HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (15) Citing Articles via Google Scholar Google Scholar Articles by Huang, J. Articles by Murthy, K. S. Search for Related Content PubMed PubMed Citation Articles by Huang, J. Articles by Murthy, K. S.

HORMONES AND SIGNALING

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC₂₀ phosphorylation by motilin receptors

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia

Submitted 12 July 2004 ; accepted in final form 30 August 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC₅₀ 0.7 ± 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G_q and G₁₃, stimulated G_q-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release, and increased cytosolic free Ca²⁺. PI hydrolysis was blocked by expression of G_q minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC₅₀ = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca²⁺-dependent contraction and myosin light-chain (MLC)₂₀ phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC₂₀ phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G_q or G₁₃ minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC₂₀ phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G_q-mediated cascade involving Ca²⁺/calmodulin activation of MLC kinase and transient MLC₂₀ phosphorylation and contraction as well as a sustained G_q- and G₁₃-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC₂₀ phosphorylation and contraction.

interdigestive gut hormone; Rho kinase; G protein signaling

Numerous studies have demonstrated the presence of motilin receptors on smooth muscle cells, which appear to facilitate or augment nerve-mediated effects of motilin (4, 6, 18, 27, 28, 34). Radioligand binding studies on gastric and intestinal membranes enriched with smooth muscle markers (5'-nucleotidase) provide evidence for the presence of motilin receptors on smooth muscle cells (3, 8, 36). Motilin activates L-type Ca²⁺ channels in dispersed human and canine intestinal muscle cells (5, 16) and enhances carbachol-induced cationic current in dispersed duodenal muscle cells (38). Motilin and related motilides such as erythromycin induce contraction of guinea pig, rat, and human gastric muscle cells, canine jejunal muscle cells, and rabbit colonic muscle cells and stimulate Ca²⁺ mobilization in cultured human colonic muscle cells (8, 15, 17, 31, 35, 39).

The signal-transduction mechanisms that mediate smooth muscle contraction by motilin have not been fully explored. Using rabbit duodenal muscle strips, Depoortere and Peeters (2) showed that motilin stimulated phosphoinositide (PI) hydrolysis to the same extent as carbachol and increased IP₃ formation. Motilin-induced PI hydrolysis was concentration dependent and paralleled motilin-induced contraction (2). In cultured, human colonic smooth muscle cells loaded with indomethacin-1, motilin caused an increase in cytosolic free Ca²⁺ {[Ca²⁺]_i}, consistent with inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release (35). Motilin-induced increase in [Ca²⁺]_i in the human medulloblastoma cell line TE671, which express motilin receptors, was retained in Ca²⁺-free medium or the presence of the Ca²⁺ channel blocker nifedipine (32).

Recent studies have shown that the contractile response to agonists consists of two phases: an initial transient phase mediated by IP₃-dependent Ca²⁺ release and Ca²⁺/calmodulin-dependent activation of myosin light chain (MLC) kinase (MLCK) leading to phosphorylation of MLC₂₀ and a sustained Ca²⁺-independent phase mediated by inhibition of MLC phosphatase (21, 25). For G_q/G₁₃-coupled receptors, inhibition of MLC phosphatase is mediated by Rho-dependent pathways involving Rho kinase-mediated phosphorylation of MYPT1, the regulatory subunit of MLC phosphatase, and/or PKC-dependent phosphorylation of CPI-17, an endogenous inhibitor of MLC phosphatase (25). Muscarinic M₃ receptors and sphingosine-1 phosphate (S1P₂) receptors engage both Rho-dependent pathways, whereas endothelin A (ET_A) and lysophosphatidic acid (LPA₃) receptors engage the MYPT1 and CPI-17 pathways, respectively (25, 39, 40).

In the present study, we have used freshly dispersed and cultured smooth muscle cells from the circular muscle layer of rabbit distal stomach and proximal small intestine to characterize the G protein-dependent signaling pathways mediating the initial and sustained phases of contraction and MLC₂₀ phosphorylation. The results demonstrated that motilin initiates a transient G_q-mediated signaling cascade involving IP₃-dependent Ca²⁺ release and MLCK-dependent MLC₂₀ phosphorylation and contraction and a sustained G_q- and G₁₃-mediated cascade involving Rho kinase- and PKC-dependent inhibition of MLC phosphatase, resulting in sustained MLC₂₀ phosphorylation and contraction.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Preparation of dispersed and cultured gastric and intestinal smooth muscle cells. Smooth muscle cells were isolated from the circular muscle layer of rabbit distal stomach and proximal small intestine by sequential enzymatic digestion, filtration, and centrifugation as previously described (21, 24, 25). Muscle strips were incubated for 30 min at 31°C in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor. The composition of the medium was (in mM) 120 NaCl, 4 KCl, 2.6 KH₂PO₄, 0.6 MgCl₂, 25 HEPES, and 14 glucose, with 2.1% Eagle's essential amino acid mixture. The partly digested tissues were washed with 100 ml of enzyme-free medium and reincubated for 30 min to allow spontaneous dispersion of muscle cells. Cells were harvested by filtration through 500 µm Nitex and centrifuged twice at 350 g for 10 min.

