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RECEPTORS AND SIGNAL TRANSDUCTION
1Laboratorio Multidisciplinario, Sección de Posgrado, Escuela Superior de Medicina, Instituto Politécnico Nacional, México City, México; 2Facultad de Ciencias Químicas, Universidad Veracruzana, Orizaba, Veracruz; 3Departamento de Biología Celular y Tisular, Escuela Médico Militar-Universidad de Falcón, México City; 4Unidad de Investigación Médica en Genética Humana, Hospital de Pediatría, Centro Médico Nacional Siglo XXI-IMSS, México City; and 5Unidad Cardiovascular del Hospital Regional 1°, De Octubre, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, México City, México
Submitted 5 October 2006 ; accepted in final form 11 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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or ERβ. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17β-estradiol. One important system related to the activity of ER and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82–101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ER and ANG II type 1 and 2 (AT1 and AT2) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca2+ kinetics and the effect of 17β-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca2+ concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca2+ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT2 receptors associate with Cav-1. Our results show that there is a close association of AT1, AT2, and ER with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling. estrogen receptors; angiotensin type 1 and 2 receptors; phosphatidylinositol 3-kinase; intracellular signaling; tissue culture; angiotensin receptors
It has been postulated that Cav-1 acts also as a scaffold protein for signaling proteins. These proteins include G protein-coupled receptors (GPCRs), heterotrimeric G proteins, receptor tyrosine kinases, components of the renin-angiotensin system (RAS)-MAPK pathway, Src family tyrosine kinases, PKC isoforms, nitric oxide (NO) synthase, epidermal cell growth factor receptor (ECGF-R), and related receptor tyrosine kinases (8, 13, 15, 19, 26, 30, 37).
Another of the proteins associated with caveolae is estrogen receptor (ER). The steroid sex hormones, involved in several physiological and pathophysiological processes, may modulate intracellular pathways (7). In the recent past, supporting evidence of the membrane effects of estrogen after its interaction with cellular surface components has been published (11). Membrane proteins involved in rapid estrogenic actions include specific ERs, ion channels, membrane enzymes, and other ligand receptors, which are all associated with caveolae (21, 34).
One of the many systems related with ER and caveolae is the RAS, which is well known for its role in the regulation of blood pressure and fluid homeostasis (42). Estrogen-induced modulation of angiotensin II (ANG II) has been recently reported; for example, ER activation is involved in protective effects of estrogen against ANG II-induced hypertension (47). In addition, estrogen induced a decrease of ANG II type 1 (AT1) receptor (AT1R) expression, effects that are mediated through the activation of ERs in SMCs (22, 23).
Estrogen acts on the RAS at different points of the cascade: 1) synthesis of ANG II, 2) ANG II receptors, and 3) ANG II-induced responses.
At the vascular smooth muscle level, ANG II has numerous actions, including modulation of vasomotor tone, cell growth, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/Akt activity (17, 18).
The actions of ANG II are the result of its binding to specific membrane receptors [i.e., AT1R and ANG II type 2 (AT2) receptor (AT2R)] (40, 41). Both of these cell surface receptors belong to the large family of GPCRs (principally, Gq/11 and Gi/o proteins), although the metabolic pathways used are completely different and with opposing functional outcomes (1, 3, 18). It has been demonstrated that Cav-1 plays a central role in the translocation of the AT1R to the plasma membrane (3). However, their functional interaction, regarding the activation of intracellular pathways, is still unclear (3), and no clear functional association of AT2Rs with Cav-1 has been reported.
Many proteins, including ANG II receptors and ER, relate with Cav-1 through their interaction with the scaffolding peptide [Cav-1 scaffolding protein 1 (CSP-1); 82–101 amino acidic residues], and it has been proposed that CSP-1 could function as a kinase inhibitor (24, 25).
