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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Department of Experimental Medical Science, Lund University, 2Department of Otorhinolaryngology, Malmö University Hospital, and 3Department of Physiology and Pharmacology, Karolinska Institute, Stockholm
Submitted 1 February 2006 ; accepted in final form 18 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTP
S was increased in KO compared with WT as shown using a pull-down assay. Following 
-toxin permeabilization, no difference in Ca2+ sensitivity or in Ca2+ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased 1-adrenergic contraction. Following inhibition of PKC, 1-adrenergic contraction was normalized. PDBu-induced Ca2+ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca2+ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca2+ sensitivity. Ca2+ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor
Compared with endothelial and adipocyte caveolae, smooth muscle caveolae have received relatively little attention. With the use of cholesterol depletion to disrupt caveolae in denuded caudal arteries from the rat, we demonstrated that serotonin (5-HT2A) as well as endothelin-1 (ET-1) receptor signaling was impaired by cholesterol depletion. Moreover, restoration of caveolae by cholesterol replenishment recovered signaling from both receptors (3, 11). Cholesterol depletion was also shown to affect phasic but not tonic contraction in ureteric muscle (2), suggesting that raft/caveolae associated signaling influences specific steps in excitation-contraction coupling. Impaired contractile responses to ET-1, ANG II, and phorbol ester were found in arteries from Cav-1-deficient (KO) mice (10). The vessels did however, have intact endothelium, and the reduced contractility was interpreted to be secondary to enhanced nitric oxide synthase activity (29). In the urinary bladder, a general reduction of force on receptor stimulation was reported (37).
Downstream of receptor activation, RhoA, as well as Rho-kinase and protein kinase C (PKC) play key roles in regulation of contractility in smooth muscle (32). RhoA (15, 22, 34) and PKC (22, 32) have been shown to reside in or to translocate to caveolae on receptor stimulation. The scaffolding domain of Cav-1 was reported to impair both membrane translocation of PKC (34) and contraction stimulated by phorbol ester and -agonist in the ferret aorta (16). Further support for a role of caveolae in PKC signaling is the finding that phosphoinositide turnover is compartmentalized in this membrane microdomain as shown using biochemical fractionation (27). Finally, Rho-kinase has been demonstrated to translocate to caveolae in a Ca2+-calmodulin-dependent manner on smooth muscle depolarization (36). Taken together, these results suggest that caveolae may play a role in contractility and in regulation of Ca2+ sensitivity of contraction in smooth muscle (4).
In the present study, using KO mice, we investigate whether PKC and RhoA/Rho-kinase-mediated contractile mechanisms depend on caveolae. We have used longitudinal smooth muscle from the small intestine, which shows robust RhoA/Rho-kinase-mediated Ca2+ sensitization, but weak sensitization in response to PKC activation with phorbol ester (8, 21, 33), and femoral artery, which displays prominent Ca2+ sensitization in response to phorbol ester (8, 38).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Preparation of intestine and femoral artery. The intestine and femoral artery were removed and placed in cold HEPES-buffered physiological saline solution (for composition, see below). Strips from the outer longitudinal small intestinal muscle layer were obtained by tearing the longitudinal layer off from the underlying circular layer (8).
Electron microscopy. Preparations were fixed in 2% glutaraldehyde and 1% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4, 300 mosM), postfixed in OsO4, dehydrated, and embedded in Durcupan ACM (Fluka, Buchs, Switzerland). Ultrathin sections were examined with the use of transmission electron microscopy (3, 11).
Western blot analysis.
