In vitro inhibition of rat small intestinal absorption by lipophilic organic cations

Cationic, lipid-soluble organic compounds may interfere with cation-mediated membrane transport processes. Thus, small intestinal absorption may be influenced by lipophilic organic cations. Therefore a series of arylalkylamines was studied in the concentration range from 0.5 to 20 mmol/l for their effect on the transport of various monosaccharides and leucine in the rat small intestine in vitro by means of the tissue accumulation technique. Whereas the monophenyl substituted monoamines (e.g. benzylamine, 2-phenylethylamine, 3-phenylpropylamine) did not show a significant effect on the active transport, the corresponding ω,ω-diphenyl derivatives exhibited a strong inhibition of the active transport of the sugars and the amino acid. These monoamines and drugs of similar structure (e.g. benzoctamine, diphenhydramine) exhibited a mixed or non-competitive type of inhibition which correlated quite well with their octanol-water partition coefficients. In contrast, di- or triamines (e.g. harmaline, imipramine, pyrilamine) revealed a rather pure competitive type of inhibition. These findings tentatively suggest a different mode of action on the active transport by lipid-soluble organic amines according to the molecular charge distribution. In addition, membrane vesicles were used to examine the effect of the different amines on the sucrase activity. Regarding the cation-dependent hydrolysis of sucrose, however, no distinct pattern developed.     

In vitro effects of ethanolamine on insulin secretion

The effects of ethanolamine on insulin secretion by the perfused rat pancreas were examined. During the second phase of glucose-induced insulin secretion 5-minute perfusions of ethanolamine at final concentrations of 0.1, 1 and 10 mM inhibited insulin release in a dose-related manner. When given throughout the experiment the highest dose of ethanolamine markedly suppressed both phases of glucose-induced insulin release. The inhibitory effect of ethanolamine was blunted in the presence of phentolamine. It is concluded that ethanolamine inhibits glucose-induced insulin secretion by the perfused rat pancreas and that alpha-adrenergic receptors play a role in its actions on insulin output.     

In vitro study of the intestinal brush border enzyme activities in developing anuran amphibian: effects of thyroxine, cortisol, and insulin

The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.     

In vitro folding/unfolding of insulin/single-chain insulin

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.     

小鼠肠道对壳寡糖的吸收率及吸收成分的影响

目的通过分析壳寡糖在小鼠肠道内的吸收率以及吸收成分,了解肠道对壳寡糖代谢的影响,初步判断壳寡糖的有效生物成分,为壳寡糖生物活性的进一步研究提供必要的实验材料和线索。方法首先用蜗牛酶将壳聚糖水解成聚合度不同的壳寡糖,并通过聚丙烯酰胺凝胶层析柱（Bio-Gel P4凝胶）将不同聚合度的壳寡糖分开,分别收集聚合度为1-3,8-11的壳寡糖,并用异硫氰酸荧光素（FITC）标记,再通过聚丙烯酰胺凝胶层析柱（Bio-Gel P-2/P4）将游离的FITC除去,随后对两组禁食24 h的小鼠分别用FITC标记的大分子量和小分子量的壳寡糖灌胃。1 h后取血清和小肠,经分离水溶成分后,通过荧光分光光度计检测血清样品和肠溶物样品中荧光的强度,进而确定壳寡糖的吸收率和吸收成分。结果通过改变酶解时间,蜗牛酶可以将壳聚糖水解成不同聚合度的壳寡糖。利用Bio-Gel PA聚丙烯酰胺凝胶层析柱可以将不同聚合度的壳寡糖分离成具有一定聚合度范围的壳寡糖。用F1TC标记的大、小分子量的壳寡糖给小鼠灌胃,从血清和肠溶物中均检测到荧光强度,两者比值平均值分别为5.68 ： 1和9.84 ： 1。结论壳寡糖在肠道的吸收率随分子量的减小而增大,除小分子壳寡糖外,吸收成分也包括部分大分子量壳寡糖。     

Differentiated intestinal epithelial cell lines asin vitro models for predicting the intestinal absorption of drugs

The oral absorption of a compound is a critical factor for the future of the compound as a drug. This absorption is mainly controlled by the passage across, the intestinal epithelium. Thus, the prediction of the intestinal absorption by means of anin vitro model may represent a powerful tool for the early selection of molecules during the process of drug development. In the present study, the differentiated human intestinal epithelial cell line HT29-18-C₁, was grown on permeable filters in dual chambers. These cells formed tight monolayers that were used to measurein vitro the transepithelial permeability coefficient (P _c) of various molecules. The results were compared within vivo data of oral absorption. A threshold value ofin vitro permeability of 2×10^–6 cm/s was found. Molecules having a permeability coefficient higher than this value were absorbed orally more than 80%, while drugs withP _c values lower than 2×10^–6 cm/s were poorly absorbed. By mathematical simulation, it was found that thisP _c value, when extrapolated to the surface area and volume of the small intestine, corresponds to an absorption of 80% for a compound with a transit time through the small intestine of 5 h. This demonstrates the predictive utility of the threshold value of the permeability coefficient derived from thein vitro model of intestinal epithelium.Abbreviations P _c transepithelial permeability coefficient - MTX methotrexate     

The role of insulin in the intestinal absorption of glucose in the rat.

