Free fatty acid receptor 3 activation suppresses neurogenic motility in rat proximal colon.

BACKGROUND: Short-chain fatty acids (SCFA) are microbial fermentation products absorbed by the colon. We recently reported that activation of the SCFA receptor termed free fatty acid receptor 3 (FFA3), expressed on cholinergic nerves, suppresses nicotinic acetylcholine receptor (nAChR)-mediated transepithelial anion secretion. This study aimed to clarify how activation of neurally expressed FFA3 affects colonic motor function. METHODS: FFA3-expressing myenteric neurons were identified by immunostaining; contractions of isolated circular muscle strips obtained from rat proximal colon were measured by isometric transducers. The effect of FFA3 agonists on defecation in vivo was examined in an exogenous serotonin-induced defecation model. KEY RESULTS: FFA3 immunoreactivity was located in nitrergic and cholinergic neurons in the myenteric plexus. In isolated circular muscle strips without mucosa and submucosa, the addition of nicotine (10 µM) or serotonin transiently relaxed the muscle through nitrergic neurons, whereas high concentrations of nicotine (100 µM) induced large-amplitude contractions that were mediated by cholinergic neurons. Pretreatment with FFA3 agonists inhibited nicotine- or serotonin-induced motility changes but had no effect on bethanechol-induced direct muscle contractions. The Gi/o inhibitor pertussis toxin reversed the inhibitory effect of an FFA3 agonist AR420626 on nicotine-evoked contractions, suggesting that FFA3 activation suppresses nAChR-mediated neural activity in myenteric neurons, consistent with an FFA3-mediated antisecretory effect. In conscious rats, exogenous serotonin increased the volume of fecal output, compared with the vehicle- or AR420626-treated groups. Pretreatment with AR420626 significantly suppressed serotonin-induced fecal output. CONCLUSION AND INFERENCES: FFA3 is a promising target for the treatment of neurogenic diarrheal disorders by suppressing nAChR-mediated neural pathways.

Luminal 5-HT stimulates colonic bicarbonate secretion in rats.

BACKGROUND AND PURPOSE: The bioactive monoamine 5-HT, implicated in the pathogenesis of functional gastrointestinal disorders, is abundantly synthesized and stored in rat proximal colonic mucosa and released to the gut lumen and subepithelial space. Despite much data regarding its expression and function, the effects of luminal 5-HT on colonic anion secretion have not been fully investigated. EXPERIMENTAL APPROACH: We measured short-circuit current (Isc ) as an indicator of ion transport in mucosa-submucosa or mucosa-only preparations of rat proximal colon. Total CO2 output was measured in vitro and in vivo. Immunohistochemistry was performed to investigate the localization of 5-HT4 , NOS1 and NOS2. KEY RESULTS: Luminal 5-HT gradually increased the amplitude and sustained the elevation of Isc . Luminal 5-HT-evoked ΔIsc was acetazolamide sensitive and HCO3 (-) dependent, consistent with cytosolic carbonic anhydrase-dependent electrogenic HCO3 (-) secretion, while not affected by tetrodotoxin (TTX), atropine or indomethacin. Pretreatment with the selective 5-HT4 antagonist GR113808, but not antagonists for 5-HT3 , 5-HT6 or 5-HT7 , inhibited luminal 5-HT-evoked ΔIsc . Furthermore, luminal cisapride and tegaserod increased Isc to the same extent as did 5-HT in the presence of indomethacin and TTX. Removal of the submucosa or pretreatment with NOS inhibitors enhanced luminal 5-HT-evoked ΔIsc , suggesting that NO synthesized in the submucosa suppresses mucosal anion secretion. NOS1 and NOS2 were immunostained in the submucosal neurons and glial cells respectively. Luminal 5-HT-evoked HCO3 (-) secretion was confirmed in vivo, inhibited by co-perfusion of GR113808, but not by ondansetron. CONCLUSIONS AND IMPLICATIONS: A novel apical 5-HT4 -mediated HCO3 (-) secretory pathway and an NO-dependent inhibitory mechanism are present in the proximal colon. Luminal 5-HT-evoked HCO3 (-) secretion may be important for the maintenance of mucosal integrity by regulating luminal pH.

