Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

Sayak Bhattacharya; Sunila Mahavadi; Othman Al-Shboul; Senthilkumar Rajagopal; John R Grider; Karnam S Murthy

doi:10.1152/ajpcell.00334.2012

. 2013 Jun 19;305(3):C334–C347. doi: 10.1152/ajpcell.00334.2012

Differential regulation of muscarinic M₂ and M₃ receptor signaling in gastrointestinal smooth muscle by caveolin-1

Sayak Bhattacharya ¹, Sunila Mahavadi ¹, Othman Al-Shboul ¹, Senthilkumar Rajagopal ¹, John R Grider ¹, Karnam S Murthy ^1,^✉

PMCID: PMC3742848 PMID: 23784544

Abstract

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of G_q- and G_i-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M₂ and M₃ receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr⁶⁹⁶) and 20-kDa myosin light chain (MLC₂₀) phosphorylation (at Ser¹⁹) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC₂₀, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M₂, but not M₃, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other G_q-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by G_q-coupled receptors and contributes to enhanced smooth muscle function.

Keywords: caveolin-1, gastric emptying, internalization, muscarinic receptors, smooth muscle

caveolae are 50- to 100-nm omega-shaped cell surface membrane invaginations found in many cell types, including smooth muscle cells. Previous studies demonstrated that several signaling molecules populate caveolar microdomains, including different types of receptors, G proteins (heterotrimeric and monomeric), protein kinases, and receptor tyrosine kinases (40, 41). A crucial role of caveolae has been implicated in many processes, such as endocytosis, transcytosis, Ca²⁺ signaling, and several other signal transduction events (4, 6). The caveolar coat proteins, caveolins, serve as markers for identification of the caveolar domains and are critical for caveolar function. Caveolins are ∼22-kDa proteins consisting of 178 amino acids with multiple acetylation and phosphorylation sites (24). Three isoforms of caveolin proteins, caveolin-1, caveolin-2, and caveolin-3, have been identified. Caveolin-1, also known as vesicular integral membrane protein, was the first member of the caveolin gene family to be identified. It shows a varied expression pattern in many tissue types, including nonmuscle and smooth muscle tissues (44). Generally, caveolin-1 alone drives the formation of caveolae and forms the major structural component that preferentially interacts with different signaling moieties to regulate their function. Caveolin-2, on the other hand, is not essential for caveolae formation and colocalizes with caveolin-1 for its stable expression. Although caveolin-2 is not a requirement for formation of caveolae, coexpression of caveolin-1 and caveolin-2 leads to deeper and more abundant caveolae, indicating a modulatory role of caveolin-2 in formation of caveolae. Caveolin-3 is a muscle-specific isoform and shares high structural and functional similarities with caveolin-1. Unlike caveolin-2, caveolin-3 can form caveolar microdomains independent of caveolin-1 (39, 56).

Caveolins interact with their client proteins through a short stretch of amino acids known as the caveolin scaffolding domain, located at its cytosolic NH₂-terminal region spanning amino acids 82–101. Similarly, interaction of proteins with caveolins is guided by a defined sequence of amino acids called the caveolin-binding motif (CBM). The most well-defined CBM contains a short tandem protein sequence with aromatic amino acids inserted in a specific pattern (35, 36, 55).

Studies from several laboratories have proposed a crucial role of caveolae in organization and regulation of smooth muscle function by modulation of cellular processes such as contraction, growth, and proliferation. Caveolin-1-knockout (KO) mice are viable; however, they are prone to develop pathophysiological complications such as cardiomyopathy, enhanced endothelial nitric oxide synthase activity, and severe pulmonary dysfunction (55). Studies examining the involvement of caveolin-1 in the regulation of gastrointestinal motility are limited and often restricted to the intact tissue or organ. An obvious conclusion from these studies is the importance of caveolins in the regulation of contractile function. Undoubtedly, the next step in the process of understanding the mechanism by which caveolins regulate muscle contraction is to understand the regulation of signal transduction pathways activated by contractile neurotransmitters in the smooth muscle. Contractile agonists induce initial contraction via activation of PLC-β, resulting in 1,4,5-inositol triphosphate (IP₃)-dependent Ca²⁺ release and myosin light chain (MLC) kinase-mediated 20-kDa MLC (MLC₂₀) phosphorylation, and sustained contraction via activation of RhoA, Rho kinase-dependent phosphorylation of myosin phosphatase 1 (MYPT1), and PKC-dependent activation of CPI-17, resulting in MLC phosphatase inhibition and MLC₂₀ phosphorylation (27, 32). Using pharmacological, molecular, and genetic approaches and three G_q-coupled receptor agonists, we report here that disruption of caveolae or ablation of caveolin-1 attenuates G_q-coupled PLC-β and RhoA signaling, leading to inhibition of muscle contraction. The results are also significant, as they describe a role of caveolin-1 in gastric emptying and, thus, are relevant to our understanding of the mechanisms that regulate gastrointestinal motility in health and disease.

EXPERIMENTAL PROCEDURES

[¹²⁵I]-labeled cAMP [γ-³²]ATP, [myo-2-³H]inositol, and [³H]scopolamine were obtained from PerkinElmer Life Sciences (Boston, MA); carbachol (CCh), methoctramine, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), forskolin, and methyl β-cyclodextrin (MβCD) from Sigma-Aldrich (St. Louis, MO); Y-27632 and U-73122 from Calbiochem (La Jolla, CA); polyclonal antibodies to caveolin-1, Rho kinase, zipper-interacting protein (ZIP) kinase, phosphorylated MYPT1, and phosphorylated MLC₂₀ from Santa Cruz Biotechnology (Santa Cruz, CA); muscarinic M₂ (WR-3721) and M₃ (WR-3741) receptor antibodies from Research and Diagnostic Antibodies (Las Vegas, NV); pSIREN-DNR-DsRed vector from Clontech Laboratories (Mountain View, CA); Lipofectamine 2000 transfection reagent, SuperScript II reverse transcriptase kit, and DH5α competent cells from Invitrogen (Carlsbad, CA); and caveolin-1 small interfering RNA (siRNA) from BD Biosciences (San Jose, CA).

Isolation of gastric muscle cells and primary culture of muscle cells.

Smooth muscle cells were isolated from the circular muscle layer of rabbit stomach by sequential enzymatic digestion, filtration, and centrifugation, as described previously (29–33). The partially digested tissue was washed twice with collagenase-free smooth muscle buffer, and the smooth muscle cells were allowed to disperse spontaneously for ∼30 min in enzyme-free medium. For some experiments, the cells were washed and cultured in DMEM containing 10% fetal bovine serum and antibiotics until they attained confluence and used after the first passage in various experiments. All animal procedures were approved and conducted in accordance with the Institutional Animal Care and Use Committee of the Virginia Commonwealth University.

Transfection of caveolin-1 siRNA.

The RNAi-Ready pSIREN-DNR-DsRed-Express vector (BD Biosciences, Clontech) encoding caveolin-1 siRNA was inserted between BamH1 and EcoR1 restriction sites and transfected into cultured gastric smooth muscle cells with Lipofectamine 2000 reagent according to the manufacturer's recommendation (43). To check the specificity of the siRNA, empty vector without the siRNA sequence was used as control. Successful knockdown of caveolin-1 protein was verified by Western blotting and immunofluorescence microscopy.

Assay for phosphatidylinositol hydrolysis.

The total pool of inositol phosphate from muscle cells was measured by anion-exchange chromatography, as described previously (19, 49). Gastric smooth muscle cells were labeled with [myo-2-³H]inositol (0.5 μCi/ml) in inositol-free medium. The cells were washed with PBS and treated with CCh (0.1 μM), methoctramine (0.1 μM), and the PLC inhibitor U-73122 (1 μM). The reaction was terminated by addition of 940 μl of chloroform-methanol-HCl (50:100:1, by volume). After extraction with 340 μl of chloroform and 340 μl of H₂O, the aqueous phase was applied to Dowex AG-1 columns, and the eluates were collected in scintillation vials and counted in the gel phase after addition of 10 ml of scintillation fluid.

Assay for cAMP.

Radioimmunoassay using [¹²⁵I]-labeled cAMP was performed to measure the level of cAMP formation (28, 29, 34). Smooth muscle cells were divided into aliquots (0.5 ml) and treated with CCh (0.1 μM), 4-DAMP (0.1 μM), and forskolin (10 μM), and the reaction was terminated with 6% trichloroacetic acid (vol/vol). The mixture was centrifuged, and the supernatant was extracted three times with diethyl ether and lyophilized. The samples were reconstituted in 50 μl of 50 mM sodium acetate (pH 6.2) and acetylated with triethylamine-acetic anhydride (2:1 vol/vol) for 30 min. cAMP was measured in triplicates using 100-μl aliquots, and the results were analyzed from standard curves using GraphPad Prism software.

Assay for Rho kinase and ZIP kinase.

Rho kinase and ZIP kinase activities were measured by immunokinase assay, as previously described (18, 35). After treatment with test reagents [CCh (0.1 μM), methoctramine (0.1 μM), and Y-27632 (1 μM)], muscle cells were permeabilized and Rho kinase and ZIP kinase were immunoprecipitated with specific antibodies. The immunokinase assay was initiated by addition of 10 μCi of [γ-³²P]ATP (3,000 Ci/mmol) and 20 μM ATP for 10 min at 37°C along with 1 μg of myelin basic protein as substrate, absorbed onto phosphocellulose disks, and washed repeatedly with 75 mM phosphoric acid to remove nonspecific radioactivity. The amount of ³²P incorporation on the disks was measured by liquid scintillation.

