| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Boston Biomedical Research Institute, Watertown 02472; 2Department of Physiology, Tufts University Graduate School of Biomedical Sciences, Boston 02111; and 3Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Submitted 22 May 2003 ; accepted in final form 4 September 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. -Agonist-induced ERK1–1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the -agonist were obtained with the cholesterol-depleting agent methyl-
-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.
signal transduction; extracellular signal-regulated kinase 1/2; caldesmon; decoy peptide
Caveolins are a family of 21- to 24-kDa integral membrane proteins. Caveolin-1 and -2 are widely expressed, whereas the expression of caveolin-3 is muscle specific (41). Although caveolins have been extensively studied in many cell types and caveolae have been purified from chicken gizzard (2), little is known about how their function relates to regulation of contractility of differentiated vascular smooth muscle (39). It has recently been reported (8) that cholesterol depletion disrupts agonist-induced contraction of vascular preparations. Cholesterol depletion is thought to disrupt caveolar function but could have other effects as well, including disruption of lipid raft function (1) and other nonspecific effects.
Thus in the present study, we took an alternative approach of introducing caveolin decoy peptides into contractile smooth muscle to test the hypothesis that the scaffolding domain of caveolins participates in the coordination of signal transduction regulating contractility in differentiated vascular smooth muscle. Results strongly argue that caveolin plays a significant role in the regulation of smooth muscle contractility and suggest that caveolin regulates the activation of PKC, ERK1/2, and the function of the downstream actin-binding protein caldesmon (CaD).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Contraction measurements. Circular strips (3-mm wide) were prepared as previously described (14) and attached to a force transducer. They were allowed to equilibrate at 37°C for at least 1 h and were then challenged with a depolarizing solution of 51 mM KCl PSS, in which 45.1 mM of NaCl had been stoichiometrically replaced by KCl. Muscle strips were then allowed to equilibrate for 1 h in PSS before beginning the experiment.
Peptide loading and organ culture. Peptides were synthesized by using an automated peptide synthesizer (model 431A; ABI) and employing 9-fluorenylmethoxycarbonyl chemistry. They were purified by reverse-phase HPLC using a Varian Prostar HPLC system and a C18 preparative column (model C-18; Higgins Analytical). Peptides included FITC tags attached to a spacer (-Ala) at their NH2 terminus to be used in confirming loading of tissue cells. The sequence synthesized was taken from the scaffolding domain of caveolin-1 (CaV1) (residues 82-101), which is highly conserved across species (5'-DGIWKASFTTFTVTKYWFYR-3'). A scrambled version of this peptide CaV1X (5'-GDAWIKYRFWTFTKSYTFTV-3') was also synthesized as a negative control.
A method originally developed to load aequorin into intact smooth muscle and referred to as a "chemical loading procedure" (28) was used to introduce the peptides into the cells of a vascular strip. Briefly, muscles were soaked for 30–120 min each in a series of four solutions at 2°C. The composition (in mM) of the solutions was as follows. Solution I: 10 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (Tes); solution II: 0.1 EGTA, 5 Na2ATP, 120 KCl, 2 MgCl2, and 20 Tes plus 50 µM peptide; solution III: 0.1 EGTA, 5 Na2ATP, 120 KCl, 10 MgCl2, and 20 Tes; solution IV: 120 NaCl, 5.8 KCl, 11 dextrose, 25 NaHCO3, 10 MgCl2, 1.4 NaH2PO4. After solution IV was added, the Ca2+ concentration ([Ca2+]) was raised to the normal value of 2.5 mM in gradual steps to avoid damage to the preparation from the "Ca2+ paradox" (47). Tissues were kept overnight in organ culture at room temperature in a 1:1 mixture of PSS and Dulbecco's modified Eagle medium in the presence of penicillin (25 U/ml), streptomycin (25 mg/ml), and nystatin (50 U/ml). The viability of the preparation and contractile function were tested on the second day by measuring the response to 51 mM KCl PSS. Success of loading was confirmed by the observation of fluorescence in all cells throughout the thickness of the strip by using digital image analysis in a confocal-like approach as we (13) previously reported for the chemical loading of oligonucleotides with the same method.
