β3-adrenoceptor stimulation ameliorates experimental colitis in rats

the enteric nervous system and potentially contributing to the cellular mechanisms of inflammatory bowel disease.

M1292 Role of Phospho-Hsp27 in the Binding of Hsp27 with Tropomyosin Sita Somara, Suresh B. Patti, Haiyan Pang, Khafil N. Bitar

M1295

BACKGROUND:Agonist-induced contraction of colonic smooth muscle cells, is accompanied by colocalization and association of HSP27 with Tropomyosin. It was postulated that agonistinduced phosphoryfation of HSP27 modulates actin-myosin interaction through thin-filament regulation of Tropomyosin. OBJECTIVE: To understand the regulation of Tropomyosin by HSP27 in modulating actin-myosin interaction, we studied the direct association of HSP27 with Tropomyosin and the role of phosphorylated HSP27 in this association. METHODS: Three forms of HSP27 were used in these studies. 1) wtHSP27 (wild type HSP27) has three phosphorylation sites viz., Ser-15, Set-78, and Ser-82.2) 3GHSP27 mutant, where all three phosphorylation sites were mutated to Glycine, to mimic non-phosphorylatable residues and 3) 3DHSP27 mutant, where all three phosphorylation sites were mutated to aspartate, to mimic constitutively phosphorylated residues. Cloning and Expression of liSP27: wtHSP27, 3DHSP27, 3GHSP27 were cloned into pGEX-KT and expressed in E.coli as GST-fusion proteins each with a mass of 54 kDa. In vitro Binding: 50 v.g each of different forms of GST-HSP27 bound to Glutathione agarose beads (200g,l) were incubated with 25 wg of Tropomyosin. After several washes, the mixture was ehited with buffer containing 10mM reduced glutathione in 50 mM tris-HC1. All the washes and eluates were spotted on a polyvinyldene fluoride (PVDF) membrane and were immuno-detected using GST, Tropomyosin, and HSP27 antibodies respectively. RESULTS: There was a co-elution of Tropomyosin wLth all the GST-HSP27(s), indicating a direct association of Tropomyosin with the three forms of GST-HSP27. The co-ehition of Tropomyosin was 200.3 _+ 44.1, P<0.05 with GSTwtHSP27; 168.0 -+ 16.7, P< 0.01 with GST-3GHSP27, and 333.0_+ 76.5, P<0.01 with GST-3DHSP27. CONCLUSIONS: 1) There is a direct association of HSP27 with Tropomyosin. 2) Non-phosphomimic, 3GHSP27 has reduced binding affinity to Tropomyosin as compared ~ t h wild type HSP27.3) Phosphomimic 3DHSP27 shows the greatest direct binding affinity to "fropomyosni. These results indicate the functional importance of phospborylated HSP27, probably due to a conformational change of the molecule, and a greater binding affinity of phospbo-HSP27 with Tropomyosin.

Lack of Evidence for The Existence of Interstitial Cells of Cajal in The Gallbladder Edward J. Parr, Audra L. Kennedy, Gary M. Mawe Intracellular electrophysiologieal studies have demonstrated that gallbladder smooth muscle cells exhibit rhythmic spontaneous action potentials that occur at a frequency of about 0.3 Hz, but the source of these events has not been determined. In the bowel, specialized cells called interstitial cells of Cajal (ICCs) act as pacemaker cells to generate electrical slow wave activity that is conducted to the smooth muscle. These cells are Kit-immunoreactive and have distinct uhrastrucntral properties, including an abundance of mitcchondria and electron-dense cytoplasm. Furthermore, slow wave depolarizations generated by 1CCs are not sensitive to blockade by inhibitors of dihydropyridine-sensitive Ca2+ channels. This study was conducted to determine whether ICCs are present in the guinea pig gallbladder, and hence could act as smooth muscle pacemaker cells. Methods: Immunohistochemistry was performed, with whole mount preparation of the guinea pig extrahepatic biliary tree, using antisera specific for c-Kit and the pan-neuronal marker, PGP 9.5. Gallbladder muscularis preparations were examined by electron microscopy, and spontaneous action potentials in intact gallbladder smooth muscle cells were studied with intracelhilar microelectrodes. Results: In the gallbladder, where the ganglionated plexus was immunostained with the anti-PGP 9.5 antiserum, the only cells that were Kit-immunoreactive were mast cells. When the remainder of the extrahepatic bihary tree was examined, Kit-immunoreactive ICCs were observed throughout the sphincter of Oddi and adjacent duodenum, coincident with PGP 9.5-immunoreactive ganglionated nerve plexus. In the bile ducts, the nerve plexus comprised sparsely distributed nerve fibers with few neurons. However, Kit-immunoreactive ICCs extended only a few micrometers along the distal end of the common bile duct. Electron microscopic examination of the gallbladder muscularis revealed a lack of cells with features characteristic of ICCs. In electrophysiologieal studies, both diltiazem (50 wM) and nifedipine (1 ~M) completely abolished the spontaneous action potentials, resulting in a flat membrane potential. Conclusions: Most of the biliary tree lacks cells characteristic of ICCs. Thus, in contrast to most of the gastrointestinal tract, rhythmic electrical activity recorded from gallbladder smooth muscle may be myogenic in origin.

