Division of Nephrology, 1 Departments of Medicine and 2 Pathology, Ralph H. Johnson Veterans Affairs Medical Center and Medical University of South Carolina, Charleston, South Carolina 29425-2227
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ABSTRACT |
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After vascular endothelial injury, angiotensin II (ANG II) plays a role in the resulting hypertrophic response, and expression of epidermal growth factor (EGF) is enhanced. Therefore, we tested the possibility that EGF regulates vascular ANG II action and receptor expression. Incubation of cultured aortic vascular smooth muscle cells (VSMC) with EGF (or basic fibroblast growth factor but not platelet-derived growth factor isoforms) resulted in concentration-dependent (1-50 ng/ml EGF), time-dependent (>8 h), and reversible decreases in ANG II surface receptor density. For example, a 50% reduction was observed after exposure to 50 ng/ml EGF for 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 h resulted in a 77% reduction in ANG II-stimulated inositol phosphate formation. EGF not only prevented but also reversed ANG II receptor upregulation by 100 nM corticosterone. The specific tyrosine kinase inhibitor tyrphostin A48 (50 µM) reduced EGF-stimulated thymidine incorporation and EGF-stimulated phosphorylation of mitogen-activated protein kinase but did not prevent EGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristate acetate (100 nM for 24 h) prevented EGF from reducing ANG II receptor density. In summary, EGF is a potent negative regulator of vascular ANG II surface receptor density and ANG II action by mechanisms that do not appear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reduced surface ANG II density cannot be eliminated by the present studies.
vascular smooth muscle cells; mitogen-activated protein kinase; G proteins
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INTRODUCTION |
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THE POTENT PRESSOR HORMONE angiotensin II (ANG II) plays an important role in the maintenance of vascular tone and, in excess, contributes to disease processes. Binding of ANG II to specific receptors on the surface of vascular smooth muscle cells (VSMC) initiates a signal transduction cascade that culminates in contraction and/or hypertrophy. Because of the importance of ANG II in homeostasis and in pathophysiological states, factors regulating ANG II action have been a topic of significant interest.
A number of growth factors including epidermal growth factor (EGF) have been demonstrated to regulate ANG II action. EGF-ANG II interactions are relevant to vascular endothelial injury, because both EGF and ANG II appear to be involved in its pathophysiology. Expression of EGF, EGF-like compounds, and EGF receptors are induced in the vascular smooth muscle after endothelial injury (8, 14), and ANG II participates in the development of neointimal hyperplasia after endothelial injury (17). However, studies on the direct regulation of ANG II action by EGF are conflicting. On one hand, EGF and ANG II are synergistic in enhancing cultured vascular smooth muscle growth (1), and EGF enhances expression of ANG II receptors transfected into naive cells (6). A third study, however, has shown that EGF reduces ANG II-stimulated signal transduction, ANG II surface receptor density, ANG II receptor mRNA transcription rate, and ANG II receptor mRNA stability in cultured VSMC (16).
To resolve this issue, we performed a detailed investigation of the effects of EGF on vascular ANG II receptor expression and signaling. Specifically, we tested the hypothesis that EGF downregulates ANG II receptors and coupled actions. In addition, we performed studies to determine whether alterations in protein tyrosine phosphorylation are involved in the regulation of ANG II receptor expression by EGF.
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METHODS |
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VSMC isolation, maintenance, and
characterization. Aortas from Sprague-Dawley rats
(Harlan Sprague Dawley, Indianapolis, IN) weighing 125-300 g were
cleaned of endothelium, fat, and adventitia. Smooth muscle strips were
incubated in collagenase (2 mg/2 ml) for 2 h, cut into
2-mm2 pieces, and allowed to adhere to a culture flask for
explantation. Then, a covering layer of growth medium [10%
(vol/vol) newborn calf serum, 1% (vol/vol) nonessential amino acids,
100 U/ml penicillin, and 100 µg/ml streptomycin in minimal essential
medium] was added. Cells were incubated in humidified 5%
CO2-95% air atmosphere until confluent. Medium was changed every 5 days. Cells were passaged every
7-10 days by harvesting with trypsin-EDTA and seeded at a ratio of
1:4. Cells exhibited characteristic stellate VSMC morphology and
stained positively for smooth muscle -actin (21). Endothelial cell
contamination was minimal (0-3%), as assessed with antibodies to
the endothelial cell marker factor VIII-related antigen. In these
studies, confluent cells from two cell lines were fixed in 3.7%
formaldehyde in phosphate-buffered saline for 15 min, treated with
0.1% Triton X-100, and then exposed to rabbit anti-factor VIII-related
antigen (1:200 dilution). The secondary antibody was labeled with
rhodamine for detection.