We hypothesized that CSP-1 can modulate ANG II-induced effects. Therefore, to explore the ability of CSP-1 to regulate ANG II function and analyze the relation between ER, AT1R, and AT2R, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca2+ kinetics and also on the role of 17β-estradiol (E2) in ANG II-induced effects in human arterial SMCs in culture. We found that CSP-1 inhibits the ANG II-mediated increase of the intracellular Ca2+ concentration ([Ca2+]i) in human arterial SMCs in culture by interfering with intracellular signaling, including the PI3K/Akt pathway. We also found that AT2Rs associate with Cav-1.
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MATERIALS AND METHODS |
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Culture of SMCs from human internal mammary arteries.
Cultures were obtained according to a previously described protocol (2). Briefly, internal mammary arteries (IMAs; 6 males) samples were received from bypass surgery patients. Samples were transported to our facility in ice-cold HBSS supplemented with 1% penicillin G potassium salt (Sigma-Aldrich, St. Louis, MO). The IMA was longitudinally sectioned, and the endothelial cell layer was enzymatically removed. The endothelium-denuded IMA was exposed to 0.125% trypsin solution (GIBCO-BRL, Grand Island, NY). IMAs were then enzymatically explanted and cultured with DMEM (GIBCO-BRL) supplemented with 1% penicillin G, 30% heat-inactivated FBS, and 1% SMC growth factor (SMC-GF). Explants were incubated in a humidified chamber at 37°C in a 5% CO2 atmosphere. SMCs from passages 2–6 were used in our experiments. Characterization of the cell type was performed by immune reactivity to smooth muscle-specific -actin antibody. More than 99% of cells expressed - actin.
CSP-1. We used a synthetic CSP-1 (Synthetic Peptides) peptide using the scaffolding domain of the Cav-1 sequence (amino acids 82–101; DGIWKASFTTFTVTKYWFYR). A 21-amino acid scramble peptide was used as the control.
[Ca2+]i measurements. SMC cultures were trypsinized and resuspended in DMEM supplemented with 1% penicillin G, 30% FBS, and 1% SMC-GF to a final concentration of 2.7 x 105 cells/ml; 25 µl were placed in the center of coverslip dishes (coverslip no. 1, glued to a perforated plastic petri dish). Cells were allowed to attach, and additional culture medium was then added to the well to a final volume of 2 ml. The cell cycle was synchronized by serum deprivation 48–72 h before experiments. Cells were loaded with 3 µM fura-2 AM (Sigma-Aldrich) for 2 h at room temperature in the dark in HEPES-Krebs-Henseleit solution (pH 7.4 at room temperature) composed of (in mM) 137 NaCl, 6 KCl, 1.8 CaCl2, 1.2 NaH2PO4, 1.2 MgSO4·7H2O, 5 dextrose, 2 sodium pyruvate, and 20 HEPES. Cells were washed and postincubated in the same buffer for an additional hour. Experimental dishes were placed on an inverted microscope (dual-wavelength fluorescence-imaging system InCytIm2, Intracellular Imaging, Cincinnati, OH) to measure the fluorescence emitted by the fura-2/Ca2+ complex when stimulated by UV light. Calibration was performed as previously reported (36).
We analyzed ANG II-induced increases in [Ca2+]i through concentration-dependent curves and found that a concentration of 1 µM induced 95% of the maximal increase in [Ca2+]i. We used this concentration in the rest of the experiments, and the kinetics induced were considered as the positive control.
Effects of agonists and antagonism on ANG II-induced increases in [Ca2+]i. A series of experiments was carried out to characterize the short-term effects of PD-123319 (an AT2 antagonist, 1 nM) (10, 39) and losartan (an AT1 antagonist, 1 and 10 µM) on the [Ca2+]i increase induced by 1 µM ANG II. We also characterized the short-term effects of losartan (10 µM), E2 (10 nM), and CSP-1 (10 nM) on CGP42112A-induced effects (an AT2 agonist, 10 nM) (31).
Experiments were conducted to characterize the short-term effects of E2 (0.1–10 nM) on the increase in [Ca2+]i induced by ANG II (1 µM).
