Rabbit Colonic Smooth Muscle Research Articles

Regulator of G protein signaling 4 is a novel target of GATA-6 transcription factor

GATA transcription factors regulate an array of genes important in cell proliferation and differentiation. Here we report the identification of regulator of G protein signaling 4 (RGS4) as a novel target for GATA-6 transcription factor. Although three sites (a, b, c) within the proximal region of rabbit RGS4 promoter for GATA transcription factors were predicted by bioinformatics analysis, only GATA-a site (16 bp from the core TATA box) is essential for RGS4 transcriptional regulation. RT-PCR analysis demonstrated that only GATA-6 was highly expressed in rabbit colonic smooth muscle cells but GATA-4/6 were expressed in cardiac myocytes and GATA-1/2/3 expressed in blood cells. Adenovirus-mediated expression of GATA-6 but not GATA-1 significantly increased the constitutive and IL-1β-induced mRNA expression of the endogenous RGS4 in colonic smooth muscle cells. IL-1β stimulation induced GATA-6 nuclear translocation and increased GATA-6 binding to RGS4 promoter. These data suggest that GATA factor could affect G protein signaling through regulating RGS4 expression, and GATA signaling may develop as a future therapeutic target for RGS4-related diseases.

Immune/Inflammatory Response and Hypocontractility of Rabbit Colonic Smooth Muscle After TNBS-Induced Colitis.

The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells. To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis. Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NFκB signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-β activity in response to acetylcholine (ACh). Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-β activity and contraction in colonic smooth muscle cells. Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-β activity and contractile responses.

Neo-innervation of a bioengineered intestinal smooth muscle construct around chitosan scaffold

Neuromuscular disorders of the gut result in disturbances in gastrointestinal transit. The objective of this study was to evaluate the neo-innervation of smooth muscle in an attempt to restore lost innervation. We have previously shown the potential use of composite chitosan scaffolds as support for intestinal smooth muscle constructs. However, the constructs lacked neuronal component. Here, we bioengineered innervated colonic smooth muscle constructs using rabbit colon smooth muscle and enteric neural progenitor cells. We also bioengineered smooth muscle only tissue constructs using colonic smooth muscle cells. The constructs were placed next to each other around tubular chitosan scaffolds and left in culture. Real time force generation conducted on the intrinsically innervated smooth muscle constructs showed differentiated functional neurons. The bioengineered smooth muscle only constructs became neo-innervated. The neo-innervation results were confirmed by immunostaining assays. Chitosan supported (1) the differentiation of neural progenitor cells in the constructs and (2) the neo-innervation of non-innervated smooth muscle around the same scaffold.

HEF-19-induced relaxation of colonic smooth muscles and the underlying mechanisms

To investigate the relaxant effect of chromane HEF-19 on colonic smooth muscles isolated from rabbits, and the underlying mechanisms. The relaxant effect and action mechanisms of HEF-19 were investigated using descending colon smooth muscle of the rabbits. Preparations 1 cm long were mounted in 15-mL tissue baths containing Tyrode's solution, maintained at 37 ± 0.5 °C and aerated with a mixture of 5% CO2 in oxygen (Carbogen). The tension and amplitude of the smooth muscle strips were recorded after adding HEF-19 (10(-6), 10(-5) and 10(-4) mol/L). After cumulative administration of four antispasmodic agents, including acetylcholine chloride (Ach) (10(-4) mol/L), histamine (10(-4) mol/L), high-K(+) (60 mmol/L) and BaCl2 (8.2 mmol/L), HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) was added to investigate the relaxant effect of HEF-19. CaCl2 (10(-4)-2.5 × 10(-3) mol/L) was added cumulatively to the smooth muscle preparations pretreated with and without HEF-19 (1 × 10(-6) or 3 × 10(-6) mol/L) and verapamil (1 × 10(-7) mol/L) to study the mechanisms involved. Finally, phasic contraction was induced with ACh (15 × 10(-6) mol/L), and CaCl2 (4 × 10(-3) mol/L) was added to the smooth muscle preparations pretreated with and without HEF-19 (3 × 10(-6) mol/L or 1 × 10(-5) mol/L) and verapamil (1 × 10(-7) mol/L) in calcium-free medium to further study the underlying mechanisms. HEF-19 (1 × 10(-6), 1 × 10(-5) and 1 × 10(-4) mol/L) suppressed spontaneous contraction of rabbit colonic smooth muscles. HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) relaxed in a concentration-dependent manner colonic smooth muscle preparations pre-contracted with BaCl2, high-K(+) solution, Ach or histamine with respective EC50 values of 5.15 ± 0.05, 5.12 ± 0.08, 5.58 ± 0.16 and 5.25 ± 0.24, thus showing a spasmolytic activity. HEF-19 (1 × 10(-6) mol/L and 3 × 10(-6) mol/L) shifted the concentration-response curves of CaCl2 to the right and depressed the maximum response to CaCl2. The two components contracted by Ach were attenuated with HEF-19 (3 × 10(-6) mol/L or 10(-5) mol/L) in calcium-free medium. HEF-19 inhibited rabbit colonic smooth muscle contraction, probably through inhibiting opening of voltage-dependent Ca(2+) channels. HEF-19 reduced inflow and intracellular release of Ca(2+) ions.

MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells

Background Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1β is mediated by the activation of NFκB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells.Methodology/Principal FindingsCultured cells at first passage were treated with or without IL-1β (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1β stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1β. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1β treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1β increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter.Conclusion/SignificanceActivation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.

Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering

Intestinal tissue engineering is an emerging field due to a growing demand for intestinal lengthening and replacement procedures secondary to massive resections of the bowel. Here, we demonstrate the potential use of a chitosan/collagen scaffold as a 3D matrix to support the bioengineered circular muscle constructs maintain their physiological functionality. We investigated the biocompatibility of chitosan by growing rabbit colonic circular smooth muscle cells (RCSMCs) on chitosan-coated plates. The cells maintained their spindle-like morphology and preserved their smooth muscle phenotypic markers. We manufactured tubular scaffolds with central openings composed of chitosan and collagen in a 1:1 ratio. Concentrically aligned 3D circular muscle constructs were bioengineered using fibrin-based hydrogel seeded with RCSMCs. The constructs were placed around the scaffold for 2 weeks, after which they were taken off and tested for their physiological functionality. The muscle constructs contracted in response to acetylcholine (Ach) and potassium chloride (KCl) and they relaxed in response to vasoactive intestinal peptide (VIP). These results demonstrate that chitosan is a biomaterial possibly suitable for intestinal tissue engineering applications.

RNA-binding protein HuR regulates RGS4 mRNA stability in rabbit colonic smooth muscle cells

Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3'-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1β-induced upregulation of RGS4 expression. We show that IL-1β treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3'-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3'-most ARE sites. IL-1β increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1β-induced upregulation of RGS4 mRNA. In addition, IL-1β increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3'-most ARE sites within RGS4 3'-UTR are essential for the instability of RGS4 mRNA and IL-1β promotes the stability of RGS4 mRNA through HuR.

Cloning and characterization of rabbit Rgs4 promoter in gut smooth muscle

Regulator of G-protein signaling 4 (Rgs4) regulates the strength and duration of G-protein signaling, and plays an important role in cardiac development, smooth muscle contraction and psychiatric disorders. Rgs4 expression is regulated at both mRNA and protein levels. In order to examine the transcriptional mechanism of Rgs4 expression, we have cloned and characterized rabbit Rgs4 promoter. The coding sequence of rabbit Rgs4 was obtained by degenerative RT-PCR and used for Northern blot and 5′-RACE analysis. A single transcript was identified in rabbit colonic smooth muscle cells. The 5′-untranslated region (UTR) extended 120 bp nucleotides upstream of the Rgs4 start codon. A putative promoter sequence (1389 bp) showed a consensus TATA box and cis-acting binding sites for several potential transcriptional factors. Reporter gene assay identified strong promoter activity in various cell types. Further analysis by deletion mutagenesis suggested that the proximal region had a highest core promoter activity while the distal region is suppressive. IL-1β significantly increased the promoter activity. The in vitro and in vivo binding activities for NF-κB transcription factor were validated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay respectively. Mutation of NF-κB site reduced the promoter activity. These data suggest that the cloned rabbit Rgs4 promoter is functionally active and NF-κB binding site possesses enhancer activity in regulating Rgs4 transcription. Our studies provide an important basis for further understanding of Rgs4 regulation and function in different diseases.

