Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

Nayoung Kim,1 Weibiao Cao,2 In Sung Song,3 Chung Yong Kim,3 Karen M. Harnett,2 Ling Cheng,2 Michael P. Walsh,4 and Piero Biancani2

1Department of Medicine, Seoul National University, Bundang Hospital, Seoungnam, Gyeronggi-Do 463-707; 3Department of Medicine, Seoul National University College of Medicine and Liver Research Institute, Seoul, Korea 110-799; 2Department of Medicine, Rhode Island Hospital, and Division of Biology and Medicine, Brown University Medical School, Providence, Rhode Island 02903; and 4Smooth Muscle Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Submitted 12 September 2003 ; accepted in final form 1 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca2+ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca2+ levels. The link between activation of the Ca2+-independent protein kinase C{epsilon} (PKC{epsilon}) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC{epsilon}-dependent, Ca2+-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC{epsilon}, but not antibodies to PKC{beta}II, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC{epsilon} from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC{epsilon}, MEK, ERK1/2, and ILK.

protein kinase C; myosin light chain kinase; phosphatase; integrin-linked kinase


SMOOTH MUSCLE CONTRACTION is regulated primarily by the reversible phosphorylation of myosin (1, 27, 58), depending on the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities, and both enzymes are subject to regulation. Most contractile stimuli elicit an increase in intracellular free Ca2+ concentration ([Ca2+]i) via entry of Ca2+ from the extracellular space and/or release from intracellular stores, whereupon Ca2+ activates calmodulin (CaM)-dependent MLCK. Activated MLCK phosphorylates the 20-kDa myosin light chains (MLC) of myosin II at Ser19 to activate cross-bridge cycling and the development of force or shortening of the muscle. Relaxation generally follows removal of the stimulus, whereupon [Ca2+]i returns to resting levels as Ca2+ is removed from the cytosol. MLCK (32) is inactive at the lower [Ca2+]i level, and myosin that was phosphorylated during the contractile phase of the cycle becomes dephosphorylated by MLCP, a serine/threonine type 1 protein phosphatase (28). Because MLCK activation depends on activation of CaM, which is critically dependent on a relatively high Ca2+ concentration, phosphatase inhibition as a mechanism for smooth muscle contraction has been invoked to explain contraction at low cytosolic Ca2+ levels.

MLCP is subject to complex regulation. The holoenzyme is a trimer of a catalytic subunit of 37–38 kDa, a 110- to 130-kDa myosin targeting subunit (MYPT1), and a 20-kDa subunit of unknown function. The best characterized mechanisms of inhibition of MLCP involve phosphorylation of MYPT1, which is accompanied by a decrease in phosphatase activity (20, 43, 46), and interaction of phosphorylated CPI-17 with the catalytic subunit of the phosphatase (51). CPI-17 is a 17-kDa cytosolic protein that becomes a potent inhibitor of type 1 protein phosphatase when phosphorylated at Thr38 (18). MYPT1 can be phosphorylated by various kinases, including Rho-associated kinase (ROK) (20, 36), MYPT1 kinase [a zipper-interacting protein (ZIP)-like kinase (44)], Raf-1 (9), and integrin-linked kinase (ILK) (46). The inhibitory phosphorylation site in MYPT1 is Thr695 (20), although phosphorylation at other sites may also have an inhibitory effect (46). Phosphorylation of CPI-17 at Thr38 is catalyzed by protein kinase C (PKC) (18), ROK (40), protein kinase N (23), MYPT1 kinase (44), and ILK (13). Inhibition of MLCP activity plays an important role in "Ca2+ sensitization" of smooth muscle contraction, i.e., agonist-induced increase in force or contraction without a change in [Ca2+]i (59).

It is becoming increasingly clear that, while smooth muscle contraction in some cases is regulated by the balance between MLCK and MLCP activities, exceptions occur. This is exemplified by comparison of the circular smooth muscles of the esophagus (ESO) and the lower esophageal sphincter (LES) of the cat, which fulfill quite different physiological roles. ESO is normally relaxed and exhibits a brief and powerful phasic contraction in response to stimulation of cholinergic nerves induced by swallowing. LES, however, maintains spontaneous myogenic tone and relaxes in response to stimulation of nonadrenergic, noncholinergic neurons. Previous work has suggested that fundamental differences exist in the signal transduction pathways involved in excitation-contraction coupling in these two smooth muscles (54, 55) for the initial phase of contraction (47). In both cases, contractile stimuli such as acetylcholine (ACh) trigger an increase in MLC phosphorylation (54). In the case of LES, both phosphorylation and contraction are inhibited by CaM and MLCK inhibitors and are unaffected by PKC inhibitors (3, 54). ACh-induced contraction of LES involves activation of M3 muscarinic receptors, which are coupled to phosphatidylinositol phospholipase C (PI-PLC) via Gq/11. PI-PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum, activation of CaM-dependent MLCK, MLC phosphorylation, and contraction (55). In sharp contrast, ACh-induced MLC phosphorylation and contraction in ESO are inhibited by PKC inhibitors but not by CaM or MLCK inhibitors (54). In this case, ACh-induced contraction involves activation of M2 muscarinic receptors, which are coupled to both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) via Gi3 (55). Both phospholipases generate DAG, PC-PLC directly via hydrolysis of phosphatidylcholine (PC) and PLD indirectly via hydrolysis of PC to phosphatidic acid (PA) and hydrolysis of PA to DAG via phosphatidate phosphohydrolase. DAG activates PKC{epsilon}, a Ca2+-independent (novel) PKC isoform (57). The link between PKC{epsilon} activation and the observed increase in MLC phosphorylation in ESO in response to ACh has not been elucidated. These results led to the unexpected conclusion that in ESO, unlike LES, MLCK is not the major kinase responsible for MLC phosphorylation and contraction.