Measurement of MLC₂₀ and MYPT1 phosphorylation by in-cell Western assay.

Phosphorylation of MLC₂₀ (Ser¹⁹) and MYPT1 (Thr⁶⁹⁶) was determined by in-cell Western assay using phosphospecific antibodies, as described previously (20). Cultured cells grown in a 96-well plate were treated with MβCD, CCh, and methoctramine and fixed at room temperature. Cells were permeabilized and incubated with phosphospecific primary antibodies and then with secondary antibodies containing Sapphire and DRAQ5 dyes for background detection. The plate was washed five times with Tween washing solution and scanned using the Odyssey imaging system at 700 and 800 nm.

[³H]scopolamine binding to smooth muscle cells.

Binding of [³H]scopolamine to dispersed gastric muscle cells was performed as described previously (3, 18). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin. Triplicate 0.5-ml aliquots (10⁶ cells/ml) were incubated for 20 min with 1 nM [³H]scopolamine alone (total binding) or with 10 μM CCh (nonspecific binding). After incubation, bound and free ligand were separated by rapid filtration under reduced pressure through 5-μm polycarbonate Nucleopore filters. The filters were washed three to four times with 5 ml of ice-cold HEPES medium containing 0.2% bovine serum albumin. Nonspecific binding (26 ± 5%) was calculated as the amount of radioactivity in the presence of 10 μM CCh and subtracted from the total binding to obtain specific binding.

Isolation of caveolar and noncaveolar fractions and Western blotting.

Smooth muscle cells were fractionated using detergent-free methods, as described previously (31, 37). Dispersed muscle cells were washed three times in ice-cold PBS and suspended in 2 ml of 500 mM sodium carbonate (pH 11.0) containing 0.2 mM phenylmethylsulfonyl fluoride and 20 μg/ml leupeptin. The suspension was homogenized with a Polytron tissue grinder (three 10-s bursts) and by sonication (three 20-s bursts), and the homogenate was adjusted to 45% sucrose in MBS (25 mM MES, pH 6.5, and 0.15 mM NaCl), placed in an ultracentrifuge tube, and overlaid with two 4-ml layers of 35% and 5% sucrose in MBS containing 250 mM sodium carbonate. After preparation of sucrose density layers, the samples were centrifuged for 16–20 h at 39,000 rpm at 4°C in a rotor (model SW41Ti, Beckman). After centrifugation, twelve 1-ml fractions were collected sequentially from the top and designated fractions 1–12. Fractions were analyzed by SDS-PAGE (15% acrylamide gels); after transfer to nitrocellulose membranes, Western blot analysis was performed with antibodies to caveolin-1.

Measurement of muscle contraction.

Cell aliquots of 0.4 ml (10⁴ muscle cells/ml) in smooth muscle buffer (pH 7.4) were treated with CCh (0.1 μM) and methoctramine (0.1 μM) for different periods of time, and the reactions were terminated with 1% acrolein. The mean length of 50 smooth muscle cells was measured by scanning micrometry, as described previously (28–33). The length of CCh-treated muscle cells was compared with the length of untreated cells, and contraction is expressed as percent decrease in mean cell length from control.

Gastric emptying.

Wild-type (WT) and caveolin-1-KO mice (Jackson Laboratories) were used for the gastric emptying experiments. The animals were deprived of food and water for 24 h prior to the experiment. After 24 h of food and water deprivation, the body weights were measured and a calculated amount of food and water was supplied. After 3 h of feeding, food and water were immediately removed and the animals were allowed to starve for 4 h. After 4 h of starvation, the body weights were measured and the mice were euthanized. The stomach was weighed with and without the contents to measure the amount of food ingested by each animal (59). Food retained in the stomach after 4 h was measured and expressed as milligrams per gram of body weight

Statistical analysis.

Values are means ± SE of n experiments. Probability was determined by an unpaired, two-tailed Student's t-test for comparison of two samples and by one-way ANOVA with Dunnett's post hoc test for comparison of more than two samples with a control using GraphPad InStat (version 3.06 for Windows) software (San Diego, CA). Each experiment was done on cells obtained from different animals. P < 0.05 was considered significant.

RESULTS

Expression of M₂ and M₃ receptors and contraction in gastric smooth muscle.

Specific primers for muscarinic M₁, M₂, M₃, and M₄ receptors were designed on the basis of the conserved sequences in human, rabbit, rat, and mouse cDNAs and used for identification of muscarinic receptor mRNA expression. Muscarinic M₂ and M₃, but not M₁ or M₄, receptors were detected by RT-PCR in RNA extracts from cultures of gastric smooth muscle cells in the first passage (Fig. 1A). When experiments were performed in the absence of reverse transcriptase, there was no amplification of M₂ or M₃ receptors. Use of M₁, M₂, M₃, and M₄ receptor cDNA as positive controls resulted in an amplification of mRNA with receptor-specific primers (Fig. 1A). As shown previously, the use of confluent cultures of smooth muscle in the first passage ensured the absence of neural, endothelial, or interstitial cells of Cajal contaminants, and the presence of PCR products in cultured muscle cells demonstrates the specific expression of M₂ and M₃ mRNA in smooth muscle cells (29, 32). Western blot analysis using antibodies for M₂ (1:1,000 dilution) or M₃ (1:2,000 dilution) receptors demonstrated the expression of M₂ and M₃ receptors of predicted size (52 and 66 kDa, respectively) in the homogenates of isolated smooth muscle cells (Fig. 1B). The results are consistent with the selective expression of M₂ and M₃ receptors in vascular and other visceral smooth muscle (3, 15, 25).

Fig. 1. — Selective expression of muscarinic M₂ and M₃ receptors in gastric smooth muscle cells. A: mRNA expression. Total RNA was isolated from cultured gastric smooth muscle cells (1st passage), and reverse-transcribed cDNA was amplified with specific primers for muscarinic receptors M₁, M₂, M₃, and M₄. Experiments were done in the presence or absence of reverse transcriptase (+RT and −RT). PCR products corresponding to M₂ and M₃ receptors were obtained with the specific primers and further confirmed with control cDNAs. Results show representative PCR products. B: protein expression. Freshly dispersed muscle cells were homogenized in lysis buffer, lysates containing equal amounts of total proteins were separated on SDS-PAGE, expression of muscarinic receptors was analyzed using selective antibody to M₂ (1:1,000 dilution) and M₃ (1:1,000 dilution) receptors, and the band corresponding to 52 and 66 kDa, respectively, was detected by chemiluminescence. Representative Western blot shows the presence of muscarinic M₂ and M₃ receptors. C: radioligand binding. Freshly dispersed gastric smooth muscle cells were incubated with [³H]scopolamine for 15 min at room temperature, and the amount of radioactive [³H]scopolamine bound to cells was measured by liquid scintillation. Cells were incubated in the presence of 0.1 μM 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) to measure binding to M₂ receptors or in the presence of 0.1 μM methoctramine (methoc) to measure binding to M₃ receptors. Values are means ± SE; n = 4–5 experiments. Significant inhibition compared with control: *P < 0.01, **P < 0.001. D: muscle contraction. Freshly dispersed gastric smooth muscle cells were treated with 0.1 μM carbachol (CCh) for 30 s in the presence or absence of 0.1 μM methoctramine or 0.1 μM 4-DAMP. Mean length of 50 muscle cells was measured by scanning micrometry and compared with the length of untreated muscle cells. Values (means ± SE) are expressed as percent decrease in cell length from control length (112 ± 4 μm); n = 6 experiments. **Significant inhibition compared with control (P < 0.001).

Selective expression of M₂ and M₃ receptors in gastric smooth muscle cells was further confirmed by radioligand-binding studies using [³H]scopolamine as a muscarinic agonist. Total binding was 2,110 ± 302 cpm/mg protein, and nonspecific binding was 22 ± 4% of total binding (456 ± 65 cpm/mg protein). Incubation of cells with the selective M₂ receptor antagonist methoctramine inhibited specific binding by 69 ± 3%, whereas incubation with the selective M₃ receptor antagonist 4-DAMP inhibited specific binding by 32 ± 4%. Incubation of cells with both methoctramine and 4-DAMP abolished the specific binding of [³H]scopolamine (Fig. 1C). The results suggest that the gastric smooth muscle cells express only M₂ and M₃ receptors and that the expression level of M₂ receptors is greater than that of M₃ receptors. Although M₂ receptors were more abundant than M₃ receptors, contraction is mainly mediated by M₃ receptors. As shown previously in intestinal muscle, treatment of dispersed muscle cells with CCh for 30 s caused muscle contraction (28 ± 3% decrease in muscle length from basal muscle length of 114 ± 4 μm) that was inhibited by 4-DAMP (4 ± 2% decrease in cell length), but not by methoctramine (25 ± 3% decrease in cell length; Fig. 1D). Physiologically, in vivo activation of the M₂ receptor augments contractions mediated by M₃ receptors. The contractile role of M₂ receptors depends on the contraction by M₃ receptors, and blockade of M₃ receptors eliminates the M₂ response. The effect of M₂ receptors is mediated indirectly via inhibition of relaxant signal (i.e., via inhibition of cAMP). This is consistent with the concept of a conditional role of the M₂ receptors in the smooth muscle (10–12).