Digestive physiology of the pig symposium: involvement of gut chemosensing in the regulation of mucosal barrier function and defense mechanisms.

Meal ingestion is followed by release of numerous hormones from enteroendocrine cells interspersed among the epithelial cells lining the intestine. Recently, the de-orphanization of G protein-coupled receptor (GPCR)-type nutrient receptors, expressed on the apical membranes of enteroendocrine cells, has suggested a plausible mechanism whereby luminal nutrients trigger the release of gut hormones. Activation of nutrient receptors triggers intracellular signaling mechanisms that promote exocytosis of hormone-containing granules into the submucosal space. Hormones released by foregut enteroendocrine cells include the glucagon-like peptides (GLP) affecting glycemic control (GLP-1) and releasing pro-proliferative, hypertrophy-inducing growth factors (GLP-2). The foregut mucosa, being exposed to pulses of concentrated HCl, is protected by a system of defense mechanisms, which includes epithelial bicarbonate and mucus secretion and augmentation of mucosal blood flow. We have reported that luminal co-perfusion of AA with nucleotides in anesthetized rats releases GLP-2 into the portal vein, associated with increased bicarbonate and mucus secretion and mucosal blood flow. The GLP-2 increases bicarbonate secretion via release of vasoactive intestinal peptide (VIP) from myenteric nerves. Luminal bile acids also release gut hormones due to activation of the bile-acid receptor known as G Protein-Coupled Receptor (GPR) 131, G Protein Bile Acid Receptor (GPBAR) 1, or Takeda G Protein-Coupled Receptor (TGR) 5, also expressed on enteroendocrine cells. The GLP are metabolized by dipeptidyl peptidase IV (DPPIV), an enzyme of particular interest to pharmaceutical, because its inhibition increases plasma concentrations of GLP-1 to treat diabetes. We have also reported that DPPIV inhibition enhances the secretory effects of nutrient-evoked GLP-2. Understanding the release mechanism and the metabolic pathways of gut hormones is of potential utility to the formulation of feedstuff additives that, by increasing nutrient absorption due to increased mucosal mass, can increase yields.

Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions.

Short chain fatty acids (SCFAs) are the major anions in the large intestine. They are produced by a bacterial fermentation of dietary fiber. SCFAs are known to have a variety of physiological and pathphysiological effects on intestine. However, the mechanisms by which intraluminal SCFAs are sensed are not known. In 2003, two orphan G protein coupled receptors (GPRs), GPR41 and GPR43, have been cloned and demonstrated to be receptors for SCFAs. Thus, we had attempted to make antibodies raised against GPR43 and GPR41 to elucidate the roles of SCFAs on colonic functions. We have also evaluated the effects of SCFAs on colonic motility to define the physiological roles on luminal SCFAs. In rat and human colon, GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extract. By immunohistochemistry, GPR43 immunoreactivity was localized with enteroendocrine cells expressing peptide YY, whereas 5-HT immunoreactive enteroendocrine cells were not immunoreactive for GPR43. GPR41 immunoreactivity was also found in human colon. In functional studies, propionate and butyrate concentration-dependently (10 microM - 10 mM) induced phasic and tonic contractions in rat colonic circular muscle. The propionate-induced phasic contraction was attenuated by atropine, tetrodotoxin and the 5-HT(4) receptor antagonists SB204070. However, acetate did not induce phasic or tonic contractions. Propionate-induced responses were not observed in mucosal free preparations. The present results suggest that the SCFA-induced physiological effects on colonic functions might be attributable to the activation of SCFA receptors on epithelial cells in the colon.

Outcome of 51 nonmalignant nodules in the liver: usefulness of aspiration cytology for diagnosis of dysplastic nodules.

Pathologic diagnostic criteria for intrahepatic nonmalignant nodules using needle biopsy are controversial. To evaluate the cytodiagnostic criteria for dysplastic nodules using aspiration biopsy, a follow-up study of nonmalignant nodules was performed. Fifty-one intrahepatic nodules diagnosed histologically and cytologically as nonmalignant using an aspiration biopsy in 39 patients were followed up without treatment. The outcomes of the nodules were investigated using the Kaplan-Meier method and Cox's multivariate analysis. The cumulative rates of development to HCC at the 2nd year were 0%, 18%, and 53% in the non-dysplastic nodules, the low-grade dysplastic nodules, and the high-grade dysplastic nodules, respectively; and significant differences were seen among them (P= 0.0001). Multivariate analysis showed that cytologic grade was a significant risk factor for development to HCC (P=0.020). In conclusion, aspiration cytology was useful for diagnosis of the dysplastic nodules to predict risks of development to HCC.

Evaluation of epithelial cell proliferation rate in normal-appearing colonic mucosa as a high-risk marker for colorectal cancer.

To determine whether the colonic epithelial proliferation rate is useful as a marker for colorectal cancer, we measured the Ki-67 labeling index (LI) in normal-appearing mucosa from the sigmoid and ascending colon in patients with two or more tumors (early cancers, which are defined as tumors the depth of invasion of which is limited to mucosal layer or submucosal layer, adenomas, or both). The association of baseline LI with the risk of development of colon tumors 2 years after endoscopic removal was assessed by cohort analysis. The presence of two or more tumors was defined as occurrence. One hundred and six specimens from the sigmoid colon and 130 from the ascending colon from 246 subjects (203 males and 43 females) were used for analysis. The patients with higher upper-third LI in the normal-appearing mucosa in the sigmoid colon, but not in the ascending colon, had significantly more tumors at follow-up colonoscopy 2 years later (risk ratio, 3.6; 95% confidence interval, 1.2-10.6). Moreover, multivariate analysis showed that it was an independent factor. We concluded that the higher upper-third Ki-67 LI of normal-appearing mucosa in the sigmoid colon indicates a high risk for colorectal cancer.

Inhibition by d-limonene of experimental hepatocarcinogenesis in Sprague-Dawley rats does not involve p21(ras) plasma membrane association.

The effects of d-limonene on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and on membrane-associated p21(ras) and labeling and apoptotic indices of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of experimental week 9, they received chow pellets containing 1% or 2% limonene. The preneoplastic and neoplastic liver lesions (cellular alteration foci, neoplastic nodules and hepatocellular carcinomas), and hepatic foci staining positive for glutathione-S-transferase, placental type (GST-P) were examined microscopically and histochemically. At week 16, quantitative histologic analysis showed that oral administration of 1% or 2% limonene resulted in significant reductions in the number and mean area of GST-P-positive hepatic foci and the number of cellular alteration foci, neoplastic nodules and hepatocellular carcinomas. Limonene, at both doses, also caused significant decreases in the labeling indices and significant increases in the apoptotic indices of cellular alteration foci, neoplastic nodules, hepatocellular carcinomas and adjacent liver. However, limonene, at both doses, had no significant influence on the production of membrane-associated p21(ras) in the visible liver white nodules. These findings indicate that limonene inhibits hepatocarcinogenesis and suggest that this effect may be clearly related to its effect in inhibiting cell proliferation and in enhancing apoptosis, but not through ras oncoprotein plasma membrane association.

Measurement of carcinoembryonic antigen in colonic effluent as a high-risk marker for colorectal carcinoma.

We evaluated the usefulness of carcinoembryonic antigen (CEA) concentrations in colonic effluent as a high-risk marker for colorectal carcinoma (CRC). After 213 patients ingested 1,800 ml of a 5% isotonic solution of magnesium citrate, colonic effluent was collected from them before routine colonoscopy from February through June 1992. Of these patients, 27 who had undergone colonoscopy after a mean of 2.6 years were selected as subjects. The relationship between the CEA concentration in the colonic effluent and the occurrence of new colorectal tumors was examined. The CEA concentration in colonic effluent was adjusted on the basis of alkaline phosphatase activity. New colorectal tumors were noted significantly more frequently (P = .006) in patients with a high CEA level in colonic effluent (5 of 5; 100%) than in those with low a CEA level (6 of 22; 27%). The CEA concentration in colonic effluent is a simple and practical biomarker for identification of patients at high risk for CRC.

Long-term responders without eradication of hepatitis C virus after interferon therapy: characterization of clinical profiles and incidence of hepatocellular carcinoma.

AIMS/BACKGROUND: The aim of this study was to determine the characteristics of patients with chronic hepatitis C showing long-term normalization of alanine aminotransferase (ALT) without eradication of HCV RNA, as well as to investigate the incidence of hepatocellular carcinoma (HCC) in such patients. METHODS: Four hundred and nineteen patients with histologically-proven chronic hepatitis C who had received interferon (IFN) therapy were studied. Complete response (CR) was defined as persistent normalization of ALT levels with eradication of serum HCV RNA (n= 126). Long-term biochemical response with positive HCV RNA (HCV-positive BR) was defined as a normal ALT level at 6 months after IFN therapy with further persistent normalization of ALT levels for 2 or more years without eradication of serum HCV RNA (n=49). All other patterns were classified as non-response (NR, n=244). RESULTS: Mean follow-up periods of CR, HCV-positive BR and NR groups were 4.9, 5.2 and 4.9 years, respectively. The HCV-positive BR group had significantly higher serum HCV RNA levels and a higher rate of HCV serological group 1 classification than the CR group. The other characteristics of the HCV-positive BR group were lower histologic activity, lower ALT levels, and a higher rate of females when compared with both the CR and NR groups. Histologic staging in the HCV-positive BR group was significantly lower than that in the NR group. Cumulative incidences of HCC estimated by the Kaplan-Meier method in both the CR and HCV-positive BR groups were significantly lower than those in the NR group (log-rank test, CR vs NR p<0.001, HCV-positive BR vs NR p=0.026). CONCLUSION: The patients with HCV-positive BR were virologically different from those with CR, and had lower ALT levels and histologic activity when compared to those with CR and NR.

Usefulness of diagnostic criteria for aspiration cytology of hepatocellular carcinoma.

OBJECTIVE: To establish new criteria for cytodiagnosis of hepatocellular carcinoma by comparing cytologic findings of hepatocellular carcinoma with those of liver cirrhosis. STUDY DESIGN: Review of cytologic findings of hepatocellular carcinoma on preoperative aspiration biopsy of 31 lesions from 27 patients who underwent surgical resection and comparison of these findings with those of liver cirrhosis in 17 patients. RESULTS: In the 11 lesions of moderately and poorly differentiated hepatocellular carcinoma, significant cytologic findings included monotonous and abundant cytoplasm, thick cytoplasm, increased nuclear/cytoplasmic ratio, irregular nuclear contours, increased chromatin density, intranuclear vacuoles and naked nuclei. In the 20 lesions demonstrating well-differentiated hepatocellular carcinoma, significant cytologic findings included monotonous and scant cytoplasm, well-defined cytoplasmic borders, thick cytoplasm, eccentric nuclei, increased nuclear/cytoplasmic ratio, thick nuclear membranes and increased chromatin density. We established the criteria for moderately and poorly differentiated hepatocellular carcinoma as including three cytologic parameters: increased nuclear/cytoplasmic ratio, irregular nuclear contours and increased chromatin density. We also established the criteria for well-differentiated hepatocellular carcinoma as including six cytologic parameters: monotonous cytoplasm, scant cytoplasm, well-defined cytoplasmic borders, thick cytoplasm, eccentric nuclei and increased nuclear/cytoplasmic ratio. For all 31 hepatocellular carcinoma lesions, including 27 lesions that were < or = 2 cm in diameter, both sensitivity and specificity were 100% by concurrently employing both criteria. CONCLUSION: The new criteria for cytodiagnosis we established were useful for differentiating hepatocellular carcinoma from liver cirrhosis. In particular, our criteria ensured appropriate diagnostic accuracy for well-differentiated hepatocellular carcinoma.

Relation of interferon therapy and hepatocellular carcinoma in patients with chronic hepatitis C. Osaka Hepatocellular Carcinoma Prevention Study Group.

BACKGROUND: The effect of interferon therapy on the incidence of hepatocellular carcinoma in chronic hepatitis C is poorly defined. OBJECTIVE: To compare the incidence of hepatocellular carcinoma in interferon-treated patients with chronic hepatitis C to that of historical controls and to examine whether response to therapy is related to incidence of hepatocellular carcinoma in patients with chronic hepatitis C. DESIGN: Retrospective cohort study. SETTING: One university hospital and seven university-affiliated hospitals. PATIENTS: 419 consecutive patients with chronic hepatitis C who started interferon therapy between January 1992 and December 1993 (interferon group) and 144 patients with chronic hepatitis C who had liver biopsy between January 1986 and December 1989 and did not receive interferon (controls). INTERVENTION: Patients in the interferon group received human lymphoblastoid interferon, recombinant interferon-alpha2a, or recombinant interferon-alpha2b for 6 months. MEASUREMENTS: The end point was development of hepatocellular carcinoma on abdominal ultrasonography or computed tomography. Sustained response was defined as persistent normalization of alanine aminotransferase (ALT) levels during interferon therapy and follow-up. Relapse was defined as a normal serum ALT level at the end of treatment with an increase to an abnormal level after cessation of treatment. Nonresponse included all other ALT patterns. RESULTS: Median follow-up in the interferon and control groups was 47.6 and 46.8 months, respectively. During follow-up, hepatocellular carcinoma was found in 28 interferon-treated patients and 19 controls. Cox proportional hazards regression analysis that included all patients revealed that interferon therapy (P=0.041), older age (P=0.003), greater histologic activity (P=0.029), and higher histologic stage (P=0.049) were independent factors associated with the development of hepatocellular carcinoma. The risk ratios for development of hepatocellular carcinoma in patients with sustained response, relapse, and nonresponse were 0.06 (95% CI, 0.01 to 0.46), 0.51 (CI, 0.20 to 1.27), and 0.95 (CI, 0.48 to 1.84), respectively, compared with controls. CONCLUSIONS: The incidence of hepatocellular carcinoma was lower in patients with sustained response to interferon therapy than historical controls and nonresponders. Interferon therapy may decrease the risk for hepatocellular carcinoma in patients with chronic hepatitis C.

Prognostic value of periodic Acid-schiff (pas) staining of fine-needle aspirates from patients with primary hepatocellular-carcinoma.

The relationships of cytological findings on periodic acid-Schiff (PAS) staining of fine-needle aspirates of hepatocellular carcinomas (HCC) with the length of survival of patients, their therapeutic response after transcatheter arterial embolization (TAE), and the tumor volume doubling time (TVDT) before chemoembolization were investigated in 74 patients with primary HCC. On the basis of cytologic findings on PAS-staining, HCC was classified into PAS-positive, mixed, and PAS-negative types. The two-year survival (70%) of patients with PAS-positive HCC was significantly longer than those of patients with the mixed or PAS-negative type of HCC. The TVDT of PAS-positive HCC was also significantly longer than that of the mixed type of HCC. These findings indicate that cytological findings on PAS-staining of HCC are useful in assessing the prognosis of patients with HCC.