In this study, we used a variety of molecular tools (phosphatase and kinase inhibitors and antibodies to signaling enzymes) in combination with intact and saponin-permeabilized isolated ESO and LES smooth muscle cells to compare the mechanisms of excitation-contraction coupling in these two smooth muscle types. Specifically, we examined the signaling pathways activated by phosphatase inhibition. LES contraction induced by phosphatase inhibition was reduced by MLCK inhibitors, supporting a central role of MLCK in this tissue. In contrast, MLCK plays no role in phosphatase inhibitor-induced contraction of ESO. Rather, contraction of ESO by phosphatase inhibitors is mediated by activation of a PKC{epsilon}-dependent pathway, which also involves mitogen-activated protein (MAP) kinase kinase (MEK), the MAP kinases ERK1 and ERK2, and activation of ILK. Thus, in ESO, phosphatase inhibitors do not evoke contraction by potentiating MLCK but likely inhibit MLCP and phosphatases that oppose the effects of activation of PKC{epsilon}. Activation of PKC{epsilon} then results in activation of a number of kinases, which include ERK1/2 and ILK.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult cats of either sex weighing between 2.5 and 4 kg after an overnight fast were anesthetized with ketamine (30 mg/kg) and then euthanized with an overdose of pentobarbital sodium in accordance with the guidelines of Brown University for the humane treatment of experimental animals. The chest and abdomen were opened with a midline incision to expose the ESO and stomach. The ESO and stomach were removed together, pinned to a wax block at their in vivo dimensions and orientation, and opened along the lesser curvature. The high-pressure zone is characterized by a visible thickening of the circular muscle layer corresponding to the squamocolumnar junction and immediately proximal to the sling fibers of the stomach. Investigators at our laboratory (4, 5) previously showed that a 5- to 8-mm band of tissue coinciding with the thickened area constitutes the LES and has distinct characteristics when examined in vivo, in the organ bath, or as single cells after enzymatic digestion. The mucosa and submucosal connective tissues were removed by sharp dissection, and the LES was excised. A 3- to 5-mm-wide strip at the junction of LES and ESO was discarded to avoid overlap. The circular muscle layer of the ESO was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA). The last slices, containing the myenteric plexus, longitudinal muscle, and serosa, were discarded, and the remaining slices were cut by hand into 2 x 2-mm tissue squares.

Dispersion of smooth muscle cells. Tissue squares were digested in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered physiological solution containing 150 U/ml collagenase type II to dissociate smooth muscle cells as described previously (5, 35). The HEPES solution contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 10.8 glucose, 24.0 HEPES, 1.9 CaCl2, and 0.6 MgCl2, as well as 0.3 mg/ml basal medium Eagle (BME) amino acid supplement and 0.08 mg/ml soybean trypsin inhibitor. The solution was oxygenated (100% O2) at 31°C, and pH was adjusted to 7.4. During the digestion period, the gas flow rate was kept low to avoid agitating the tissue. At the end of the digestion period, the tissue was poured over a 500-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and incubated in this solution at 31°C, gassed with 100% O2. The cells were allowed to dissociate freely for 10–20 min. Care was taken not to agitate the fluid to avoid cell contraction in response to mechanical stress. All glassware used in this procedure was siliconized with a 0.05% silicon solution (Sigma, St. Louis, MO) to prevent the cells from adhering to the glass.

The isolated cells were relaxed, as their unstimulated length did not change after prolonged incubation in Ca2+-free medium. For instance, in intact LES cells, the initial cell length was 67–72 µm in normal buffer and 68–72 µm in Ca2+-free medium.

Preparation of permeable smooth muscle cells. Cells were permeabilized to allow the use of microcystin-LR and various antibodies, which do not cross the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with an enzyme-free cytosolic buffer (buffer A) composed of (in mM) 20 NaCl, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 25 NaHCO3, 0.61 CaCl2, and 1 EGTA, as well as 2% bovine serum albumin, yielding 0.36 µM free Ca2+, calculated according to the method of Fabiato and Fabiato (19a). Investigators at our laboratory (3) previously showed that the maximal contractile response of permeabilized ESO smooth muscle cells to ACh requires 0.36 µM Ca2+ in buffer A. Buffer A was equilibrated with 95% O2-5% CO2 to maintain pH 7.2 at 31°C. Smooth muscle cells dispersed spontaneously in this medium. After dispersion, the cells were permeabilized by incubation for 3 min in buffer A containing saponin (75 µg/ml). After exposure to saponin, the cell suspension was centrifuged at 500 g, and the resulting pellet was resuspended in saponin-free buffer A containing 10 µM antimycin A, 1.5 mM ATP, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase (6, 55). After the cells were washed free of saponin, they were resuspended in the same buffer. Permeabilization did not affect the unstimulated cell length. For instance, the length of permeable unstimulated ESO cells was ~70 µm and was the same as the length of unstimulated intact cells (70–75 µm).

To measure phosphatase inhibitor-induced contractions, ESO and LES circular smooth muscle cells were exposed to okadaic acid for 2 min, to microcystin-LR for 1 min, or to antibodies against the catalytic subunit of type 1 protein phosphatase for 1 min. Cells were preincubated with appropriate concentrations of kinase inhibitors (ML-7, ML-9, chelerythrine, or SM-1) for 1 min before the addition of phosphatase inhibitor. Cells were incubated with appropriate concentrations of the MEK antagonist PD-98059 (66) for 1 h before the addition of phosphatase inhibitor. When PKC or MLCK antibodies were used, the permeabilized cells were incubated in antiserum for 1 h at 31°C before the addition of phosphatase inhibitor (7, 55). After exposure to phosphatase inhibitors, the cells were fixed in 1% acrolein. A drop of the cell-containing medium was placed on a glass slide and covered by a coverslip, and the edges were sealed with clear nail enamel to prevent evaporation.

Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany) and a closed-circuit television camera (model WV-CD51; Panasonic, Secaucus, NJ) connected to a Macintosh computer (Apple, Cupertino, CA). The Image software program (National Institutes of Health, Bethesda, MD) was used to measure cell length and for data accumulation. The average length of 30 cells, measured in the absence of agonists, was taken as a "control" length and compared with the length after the addition of test agents. The initial length of isolated ESO and LES cells was 65 ± 2 µm (mean ± SE). "Shortening" was defined as the percentage decrease in average length after agonist treatment compared with control length.

The cell shortening model used in this study has been used extensively by different investigators in our laboratory and elsewhere. Over the years, data obtained in isolated ESO and LES cells have been consistent and reproducible. Nevertheless, the degree of isolated cell shortening is less than the shortening capacity of other smooth muscles in intact tissues, and growing appreciation of the importance of cell matrix attachment and cytoskeletal structure in smooth muscle mechanics may suggest caution in extrapolating conclusions obtained from untethered cell preparations to in vivo muscle behavior.

Measurement of PKC activity. PKC activity in immunoprecipitates from ESO was measured with the Pierce colorimetric PKC assay (no. 29510; Rockford, IL). Briefly, a peptide substrate that was labeled with brightly colored fluorescent dye was incubated with the kinase-containing sample. The reaction mixture was applied to an affinity column that binds phosphorylated peptides. To wash nonphosphorylated peptide from the membranes, the column was washed twice with phosphopeptide binding buffer. To elute the phosphorylated peptide from the affinity membranes, the membranes were washed twice with phosphopeptide elution buffer. The phosphorylated product was quantified by measurement of its absorbance at 570 nm.

Measurement of ILK activity. ESO circular smooth muscle was homogenized (3- to 20-s burst, Tissue Tearer; Biospec Products, Bartlesville, OK) in a 3- to 20-s burst in 50 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 µg/ml leupeptin, 2.5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium fluoride, and 1 mM sodium orthovanadate. The sample was centrifuged with a Fisher microfuge (15,000 g, 10 min, 4°C; Pittsburgh, PA), and the supernatant was collected. An ILK antibody (Upstate Biotechnology, Lake Placid, NY) was purified by dialysis with Slide-A-Lyzer cassettes (Pierce).

ILK was immunoprecipitated from the ESO sample with the Seize primary immunoprecipitation kit (no. 45335; Pierce). Briefly, purified ILK antibody was coupled to an aminolink coupling gel in a Handee spin cup column (Pierce). The ESO sample was applied to the ILK antibody-coupled spin cup column. ILK was eluted with immunopure elution buffer, and the eluent was neutralized with 1 M Tris, pH 9.5.

ILK activity was measured by adding 50 µl of ESO sample and 50 µl of kinase reaction buffer (in mM: 50 HEPES, pH 7.0, 10 MnCl2, 10 MgCl2, 2 NaF, and 1 Na3VO4) containing 5 µCi of [{gamma}-32P]ATP (BLU502A; NEN Life Sciences, Boston, MA) to two plates coated with myelin basic protein (SMP106; NEN Life Sciences). The plates were incubated for 20 min at room temperature, and the reaction was stopped by washing the plates five times with ice-cold reaction buffer. One plate was washed after 0-min incubation, and the other plate was washed after 20-min incubation. The radioactivity of the plates was measured, and the difference between the 0- and 20-min plates in counts per minute was determined.

Protein determination. Protein content of ESO tissue was determined by colorimetric assay (Bio-Rad, Melville, NY) according to the method of Bradford (8a). Briefly, samples were diluted 1,000 times in distilled water and mixed with Bio-Rad protein assay dye reagent (1:4, vol/vol) before quantification of the color change of Coomassie brilliant blue G-250 dye measured at 595 nm.

Drugs and chemicals. We purchased microcystin-LR and PD-98059 from Calbiochem (San Diego, CA), collagenase type II and soybean trypsin inhibitor from Worthington Biochemicals (Freehold, NJ), chelerythrine from LC (Boston, MA), PKC{epsilon} and PKC{beta}II antibodies from GIBCO-BRL (Gaithersburg, MD), and ERK1, ERK2, and MLCK antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). [{gamma}-32P]ATP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Okadaic acid, ML-7, ML-9, saponin, BME amino acid supplement, HEPES, creatine phosphate, creatine kinase, ATP, antimycin A, CaM, MLC, and other reagents were purchased from Sigma. SM-1 (AKKLSKDRMKKYMARRKWQKTG) was synthesized in the Peptide Synthesis Core Facility at the University of Calgary (Calgary, AB, Canada). The purity of the peptide (>95%) was confirmed by analytical high-performance liquid chromatography and amino acid analysis.