Functional studies were done on dispersed intestinal smooth muscle cells, whereas radioligand binding and transfection studies were on gastric smooth muscle cells that can be more readily cultured. The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they attained confluence and were then passaged once for use in various studies.

Minigene construction and transfection into cultured gastric smooth muscle cells. Activation of specific G protein subunits was blocked by the expression of cDNA encoding the last COOH-terminal 11 amino acids of mouse G_q and G₁₃ and human G_i as described previously (40). The cDNA sequences were amplified by PCR and verified by DNA sequencing. All G minigene constructs used for transfection experiments were purified with an endotoxin-free maxi-prep kit (Qiagen). Cultured rabbit gastric smooth muscle cells were transiently transfected with minigene plasmid DNA using Effectene transfection reagent (Qiagen). Transfection efficiency was monitored by cotransfection of pGreen Lantern-1. Analysis by fluorescence microscopy showed that 80% of the cells were transfected.

Expression of dominant negative GRK2 and RGS4 in cultured gastric smooth muscle cells. Dominant negative G protein-coupled receptor kinase 2 (GRK2) (K220R) or RGS4(N88S) was subcloned into the multiple cloning site (EcoRI) of the eukaryotic expression vector pEXV, and a myc tag was incorporated into the NH₂ terminus. Recombinant plasmid DNAs (2 µg each) were transiently transfected into smooth muscle cells in primary culture using Effectene transfection reagent for 48 h. The cells were cotransfected with 1 µg of pGreen Lantern-1 to monitor expression. Control cells were cotransfected with 2 µg of vector (pEXV) and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency (85%) was monitored microscopically by the expression of green fluorescent protein using FITC filters.

Identification of motilin-activated G proteins. G proteins activated by motilin were identified by an adaptation of method of Okamoto et al. (26), as described previously (23, 24). Dispersed intestinal muscle cells were homogenized in 20 mM HEPES (pH 7.4) containing 2 mM MgCl₂, 1 mM EDTA, and 2 mM DTT, centrifuged at 30,000 g for 30 min at 4°C, and solubilized at 4°C in 20 mM HEPES (pH 7.4) buffer containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized membranes were incubated for 20 min at 37°C with 100 nM [³⁵S]GTPS in 10 mM HEPES (pH 7.4) in the presence or absence of 1 µM motilin. After the reaction was stopped, the membranes were incubated for 2 h on ice in wells precoated with specific antibodies to G_q, G_i1, G_i2, G_i3, and G₁₃. The wells were washed with phosphate buffer containing 0.05% Tween 20, and radioactivity from each well was counted by liquid scintillation.

Characterization of motilin receptors by radioligand binding. Cultured gastric smooth muscle cells were redispersed and suspended in HEPES medium containing 1% BSA, amastatin (10 µM), phospharamidon (1 µM), and bacitracin (0.7 mM). Triplicate aliquots (0.3 ml) of cell suspension (10⁶ cells/ml) were incubated for 15 min at 25°C with ¹²⁵I-labeled motilin (50 pM) alone or in the presence of unlabeled motilin (10 µM). Bound and free radioligand were separated by rapid filtration through 5-µm polycarbonate nucleopore filters followed by washing three times with HEPES medium. Nonspecific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of unlabeled motilin (10 µM). Specific binding was calculated as the difference between total and nonspecific binding. Nonspecific binding was 20 ± 2% of total binding. Binding was also measured in cells expressing vector or dominant negative GRK2(K220R). The cells were first treated with 1 µM cold motilin for 20 min and washed rapidly for 5 min before specific ¹²⁵I-labeled motilin binding was determined.

Measurement of Ca²⁺ release and [Ca²⁺]_i in muscle cells. Ca²⁺ release was measured in dispersed intestinal muscle cells as described previously (23, 24). The cells were first permeabilized and suspended in a medium containing Ca²⁺ (100 nM), ⁴⁵Ca²⁺ (10 µCi/ml), antimycin (10 µM), and an ATP regenerating system (1.5 mM ATP, 5 mM creatine phosphate, and 10 U/ml creatine kinase). Ca²⁺ uptake was measured at intervals for 60 min when a steady state (2.53 ± 0.28 nmol/10⁶ cells) was attained; IP₃ or motilin was then added, and the reaction was terminated after 15 s. U-73122 or heparin was added 60 s before IP₃ or motilin. The decrease in steady-state ⁴⁵Ca²⁺ cell content represented net Ca²⁺ release expressed in nanomoles per 10⁶ cells.