ANG II radioligand binding. Binding studies were performed on confluent cells in duplicate wells of 24-well plates. Binding buffer consisted of 50 mM tris(hydroxymethyl)aminomethane (Tris), 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.25% bovine serum albumin, and 0.5 mg/ml bacitracin, pH 7.4. Incubation volume was 300 µl. We performed single-concentration ANG II receptor binding studies by adding 50 fmol of 125I-labeled ANG II (New England Nuclear, Boston, MA for all isotopes) to all wells and 1 µM unlabeled ANG II (Peninsula Laboratories, Belmont, CA) to certain wells (for the determination of nonspecific binding, <15% of total binding). Competition binding studies were performed by adding 125I-ANG II (50 fmol) to all wells and nine concentrations of unlabeled ANG II (0.5 nM-10 µM) to various wells. Studies were performed at 4°C for 90 min to obtain binding equilibrium and prevent receptor internalization (22). We removed free hormone by washing monolayers three times with ice-cold saline. Cells were solubilized with 0.1% sodium dodecyl sulfate (SDS)-0.1 N NaOH, and gamma radioactivity was counted. Receptor density and binding affinity were determined by weighted four-parameter curve fitting (SigmaPlot, Jandel Scientific, Corte Madera, CA). In previous studies, we found that all ANG II receptors in cultured VSMC are of the AT1 subtype (20). ANG II-receptor binding parameters are not affected by cell passage or by body weight at which animals are killed (125-300 g).
AT1a receptor mRNA blotting.
Total RNA from confluent VSMC was isolated with TRIzol reagent (GIBCO
Laboratories, Grand Island, NY), a modification of the guanidinium
isothiocyanate method. Twenty micrograms of RNA were loaded on each
lane of a formaldehyde-agarose gel and separated by electrophoresis.
The gels were blotted on nylon membranes, and RNA was cross-linked to
the membranes with ultraviolet light (1,200 J). The plasmid pCA18
containing the complete coding sequence of the rat ANG II
AT1a receptor (15) was kindly
provided by Kenneth E. Bernstein, Emory University School of Medicine.
The insert was subcloned into pBluescript (Stratagene, La Jolla, CA), and the resulting plasmid was digested with Cla1 or Ssp1. The insert
was oriented such that T3 polymerase directed transcription of a
full-length (Cla1) or partial (Ssp1) antisense RNA. Riboprobes were
then prepared by in vitro transcription in the presence of [32P]UTP with the
MaxiScript kit (Ambion, Austin, TX). As a control for loading, a
375-base pair antisense riboprobe for rat glyceraldehyde-3 phosphate
dehydrogenase was also used. Cross-linked, prehybridized nylon
membranes were hybridized overnight with the probes at 68°C in
Quik-Hyb (Stratagene), then washed [once for 20 min at room temperature with 1× saline sodium citrate (SSC) in 0.1% SDS,
three times for 20 min at 68°C with 0.2× SSC in 0.1%
SDS], rinsed with distilled water, blotted dry, and exposed to
X-ray film at 70°C for 15-60 min using an intensifying
screen. Autoradiograms were imaged on a flatbed scanner
and quantitated with densitometry software (ScanAnalysis,
Elsevier/BIOSOFT, Nottingham, UK) for the Apple Macintosh.
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RESULTS |
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EGF effects on growth and integrity of VSMC. Incubation of serum-deprived cells for 24 h with EGF resulted in concentration-dependent (1-50 ng/ml) incorporation of thymidine. EGF (50 ng/ml) elicited 8- to 10-fold increases in thymidine incorporation over control (n = 8 plates from 3 different cell lines). Basic fibroblast growth factor (bFGF) was similarly potent, but platelet-derived growth factor isoforms (PDGF-AB and PDGF-BB) elicited only two- to threefold increases in thymidine incorporation (data not shown). Treatment for 24 h with 50 ng/ml EGF did not result in deficits in cell integrity as assessed (6 wells on 2 plates) by gross inspection, protein content [0.55 ± 0.02 (control) vs. 0.61 ± 0.02 (EGF) µg/µl], cell count [1,146 ± 212 (control) vs. 1,246 ± 117 (EGF) × 104 cells/ml], and dye exclusion [85 ± 3 (control) vs. 84 ± 4 (EGF) percentage of cells that exclude dye].