Statistical analysis. Data are expressed as means ± SE. Statistical differences between multiple groups were tested by analysis of variance (ANOVA) for repeated measures and checked for significance by performing Scheffé's F-test.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effects of okadaic acid, microcystin-LR, and anti-PP1c on ESO and LES. The type 1 and type 2A protein serine/threonine phosphatase inhibitors okadaic acid and microcystin-LR elicited contractile responses in ESO and LES in a concentration-dependent manner (P < 0.05, ANOVA) (Fig. 1). Okadaic acid, being membrane-permeant, was used with intact cells, whereas microcystin-LR, which is not membrane-permeant, was used with permeabilized cells. Okadaic acid elicited a maximal response at 10–9 M, with ESO and LES shortening of 18.7 ± 2.1 and 18.6 ± 1.6%, respectively (Fig. 1A). Similar results were obtained with microcystin-LR: maximal contractions of ESO and LES were 21.0 ± 1.8 and 18.8 ± 2.2%, respectively (Fig. 1B). There were no statistically significant differences between ESO and LES contractions induced by either okadaic acid or microcystin-LR. The sensitivities of the contractile responses to both phosphatase inhibitors suggest that contraction results from inhibition of type 2A phosphatase, which is more sensitive than type 1 phosphatase to these inhibitors: type 2A phosphatase is inhibited completely by 1–2 nM okadaic acid, whereas these concentrations have little effect on type 1 phosphatase, which is completely inhibited at 1 µM okadaic acid (12); the Ki values for inhibition of type 1 and type 2A phosphatases by microcystin-LR are 0.18 nM and 9 pM, respectively (31). However, incubation of permeabilized ESO and LES smooth muscle cells for 1 min with antibodies against the catalytic subunit of type 1 smooth muscle phosphatase (anti-PP1c) also elicited a contractile response in a concentration-dependent manner (P < 0.01, ANOVA) (Fig. 2). The maximal contractions induced by the antibody or by the phosphatase inhibitors okadaic acid and microcystin-LR were comparable to the maximal contractile responses to ACh (Fig. 2, right). These results indicate that inhibition of types 1 and 2A protein phosphatases triggers a contractile response in both ESO and LES.



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Fig. 1. Phosphatase inhibitor-induced contraction of isolated esophagus (ESO) and lower esophageal sphincter (LES) circular smooth muscle cells. Cells were contracted by 2-min exposure to okadaic acid (A) or by 1-min exposure to microcystin-LR (B) at the indicated concentrations for ESO ({circ}) or LES ({bullet}). Okadaic acid experiments were conducted in intact cells, and microcystin-LR experiments were performed in permeabilized cells. Values are means ± SE for 6 animals, with 30 cells counted for each animal.

 


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Fig. 2. Effect of antibody against catalytic subunit of type 1 protein phosphatase (anti-PP1c) on saponin-permeabilized LES ({blacksquare}) and ESO ({square}) circular smooth muscle cells. Contraction was elicited by addition of anti-PP1c at the indicated dilutions to permeabilized cells. For comparison, the contractile responses of intact cells to acetylcholine (Ach; 10–9 M) are shown at right. Values are means ± SE for 3 animals, with 30 cells counted for each animal. All values are significantly different (P < 0.01) from control (no anti-PP1c).

 
Contraction induced by phosphatase inhibition is mediated by MLCK in LES but not in ESO. When added to intact LES smooth muscle cells before a maximally effective concentration of okadaic acid (10–9 M), the MLCK inhibitors ML-7 and ML-9 caused concentration-dependent inhibition of contraction (P < 0.001, ANOVA), with near-complete inhibition at 10–5 M. Similarly, microcystin-LR (10–9 M)-induced contraction of permeabilized LES smooth muscle cells was inhibited by ML-7 and ML-9 in a concentration-dependent manner (P < 0.001, ANOVA). In contrast, in ESO, ML-7 and ML-9 had little or no effect on okadaic acid- or microcystin-LR-induced contractions (data not shown).

Because ML-7 and ML-9 may not be entirely selective for MLCK (64), we used a selective anti-MLCK antibody to inhibit okadaic acid-induced contraction of permeabilized LES and ESO cells. The antibody inhibited LES cell contraction (P < 0.001, ANOVA) but had no effect on contraction of ESO cells (Fig. 3). Similarly, the effect of an MLCK inhibitor peptide (SM-1) derived from the autoinhibitory domain of MLCK was tested on contraction of permeabilized cells induced by anti-PP1c. SM-1 inhibited LES contraction in a concentration-dependent manner (P < 0.001, ANOVA) but did not affect contraction of ESO (Fig. 4). These data clearly demonstrate that contraction induced by phosphatase inhibition is MLCK dependent in LES but not in ESO circular smooth muscle cells.



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Fig. 3. Effect of anti-myosin light chain kinase (MLCK) antibody on okadaic acid-induced contraction of ESO (A) and LES (B) circular smooth muscle cells. Cell shortening was activated by addition of okadaic acid in the absence ({bullet}) or presence ({circ}) of anti-MLCK (1:200 dilution). Values are means ± SE for 3 animals, with 30 cells counted for each animal.

 


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Fig. 4. Effect of the MLCK inhibitor peptide SM-1 on anti-PP1c-induced contraction of saponin-permeabilized ESO ({circ}) and LES ({bullet}) cells. Permeabilized cells were treated with a maximally effective concentration (1:200 dilution) of anti-PP1c (see Fig. 2) in the absence (control) or presence of the indicated concentrations of SM-1. Values are means ± SE for 3 animals, with 30 cells counted for each animal.

 
Contraction induced by phosphatase inhibition is mediated by PKC in ESO but not in LES. The PKC inhibitor chelerythrine inhibited contraction of intact or permeabilized ESO (as appropriate) induced by a maximally effective concentration of okadaic acid (10–9 M) or microcystin-LR (10–9 M) in a concentration-dependent manner (P < 0.001, ANOVA) (Fig. 5). In contrast, over the same concentration range, chelerythrine had no effect on LES contractions elicited by the phosphatase inhibitors. These results suggest that phosphatase inhibitor-induced contraction of ESO, but not LES, involves PKC.



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Fig. 5. Effect of the protein kinase C (PKC) inhibitor chelerythrine on phosphatase inhibitor-induced contraction of ESO ({circ}) and LES ({bullet}) cells. Cell shortening was activated in intact cells by 10–9 M okadaic acid (A) or in permeabilized cells by 10–9 M microcystin-LR (B) in the absence (control) or presence of the indicated concentrations of chelerythrine. Values are means ± SE for n animals, with 30 cells counted for each animal; n = 4 in A and n = 6 in B.