Tissue preparations were frozen using clamps precooled in liquid N2. Frozen tissue was pulverized and mixed with cold (4°C) SDS sample buffer, boiled, briefly centrifuged, and loaded on 15% polyacrylamide gels after the protein concentration using the Bio-Rad BC protein assay was determined. The SDS sample buffer contained 25 mM Tris·HCl (pH 6.8), 10% glycerol, 5% mercaptoethanol, 1 mg/ml bromophenol blue, 2% SDS, 30 mM DTT. Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). After blocking, membranes were incubated with monoclonal antibodies against Cav-1, -2 or -3 (clones 2297, 65, and 26; BD Biosciences, Stockholm, Sweden). The 
-actin antibody (1:5,000 dilution) was from Sigma (St. Louis, MO). ET-1 was detected using a monoclonal antibody from BD Biosciences (1:1,000). The PKC- antibody (ab11723) was from Abcam. Anti-CPI-17 and anti-phosphorylated CPI-17 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). P-CPI-17 was normalized to total CPI-17 in the same homogenate, and then, in each experiment, to the average CPI-17-P/CPI-17 ratio for all WT mice. Horseradish peroxidase-conjugated secondary antibodies were detected using enhanced chemiluminescence (the West Pico reagent for Cav-1, and the West Femto for Cav-2 and Cav-3; Pierce, Rockford, IL). For caveolins, identical exposure times were used to allow expression levels in the different tissues to be roughly compared.
mRNA study. Longitudinal muscle and lung parenchyma (for primer testing, see below) were rinsed with cold PBS and stored in RNAlater (QIAGEN, Hilden, Germany) at –20°C until homogenization and extraction of the total RNA using the RNeasy Mini kit (QIAGEN). The purity of total RNA was checked by a spectrophotometer and the wavelength absorption ratio (260/280 nm) was between 1.6 and 2.0 in all preparations. Reverse transcription of total RNA to cDNA was carried out using Omniscript reverse transcriptase kit (QIAGEN) in a 20-µl reaction volume at 37°C for 1 h by using Mastercycler personal PCR machine (Eppendorf, Hamburg, Germany).
Specific primers were designed by using Prime Express 2.0 software (Applied Biosystems, Foster City, CA) and synthesized by DNA Technology (Aarhus, Denmark). The sequences for Cav-1 (forward 5'-TGT ATG ACG CGC ACA CCA A-3' and reverse 5'-TCC CTT CTG GTT CTG CAA TCA-3'), Cav-2 (forward 5'-GGC GTT GAC TAC GCA GAT CC-3' and reverse 5'-GCA ATC AGA TCC TCG AAG CCT A-3'), Cav-3 (forward 5'-CCC AAG AAC ATC AAT GAG GAC A-3'and reverse 5'-GTG GTG AAG CTC ACC TTC CAT AC-3') and -actin (forward 5'-TGG GTC AGA AGG ACT CCT ATG TG-3' and reverse 5'-CGT CCC AGT TGG TAA CAA TGC-3') were all spanning over an intron.
Real-time PCR was performed on a Smart Cycler (Cepheid, Sunnyvale, CA) using QuantiTectTM SYBR Green PCR kit (QIAGEN). The thermal cycler was programmed to start with heating to 95°C for 15 min, followed by touchdown PCR, i.e., denature at 94°C for 30 sec and annealing at 66°C for 1 min for the first PCR cycle; thereafter, a decrease of 2°C for the annealing temperature in every cycle until 56°C was reached. Finally, 40 thermal cycles with 94°C for 30 s and 55°C for 1 min were performed. For primer testing, cDNA from lung parenchyma was diluted in half 10log concentrations and the primers showed that the amplification was close to 2 (1.89–1.93).
Quantification of the gene expression was assessed with the comparative cycle threshold (Ct) method (http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). The relative amounts of mRNA for the caveolins were determined by subtracting the Ct values for these genes from the Ct value for the housekeeping gene -actin (
Ct). Data are depicted as 2Ct x 105.
Immunofluorescence staining of Cav-1 and -3.