In some experiments, motilin-induced increase in [Ca²⁺]_i was measured by fluorescence in single smooth muscle cell loaded with fluorescent Ca²⁺ dye fura 2 (24). Dispersed muscle cells were plated on coverslips for 12 h in DMEM. After being washed with PBS, the cells were loaded with 5 µM fura 2-AM for 1 h at room temperature. The cells were visualized through a x40 objective (ZEISS; 0.9 NA) with a Zeiss Axioskop 2 plus upright fluorescence microscope and imaged with a setup consisting of a charge coupled device camera (Imago, TILL Photonics, Applied Scientific Instrumentation, Eugene, OR) attached to an image intensifier. The cells were alternately excited at 380 and 340 nm. The background and autofluorescence were corrected from images of a cell without the fura 2.

Assay for PI hydrolysis. PI hydrolysis was determined from the formation of total inositol phosphates using ion-exchange chromatography as described previously (23, 24). Cultured smooth muscle cells were labeled with myo-[³H]inositol (0.5 µCi/ml) for 24 h in inositol-free Dulbecco's modified Eagle's medium. The cultured cells were washed with PBS and treated with motilin (1 µM) for 30 s in 1 ml of 25 mM HEPES buffer (pH 7.4) containing (in mM) 115 NaCl, 5.8 KCl, 2.1 KH₂PO₄, 2 CaCl₂, and 14 glucose. The reaction was terminated by the addition of 940 µl of chloroform-methanol-HCl (50:100:1). The samples were extracted with 310 µl chloroform and 340 µl of H₂O, and then phases were separated by centrifugation at 1,000 g for 15 min. The upper aqueous phase was applied to DOWEX AG-1 column, and [³H]inositol phosphates were eluted with 0.8 M ammonium formamate-0.1 M formic acid. Radioactivity was determined by liquid scintillation and was expressed as counts per minute (cpm) per milligram of protein.

Assay for Rho kinase activity. Rho kinase activity was determined by immunokinase assay in cell extracts as described previously (25, 40). Rho kinase immunoprecipitates were washed twice with a phosphorylation buffer containing 10 mM MgCl₂ and 40 mM HEPES (pH 7.4) and then incubated for 5 min on ice with 5 µg of myelin basic protein. Rho kinase assay was initiated by the addition of 10 µCi of [-³²P]ATP (3,000 Ci/mmol) and 20 µM ATP, followed by incubation for 10 min at 37°C. ³²P-labeled myelin basic protein was absorbed onto phosphocellulose disks, and free radioactivity was removed by repeated washings with 75 mM phosphoric acid. The amount of radioactivity on the disks was measured by liquid scintillation.

Immunoblot analysis of MLC₂₀, MYPT1, and CPI-17 phosphoproteins. Phosphorylation of MLC₂₀, MYPT1, and CPI-17 was determined by immunoblot analysis with phosphospecific antibodies as described preciously (25, 40). Muscle cells were treated with motilin for 30 s or 5 min in the presence or absence of various inhibitors and solubilized on ice in a medium containing 20 mM Tris·HCl (pH 8.0), 1 mM DTT, 100 mM NaCl, 0.5% SDS, 0.75% deoxycholate, 1 mM PMSF, 10 µg/ml leupeptin, and 100 µg/ml aprotinin. The lysate proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were incubated for 12 h with phosphospecific antibodies to MLC₂₀ (Ser¹⁹), MYPT1 (Thr⁶⁹⁶), or CPI-17 (Thr³⁸) and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies. The bands were identified by enhanced chemiluminescence.

Measurement of contraction in dispersed smooth muscle cells. Contraction was determined in freshly dispersed gastric and intestinal circular muscle cells by scanning micrometry as described previously (21, 23, 24). A cell aliquot containing 10⁴ cells/ml was treated with motilin in the presence or absence of various inhibitors; the reaction was terminated with 1% acrolein at a final concentration of 0.1%. The lengths of muscle cells treated with motilin were measured and compared with the lengths of untreated cells. Contractile response was expressed as percent decrease in mean cell length from control.

Materials. Motilin was obtained from Bachem (King of Prussia, PA), [³⁵S]GTPS, myo-[³H]inositol, ¹²⁵I-labeled motilin, and ⁴⁵Ca²⁺ were from New England Nuclear (Boston, MA); polyclonal antibodies to G subunits, MLC₂₀, MYPT1, and CPI-17 were from Santa Cruz Biotechnology (San Cruz, CA); U-73122, ML-9, Y27632, and bisindolylmalemide were from Calbiochem (San Diego, CA); pGreen Lantern-1 and Lipofectamine Plus reagent were from Life Technologies GIBCO-BRL (Rockville, MD); and all other reagents were from Sigma.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Identification of motilin receptors. Motilin inhibited specific ¹²⁵I-labeled motilin binding to cultured gastric smooth muscle in a concentration-dependent fashion with an IC₅₀ of 0.7 ± 0.2 nM (Fig. 1A). The binding of ¹²⁵I-labeled motilin to vector-transfected gastric muscle cells decreased by 87 ± 2% following exposure of the cells for 20 min to 1 µM motilin but decreased by only 21 ± 10% in cells expressing dominant negative GRK2(K220R) (Fig. 1B). The results implied that motilin receptors are internalized via a mechanism involving GRK2-mediated phosphorylation.

