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Am J Physiol Gastrointest Liver Physiol 293: G240-G249, 2007. First published April 12, 2007; doi:10.1152/ajpgi.00064.2007
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HORMONES AND SIGNALING

Ectopic expression of caveolin-1 restores physiological contractile response of aged colonic smooth muscle

Sita Somara,1 Robert R. Gilmont,1 Jeffrery R. Martens,2 and Khalil N. Bitar1

1Department of Pediatrics-Gastroenterology and 2Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan

Submitted 6 February 2007 ; accepted in final form 6 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reduced colonic motility has been observed in aged rats with a parallel reduction in acetylcholine (ACh)-induced myosin light chain (MLC20) phosphorylation. MLC20 phosphorylation during smooth muscle contraction is maintained by a coordinated signal transduction cascade requiring both PKC-{alpha} and RhoA. Caveolae are membrane microdomains that permit rapid and efficient coordination of different signal transduction cascades leading to sustained smooth muscle contraction of the colon. Here, we show that normal physiological contraction can be reinstated in aged colonic smooth muscle cells (CSMCs) upon transfection with wild-type caveolin-1 through the activation of both the RhoA/Rho kinase and PKC pathways. Our data demonstrate that impaired contraction in aging is an outcome of altered membrane translocation of PKC-{alpha} and RhoA with a concomitant reduction in the association of these molecules with the caveolae-specific protein caveolin-1, resulting in a parallel decrease in the myosin phosphatase-targeting subunit (MYPT) and CPI-17 phosphorylation. Decreased MYPT and CPI-17 phosphorylation activates MLC phosphatase activity, resulting in MLC20 dephosphorylation, which may be responsible for decreased colonic motility in aged rats. Importantly, transfection of CSMCs from aged rats with wild-type yellow fluorescent protein-caveolin-1 cDNA restored translocation of RhoA and PKC-{alpha} and phosphorylation of MYPT, CPI-17, and MLC20, thereby restoring the contractile response to levels comparable with young adult rats. Thus, we propose that caveolin-1 gene transfer may represent a promising therapeutic treatment to correct the age-related decline in colonic smooth muscle motility.

aging; myosin phosphorylation; RhoA; PKC

A VARIETY OF PHYSIOLOGICAL FUNCTIONS in tissues and organs are altered during the process of aging. The overall physiology of the gut requires coordinated control of motility. With aging, colonic motility, regulated by the smooth muscle contraction and relaxation cycle, declines (5, 39).

Agonist-induced contraction of colonic smooth muscle results from sequential activation of signal transduction pathways regulated by phosphorylated and dephosphorylated proteins. Myosin light chain (MLC20) phosphorylation is essential for force production, and the amount of force generated is related to the level of its phosphorylation (42, 43). A critical factor regulating MLC20 phosphorylation is the balance between the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP) (26). Different signaling transduction mechanisms regulate smooth muscle contraction by modulating either MLCK or MLCP. The predominant contractile pathway modulating the initiation of contraction and regulating kinase activity of MLCK is a rise in intracellular Ca2+ concentration (41). The initial contractile response (30 s) is suggested to be dependent on MLCK-mediated MLC20 phosphorylation, whereas the sustained contractile response (4 min) is suggested to be associated with maintenance of MLC20 phosphorylation mediated by inhibition of MLCP (8).

MLCP activity is regulated by PKC and RhoA/ROCK pathways in smooth muscle (26, 44). MLCP is a trimeric enzyme consisting of a 110- to 130-kDa myosin phosphatase-targeting subunit (MYPT1) that anchors MLCP to phosphorylated MLC20; a 37-kDa catalytic subunit (PP1c) of the type 1 protein serine/threonine phosphatase family, specifically, the {delta}-isoform; and a 20-kDa small subunit of unclear function (1, 16, 35, 36). Activation of the RhoA/ROCK pathway results in phosphorylation of MYPT, which results in the inhibition of MLCP activity, whereas activation of the PKC pathway predominantly results in the phosphorylation of CPI-17, which is a potent inhibitor of PP1c and also results in the inhibition of MLCP activity. Activation of the RhoA/ROCK and PKC pathways is associated with translocation of RhoA and PKC-{alpha} to the membrane. Agonist-induced contraction of colonic smooth muscles cells (CSMCs) suggests that RhoA and PKC-{alpha} translocate to caveolae microdomains on the membrane (47).

An emerging concept is that functional microdomains exist within the fluid bilayer of the plasma membrane. These dynamic microdomain structures, termed lipid rafts, are rich in tightly packed sphingolipids and cholesterol (37). One well-studied subpopulation of lipid rafts is caveolae that have an invaginated morphology and contain the scaffolding protein caveolin (2, 15, 17, 28, 37, 49). The caveolin gene family consists of three members: caveolin-1 (Cav-1), Cav-2, and Cav-3. This gene family is conserved across species from Caenorhabditis elegans to humans (13) and represent the functional assembly units of caveolae (13). Caveolae are abundant in smooth muscle and cardiac myocytes (12, 34). Multiple cell signaling pathways converge through interactions with caveolin to regulate excitation-contraction coupling in smooth muscle. In vitro studies have illustrated that caveolins interact with a variety of signaling molecules including G{alpha} subunits, MAPK, Src tyrosine kinases, endothelial nitric oxide synthase, PKC-{alpha}, and RhoA (28, 34). Taggart et al. (47) have reported that both PKC-{alpha} and RhoA exhibited a stimulus-dependent translocation to the plasma membrane of SMCs, where they may interact with caveolins in caveolae. Indeed, Cav-1, in particular, has been found to interact with a wide variety of signal transducing molecules, including PKC-{alpha} and RhoA (6, 14). Translocation of PKC-{alpha} and RhoA to the plasma membrane in vascular SMCs was inhibited upon introduction of Cav-1 scaffolding domain peptide, suggesting that Cav-1 directly interacts with PKC-{alpha} and RhoA via its scaffolding domain (20). There is increasing evidence that caveolae may contain key components for Ca2+ handling and may serve as initiation sites for Ca2+ sparks in cardiac and smooth muscle myocytes (7, 19, 24). In addition, in cardiac myocytes, specific beta-adrenergic receptor subtypes and adenylate cyclase are localized to caveolae (31) and various PKC isoforms translocate to caveolae following activation (32). In contrast, activated adenosine receptors translocate out of caveolae (22). Thus, caveolae are essential modulators of signal transduction pathways. Changes in caveolin levels affect the formation of caveolae, resulting in disarray in the activation of caveolae-linked signal transduction pathways. Cav-1 knockout mice have been reported to have 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 (9). Shakirova et al. (33) reported that Rho activation and PKC-mediated contraction of smooth muscle was increased in the absence of caveolae and Cav-1. Ablation of caveolae attenuated endothelin-induced contraction in the ileum, without influencing muscarinic or serotonergic force (33).

The age-dependent decrease in agonist-induced contraction is attributed to impairment in the signal transduction pathway of the SMC. The possible site for the impairment has been suggested to be downstream of the rise in intracellular Ca2+ levels (21). We investigated the effect of aging on the signal transduction pathways mediated by RhoA and PKC-{alpha}. The results presented herein may provide a subcellular and molecular basis for the age-related decline of the contractile response in CSMCs. Our present study also shows that the age-related decline in physiological contraction responsible for colonic motility could be reinstatement by Cav-1 gene transfer, suggesting that Cav-1 could be a critical factor affected by aging and a putative therapeutic target.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. G-418, penicillin-streptomycin, FBS, and DMEM were from GIBCO-BRL (Grand Island, NY). Collagenase type II was from Worthington Biochemical (Lakewood, NJ). Protein G-Sepharose was from Pharmacia Biotech (Uppsala, Sweden). Polyvinylidene fluoride (PVDF) membranes were from Bio-Rad (Hercules, CA). Enhanced chemiluminescence (ECL) detection reagents were from Amersham Biosciences. ACh chloride, MLCK antibody, M3 receptor antibody, and all other reagents were from Sigma (St. Louis, MO). Mouse monoclonal phospho-MLC20 (Ser19) and mouse monoclonal anti-RhoA antibody were from Cytoskeleton. Rabbit polyclonal PKC-{alpha} was from Abcam (Cambridge, MA), and phospho-PKC-{alpha} antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). MYPT, phospho-MYPT (T696), and phospho-CPI-17 (T38) antibody was from Upstate Biotechnology. Peroxidase-conjugated secondary anti-mouse antibody and anti-rabbit antibody were purchased from Bio-Rad (Irvine, CA). Aged (28–33 mo old) rats were obtained National Institute on Aging (Bethesda, MD), and adult (12–18 mo old) rats were obtained from Harlan (Madison, WI).

Isolation of SMCs. All procedures were performed according to "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. SMCs from the rat colon were isolated as previously described (18). Briefly, the circular smooth muscle layer from the distal colon of the rat was removed by sharp dissection. A 5-cm length of the recto sigmoid, distal to the junction of the jejunum, was dissected and digested with collagenase to yield isolated SMCs. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml HEPES buffer (pH 7.4) [containing (in mM) 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 5.6 glucose, with 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II), 0.01 (wt/vol) soybean trypsin inhibitor, and 0.184 (wt/vol) DMEM]. After the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex. The partially digested tissue left on the filter was washed four times with 10 ml of collagenase-free buffer solution. The tissue was then transferred into 15 ml of fresh collagenase-free buffer solution, and cells were gently dispersed. After a hemocytometric cell count, the harvested cells were resuspended in collagenase-free HEPES buffer.

Preparation of particulate fractions. Particulate fractions from freshly isolated SMCs from adult and aged rat colons were prepared as previously described (30). Briefly, freshly isolated SMCs were treated with ACh (0.1 µM) for 30 s and for 4 min followed by immediate freezing in an acetone-dry ice slurry. After stimulation, cells were washed twice with buffer A[containing (in mM) 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate (pH 7.4)] and sonicated in buffer B [containing (in mM) 1 Na3VO4, 1 NaF, 2 PMSF, 5 EDTA, 1 Na4MoO4, 1 DTT, 20 NaH2PO4, 20 Na2HPO4, and 20 Na4P2O7·10H2O, with 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml antipain-HCl (pH 7.4), 0.08 mg/ml soybean trypsin inhibitor, 60 µg/ml phosphor-amidon, and 5 mg/ml Pefbloc]. Sonicated cells were centrifuged at 100,000 g for 60 min. The supernatant material was collected as the cytosolic fraction while the pellet material was resuspended in lysis buffer with 1% Triton X-100 followed by sonicating twice for 30 s each time and collected as the particulate fraction. The protein content was determined using Bio-Rad protein assay reagent.

Transfection of SMCs. Isolated CSMCs were cultured in DMEM with 10% FBS and 3% penicillin-streptomycin on collagen type IV-coated dishes. Cells were passaged on the day before transfection and allowed to reach 70% confluence on the day of transfection. Cells were washed with PBS twice. Aged rat CSMCs were transfected with yellow fluorescent protein (YFP)-tagged wild-type (WT) Cav-1 cDNA (a gift from Jeffrery R. Martens), whereas rabbit CSMCs were transfected with dominant negative (DN) Cav-1 cDNA (a gift from Jeffrery R. Martens) using a QiaGen Effectene transfection kit. DN Cav-1 cDNA is a Cav-1/{Delta}TFT deletion mutation that has deletion of a 9-bp microdeletion corresponding to three amino acids: threonine-phenylalanine-threonine (amino acids 91–93) in the scaffolding domain of rabbit Cav-1 cDNA. The correctness of the deletion was verified by DNA sequencing. The caveolin scaffolding domain of Cav-1 from the rat and rabbit share 100% sequence homology. Rabbit and rat Cav-1 sequences share 98% sequence identity. The only two differences that occur are at positions 170 and 174 on the COOH terminus of the protein.

Briefly, the cDNA was diluted with buffer EC and mixed with enhancer followed by incubation at room temperature for 5 min. The DNA-enhancer mixture was mixed with Effectene transfection reagent followed by an incubation at room temperature for 10 min to allow complex formation. The transfection complex was then mixed with cell culture medium and overlaid on the cells. After 2 days of transfection, cells were selected with G-418 (0.5 mg/ml) for 2 days. The estimated transfection efficiency was ~40%, as measured by the numbers of cells remaining attached to the plate upon G-418 treatment. These cells were maintained in G-418 (0.3 mg/ml) media and were grown until confluent (~2 wk) as stable transfection for further experiments.

Immunoprecipitation and immunoblot analysis. Antibody (1–2 µg) was added to 200 µg of sample protein in 200 µl of lysis buffer and rocked overnight at 4°C; 50 µl of 50% Protein G-Sepharose bead slurry was added to the overnight mixture and rocked at 4°C for 2 h. The beads bound with proteins were then collected by centrifuging at 14,000 g for 3 min at 4°C. The supernatant was discarded, and the bead pellet was washed three times at room temperature with Tris-buffered saline (TBS) bead wash buffer (20 mM Tris·HCl and 150 mM NaCl; pH 7.6). Beads were then resuspended in 25 µl of 2x sample buffer and boiled for 5 min. Proteins from the immunoprecipitates were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was immunoblotted with the desired antibodies. Replicates of experiments were performed using completely separate sets of cells.

SDS-PAGE and Western blot analysis. For one-dimensional SDS-PAGE, samples were mixed in an equal volume of 2x sample buffer [50 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.1% (wt/vol) bromophenol blue; pH 6.8]. Proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes. The PVDF membrane was then blocked with 5% nonfat dry milk for 1 h. After being blocking, the membrane was incubated in an appropriate dilution of primary antibody in 5% nonfat dry milk in TBS-Tween 20 (TBST) for 1 h. The membrane was washed three times with TBS to remove unbound primary antibody for 15 min each wash at room temperature. The membrane was then incubated in an appropriate dilution of secondary antibody in 5% nonfat dry milk in TBST for 1 h at room temperature. The membrane was washed three times with TBST for 15 min each wash at room temperature to remove unbound secondary antibody. The membrane was then incubated with ECL reagent for 1 min. Proteins were detected on the membrane by immediately exposing the membrane to the film for 30 s and 1 min.

Lipid raft preparation. Detergent-free purification of caveolae-enriched membrane fractions was carried out following the method described by Lisanti et al. (23) with slight modifications. Briefly, rabbit CSMCs were washed twice with cold 1x PBS (pH 7.4) and scraped into 500 mM sodium carbonate (pH 11; Sigma) plus caveolin-enriched membrane buffer (CEM buffer; 25 mM MES, 150 mM NaCl, 1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin). Cells were homogenized and sonicated for 20–30 s, and the sample (3 ml) was adjusted to 40% sucrose by the addition of 3 ml of 80% sucrose in CEM buffer before placement at the bottom of an ultracentrifuge tube (Beckman Coulter, Hayward, CA). Two milliliters each of 25%, 15%, and 5% sucrose in 250 mM sodium carbonate (pH 11) plus CEM buffer were then sequentially layered above. The discontinuous gradient was centrifuged at 36,000 rpm for 22 h using a Beckman SW50.1 rotor, after which twelve 1-ml fractions were collected from the top to the bottom of the tube. The pellet fraction (fraction 12) was separately sonicated in 12 ml MES buffer. Equal volume aliquots were analyzed by SDS-PAGE and immunoblot analysis.

Contraction. Contraction experiments were performed as previously described (30). Briefly, isolated CSMCs were cultured to confluence. Fresh medium was added to the culture flask. Cells were then scraped off with a "policeman" and allowed to float freely for 48 h in a standing flask with occasional shaking to prevent further settling and sticking to the bottom of the flask. Aliquots of cultured cell suspension (2.5 x 104 cells/0.5 ml) were stimulated with ACh (10–7 M) for 30 s or 4 min. The reaction was allowed to proceed for 30 s or 4 min and stopped by the addition of 0.1 ml acrolein at a final concentration of 1% (vol/vol). Individual cell length was measured by computerized image micrometry. The average length of cells in the control state or after the addition of test agents was obtained from 50 cells encountered randomly in successive microscopic fields. The contractile response was defined as the decrease in the average length of the 50 cells and is expressed as the absolute change or the percent change from control length (4).

Data analysis. Data analysis was done as previously described (40). Briefly, Western blot bands were quantitated using a densitometer (model GS-700, Bio-Rad), and band volumes (absorbance units x mm2) were calculated and expressed as a percentage of the total volume. Each set of experiments had an internal control of its own. Control band intensities were standardized to 100%. Band intensities of samples from treated cells were compared with the control and expressed as the percent change from the control. Blotting data are within the linear range of detection for each antibody used. Statistical analysis results were same whether data were analyzed using percent changes or absolute values. All means were compared and analyzed using Student's t-test. Difference between control and experimental values were compared using Student's t-test and considered significant at P < 0.05. All observations were done with three separate sets of experiments; n values refer to numbers of animals.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of aging on ACh-induced contraction of colonic smooth muscle. The key event for smooth muscle contraction is phosphorylation of MLC20 at Ser19 (45, 46). We investigated the effect of aging on the phosphorylation of MLC20 in rat CSMCs. CSMCs isolated from adult and aged rat were stimulated with ACh (10–7 M) followed by immunoblot analysis of whole cell lysates with anti-phospho-MLC20 (Ser19). Aged rat colonic smooth muscle showed reduced ACh-induced MLC20 phosphorylation at Ser19 (117.13 ± 3.14% and 122.77 ± 1.67% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with adult counterparts (180.94 ± 3.26% and 194.02 ± 3.42% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 1A). To examine whether the decreased levels of MLC20 phosphorylation on the membrane were due to reduced expression of any of the components modulating MLC20 phosphorylation, we examined the expression profiles of PKC-{alpha} and RhoA and their respective downstream signaling pathways. The levels of expression of MLCK, PKC-{alpha}, RhoA, MYPT, and M3 receptor did not change significantly between adult and aged CSMCs (Fig. 1B).


Figure 1
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  		Fig. 1. A: effect of aging on ACh-induced myosin light chain (MLC20) phosphorylation in rat colonic smooth muscle cells (CSMCs). CSMCs isolated from adult and aged rats were stimulated with ACh (10–7 M) followed by immunoblot (IB) analysis of whole cell lysates with anti-phospho-MLC20 (Ser19). Aged rat CSMCs showed reduced ACh-induced MLC20 phosphorylation at Ser19 compared with adult counterparts. B: effect of aging on expression of different contractile components in rat CSMCs. Whole cell lysates of SMCs from old and adult rabbit colons were immunoblotted and probed for different contractile signaling molecules. No significant differences in the expression of MLC kinase (MLCK), RhoA, PKC-{alpha}, myosin phosphatase-targeting subunit (MYPT1), and M3 receptor between aged and adult CSMCs were observed. C: effect of aging on ACh-induced phosphorylation of CPI-17 in rat CSMCs. CSMCs isolated from adult and aged rats were stimulated with ACh (10–7 M) followed by IB analysis of whole cell lysates with anti-phospho-CPI-17 (T38) antibody showed that there was a significant and sustained decrease in ACh-induced CPI-17 phosphorylation in aged rat CSMCs compared with adult rat CSMCs. D: effect of aging on ACh-induced phosphorylation of MYPT in rat CSMCs. Simultaneously, stimulation of CSMCs isolated from adult and aged rats with ACh (10–7 M) followed by IB analysis of cell lysates with anti-phospho-MYPT (T696) showed that a significant and sustained decrease in ACh-induced MYPT phosphorylation in aged rat CSMCs compared with adult counterparts. E: effect of aging on ACh-induced translocation of PKC-{alpha} in rat CSMCs. CSMCs isolated from adult and aged rats were stimulated with ACh (10–7 M) followed by a separation of particulate and cytosolic fractions as described in MATERIALS AND METHODS. IB analysis of the particulate fraction from adult and aged rat CSMCs with anti-PKC-{alpha} antibody showed a significant and sustained decrease in ACh-induced PKC-{alpha} translocation in aged rat CSMCs compared with adult rat CSMCs. F: effect of aging on ACh-induced translocation of RhoA in rat CSMCs. IB analysis of the particulate fraction from adult and aged rat CSMCs with anti-RhoA antibody showed that significant and sustained ACh-induced RhoA translocation was decreased in aged rat CSMCs compared with adult rat CSMCs. *P < 0.01.

 
Reduced ACh-induced MLC20 phosphorylation indicates increased phosphatase activity. We thus examined the phosphorylation of CPI-17 and of MYPT, which modulate phosphatase activity. Stimulation of CSMCs isolated from adult and aged rats with ACh (10–7 M) and immunoblot analysis with anti-phospho-CPI-17 (T38) antibody showed that there was a significant and sustained decrease in ACh-induced CPI-17 phosphorylation in aged rat CSMCs (123.33 ± 4.57% and 136.14 ± 3.33% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3) compared with that in adult rat colonic smooth muscle (195.99 ± 2.23% and 213.17 ± 1.65% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 1C). Simultaneously, stimulation of CSMCs isolated from adult and aged rats with ACh (10–7 M) followed by immunoblot analysis with anti-phospho-MYPT (T696) antibody showed a significant and sustained decrease in ACh-induced MYPT phosphorylation in aged rat colonic smooth muscle (115.18 ± 1.23% and 121.39 ± 1.03% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3) compared with adult counterparts (169.15 ± 9.99% and 177.30 ± 10.48% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 1D). Our data thus demonstrate that aging reduces the phosphorylation of CPI-17, a PKC-{alpha} substrate, and of MYPT, a RhoA/ROCK substrate, resulting in the activation of MLCP activity, leading to an age-related decline in the phosphorylation of MLC20.

Phosphorylation of CPI-17 and of MYPT is modulated by PKC-{alpha} and RhoA/ROCK pathways, respectively. PKC-{alpha} and RhoA/ROCK pathways are activated at the membrane upon ACh-induced translocation of PKC-{alpha} and RhoA in CSMC. We thus investigated ACh-induced translocation of PKC-{alpha} and RhoA to study whether age-related effects are due to alterations in either PKC-{alpha} or RhoA translocation and subsequent activation of the PKC-{alpha} or RhoA-ROCKII pathway. CSMCs isolated from adult and aged rats were stimulated with ACh (10–7 M) followed by a separation of particulate and cytosolic fractions as described in MATERIALS AND METHODS. Immunoblot analysis of the particulate fraction from adult and aged rats CSMCs with anti-PKC{alpha} antibody showed that there was a significant and sustained decrease in ACh-induced PKC-{alpha} translocation in aged rat CSMCs (118.25 ± 2.14% and 123.87 ± 1.03% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with that in adult rat CSMCs (176.63 ± 1.79% and 187.74 ± 1.36% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 1E). Immunoblot analysis of the particulate fraction from adult and aged rat CSMCs with anti-RhoA antibody showed a significant and sustained decrease in ACh-induced RhoA translocation in aged rat CSMCs (114.80 ± 1.15% and 125.31 ± 1.01% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with that in adult rat CSMCs (183.59 ± 2.20% and 191.02 ± 4.38% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 1F). Thus, our data show that the age-related reduction in ACh-induced MLC20 phosphorylation is the consequence of MLCP activation, which, in turn, is due to reduced activation of the PKC-{alpha} and RhoA-ROCKII pathway due to an age-related decrease in the translocation of PKC-{alpha} and RhoA.

Effect of aging on caveolin expression and ACh-induced association of Cav-1 with PKC-{alpha} and RhoA in colonic smooth muscle. Taggart et al. (47) reported that both PKC-{alpha} and RhoA exhibited a stimulus-dependent translocation to caveolae on the plasma membrane of SMCs. Reduced translocation of PKC-{alpha} and RhoA could be due to reduced numbers of caveolae on the membrane. We thus investigated the levels of the caveolae marker protein Cav-1 in the particulate fraction of CSMCs. Immunoblot analysis with anti-Cav-1 antibody showed that there was a significant reduction in the levels of Cav-1 protein in aged rat CSMCs (56.24 ± 0.44%, P ≤ 0.001, n = 3; Fig. 2A) compared with adult rat CSMCs (100%). Our data showed that Cav-1 expression in the aged CSMC is reduced.


Figure 2
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  		Fig. 2. A: effect of aging on caveolin levels in rat CSMCs. IB analysis of the particulate fraction of unstimulated CSMCs with anti-caveolin-1 (Cav-1) antibody showed that there was a significant reduction in the levels of Cav-1 protein in aged rat CSMCs (56.24 ± 0.44%, P ≤ 0.001, n = 3) compared with adult rat CSMCs (100%). B: effect of aging on ACh-induced translocation of PKC-{alpha} to caveolae in CSMCs. Lipid raft membrane microdomains on the plasma membrane were purified from adult and aged CSMCs by sucrose density gradient as described in MATERIALS AND METHODS. Membrane fractions of stimulated CSMCs were immunoblotted with either anti-Cav-1 antibody or anti-phospho-PKC-{alpha} (Ser657) antibody. ACh stimulation increased PKC-{alpha} levels in the fractions (fractions 5–7) that had caveolin in lipid raft fractions (fractions 5–7) from adult CSMCs. IB analysis of lipid raft fractions from aged CSMCs with anti-PKC-{alpha} antibody and anti-Cav-1 antibody showed an absence of phospho-PKC-{alpha} and depletion of Cav-1 in lipid raft fractions 5–7 with only traces of Cav-1 found outside the lipid raft fractions. C: effect of aging on ACh-induced association of Cav-1 with PKC-{alpha} in rat CSMCs. Particulate fractions from unstimulated and ACh-stimulated CSMCs from aged and adult rats were immunoprecipitated (IP) with anti-Cav-1 antibody followed by IB analysis with anti-PKC-{alpha} antibodies separately. CSMCs from aged rats showed a significant decrease in the ACh-induced association of Cav-1 with PKC-{alpha} compared with adult CSMCs. D: effect of aging on ACh-induced association of Cav-1 with RhoA in rat CSMCs. Particulate fractions from unstimulated and ACh-stimulated CSMCs from aged and adult rats were immunoprecipitated with anti-Cav-1 antibody followed by IB analysis with anti-RhoA antibodies separately. CSMCs from aged rats showed a significant decrease in the ACh-induced association of Cav-1 with RhoA compared with adult CSMCs. *P < 0.01.

 
We further confirmed reduced Cav-1 expression by analyzing the caveolin-rich lipid raft membrane. Lipid raft membrane fractions along with the pellet fraction (fraction 12) of CSMCs were immunoblotted with anti-Cav-1 antibody. Fractions 5–7 are the caveolin-rich lipid raft fractions. In adult rat CSMCs, caveolin is associated with lipid rafts and can be thus seen in fractions 5–7, whereas in aged rat CSMC, caveolin is not associated with lipid rafts (fractions 5–7) and thus all of the cav-1 expressed in aged rat CSMCs was present in the pellet fraction (fraction 12) (Fig. 2B). Figure 2A shows that there was also reduced expression of Cav-1 in aged CSMCs. This suggests that aging not only affects the expression of Cav-1 but also impairs the translocation of Cav-1 to lipid raft membrane microdomains and caveolae formation. This results in impairment in the activation of signaling pathways (PKC and RhoA) whose activation is caveolae mediated.

We therefore examined the effect of aging on the sequestration of translocated PKC-{alpha} in the caveolae. Lipid raft membrane fractions of CSMCs were immunoblotted with anti-phospho-PKC-{alpha} (Ser657) antibody. In the adult CSMC lipid raft fraction, ACh stimulation increased PKC-{alpha} levels in the fractions (fractions 5–7) that had caveolin, suggesting that ACh induces PKC-{alpha} translocation to caveolae microdomains (Fig. 2B). In aged CSMCs, isolated lipid raft fractions (fractions 5–7) showed a loss of phospho-PKC-{alpha}. Absence of phospho-PKC-{alpha} in lipid raft fractions 5–7 of aged CSMC indicated that the translocation of PKC-{alpha} is dependent on an interaction with Cav-1. Both translocated PKC-{alpha} and RhoA have been reported to interact with caveolins in caveolae (10, 47). We thus investigated the effect of aging on the association of Cav-1 with the signaling molecules PKC-{alpha} and RhoA in the CSMC particulate fraction in response to ACh stimulation. Particulate fractions from unstimulated and ACh-stimulated CSMCs from aged and adult rats were immunoprecipitated with anti-Cav-1 antibody followed by immunoblot analysis with either anti-PKC-{alpha} or anti-RhoA antibody. CSMCs from aged rats showed a significant decrease in ACh-induced association of Cav-1 with PKC-{alpha} (112.16 ± 5.46% and 118.30 ± 0.83% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with adult CSMCs (156.94 ± 1.29% and 166.59 ± 1.94% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 2C). Similarly, CSMC from aged rats showed a significant decrease in the ACh-induced association of Cav-1 with RhoA (116.82 ± 1.59% and 118.26 ± 2.90% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with adult CSMCs (157.87 ± 2.88% and 168.68 ± 1.98% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 2D). The aged-related decrease in the association of Cav-1 with PKC-{alpha} and with RhoA can be attributed to reduced levels of Cav-1 expression.

Effect of aging on the ACh-induced contractile response in rat CSMCs and its restoration upon ectopic expression of Cav-1. The normal physiological functioning of the gastrointestinal tract requires overall coordinated control of motility. With aging, colonic motility regulated by the smooth muscle contraction and relaxation cycle is reported to be declined. The effects of aging on gastric and colonic motility in rats include slow gastric emptying of liquids with decreased fecal pellet transit and production (39, 48). We examined the effect of aging on the ACh-induced colonic contractile response by studying SMC length shortening. ACh-induced shortening of cell length in aged rat CSMCs was decreased (18.88 ± 1.43% and 17.46 ± 1.30% decrease in cell length at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with adult rat CSMCs (42.91 ± 1.41% and 56.33 ± 0.98% decrease in cell length at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 3A). Importantly, transfection of Cav-1 cDNA into aged rat CSMCs restored ACh-induced contraction to levels comparable with control adult rats. As shown in Fig. 3A, ectopic expression of Cav-1 dramatically restored the degree of contractile response of aged rat CSMCs to ACh (31.85 ± 1.29% and 35.05 ± 0.85% decrease in cell length at 30 s and 4 min, respectively, P ≤ 0.01, n = 3).


Figure 3
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  		Fig. 3. A: effect of aging on the ACh-induced contractile response in rat CSMCs. ACh- induced shortening of cell length in aged rat CSMCs was decreased compared with adult rat CSMCs. Introduction of wild-type (WT) Cav-1 cDNA into aged rat CSMCs restored the physiological ACh-induced contraction. B: restoration of physiological MLC20 phosphorylation in CSMCs from aged rats upon introduction of WT Cav-1 cDNA. Aged rat CSMCs transfected with WT Cav-1 cDNA showed restoration of ACh-induced MLC20 phosphorylation at Ser19. *P < 0.01.

 
To investigate the mechanism of this Cav-1-mediated restoration of contractile function, we first measured MLC20 phosphorylation in aged rat CSMCs transfected with WT Cav-1 cDNA. Our data showed restoration of ACh-induced MLC20 phosphorylation at Ser19 (165.28 ± 4.27% and 173.73 ± 8.53% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 3B), which were significantly similar to the percentages of ACh-induced MLC20 phosphorylation in adult rats. Reinstatement of contraction and restoration of MLC20 phosphorylation suggested restored activation of PKC-{alpha} and RhoA/ROCK pathways. We thus measured the translocation of both PKC-{alpha} and RhoA and the activation of their subsequent downstream effectors.

Aged rat CSMCs transfected with WT Cav-1 cDNA showed restoration of ACh-induced translocation of PKC-{alpha} (167.25 ± 3.00% and 178.13 ± 4.26% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 4A), restoration of ACh-induced association of Cav-1 with PKC-{alpha} (135.78 ± 0.90% and 142.63 ± 1.41% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 4B), and consequent restoration of ACh-induced CPI-17 phosphorylation (190.00 ± 2.05% and 203.32 ± 2.62% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 4C) to physiological levels. Similarly, aged rat CSMCs transfected with WT Cav-1 cDNA showed restoration of ACh-induced translocation of RhoA (170.42 ± 6.77% and 181.37 ± 10.63% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 4A), restoration of ACh-induced association of Cav-1 with RhoA (146.54 ± 8.57% and 157.27 ± 1.12% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 4D), and consequent restoration of ACh-induced MYPT phosphorylation (160.84 ± 8.71% and 165.19 ± 9.42% at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 4E) to physiological levels.


Figure 4
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  		Fig. 4. Restoration of physiological contraction in CSMCs from aged rats upon introduction of WT Cav-1 cDNA. CSMCs from aged and aged rats transfected with functional WT Cav-1 cDNA were stimulated with ACh and used for restoration experiments. A: particulate fractions of unstimulated and ACh-stimulated CSMCs were immunoblotted with either anti-PKC-{alpha}, anti-RhoA, or anti-actin antibody. Aged rat CSMCs transfected with WT Cav-1 showed restoration of ACh-induced PKC-{alpha} translocation and restoration of ACh-induced RhoA translocation to the particulate fraction. B: ACh-induced association of Cav-1 with PKC-{alpha} was restored to physiological levels in aged CSMCs transfected with WT Cav-1 cDNA. C: ACh-induced CPI-17 phosphorylation was restored to physiological levels in aged CSMCs transfected with WT Cav-1 cDNA. D: ACh-induced association of Cav-1 with RhoA was restored to physiological levels in aged CSMCs transfected with WT Cav-1 cDNA. E: ACh-induced MYPT phosphorylation was restored to physiological levels in aged CSMCs transfected with WT Cav-1 cDNA. *P < 0.01.

 
Our results thus suggest that PKC-{alpha} and RhoA/ROCK pathways are restored upon ectopic expression of Cav-1 in aged CSMCs. Restoration of PKC-{alpha} and RhoA/ROCK pathways leads to inhibition of MLCP activity, resulting in the maintenance of ACh-induced MLC20 phosphorylation. Together, these data suggest that the physiological contractile response of aged CSMCs can be restored by ectopic expression of functional WT Cav-1 cDNA.

Effect of DN Cav-1 cDNA on ACh-induced translocation of PKC-{alpha} and RhoA and their association with Cav-1. To further confirm the role of caveolin in the translocation and activation of PKC-{alpha} and RhoA, we studied the translocation of PKC-{alpha} and RhoA and their association with Cav-1 in rabbit CSMCs transfected with DN Cav-1 cDNA (11). Cav-1 proteins form homooligomeric complexes and form caveolae. DN Cav-1 cDNA has a 9-bp deletion mutant, cav-1-{Delta}TFT, in which three amino acids (91–93 aa) from the scaffolding region of the protein were removed. Mutant caveolin constructs have the potential to behave in a DN fashion by binding endogenous caveolin and preventing normal cell surface expression, resulting in impaired caveolae formation. Thus, cells transfected with DN Cav-1 cDNA have impaired caveolae. Untransfected and transfected cells were stimulated with ACh for 30 s and 4 min. Particulate fractions of ACh-stimulated and unstimulated SMCs were separated from the cytosolic fraction by ultracentrifugation. Immunoblot analysis of particulate fractions from rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced levels of PKC-{alpha} (105.18 ± 3.10% and 114.27 ± 4.25% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with untransfected CSMCs (150.77 ± 2.74% and 161.47 ± 2.06% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 5A). Similarly, immunoblot analysis of particulate fractions from rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced levels of RhoA (111.89 ± 3.53% and 117.28 ± 4.29% at 30 s and 4 min, respectively, P ≤ 0.01; n = 3) compared with untransfected CSMCs (166.17 ± 3.51% and 176.64 ± 1.82% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 5B). We further confirmed our data by studying the association of PKC-{alpha} and RhoA with Cav-1. Equal amounts of the particulate fraction were immunoprecipitated with anti-Cav-1 antibody followed by immunoblot analysis with either anti-PKC-{alpha} or anti-RhoA antibody. Rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced ACh-induced association of Cav-1 with translocated PKC-{alpha} (109.98 ± 4.08% and 121.34 ± 3.10% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with untransfected rabbit CSMCs (160.54 ± 2.78% and 167.35 ± 3.34% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 5C). Similarly, rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced ACh-induced association of Cav-1 with translocated RhoA (114.20 ± 3.67% and 128.64 ± 6.67% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3) compared with untransfected CSMCs (164.80 ± 3.18% and 173.66 ± 7.50% at 30 s and 4 min, respectively, P ≤ 0.01, n = 3; Fig. 5D). These data suggest that impaired caveolae impair the translocation of PKC-{alpha} and RhoA, resulting in reduced association of Cav-1 with PKC-{alpha} and RhoA. The data suggest that the presence of Cav-1, i.e., caveolae, is essential for the translocation and activation of PKC-{alpha} and RhoA.


Figure 5
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  		Fig. 5. ACh-induced association of translocated PKC-{alpha} and RhoA with Cav-1. Rabbit CSMCs and CSMCs transfected with dominant negative (DN) Cav-1 cDNA were stimulated with ACh for 30 s and 4 min. The particulate fraction of ACh-stimulated and unstimulated SMCs was separated from the cytosolic fraction by ultracentrifugation. IB analysis of the particulate fraction from rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced levels of PKC-{alpha} (A) and reduced level of RhoA (B) compared with the particulate fraction from untransfected CSMCs. Equal amount of particulate fractions were immunoprecipitated with anti-Cav-1 antibody followed by IB analysis with either anti-PKC{alpha} or anti-RhoA antibody. Rabbit CSMCs transfected with DN Cav-1 cDNA showed reduced ACh-induced association of Cav-1 with translocated PKC-{alpha} (C) and reduced ACh-induced association of Cav-1 with translocated RhoA (D). These data suggest that downregulation of Cav-1 impairs the translocation of PKC-{alpha} and RhoA resulting in reduced association of Cav-1 with PKC-{alpha} and RhoA. *P < 0.01.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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In present study, we showed that the age-related decline in colonic motility is associated with a decrease in Cav-1 expression formation on the membrane. We further showed that the introduction of functional WT Cav-1 in CSMCs can restore the physiological contractile response in aged CSMCs.

Caveolae are omega-shaped membrane invaginations that are abundant in SMCs and integrate important signaling pathways (3). Aging is characterized by diminished colonic contractility in response to ACh (4, 38, 39). The contractile response, as indicated by shortening of cell length, is reduced in aged CSMCs compared with those of the adult (4). Munro et al. (25) have previously shown that longitudinal smooth muscle from the urinary bladder of aged rats showed decreased maximal force and lower maximal velocity of cell length shortening. Aged rat CSMCs showed a decrease in ACh-induced phosphorylation of MLC20 compared with adult CSMCs. Imbalanced basal protein kinase and phosphatase activities have been shown to be responsible for age-related reduced synaptic strength during aging (27). Our present study showed that the age-related decline is due to reduced or lack of compartmentalization of signaling molecules on the membrane.

Downstream of receptor activation, RhoA as well as Rho kinase and PKC play key roles in the regulation of contractility in smooth muscle (41). RhoA and PKC have been shown to reside in or to translocate to caveolae upon receptor stimulation (14, 47). The scaffolding domain of Cav-1 was reported to impair both membrane translocation of PKC (47) and contraction stimulated by phorbol ester and {alpha}-agonist in the ferret aorta (20). Rho kinase has been demonstrated to translocate to caveolae in a Ca2+/calmodulin-dependent manner on smooth muscle depolarization (10). The present investigation indicates that aged rat colonic smooth muscle exhibits a significant and sustained reduction in ACh-induced MYPT and CPI-17 phosphorylation, which correlates with a significant and sustained decrease in ACh-induced translocation of RhoA and PKC-{alpha} to the particulate fraction of aged rat CSMCs compared with adult counterparts. The present data show an attenuation of both the PKC pathway and RhoA pathway in aged rats compared with adult rats. Our results thus suggest that in aged CSMCs, decreased translocation of RhoA and PKC-{alpha}, resulting in decreased phosphorylation of MYPT and CPI-17, may lead to increased MLCP activity and thus to decreased MLC20 phosphorylation. We thus propose that the changes in the upstream cascade leading to sustained MLC20 phosphorylation and sustained smooth muscle contraction can affect colonic motility in aging.

RhoA and PKC-{alpha} have been reported to be translocated to caveolin-rich membrane microdomains (caveolae). Analysis of lipid raft membrane fractions showed ACh-induced sequestration of PKC-{alpha} in the same fractions that are enriched with Cav-1 in adult rat CSMCs. Analysis of similar fractions from aged CSMCs showed reduced ACh-induced sequestration of PKC-{alpha} in the fractions enriched with Cav-1. Our results suggest that the age-related reduction in ACh-induced sequestration was due to reduced expression of Cav-1 in aged CSMCs. Furthermore, we observed a reduction in the association of Cav-1 with RhoA and with PKC-{alpha}, which is also consistent with reduced expression of Cav-1. Our study indicates that ACh-induced membrane translocation of PKC-{alpha} and RhoA is reduced in aged rat CSMCs, which can be attributed to unavailability of caveolae for sequestration. The reduction in Cav-1 levels in the particulate fraction and depletion of Cav-1 in the lipid rafts of aged rat CSMCs confirm that caveolae formation is impaired. Caveolae play a major role in contractility and in the regulation of Ca2+ sensitivity of contraction in smooth muscle (3). CSMCs transfected with DN Cav-1 cDNA showed reduced translocation of PKC-{alpha} and RhoA with a concomitant reduction in the association of Cav-1 with PKC-{alpha} and RhoA in the particulate fraction (Fig. 5). A reduced availability of caveolae for docking of PKC-{alpha} and RhoA can explain the reduced translocation of PKC-{alpha} and RhoA. We also observed that CPI-17 and MYPT1, the downstream effectors of PKC-{alpha} and RhoA, are also affected in aged rat CSMCs. Significant and sustained reduction in the phosphorylation of CPI-17 and MYPT1 was exhibited by aged CSMCs, suggesting reduced activation of PKC-{alpha} and RhoA pathways. Thus, inhibition of MLCP activity by phosphorylation of CPI-17 and MYPT1 was also impaired, which seems to be accountable for the reduced MLC20 phosphorylation observed in aged rat CSMCs. Ectopic expressions of WT Cav-1 in aged CSMCs demonstrate increases in levels of ACh-induced phosphorylated MLC20 comparable with levels in control adult CSMCs. Simultaneously, aged CSMCs expressing endogenous WT Cav-1 exhibited restoration of ACh-induced PKC-{alpha} and RhoA translocation along with ACh-induced CPI-17 and MYPT1 phosphorylation, suggesting restoration of ACh-induced inhibition of MLCP activity. Taken together, these results suggest that in aged rat CSMCs, contractility of smooth muscle is affected due to impaired or reduced caveolae formation.

There is no clear information available on changes in the regulation of smooth muscle contraction during aging. It has been suggested that age-related differences in basal enzyme activities may represent either a cause or consequence of disrupted calcium homeostatic mechanisms with age (27). Aging has been suggested to impair the ability of contraction to stimulate mammalian target of rapamycin, p70s6k, and ERK1/2 phosphorylation in rat skeletal muscle (29). Interestingly, aging has been shown to affect signal transduction pathways, such as of the tyrosine kinase-Src kinase pathway, which may participate in cross talk with the PKC pathway (4). This article demonstrates an understanding of the molecular mechanisms responsible for the decline in aging and a putative reinstatement of physiological function by reinstating the levels of phosphorylated MLC20.

In summary, introduction of WT Cav-1 cDNA into aged rat CSMCs restored ACh-induced translocation of PKC-{alpha} and RhoA and ACh-induced phosphorylation of MYPT and CPI-17, consistent with restoration of ACh-induced MLC20 phosphorylation. Shortening of cell length was also restored in aged SMCs upon introduction of WT Cav-1 cDNA. The present investigation thus provides insights regarding the age-related decline of contraction and a possible solution to restore the physiological level of contraction. In view of our present investigation, we propose that the age-related decline in smooth muscle contraction can be restored by introduction of WT Cav-1 cDNA.


   GRANTS
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 ABSTRACT
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5-RO1-DK-042876.


   ACKNOWLEDGMENTS
 
We thank Bhushan M. Kanumuri for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., MSRB I, Rm. A520, Ann Arbor, MI 48109-0658 (e-mail: bitar{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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