 
We then used isoenzyme-specific antibodies to identify the PKC isoenzyme that mediates contraction of ESO smooth muscle in response to phosphatase inhibitors. Contraction of permeabilized ESO induced by okadaic acid (10–9 M) or microcystin-LR (10–9 M) was inhibited in a concentration-dependent manner by anti-PKC{epsilon} (P < 0.001, ANOVA) (Fig. 6) but not by anti-PKC{beta}II (Fig. 7). Anti-PKC{epsilon} caused almost complete inhibition of ESO contraction at a 1:200–1:100 dilution. In contrast, anti-PKC{epsilon} or anti-PKC{beta}II had no effect on LES contractions elicited by okadaic acid or microcystin-LR (Figs. 6 and 7).



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Fig. 6. Effect of PKC{epsilon} antibodies on phosphatase inhibitor-induced contraction of ESO ({circ}) and LES ({bullet}) permeabilized cells. Cell shortening was activated by 10–9 M okadaic acid (A) or by 10–9 M microcystin-LR (B) in the absence (control) or presence of the indicated concentrations of anti-PKC{epsilon}. Values are means ± SE for n animals, with 30 cells counted for each animal; n = 5 in A and n = 4 in B.

 


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Fig. 7. Lack of effect of PKC{beta}II antibodies on phosphatase inhibitor-induced contraction of permeabilized ESO cells. Cell shortening was activated in permeabilized cells by 10–9 M okadaic acid ({circ}) or by 10–9 M microcystin-LR ({bullet}) in the absence (control) or presence of the indicated concentrations of anti-PKC{beta}II. Values are means ± SE for 3 animals, with 30 cells counted for each animal.

 
Similarly, contraction induced by anti-PP1c in permeabilized LES cells was inhibited by preincubation with anti-MLCK or the MLCK autoinhibitory peptide SM-1 (P < 0.001, ANOVA) but was not affected by anti-PKC{epsilon} or anti-PKC{beta}II (Fig. 8). In permeabilized ESO cells, however, contraction elicited by anti-PP1c was not affected by anti-MLCK or SM-1 or by anti-PKC{beta}II, but it was inhibited by anti-PKC{epsilon} (P < 0.001, ANOVA). Taken together, these results support the conclusion that phosphatase inhibitor-induced contraction of ESO involves PKC, specifically the PKC{epsilon} isoform, but not MLCK, whereas contraction of LES involves MLCK but not PKC.



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Fig. 8. Effects of anti-MLCK, SM-1, anti-PKC{epsilon}, and anti-PKC{beta}II antibodies (Ab) on contraction of saponin-permeabilized ESO ({square}) and LES ({blacksquare}) cells in response to a maximally effective concentration of anti-PP1c. Cell shortening was activated by anti-PP1c (1:200 dilution) in the absence or presence of anti-MLCK (1:200 dilution), SM-1 (10–5 M), anti-PKC{epsilon} (1:200 dilution), or anti-PKC{beta}II (1:200 dilution). Values are means ± SE for 3 animals, with 30 cells counted for each animal.

 
The importance of PKC, specifically the PKC{epsilon} isoform, in ESO is confirmed in Fig. 9, which shows that okadaic acid treatment of intact ESO cells induces transient translocation of PKC{epsilon} from the cytosolic to the particulate fraction, which reflects activation of the kinase. The cytosolic and membrane fractions were immunoprecipitated with anti-PKC{epsilon} at the indicated times after the addition of okadaic acid, and PKC activity was measured in the immunoprecipitates (Fig. 9, left). The membrane-to-cytosolic activity ratio of PKC was significantly increased 1, 2, and 5 min after okadaic acid stimulation (P < 0.01, ANOVA) (Fig. 9, right). These data clearly demonstrate that contraction induced by phosphatase inhibition is PKC{epsilon} dependent in ESO, but not LES, circular muscle.



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Fig. 9. Translocation of PKC{epsilon} in response to okadaic acid treatment of ESO cells. Intact cells were treated with 10–9 M okadaic acid at time 0. PKC{epsilon} was immunoprecipitated from the cytosolic and membrane fractions at the indicated times after okadaic acid addition, and PKC{epsilon} activity was measured in the immunoprecipitate. A: PKC activity in the cytosolic ({bullet}) and particulate ({blacksquare}) fractions as a function of time of exposure to okadaic acid. B: particulate-to-cytosolic ratio ({blacklozenge}) of PKC activity as a function of time of exposure to okadaic acid. Values are means ± SE for 3 animals.

 
Investigators at our laboratory (10) and elsewhere (52, 53) previously showed that PKC-dependent contraction of ESO is linked to activation of ERK1 and ERK2. To test the hypothesis that contraction elicited by phosphatase inhibitors is similarly mediated by ERK1 and ERK2 in ESO but not in LES, we examined the role of these kinases in okadaic acid- and microcystin-LR-induced contractions.

MAP kinases mediate okadaic acid- and microcystin-LR-induced contraction of ESO but not of LES. Antibodies to ERK1 and ERK2 inhibited okadaic acid (10–9 M)- and microcystin-LR (10–9 M)-induced contractions of ESO in a concentration-dependent manner (P < 0.001, ANOVA) but had no significant effect on LES contractions (Figs. 10 and 11). Similarly, the MEK antagonist PD-98059 inhibited okadaic acid- and microcystin-LR-induced contractions of ESO in a concentration-dependent manner (P < 0.001, ANOVA) but had no effect on LES contractions (Fig. 12). These data suggest that ERK1 and ERK2 and the upstream kinase MEK are involved in okadaic acid- and microcystin-LR-induced contraction of ESO but not of LES.