Tubular ileum segments were pulled over 20-µl plastic pipette tips, which were immersed in Histochoice (Amresco) for at least 4 h. After incubation in fixative, tissues were thoroughly washed in 70% ethanol and maintained therein at 4°C for at least 3 days, with three solution changes, until further processing. Following incubation in 96% (2 h) and 100% ethanol (1 h), 1:1 ethanol:xylene (30 min) and xylene (1 h), tissues were immersed in paraffin (2 x 1 h) and embedded. Sections measuring 10 µm were cut and deparaffinized. The sections were washed (2 x 30 min in PBS), permeabilized with 0.2% Triton X-100 for 15 min, blocked with 2% bovine serum albumin in PBS for 2 h, and incubated with primary antibodies in the same solution overnight at 4°C. Cav-1 and -3 antibodies (identical to those used for Western blot analysis) were used at dilutions of 1:125, and 1:1,000, respectively. After being washed in PBS, the sections were incubated with Cy5-labeled anti-mouse IgG for 1 h at room temperature. Nuclei were stained with DNA dye Sytox Green (1:3,000, Molecular Probes) after a brief washing in PBS. Pictures were obtained in a Zeiss LSM 510 confocal microscope. Caveolin proteins were detected by monitoring Cy5 fluorescence upon excitation at 633 nm. Sytox Green fluorescence was monitored upon excitation at 488 nm. Primary and secondary antibody omission controls verified specificity of staining (
Fig. 3).
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S was added to one of them. Activated RhoA was precipitated using the Rho-binding domain from Rhotekin coupled via GST to GSH beads. Loads and precipitates were analyzed by Western blotting using the antibody against Rho (A-C) supplied with the kit, or the Cav-1 and ET-1 antibodies described above.
Force recording, intact small intestinal preparations.
Strips from the longitudinal muscle of the small intestine (7 mm long, 1.5 mm wide, and 50 µm thick) were mounted in organ baths with the use of a thin silk thread between a stainless steel pin on an adjustable stand and a transducer (model FT03, Grass Instruments, Freeport, IL). Preparations were equilibrated in Krebs solution (for composition, see below) with 2.5 mM Ca2+ for 1 h at the optimal length for active force. The Krebs solution, pH of 7.4 at 37°C, was gassed with 95% O2-5% CO2. Force responses were expressed relative to the peak force of the high-K+ (80 mM) contraction, recorded at the beginning of the experiment.
Force recording, intact femoral artery preparations.
Intact femoral arteries were mounted in four-channel wire myographs (model 610M, Danish Myotechnology, Aarhus, Denmark), as described previously (3). A human hair was pulled through the arterial lumen to remove the endothelium and 300 µM N
-nitro-L-arginine methyl ester (L-NAME) was included in all solutions. Force was normalized to the tube length of the arterial segment. To get an estimate of the force per cross-sectional area (stress), force was multiplied by length (wire distance) and divided by wet weight. A complication was that the individual preparations weighed too little (60 µg) to weigh reliably with standard equipment. We therefore recorded force and length from four preparations and weighed them pooled together for one stress determination. Wet weight was obtained using a Mettler M3 balance after gentle blotting of the tissue on a filter paper.
Force recording, -toxin-permeabilized preparations.
Intestinal strips, 4-mm long, 1-mm wide, and 50-µm thick, were wrapped at both ends with aluminum foil. Femoral artery segments (2 mm long, cut as spiral strips from the media) were mounted on two 30 µm steel wires (31). All preparations were mounted horizontally between two tungsten wires, one of which was attached to a force transducer (model AE 801, SensoNor, Horten, Norway) and the other to a micrometer screw (8). Experiments were performed on "bubble plates" (140 µl solution) with stirring. Preparations were equilibrated in HEPES-buffered physiological saline solution and a high-K+ (80 mM) test contraction was induced. Following relaxation, preparations were permeabilized with Staphylococcus aureus -toxin (10,000 U/ml, Calbiochem-EMD Biosciences, San Diego, CA) for 60 min. Permeabilized preparations were treated with 10 µM A23187
[GenBank]
for 20 min to deplete intracellular Ca2+ stores. Experiments were run at room temperature (22°C).