Regulation of ANG II receptor density by EGF. Studies were performed to characterize regulation of ANG II receptor binding by EGF in cultured VSMC. Serum-deprived VSMC were incubated with EGF for 24 h, and then ANG II radioligand binding (125I-ANG II ± a single excess concentration of unlabeled ANG II) was performed. As demonstrated in Fig. 1, exposure to EGF resulted in concentration-dependent reductions in specific ANG II binding, with a threshold effect at <10 ng/ml and maximal reductions (50% of control) at 50 ng/ml. The EGF effect on ANG II binding was of similar magnitude regardless of how the binding data were expressed, i.e., percentage of control, counts per minute per well, or counts per minute per milligram cell protein. bFGF but not PDGF-BB or PDGF-AB also reduced ANG II binding in VSMC (Fig. 1). EGF reduced ANG II binding in serum-repleted VSMC as well, but the threshold concentration in the serum-repleted setting was two- to threefold higher. To determine whether the reduction in ANG II binding was a function of binding affinity or receptor density, VSMC were incubated with 0 or 50 ng/ml EGF for 24 h, and then ANG II competition binding studies (125I-ANG II ± 9 concentrations of unlabeled ANG II) were performed. Analysis of binding data (n = 7 pairs of plates from 3 different cell lines, analysis of variance) revealed that binding affinities were not different between control cells [pKi = 7.83 ± 0.08, dissociation constant (Kd) = 14.9 nM] and EGF-treated cells (pKi = 7.80 ± 0.14, Kd = 15.8 nM), whereas receptor density in EGF-treated cells (2.00 ± 0.64 pmol/mg protein) was reduced by 43% (P < 0.05) compared with that from control cells (3.53 ± 0.91 pmol/mg protein). To determine whether EGF can accentuate homologous downregulation of ANG II receptors, VSMC were incubated with 50 ng/ml EGF alone, 100 nM ANG II alone, or EGF and ANG II together for 24 h. EGF with ANG II did not reduce ANG II binding to a greater extent than that of ANG II alone: 49 ± 4% of control (EGF), 35 ± 4% of control (ANG II), 33 ± 3% of control (EGF + ANG II) (n = 9 plates from 3 different lines).
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Figure 2 depicts the time dependence of EGF-mediated reduction in ANG II binding. Exposure to 50 ng/ml EGF for 1, 2, 4, or 8 h did not alter ANG II binding, whereas the effect was observed at 12 h and after (Fig. 2A). Removal of EGF after 24 h allowed 50% recovery of binding. Figure 2B demonstrates that it was necessary for EGF to be present for the entire 24 h for the full EGF effect to be manifested, although ANG II binding was perceptibly decreased 24 h later by even 1 h of EGF exposure. AT1a-receptor mRNA levels were measured after various durations of EGF exposures. Figure 3 demonstrates that after a 4-h EGF exposure (when EGF had not yet reduced ANG II binding), AT1a-receptor mRNA levels were greatly reduced, whereas after a 24-h EGF exposure (when EGF had greatly reduced ANG II binding), AT1a-receptor mRNA levels were returned nearly to control.
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We investigated the interaction of EGF with corticosteroids on the regulation of ANG II receptor binding. Glucocorticoids and mineralocorticoids effect increases in vascular ANG II AT1a-receptor mRNA levels, synthesis of AT1a receptors, and upregulation of ANG II surface receptor density (18, 20, 24, 25). These studies were performed to determine how EGF, which decreases ANG II surface binding by as yet unknown means, would affect glucocorticoid-mediated upregulation of ANG II binding, which occurs by AT1a-receptor gene induction. Figure 4A demonstrates that 24 h of incubation of VSMC with 100 nM corticosterone increased ANG II binding by >100% and that a similar duration of incubation with 50 ng/ml EGF decreased ANG II binding by >50%. When administered together, corticosterone and EGF resulted in ANG II binding that was not different from control. In a follow-up experiment, we investigated the effects of EGF in VSMC in which ANG II receptor upregulation by corticosterone had already been established (Fig. 4B). In cells in which ANG II receptors were upregulated by 24 h incubation with corticosterone, addition of EGF in the continued presence of corticosterone for an additional 24 h completely reversed the upregulation.