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Fig. 10. Effects of antibodies to ERK1 and ERK2 on okadaic acid-induced contraction of permeabilized ESO ({circ}) and LES ({bullet}) cells. Cell shortening was activated by 10–9 M okadaic acid in the absence (control) or presence of the indicated concentrations of anti-ERK1 (A) or anti-ERK2 (B). Values are means ± SE for 4 animals, with 30 cells counted for each animal.

 


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Fig. 11. Effects of antibodies to ERK1 and ERK2 on microcystin-LR-induced contraction of permeabilized ESO ({circ}) and LES ({bullet}) cells. Cell shortening was activated by 10–9 M microcystin-LR in the absence (control) or presence of the indicated concentrations of anti-ERK1 (A) or anti-ERK2 (B). Values are means ± SE for 5 animals, with 30 cells counted for each animal.

 


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Fig. 12. Effects of the MAPK kinase (MEK) inhibitor PD-98059 on phosphatase inhibitor-induced contraction of intact (A) or permeabilized (B) ESO ({circ}) and LES ({bullet}) cells. Cell shortening was activated by 10–9 M okadaic acid (A) or 10–9 M microcystin-LR (B) in the absence (control) or presence of the indicated concentrations of PD-98059. Values are means ± SE for 8 animals, with 30 cells counted for each animal.

 
Phosphatase inhibitor-mediated contraction involves activation of ILK. ILK has been implicated in Ca2+-independent contraction of vascular smooth muscle elicited by phosphatase inhibition (14, 65). We therefore examined the possibility that activation of ILK may play a role in phosphatase inhibitor/PKC{epsilon}-mediated contraction of ESO smooth muscle.

ILK antibodies inhibited okadaic acid-induced contraction of ESO permeable cells (Fig. 13), and ILK activity increased in a concentration-dependent manner in response to okadaic acid (Fig. 14). Finally, the MEK inhibitor PD-98059 inhibited both okadaic acid-induced contraction (as shown in Fig. 12) and ILK activity (Fig. 15).



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Fig. 13. Effects of anti-integrin-linked kinase (ILK) antibody on okadaic acid-induced contraction of permeabilized ESO cells. Cell shortening was activated by 10–9 M okadaic acid in the absence ({bullet}) or presence ({circ}) of ILK antibody (1:200 dilution). Values are means ± SE for 3 animals, with 30 cells counted for each animal.

 


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Fig. 14. ILK activation in response to okadaic acid treatment of ESO. ESO circular smooth muscle was treated with 10–6 M okadaic acid for the indicated times. ILK was immunoprecipitated from ESO circular smooth muscle, and kinase activity was measured in the immunoprecipitate as described in METHODS. Values are means ± SE for 3 animals.

 


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Fig. 15. ILK was immunoprecipitated from ESO circular smooth muscle, and kinase activity was measured in the immunoprecipitate from untreated ESO (open bar) or from ESO stimulated with okadaic acid (10–6 M) for 2 min in the absence (solid bar) or presence (shaded bar) of the MEK inhibitor PD-98059 (10–5M) as described in METHODS. Values are means ± SE for 3 animals.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have suggested that the signaling pathways involved in excitation-contraction coupling differ between ESO and LES circular smooth muscles of the cat. In its initial response to agonists, LES resembles most smooth muscles in that MLCK plays a key role in the contractile response to a variety of stimuli. Contraction of ESO, however, does not appear to involve activation of MLCK. Nevertheless, the initial ACh-induced contraction of both smooth muscles correlates with an increase in MLC phosphorylation of ~40%, suggesting that myosin phosphorylation in ESO is catalyzed by a kinase different from MLCK. Indeed, the addition of activated MLCK in permeabilized ESO cells elicited a small contraction and a minor increase in MLC phosphorylation (from 11 to 18%) compared with LES (from 6 to 25%) (54). Similarly, ACh-induced MLC phosphorylation and contraction in LES were inhibited by CaM and MLCK inhibitors but not by PKC inhibitors, whereas in ESO, they were inhibited by PKC inhibitors but not by CaM or MLCK inhibitors (54). The PKC isoenzyme involved in ACh-induced contraction of ESO appears to be PKC{epsilon}, since 1) only this isoenzyme translocated from the soluble to the particulate fraction in response to ACh treatment, 2) contraction induced by the synthetic DAG PKC activator 1,2-dioctanoylglycerol was inhibited by anti-PKC{epsilon} but not by antibodies to other PKC isoenzymes, and 3) an N-myristoylated peptide corresponding to the autoinhibitory domain of PKC{epsilon} but not to the autoinhibitory domain of PKC{alpha}, PKC{delta}, or PKC{alpha}{beta}{gamma} inhibited contraction of permeabilized ESO in response to 1,2-dioctanoylglycerol (57).

Several kinases in addition to MLCK have been shown to be capable of phosphorylating MLC at the activating site (Ser19); however, in most instances, phosphorylation has been demonstrated with the isolated light chain, but not intact myosin, as the substrate. Nevertheless, several kinases are capable of phosphorylating intact myosin at Ser19 of MLC: CaM-dependent PKII (16), MAP kinase-activated protein kinase-2 (39), ROK (2), ILK (13), and ZIP kinase (49). Of these candidates, ILK has been shown to associate with the contractile machinery of smooth muscle and therefore is likely to be a kinase responsible for Ca2+-independent myosin phosphorylation and contraction of rat tail arterial smooth muscle in response to the phosphatase inhibitor microcystin-LR (13, 65). However, we cannot rule out the involvement of other candidate kinases.