Solutions. The HEPES-buffered physiological saline solution for dissection contained (in mM) 135.5 NaCl, 5.9 KCl, 1.2 MgCl2, 11.6 glucose, and 11.6 HEPES, pH 7.4. The Krebs solution, for experiments on intact small intestinal muscle tissue, contained (in mM) 122 NaCl, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, and 11.5 glucose. For permeabilized preparations, the relaxation solution (pCa 9) contained (in mM) 30 N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 10 phosphocreatine, 5.14 Na2ATP, 7.92 Mg-acetate, 46.6 K+-methanesulfonate, 10 EGTA, and 1 DTE. The contraction solution (pCa 4.5) was made by replacing EGTA with Ca-EGTA. Ionic strength (0.15 M) and free [Mg2+] (2 mM) were held constant by adjusting [K+-methanesulfonate] and [Mg-acetate]. pH was adjusted to 7.1. Fixed free Ca2+ concentrations were obtained by mixing relaxation and contraction solutions.
Drugs. Y-27632 and microcystin-LR was obtained from Calbiochem-EMD Biosciences. All other drugs used were obtained from Sigma.
Statistics. Values are means ± SE. Unless stated otherwise, n refers to the number of mice. Statistical significance was determined using Student’s t-test for unpaired data.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Caveolin detection by Western blot analysis.
Cav-1 ( and ), Cav-2 ( and ), and Cav-3 were detected by Western blot analysis in WT longitudinal smooth muscle from the ileum (Fig. 2A). Cav-1 and Cav-2 were not detected in KO ileum (Fig. 2A). Reduced Cav-2 content in Cav-1–/– tissues has been reported previously (10, 28). In the KO ileum, expression of Cav-3 was also reduced (by 76 ± 6% vs. WT, n = 7, P < 0.001; Fig. 2A). Cav-1, Cav-2, and Cav-3 were absent, or exhibited considerably reduced expression, in the denuded femoral artery from KO (Fig. 2B). To verify reduced expression of Cav-3, which was unexpected, Cav-3 expression in the ileum was normalized to -actin (Fig. 2C). This confirmed reduced contents in relation to a housekeeping protein in KO relative to WT (n = 4, P < 0.05). Dilutions of extracts, blotted for Cav-3, also verified reduced Cav-3 expression in the longitudinal smooth muscle from ileum (Fig. 2D).
Immunofluorescence staining of Cav-1 and Cav-3 in ileum. Cav-1 and Cav-3 were visualized by immunofluorescence. Cav-1 staining revealed 7–15 clusters per cell membrane profile (red in Fig. 3, top left) in ileum longitudinal smooth muscle. Cav-3 staining showed some degree of clustering along cell membrane profiles in WT muscle (Fig. 3). In KO, Cav-3 clustering was lost and staining appeared inside cells (Fig. 3).
Quantitative real-time PCR.
In WT longitudinal muscle Cav-1 mRNA levels were 15-fold higher than those of Cav-2 and Cav-3 (Fig. 4). No differences in mRNA between WT and KO tissues were obtained for Cav-2. The mRNA levels for Cav-3 were lower in KO compared with WT longitudinal smooth muscle preparations but this difference did not reach significance (P = 0.091). The tissues used for PCR (Fig. 4) correspond with that used for blotting in Fig. 2A.
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Force responses to receptor agonists were examined in intact ileum strips. Original records are shown in Fig. 5A. Amplitudes of the carbachol and 5-HT responses were unchanged in the KO preparations (Fig. 5B). The EC50 values for carbachol in KO and WT ileum were not different (WT: 0.35 ± 0.08; KO: 0.34 ± 0.03 µM, n = 8), and contractions exhibited a rapid phase followed by sustained contraction in both KO and WT. Contractions in response to 10 nM ET-1 were reduced by 50% in small intestinal tissue from KO compared with WT mice (Fig. 5).
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S (Fig. 6A). Basal Rho-activity was below the detection limit (Fig. 6B). The Rho-activation level was increased in KO vs. WT muscle after incubation with GTPS (n = 5, P < 0.05). Moreover, Cav-1 was precipitated together with Rho in WT but not in KO. Some ETA receptor also coprecipitated with Rho. The amount of Rho (A, B, C) and ETA was similar in WT and KO homogenates (Fig. 6A, left lanes). In six experiments on independent homogenates, ETA receptor expression was 114 ± 13% in KO relative to WT (P > 0.05).