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Effects of EGF on ANG II action. Effects of EGF on ANG II actions were investigated in cultured VSMC, so that correlation of ANG II responses with ANG II receptor density might be assessed. We measured total inositol phosphate formation, a well-described second messenger response to ANG II in cultured VSMC (Fig. 5). EGF (50 ng/ml) elicited a 20% increase in total inositol phosphates over control after 1 min, which resolved by 5 min (Fig. 5A). In comparison, 100 nM ANG II elicited a 75% increase in total inositol phosphates over control after 0.5 min. When cells were exposed to 50 ng/ml EGF for 5 min and then 100 nM ANG II for an additional 0.5 min, total inositol phosphates were increased by 112% over control. In contrast, 24 h (i.e., a more prolonged) EGF exposure blunted ANG II-stimulated inositol phosphate responses by 77% (Fig. 5B). These studies demonstrate that prolonged but not brief exposure to EGF reduces ANG II-stimulated inositol phosphate formation. The time courses of the reduction in receptor binding and of the attenuation of ANG II action are similar.
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Mechanisms of EGF-mediated reduction in ANG II receptor density. Studies were performed to determine whether EGF-mediated reductions in ANG II binding were dependent on tyrosine phosphorylation, an event requisite for most EGF actions. These studies were performed with A48, which has a low half-maximal inhibitory concentration for inhibition of tyrosine phosphorylation (4, 26). Initial studies were performed to determine whether tyrphostin A48 was able to block EGF actions (thymidine incorporation, tyrosine phosphorylation of MAP kinase), which are known to be dependent on tyrosine kinase activity. One-hour preincubation with tyrphostin A48 (50-75 µM) decreased EGF-mediated thymidine incorporation (Fig. 6A) and tyrosine phosphorylation of p44/42 MAP kinase (Fig. 6B). Figure 6C demonstrates that 50 µM tyrphostin A48 alone did not alter ANG II binding and could not reverse the inhibition by 50 ng/ml EGF of ANG II binding. Studies were not performed with 75 µM tyrphostin A48, since tyrphostin A48 alone in this concentration reduced ANG II binding (data not shown). Similarly, concentrations of the tyrosine kinase inhibitor genistein that inhibited EGF-stimulated thymidine incorporation reduced ANG II binding.
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From the results of the experiments outlined above, we sought alternate biochemical pathways by which EGF might reduce ANG II receptor binding. To determine whether protein kinase C is involved, serum-deprived VSMC were exposed to 0 or 50 ng/ml EGF for 24 h after protein kinase C had been downregulated by a previous 24-h incubation period with 100 nM phorbol myristate acetate (PMA). Figure 7 demonstrates that PMA alone did not alter ANG II binding and that EGF reduced ANG II binding even when protein kinase C was downregulated. In preliminary studies, we documented that long-term PMA treatment downregulated protein kinase C, in that 24-h treatment with 100 nM PMA prevented acute PMA treatment (10 min, 100 nM) from phosphorylating MAP kinase (n = 2 in duplicate, data not shown). These studies suggest that protein kinase C is not involved in the intracellular signaling pathway by which EGF reduces ANG II surface receptor density.
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It has been reported that EGF acts through a pertussis toxin-sensitive G protein (Gi) rather than tyrosine phosphorylation in cultured liver cells (11). Therefore, we performed studies to determine whether pertussis toxin can prevent EGF from reducing ANG II binding. Serum-deprived VSMC were treated with 0 or 50 ng/ml EGF either at the same time as or 24 h after exposure to 100 ng/ml pertussis toxin. Figure 8 demonstrates that pertussis toxin alone did not alter ANG II binding and that pertussis toxin was unable to negate the effects of EGF on ANG II binding. In preliminary studies, we documented that pertussis toxin blocks the Gi-signaling pathway in VSMC in that preincubation with pertussis toxin (100 ng/ml overnight) prevented ligands that signal through Gi (lysophosphatidic acid, sphyngosine-1-phosphate) from phosphorylating MAP kinase (n = 2 in duplicate, data not shown). These studies suggest that pertussis toxin-sensitive targets such as Gi are not involved in the intracellular signaling pathway by which EGF reduces ANG II surface receptor density.