A series of experiments was carried out to characterize the short-term effects of increasing concentrations of CSP-1 (1 pM–10 nM) on [Ca2+]i increases induced by 1 µM ANG II.
Immunoexpression of AT1R, AT2R, ER, and Cav-1.
To determine the expression of AT1, AT2, ER, and Cav-1 on SMCs, we used the following immunocytochemical protocol. After 48–72 h of serum deprivation, cells were washed with ice-cold PBS and fixed for 10 min in methanol at 4°C. Cells were washed and incubated for 30 min with blocking solution (2% IgG-free BSA) and incubated for 24 h at 4°C with AT1, AT2, ER, or Cav-1 antibodies (1:200, developed in the rabbit, Santa Cruz Biotechnology, Santa Cruz, CA). After this period, cells were washed with PBS and incubated for 1 h at room temperature in a dark chamber with fluorescein-conjugated secondary antibodies (goat anti-rabbit IgG, 1:250 dilution, ZYMED, San Francisco, CA). To evaluate the coexpression of AT1, AT2, ER, and Cav-1, we performed a second incubation with an alternative antibody (AT2, AT1, and Cav-1 or ER) and used rhodamine-labeled secondary antibody. Finally, immunoexpression and coexpression patterns were evaluated by confocal microscopy (confocal TCS SP2, Leica).
Immunocoprecipitation assay.
Immunocoprecipitation assays were developed according to previously described protocols (5, 29). SMCs (107 cells) were lysated with 50 µl of nondenaturing extraction buffer composed of 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 2 mM Na3VO4, 1 mM NaF, and mini-complete protease inhibitor (Roche Diagnostics, Mannheim, Germany), all diluted in 1x PBS. The homogenate was incubated 3 min on ice and passed five times through an insulin syringe. The homogenate was then incubated on ice for an additional 10 min, and the protein concentration was measured by the DC protein assay (Bio-Rad Laboratories, Hercules, CA). A total of 0.5–1 mg protein was incubated for 3 h at 4°C under mild orbital agitation with 3 µg of immunoprecipitating antibody (anti Cav-1 H-97, Santa Cruz Biotechnology). Next, 20 µl of protein G-Sepharose (Sigma-Aldrich) were added, and the mixture was incubated overnight at 4°C. The immunoprecipitation reaction was centrifuged for 15 min at 12,000 g, and the supernatant was recovered and stored at 4°C. The pellet, containing immunoprecipitated proteins, was washed three times with extraction buffer for 15 min at 12,000 g. Coimmunoprecipitation was performed at least three times with immunoprecipitating antibody for the confirmation of results. Finally, to determinate the association beetwen Cav-1-AT1, Cav-1-AT2, and Cav-1-ER, Western blots from the Cav-1 immunoprecipitation fraction were performed according to previously described procedures. Briefly, immunoprecipitating fraction proteins were applied to a 12.5% SDS-PAGE gel, electrotransferred to nitrocellulose membranes (Bio-Rad Laboratories), incubated 1 h at 37°C in blocking buffer [5% nonfat dry milk in PBS plus 0.1% Tween 20 (PBS-T)], and then incubated for 3 h at room temperature with the following different primary antibodies: Cav-1 (C-97), AT1 (N-10), AT2 (H-143), and ER (H-184). All the primary antibodies were purchase from Santa Cruz Biotechnology. All primary antibodies were diluted 1:100 in PBS-T. Membranes were washed three times during 5 min in PBS-T and incubated for 1 h at room temperature in the presence of horseradish peroxidase-conjugated secondary antibodies diluted 1:1,000 in PBS-T. Membranes were washed three times in PBS-T, and immunoblots were then developed with a diaminobenzidine (DAB) colorimetric method (Bio-Rad Laboratories).