M1293 ROLE OF PGEz IN MAINTENANCE OF TONIC CONTRACTION OF GUINEA PIG GALLBLADDER Wen-Zbong Liu, Zuo-Liang Xiao, Piero Biancani, Jose Behar

M1296

The factors that contribute to the genesis of myogenic gallbladder tonic contraction are unknown. The present studies were aimed at determining whether prostaglandins were involved in the maintenance of tonic contraction. They were conduced in muscle strips and dissociated muscle cells. Muscle strips were placed in the muscle bath and equilibrated for 3 hours. Single cells were obtained by enzymatic digestion. Muscle strips developed tonic contraction that was unaffected by tetrodotoxin (105 M). PGE2 caused a dose-dependent contraction with a maximal respons of 2+/-0.2 g with a concentration of 106 M greater than that of PGF2a of 1.4 +/-0.15 g. Gallbladder tonic contraction gradually declined with increasing concentrations of indomethacni (10" to l0 5 M) from 1.1 +/-0.1 to 0. lg and with the prostaglandin E2 receptor subtype 1 (EP1) antagonist SC-19220 (10 5 M) from 1.2+/0.ho 0.1 g. PGE2 induced contraction was also completely blocked by the EP1 receptor antagonist [105 M[ and by the RhoA inhibitor Y-27632 [10SM]. The signal transduction pathway that mediates PGE2 induced contraction was then determined in muscle cells. PGE2 caused a dose dependent contraction that was blocked by EP1 receptor antagonist (10 5 M), by the anti-Go/it antibody (1:200) and by the anti-RhoA antibody (1:200). PGE2 increased ~SS-GTP36 binding to Go/H by 74.9% over basal level.This increased binding was reduced by EP1 receptor antagonist to 22.7%. Immuno-precipitation studies using an EP1 receptor antibody revealed that the EP1 receptor co-immunopecipitates with RhoA protein determined by Westem blot. It is concluded that PGE2 contributes to the maintenance of gallbladder tonic contraction primarily through the EP1 receptor subtype that couples to Gore and to the small G protein RhoA

~3-Adrenoceptor Stimulation Ameliorates Experimental Colitis in Rats Giovanni Barbara, Eman Abu-Gharbieh, Valentina Vasina, Roberto De Giorgio, Cesare Cremon, Vincenzo Stanghellini, Roberto Corinaldesi, Fabriaio De Ponti BACKGROUND: Neural supply to the gastrointestinal tract is known to modulate gut inflammatory processes. Colonic myenteric plexus nomdrenaline release is reduced in experimental colitis and adrenoceptor stimulation is known to ameliorate human ulcerative colitis. These data suggest that adrenergic pathways are protective in colonic inflammation. 133adrenoceptorts are widely distributed in the gastrointestinal tract and their activation may mediate anti-inflammatory activities. AIM: To investigate the role of 133-adrenoceptors in a rat model of colonic inflammation. METHODS: Colitis was induced in male Sprague Dawley rats by intrarectal administration of 15 mg/rat dinitrobenzene sulphonic acid (DNBS). The ~3-adrenoceptor agonist SR58611A (3 mg/kg or 10 mg/kg) was administered oraUy starting from the day before the induction of colitis for 7 days. Colitis was assessed by means of macroscopic and histologtcal scores and measurement of myeloperoxidase activity (MPO) on day 6 post-colitis. RESULTS: The main results of the study are reported in Table. In non colitic animals, SR58611A did not affect body weight as well scores for colonic damage and inflammation. Colitis impaired body weight gain throughout the study and increased colonic damage and inflammation. SR58611A at a dose of 3 mg/kg or 10 mg/kg significantly reduced the impairment of body weight gain following colitis, decreased colonic damage by about 35% and inflammation by about 45%. CONCLUSIONS: These results provide further evidence of adrenergic modulation of inflammation and indicate that f33-adrenoceptor agonists may be of potential therapeutic value in inflammatory bowel diseases. Supported by Sanofi-Synthelabo