View larger version (16K): [in this window] [in a new window]   Fig. 1. 125I-labeled motilin binding to cultured muscle cells. A: muscle cells were incubated with 125I-labeled motilin alone and in the presence of various concentrations of unlabeled motilin. Specific 125I-labeled motilin binding was competitively inhibited by unlabeled motilin. B: 125I-labeled motilin binding in cells expressing vector or dominant negative G protein-coupled receptor kinase 2 (GRK2) (K220R) before and after treatment for 20 min with 1 µM motilin. Values are means ± SE of 8 experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Time course and concentration dependence of motilin-induced contraction of dispersed smooth muscle cells. A: motilin (1 µM) was added to freshly dispersed intestinal circular smooth muscle cells for various intervals. Contraction consisted of a transient initial peak followed by a sustained contraction. B: motilin was added to dispersed smooth muscle cells at various concentrations for 30 s, and contraction was measured by scanning micrometry. Results are expressed as %decrease in cell length from control (mean control cell length: 121 ± 3 µm). Values are means ± SE of 4 experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Activation of G proteins by motilin in dispersed muscle cells. Membranes isolated from freshly dispersed intestinal smooth muscle cells were incubated for 20 min with [35S]GTPS in the presence or absence of motilin (1 µM) in wells coated with various G antibodies. Motilin caused selective activation of Gq and G13. Results are expressed as motilin-induced increase in [35S]GTPS binding to specific G isoforms. Values are means ± SE of 4 experiments. **Significant increase in the G activation (P < 0.01).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Motilin-induced phosphoinositide (PI) hydrolysis in cultured smooth muscle cells. Cultured gastric circular smooth muscle cells were prelabeled with myo-[3H]inositol, and PI hydrolysis in response to motilin was measured after 60 s by ion-exchange chromatography. Results are expressed as total [3H]inositol phosphate formation in counts per minute (cpm) per milligram of protein. PI hydrolysis was inhibited in cells expressing Gq minigene but not G13 minigene. Values are means ± SE of 4 experiments. **Significant inhibition from control response (P < 0.01).

View larger version (30K): [in this window] [in a new window]   Fig. 5. Motilin-induced PI hydrolysis in cells expressing dominant negative RGS4(N88S) or GRK2(K220R). A: cultured gastric circular smooth muscle cells overexpressing RGS4(N88S), wild-type (WT) RGS4, or vector alone were prelabeled with myo-[3H]inositol, PI hydrolysis in response to 1 µM motilin was measured after 60 s, and the results were expressed as total [3H]inositol phosphate formation in cpm/mg protein. Values are means ± SE of 4 experiments. **Significant inhibition from control response (P < 0.01); ##significant increase from control response (P < 0.01). B: cultured muscle overexpressing GRK2(K220R), WT GRK2, or vector alone were pretreated for 20 min with 1 µM motilin, after which the PI hydrolysis in response to 1 µM motilin was determined. Values are means ± SE of 4 experiments. **Significant difference from response in vector-expressing cells (P < 0.01).

Motilin-stimulated IP₃-dependent Ca²⁺ release. Motilin stimulated Ca²⁺ release (31 ± 2% decrease in steady-state ⁴⁵Ca²⁺ content in 15 s) in freshly dispersed permeabilized intestinal smooth muscle cells to the same extent as 1 µM IP₃ (32 ± 3%; Fig. 6). Ca²⁺ release was strongly inhibited by heparin, a blocker of IP₃ receptors, and U-73122, an inhibitor of PI hydrolysis (5 ± 1 and 3 ± 2% decrease in steady-state ⁴⁵Ca²⁺ content, respectively; P < 0.001 from control), implying that motilin induces a prompt Ca²⁺ release mediated by IP₃ (Fig. 5). Ca²⁺ release was accompanied by increase in cytosolic Ca²⁺ measured in fura 2-loaded single muscle cells (Fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Motilin-induced Ca2+ release and cytosolic free Ca2+ {[Ca2+]i} in smooth muscle cells. Ca2+ release was measured in permeabilized muscle cells loaded with 45Ca2+ as described in MATERIALS AND METHODS. U-73122 (1 µM) or heparin (100 ng/ml) were added 10 min before addition of a maximal concentration of motilin (1 µM). Results are expressed as the maximal decrease in steady-state 45Ca2+ cell content (2.53 ± 0.28 nmol/106 cells) 15 s after addition of motilin or inositol 1,4,5-trisphosphate (IP3). Values are means ± SE of 4 experiments. **Significant inhibition of Ca2+ release (P < 0.01). Inset: fluorescence ratio in fura 2-loaded single smooth muscle cell exposed to motilin (1 µM).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Inhibition of motilin-induced initial and sustained muscle contraction in intestinal circular muscle cells. Freshly dispersed intestinal circular muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), and Y27632 (1 µM) for 10 min and then treated with motilin (1 µM). Contraction was measured after 30 or 300 s. Muscle contraction was measured by scanning micrometry and expressed as %decrease in cells length from control (mean control cell length: 121 ± 3 µm). Values are means ± SE of 4 experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Motilin-induced phosphorylation of MLC20. Freshly dispersed intestinal circular muscle cells were treated for 30 and 300 s with 1 µM motilin after the addition of 1 µM ML-9, 1 µM Y27632, or 1 µM bisindolylmaleimide. MLC20 phosphorylation was determined using phosphospecific Ser19 MLC20 antibody. Equal amounts of protein (50 µg) were loaded and confirmed by Western blot analysis using MLC20 antibody. Values are means ± SE of 3 experiments.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Inhibition of motilin-induced initial and sustained muscle contraction in gastric circular muscle cells. Freshly dispersed gastric circular muscle cells were incubated with U-73122 (1 µM), ML-9 (10 µM), bisindolylmaleimide (1 µM), or Y27632 (1 µM) for 10 min and then treated with motilin (1 µM). Contraction was measured after 30 or 300 s. Muscle contraction was measured by scanning micrometry and expressed as %decrease in cell length from control (mean control cell length: 114 ± 4 µm). Values are means ± SE of 4 experiments.