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DISCUSSION |
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The results of the present study demonstrate that EGF inhibits ANG II action in vascular smooth muscle. Because ANG II-stimulated inositol phosphate formation and ANG II receptor density were reduced after prolonged EGF exposure (>8 h), but neither were reduced after brief EGF exposure, it is likely that the reduction in ANG II action by EGF is at least partially mediated through the reduction in ANG II receptor density. Dependence of ANG II action on ANG II surface receptor number has been demonstrated consistently in our laboratory under a wide variety of circumstances (20, 23, 24). Our results are consistent with those from another set of investigators, who reported that EGF reduced ANG II receptor density and ANG II-stimulated inositol phosphate formation in cultured VSMC (16).
These studies elucidate to some degree the mechanisms of regulation of ANG II receptors by EGF. The time-course experiments are informative, in that >8 h of EGF exposure was necessary for downregulation of ANG II receptor density (Fig. 2A). This time course eliminates the possibility that EGF directly interferes with the ANG II-ANG II receptor binding reaction. If a steric effect of EGF were involved in reducing ANG II binding, this would occur after much shorter EGF exposures. Another potential mechanism, which the current studies do not address, is that EGF alters the phenotype of the cultured VSMC (i.e., secretory) to a form (contractile or fibroblastic) that expresses fewer surface ANG II receptors.
We confirmed prior data (16) demonstrating that EGF exposure results in significant reductions in AT1a-receptor mRNA levels after 4 h exposure that were virtually resolved by 24 h. These investigators determined that EGF reduces AT1a-receptor mRNA levels by slowing the rate of transcription as well as destabilizing mRNA. It is possible that a reduced amount of AT1a-receptor mRNA at 4 h after EGF exposure results in reduced surface receptor density and reduced ANG II action 24 h after EGF exposure (16). In our studies, EGF offset the ability of corticosterone (which induces AT1a receptor mRNA) to increase ANG II receptor binding (Fig. 4A) and reversed established upregulation of ANG II receptors (Fig. 4B). Although all these data are consistent with regulation by EGF of ANG II receptor density via modulation of AT1a-receptor mRNA levels, it is also possible that EGF regulates ANG II receptor density by effects on existing ANG II receptor protein. EGF could enhance ANG II receptor internalization to increase the internal receptor pool or hasten the degradation rate of existing receptors. To make these distinctions, future studies will be performed to determine the turnover rate of ANG II receptors and to measure the total cell pool of ANG II receptor protein in the unperturbed state and on administration of EGF.
EGF was unable to reduce ANG II receptor density beyond the level reached by homologous downregulation (by ANG II itself). This result may be a manifestation of reaching the point of maximal downregulation on exposure to 100 nM ANG II for 24 h. ANG II and EGF may downregulate ANG II receptors through similar mechanisms, whereas additive effects of different mechanisms on ANG II binding would have allowed EGF to lower ANG II receptor density below that reached by exposure to ANG II alone. The report that ANG II causes cultured VSMC to release EGF-like growth factor (19) raises the intriguing possibility that a component of homologous downregulation of ANG II receptors by ANG II may be mediated through autocrine EGF or related compounds.
It is of interest that EGF and bFGF reduced ANG II receptor density but PDGF isoforms did not (Fig. 1). This result is in direct contradistinction to an earlier study in which PDGF-BB (50 ng/ml for 24 h) as well as EGF and bFGF produced robust reductions in ANG II binding (16). Reasons for this difference are not clear. All three polypeptides were indeed growth factors in our VSMC, but EGF and bFGF effected significantly greater thymidine incorporation than did either of the PDGF isoforms. It is possible that densities of various growth factor receptors vary among animals or cell lines and that cells with more receptors for a particular growth factor can respond to that growth factor with reduced ANG II receptor density. Similarly, differences between experimental preparations might explain the conflict in the literature with regard to regulation of ANG II receptors by EGF. Our results demonstrating reductions in ANG II density by EGF are concordant with another study performed in VSMC derived from rat vessels (16) and discordant with a study performed in Cos cells transfected with ANG II receptors (6).
