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INTRODUCTION |
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CARDIAC FIBROBLASTS (CFs) are an important site of ANG II action in the heart (reviewed in Refs. 2 and 24). CFs are the major nonmyocyte constituent of cardiac tissue, comprising two-thirds of the cell number and one-fifth of the cell mass in the heart. CFs respond to ANG II by increased proliferation (4), by increased extracellular matrix production (6, 8), and by synthesis and release of paracrine factors that mediate cardiac myocyte hypertrophy and function (12, 13). An intra-cardiac renin-angiotensin system exists that contributes to the effects of ANG II on heart cells (7).
Our laboratory has observed several differences in the functional coupling of angiotensin receptors in CFs vs. cardiac myocytes. For instance, the ANG II-induced production of inositol phosphates (IPs) and Ca2+ transients is negligible in freshly isolated adult rat cardiac myocytes, whereas these functional parameters are very responsive to ANG II stimulation in cultured adult CFs (9, 15). ANG II binds to AT1 receptors on CFs (6, 26) and activates G protein-coupled signaling (via Gq) and tyrosine kinases (19, 20, 21, 28). The Gq-linked signaling includes activation of phospholipase C (PLC), production of inositol phosphates (IPs), mobilization of intracellular Ca2+, and consequent activation of protein kinase C (PKC) (4). Data in the literature suggest that PKC mediates multiple cellular responses to ANG II and has numerous downstream signaling effects.
The AT1 receptor was shown to be serine/threonine phosphorylated in vascular smooth muscle (11), but the kinases involved in phosphorylation and the effects on receptor desensitization were not clear. Many investigators have studied the effects of PKC on AT receptor desensitization using heterologous expression systems, although initial reports differed as to whether PKC was important in AT receptor desensitization. Currently, evidence exists both for and against involvement of PKC in desensitization of AT1 receptors. Studies performed using isolated neonatal CFs demonstrated a lack of effect of PKC inhibition on ANG II-induced Ca2+ signaling (23). AT1A receptors transfected into human embryonic kidney cells were phosphorylated by PKC, but desensitization did not appear to be influenced by this phosphorylation event (16). Recent evidence has demonstrated the involvement of PKC in desensitization of heterologously expressed AT1A receptors using nanomolar concentrations of ANG II (3). Additionally, mutagenesis of the COOH terminus of the AT1A receptor has identified specific residues that are phosphorylated by PKC that can lead to receptor desensitization (22). The lack of consistency in these reports suggests that the mechanisms governing desensitization of AT receptors may differ among cell types and expression systems.
We have tried to examine physiologically relevant signaling systems by using adult rat CFs containing only endogenously expressed AT1 receptors. We have observed a rapid desensitization of ANG II-linked signaling in CFs. The data demonstrate that Gq-coupled ANG II signaling in CFs desensitizes by a mechanism that involves PKC and that desensitization is homologous and distinct from that of purinergic signaling. It seems likely that desensitization involves effects of PKC on the AT1 receptor.
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MATERIALS AND METHODS |
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Materials.
Staurosporine, GF-109203X, ANG II, UTP, phorbol 12-myristate 13-acetate
(PMA), 4-PMA, and the acetoxymethyl ester of indo 1 (indo 1-AM) were
supplied by Calbiochem (La Jolla, CA). Unless otherwise noted, all
other chemicals used were of reagent grade and supplied by Sigma (St.
Louis, MO).
Preparation and culture of adult rat CFs. Fibroblasts were prepared as previously described (15). Briefly, the ventricles of three to five hearts from adult male 300- to 350-g Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were minced, pooled, and placed in a collagenase/pancreatin digestion solution. Fibroblasts were isolated from enriched fractions on Percoll and suspended in DMEM (GIBCO BRL, Life Technologies) supplemented with penicillin, streptomycin, Fungizone, and 10% FBS (Gemini Bio-Products, Calabasas, CA). After a 30-min period of attachment to uncoated culture plates, cells that were weakly attached or unattached were rinsed free and discarded. After 2-3 days, confluent cultures were amplified by trypsinization and seeding onto new dishes. For signaling assays, only early passage (<5) cells were used. Cells were seeded onto 35-mm (0.5-1.0 × 105 cells/plate) or 60-mm plates (1.5-2.0 × 105 cells/plate), and grown to 80-90% confluence (3-4 days). The purity of these cultures was greater than 95% CFs as previously described (25, 26).