Because MLCK activation depends on activation of CaM, which requires a high Ca2+ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca2+ levels. The link between activation of the Ca2+-independent PKC{epsilon} and MLC phosphorylation in the ESO, however, has not been elucidated.

Inhibitors of protein serine/threonine phosphatases are useful tools for studying signaling pathways involved in the regulation of smooth muscle contraction. Okadaic acid is a widely used phosphatase inhibitor that is more potent toward type 2A than toward type 1 phosphatase (60), while microcystin-LR is less discriminating between types 1 and 2A phosphatases (61). In the present study, we found that okadaic acid and microcystin-LR caused a similar concentration-dependent contraction of ESO and LES, consistent with the involvement of a type 2A phosphatase in the contractile responses (Fig. 1). Concentration-dependent contraction of ESO and LES was also observed in response to anti-PP1c, implicating type 1 phosphatase in the contractile responses (Fig. 2). We conclude that both types 1 and 2A phosphatases are involved in the contractile responses of ESO and LES to phosphatase inhibitors.

Phosphatase inhibitor-induced contraction of LES was mediated by activation of MLCK, since the contraction was inhibited by the selective MLCK inhibitors ML-7 and ML-9 or an antibody to MLCK (Fig. 3) in a concentration-dependent manner. These data indicate that phosphatase (MLCP) inhibition in LES induces contraction through the classic CaM-MLCK pathway. In ESO, however, contraction induced by phosphatase inhibitors was not mediated by activation of MLCK, since it was unaffected by MLCK inhibitors or anti-MLCK.

To ensure the specificity of MLCK inhibition, we used SM-1, a synthetic peptide corresponding to the autoinhibitory sequence of MLCK (residues 783–804 of chicken gizzard MLCK) (65), which is the most selective inhibitor of Ca2+/CaM-dependent MLCK (21, 30, 38). As observed with the other phosphatase inhibitors, contraction induced by anti-PP1c was inhibited by SM-1 in LES but not in ESO smooth muscle cells (Figs. 4 and 8). Taken together, these data strongly support the importance of MLCK in the initial contraction of LES smooth muscle and indicate that contraction of ESO smooth muscle is MLCK independent. In the case of ESO, phosphatase inhibitors must enhance the effect of kinases other than MLCK (e.g., ILK).

Because PKC{epsilon} has been implicated in ESO contraction (57), we examined the effect of the PKC inhibitor chelerythrine on okadaic acid- and microcystin-LR-induced contraction (Fig. 5). Chelerythrine inhibited ESO contraction in a concentration-dependent manner but had no effect on LES contraction in response to these phosphatase inhibitors, suggesting that in ESO, the phosphatase inhibitors inhibit a phosphatase that opposes the action of PKC. We verified that the PKC isoenzyme involved was PKC{epsilon}, since anti-PKC{epsilon} inhibited okadaic acid- and microcystin-LR-induced contraction in ESO but not in LES (Fig. 6), whereas anti-PKC{beta}II had no effect on phosphatase inhibitor-induced contraction in either LES or ESO (Fig. 7). To resolve possible concerns regarding the specificity of these inhibitors, we also used anti-PP1c as a contractile agent, antibodies against MLCK, PKC{epsilon}, and PKC{beta}II (as a negative control), and the highly selective MLCK peptide inhibitor SM-1 (Fig. 8). ESO contraction induced by the PP1c antibody was not affected by SM-1, anti-MLCK, or anti-PKC{beta}II but was inhibited by anti-PKC{epsilon}. LES contraction, however, was inhibited by anti-MLCK and SM-1 but was unaffected by anti-PKC{epsilon} or anti-PKC{beta}II. These data confirm that anti-PP1c-induced phosphatase inhibition contracts LES through an MLCK-dependent pathway but contracts ESO through an MLCK-independent pathway. In addition, we found that phosphatase inhibition by okadaic acid stimulates PKC{epsilon} activity in ESO, as demonstrated by translocation of the enzyme activity from the cytosol to the particulate fraction (Fig. 9).

Several possible mechanisms could be responsible for PKC activation and ESO contraction in response to phosphatase inhibition. Phosphatases may reverse phosphorylation-dependent activation of PKC, PKC-dependent phosphorylation of key substrates, or both. Phosphorylation of PKC regulates the activity and subcellular localization of the enzyme (48). The kinase, which is synthesized as a soluble unphosphorylated protein (8), must be phosphorylated before it is competent to respond to second messengers (17). The first phosphorylation of PKC (conventional, novel, and atypical PKC isoforms) occurs in the activation loop and is catalyzed by the phosphoinositide-dependent protein kinase PDK-1 (15). This phosphorylation is necessary for the catalytic activity of PKC (48). Phosphorylation by PDK-1 is followed by two autophosphorylations near the COOH terminus of PKC that stabilize the structure of the active site and the regulatory domain, allowing tighter binding of substrates and cofactors (17) and increasing the phosphatase resistance of PKC (8). The mature, phosphorylated species is half-phosphorylated at the activation loop and quantitatively phosphorylated at the two COOH-terminal sites, suggesting that dephosphorylation and/or phosphorylation at the active site may regulate kinase activity in response to stimuli (48). The phosphorylations act cooperatively to maintain the active (latent) conformation. The phosphorylated species is found in the cytosol or, on activation, is associated with membranes (26). PKC signaling is desensitized through dephosphorylation and proteolysis. After activation, PKC is dephosphorylated by a membrane-bound type 2A phosphatase. Okadaic acid has been shown to inhibit phorbol ester-induced dephosphorylation of PKC in intact COS cells (25). It is possible, therefore, that contraction induced by phosphatase inhibition may result from inactivation of membrane-bound type 2A phosphatase, leading to increased phosphorylation of PKC.