Signaling by M₂ and M₃ receptors in gastric smooth muscle.

Incubation of freshly dispersed smooth muscle cells with CCh (0.1 μM) in the presence of the M₂ receptor antagonist methoctramine for 60 s caused a significant increase in phosphatidylinositol (PI) hydrolysis (3,075 ± 532 cpm/mg protein compared with basal PI hydrolysis of 400 ± 83 cpm/mg protein; Fig. 2A). The increase in PI hydrolysis was significantly inhibited in the presence of the selective M₃ receptor antagonist 4-DAMP (84 ± 5% inhibition, P < 0.001) or the PLC inhibitor U-73122 (1 μM, 87 ± 6% inhibition, P < 0.001; Fig. 2A). Treatment of cells with CCh (0.1 μM) for 10 min caused a significant stimulation of Rho kinase activity (33,580 ± 2,410 cpm/mg protein compared with basal activity of 9,006 ± 1,680 cpm/mg protein) and ZIP kinase activity (21,330 ± 2,760 cpm/mg protein compared with basal activity of 4,446 ± 1,210 cpm/mg protein; Fig. 2, B and C). The increase in Rho kinase and ZIP kinase activity was significantly inhibited in the presence of 4-DAMP (85 ± 7% and 80 ± 5% inhibition, respectively, P < 0.001) or the Rho kinase inhibitor Y-27632 (1 μM, 88 ± 5% and 80 ± 6% inhibition, respectively, P < 0.001; Fig. 2, B and C). Methoctramine had no significant effect on CCh-stimulated Rho kinase and ZIP kinase activity (Fig. 2, B and C). These results suggest that CCh-stimulated PI hydrolysis and Rho kinase and ZIP kinase activity were mediated via M₃ receptors and that ZIP kinase activity was dependent on Rho kinase activity.

Fig. 2. — Signaling by M₃ and M₂ receptors in gastric muscle. A: phosphatidylinositol (PI) hydrolysis. Freshly dispersed muscle cells labeled with [*myo*-³H]inositol were incubated with 0.1 μM CCh + 0.1 μM methoctramine for 60 s. In some experiments, cells were pretreated with 0.1 μM 4-DAMP or the selective PLC inhibitor U-73122 (1 μM) for 10 min. Total [³H]inositol phosphates were separated by ion-exchange chromatography, and radioactivity was counted by liquid scintillation. Values (means ± SE) are expressed as total [³H]inositol phosphate formation; n = 4 experiments. **Significant increase in response to CCh (P < 0.001). B and C: Rho kinase and zipper-interacting protein (ZIP) kinase activity. Freshly dispersed gastric muscle cells were incubated with 0.1 μM CCh for 10 min in the presence or absence of 0.1 μM 4-DAMP or 0.1 μM methoctramine. Rho kinase and ZIP kinase activity was measured using [γ-³²P]ATP by immunokinase assay in the presence or absence of the selective Rho kinase inhibitor Y-27632 (1 μM). Values are means ± SE; n = 4–5 experiments. **Significant increase in response to CCh (P < 0.001). D: cAMP assay. Muscle cells were treated with forskolin (Fsk; 10 μM) in the presence or absence of 0.1 μM CCh for 60 s, and cAMP was measured by radioimmunoassay. In some experiments, cells were pretreated with 4-DAMP or methoctramine for 10 min or pertussis toxin (PTx; 400 ng/ml) for 60 min and then treated with 10 μM forskolin and CCh for 60 s. Results were computed from a standard curve using Prism software. Values are means ± SE; n = 4 experiments. **Significant inhibition of forskolin-stimulated cAMP formation (P < 0.001).

Previous studies in vascular and visceral smooth muscle showed that muscarinic M₂ receptors are coupled to G_i protein and inhibition of adenylyl cyclase (AC) activity. The increase in cAMP formation by forskolin (24.0 ± 2.3 pmol/mg protein compared with basal levels of 2.5 ± 0.5 pmol/mg protein) was significantly inhibited (8.0 ± 1.7 pmol/mg protein, 74 ± 3% inhibition, P < 0.001) in the presence of CCh (0.1 μM), and the inhibition was significantly reversed by pretreatment of cells with methoctramine (9 ± 4% inhibition) or pertussis toxin (4 ± 2% inhibition), but not 4-DAMP (67 ± 7% inhibition; Fig. 2D).

Effect of caveolae/caveolin-1 on G_q-coupled receptor signaling.

The role of caveolae and its main structural protein caveolin-1 in the regulation of G_q-coupled M₃ receptor signaling was investigated using three complementary approaches: MβCD, caveolin-1-specific siRNA, and caveolin-1-KO mice. Responses to CCh in dispersed muscle cells were measured after cholesterol depletion with MβCD (1 mM) and after replenishment of cholesterol (5 mM) for 1 h to restore membrane caveolae (2). Control responses to CCh were measured in the absence of MβCD or cholesterol treatment.

Inhibition of PI hydrolysis and Rho kinase and ZIP kinase activity.

CCh-induced PI hydrolysis was significantly inhibited by pretreatment with MβCD in dispersed muscle cells (42 ± 3% inhibition, P < 0.01 compared with control DMSO-treated cells) or transfection of caveolin-1 siRNA in cultured muscle cells (46 ± 4% inhibition compared with cells transfected with control siRNA; Fig. 3, A and B). Similarly, CCh-induced PI hydrolysis was significantly inhibited (38 ± 7% inhibition, P < 0.01) in gastric muscle cells from caveolin-1-KO mice compared with WT mice (Fig. 3C).

Fig. 3. — Inhibition of CCh-stimulated PI hydrolysis by caveolin-1 (Cav-1). Freshly dispersed gastric smooth muscle cells treated with DMSO or 1 mM methyl β-cyclodextrin (MβCD; A), cultured muscle cells transfected with control vector or vector containing caveolin-1 small interfering RNA (siRNA; B), or muscle cells isolated from wild-type (WT) or caveolin-1-knockout (KO, Cav-1^−/−) mice (C) were labeled with [*myo*-³H]inositol. Cells were treated with 0.1 μM CCh + 0.1 μM methoctramine for 60 s, and PI hydrolysis was measured as formation of water-soluble [³H]inositol phosphates by ion-exchange chromatography and radioactivity was counted by liquid scintillation. *Inset*: Western blot showing siRNA-mediated suppression of caveolin-1 in cultured muscle cells. ctrl, Control. Values are means ± SE; n = 4 experiments. **Significant inhibition compared with control CCh response (P < 0.01).

The CCh-induced increase in Rho kinase and ZIP kinase activity was also significantly inhibited by MβCD in dispersed muscle cells (54 ± 6% and 48 ± 6% inhibition, respectively, P < 0.01) or transfection of caveolin-1 siRNA in cultured muscle cells (31 ± 4% and 59 ± 6% inhibition, respectively, P < 0.01; Fig. 4, A and B, and Fig. 5, A and B). Similar inhibition of CCh-stimulated Rho kinase and ZIP kinase activity (45 ± 8% and 54 ± 6% inhibition, respectively, P < 0.01) was obtained in muscle cells from caveolin-1-KO mice compared with WT mice (Figs. 4C and 5C).

Fig. 4. — Inhibition of CCh-stimulated Rho kinase activity by caveolin-1. Freshly dispersed gastric smooth muscle cells treated with DMSO or 1 mM MβCD (A), cultured muscle cells transfected with control vector or vector containing caveolin-1 siRNA (B), or muscle cells isolated from WT or caveolin-1-KO mice (C) were treated with 0.1 μM CCh for 10 min, and Rho kinase activity was measured by immunokinase assay. Suppression of caveolin-1 in cultured muscle cells was verified by Western blot analysis. Values are means ± SE; n = 4–5 experiments. **Significant inhibition compared with control CCh response (P < 0.01).

Fig. 5. — Inhibition of CCh-stimulated ZIP kinase activity by caveolin-1. Freshly dispersed gastric smooth muscle cells treated with DMSO or 1 mM MβCD (A), cultured muscle cells transfected with control vector or vector containing caveolin-1 siRNA (B), or muscle cells isolated from WT or caveolin-1-KO mice (C) were treated with 0.1 μM CCh for 10 min, and ZIP kinase activity was measured by immunokinase assay. Suppression of caveolin-1 in cultured muscle cells was verified by Western blot analysis. Values are means ± SE; n = 4–5 experiments. **Significant inhibition compared with control CCh response (P < 0.01).

Replenishment of cholesterol to restore caveolae reversed the inhibitory effect of MβCD on CCh-induced PI hydrolysis (13 ± 3% inhibition vs. 55 ± 6% inhibition with MβCD treatment) and Rho kinase (11 ± 2% inhibition vs. 62 ± 8% inhibition with MβCD treatment) and ZIP kinase (6 ± 2% inhibition vs. 50 ± 7% inhibition with MβCD treatment) activity (Fig. 6).