M1294

Astestnte.t of colitis following stimulation of p 3 - M l r e n o c ~

Human Enteric Gila Have the Potential to Modulate Inflammatory Processes Nicola Boguth, Wolfgang Stremmel, Anne Ruhl

Group

In transgenic animal models, enteric gila have been shown to be essential in maintainmg the integritiy of the bowel (Cell, 1998: PNAS 2002). Despite increasing knowledge of enteric ghal function in rodent models, almost nothing is known about physiological properties of enteric gila in man. Therefore, we have developed a tissue culture system that for the first time allows to grow purified enteric gha from human gut. Primary cultures were generated from surgical specimens obtained from patients who had given informed consent prior to colonic resections for colorectal cancer. Whole mount preparations of myenteric plexus from disease-flee pieces of colon were enzymatically dissociated and grown in DMEM with 10% FCS. After about 4-6 weeks, half of the cultures were subjected to antibody-mediated complement lysls of fibroblasts until they were 100% homogeneous enteric glial cells (EGC) as evidenced morphologically and immnnocytochemically. Untreated cultures were completely overgrown by fibroblasts. Both purified EGC and enteric fibroblast cultures were stimulated with human recombinant interleukin-l[3 (rHulL-l[3; 0-100 ng/mL). After 24 hrs, total cellular RNA was extracted and interleukin-6 (IL-6) mRNA expression was assessed by semiquantitative RT-PCR with GAPDH as honse-keeping gene. PCR products were analyzed on agarose gels and quantification was performed using optical densitometry on ethidium bromide stained gel pictures. Both unstimulated EGC and fibroblasts displayed some constitutive IL-6 mRNA expression. Cytokine-stimulation significantly and dose-dependently augmented [L-6 mRNA synthesis in EGC cultures (p
TreMme~

Wtdght gain %

t.tramta~

o~t

Macrmmoplc Mk:oscopic score score

MPO (g/rag)

t

Saline

Vehicle

26.5q.8

1.1~0.1

0.5~02

2

Saline

m~g

SR10

27.8,1.0

1.1~0.1

1.3~0.3

1.4~0.2

3

DNBS

Vehicle

15.3q.2"

6.9~0.8'

5.5~0.4"

58.0~9.6"

4

DNBS

SR3 m ~

18.1~1,3"*

4,3~0,4"

3.8~0.6"

5

DNBS

SR10

20.61.9"

4,1~0,7"*

3.6~0.6"*

rn~

1.9~'0.3

35,5~7,3 29.8~8,6~

*P less than 0.01 vs. Group 1; ~P tessthan 0.01 vs. Group 3; "*P less than 0.05 vs. Group3

M1297 Glial Cells, but not Interstitial Cells, Express P2xT, a lonotropic Purinergic Receptor, in the Rat GI Musculature Jean-Marie Vanderwinden, Jean-Pierre Timmermans, Serge N. Schiffraann Purinerg~c (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal (GI) tract. So far seven ionotropic purinergic receptors (P2X) have been cloned. As specific antibodies become available, their respective distribution in the GI tract can be elucidated. Here we used high resolution tricolor confoeal microscopy as previously described to study the distribution of P2X7 immunoreactivity (-Jr) in the muscularis propria of the rat stomach, small intestine and colon. In all the regions

A-347

AGA Abstracts