Similar data were obtained in dispersed gastric muscle cells, where sustained contraction was partly inhibited by Y27632 and bisindolylmaleimide and was abolished by a combination of both inhibitors but was not affected by U-73122 or ML-9 (Fig. 9).

G_q- and G₁₃-dependent activation of Rho kinase by motilin. Depending on the agonist, RhoA is activated via G₁₃ alone or via both G₁₃ and G_q (9, 25, 40). Treatment of cultured gastric smooth muscle cells with motilin for 5 min caused a fourfold increase in Rho kinase activity above basal level (Fig. 10). Rho kinase activity was partly inhibited in cells expressing G_q minigene or G₁₃ minigene and was virtually abolished in cells coexpressing both minigenes (Fig. 10), implying participation of both G proteins in motilin-induced activation of RhoA.

View larger version (23K): [in this window] [in a new window]   Fig. 10. Motilin-induced activation of Rho kinase. Cultured gastric smooth muscle cells expressing Gq, G13, or both Gq and G13 minigenes were treated with motilin (1 µM) for 300 s, and Rho kinase activity was measured as described in MATERIALS AND METHODS. Results are expressed as cpm/mg protein. There is partial inhibition by each minigene and complete inhibition by both minigenes. Values are means ± SE of 4 experiments. **Significant inhibition from motilin-stimulated Rho kinase activity (P < 0.01).

View larger version (24K): [in this window] [in a new window]   Fig. 11. Motilin-induced phosphorylation of MYPT1 and CPI-17. Dispersed muscle cells were treated with motilin (1 µM) for 300 s. MYPT1 and CPI-17 phosphorylation was measured using phosphospecific Thr696 MYPT1 antibody and phosphospecific Thr38 CPI-17 antibody, respectively. Equal amounts of protein (50 µg) were loaded and confirmed by Western blot analysis using MYPT1 or CPI-17 antibody.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

In the present study, we provide a detailed analysis of the signaling pathways that mediate Ca²⁺-dependent and independent contraction by motilin receptors in gastric and intestinal smooth muscle cells. Motilin bound with high affinity (IC₅₀ 1 nM) to motilin receptors on smooth muscle cells. The receptors could be rapidly internalized and desensitized via a GRK2-mediated mechanism: expression of a dominant negative GRK2(K220R) blocked internalization as determined by the binding of ¹²⁵I-labeled motilin to surface receptors and desensitization as determined by measurement of PI hydrolysis. Treatment of the cells with motilin induced a concentration-dependent contraction consisting of an initial transient phase followed by a sustained phase. Only the initial phase was Ca²⁺ dependent, reflecting G_q-mediated stimulation of PI hydrolysis and IP₃-dependent Ca²⁺ release. The response was attenuated by wild-type RGS4 and augmented in cells overexpressing RGS4(N88S). Initial contraction and MLC₂₀ phosphorylation were inhibited by the MLCK inhibitor ML-9 but were not affected by Rho kinase or PKC inhibitors. In this respect, motilin receptors conformed to a pattern observed with other G_q-coupled receptors in gastrointestinal smooth muscle, including muscarinic M₃, CCK-A (23), endothelins ET_A and ET_B (9), 5-HT₂ (13), histamine H₁ (20), S1P₂ (40), neuropeptide Y/peptide YY Y₂, pancreatic polypeptide Y₄ (unpublished observation), UTP P2Y₂ (24), and LPA₃ (39) receptors, all of which stimulate PI hydrolysis by activating PLC-1 via G_q.