Immunoblot analysis for activated Akt. Homogenates of SMCs were prepared by their mechanical separation from plates and low-speed centrifugation for 5 min, and the pellet was washed with ice-cold RIPA buffer (PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS) and freshly prepared protease inhibitors (Roche Diagnostics), incubated on ice for 60 min at 4°C, and disrupted additionally by sonication (Bransonic 1210). The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was recovered. A total of 30 µg protein was mixed with Laemmli sample buffer (containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris·HCl; pH 6.8; Bio-Rad) and boiled for 5 min. Samples were gel resolved by 10% SDS-PAGE and electrotransferred to 0.45-µm nitrocellulose membranes (Bio-Rad). Nonspecific binding was blocked by incubating the membranes in Blotto (PBS, 8% nonfat milk, and 0.05% Tween 20) for 1 h at room temperature. Blocked membranes were incubated in primary antibody [polyclonal antibody to phosphorylated (p-)Akt1/2/3, Santa Cruz Biotechnology] diluted in Blotto (1:200 dilution) for 3 h, washed, incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted in Blotto (1:500 dilution), washed three times with PBS-T, and finally stained with the use of the DAB substrate kit (Vector Laboratories).
Statistical analysis.
Values that represent changes in [Ca2+]i are expressed as means ± SE and were analyzed by one-way ANOVA. Ca2+ kinetic measurements were performed in 
20 randomly selected SMCs; the cell number (n) was limited only by the view field on the microscope objective, but it was representative of a total population of 6 x 103 cells in each plate. Three plates were used for each condition.
Data from densitometry of blots were analyzed by EpiChemi Darkroom from UVP Bioimaging System and are expressed as means ± SE. Differences were analyzed by one-way ANOVA and a Bonferoni post test for individual differences.
Values are expressed as means ± SE. Data were compared versus each control experiment, and statistical differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Figure 1A shows a representative tracing of the changes in [Ca2+]i induced by ANG II at 1 µM; this concentration elicited 95% of the maximal effect of ANG II. The [Ca2+]i increase elicited by 1 µM ANG II was partially abolished by the preincubation of cells (30 min) with 1 µM losartan and completely blocked in the presence of 10 µM losartan. We assume that this represents a complete antagonism of the AT1R population. We used 1 µM ANG II in the rest of the experiments as a positive control.
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Effects of AT2R blockage and stimulation on the ANG II-induced increase in [Ca2+]i.
To evaluate the participation of AT2 in angiotensin-induced effects, we performed a series of experiments in the presence of PD-123319 (1 nM), an AT2 antagonist. Our results showed no significant differences with the changes induced by ANG II (Fig. 2, A and B). The analysis of the area under the curve as a measure of the increase in [Ca2+]i showed no statistical differences between ANG II-induced effects in the absence and presence of PD-123319 (Table 1). However, when we stimulated AT2Rs with the specific agonist, CGP42112A (10 nM), in the presence of losartan (10 µM), we observed a significant increase in [Ca2+]i, 20% of the ANG II maximal effect (Fig. 2, A and B). Preincubation with E2 completely blocked this effect.
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Immunoexpression of AT1R, AT2R, and Cav-1 on SMCs.
We found intense immunoexpression of AT1R, AT2R, ER, and Cav-1 (Fig. 4) in human SMCs in culture. Our result showed strong coexpression of AT1 and Cav-1; AT2 and Cav-1 seemed to be expressed preferentially but not exclusively near the periphery of cells. ER and Cav-1 also showed coexpression sites. The pixel-by-pixel analysis (Fig. 4) strongly suggested the coexpression of these molecules; however, to assure this, we performed an immunocoexpression analysis.
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on SMCs.
We found an intense level of expression of AT1R, AT2R, and ER (Fig. 5) in human SMCs in culture. The fluorescence did not appear to be particularly associated with the cytoplasm or plasmalemma. These results demonstrate the presence of AT2Rs in SMCs in culture and support the responses found with pharmacological stimuli of those receptors (Fig. 2, A and B).
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are shown in yellow in Fig. 5. Our results showed that there was a higher association between AT1 and ER than between AT2 and ER, as shown by the differential intensity levels of the merged images. No unspecific fluorescence was observed when human SMCs were incubated without the primary antibody (data not shown).