Phosphoinositide hydrolysis.
Cells in 60-mm dishes were labeled for 18 h with
[3H]myo-inositol (5 µCi/ml) (Amersham,
Arlington Heights, IL) in DMEM without serum. Unincorporated
[3H]inositol was removed by a series of three washes,
after which cells were placed in DMEM containing 10 mM LiCl, 10 mg/ml
leupeptin, and 1 mg/ml BSA. After a 10-min incubation, agonists were
added, and incubation continued at 37°C for 10 min or as indicated in legends to Figs. 1-6. Reactions were stopped by aspiration of
medium and addition of cold 5% trichloroacetic acid.
[3H]IPs were purified using Dowex columns and quantified
as previously described (15). Total [3H]IPs
were counted by liquid scintillation spectrometry and data expressed as
the fold change over controls (calculated using cpm/mg of
protein).
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Intracellular Ca2+
measurements.
Adult rat CFs (0.25 × 105 cells) were plated on 22-mm
glass coverslips and serum-deprived overnight prior to experiments.
Briefly, the cells were washed twice in HEPES-buffered saline (HBS: 130 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl2, 1 mM
CaCl2, and 25 mM HEPES, pH 7.4) and incubated in 2 ml HBS
containing 1 µM indo 1-AM at 37°C for 30 min. Cells were then
washed twice with HBS and placed in a 37°C chamber containing HBS
where groups of 5-8 cells were viewed using an inverted Nikon
Diaphot microscope. Fluorometric measurements were collected using the
DX-1000 System (Solamere Technology, Salt Lake City, UT), where the
field was excited at 385 nm and the emission ratio was collected at 405 nm and 495 nm, as previously described (14, 15). Nanomolar Ca2+ concentrations were calculated using the following
formula
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Estimation of protein content. Acid precipitable material was suspended in 0.4 N NaOH, and the protein content was estimated by the method of Bradford (5).
Analysis of data. Statistical comparisons (t-tests and one-way ANOVA) and graphics were performed using the programs GraphPad Prism 2.0 (GraphPad Software, San Diego, CA) and MacLab 3.5.1 (ADInstruments, Mountain View, CA).
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RESULTS |
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Inhibition of PKC enhances ANG II-induced production of IPs and Ca2+ transients. ANG II stimulated a two- to fourfold increase in IP production by CFs. IP production was significantly increased in CFs pretreated for 30 min with 0.1 µM of the PKC inhibitors staurosporine (223%, Fig. 1A) and GF-109203X (178%, Fig. 1B) compared with those treated with ANG II alone. For comparison, UTP-stimulated IP production was unaffected by staurosporine. Pretreatment with GF-109203X (0.1 µM for 30 min) also led to increases in peak Ca2+ transients vs. ANG II alone (Fig. 2); the fluorescent properties of staurosporine prevented its use in experiments involving assessment of intracellular Ca2+.
The enhancement of the ANG II signal by PKC inhibitors in both IP assays and intracellular Ca2+ measurements yielded the initial evidence that PKC was an important modulator of ANG II-induced signaling in CFs. The fact that the IP and Ca2+ signals to ANG II were so tightly coupled and responses to UTP were unaffected suggested that the effects of PKC were likely upstream of these signaling molecules, possibly at the level of the AT1 receptor. To further establish the specificity of PKC in the increased Gq-linked IP production, we treated CFs overnight with PMA (200 nM) to deplete the cells of PKC. In agreement with the PKC inhibitor data, this treatment led to significant increases in ANG II-induced IP production (210% over ANG II alone, Fig. 3A). Since inhibition of PKC effects on ANG II signaling augmented IP accumulation, we predicted that acute activation of PKC would reduce ANG II signaling. Activation of PKC by a 10-min exposure to PMA produced a concentration-dependent reduction in IP formation in response to ANG II (Fig. 3B). The EC50 for this effect wasDesensitization of ANG II-induced signaling is rapid and is slowed by inhibition of PKC. A time course for ANG II-induced signaling was performed to better understand the influence of PKC inhibition on desensitization (Fig. 4). ANG II-stimulated IP production desensitized within 30 s, with no further increases in IP signal occurring over a 15-min time course. PKC inhibition by staurosporine led to increases in IP production at every time point between 1 and 15 min and dramatically slowed desensitization of the ANG II signal. If one compares IP production in the presence and absence of PKC inhibition, it appears as though ANG II-induced IP production exhibits biphasic desensitization, i.e., a rapid phase, and a slower desensitization that becomes apparent following PKC inhibition.