During the sustained Ca²⁺-independent phase, MLC₂₀ phosphorylation and muscle contraction are mediated by G protein-dependent inhibition of MLC phosphatase. The pathways that lead to inhibition of MLC phosphatase vary with the agonist, but they usually involve phosphorylation of the regulatory subunit of MLC phosphatase via Rho kinase, phosphorylation of CPI-17, an endogenous inhibitor of MLC phosphatase via PKC, or phosphorylation of both MYPT1 and CPI-17 (9, 25, 39, 40). G_i-coupled receptor agonists trigger a distinct pathway involving phosphorylation of both CPI-17 and MLC₂₀ via integrin-linked kinase (ILK) (22). A role for ILK has been suggested also for the transient contraction induced in esophageal smooth muscle by phosphatase inhibitors (12). Inhibition of MLC phosphatase and stimulation of MLC₂₀ phosphorylation by motilin receptors involved phosphorylation of both MYPT1 by Rho kinase and CPI-17 by PKC. The upstream pathway involves G_q- and/or G₁₃-dependent activation of RhoA, which results in activation of both Rho kinase and PLD (25). Dephosphorylation of phosphatidic acid, the primary product of PLD activity, yields diacylglycerol, which, in turn, activates PKC. Sustained contraction and MLC₂₀ phosphorylation by motilin were partially inhibited by the Rho kinase inhibitor Y27632 and the PKC inhibitor bisindolylmaleimide, and they were abolished by a combination of both inhibitors. In this respect, signaling by motilin receptors closely mimicked signaling by M₃ and S1P₂ receptors, where MLC₂₀ phosphorylation results from coordinate inhibition of MLC phosphatase via MYPT1 and CPI-17 (25, 40). It differs from signaling by ET_A receptors (9), which involves selective phosphorylation of MYPT1, or signaling by LPA₃ receptors (39), which involves selective phosphorylation of CPI-17.

In summary, motilin receptors located on circular smooth muscle cells of the stomach and intestine are coupled to both G_q and G₁₃. The receptors are rapidly desensitized and internalized via a GRK2-dependent mechanism, and the initial G_q-mediated response (PI hydrolysis) is modulated by RGS4. Initial contraction and MLC₂₀ phosphorylation by motilin is mediated by Ca²⁺/calmodulin-dependent MLCK. In contrast, sustained contraction and MLC₂₀ phosphorylation are mediated by a dual pathway involving G_q- and G₁₃-mediated activation of RhoA, leading to phosphorylation of MYPT1 and CPI-17 by Rho kinase and PKC, respectively. Phospshorylation of both MYPT1 and CPI-17 caused cooperative inhibition of MLC phosphatase and resulted in sustained MLC₂₀ phosphorylation and contraction. A model depicting the signaling pathways initiated by motilin receptors in gastrointestinal smooth muscle is depicted in Fig. 12.