Immunocoprecipitation. To demonstrate the coexpression of AT2R and Cav-1, we performed an immunocoprecipitation analysis using Cav-1 antibodies as the primary immunoprecipitant.
We found positive immunocoprecipitation between AT1, AT2, ER, and Cav-1 (Fig. 6, A–C). These results confirm the close relationship between those receptors in or near caveolae.
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Immunoreactivity was made evident in protein bands with apparent molecular masses of 56 and 60 kDa for p-Akt2 and p-Akt1/3, respectively (Fig. 7A). Our results showed increases in p-Akt1, p-Akt2, and p-Akt3 as a result of ANG II stimuli. In the presence of E2 or CSP-1, we observed significantly decreases in p-Akt1, p-Akt2, and p-Akt3 (Fig. 7, B and C, respectively).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. A wide variety of cellular signaling molecules have been shown to associate within caveolae, leading to the hypothesis that caveolae may serve as cell-surface microdomains that concentrate, organize, and modulate cellular signaling pathways. Similarly, various G protein-associated signaling components, such as β2-adrenergic, B2-bradykinin, and ANG II receptors, and downstream signaling proteins, such as adenylyl cyclase, PKC, and endothelial NO synthase, interact with Cav isoforms, showing a potential role for caveolae in the regulation of such signaling pathways (4). In this work, ANG II, an agonist of human SMC activity, was used as a positive control, since it induces acute increases in [Ca2+]i by interacting with its membrane receptors and triggering intracellular mechanisms, leading to the activation of the phosphoinositide-PLC enzymatic pathway and Ca2+ signaling (14, 17, 35, 40, 43).
In relation to the analysis of E2 effects, our results indicate that this hormone blocked, in a concentration-dependent manner, ANG II-induced increases in [Ca2+]i. This inhibition seemed to initially affect the second component of intracellular Ca2+ kinetics, which is primarily dependent on Ca2+ entry into the cell [due to synthesis of diacylglycerol (DAG) by PLC-β activity; DAG acts by stimulating PKC, and, finally, this phosphorylates a voltage-dependent-Ca2+ channel, allowing the entry of Ca2+] and, in a slower manner, the spike-like first component of Ca2+ kinetics, which is due to IP3 synthesis and Ca2+ release from intracellular stores [due to the synthesis of IP3 by PLC-β activity; IP3 acts by interacting with this receptor in intracellular (sarcoplasmic reticulum) store, inducing Ca2+ release].
As reported previously, we found that the maximum effect elicited by ANG II (1 µM) was blocked by the addition of losartan (AT1 antagonist; 10 µM) and that PD-123319 (an AT2 antagonist) (16) did not significantly block the elicited effects of ANG II. There were no differences in the areas under the curves of Ca2+ kinetics in the absence or presence of the antagonist, and we found no differences in the slopes of the first (spike-like) components.
Interestingly, when we stimulated these cells with CGP42112A (an AT2R agonist) in the presence of losartan (complete blockage of AT1Rs), we observed a small increase in [Ca2+]i, which was abolished by E2 and CSP-1. We do not have any experimental explanation for this Ca2+ increase, but it might be associated with increases in [Ca2+]i related to relaxation, as reported by Tsutsumi et al. (44). Tsutsumi et al. (44) showed that AT2 stimuli in aortic vascular SMCs stimulated the production of bradykinin, which, in turn, activated the NO/cGMP system in a paracrine manner, thereby promoting vasodilatation; however, more work is necessary to clarify this phenomena.