For the majority of EGF actions, phosphorylation of protein tyrosine is requisite. EGF phosphorylates tyrosine residues on EGF receptors (9), phospholipase C (13), G proteins (7), and MAP kinase (Fig. 6B). Therefore, we performed studies to determine whether tyrosine phosphorylation is necessary for reduction of ANG II binding by EGF. Tyrphostin A48 (a member of a family of tyrosine kinase inhibitors derived from erbstatin) at 50 µM was chosen to inhibit tyrosine kinases for these reasons: 1) it has a very low inhibitory concentration for EGF receptor kinase among the tyrphostins (4); 2) it reduced EGF-stimulated thymidine incorporation and tyrosine phosphorylation of MAP kinase by 50-80% (Fig. 6, A and B); and 3) it did not itself alter ANG II binding (Fig. 6C). Figure 6C shows that reductions in ANG II binding by EGF could not be prevented to any degree when tyrosine kinases were inhibited by 50 µM tyrphostin A48. These results suggest that tyrosine phosphorylation may not be necessary for EGF to reduce ANG II surface receptor density in cultured VSMC. This is the major finding of the present study. On the other hand, 50 µM tyrphostin A48 was not complete in its ability to prevent EGF-stimulated thymidine incorporation and tyrosine phosphorylation of MAP kinase. It is possible, therefore, that a minimal amount of protein tyrosine phosphorylation may be sufficient for EGF to reduce ANG II binding. This possibility is not testable with the existing reagents, because concentrations of tyrosine kinase inhibitors that completely prevented EGF effects on thymidine incorporation and tyrosine phosphorylation of MAP kinase reduced ANG II binding on their own. However, the fact that the EGF effect on ANG II binding was so completely unaffected by 50 µM tyrphostin A48 points to a mechanism(s) other than tyrosine phosphorylation.
There have been occasional reports of EGF actions that do not require protein tyrosine phosphorylation. In human pancreatic carcinoma cells, tyrosine phosphorylation was necessary for EGF-stimulated migration but not EGF-stimulated adhesion (10). Therefore, other mechanisms of reduction of ANG II receptor density by EGF were sought. We postulated that serine-threonine phosphorylation rather than tyrosine phosphorylation might be necessary for EGF to reduce ANG II binding. Therefore, protein kinase C, a serine-threonine kinase, was downregulated by standard means (prolonged exposure to PMA) before and during EGF exposure. However, EGF was still able to reduce ANG II binding (Fig. 7). Another possibility is that EGF action is mediated through a pertussis toxin-sensitive G protein. The precedent for this mechanism derives from the fact that pertussis toxin blocks activity of phospholipase C but not tyrosine phosphorylation of phospholipase C in rat hepatocytes (11). However, pertussis toxin had no effect on reduction in ANG II binding by EGF (Fig. 8). Therefore, at this point in time, mechanisms by which EGF depresses ANG II action and ANG II surface receptor density in cultured VSMC are unknown, and further studies will be necessary.
The clinical implications of these studies may be relevant to vascular endothelial injury. Binding sites for EGF in vascular smooth muscle are well documented (3, 5), and vascular injury induces the expression of EGF, EGF-like compounds, and EGF receptors in vascular smooth muscle (8, 14). Mechanically injured VSMC in culture also release growth factors, including EGF (2). Because vasorelaxing factors are lost on endothelial injury, there is risk for development of unopposed vasoconstriction, i.e., vascular spasm. Reduction of ANG II receptor density on induction of the EGF system may reduce the likelihood of extreme vasoconstriction during the first few days after vascular endothelial injury.
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ACKNOWLEDGEMENTS |
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We sincerely thank Jana J. Fine, Pamela S. Wackym, and Georgiann Collinsworth for expert technical assistance.
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FOOTNOTES |
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This work was supported by a grant-in-aid from the South Carolina affiliate of the American Heart Association, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52448, a Veterans Affairs Merit Award, and biomedical research funds from Dialysis Clinic, Inc.
Address for reprint requests: M. E. Ullian, Division of Nephrology, Dept. of Medicine, Medical University of South Carolina, Clinical Sciences Bldg. 829, 171 Ashley Ave., Charleston, SC 29425-2227.
Received 15 January 1997; accepted in final form 16 June 1997.
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