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-toxin permeabilized longitudinal smooth muscle from the ileum.
To examine whether Ca2+ sensitization might be affected in the absence of caveolae, we used -toxin permeabilized smooth muscle. In the ileum, Rho/Rho-kinase mediated modulation of Ca2+ sensitivity is prominent and well characterized (8, 18, 33). The pCa-force relationships were identical in -toxin permeabilized strips from WT and KO (Fig. 7A). Following a submaximal Ca2+ concentration (pCa 6.0), strips were stimulated with carbachol (10 µM; in the presence of 30 µM GTP) and GTPS (10 µM). A maximal contraction was finally achieved by inhibiting the myosin phosphatase with 1 µM microcystin-LR. Ca2+ sensitization was not affected in KO compared with WT, irrespective of stimulus (Fig. 7B). Separate experiments showed that ET-1 (10 nM following 30 µM GTP) induced little or no Ca2+ sensitization in the mouse ileum (not shown).
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S-induced contraction completely in both WT and KO mice (not shown).
PKC-mediated contraction in intact femoral arteries.
Since PKC responses have been shown to depend on CPI-17 (19, 38), and the CPI-17 content is low in longitudinal smooth muscle from the ileum, we investigated whether PKC-mediated contraction was different using femoral arteries. In the femoral artery, the CPI-17 content is high and phorbol 12,13-dibutyrate (PDBu), which activates PKC, gives rise to a sizeable contraction (8). Experiments on denuded, but otherwise intact, ring preparations of the femoral artery were made in the continuous presence of 300 µM L-NAME to avoid residual nitric oxide synthesis. Following relaxation from contraction induced by 80 mM K+ (HK), 1 µM PDBu was added (Fig. 8A). The time course of contraction in response to PDBu was slower in KO, but the level of force reached after 30 min was increased (Fig. 8, A and B, n = 16 WT and 16 KO preparations from 4 WT/KO pairs of mice, P < 0.05). After 30 min in the PDBu solution, the PKC inhibitor GF 109203X (GFX) was added in a cumulative manner (Fig. 8, A and C). The IC50 value for GFX was not different in KO vs. WT (0.31 ± 0.05 µM for WT and 0.40 ± 0.07 µM for KO, P > 0.05). Expression of PKC-, as assessed by Western blot analysis, was not different in KO (KO: 106 ± 12% of WT, n = 8).
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1-Adrenergic responses in rabbit femoral artery are mediated in part by PKC (17). We compared the concentration response relations for the selective 1-adrenergic receptor agonist cirazoline in WT and KO femoral arteries in the presence of L-NAME. The typical pattern was sustained contraction with little inactivation, such as that shown in Fig. 9A. Phasic repetitive activity sometimes occurred, and contraction often inactivated faster in WT than in KO in those cases. Force was averaged during the time that each concentration was maintained. Consistent with increased PKC-mediated contraction in KO, cirazoline responses were increased, both when expressed as absolute force (Fig. 9B), and following normalization to contraction induced by 80 mM K+ (Fig. 9C), which was unchanged (bars in Fig. 9B). EC50 values for cirazoline were not different (0.17 ± 0.08 in KO vs. 0.36 ± 0.19 µM in WT, n = 16 preparations from 4 animals of each genotype; P > 0.05).
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1-Adrenergic contraction was next examined in the presence of the PKC inhibitor GFX. Cirazoline-induced force was not different following PKC inhibition (n = 8 preparations from 2 WT/KO pairs; Fig. 9E). Vehicle-treated preparations from the same animals confirmed the difference in cirazoline-induced force between WT and KO.
PKC mediated contraction in permeabilized femoral arteries.