We compared the ANG II response to that of the P2Y agonist, UTP. Although AT and purinergic receptors both couple to the Gq signaling cascade, their desensitization time courses are distinct, with the response to UTP resembling that of ANG II when PKC is inhibited (Fig. 4). The UTP response did not appear to be modulated by PKC: staurosporine did not alter the IP response to UTP after 15-min treatments (Fig. 1A) or at intermediate times (1, 2, 5, and 10 min; data are not shown but are superposable on UTP data in Fig. 4).Buffering intracellular Ca2+ has no effect on ANG II-induced IP production. Since Ca2+ mobilization contributes to the activation of many isoforms of PKC, we wanted to determine whether buffering the ANG II-induced Ca2+ transient would mimic inhibition of PKC. This possibility was tested using the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) (30 µM), which was able to effectively buffer agonist-induced changes in intracellular Ca2+ levels in CFs (data not shown). Surprisingly, there was no effect of BAPTA on ANG II-induced IP production (Fig. 5). We expected that blocking the Ca2+ transient would have an effect similar to the pharmacological blockade of PKC activity (enhanced signaling). The lack of a BAPTA effect suggests that a Ca2+-independent form of PKC may be responsible for the enhanced ANG II signaling. In contrast to ANG II signaling, BAPTA inhibited IP production in response to UTP by 35% (Fig. 5), pointing out mechanistic differences in regulation of these distinct Gq-linked signals. For purinergic responses, it appears likely that elevations in intracellular Ca2+ are necessary to regulate one or more components of the IP signaling pathway, whereas ANG II-linked IP production does not exhibit Ca2+ dependence.
Desensitization of ANG II signaling in CFs is homologous and reversible. To extend our information on desensitization beyond the formation of IPs, we measured increases in intracellular calcium concentration following administration of ANG II and UTP (Fig. 6). Indo-1 emission ratios (405 nm/495 nm) were collected continuously during sequential additions of ANG II (Fig. 6A) or ANG II followed by UTP (Fig. 6B). These data clearly demonstrate that the ANG-II-induced Ca2+ signal was homologously desensitized within the 2 min, since the second addition of ANG II resulted in no signal, whereas the addition of UTP induced a robust transient. Furthermore, experiments performed in the absence of extracellular Ca2+ (by chelation with EGTA) indicated that the Ca2+ transients were mainly due to Ca2+ release from IP3-sensitive internal stores (Fig. 6C). These experiments also indicate that desensitization of the ANG II response is not due to depletion of Ca2+ storage pools, since UTP caused a large transient when given 2 min after ANG II.
Resensitization of the ANG II-induced Ca2+ signal was assessed by sequential administration of two doses of ANG II to indo 1-labeled cultures. CF cultures were exposed to 1 µM ANG II for 1 min, washed with HBS, and incubated for 2-30 min prior to a second exposure to 1 µM ANG II. At 2 min, no resensitization had occurred; transients measured 10 and 20 min after the first exposure to ANG II were partially resensitized (Fig. 6, D and inset). The peak Ca2+ responses to ANG II were fully restored when consecutive doses of ANG II were given 30 min apart (Fig. 6, D and inset). ![]() |
DISCUSSION |
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In this report we have examined the desensitization of ANG II-linked signaling in the major nonmyocyte component of the heart, the CF. We have determined that ANG II signaling is rapidly desensitized in isolated CFs and that this rapid phase of desensitization is homologous and requires the activity of a phorbol ester-sensitive PKC. The desensitization of the ANG II signal may be an important event in CFs, since it effectively limits the responsiveness of the cells to ANG II. Thus, although CFs are an important target for ANG II action in the myocardium and contribute to Gq-linked hypertrophic responses (12, 13), the effect of ANG II in CFs may be short-lived, because of the sensitivity of the AT1 receptor to desensitization.
This study represents the first complete evidence that PKC is involved
in the desensitization of endogenous AT receptor-linked signaling in
adult rat CFs. The evidence comes from several approaches. Using
multiple means of inhibiting PKC activity in CFs, we have observed an
enhancement of Gq-linked signaling in response to ANG II.