Alternatively, contraction induced by phosphatase inhibition may result from an increased pool of phosphorylated PKC substrates. Phosphatases (mostly type 2A but also type 1) have been shown to dephosphorylate several PKC substrates in a variety of cell preparations (19, 22, 62). For example, in intact Swiss 3T3 cells, okadaic acid prevented the dephosphorylation of the PKC substrate MARCKS that occurs after PKC activation by bombesin (11). In SH-SY5Y cells, a protein phosphatase (possibly type 2A) with high basal activity counteracts PKC-induced phosphorylation of myelin basic protein and other PKC substrates (41). A potential PKC substrate that has been implicated in the regulation of smooth muscle contraction is CPI-17, a heat-stable protein that potently inhibits MLCP activity when phosphorylated at Thr38. Thiophosphorylated CPI-17 causes Ca2+-independent contraction in {beta}-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle (14, 37, 42). Thus it is possible that phosphatase inhibitors may facilitate phosphorylation of CPI-17 by PKC by preventing its dephosphorylation.

A connection between MAP kinase activation and PKC-mediated smooth muscle contraction has been proposed for some time (33, 34). In the ferret aorta, ERK1, ERK2, and possibly p38 MAP kinase are activated in response to phenylephrine (45). In colonic smooth muscle, MAP kinase is activated during PKC-dependent contraction and cotranslocated with heat shock protein 27 (HSP27) (68). Investigators at our laboratory (10) recently showed that PKC-dependent contraction, exemplified by ACh-induced contraction of ESO, may be associated with activation of MAP kinases. Contraction in response to ACh depends on ERK phosphorylation and/or phosphorylation of the HSP27-linked p38 MAP kinase. In this study, we observed that ERK1 and ERK2 antibodies nearly abolished okadaic acid- and microcystin-LR-induced contraction of permeabilized ESO, but not LES, cells (Figs. 10 and 11). In addition, the MEK antagonist PD-98059 inhibited, in a concentration-dependent manner, okadaic acid- or microcystin-LR-induced contraction in ESO but not in LES (Fig. 12). These data suggest that phosphatase inhibitor-induced contraction of ESO, but not LES, is associated with activation of ERKs, which are likely to participate in PKC{epsilon}-dependent contractile pathways.

One of the intermediate proteins in the PKC{epsilon}-MAP kinase contractile pathway may be ILK (13). The Ca2+ independence of ILK activity makes it eligible as a candidate intermediate protein in the Ca2+-independent PKC{epsilon}-mediated contraction. ILK was identified and cloned on the basis of its interaction with the {beta}1-integrin cytoplasmic domain (24), and its catalytic domain is homologous to other protein kinase catalytic domains. ILK interacts with various proteins, several of which are connected to the actin cytoskeleton and anchor it to the extracellular matrix. Our data show a link between the PKC{epsilon}-MAP kinase contractile pathway and activation of ILK, because okadaic acid-induced contraction of ESO muscle cells is inhibited by ILK antibodies, and okadaic acid causes increased ILK activity, which is inhibited by the MEK inhibitor PD-98059 (Figs. 1315). Whether activation of ILK is a direct result of phosphorylation by ERK1/2 or whether other intermediate events may be involved remains to be demonstrated.

Other PKC substrates that have been implicated in the regulation of smooth muscle contraction are calponin (67) and caldesmon (63). Phosphorylation of these proteins alleviates their inhibition of the actin-activated MgATPase activity of phosphorylated smooth muscle myosin. Investigators at our laboratory (10) previously showed that PKC-dependent contraction, exemplified by ACh-induced contraction of ESO, is mediated by activation of two distinct pathways, one dependent on activation of ERK1/2 MAP kinases, as illustrated in the present study, and a distinct one dependent on phosphorylation of the HSP27-linked p38 MAP kinase, which has been shown to involve caldesmon and calponin (29, 50). ILK may be the kinase responsible for MLC phosphorylation in the ERK1/2 pathway. The kinase responsible for MLC phosphorylation in the p38-HSP27 pathway has not yet been defined. These possible contractile signal transduction pathways are illustrated in Fig. 16.



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Fig. 16. We propose two distinct contractile pathways for PKC-mediated contraction of esophageal muscle: one depending on activation of ERK1/2 and activation of ILK, as illustrated in the present study, and the other one involving p38-heat shock protein 27 (HSP27) and caldesmon-calponin, as explored by Ibitayo et al. (29) and Patil et al. (50), respectively.

 
In summary, contraction induced by phosphatase inhibition has been invoked to explain MLCP and contraction at low Ca2+ levels (i.e., under conditions that do not allow for full CaM activation). Our data in LES are consistent with the following contractile mechanism: inhibition of MLCP by okadaic acid, microcystin-LR, or anti-PP1c tilts the kinase-to-phosphatase activity ratio in favor of Ca2+/CaM-dependent MLCK, resulting in MLC phosphorylation and contraction. In sharp contrast, MLCK does not appear to play an important role in contraction of ESO. Nevertheless, MLC phosphorylation does correlate with contraction in this tissue (54), suggesting that a kinase distinct from MLCK is involved. Our data suggest that ILK may be involved in the PKC{epsilon}-activated ERK1/2 contractile pathway (54) and support ILK as a likely candidate kinase for MLC phosphorylation in ESO.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a Korean Society of Gastrointestinal Motility Grant (to N. Kim), National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-28614 (to P. Biancani), and Canadian Institutes of Health Research Grant MOP13101(to M. P. Walsh). M. P. Walsh is an Alberta Heritage Foundation for Medical Research Medical Scientist and recipient of a Canada Research Chair (Tier I) in Biochemistry.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Biancani, Gastrointestinal Motility Research, 5SWP, Rm. 20, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903 (E-mail: piero_biancani{at}brown.edu).

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


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