Fig. 6. — Reversal of MβCD-induced inhibition of PI hydrolysis and Rho kinase and ZIP kinase activity in response to CCh by cholesterol. Freshly dispersed gastric smooth muscle cells were treated with DMSO, 1 mM MβCD, or MβCD followed by 5 mM cholesterol. Cells were treated for 60 s with 0.1 μM CCh + 0.1 μM methoctramine to measure PI hydrolysis or for 10 min with 0.1 μM CCh to measure Rho kinase and ZIP kinase activity. PI hydrolysis was measured as formation of water-soluble [³H]inositol phosphates by ion-exchange chromatography, and radioactivity was counted by liquid scintillation. Rho kinase and ZIP kinase activity was measured by immunokinase assay. Values are means ± SE; n = 4 experiments. **Significant inhibition compared with control CCh response (P < 0.01).

Stimulation of PI hydrolysis in response to G_q-coupled histamine and substance P (SP) receptor activation was measured to examine the effect of caveolae/caveolin-1 on other G_q-coupled receptors (26, 27). Histamine (1 μM)- and SP (1 μM)-induced PI hydrolysis was significantly inhibited by pretreatment with MβCD in dispersed muscle cells (47 ± 4% and 61 ± 8%, respectively, P < 0.01 compared with control DMSO-treated cells) and transfection of caveolin-1 siRNA in cultured muscle cells (51 ± 5% and 47 ± 4%, respectively, P < 0.01 compared with cells transfected with control siRNA; Fig. 7). These results suggest that regulation of PI hydrolysis by caveolin-1 is not limited to M₃ receptors but general to other G_q-coupled receptors, such as histamine (H₁) and neurokinin receptors.

Fig. 7. — Inhibition of histamine- and substance P (SP)-stimulated PI hydrolysis by caveolin-1. Freshly dispersed gastric smooth muscle cells were treated with DMSO or 1 mM MβCD (A), or cultured muscle cells were transfected with control vector or vector containing caveolin-1 siRNA (B). Cells were treated with 1 μM SP or 1 μM histamine for 60 s, and PI hydrolysis was measured as formation of water-soluble [³H]inositol phosphates by ion-exchange chromatography and radioactivity was counted by liquid scintillation. Values are means ± SE; n = 4 experiments. **Significant inhibition compared with control response to histamine or SP (P < 0.01).

Inhibition of MYPT1 and MLC₂₀ phosphorylation and muscle contraction.

The effect of caveolae and caveolin-1 on phosphorylation of MYPT1 (at Thr⁶⁹⁶), the regulatory subunit of MLC phosphatase, and MLC₂₀ (at Ser¹⁹), the biochemical correlate of smooth muscle contraction, was examined in dispersed muscle cells using MβCD. Treatment of dispersed muscle cells with CCh significantly increased phosphorylation of MYPT1 and MLC₂₀, which was significantly attenuated by MβCD in dispersed muscle cells (46 ± 9% and 58 ± 10%, respectively, P < 0.01; Fig. 8A). In contrast, increase in MYPT1 and MLC₂₀ phosphorylation in response to KCl (20 mM) was not significantly affected by MβCD in dispersed muscle cells: 178 ± 23% vs. 162 ± 26% increase in MYPT1 phosphorylation and 268 ± 35% vs. 253 ± 31% increase in MLC₂₀ phosphorylation.

Fig. 8. — Inhibition of CCh-stimulated phosphorylation of 20-kDa myosin light chain (MLC₂₀) and myosin phosphatase 1 (MYPT1) and muscle contraction by caveolin-1. A: freshly dispersed control (DMSO-treated) or MβCD (1 mM)-treated smooth muscle cells were treated with 0.1 μM CCh + 0.1 μM methoctramine for 30 s (for MLC₂₀ phosphorylation) or 10 min (for MYPT1 phosphorylation), and phosphorylation was measured by in-cell Western assay using phosphospecific antibody to MLC₂₀ (Ser¹⁹) or MYPT1 (Thr⁶⁹⁶). Values are means ± SE; n = 3 experiments. *Significant inhibition compared with response to CCh + methoctramine in the absence of MβCD (P < 0.05). B: dispersed control or MβCD-treated smooth muscle cells or muscle cells isolated from WT or caveolin-1-KO mice were treated with 0.1 μM CCh for 30 s (initial contraction) or 10 min (sustained contraction). Mean length of 50 muscle cells was measured by scanning micrometry, and results are expressed as percent decrease in cell length (control cell lengths = 115 ± 4 and 111 ± 5 μm for DMSO- and MβCD-treated cells, respectively, and 92 ± 5 and 98 ± 4 μm for WT and caveolin-1-KO mice, respectively). Values are means ± SE; n = 5–6 experiments. **Significant inhibition compared with response to CCh in the absence of MβCD or response in WT mice (P < 0.01).

Previous studies showed that CCh-induced initial contraction was dependent on PI hydrolysis, whereas sustained contraction was dependent on the RhoA/Rho kinase pathway (32). CCh-induced initial (29 ± 3% decrease in cell length) and sustained (22 ± 3% decrease in cell length) contraction were significantly inhibited by MβCD (51 ± 4% and 49 ± 3% inhibition, respectively, P < 0.01) compared with control cells (Fig. 8B). Similar inhibition of CCh-induced initial (44 ± 4% inhibition) and sustained (39 ± 3% inhibition) contraction was obtained in cells from caveolin-1-KO mice compared with WT mice (Fig. 8B). In contrast, contraction in response to 20 mM KCl (28 ± 4% decrease in cell length) was not significantly affected by MβCD treatment (27 ± 3% decrease in cell length). Similarly, contraction in response to KCl in WT mice (30 ± 4% decrease in cell length) was not significantly different from that in caveolin-1-KO mice (26 ± 3% decrease in cell length).

Regulation of gastric emptying by caveolin-1.

The physiological significance of the agonist-induced decrease in muscle contraction in caveolin-1-KO mice was also examined. As shown in Fig. 9, caveolin-1-KO mice exhibited a significant delay in gastric empting compared with WT mice. There was no significant difference in the body weight or the amount of food consumed in WT and caveolin-1-KO mice (Table 1). The decrease in gastric emptying correlates with the decrease in CCh-induced PI hydrolysis, Rho and ZIP kinase activity, and contraction by disruption of caveolae in vitro or lack of caveolin-1 in vivo.

Table 1.

Body weight, food intake, and gastric emptying parameters in control and caveolin-1-KO mice

	WT	Caveolin-1-KO
Body wt, g	28.68 ± 0.6	25.70 ± 1.4
Food intake, g	2.21 ± 0.05	2.01 ± 0.06
Stomach wt, g
With contents	0.243 ± 0.2	0.312 ± 0.01
Without contents	0.172 ± 0.01	0.198 ± 0.01
Food retained in the stomach
g	0.08 ± 0.01	0.114 ± 0.003
mg/g body wt	2.7 ± 0.5	4.5 ± 0.3^∗

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Values are means ± SE. WT, wild-type; KO, knockout.

^∗

P < 0.05 vs. WT.

Lack of effect of caveolin-1 on M₂-mediated inhibition of cAMP.

Forskolin-stimulated increase in cAMP was not affected by MβCD in dispersed muscle cells (21.2 ± 2.8 and 20.4 ± 2.3 pmol/mg protein in MβCD-treated and control cells, respectively) or by transfection of caveolin-1 siRNA in cultured muscle cells (20.5 ± 2.1 and 19.4 ± 2.8 pmol/mg in caveolin-1 siRNA-transfected and control cells, respectively; Fig. 10, A and B). Forskolin-induced cAMP formation was also similar in muscle cells from WT and caveolin-1-KO mice (20.9 ± 3.2 and 18.9 ± 1.6 pmol/mg protein, respectively; Fig. 10C). In contrast to the effect on M₃ receptor-mediated signaling, inhibition of forskolin-stimulated cAMP formation by CCh was not affected by MβCD (68 ± 5% and 71 ± 6% in control and MβCD-treated cells, respectively), caveolin-1 siRNA (70 ± 6% and 67 ± 6% in control and siRNA-transfected cells, respectively), or caveolin-1 KO (73 ± 4% and 72 ± 9% in WT and caveolin-1-KO mice, respectively; Fig. 10).

Fig. 10. — Lack of effect on cAMP formation by caveolin-1. Freshly dispersed gastric smooth muscle cells treated with DMSO or 1 mM MβCD (A), cultured muscle cells transfected with control vector or vector containing caveolin-1 siRNA (B), or muscle cells isolated from WT or caveolin-1-KO mice (C) were treated with 10 μM forskolin in the presence or absence of 0.1 μM CCh for 1 min, and cAMP formation was measured by radioimmunoassay. Suppression of caveolin-1 in cultured muscle cells was verified by Western blot analysis. Values are means ± SE; n = 4–5 experiments. **Significant inhibition of forskolin-stimulated cAMP formation (P < 0.01).

Effect of caveolae/caveolin-1 on M₂ and M₃ receptor internalization.

Differential regulation of M₂ and M₃ receptor signaling raised the possibility that M₃, but not M₂, receptors are colocalized with caveolin-1. To examine this notion, colocalization of M₂ and M₃ receptors with caveolin-1 was examined by Western blot analysis of muscle homogenates fractionated by sucrose density gradient centrifugation. As shown previously, caveolin-1 was selectively present in low-density fractions 4 and 5 (31). In the unstimulated state, M₂ and M₃ receptors are present in high-density gradient fractions 9–12 and absent from caveolar fractions 4 and 5. However, treatment of cells with CCh for 10 min caused translocation of M₂, but not M₃, receptors to caveolar fractions (Fig. 11A). Treatment of cells with MβCD blocked translocation of M₂ receptors to caveolar fractions (Fig. 11A). We tested the hypothesis that M₂ receptors are selectively internalized via a caveolae-dependent pathway. Internalization was measured as decrease in radioligand ([³H]scopolamine) binding to surface receptors after pretreatment of cells with CCh for 20 min. Binding to M₂ receptors was examined in the presence of the M₃ receptor antagonist 4-DAMP (0.1 μM), whereas binding to M₃ receptors was examined in the presence of the M₂ receptor antagonist methoctramine (0.1 μM). Binding to M₂ or M₃ receptors was expressed as percentage of total (M₂ and M₃ receptor) specific binding.

Fig. 11. — Selective inhibition of M₂ receptor internalization by caveolin-1. A: translocation of M₂ receptors to caveolar fractions. Dispersed muscle cells of control and MβCD-treated groups were treated with CCh for 15 min, and cell homogenates were subjected to sucrose density gradient centrifugation. Fractions were analyzed by SDS-PAGE and probed with caveolin-1-specific antibody. Results are representative of 3 different experiments. B and C: receptor internalization. Dispersed control or MβCD-treated gastric smooth muscle cells or cultured control or caveolin-1 siRNA-transfected (suppression of caveolin-1 was verified by Western blot analysis) muscle cells were incubated with 0.1 μM CCh for 20 min in the presence of 0.1 μM 4-DAMP to induce M₂ receptor internalization or 0.1 μM methoctramine to induce M₃ receptor internalization. Cells are washed and incubated with [³H]scopolamine for 15 min in the presence of 4-DAMP to measure binding to M₂ receptors or methoctramine to measure binding to M₃ receptors. Decrease in specific binding after CCh pretreatment represents receptor internalization. Values (means ± SE) are expressed as percentage of total (M₂ + M₃) specific binding; n = 3 experiments. **Significant decrease in binding compared with binding before CCh pretreatment (P < 0.001).

Inhibition of M₂ receptor internalization by caveolin-1.

Treatment of freshly dispersed cells with MβCD or transfection of caveolin-1 siRNA in cultured muscle cells had no effect on M₂ receptor binding (66 ± 3% to 68 ± 5% of total muscarinic binding). Pretreatment of control cells with CCh attenuated binding to M₂ receptors to 21 ± 4% of total binding (69 ± 5% compared with control) in freshly dispersed cells and 26 ± 5% of total binding (61 ± 6% compared with control) in cultured muscle cells, reflecting internalization of M₂ receptors (Fig. 11B). In contrast, when cells were pretreated with MβCD or transfected with caveolin-1 siRNA, CCh treatment had no significant effect on M₂ receptor binding (62 ± 7% to 64 ± 6% of total specific binding; Fig. 11B). These results suggest that M₂ receptor internalization was mediated via a caveolae-dependent pathway in gastric smooth muscle. An essential step in the caveolae-dependent internalization process involves activation of Src kinase and phosphorylation of caveolar coat proteins at Tyr¹⁴ (50). In cells pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, an Src family kinase inhibitor), CCh treatment had no significant effect on binding to the M₂ receptor (59 ± 7% of total specific binding).

Lack of effect of caveolin-1 on M₃ receptor internalization.

Treatment of freshly dispersed cells with MβCD or transfection of caveolin-1 siRNA in cultured muscle cells had no effect on M₃ receptor binding (28 ± 4% to 33 ± 4% of total muscarinic binding). Pretreatment of control cells with CCh attenuated binding to M₃ receptors to 10 ± 2% of total binding (66 ± 4% compared with control) in freshly dispersed cells and 10 ± 3% of total binding (69 ± 5% attenuation compared with control) in cultured muscle cells, reflecting internalization of M₃ receptors (Fig. 11C). Pretreatment with MβCD or transfection with caveolin-1 siRNA had no effect on CCh-induced internalization of M₃ receptors (11 ± 2% to 11 ± 4% of total binding, reflecting 61 ± 3% to 64 ± 4% attenuation; Fig. 11C). Treatment of cells with PP2 attenuated M₃ receptor internalization minimally, but significantly (16 ± 4% of total binding, reflecting 45 ± 5% attenuation), suggesting that M₃ receptor internalization was partly mediated via the Src kinase-dependent pathway in gastric smooth muscle.

DISCUSSION

Excitation-contraction coupling in smooth muscle is regulated by cell-extrinsic (extracellular matrix) and cell-intrinsic proteins. Caveolae contain the protein caveolin, and all three gene products of the caveolin proteins (caveolin-1, caveolin-2, and caveolin-3) are expressed in smooth muscle. However, caveolin-1, which drives the formation of caveolae, is more abundant than caveolin-3. Our studies indicate that the effect of caveolin-1 appears to be receptor- and signaling-specific. Caveolin-1 facilitates G_q-coupled receptor signaling activated by CCh, histamine, and SP, but not G_i-coupled receptor signaling by CCh. In contrast, caveolin-1 facilitates M₂, but not M₃, receptor internalization.

The role of caveolin-1 was examined using three complementary approaches: 1) MβCD to deplete cholesterol in freshly dispersed muscle cells, 2) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and 3) caveolin-1-KO mice. A novel aspect of these studies is the similarity of results obtained with all three approaches. Depletion of cholesterol by MβCD results in disruption of caveolae and affects plasma membrane signaling pathways. The exact mechanism by which cholesterol removal affects signaling pathways is not clear; however, cholesterol replenishment restores the plasma membrane signaling (41). The efficiency and specificity of MβCD treatment were confirmed in vascular and airway smooth muscle by the significant decrease in caveolae and lack of effect of MβCD on other cellular structures (16) and functions, including intact response of smooth muscle to KCl (8). To more precisely understand the role of caveolin-1, the most abundant isoform of caveolin in smooth muscle, we used caveolin-1 siRNA to suppress caveolin-1 expression and caveolin-1-KO mice. Caveolin-1-KO mice have been used in previous studies to examine the role of caveolin-1 in the regulation of vascular and visceral muscle function (21, 45).

Regulation of G_q-coupled receptor signaling by caveolin-1.

PI hydrolysis in response to CCh, histamine, and SP was significantly inhibited by the disruption of caveolae in freshly dispersed cells and by the suppression of caveolin-1 in cultured muscle cells. Replenishment of cholesterol following MβCD treatment reversed the inhibitory effect of MβCD on PI hydrolysis and Rho kinase and ZIP kinase activity, suggesting that the effects are cholesterol-specific and that restoration of caveolae with cholesterol restores G_q-receptor signaling. A similar decrease in PI hydrolysis in response to CCh was obtained in gastric muscle from caveolin-1-KO mice. Stimulation of Rho kinase and ZIP kinase activity in response to CCh, histamine, and SP was also significantly inhibited by the disruption of caveolae in freshly dispersed cells and by the suppression of caveolin-1 in cultured muscle cells. A similar decrease in Rho kinase and ZIP kinase activity in response to CCh was obtained in gastric muscle from caveolin-1-KO mice. It is noteworthy that all three manipulations caused only partial inhibition of PI hydrolysis, Rho kinase and Zip kinase activity, and muscle contraction in response to contractile agonists, suggesting that responses are not dependent on caveolin-1 but that caveolin-1 seems to facilitate G_q/13-coupled receptor signaling.

Inhibition of PI hydrolysis could lead to a decrease in IP₃ formation, IP₃-induced Ca²⁺ release, and Ca²⁺/calmodulin-dependent MLC kinase activity, MLC₂₀ phosphorylation, and muscle contraction in response to CCh. Consistent with this notion, CCh-induced MLC₂₀ phosphorylation and contraction were significantly reduced in cells treated with MβCD or in cells lacking caveolin-1. One of the downstream targets of Rho kinase and ZIP kinase is MYPT1. Both kinases phosphorylate MYPT1 at Thr⁶⁹⁶, leading to inhibition of MLC phosphatase and increase in MLC₂₀ phosphorylation and muscle contraction (27). Thus inhibition of Rho kinase and ZIP kinase activity could lead to a decrease in MYPT1 phosphorylation and muscle contraction in response to CCh. Consistent with this notion, CCh-induced MYPT1 phosphorylation at Thr⁶⁹⁶ and contraction were significantly reduced in cells treated with MβCD. A similar decrease in contraction was also observed in muscle cells obtained from caveolin-1-KO mice. Our results are similar to recent data from studies with vascular and airway smooth muscle cells, where disruption of caveolae with MβCD led to reduced contraction in response to contractile agonists such as endothelin-1, serotonin, α₁-adrenergic receptor agonist, and acetylcholine (5, 16). However, Shakirova et al. (47) showed that the effect of caveolin-1 was agonist- and tissue-specific. The response of ileal longitudinal muscle to endothelin-1, but not to CCh or serotonin, was reduced in caveolin-1-KO mice, whereas the response of femoral arterial muscle to α₁-adrenergic agonist was increased (35, 47).

Consistent with the inhibition of agonist-induced muscle contraction, gastric emptying in vivo was significantly inhibited in caveolin-1-KO mice. Gastric emptying involves coordinated activity of the fundus, antrum, pyloric sphincter, and duodenum and is regulated by the activity of the autonomic and enteric nervous system, interstitial cells of Cajal, and smooth muscle. Intact muscle contraction in response to the excitatory transmitter acetylcholine is important for proper emptying of gastric contents. The singular contribution of smooth muscle caveolin-1 to the regulation of gastric emptying is difficult to predict from the studies with caveolin-1-KO mice. However, the decrease in muscle contraction in response to CCh and the significant delay in gastric emptying in caveolin-1-KO mice suggest that caveolin-1 plays an important role in physiological motor functions of the stomach.

Analysis of M₃ receptor expression in sucrose density gradient fractions showed that M₃ receptors are not associated with caveolin-1. This suggests that distal components of the M₃ receptor may be spatially associated with caveolin-1. Recent studies demonstrated that KCl-induced contraction was similar in WT and caveolin-1-KO mice, suggesting that caveolin-1 KO does not affect the membrane depolarization process (47). Our results suggest that caveolin-1 is necessary for optimal PLC-β- and RhoA-dependent signaling in gastric smooth muscle. Although our studies did not identify the loci of caveolin-1 interaction to regulate PI hydrolysis and RhoA-dependent pathways, several possible loci of interaction have been implicated in previous studies. We and others have shown that Gα_q protein contains a CBM and directly interacts with caveolin proteins (1, 31, 46). Studies also suggest that interaction of caveolin-1, β-dystroglycan, and the actin cytoskeleton is important for the structural and spatial distribution of caveolae and plays an important role in Gα_q/PLC-β signaling (48).

RhoA, a small GTP-binding protein, exists in active GTP-bound and inactive GDP-bound states. PKC, in addition to Rho kinase and ZIP kinase, plays an important role in the inhibition of MLC phosphatase and regulation of muscle contraction via phosphorylation of the endogenous MLC phosphatase inhibitor CPI-17 (57). Further indication of a potential interaction is the fact that CBMs are contained within the catalytic domain of PKC-α and Rho kinase and the switch region of RhoA. Sucrose gradient fractionation, coimmunoprecipitation, and immunogold labeling studies demonstrated interaction between RhoA and caveolae. Activation-induced translocation and colocalization of RhoA with caveolae were inhibited by MβCD treatment or by blocking the caveolin-1 scaffolding domain with a synthetic peptide in ferret aorta (9, 51). Translocation of Rho kinase has also been reported in rabbit arteries (52). PKC, in addition to Rho kinase and ZIP kinase, plays an important role in the inhibition of MLC phosphatase and regulation of muscle contraction via phosphorylation of CPI-17 (57). Previous studies in vascular muscle have identified caveolae as regulators of PKC signaling (22, 51). Translocation of PKC-α from cytosol to membrane was inhibited by chemical loading of peptide corresponding to the scaffolding domain of caveolin-1 (22, 50). These studies suggest a direct interaction between the scaffolding domain of caveolin and PKC-α and RhoA, and caveolae may be involved in the recruitment of signaling molecules downstream of G_q-coupled receptor activation. Further studies examining localization of PLC-β₁ and RhoA, Rho kinase, and PKC isoforms in gastrointestinal smooth muscle may reveal more precise understanding of the regulation of Ca²⁺-dependent initial contraction and Ca²⁺-independent sustained contraction.

Regulation of G_i-coupled receptor signaling by caveolin-1.

In contrast to the effect of caveolae and caveolin-1 on the G_q-coupled receptor signaling, disruption of caveolae with MβCD in freshly dispersed muscle cells or suppression of caveolin-1 in cultured muscle cells with caveolin-1 siRNA did not affect the inhibition of AC activity in response to CCh. Similar results were obtained using caveolin-1-KO mice. Our studies also demonstrated that the increase in AC activity in response to the direct activator, forskolin, was similar in control cells and in cells treated with MβCD or caveolin-1 siRNA. Stimulation of AC activity by forskolin was also similar in gastric muscle from WT and caveolin-1-KO mice. These results suggest that caveolin-1 had no effect on AC activity. Our results differ from results from previous studies, which showed that suppression of caveolin-1 in C6 glioma cells or lack of caveolin-1 in the brain of caveolin-1-KO mice significantly augmented forskolin-stimulated AC activity (1, 17). These studies suggest that the effect of caveolin-1 on AC activity varies within different tissues and/or with the expression of specific AC isoforms. In addition, given that cAMP signaling is compartmentalized by the expression of phosphodiesterase, we speculate that such intermolecular association with other signaling partners in caveolar microdomains is also relevant to understanding the role of caveolin-1 in cAMP signaling.

Regulation of M₂ and M₃ receptor internalization by caveolin-1.

Compelling evidence showing selective regulation of M₃ receptor signaling by caveolin-1 underscored the importance of determining the probability of occurrence of muscarinic receptors in caveolar fractions. Western blot analysis of M₂ and M₃ receptors showed that, in the basal state, M₂ and M₃ receptors are absent in the caveolar fraction. However, upon treatment with CCh, M₂, but not M₃, receptors were translocated to the caveolar fractions, raising the possibility that caveolae could facilitate M₂, but not M₃, receptor internalization. The molecular events that drive the receptor internalization process can be either clathrin-dependent or clathrin-independent and caveolae-dependent. The essential step in the internalization process via caveolae involves phosphorylation of caveolin-1 at Tyr¹⁴ via Src kinase (50).

The role of caveolin-1 in the regulation of M₂ and M₃ receptor internalization was examined using MβCD in freshly dispersed muscle cells and in cultured muscle cells transfected with caveolin-1 siRNA. Limited yield of muscle cells from mice precluded the use of caveolin-1-KO mice for binding studies. Disruption of caveolae or suppression of caveolin-1 had no effect on the maximal binding of M₂ or M₃ receptors but selectively attenuated M₂ receptor internalization. Internalization of M₂ receptors was also attenuated by the blockade of Src kinase. The results suggest that M₂ receptor internalization was facilitated by caveolin-1 and consistent with the translocation of M₂ receptors to caveolar fractions. This is in contrast to the lack of effect of caveolin-1 on the rapid responses (within 60 s) to M₂ receptor activation, such as inhibition of AC activity. However, caveolin-1 may affect receptor internalization-dependent signaling, such as stimulation of ERK1/2 and Src activities (7, 42). The results also suggest that M₃ receptor internalization is caveolae-independent and may involve clathrin-dependent pathways. This is in contrast to the positive effect of caveolin-1 on the rapid responses of M₃ receptor signaling, such as stimulation of PI hydrolysis and Rho kinase and ZIP kinase activity. These results also suggest that caveolae-dependent internalization is receptor-specific.

Several lines of evidence suggest involvement of caveolae in the internalization of M₂ receptors and β-arrestins and dynamins in the internalization of M₃ receptors (38, 53, 54, 58). In cardiac myocytes, agonist-induced translocation of M₂ receptors has been implicated in the regulation of M₂ receptor functions, such as endothelial nitric oxide synthase activation (13, 14). Studies in HEK-tsA201 cells showed that phosphorylation-dependent internalization of M₂ receptors was β-arrestin- and clathrin-independent. (38), whereas internalization of M₄, M₁, and M₃ receptors was β-arrestin- and dynamin-dependent (53, 54). These different pathways of receptor endocytosis seem to target receptors to different intracellular compartments. Krudewig et al. (23) reported that β-arrestin- and dynamin-independent M₂ receptor internalization leads to receptor downregulation, whereas β-arrestin- and dynamin-dependent internalization of M₁ and M₃ receptors leads to receptor recycling to the membrane. Our studies demonstrate that caveolin-1 positively regulates M₂, but not M₃, receptor internalization in gastric muscle. Further studies are necessary to examine the role of β-arrestins and other proteins in the regulation of M₂ and M₃ receptor internalization and their physiological significance in the smooth muscle.

In summary, our results reveal that caveolae/caveolin-1 facilitates G_q-coupled receptor signaling and contraction in gastrointestinal smooth muscle (Fig. 12). The effects of caveolin-1 are not associated with its interaction with the receptor or changes in the binding of ligand to the receptor and are likely the result of its interaction with key signaling molecules distal to the receptor, such as Gα_q/PLC-β₁ and RhoA/Rho kinase. The molecular loci of caveolin interaction in the Gα_q/PLC-β₁ and RhoA/Rho kinase pathways remain to be determined. It is noteworthy that caveolin-1 contributes to the facilitation of gastric emptying in vivo, suggesting that the mechanisms related to the interactions of caveolin-1 with the signaling molecules are determinants of the functional significance of caveolae. The participation of caveolin-1 in the regulation of gastric emptying identified in the present study suggests that caveolins may have a role in the pathophysiological changes in gastrointestinal motility.

Fig. 12. — Regulation of G_q- and G_i-coupled receptor signaling by caveolin-1 in gastric smooth muscle. Caveolin-1 facilitates G_q/PLC-β₁- and RhoA/Rho kinase-dependent pathways that lead to Ca²⁺/calmodulin-dependent stimulation of myosin light chain kinase (MLCK) activity and inhibition of myosin light chain phosphatase (MLCP) activity, respectively, and increase in MLC₂₀ phosphorylation, an essential step in muscle contraction. Loci of interaction of caveolin-1 with the signaling molecules in the pathways distal to the receptor were not identified in the present study. Previous studies have shown that caveolae/caveolin-1 interacts with several molecules in the pathway, including Gα_q, PLC-β, RhoA, and Rho kinase. Caveolin-1 had no effect on G_i-mediated inhibition of adenylyl cyclase (AC) activity.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-28300 and DK-15564 (to K. S. Murthy).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.B. and K.S.M. conceived and designed the research; S.B. performed the experiments; S.B. and K.S.M. analyzed the data; S.B. and K.S.M. interpreted the results of the experiments; S.B. prepared the figures; S.B. and K.S.M. drafted the manuscript; S.B., S.M., J.R.G., and K.S.M. edited and revised the manuscript; S.B., S.M., O.A.-S., S.R., J.R.G., and K.S.M. approved the final version of the manuscript.

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21. Insel PA, Patel HH. Do studies in caveolin-knockouts teach us about physiology and pharmacology or instead, the ways mice compensate for “lost proteins”? Br J Pharmacol 150: 251–254, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Je HD, Gallant C, Leavis PC, Morgan KG. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91–H98, 2004 [DOI] [PubMed] [Google Scholar]
23. Krudewig R, Langer B, Vogler O, Markschies N, Erl M, Jackobs KH, van Koppen CJ. Distinct internalization of M₂ muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. J Neurochem 74: 1721–1730, 2000 [DOI] [PubMed] [Google Scholar]
24. Lajoie P, Nabi IR. Lipid rafts, caveolae, and their endocytosis. Int Rev Cell Mol Biol 282: 135–163, 2010 [DOI] [PubMed] [Google Scholar]
25. Lin S, Kajimura M, Takeuchi K, Kodaira M, Hanai H, Kaneko E. Expression of muscarinic receptor subtypes in rat gastric smooth muscle: effect of M₃ selective antagonist on gastric motility and emptying. Dig Dis Sci 42: 907–914, 1997 [DOI] [PubMed] [Google Scholar]
26. Morini G, Kuemmerle JF, Impicciatore M, Grider JR, Makhlouf GM. Coexistence of histamine H₁ and H₂ receptors coupled to distinct signal transduction pathways in isolated intestinal muscle cells. J Pharmacol Exp Ther 264: 598–603, 1993 [PubMed] [Google Scholar]
27. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 68: 345–374, 2006 [DOI] [PubMed] [Google Scholar]
28. Murthy KS, Coy DH, Makhlouf GM. Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase Cβ₃ by Gβγ and inhibition of adenylyl cyclase by Gα_i1 and Gα_o. J Biol Chem 271: 23458–23463, 1996 [DOI] [PubMed] [Google Scholar]
29. Murthy KS, Mahavadi S, Huang J, Zhou H, Sriwai W. Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle. Am J Physiol Cell Physiol 294: C477–C487, 2008 [DOI] [PubMed] [Google Scholar]
30. Murthy KS, Makhlouf GM. Adenosine A₁ receptor-mediated activation of phospholipase Cβ₃ in intestinal muscle: dual requirement for α, β, and γ subunits of G_i3. Mol Pharmacol 47: 1172–1179, 1995 [PubMed] [Google Scholar]
31. Murthy KS, Makhlouf GM. Heterologous desensitization mediated by G protein-specific binding to caveolin. J Biol Chem 275: 30211–30219, 2000 [DOI] [PubMed] [Google Scholar]
32. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, Makhlouf GM. Differential signalling by muscarinic receptors in smooth muscle: M₂-mediated inactivation of myosin light chain kinase via G_i3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and M₃-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Murthy KS, Zhou H, Grider JR, Makhlouf GM. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. Am J Physiol Gastrointest Liver Physiol 284: G1006–G1016, 2003 [DOI] [PubMed] [Google Scholar]
34. Murthy KS, Zhou H, Makhlouf GM. PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle. Am J Physiol Cell Physiol 282: C508–C517, 2002 [DOI] [PubMed] [Google Scholar]
35. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J Biol Chem 273: 5419–5422, 1998 [DOI] [PubMed] [Google Scholar]
36. Ostrom RS, Insel PA. The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol 143: 235–245, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Ostrom RS, Insel PA. Methods for the study of signaling molecules in membrane lipid rafts and caveolae. Methods Mol Biol 332: 181–191, 2006 [DOI] [PubMed] [Google Scholar]
38. Pals-Rylaarsdam R, Gurevich V, Lee KB, Ptasienski JA, Benovic JL, Hosey MM. Internalization of the M₂ muscarinic acetylcholine receptor: arrestin-independent and -dependent pathways. J Biol Chem 272: 23682–23689, 1997 [DOI] [PubMed] [Google Scholar]
39. Parton RG, Hanzal-Bayer M, Hancock JF. Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci 119: 787–796, 2006 [DOI] [PubMed] [Google Scholar]
40. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 8: 185–194, 2007 [DOI] [PubMed] [Google Scholar]
41. Patel HH, Murray F, Insel PA. Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol 48: 359–391, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3: 639–650, 2002 [DOI] [PubMed] [Google Scholar]
43. Qin Y, Thomas D, Fontaine CP, Colvin RA. Silencing of ZnT1 reduces Zn²⁺ efflux in cultured cortical neurons. Neurosci Lett 450: 206–210, 2009 [DOI] [PubMed] [Google Scholar]
44. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002 [DOI] [PubMed] [Google Scholar]
45. Sathish V, Yang B, Meuchel LW, VanOosten SK, Ryu AJ, Thompson MA, Prakash YS, Pabelick CM. Caveolin-1 and force regulation in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 300: L920–L929, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Sengupta P, Philip F, Scarlata S. Caveolin-1 alters Ca²⁺ signal duration through specific interaction with the Gα_q family of G proteins. J Cell Sci 121: 1363–1372, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Shakirova Y, Bonnevier J, Albinsson S, Adner M, Rippe B, Broman J, Arner A, Swärd K. Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol 291: C1326–C1335, 2006 [DOI] [PubMed] [Google Scholar]
48. Sharma P, Ghavani S, Stelmack GL, McNeil KD, Mutawe MM, Klonish T, Unruh H, Halayko AJ. β-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca²⁺ release. Cell Sci 123: 3061–3070, 2010 [DOI] [PubMed] [Google Scholar]
49. Sriwai W, Zhou H, Murthy KS. G_q-dependent signalling by the lysophosphatidic acid receptor LPA₃ in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA. Biochem J 411: 543–551, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Sverdlov M, Shajahan AN, Minshall RD. Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis. J Cell Mol Med 11: 1239–1250, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]
51. Taggart MJ, Leavis P, Feron O, Morgan KG. Inhibition of PKCα and RhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res 258: 72–81, 2000 [DOI] [PubMed] [Google Scholar]
52. Urban NH, Berh KM, Ratz PH. K⁺ depolarization induces RhoA kinase translocation to caveolae and Ca²⁺ sensitization of arterial muscle. Am J Physiol Cell Physiol 285: C1377–C1385, 2003 [DOI] [PubMed] [Google Scholar]
53. Vogler O, Bogatkewitsch G, Wriske C, Krummenerl P, Jackobs KH, van Koppen CJ. Receptor subtype-specific regulation of muscarinic acetylcholine sequestration by dynamin. J Biochem 273: 12155–12160, 1998 [DOI] [PubMed] [Google Scholar]
54. Vogler O, Nolete B, Voss M, Jackobs KH, van Koppen CJ. Regulation of muscarinic acetylcholine receptor sequestration function by β-arrestin. J Biol Chem 274: 12333–12338, 1999 [DOI] [PubMed] [Google Scholar]
55. Williams TM, Lisanti MP. The caveolin genes: from cell biology to medicine. Ann Med 36: 584–595, 2004 [DOI] [PubMed] [Google Scholar]
56. Williams TM, Lisanti MP. The caveolin proteins. Genome Biol 5: 214, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
57. Woodsome TP, Eto M, Everett A, Brautigan DL, Kitazawa T. Expression of CPI-17 and myosin phosphatase correlates with Ca²⁺ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol 535: 553–564, 2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
58. Wu G, Krupnick JG, Benovic JL, Lanier SM. Interaction of arrestins with intracellular domain of muscarinic and α₂-adrenergic receptors. J Biol Chem 272: 17836–17842, 1997 [DOI] [PubMed] [Google Scholar]
59. Yeung CK, McCurrie JR, Wood D. A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo. J Pharmacol Toxicol Methods 45: 235–240, 2001 [DOI] [PubMed] [Google Scholar]

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[B21] 21. Insel PA, Patel HH. Do studies in caveolin-knockouts teach us about physiology and pharmacology or instead, the ways mice compensate for “lost proteins”? Br J Pharmacol 150: 251–254, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Je HD, Gallant C, Leavis PC, Morgan KG. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91–H98, 2004 [DOI] [PubMed] [Google Scholar]

[B23] 23. Krudewig R, Langer B, Vogler O, Markschies N, Erl M, Jackobs KH, van Koppen CJ. Distinct internalization of M₂ muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. J Neurochem 74: 1721–1730, 2000 [DOI] [PubMed] [Google Scholar]

[B24] 24. Lajoie P, Nabi IR. Lipid rafts, caveolae, and their endocytosis. Int Rev Cell Mol Biol 282: 135–163, 2010 [DOI] [PubMed] [Google Scholar]

[B25] 25. Lin S, Kajimura M, Takeuchi K, Kodaira M, Hanai H, Kaneko E. Expression of muscarinic receptor subtypes in rat gastric smooth muscle: effect of M₃ selective antagonist on gastric motility and emptying. Dig Dis Sci 42: 907–914, 1997 [DOI] [PubMed] [Google Scholar]

[B26] 26. Morini G, Kuemmerle JF, Impicciatore M, Grider JR, Makhlouf GM. Coexistence of histamine H₁ and H₂ receptors coupled to distinct signal transduction pathways in isolated intestinal muscle cells. J Pharmacol Exp Ther 264: 598–603, 1993 [PubMed] [Google Scholar]

[B27] 27. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 68: 345–374, 2006 [DOI] [PubMed] [Google Scholar]

[B28] 28. Murthy KS, Coy DH, Makhlouf GM. Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase Cβ₃ by Gβγ and inhibition of adenylyl cyclase by Gα_i1 and Gα_o. J Biol Chem 271: 23458–23463, 1996 [DOI] [PubMed] [Google Scholar]

[B29] 29. Murthy KS, Mahavadi S, Huang J, Zhou H, Sriwai W. Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle. Am J Physiol Cell Physiol 294: C477–C487, 2008 [DOI] [PubMed] [Google Scholar]

[B30] 30. Murthy KS, Makhlouf GM. Adenosine A₁ receptor-mediated activation of phospholipase Cβ₃ in intestinal muscle: dual requirement for α, β, and γ subunits of G_i3. Mol Pharmacol 47: 1172–1179, 1995 [PubMed] [Google Scholar]

[B31] 31. Murthy KS, Makhlouf GM. Heterologous desensitization mediated by G protein-specific binding to caveolin. J Biol Chem 275: 30211–30219, 2000 [DOI] [PubMed] [Google Scholar]

[B32] 32. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, Makhlouf GM. Differential signalling by muscarinic receptors in smooth muscle: M₂-mediated inactivation of myosin light chain kinase via G_i3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and M₃-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155, 2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Murthy KS, Zhou H, Grider JR, Makhlouf GM. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. Am J Physiol Gastrointest Liver Physiol 284: G1006–G1016, 2003 [DOI] [PubMed] [Google Scholar]

[B34] 34. Murthy KS, Zhou H, Makhlouf GM. PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle. Am J Physiol Cell Physiol 282: C508–C517, 2002 [DOI] [PubMed] [Google Scholar]

[B35] 35. Okamoto T, Schlegel A, Scherer PE, Lisanti MP. Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J Biol Chem 273: 5419–5422, 1998 [DOI] [PubMed] [Google Scholar]

[B36] 36. Ostrom RS, Insel PA. The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol 143: 235–245, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Ostrom RS, Insel PA. Methods for the study of signaling molecules in membrane lipid rafts and caveolae. Methods Mol Biol 332: 181–191, 2006 [DOI] [PubMed] [Google Scholar]

[B38] 38. Pals-Rylaarsdam R, Gurevich V, Lee KB, Ptasienski JA, Benovic JL, Hosey MM. Internalization of the M₂ muscarinic acetylcholine receptor: arrestin-independent and -dependent pathways. J Biol Chem 272: 23682–23689, 1997 [DOI] [PubMed] [Google Scholar]

[B39] 39. Parton RG, Hanzal-Bayer M, Hancock JF. Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci 119: 787–796, 2006 [DOI] [PubMed] [Google Scholar]

[B40] 40. Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 8: 185–194, 2007 [DOI] [PubMed] [Google Scholar]

[B41] 41. Patel HH, Murray F, Insel PA. Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol 48: 359–391, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3: 639–650, 2002 [DOI] [PubMed] [Google Scholar]

[B43] 43. Qin Y, Thomas D, Fontaine CP, Colvin RA. Silencing of ZnT1 reduces Zn²⁺ efflux in cultured cortical neurons. Neurosci Lett 450: 206–210, 2009 [DOI] [PubMed] [Google Scholar]

[B44] 44. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002 [DOI] [PubMed] [Google Scholar]

[B45] 45. Sathish V, Yang B, Meuchel LW, VanOosten SK, Ryu AJ, Thompson MA, Prakash YS, Pabelick CM. Caveolin-1 and force regulation in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 300: L920–L929, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46. Sengupta P, Philip F, Scarlata S. Caveolin-1 alters Ca²⁺ signal duration through specific interaction with the Gα_q family of G proteins. J Cell Sci 121: 1363–1372, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B47] 47. Shakirova Y, Bonnevier J, Albinsson S, Adner M, Rippe B, Broman J, Arner A, Swärd K. Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol 291: C1326–C1335, 2006 [DOI] [PubMed] [Google Scholar]

[B48] 48. Sharma P, Ghavani S, Stelmack GL, McNeil KD, Mutawe MM, Klonish T, Unruh H, Halayko AJ. β-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca²⁺ release. Cell Sci 123: 3061–3070, 2010 [DOI] [PubMed] [Google Scholar]

[B49] 49. Sriwai W, Zhou H, Murthy KS. G_q-dependent signalling by the lysophosphatidic acid receptor LPA₃ in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA. Biochem J 411: 543–551, 2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50. Sverdlov M, Shajahan AN, Minshall RD. Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis. J Cell Mol Med 11: 1239–1250, 2007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51. Taggart MJ, Leavis P, Feron O, Morgan KG. Inhibition of PKCα and RhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res 258: 72–81, 2000 [DOI] [PubMed] [Google Scholar]

[B52] 52. Urban NH, Berh KM, Ratz PH. K⁺ depolarization induces RhoA kinase translocation to caveolae and Ca²⁺ sensitization of arterial muscle. Am J Physiol Cell Physiol 285: C1377–C1385, 2003 [DOI] [PubMed] [Google Scholar]

[B53] 53. Vogler O, Bogatkewitsch G, Wriske C, Krummenerl P, Jackobs KH, van Koppen CJ. Receptor subtype-specific regulation of muscarinic acetylcholine sequestration by dynamin. J Biochem 273: 12155–12160, 1998 [DOI] [PubMed] [Google Scholar]

[B54] 54. Vogler O, Nolete B, Voss M, Jackobs KH, van Koppen CJ. Regulation of muscarinic acetylcholine receptor sequestration function by β-arrestin. J Biol Chem 274: 12333–12338, 1999 [DOI] [PubMed] [Google Scholar]

[B55] 55. Williams TM, Lisanti MP. The caveolin genes: from cell biology to medicine. Ann Med 36: 584–595, 2004 [DOI] [PubMed] [Google Scholar]

[B56] 56. Williams TM, Lisanti MP. The caveolin proteins. Genome Biol 5: 214, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57. Woodsome TP, Eto M, Everett A, Brautigan DL, Kitazawa T. Expression of CPI-17 and myosin phosphatase correlates with Ca²⁺ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol 535: 553–564, 2001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B58] 58. Wu G, Krupnick JG, Benovic JL, Lanier SM. Interaction of arrestins with intracellular domain of muscarinic and α₂-adrenergic receptors. J Biol Chem 272: 17836–17842, 1997 [DOI] [PubMed] [Google Scholar]

[B59] 59. Yeung CK, McCurrie JR, Wood D. A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo. J Pharmacol Toxicol Methods 45: 235–240, 2001 [DOI] [PubMed] [Google Scholar]

PERMALINK

Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

Sayak Bhattacharya

Sunila Mahavadi

Othman Al-Shboul

Senthilkumar Rajagopal

John R Grider

Karnam S Murthy

Abstract

EXPERIMENTAL PROCEDURES

Isolation of gastric muscle cells and primary culture of muscle cells.

Transfection of caveolin-1 siRNA.

Assay for phosphatidylinositol hydrolysis.

Assay for cAMP.

Assay for Rho kinase and ZIP kinase.

Measurement of MLC20 and MYPT1 phosphorylation by in-cell Western assay.

[3H]scopolamine binding to smooth muscle cells.

Isolation of caveolar and noncaveolar fractions and Western blotting.

Measurement of muscle contraction.

Gastric emptying.

Statistical analysis.

RESULTS

Expression of M2 and M3 receptors and contraction in gastric smooth muscle.

Fig. 1.

Signaling by M2 and M3 receptors in gastric smooth muscle.

Fig. 2.

Effect of caveolae/caveolin-1 on Gq-coupled receptor signaling.

Inhibition of PI hydrolysis and Rho kinase and ZIP kinase activity.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Inhibition of MYPT1 and MLC20 phosphorylation and muscle contraction.

Fig. 8.

Regulation of gastric emptying by caveolin-1.

Fig. 9.

Table 1.

Lack of effect of caveolin-1 on M2-mediated inhibition of cAMP.

Fig. 10.

Effect of caveolae/caveolin-1 on M2 and M3 receptor internalization.

Fig. 11.

Inhibition of M2 receptor internalization by caveolin-1.

Lack of effect of caveolin-1 on M3 receptor internalization.

DISCUSSION

Regulation of Gq-coupled receptor signaling by caveolin-1.

Regulation of Gi-coupled receptor signaling by caveolin-1.

Regulation of M2 and M3 receptor internalization by caveolin-1.

Fig. 12.