W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells

W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells

Interleukin-1β up-regulates RGS4 through the canonical IKK2/IκBα/NF-κB pathway in rabbit colonic smooth muscle

Initial Ca2+-dependent contraction of the intestinal smooth muscle mediated by G(q)-coupled receptors is attenuated by RGS4 (regulator of G-protein signalling 4). Treatment of colonic muscle cells with IL-1beta (interleukin-1beta) inhibits acetylcholine-stimulated initial contraction through increasing the expression of RGS4. NF-kappaB (nuclear factor kappaB) signalling is the dominant pathway activated by IL-1beta. In the present study we show that RGS4 is a new target gene regulated by IL-1beta/NF-kappaB signalling. Exposure of cultured rabbit colonic muscle cells to IL-1beta induced a rapid increase in RGS4 mRNA expression, which was abolished by pretreatment with a transcription inhibitor, actinomycin D, implying a transcription-dependent mechanism. Existence of the canonical IKK2 [IkappaB (inhibitor of NF-kappaB) kinase 2]/IkappaBalpha pathway of NF-kappaB activation induced by IL-1beta in rabbit colonic muscle cells was validated with multiple approaches, including the induction of reporter luciferase activity and endogenous NF-kappaB-target gene expression, NF-kappaB-DNA binding activity, p65 nuclear translocation, IkappaBalpha degradation and the phosphorylation of IKK2 at Ser(177/181) and p65 at Ser(536). RGS4 up-regulation by IL-1beta was blocked by selective inhibitors of IKK2, IkappaBalpha or NF-kappaB activation, by effective siRNA (small interfering RNA) of IKK2, and in cells expressing either the kinase-inactive IKK2 mutant (K44A) or the phosphorylation-deficient IkappaBalpha mutant (S32A/S36A). An IKK2-specific inhibitor or effective siRNA prevented IL-1beta-induced inhibition of acetylcholine-stimulated PLC-beta (phopsholipase C-beta) activation. These results suggest that the canonical IKK2/IkappaBalpha pathway of NF-kappaB activation mediates the up-regulation of RGS4 expression in response to IL-1beta and contributes to the inhibitory effect of IL-1beta on acetylcholine-stimulated PLC-beta-dependent initial contraction in rabbit colonic smooth muscle.

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA 2 value of 9.17 ± 0.01 ( n = 5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 μM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [ 125I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The p Ki of MA-2029 was 8.58 ± 0.04 in the rabbit colon homogenate ( n = 4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 μg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 μg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.

RhoA- and PKC-α-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells.

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-alpha. Presently, we examined the cross talk between RhoA and PKC-alpha in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-alpha, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-alpha by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-alpha pathways, suggesting cross talk between RhoA and PKC-alpha resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.

Translocation and association of ROCK-II with RhoA and HSP27 during contraction of rabbit colon smooth muscle cells

The focus of the paper is to understand the role of HSP27 in mediating the association of RhoA with ROCK-II in sustained contraction of smooth muscle cells from the rabbit colon. In circular smooth muscle cells; acetylcholine-induced contraction (10−7 M) was associated with translocation of ROCK-II to the particulate fraction, which remained sustained at 4 min after stimulation (135.1 ± 8.1% increase, P⩽0.05). There was also an increased association of ROCK-II with RhoA particulate fraction (147.46 ± 9.31 and 148.22 ± 9.41, n=3, P⩽0.05) and with HSP27 (155.6 ± 10.7% increase, P⩽0.05) in the particulate fraction. Pre-incubation of cells with Y27632 resulted in the inhibition of the association of ROCK-II with RhoA in the particulate fraction. Acetylcholine (10−7 M) induced sustained phosphorylation of MLC (122.75 ± 9.97%, P⩽0.05 and 174.65 ± 28.36%, P⩽0.05 increase in the di phospho-MLC at 30 s and 4 min, respectively), which was inhibited upon pre-incubation with Y27632. Results suggest that ROCK-II undergoes a translocation to the particulate fraction with RhoA and with HSP27, suggesting that translocation and association of ROCK-II with RhoA is mediated by HSP27. Maintenance of the functional association of RhoA with ROCK-II in the particulate fraction mediated by HSP27 appears to be important to retain MLC in the phosphorylated state and hence the sustained contraction.

Translocation and association of ROCK-II with RhoA and HSP27 during contraction of rabbit colon smooth muscle cells

The focus of the paper is to understand the role of HSP27 in mediating the association of RhoA with ROCK-II in sustained contraction of smooth muscle cells from the rabbit colon. In circular smooth muscle cells; acetylcholine-induced contraction (10 −7 M) was associated with translocation of ROCK-II to the particulate fraction, which remained sustained at 4 min after stimulation (135.1 ± 8.1% increase, P⩽0.05). There was also an increased association of ROCK-II with RhoA particulate fraction (147.46 ± 9.31 and 148.22 ± 9.41, n=3, P⩽0.05) and with HSP27 (155.6 ± 10.7% increase, P⩽0.05) in the particulate fraction. Pre-incubation of cells with Y27632 resulted in the inhibition of the association of ROCK-II with RhoA in the particulate fraction. Acetylcholine (10 −7 M) induced sustained phosphorylation of MLC (122.75 ± 9.97%, P⩽0.05 and 174.65 ± 28.36%, P⩽0.05 increase in the di phospho-MLC at 30 s and 4 min, respectively), which was inhibited upon pre-incubation with Y27632. Results suggest that ROCK-II undergoes a translocation to the particulate fraction with RhoA and with HSP27, suggesting that translocation and association of ROCK-II with RhoA is mediated by HSP27. Maintenance of the functional association of RhoA with ROCK-II in the particulate fraction mediated by HSP27 appears to be important to retain MLC in the phosphorylated state and hence the sustained contraction.