Initially, we used staurosporine to pharmacologically inhibit PKC,
finding that staurosporine significantly enhanced ANG II-stimulated IP.
Because of the nonselective nature of staurosporine, we also employed
GF-109203X, to confirm our observation that inhibition of PKC enhanced
IP production. Specific inhibitors of PKA and calcium/calmodulin kinase
II did not affect the ANG II-induced IP signal. Thus the effects seen
with staurosporine were likely due to PKC inhibition rather than to
nonselective inhibition of other protein kinases. Consistent with
pharmacological inhibitor studies, depletion of cellular PKC by
overnight treatment of CFs with PMA substantially enhanced the response
to ANG II. Without the influence of
12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive PKC,
the response desensitized slowly, presumably due to the action of G
protein-coupled receptor kinases (GRK) (16). Conversely, acute addition of PMA reduced the ANG II response in CFs, with an
EC50 (1 nM) consistent with PKC activation; the
PKC-inactive congener 4-PMA was without effect. Enhancement of the
ANG II response following PKC inhibition has also been described in
neonatal myocytes (1), although desensitization of the
signal was not affected by PKC inhibition (discussed below). The
influence of PKC on carbamylcholine-stimulated IP production has also
been reported in human astrocytoma cells, where it is heterologous and
appears to result from interference with the interaction of Gq with PLC (17).
Intracellular Ca2+ mobilization occurs downstream of PLC activation and IP production. In CFs, this increase in hormone-induced cytosolic Ca2+ occurs largely from release from internal stores, with very little capacitative Ca2+ entry (15). The peak Ca2+ transients from CFs pretreated with the PKC inhibitor GF-109203X were larger than those in control cells, in agreement with our observations in the IP assays. Other than the difference in the magnitude of the peaks, the profiles from the Ca2+ measurements were similar ± PKC inhibitor. Both transients peaked and returned to baseline with no elevated plateau phase, which is characteristic of an IP3-mediated transient with little influence from extracellular Ca2+ entry. Additionally, the pharmacological blockade of PKC did not appear to affect the ability of the fibroblasts to extrude Ca2+ from the cytosol, as signal decay was comparable in the two groups.
How quickly and to what extent do IP production and the release of intracellular Ca2+ desensitize following addition of ANG II? The time course of ANG II-stimulated IP production indicates that this signal peaks and is fully desensitized within 30 s of ANG II administration. The rapid desensitization of the ANG II signal can be significantly slowed by PKC inhibition, such that full desensitization occurs only after 15 min of ANG II treatment. We hypothesize that this second and slower phase of desensitization that occurs in the absence of PKC activity (i.e., with inhibition or depletion of PKC) is due to GRK activity (16). We conclude that blockade of PKC prevents a rapid phase of desensitization of the ANG II signal and enhances the Gq-linked signaling events that occur downstream of AT receptor activation.
The majority of studies to date examining PKC involvement in ANG II/Gq desensitization have used neonatal cardiac myocytes as a cellular model; the results of these studies differ from our data. It has been demonstrated that hormone-stimulated PKC activity and intracellular Ca2+ transients were diminished by 20-h treatment with PMA, suggesting that PKC plays a role in desensitization of ANG II signaling in neonatal myocytes (27). Abdellatif et al. (1), on the other hand, concluded that desensitization of the ANG II signal likely occurs at the level of the receptor and is independent of PKC. This was concluded after depleting PKC with TPA and observing that desensitization of ANG II-induced IP production still occurred within a similar time frame as the control (PKC replete) cells. These findings contrast with our results: we observe a substantial delay in desensitization of the ANG II signal in CFs when PKC activity is blocked pharmacologically. CFs also differ from myocytes in their resensitization to ANG II. CFs fully resensitize within 30 min (Fig. 6D), whereas neonatal cardiac myocytes require 50 min or more for full resensitization of the ANG II signal (1). It is plausible that the responses of CFs more closely and quickly mirror changing plasma and local ANG II levels than do responses of myocytes, perhaps reflecting physiological needs and capacities of each cell type.
In previous studies, we have noted differences between signaling in cardiac myocytes and fibroblasts. We have found that the ANG II produces a much larger Gq signal in fibroblasts than it does in myocytes (15). In particular, we measure very little ANG II-induced IP production in adult cardiac myocytes. Since ANG II can stimulate phosphoinositide hydrolysis in the intact adult rat heart (10), it is likely that the majority of ANG II-induced IP accumulation occurs in the CFs. This may not be the case in the neonatal myocardium, where significant increases in IP production occur following ANG II stimulation of isolated myocytes (1), suggesting that significant temporal and developmental changes occur in the signaling properties of isolated cardiac myocytes and fibroblasts.
Functional expression of cloned human and rat AT1 receptor subtypes (AT1A and AT1B) in heterologous expression systems has enabled investigators gain a clearer picture of the molecular events that govern AT receptor desensitization. Recent studies have utilized truncation mutants of the AT1 receptor that lack COOH-terminal amino acids in attempts to define the structurally important regions that control receptor function. The COOH terminus has been identified as a likely candidate for phosphorylation by PKC and other kinases based on the existence of multiple serine and threonine residues in this region (3, 22). Direct phosphorylation of the AT1A receptor by PKC has been demonstrated (3); however, definition of the functional consequences of this phosphorylation event has not been clearly established. AT1A receptors overexpressed in human embryonic kidney 293 cells are phosphorylated by PKC and GRK isoforms 2, 3, and 5, but desensitization of the receptor appears to depend upon the GRKs rather than PKC (16). Thomas et al. (23) found that Gq-linked signaling in nontransfected neonatal myocytes was not affected by PKC inhibition and that PKC did not appear to phosphorylate a synthetic peptide corresponding to the COOH-terminal region of the AT1A receptor. Since these reports, evidence has emerged demonstrating the involvement of PKC in phosphorylation and desensitization of heterologously expressed AT1A receptors (3). Our data support a role for PKC in AT1 desensitization. Although we cannot rule out the possibility that the PKC effects on ANG II desensitization in fibroblasts are distal to receptor, the data from other cell types lends support to the hypothesis that PKC can phosphorylate and desensitize these receptors. Our data are consistent with an effect of PKC on the AT1 receptor.
CFs express at least four isoforms of PKC: ,
,
, and
(4). The majority of these PKC isoforms are
Ca2+ independent (
,
, and
), whereas only the
-isoform is Ca2+ dependent. PKC isoform
specificity is observed in activation of MAP kinases and regulation of
CF growth and proliferation (4). Our data with BAPTA/AM
indicate that ANG II-induced IP production does not require an
elevation of intracellular Ca2+, whereas the PKC inhibitor
data demonstrate that rapid desensitization requires PKC. It is
possible that one or more of the Ca2+-independent PKC
isoforms mediates desensitization of the ANG II response in CFs,
although further studies will be required to determine which PKC
isoforms regulate desensitization. It is also of interest that cardiac
myocytes express similar isoforms of PKC as the CFs and that they are
differentially responsive to neurohormones (18).
Our data demonstrate that ANG II signaling in CFs undergoes rapid, homologous desensitization in a PKC-dependent manner. The response to ANG II resensitizes rapidly and completely within 30 min. Production of IPs and release of intracellular Ca2+ are Gq-linked events that are sensitive to modulation by PKC in CFs. Although PKC seems to account for a large portion of the ANG II-induced desensitization in CFs, it is possible that other kinases, such as GRKs, may be involved. There may be both biological and experimental reasons that GRKs dominate in some experimental systems and PKCs dominate in others in which ANG II responsiveness and desensitization are studied. Our data on CFs represent responsiveness and desensitization of endogenous receptors in a primary culture of mammalian cells. Systems in which the AT1A receptor or a particular GRK is overexpressed heterologously may yield different data that reflect the concentration of the overexpressed protein or the differentiated properties of the host cell (e.g., different expression levels of the appropriate PKC isoforms). The adult CF may be a good model cell in which endogenous PKC and GRK-dependent systems can be advantageously studied with respect to their participation in modulating responsiveness to ANG II.
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ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-41307 and HL-09887 and by a grant from the University of California, San Diego, Faculty Senate.
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FOOTNOTES |
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Present address of J. G. Meszaros: Dept. of Physiology, NE Ohio Univ. College of Medicine, Rootstown, OH 44272.
Address for reprint requests and other correspondence: L. L. Brunton, UCSD School of Medicine, Dept. of Pharmacology, La Jolla, CA 92093-0636 (E-mail: lbrunton{at}ucsd.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.
Received 17 December 1999; accepted in final form 17 July 2000.
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