To address whether increased PDBu-induced and PKC-mediated contraction was due to increased Ca2+ sensitization, -toxin-permeabilized spiral strips from the femoral artery were used. Force was normalized to a reference (pCa 4.5) contraction. The response to an intermediate Ca2+ concentration (pCa = 6.5) was similar in strips from WT and KO mice. Moreover, the addition of increasing amounts of PDBu caused Ca2+ sensitization that was identical in WT and KO (Fig. 10, A and B). Results were the same when data was normalized to depolarization-induced contraction obtained before permeabilization (not shown). Phosphorylation of CPI-17 was similar in intact preparations from WT and KO after 30-min incubation with 1 µM PDBu (WT: 100 ± 9%, KO: 102 ± 10%, n = 4).
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DISCUSSION |
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All caveolin family members were expressed in smooth muscle. In agreement with previous studies (10, 28, 37), ablation of Cav-1 was associated with reduction of the Cav-2 protein(s). We also made the unexpected observation that the Cav-3 protein content was reduced and its distribution altered in KO mice. The relative mRNA abundance for Cav-1, -2, and -3 was found to be 15:1:1 in the ileum. This suggests that an absolute minority (7%) of caveolae in WT intestinal smooth muscle are Cav-3 driven. The relative levels of expression, the intracellular Cav-3 accumulation, and the observed reduction of Cav-3 protein, probably all explain the apparent absence of caveolae in electron micrographs from KO tissue. The basis of the reduced Cav-3 protein expression is unclear. Cav-3 mRNA levels were not different, which suggests a mechanism involving Cav-3 protein degradation. Importantly, however, the apparent absence of caveolae and the reduced membrane association of Cav-3 in the KO argue against the possibility that the remaining Cav-3 compensates for the lack of Cav-1 in smooth muscle.
Caveolae have been suggested to play a role in signaling from smooth muscle ET-1 and 5-HT2A receptors (3, 6, 11). M2 muscarinic receptors have been shown to translocate to caveolae on agonist binding which was suggested to be important for downstream signaling (12). Finally, caveolae have been proposed to play a role in M2 and M3 receptor desensitization (25). In support of a role of caveolae in muscarinic signaling, contraction was found to be selectively impaired (by 70%) in the bladder of KO mice (20), although another study suggested a reduction of contraction elicited by both carbachol and KCl in the bladder (37). We find that contractions in response to 5-HT and the muscarinic agonist carbachol are unchanged in intestinal muscle lacking caveolae when expressed relative to a reference high-K+ contraction. Serotonergic contractions of longitudinal smooth muscle from the small intestine have been reported to be due to 5-HT1 and 5-HT3 receptors (39). The contribution to contraction of 5-HT2A receptors, which may be caveolae associated (6, 11, 13), could thus be small in the ileum longitudinal smooth muscle.
It may be relevant in regard to endothelin-induced contraction that both ETA and ETB receptors have been proposed to depend on caveolae (3, 35). The present results showed that impaired contractility in response to ET-1 in the ileum was not associated with reduced ET-1 receptor expression, nor was it due to reduced Ca2+ sensitization. Further studies of endothelin signaling in the absence of caveolae seem warranted.
On the basis of the reported translocation to caveolae of key components of Ca2+ sensitization, we formulated the hypothesis that Ca2+ sensitization would be affected in the absence of caveolae. Our results confirm the association between Rho and Cav-1 that was previously observed in endothelial cells and fibroblasts (15, 22). This interaction appears to be functionally inhibitory since Rho activation was greater in KO. Our data on permeabilized ileum suggest, however, that Ca2+-sensitization proceeds normally in permeabilized muscle without caveolae and Cav-1. It therefore has to be assumed that GTPS-induced Ca2+ sensitization is not limited by Rho activation in the mouse ileum. Alternatively, Rho activation by ligand plus GTP may not be changed. We also failed to detect a difference in the sustained phase of carbachol contraction, which is known to depend on RhoA/Rho-kinase (21, 33). This indicates that Ca2+ sensitization is unchanged in situ in the absence of caveolae. A modest change in stress (force per cross-sectional area) cannot be ruled out, nor can it be ruled out that Ca2+-sensitization mechanisms have adapted by a fine tuning of the expression of downstream intermediaries, but caveolae and Cav-1 are clearly not required for a functional pathway, albeit subtly affecting maximal RhoA activation.
PKC-mediated contraction in the intact femoral artery was increased in the KO relative to the WT. PKC protein expression was not changed as shown directly for PKC-, and as suggested by the Ca2+-sensitization study. The data is thus compatible with the removal of an inhibitory influence of Cav-1 similar to the situation with Rho. Moreover, 1-adrenergic contraction was increased as expected for a response mediated partly by PKC. 1-Adrenergic contraction was not enhanced following inhibition of PKC; thus creating a strong argument that enhanced PKC signaling in KO compared with WT indeed underlies the increased 1-adrenergic contraction. The unchanged Ca2+ sensitization following permeabilization, and the unchanged phosphorylation of CPI-17, demonstrate that the increased PKC-induced contraction is due to a membrane-delimited mechanism. It seems reasonable to propose that Cav-1 is effective as a kinase inhibitor only in the sarcolemma, where it is located, and not intracellularly, where substrates such as CPI-17 are found. A variety of membrane-associated effector mechanisms, which could mediate the PKC effect, have been described. Examples include the delayed rectifier current (1), KATP channels (5), and Ca2+ sparks (7), all of which are bypassed in the -toxin permeabilized preparations. It is notable that the frequency of spontaneous transient outward currents, which are activated by Ca2+ sparks, are reduced in cerebral arterial myocytes from Cav-1-deficient mice (10). Whether this reduction is normalized by PKC inhibition is not known.
The possible association of 1-adrenergic receptors with caveolae is controversial. We previously examined the distribution of 1A-receptors in sucrose density gradients of smooth muscle homogenates from the rat caudal artery, and found that the 1A-receptors were present in the fractions of highest density, contrasting with 5HT2A receptors which were present in the lighter caveolin-containing fractions (11). On the other hand, binding of isotope-labeled phenylephrin and prazosin to caveolin-containing sucrose gradient fractions from heart and aorta was observed by others (14, 24). The latter studies support a caveolar association of 1-receptors in those specific cells and tissues. The present data does not distinguish between the possible 1-receptor locales, which may be tissue and receptor subtype specific. Alleviated inhibition of PKC by caveolin at the membrane is sufficient to explain the present results in the femoral artery.
Measurements of stress (force per cross-sectional area), using traditional methodology involving weighing, showed that depolarization-induced stress was reduced in KO compared with WT femoral arteries. This was due to a significant increase in the wet weight per mm length of femoral artery rather than a change in depolarization induced force. A detailed morphometric analysis needs to be made to justify such a stress calculation, because the increased wet weight may be due to increased extracellular matrix or increased adventitial cell populations. Moreover, force per length of arterial tube, not stress, is the relevant variable for regulation of peripheral resistance. In permeabilized tissue it is notoriously difficult to minimize experimental variation in stress and absolute force determinations. This is because cutting of strips involves considerable handling that may harm the tissue, the small weight of the preparations (
60 µg), and because the success of permeabilization varies. We can therefore not rule out changes in stress or absolute force in the permeabilized strips. Our data in intact tissue justifies normalization to a reference contraction, however, and the unchanged phosphorylation of CPI-17 independently shows that PKC-driven Ca2+ sensitization is unaltered in the absence of caveolae.
Our results concerning PKC-mediated contraction are fully compatible with the inhibitory effect seen after chemical loading of the scaffolding domain peptide from Cav-1 in the intact ferret aorta (16).
In conclusion, PKC mediated contraction and Rho activation but not Ca2+-sensitization are increased in smooth muscle following genetic ablation of Cav-1. Increased RhoA activation and PKC-driven force generation in the absence of caveolae may play a role in contractility or motility in many cell types, including, apart from smooth muscle cells, endothelial cells and fibroblasts.
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ACKNOWLEDGMENTS |
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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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) and KO (
). Slopes were significantly reduced in KO vs. WT (P < 0.05, n = 4) verifying reduced Cav-3 in Cav-1-deficient smooth muscle.
) and absence (