View larger version (25K): [in this window] [in a new window]   Fig. 12. Pathways mediating initial and sustained contraction and MLC20 phosphorylation by motilin. Initial contraction and MLC20 phosphorylation induced by motilin involve Gq-dependent PLC- activation, IP3 generation and Ca2+ release, and Ca2+/calmodulin-dependent activation of MLC kinase. Sustained contraction and MLC20 phosphorylation involve Gq- and G13-dependent activation of Rho kinase, phosphorylation of MYPT1 and CPI-17, and inhibition of MLC phosphatase.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. S. Murthy, P.O. Box 908711, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail: skarnam{at}hsc.vcu.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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Brown JC, Mutt V, and Dryburgh JR. The further purification of motilin, a gastric motor activity stimulating polypeptide from the mucosa of the small intestine of hogs. Can J Physiol Pharmacol 49: 399–405, 1971.[ISI][Medline]
2. Depoortere I and Peeters TL. Transduction mechanism of motilin and motilides in rabbit duodenal smooth muscle. Regul Pept 55: 227–235, 1995.[CrossRef][ISI][Medline]
3. Depoortere I, Peeters TL, and Vantrappen G. Distribution and characterization of motilin receptors in the cat. Peptides 14: 1153–1157, 1993.[CrossRef][ISI][Medline]
4. Depoortere I, Peeters TL, and Vantrappen G. Motilin receptors of the rabbit colon. Peptides 12: 89–94, 1991.[CrossRef][ISI][Medline]
5. Farrugia G, Macielag MJ, Peeters TL, Sarr MG, Galdes A, and Szurszewski JH. Motilin and OHM-11526 activate a calcium current in human and canine jejunal circular smooth muscle. Am J Physiol Gastrointest Liver Physiol 273: G404–G412, 1997.[Abstract/Free Full Text]
6. Feighner SD, Tan CP, McKee KK, Palyha OC, Hreniuk DL, Pong SS, Austin CP, Figueroa D, Macneil D, Cascieri MA, Nargund R, Bakshi R, Abramovitz M, Stocco R, Kargman S, O'Neill G, Vander Ploeg LHT, Evans J, Patchett AA, Smith RG, and Howard AD. Receptor for motilin indentified in the human gastrointestinal system. Science 284: 2184–2188, 1999.[Abstract/Free Full Text]
7. Harada N, Chijiiwa Y, Misawa T, Yoshinaga M, and Nawata H. Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine. Life Sci 51: 1381–1387, 1992.[CrossRef][ISI][Medline]
8. Hasler WL, Heldsinger A, and Owyang C. Erythromycin contracts rabbit colon myocytes via occupation of motilin receptors. Am J Physiol Gastrointest Liver Physiol 262: G50–G55, 1992.[Abstract/Free Full Text]
9. Hersch E, Huang J, Grider JR, and Murthy KS. G_q/G₁₃ signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ET_A and CPI-17 dephosphorylation via ET_B. Am J Physiol Cell Physiol 287: C1209–C1218, 2004.[Abstract/Free Full Text]
10. Itoh Z. Motilin and clinical aplplication. Peptides 18: 593–608, 1997.[CrossRef][ISI][Medline]
11. Itoh Z, Honda R, Hiwatashi K, Takeuchi S, Aizawa I, Takayanagi R, and Couch EF. Motilin-induced mechanical activity in the canine alimentary tract. Scand J Gastroenterol 11: 93–110, 1976.[ISI][Medline]
12. Kim N, Cao W, Song IS, Kim CY, Harnett KM, Cheng L, Walsh MP, and Biancani P. Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles. Am J Physiol Cell Physiol 287: C384–C394, 2004.[Abstract/Free Full Text]
13. Kuemmerle JF, Martin DC, Murthy KS, Kellum JM, Grider JR, and Makhlouf GM. Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca²⁺ mobilization. Mol Pharmacol 42: 1090–1096, 1992.[Abstract]
14. Lee KY, Chang TM, and Chey WY. Effect of rabbit antimotilin serum on myoelectric activity and plasma motilin concentrations in the fasting dog. Am J Physiol Gastrointest Liver Physiol 245: G547–G553, 1983.[Abstract/Free Full Text]
15. Louie DS and Owyang C. Motilin receptors on isolated gastric smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 254: G210–G216, 1988.[Abstract/Free Full Text]
16. Lu G, Sarr MG, and Szurszewski JH. Effect of motilin and erythromycin on calcium-activated potassium channels in rabbit colonic myocytes. Gastroenterology 114: 748–754, 1998.[CrossRef][ISI][Medline]
17. Matthijs G, Peeters TL, and Vantrappen G. The role of intracellular calcium stores in motilin induced contractions of the rabbit duodenum. Naunyn Schmiedebergs Arch Pharmacol 339: 332–339, 1989.[ISI][Medline]
18. Miller P, Roy A, St-Pierre S, Dagenais M, Lapointe R, and Poitras P. Motilin receptors in the human antrum. Am J Physiol Gastrointest Liver Physiol 278: G18–G23, 2000.[Abstract/Free Full Text]
20. Morini G, Kuemmerle JF, Impicciatore M, Grider JR, and Makhlouf GM. Coexistance of Histamine H₁ and H₂ receptors coupled to distinct signal transduction pathways in isolated intestinal muscle cells. J Pharmacol Exp Ther 264: 598–603, 1992.
21. Murhy KS, Grider JR, Kuemmerle JF, and Makhlouf GM. Sustained muscle contraction induced by agonist, growth factors and Ca²⁺ mediated by distinct PKC isozymes. Am J Physiol Gastrointest Liver Physiol 279: G201–G210, 2000.[Abstract/Free Full Text]
22. Murthy KS, Huang J, Zhou H, Kuemmerle JF, and Makhlouf GM. Receptors coupled to inhibitory G proteins induce MLC20 phosphorylation and muscle contraction via PI 3-kinase-dependent activation of integrin-linked kinase (ILK) (Abstract). Gastroenterology 126: A413, 2004.
23. Murthy KS and Makhlouf GM. Opioid µ, and receptor-induced activation of phospholipase C-3 and inhibition of adenylyl cyclase is mediated by Gi2 and Go in smooth muscle. Mol Pharmacol 50: 870–877, 1996.[Abstract]