Western blot analysis. Muscle strips were quick frozen by immersion for 60 min in a dry ice-acetone slurry containing 10% TCA and 10 mM DTT. Muscles were stored at –80°C until used. Samples were homogenized in a buffer containing 20 mM MOPS, 4% SDS, 10% glycerol, 10 mM DTT, 20 mM -glycerophosphate, 5.5 µM leupeptin, 5.5 µM pepstatin, 20 KIU aprotinin, 2 mM Na3VO4, 1 mM NaF, 100 µM ZnCl2, 20 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 5 mM EGTA and heated to 70°C for 10 min or boiled for 10 min. Protein-matched samples (modified Lowry protein assay, DC protein assay kit; Bio-Rad, Hercules, CA) were electrophoresed on SDS-PAGE (Protogel; National Diagnostics), transferred to polyvinylidene difluoride membranes, and subjected to immunostaining by using the appropriate antibody and densitometry. Success of protein matching was confirmed by naphthol blue-black staining of the membrane and densitometry of the actin band. Any mismatch of lane loading was corrected by normalization to actin staining. Each set of samples (CaV1, CaV1X, sham) from an individual experiment was run on the same gel, and densitometry was performed on the same film. Between experiments, films of similar exposure times/intensities were used so that the quantitative values could be combined for analysis. Care was taken to ensure that saturation of the signal did not occur at any step in the processing. Densitometry was performed with NIH Image software (National Institutes of Health, Bethesda, MD).
Preparation of single cells. Single vascular smooth muscle cells from ferret aorta were enzymatically isolated by using a modification of a previously published method (27). The aorta was cut into small pieces (2 x 2 mm) and placed in a siliconized flask containing digestion medium. For each 100 mg wet wt of aorta, digestion medium A consisted of 1.4 mg class 2 collagenase (type II, 390 U/mg, lot no. M1E4817), and 1.6 mg elastase (grade II, 4.0 U/mg) in 7.5 ml of Ca2+/Mg2+-free HBSS with 0.2% BSA. Tissue pieces were incubated in a shaking water bath (9.5 cycles/min) at 34°C under an atmosphere of oxygen for 90 min. The pieces were then filtered on a nylon mesh (526-µm mesh opening), rinsed with 10 ml of HBSS, and reincubated for 20 min in digestion medium B, i.e., the same digestion solution as medium A except for a decrease in the amount of elastase to 0.74 mg. Pieces were filtered, rinsed again, and reincubated in digestion medium C, i.e., medium B, plus 0.5 mg of 5,000 units soybean trypsin inhibitor (Type II-S). After being filtered and rinsed with 10 ml of HBSS, the dissociated cells were poured over glass coverslips and plated for 40 min on the ice. For all experiments, isolated cells were tested to confirm that they shortened in response to phenylephrine (PE).
Digital imaging. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 10% goat serum, and incubated with a mouse monoclonal CaV1 antibody (1:1,000; BD Transduction Laboratories, Lexington, KY), followed by a goat anti-mouse Oregon Green secondary antibody (1:250; Molecular Probes, Eugene, OR), and mounted with Fluorosave (Calbiochem, San Diego, CA). Images were obtained by using a Kr/Ar laser (Radiance 2000; Bio-Rad) scanning confocal microscope equipped with an oil immersion objective (x60, model NA1.4; Nikon). Images were recorded with Laser sharp 2000 for Windows NT.
Antibodies. Rabbit polyclonal phospho-CaD antibody (1:1,000) and rabbit polyclonal phosphoadducin antibody (1:2,000) were obtained from Upstate Biotechnology (Lake Placid, NY). The mouse monoclonal phospho-p44/p42 (Thr202/Tyr204) MAPK antibody (1: 2,000) was obtained from New England Biolabs (Beverly, MA). CaV1, caveolin-2 (CaV2), and caveolin-3 (CaV3) mouse monoclonal antibodies were obtained from BD Transduction Laboratories and used at the indicated dilutions.