Coupling of M2 muscarinic receptor to L-type Ca2+ channel via c-src kinase in rabbit colonic circular smooth muscle

Background & Aims: The L-type Ca2+ channel is a major pathway for Ca2+ influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein–coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca2+ channels via c-src kinase. Methods: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of α subunit of Ca2+ channel was determined by a GST pull-down assay. Results: Methacholine (10 μmol/L) enhanced Ca2+ channel currents by 30% under conditions whereby the M3 receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Gαq antibody. Similar effects were observed by blocking intracellular Ca2+ release with heparin. Enhancement was abolished by intracellular anti-Gαi antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of α1c subunit of the Ca2+ channel. In M2 receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol. Conclusions: These studies demonstrate stimulation of Ca2+ current in colonic smooth muscle cells by M2 receptor coupled to Gαi–G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.GASTROENTEROLOGY 2002;123:827-834

Pp68c-sπ-dependent regulation of Ca2+ channels in rabbit colonic smooth muscle

pp68c-sπ-dependent regulation of Ca2+ channels in rabbit colonic smooth muscle

Pp68c-sπ-dependent regulation of Ca2+ channels in rabbit colonic smooth muscle

pp68c-sπ-dependent regulation of Ca2+ channels in rabbit colonic smooth muscle

The human ether-a-go-go related gene (HERG)-like potassium channel in rabbit colonic smooth muscle cells: A potential target for the actions of cisapride

We have recently identified HERG-like K+ currents in esophageal smooth muscle where it importantly contributes to the resting membrane potential (RMP)(Akbarali et aI., Am.J.Phys. 277: C1284, 1999). Drugs that block cardiac HERG channels cause abnormalities such as long Q-T syndrome, an effect that can be induced by the gastrointestinal prokinetic agent, cisapride. The aim of this study was, therefore, to determine a) the presence and role of HERG-like currents in colonic smooth muscle and b) the potential effect of cisapride on these K+ currents. Whole cell patch-clamp experiments were carried out on freshly isolated rabbit colonic circular muscle cells. The conductance of inwardly rectifying HERG-like K+ channels in cells was enhanced by perfusing with isotonic extracellular K+ solution (140 mM K+). Under these conditions, when cells were voltageclamped at 0 mV (EK ) , step hyperpolarizations to -120 mV in 10 mV increments resulted in large inward currents that activated rapidly and then slowly inactivated in a timeand voltage-dependent fashion (Tinac .= 55 ± 5ms at -120 mY; n=4). The peak current-voltage relationship showed marked inward rectification with a reversal at EK . The amplitude of peak currents at -120 mV was -170 ± 14 pA (n=26). At potentials negative to -60 mV, prominent inactivation resulting in cross-over of the currents was observed. A U-shaped voltage-dependence for non-inactivating (window) currents occured between -80 mV and -40 mV (peak amplitude of -80 ± 4 pA; n=3) suggesting contribution of the HERG-like channels to the resting potential of the smooth muscle cells. These K+ currents were completely abolished by the selective HERG channel blocker, E-4031 (I /LM; n= 10). Cisapride also dose-dependently blocked (0.001 10 /LM; n=24) the HERG-like K+ current with an ICso of I/LM. E-4031 and cisapride-sensitive currents demonstrated strong inward rectification. In further studies, the RMP of whole muscle strips was measured using high resistance microelectrodes. Cisapride (I/LM) and E-4031 (3/LM), in the presence of atropine (I/LM) and nifedipine (I/LM), depolarized the RMP from -47 ± 2 mV to -34 ± 4 mV and from -54mV ± 3mV to -37 ± 3 mY, respectively (n=3). These studies demonstrate a) that HERG-like currents in rabbit colonic smooth muscle are involved in maintaining resting potential and b) these gastrointestinal smooth muscle K+ channels, like in the heart, are potential targets for the prokinetic agent, cisapride. Supported by NIH(DK46367)

Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27.

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

Modulation of Voltage-dependent Ca2+Channels in Rabbit Colonic Smooth Muscle Cells by c-Src and Focal Adhesion Kinase

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.