24. Murthy KS and Makhlouf GM. Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-1 via Gq/11 and to PLC-3 via Gi3. J Biol Chem 273: 4695–4704, 1998.[Abstract/Free Full Text]
25. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM. Differential signaling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1, and p21-activated kinase pathway, and m3-mediated MLC₂₀ phosphorylation via Rho kinase/myosin phosphatase targeting subunit I and protein kinase C/CPI-17 pathway. Biochem J 374: 145–155, 2003.[CrossRef][ISI][Medline]
26. Okamoto T, Ikezu T, Murayama Y, Ogata E, and Nishimoto I. Measurement of GTPS binding to specific G proteins in membranes using G protein antibodies. FEBS Lett 305: 125–128, 1992.[CrossRef][ISI][Medline]
27. Parkman HP, Pagano AP, Vozzelli MA, and Ryan JP. Gastrokinetic effects of erythromycin: myogenic and neurogenic mechanisms of action in rabbit stomach. Am J Physiol Gastrointest Liver Physiol 269: G418–G426, 1995.[Abstract/Free Full Text]
28. Peeters TL, Matthijs G, and Vantrappen G. Ca²⁺ dependence of motilide-induced contractions in rabbit duodenal muscle strips in vitro. Naunyn Schmiedebergs Arch Pharmacol 343: 202–208, 1991.[CrossRef][ISI][Medline]
29. Poitras P. In: Gut Peptides: Biochemistry and Physiology, edited by Walsh JH and Dockray GJ. New York: Raven, 1994, p. 261–304.
30. Sarna SK, Gonzalez A, and Ryan RP. Enteric locus of action of prokinetics: ABT-229, motilin, and erythromycin. Am J Physiol Gastrointest Liver Physiol 278: G744–G752, 2000.[Abstract/Free Full Text]
31. Satoh M, Sakai T, Sano I, Fujikura K, Koyama H, Ohshima K, Itoh Z, and Omura S. EM574, an erythromycin derivative, is a potent motilin receptor agonist in human gastric antrum. J Pharmacol Exp Ther 271: 574–579, 1994.[Abstract/Free Full Text]
32. Thielemans L, Depoortere I, Van Assche G, Bender E, and Peeters TL. Demonstration of a functional motilin receptor in TE671 cells from human cerebellum. Brain Res 895: 119–128, 2001.[CrossRef][ISI][Medline]
33. Van Assche G, Depoortere I, and Peeters TL. Localization of motilin binding sites in subcellular fractions from rabbit antral and colonic smooth muscle tissue. Regul Pept 77: 89–94, 1998.[CrossRef][ISI][Medline]
34. Van Assche G, Depoortere I, Thijs T, Janssens JJ, and Peeters TL. Concentration-dependent stimulation of cholinergic motor nerves or smooth muscle by [Nle13] motilin in the isolated rabbit gastric antrum. Eur J Pharmacol 337: 267–274, 1997.[CrossRef][ISI][Medline]
35. Van Assche G, Depoortere I, Thijs T, Missiaen L, Penninckx F, Takanashi H, Geboes K, Janssens J, and Peeters TL. Contractile effects and intracellular Ca²⁺ signaling induced by motilin and erythromycin in the circular smooth muscle of human colon. Neurogastroenterol Motil 13: 27–35, 2001.[CrossRef][ISI][Medline]
36. Van Lier Ribbink JA, Sarr MG, and Tanaka M. Neural isolation of the entire canine stomach in vivo: effect on motility. Am J Physiol Gastrointest Liver Physiol 257: G30–G40, 1989.[Abstract/Free Full Text]
37. Vantrappen G and Peeters TL. Motilin. In: Handbook of Physiology. The Gastrointestinal System. Neural and Endocrine Biology. Bethesda, MD: Am Physiol Soc, 1989, sect. 6, vol. II, chapt. 22, p. 545–558.
38. Yamada K, Yanagida H, Ito Y, and Inoue R. Postsynaptic enhancement by motilin of muscarinic receptor cation currents in duodenal smooth muscle. Am J Physiol Gastrointest Liver Physiol 274: G487–G492, 1998.[Abstract/Free Full Text]
39. Zhou H, Huang J, and Murthy KS. Lysophosphatidic acid (LPA) interacts with LPA₃ receptors to activate selectively G_q and induce initial and sustained MLC₂₀ phosphorylation and contraction (Abstract). Gastroenterology 126: A278, 2004.[CrossRef]
40. Zhou H and Murthy KS. Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P₁ and S1P₂. Am J Physiol Cell Physiol 286: C1130–C1138, 2004.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. S. Murthy and W. Sriwai Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A Am J Physiol Gastrointest Liver Physiol, January 1, 2008; 294(1): G327 - G335. [Abstract] [Full Text] [PDF]

				W. Hu, S. Mahavadi, F. Li, and K. S. Murthy Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1 Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1991 - C2000. [Abstract] [Full Text] [PDF]

				H. K. Kim, W. S. Park, S. H. Kang, M. Warda, N. Kim, J.-H. Ko, A. E.-b. Prince, and J. Han Mitochondrial alterations in human gastric carcinoma cell line Am J Physiol Cell Physiol, August 1, 2007; 293(2): C761 - C771. [Abstract] [Full Text] [PDF]

				J. Huang, H. Zhou, S. Mahavadi, W. Sriwai, and K. S. Murthy Inhibition of G{alpha}q-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2 Am J Physiol Cell Physiol, January 1, 2007; 292(1): C200 - C208. [Abstract] [Full Text] [PDF]

				S. K. Sarna Molecular, functional, and pharmacological targets for the development of gut promotility drugs Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G545 - G555. [Abstract] [Full Text] [PDF]

				M. A. F. De Godoy and S. Rattan Autocrine regulation of internal anal sphincter tone by renin-angiotensin system: comparison with phasic smooth muscle Am J Physiol Gastrointest Liver Physiol, December 1, 2005; 289(6): G1164 - G1175. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire