Hepatocyte nuclear factor-4 regulates intestinal expression of the guanylin/heat-stable toxin receptor.
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We have investigated the regulation of gene transcription in the intestine using the guanylyl cyclase C (GCC) gene as a model. GCC is expressed in crypts and villi in the small intestine and in crypts and surface epithelium of the colon. DNase I footprint, electrophoretic mobility shift assay (EMSA), transient transfection assays, and mutagenesis experiments demonstrated that GCC transcription is regulated by a critical hepatocyte nuclear factor-4 (HNF-4) binding site between bp -46 and -29 and that bp -38 to -36 were essential for binding. Binding of HNF-4 to the GCC promoter was confirmed by competition EMSA and by supershift EMSA. In Caco-2 and T84 cells, which express both GCC and HNF-4, the activity of GCC promoter and/or luciferase reporter plasmids containing 128 or 1973 bp of 5'-flanking sequence was dependent on the HNF-4 binding site in the proximal promoter. In COLO-DM cells, which express neither GCC nor HNF-4, cotransfection of GCC promoter/luciferase reporter plasmids with an HNF-4 expression vector resulted in 23-fold stimulation of the GCC promoter. Mutation of the HNF-4 binding site abolished this transactivation. Transfection of COLO-DM cells with the HNF-4 expression vector stimulated transcription of the endogenous GCC gene as well. These results indicate that HNF-4 is a key regulator of GCC expression in the intestine. (+info)
Effect of motilin on the lower oesophageal sphincter.
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The effect of motilin on lower oesophageal sphincter (LES) pressure has been studied in unanesthetised specially trained dogs using an infusion manometric technique. Motilin produced significant rises in resting pressure and contractions of the LES after doses ranging from 0-009 mug/kg to 0-05 mug/kg. Doses greater than 0-05 mug/kg resulted in repetitive high amplitude contractions. Atropine 30 mug/kg completely abolished the effect of the lower doses of motilin. Higher doses of motilin in atropinised dogs still caused a small rise in baseline pressure and contractile activity still appeared. Hexamethonium 2 mg/kg resulted in both a diminished rise in LES pressure and the disappearance of contractions after motilin. Hexamethonium and atropine together completely abolished the LES response to motilin. We conclude that motilin increases LES pressure by acting on preganglionic cholinergic neurones to release acetylcholine which excites other cholinergic neurones supplying the circular muscle of the LES. (+info)
Cellular microbiology: can we learn cell physiology from microorganisms?
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Cellular microbiology is a new discipline that is emerging at the interface between cell biology and microbiology. The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yielding promising applications and striking results. (+info)
Glu-857 moderates K+-dependent stimulation and SCH 28080-dependent inhibition of the gastric H,K-ATPase.
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The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-ATPase. (+info)
Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides.
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Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP. (+info)
Relationship between the actions of atrial natriuretic peptide (ANP), guanylin and uroguanylin on the isolated kidney.
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Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 microg/ml, N = 12) or uroguanylin (0.5 microg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 microg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 +/- 0.86 to 64.62 +/- 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 +/- 0.01 to 0.15 +/- 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 +/- 0. 86 to 85.19 +/- 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins. (+info)
Immunohistochemical survey of the gut endocrine cells in the common tree shrew (Tupaia belangeri).
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Regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the common tree shrew (Tupaia belangeri) were studied immunohistochemically. Ten types of immunoreactive endocrine cells were localized in the gastric mucosa, i.e., chromogranin-, serotonin-, gastrin-, somatostatin-, bovine pancreatic polypeptide (BPP)-, enteroglucagon-, pancreatic glucagon-, peptide tyrosine tyrosine (PYY)-, motilin-, and substance P (SP)-immunoreactive (IR) cells. In the intestine, 13 types of immunoreactive cells were observed, i.e., chromogranin-, serotonin-, somatostatin-, gastrin-, BPP-, enteroglucagon-, PYY-, secretin-, cholecystokinin (CCK)-, gastric inhibitory peptide (GIP)-, motilin-, neurotensin-, and SP-IR cells. The regional distribution and relative frequency of the cell types varied along the gastrointestinal tract. Basically, the types, distribution, and relative frequency of the gut endocrine cells were similar to those reported in other mammalian species. However, some characteristic findings were noted in the present study: (1) the considerably large number of gastrin-IR cells in the pyloric region; (2) numerous serotonin-IR cells in the stomach; (3) appreciable number of BPP-IR cells in the transitional region of the stomach; and (4) wide distribution of PYY- and motilin-IR cells in the gut. (+info)
Impaired meal stimulated glucagon-like peptide 2 response in ileal resected short bowel patients with intestinal failure.
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BACKGROUND: Glucagon-like peptide 2 (GLP-2) is a growth factor for the intestinal epithelium in rodents and may affect intestinal transit. AIMS: To study the GLP-2 response to nutrient ingestion in seven short bowel patients with intestinal failure and seven controls. METHODS: The patients and controls were admitted twice for two test meals after a night of fasting. Meal A was liquid (300 ml, 1.88 MJ); meal B was a regular breakfast (755 g, 3.92 MJ). Plasma samples were collected for 180 minutes; GLP-2 immunoreactivity was measured with an NH(2) terminal specific radioimmunoassay. RESULTS: Both meals elicited significant increases in plasma GLP-2 in controls. The magnitude and duration of the responses were dependent on the meal size: the maximum median (25-75%) increases after meal A and B were 24 (3-28) and 48 (33-56) pmol/l. Plasma GLP-2 returned to basal concentrations 180 minutes after meal A, but remained at 50% of peak values after meal B. In the patients neither meal significantly changed the GLP-2 concentration; the maximum median elevation after meal B was 5 (2-8) pmol/l. There were significant differences between patients and controls with respect to the GLP-2 responses to meals A and B. CONCLUSION: Identification of GLP-2 as a tissue specific intestinal growth factor and demonstration of an impaired meal stimulated GLP-2 response in short bowel patients raises the possibility that GLP-2 administration may constitute a new therapeutic strategy, enhancing jejunal adaptation in ileum resected short bowel patients with intestinal failure. (+info)