Materials. Methyl--cyclodextrin (CD) was purchased from Sigma. 12-Deoxyphorbol 13-isobutyrate 20-acetate (DPBA) was purchased from ICN Biomedicals (Costa Mesa, CA).
Statistics. Each set of data was expressed as means ± SE. Student's t-test was used to determine the statistical significance of the means between groups of two, with P < 0.05 taken as significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-isoform and the lower band the -isoform. The -variant appears to be far more abundant in this smooth muscle tissue. Note that a CaV1-immunoreactive high-molecular-mass band was seen at 
300 kDa in samples homogenized in SDS-containing buffer and heated to 70°C for 10 min (Fig. 1B), but this band was essentially eliminated by boiling the sample (Fig. 1A). This band is likely to be an oligomerized form of CaV1 because it has previously been reported that CaV1 forms large oligomeric structures resistant to SDS but broken down by boiling (35). Thus it appears that the predominant caveolin isoform in this tissue is CaV1.
|
Images obtained by staining freshly enzymatically isolated cells with the same CaV1 antibody as that shown for the Western blot analysis indicate that CaV1 is localized to punctate spots (some of which are marked by white arrows in Fig. 1C) at the cell edge. These distinct punctate spots were consistently observed although caveolae are known to be only 50 nm in diameter, well below the limit of resolution of the confocal microscope. A similar, distinct punctate pattern has been reported before (12) and is assumed to represent a clustering of caveolae as has been observed in electron micrographs of smooth muscle (40).
Chemically loaded CaV1 scaffolding domain peptides inhibit PKC-induced contractility but not KCl-induced contractility. Preparations of the aorta of the ferret were chemically loaded with a synthetic CaV1 scaffolding domain peptide, a scrambled version of this peptide (CaV1X), or sham loaded. As is shown in Fig. 2A, the increase in contractility in response to PKC activation with 3 µM of the phorbol ester DPBA for 10 min was significantly inhibited in CaV1-loaded preparations compared with CaV1X-loaded preparations. This time point was approximately at the midpoint of the development of the contraction. At 1 h of exposure to DPBA, a steady-state time point, the magnitude of the inhibition was less (14%) but was also statistically significant (P < 0.01). This concentration of DPBA was previously found to be maximally effective in this tissue (33).
|
The -agonist PE is a more physiological agonist and has been suggested to contract ferret aorta by both PKC-dependent and PKC-independent mechanisms. The PKC-dependent fraction of the contraction has been shown to be Ca independent, but the PKC-independent fraction is Ca dependent (16, 27). Thus we also determined the effect of CaV1 decoy peptides on PE contractions. As shown in Fig. 2, C and D, the contraction to PE both in the presence and absence of extracellular Ca is inhibited significantly by the CaV1 peptide but not the CaVX peptide.
To investigate whether the effect of the CaV1 scaffolding domain peptide was selective, we also measured contractility in response to a nonreceptor-mediated stimulus, depolarization with a PSS containing an elevated concentration of KCl. As is seen in Fig. 2B, there was no effect of CaV1 or CaVX peptide loading on the contraction in response to KCl PSS.
Activation with a phorbol ester sequentially increases PKC substrate phosphorylation, ERK1/2 phosphorylation, and CaD phosphorylation. As a first step in the investigation of the mechanism of the effect on contractility of CaV1 scaffolding domain peptides, we determined the temporal profile of the activation of signaling molecules that have been implicated in a PKC-dependent pathway leading to contraction of this tissue (29). Ca/CaM/myosin light-chain kinase-mediated phosphorylation of the 20 kDa myosin light chains (LC20) is an important mechanism of regulation of smooth muscle contraction, but it has previously been shown that phorbol ester activation of this tissue causes negligible increases in either the intracellular [Ca2+] or the LC20 phosphorylation level. Rather, the PKC-activated pathway in this tissue has been reported to involve activation of ERK1/2 and phosphorylation of a downstream substrate, CaD (17). CaD is reported to interfere with the availability of actin for interaction with myosin, and phosphorylation of CaD is thought to reverse this inhibitory action (29).
Time courses for the activation of ERK1/2 (as monitored with phosphospecific antibodies), phosphorylation of CaD, and the increase in contractile force in response to DPBA are shown in Fig. 3. Additionally, for comparison, the time course for the site-specific PKC-mediated phosphorylation of 
-adducin at Ser662 (numbering according to the human sequence) was monitored as a reporter of endogenous PKC kinase activity (10). The peak increase in endogenous PKC kinase activity (adducin phosphorylation) is seen within 5 min. ERK1/2 activation peaks at 10–15 min. Much later, by 60 min, phosphorylation of CaD on Ser789, an ERK1/2 phosphorylation site (5, 6), reaches a peak. It is of interest that only CaD phosphorylation has a time course that parallels the slow time course of the contractile event itself (Fig. 3). This finding is consistent with past suggestions that the phosphorylation of CaD at Ser789 is the final signaling event that causes the increased contractility (6). The reason for the pronounced delay between ERK1/2 activation and CaD phosphorylation is not known but may relate, in part, to the time required for the previously described translocation of ERK1/2 from the plasmalemma to the contractile filaments (15). On the basis of these time-course curves, CaV1 peptide-loaded preparations were quick frozen at 10 min and 1 h after exposure to DPBA to evaluate the effects on these signaling events.
|
CaV1 scaffolding domain peptides disrupt phorbol ester-dependent PKC activation, ERK1/2 activation, and CaD phosphorylation. Preparations loaded with the CaV1 scaffolding domain peptide, the CaV1X peptide, or sham loaded were quick frozen at 10 min to monitor -adducin Ser662 phosphorylation and ERK1/2 activation. Phospho-Ser662 -adducin antibody also detects phosphorylation of Ser724 of -adducin (numbering according to the human sequence). As can be seen in Fig. 4A, loading with the CaV1 scaffolding domain peptide caused a significant decrease in the phosphorylation at the PKC site of both isoforms of adducin, compared with that in either sham-loaded muscles or muscles loaded with CaV1X. The phosphoadducin levels in muscles not activated with DPBA (resting) are shown for reference. Levels in CaV1X-loaded muscles were not significantly different from those in sham-loaded muscles. These results imply that PKC interacts with caveolin in the caveoli before it acquires kinase activity and before it can be targeted to and phosphorylate substrates, such as adducin or the yet to be determined upstream molecule that leads to ERK1/2 activation, and subsequent contraction.
|
ERK1/2 activity, as monitored by phosphospecific antibodies, was also measured in the preparations quick frozen at 10 min. As shown in Fig. 4B, loading of muscles with the CaV1 scaffolding domain peptide caused a significant inhibition of both ERK1 and ERK2 phosphorylation at the 10-min time point compared with that in either CaV1X-loaded or sham-loaded muscles.
In muscles quick frozen at the 1-h time point, both ERK1/2 activity (Fig. 4C) and Ser789 CaD phosphorylation (Fig. 4D) were measured. It is worth noting that in these differentiated smooth muscle cells, ERK1/2 activation is quite prolonged compared with some other cell types (3, 45), and significant activation persists at 60 min. This activity was significantly inhibited by loading with the CaV1 scaffolding domain peptide but not with the CaV1X peptide or sham loading (Fig. 4C). Similarly, Ser789 phosphorylation of CaD was inhibited by loading with the CaV1 scaffolding domain peptide but not with the CaV1X peptide or sham loading (Fig. 4D).
We additionally measured the effect of the CaV1 scaffolding domain peptide on ERK activation during the Ca-independent PE contraction. Muscles were placed in a Ca-free PSS containing 2 mM EGTA and quick frozen after being exposed to PE for a time previously determined to be appropriate (6) for the measurement of ERK activation (5–7 min). Phospho-ERK signals from muscles loaded with the CaV1 peptide were inhibited by 43 ± 16% compared with sham-loaded muscles (P < 0.05, n = 3). In contrast, the phospho-ERK signal from CaV1X-loaded muscles was inhibited only 20 ± 14% compared with the sham-loaded muscles (P > 0.05 n = 3).
A cholesterol-depleting agent inhibits contractility and agonist-induced signaling. Previous studies (46) have reported that CaV1 (4) and CaV3 knockout in the mouse leads to a hyperactivation of ERK1/2 in cardiac muscle. The same group (9) has also suggested that the scaffolding domain peptides might act as nonspecific kinase inhibitors. This suggestion is actually not consistent with the lack of effect of these peptides on KCl contractions in this tissue, because previous studies have shown that KCl contractions are dependent on the activity of both MLCK (11) and CaMKII (20). However, to confirm that disruption of caveolin-dependent signaling is associated with ERK inhibition in this smooth muscle tissue, we also used an alternative approach to disrupt caveolar function by using a cholesterol-depleting agent, CD (8, 42). Although the use of a cholesterol-depleting agent raises additional concerns, such as a possible detergent-like effect, which might increase cell leakiness, the potential artifacts with the two approaches are expected to differ. Thus if similar results are obtained, this argues strongly for a true caveolin-dependent signaling effect. As can be seen in Fig. 5, this agent was effective in inhibiting DPBA- and PE- but not KCl-induced contractility. Also, treatment with CD was effective in inhibiting DPBA- and PE-induced increases in ERK2 phosphorylation (Fig. 6).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because CaV1 appeared to be the most abundant isoform in this tissue, we used the CaV1 scaffolding domain sequence to synthesize the scaffolding domain peptide used in this study. Chemical loading of this peptide into contractile smooth muscle indicated that the CaV1 scaffolding domain (but not a scrambled version of the domain) was able to alter agonist-induced contractility of vascular smooth muscle tissue. Although such peptides have been used in many nonmuscle tissues in a dominant-negative-type approach to probe caveolar function (25, 38, 43), to the best of our knowledge, this approach has not previously been used in differentiated vascular smooth muscle to probe contractile function. However, the concept that CaV1 may regulate agonist-induced contraction of vascular smooth muscle is also consistent with a report in which cholesterol depletion to cause caveolar disruption was shown to impair agonist-induced contractions of vascular smooth muscle (8).
In the present study, we found that chemical loading of CaV1 peptides into contractile smooth muscle resulted in a significant inhibition of the contraction in response to the phorbol ester DPBA or the -agonist PE. Interestingly, the peptides had no effect on the contraction in response to an elevation of the KCl concentration in the PSS. Many receptors and their downstream signaling molecules have been reported to localize in caveolae and are thought to interact with caveolin (22, 32). Thus the lack of inhibition of KCl contractions by the caveolin peptides could be due to the fact that this depolarization-mediated contraction is not thought to involve a receptor-mediated signaling pathway. However, it has also been suggested that most kinases directly interact with caveolin and that the scaffolding domain peptides used here might inhibit all kinases (9). Lack of an effect of both the scaffolding domain peptide and CD on KCl-induced contractions is inconsistent with this view, because both MLCK and CaMKII are reported to be required for KCl-induced contractions in this tissue (11, 20). The implication of the present study is that neither MLCK nor CaMKII interact with caveolin in the signaling pathways that lead to KCl-induced alterations in contractility.
It is to be noted that both the decoy peptide approach and the depletion of cholesterol with CD have limitations. However, any potential artifacts caused by these two very different approaches would be expected to differ. Thus the fact that similar inhibitions of phorbol ester and -adrenergic agonist-induced contractions were seen with both approaches strongly argues for an involvement of caveolin in agonist-induced activation of smooth muscle.