Our immunological experiments showed the expression of AT1Rs and AT2Rs; this was made evident by the strong fluorescence located in the periphery of the cells as well as the cytoplasm. Intense ER expression was also observed. The coexpression of both receptors was observed as a yellow stain (FITC + rhodamine fluorescence). The presence of a strong yellow color in the merged images using confocal analysis suggests that AT1R, AT2R, and ER colocalize or at least that they are located in similar cellular subcompartments. Using these immunocoexpression experiments, we demonstrated, as reported, the relationship between AT1 and ER with Cav-1 and found that the AT2R is also in a close relationship with Cav-1 and relates with AT1R and ER in the plasmalemma. in this regard, it has been proposed that AT1, a nonpalmytoylated GPCR expressed in SMCs, coimmunoprecipitates with Cav-1 and, when activated, cofractionates with Cav-1 on sucrose gradients. The interaction between AT1 and Cav-1 can be made by the Cav-1 scaffolding domain sequence at the COOH terminus of the receptor. These observations led to the hypothesis that an AT1-Cav-1 complex in caveolae may coordinate ANG II-induced signaling. Therefore, Cav-1 acts as a molecular chaperone rather than a plasma membrane scaffold for AT1 (46).
In relation to the analysis of CSP-1 effects, our results indicated that this peptide did not induce changes in [Ca2+]i by itself but rather blocked increases in [Ca2+]i elicited by the addition of ANG II in a concentration-dependent manner.
CSP-1 has a regulatory inhibitory effect. The COOH-terminal half of the oligomerization domain (residues 82–101 in Cav-1) binds to and regulates the activity of signaling molecules, including receptor tyrosine kinases and their downstream targets (EGFR, c-Neu, Ha-Ras, MEK, and ERK1/2), nonreceptor tyrosine kinases (Src and Fyn), GPCRs and their downstream signaling molecules (endothelin receptor, various G subunits, and adenylyl cyclase), and regulated enzymes (endothelial NO synthase). Because of its ability to bind these and other molecules, this region is called CSP-1 (33) and is implicated in membrane attachment, which is both necessary and sufficient for membrane attachment in vitro (32, 33).
Our results showed that CSP-1 inhibits the effects evoked by ANG II. These effects are exerted intracellularly since CSP-1 diffuse into the cell in <1 min after its addition into the medium (in separated experiments, we used FITC-labeled CSP-1 and explored the entry kinetics of the peptide into cells through confocal-dynamic microscopy analysis; data not shown) and seem to remain intracellular, as judged by no changes in the inhibition of ANG II-induced increases in [Ca2+]i, at least during the experimental times employed.
The acute vasoconstrictor effects of ANG II involve the activation of Ca2+ channels and the release of Ca2+ from the sarcoplasmic reticulum, mainly through the synthesis of IP3 and a Ca2+-release mechanism activated by Ca2+ influx through L-type Ca2+ channels. In vascular SMCs, ANG II also activates several other kinases, such as tyrosine protein kinases and PI3K. It has been suggested that PI3K mediates the stimulation of Ca2+ channels by ANG II (35). PI3K is phosphorylated in response to ANG II in vascular SMCs (38), and PI3K promotes voltage-dependent Ca2+ channel trafficking to the plasma membrane (45). Furthermore, Akt/PKB has recently been identified as an important PI3K downstream target in ANG II-activated vascular SMCs. Akt/PKB contains a catalytic domain closely related to both PKA and PKC (26). CSP-1 physically interacts with PKC and regulates its function (25).
Our results suggest that CSP-1 inhibits downstream pathways induced by AT1 activation. When we assayed ANG II in the presence of CSP-1, the activation of Akt1, Akt2, and Akt3 (measured as their phosphorylated forms) was inhibited. These effects seem to be relatively specific, since even when E2 inhibited the intracellular Ca2+ kinetics induced by ANG II, the presence of the steroid decreased, but only partially, Akt1, Akt2, and Akt3 activation.
In conclusion, our results demonstrate that, in human arterial SMCs in culture, exogenous CSP-1 blocks ANG II-induced [Ca2+]i increases. This effect may be a direct result of inhibition of the release of Ca2+ from intracellular stores and indirect inhibition of Akt1, Akt2, and Akt3 activation. Our results also showed a close relationship between AT1, AT2, ER, and Cav-1 in this cell type. We believe that this might be relevant in the physiological or physiopathological modulation of their function.
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FOOTNOTES |
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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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