Increased cholinergic contractions of jejunal smooth muscle caused by a high cholesterol diet are prevented by the 5-HT(4 )agonist – tegaserod

BACKGROUND: Excess cholesterol in bile and in blood is a major risk factor for the respective development of gallbladder disease and atherosclerosis. This lipid in excess negatively impacts the functioning of other smooth muscles, including the intestine. Serotonin is an important mediator of the contractile responses of the small intestine. Drugs targeting the serotonin receptor are used as prokinetic agents to manage intestinal motor disorders, in particular irritable bowel syndrome. Thus, tegaserod, acting on 5-HT(4 )receptor, ideally should obviate detrimental effects of excessive cholesterol on gastrointestinal smooth muscle. In this study we examined the effect of tegaserod on cholesterol-induced changes in the contractile responses of intestinal smooth muscle. METHODS: The effects of a high cholesterol (1%) diet on the in vitro contractile responses of jejunal longitudinal smooth muscle from Richardson ground squirrels to the cholinergic agonist carbachol were examined in the presence or absence of tetrodrodotoxin (TTX). Two groups of animals, fed either low (0.03%) or high cholesterol rat chow diet, were further divided into two subgroups and treated for 28 days with either vehicle or tegaserod. RESULTS: The high cholesterol diet increased, by nearly 2-fold, contractions of the jejunal longitudinal smooth muscle elicited by carbachol. These cholinergic contractions were mediated by muscarinic receptors since they were blocked by scopolamine, a muscarinic receptor antagonist, but not by the nicotinic receptor antagonist, hexamethonium. Tegaserod treatment, which did not affect cholinergic contractions of tissues from low cholesterol fed animals, abrogated the increase caused by the high cholesterol diet. With low cholesterol diet TTX enhanced carbachol-evoked contractions, whereas this action potential blocker did not affect the augmented cholinergic contractions seen with tissues from animals on the high cholesterol diet. Tegaserod-treatment removed the effects of a high cholesterol diet on neuronal muscarinic receptors, as the potentiating effect of TTX on carbachol-elicited contractions was maintained in these animals. CONCLUSION: A high cholesterol diet causes significant changes to cholinergic neurotransmission in the enteric nerves of the jejunum. The mechanisms by which these effects of cholesterol are reversed by tegaserod are unknown, but relate to removal of an inhibitory effect of cholesterol on enteric nerves

Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD

A murine model of ulcerative colitis: induced with sinusitis-derived superantigen and food allergen

BACKGROUND: The etiology of ulcerative colitis (UC) is to be understood. The basic pathological feature of UC is intestinal chronic inflammation. Superantigen, such as Staphylococcus enterotoxin B (SEB), is reported to compromise intestinal barrier function by increasing epithelial permeability and initiate inflammation in the intestinal mucosa. Inasmuch as anatomic position of the sinus, chronic sinusitis-derived SEB may follow the secretion and to be swallowed down to the gastrointestinal tract and induce lesions to the intestinal mucosa. METHODS: Sinus wash fluid (SWF, containing SEB) was collected from a group of patients with both chronic sinusitis (CS) and UC. A group of mice were sensitized to ovalbumin (OVA) in the presence of SWF. The sensitized mice were challenged with the specific antigen OVA. The inflammatory status of the colonic tissue was determined with histology, serology and electron microscopy. Using horseradish peroxidase (HRP) as a tracer, another group of mice was stimulated with SWF for 2 hours. The HRP activity was detected in the colonic tissue with enzymatic approaches and electron microscopy. RESULTS: Epithelial hyperpermeability in colonic epithelium was induced by stimulating with SWF. The HRP activity in the colonic mucosa was almost 11 times more in the SWF treated group (3.2 ± 0.6 μg/g tissue) than the control group (0.3 ± 0.1 μg/g tissue). Mice were sensitized using a mixture of SWF and OVA (serum OVA-specific IgE was detected with a highest titer as 1:64). Challenge with OVA induced extensive inflammation in the colonic mucosa by showing (1) marked degranulation in mast cells (MC, 46.3 ± 4.5%) and eosinophils (Eo, 55.7 ± 4.2%); (2) inflammatory cell infiltration (MC = 145.2 ± 11.4; Eo = 215.8 ± 12.5; mononuclear cell = 258.4 ± 15.3/mm(2 )tissue); (3) increased MPO activity (12.9 ± 3.2 U/g tissue) and inflammatory scores (1.8 ± 0.3); (4) mucosal surface ulcers; (5) edema in the lamina propria; (6) bacterial translocation and abscess formation in the subepithelial region. CONCLUSION: Introducing Sinusitis-derived SEB-containing SWF to the gastrointestinal tract compromised colonic mucosal barrier function increasing epithelial permeability to luminal macromolecular protein in mice. The SWF facilitated colonic mucosal sensitization to luminal antigen. Multiple challenging the sensitized colonic mucosa with specific antigen OVA induced inflammation, induced a condition similar to human ulcerative colitis

Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination

Placental deiodination of T-4 to rT(3) has been proposed as the factor controlling materno-fetal transmission of T-4. We investigated T-4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T-4 (150 nnol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T-4 in the fetal circuit was very low, with fetal T-4 levels reaching only 4.1 +/- 0.04 pmol/L at 6 h. Levels of rT, rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T-3 could be measured in either circuit. Addition of 0.5 mmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT(3) [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 mmol/L (2.7% of control values)] and a major (similar to 2700-fold) increase in T-4 appearance in the fetal circuit, with fetal T-4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T-4

Hypothyroidism and Graves-Disease After Mantle Irradiation - a Follow-Up-Study

Abstract Fifty of 66 patients whose thyroid function had previously been assessed 7–139 months after irradiation for Hodgkin's disease were re‐evaluated 35 ± 3 months later. They could be divided into three groups: those whose thyroid function had been normal in the first study (N= 26), those who had had asymptomatic impaired thyroid reserve (N= 19), and those in whom evidence of Graves' disease had developed (N= 5). The 26 patients who had been euthyroid when first studied had developed significant increases in mean throid‐stimulating hormone (TSH) levels basal and following thyrotrophin releasing hormone) without changes in mean free thyroxine index (FTI). In three of these patients, each studied within six years of irradiation, basal TSH had risen to hypothyroid levels. There were no significant changes in mean FTI or basal and peak TSH in 19 patients who had demonstrated impaired thyroid reserve in the first study. The cumulative incidence of impaired thyroid reserve in the total cohort is now 30/66 (45%) but only one of these 30 has developed clinical hypothyroidism. Five patients developed evidence of Graves' disease. Two patients with thyrotoxicosis and one with euthyroid Graves' disease were found in the initial study. On re‐evaluation, a third patient had developed frank thyrotoxicosis and another euthyroid Graves' disease, giving a cumulative incidence of Graves' disease of 5/66 (7%). Three of these five were HLA‐DR3 and three had measurable thyrotrophin binding inhibiting immunoglobulins. We conclude that impaired thyroid reserve continues to develop within six years of mantle irradiation in adults but once established appears to remain stable. There also appears to be a high incidence of Graves' disease after mantle irradiation

Single-Dose Intravenous Pamidronate Is Effective Alternative Therapy for Pagets-Disease Refractory to Calcitonin

We have conducted an open, prospective study to investigate the efficacy of a single 60 mg infusion of pamidronate as alternative therapy in 15 subjects with severe Paget's bone disease refractory to calcitonin. Disease activity was assessed with a visual-analogue score of symptom severity, plasma alkaline phosphatase and quantitative estimation of Tc-99m-methylene biphosphonate uptake on bone scan. All indices of disease activity fell after pamidronate, reaching a nadir at 3 months. Although disease activity increased thereafter, only 3 subjects required retreatment within 12 months. Plasma calcium fell after 3 days and remained below baseline levels for 6 months associated with evidence of secondary hyperparathyroidism. Pamidronate was well tolerated; femoral neck fractures occurred in 2 subjects with severe local Paget's disease but were unlikely to be due to the drug. We conclude that pamidronate is an effective and promising alternative for treatment of patients with severe Paget's disease no longer adequately controlled by calcitonin. Calcium supplementation may be prudent to prevent secondary hyperparathyroidism associated with the use of this agent

Blunted Parathyroid Response to Correction of Hypercalcemia in Subjects with Squamous-Cell Carcinoma

In malignancy-associated hypercalcemia (MAH) elevated plasma calcium levels are believed to inhibit parathyroid secretion independently of the underlying tumor. This predicts that correction of hypercalcemia should disinhibit circulating parathyroid hormone (PTH) levels, irrespective of the underlying disease. We have tested this hypothesis in subjects with multiple myeloma (MM) and squamous cell carcinoma (SCC) treated with pamidronate. In the MM group, PTH levels returned to normal as hypercalcemia was corrected. In contrast, PTH levels remained low in the SCC group despite a similar fall, in plasma calcium. Calcitriol levels were significantly higher and magnesium levels slightly lower in the SCC group than those in the MM group. We conclude that the parathyroid response to the correction of hypercalcemia is blunted in subjects with SCC but not MM. In addition to hypercalcemia, other factors, perhaps related to tumor secretion of PTH-related protein, may therefore contribute to suppressing PTH secretion in MAH due to SCC