Previous studies on this tissue have shown that phorbol ester stimulation causes negligible changes in myosin phosphorylation (14) and have pointed to a pathway that involves activation of PKC and ERK1/2 and phosphorylation of the actin-binding protein CaD (29). However, this issue has generated some controversy in the field and has been recently reviewed (29). It appears quite likely that the relative importance of this pathway is tissue and agonist dependent, but in ferret aorta, all data currently available are consistent with a pathway linking PKC to ERK and CaD. Thus we determined the effect of CaV1 scaffolding domain peptides on the PKC-dependent regulation of these signaling molecules.
The -agonist PE has been reported to be able to activate this pathway in ferret aorta as well, and PE is a more physiological agonist. Thus we also determined the effect of CaV1 decoy peptides and cholesterol depletion on PE contractions and found a similar inhibition of contraction. However, PE has multiple actions, including the activation of MLCK. So for further investigation of the mechanism of interaction PKC with caveolin, we focused on direct phorbol ester-dependent signaling.
The direct downstream target of PKC that leads to activation of MEK/ERK in this tissue is not known. For this reason, and because of the availability of phospho-antibodies to a PKC-specific site on adducin, we monitored adducin phosphorylation at this site as an intracellular PKC kinase assay. Phosphorylation at this site is unique to PKC and the phospho-antibody raised against this site does not recognize adducin phosphorylated by Ca-, cAMP-, or rho-dependent kinases (10, 21). In the tissue used in the present study, PKC-dependent contractions have been linked with the epsilon isoform of PKC (23). Although phosphorylation at this site on adducin has not been demonstrated specifically for PKC-
, it has been shown that other novel isoforms of PKC, as well as the classical isoforms, do specifically phosphorylate adducin, and thus it is likely that PKC- as well can use adducin as a substrate (18, 19, 26).
Adducins are a family of highly related proteins that mediate the association of spectrin and actin to form the subcortical membrane skeleton and are generally not thought to be associated with caveolae (10). Thus the finding that CaV1 peptides inhibit adducin phosphorylation indicates that PKC may have dissociated from the caveolae and translocated to the membrane cytoskeleton at the time that adducin phosphorylation is detected.
DPBA-induced phosphorylation of ERK1/2 and CaD peaks at 10–15 and 60 min, respectively. The time course of the phosphorylation of CaD seen in the present study closely follows the time course of the agonist-induced contraction. The question arises as to the mechanism by which the significant delay between the phosphorylation of ERK1/2 and CaD occurs. ERK1/2 activation is known to occur at the cell membrane, but CaD phosphorylation by ERK1/2 requires a subsequent translocation of active ERK1/2 from the cell membrane to the contractile filaments in which CaD is bound (15). Thus the delay between ERK1/2 activation and CaD phosphorylation appears to relate to the little-understood mechanisms by which ERK1/2 translocates to the contractile filaments.
Recently, CaV1 knockout mice have been bred (7). These mice have a decreased cardiovascular endurance, reduced myogenic tone of their blood vessels and aortic rings, and display decreased contractile force to agonists such as the phorbol ester PMA. Results presented in the present study may provide a subcellular basis for the vascular defects reported in these CaV1 knockout mice.
In a CaV3 knockout mouse (46), results somewhat different from those reported here were found in that a hyperactivation of cardiac ERK1/2 accompanied an observed cardiomyopathy. Also, although the heart is reported to contain little CaV1, similar findings have been reported in the CaV1 knockout mouse, i.e., a cardiac hypertrophy and a hyperactivation of cardiac ERK1/2 (4). ERK1/2 activity was not measured in the vasculature. It is likely that the cardiac hypertrophy and the cardiac ERK1/2 hyperactivation in the CaV1 knockout mouse were secondary to vascular defects (4). In the present study, we observed a hypoactivation of ERK1/2 on the addition of the CaV1 scaffolding domain peptide, indicating that either the interaction of CaV1 with ERK1/2 differs in smooth muscle from that of CaV3 in cardiac muscle, or the addition of the peptide is not acting as a dominant-negative sort of inhibitor. To further resolve the issue, we also took a second approach to block caveolin's action in this tissue. Cholesterol-depleting agents have previously been found to downregulate caveolae in smooth muscle and in other tissues (8, 42). When we used these agents, we found that ERK and contractility were both inhibited, confirming the results of the peptide approach.
Thus in summary, activation of PKC in the presence of a phorbol ester and the subsequent downstream activation of ERK1/2, phosphorylation of CaD, and contraction of vascular smooth muscle require interaction with caveolin, most likely, CaV1. Disruption of CaV1-dependent function leads to a hypocontractile state and a hypoactivation of ERK1/2 both in the presence of a phorbol ester and in the presence of an -agonist.
|
ACKNOWLEDGMENTS |
|---|
GRANTS
This study was supported by National Heart, Lung, and Blood Institute Grants HL-31704 and HL-42293 (to K. G. Morgan).
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-adrenoceptor stimulation. Am J Physiol Cell Physiol 275: C1081–C1086, 1998. subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J Biol Chem 271: 29182–29190, 1996. and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res 258: 72–81, 2000.[CrossRef][ISI][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. J. Halayko, T. Tran, and R. Gosens Phenotype and Functional Plasticity of Airway Smooth Muscle: Role of Caveolae and Caveolins Proceedings of the ATS, January 1, 2008; 5(1): 80 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Gosens, G. L. Stelmack, G. Dueck, M. M. Mutawe, M. Hinton, K. D. McNeill, A. Paulson, S. Dakshinamurti, W. T. Gerthoffer, J. A. Thliveris, et al. Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1406 - L1418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Mendez-Bolaina, J. Sanchez-Gonzalez, I. Ramirez-Sanchez, E. Ocharan-Hernandez, M. Nunez-Sanchez, E. Meaney-Mendiolea, A. Meaney, J. Asbun-Bojalil, A. Miliar-Garcia, I. Olivares-Corichi, et al. Effect of caveolin-1 scaffolding peptide and 17 -estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1953 - C1961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. S. Prakash, M. A. Thompson, B. Vaa, I. Matabdin, T. E. Peterson, T. He, and C. M. Pabelick Caveolins and intracellular calcium regulation in human airway smooth muscle Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1118 - L1126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Linder, K. M. Thakali, J. M. Thompson, S. W. Watts, R. C. Webb, and R. Leite Methyl-beta-cyclodextrin Prevents Angiotensin II-Induced Tachyphylactic Contractile Responses in Rat Aorta J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 78 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Albinsson, Y. Shakirova, A. Rippe, M. Baumgarten, B.-I. Rosengren, C. Rippe, R. Hallmann, P. Hellstrand, B. Rippe, and K. Sward Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1222 - R1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Somara, R. R. Gilmont, J. R. Martens, and K. N. Bitar Ectopic expression of caveolin-1 restores physiological contractile response of aged colonic smooth muscle Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G240 - G249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kamishima, T. Burdyga, J. A. Gallagher, and J. M. Quayle Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H204 - H214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Clarke, V. Ohanian, and J. Ohanian Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2248 - H2256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Adebiyi, G. Zhao, S. Y. Cheranov, A. Ahmed, and J. H. Jaggar Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1584 - H1592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dubroca, X. Loyer, K. Retailleau, G. Loirand, P. Pacaud, O. Feron, J.-L. Balligand, B. I. Levy, C. Heymes, and D. Henrion RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries Cardiovasc Res, January 1, 2007; 73(1): 190 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shakirova, J. Bonnevier, S. Albinsson, M. Adner, B. Rippe, J. Broman, A. Arner, and K. Sward Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1326 - C1335. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Prisby, M. K. Wilkerson, E. M. Sokoya, R. M. Bryan Jr., E. Wilson, and M. D. Delp Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms J Appl Physiol, July 1, 2006; 101(1): 348 - 353. [Abstract] [Full Text] [PDF]
|
|
0.05; **P 