1. Acute pre-treatment with either mannoheptulose or streptozotocin--both compounds acting as powerful suppressors of insulin secretion--caused a significant decrease on the in vivo rate of intestinal glucose absorption following an intragastric [U-14C]glucose administration. 2. Mannoheptulose treatment also lowered the rate of whole-body oxidation of the administered tracer. 3. Insulin had no effect on the metabolic fate of [U-14C]glucose by isolated enterocytes. 4. However, the rate of glucose uptake, measured by the oxidation of [1-14C]glucose to 14CO2 in the presence of phenazine methosulphate, was decreased by insulin at concentrations of 50-200 munits/ml. 5. In addition, the rate of transport of [U-14C]glucose by brush-border membrane vesicles was also inhibited by insulin at high concentrations (100-1000 munits/ml). 6. This indicated that insulin acts by inhibiting glucose transport in isolated in vitro preparations. 7. Acute pre-treatment with either mannoheptulose or streptozotocin caused a significant decrease in the rate of gastric emptying, measured as the distribution of [3H]insulin along the gastrointestinal tract, following an intragastric glucose load. 8. It is concluded that insulin secretion modulates intestinal glucose absorption in vivo by enhancing gastric emptying in spite of the inhibitory effects of glucose transport observed with in vitro preparations.     

In vitro appraisal of the probiotic value of intestinal lactobacilli

Lactobacilli play a distinctive role in the microbial balance of the chicken gut. In experiments simulating the chicken crop, the antagonism of lactobacilli against Enterobacteriaceae and Salmonella typhimurium was demonstrated and was attributed to lactic acid production. Moreover, adhesion to the crop epithelium was a common characteristic of intestinal lactobacilli. As opposed to salmonellas, lactobacilli were sensitive to deconjugated bile salts at 2.5mm. This sensitivity could lower their chance of proliferation in the small bowel of the chicken tract.The authors are at the Laboratory of Microbial Ecology, Faculty of Agricultural Sciences, State University of Gent, Coupure L., 653, B-9000 Gent, Belgium.     

In vitro translation of intestinal sucrase-isomaltase and glucoamylase

It is has been proposed that both sucrase-isomaltase and glucoamylase are initially synthesized as large single-chain precursors which are then processed to heterodimers. We have tested this hypothesis by in vitro translation of their mRNAs. The primary translation product of sucrase-isomaltase mRNA was a single polypeptide of Mr = 190,000. Similar experiments using antiserum against glucoamylase revealed a single polypeptide of Mr = 145,000. These results are consistent with the single chain precursor hypothesis for sucrase-isomaltase. However, the glucoamylase product (145 kDa) is too small to be processed to a heterodimer of Mr = 230,000. Moreover, the mature subunits (Mr = 135,000 and 125,000) probably are derived from the 145 kDa precursor by proteolytic trimming and must include and share most of the precursor protein.     

In vitro models of intestinal epithelial cell differentiation

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.     

In vitro studies on active calcium absorption from ovine rumen

From various in vivo and in vitro studies it has been shown that the rumen represents a significant site of Ca²⁺ absorption in sheep and goats. It was the aim of the present study to further characterize the underlying mechanisms. Unidirectional flux rates of Ca²⁺ across rumen wall epithelia of sheep were measured in vitro by applying the Ussing-chamber technique in the absence of electrochemical gradients. Under these conditions, significant Ca²⁺ net flux rates (J_net) clearly indicate the presence of active mechanisms for Ca²⁺ transport. Short chain fatty acids (SCFAs) caused highest stimulation of Ca²⁺ J_net (6.3 ± 1.9 nmol · cm⁻² · h⁻¹) when used as a mixture of acetate, proprionate and butyrate in physiological proportions (36, 15, 9 mmol · l⁻¹, respectively). The effect of 30 mmol · l⁻¹ butyrate (3.2 ± 0.6 nmol · cm⁻² · h⁻¹) was higher than respective amounts of propionate and acetate (0.6 ± 0.8 nmol · cm⁻² · h⁻¹ and 0.9 ± 0.8 nmol · cm⁻² · h⁻¹, respectively). Eliminating SCFAs resulted in Ca²⁺ J_net of 0.4 ± 1.1 nmol ^. cm^−2 .h⁻¹. Addition of Ca channel blocker verapamil (mucosal 1 mmol · l⁻¹) had no significant effect on SCFA-stimulated J_net of Ca²⁺, whereas application of Na⁺/H⁺ inhibitor amiloride (mucosal 1 mmol · l⁻¹) further enhanced the Ca²⁺ J_net by >65%. The Ca²⁺-pump inhibitor vanadate had no significant effect on J_net of Ca²⁺. Dietary Ca depletion enhanced calcitriol plasma concentrations but had no effect on active Ca²⁺ absorption across the rumen wall of sheep. In addition, no effect on active Ca²⁺ absorption could be observed during early lactation. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in sheep. Our results suggest the presence of a Ca²⁺/H⁺ exchange mechanism in the apical membrane of rumen epithelial cells which depends on SCFA absorption and which does not seem to be under the control of calcitriol. Basolateral Ca²⁺ extrusion occurs independently from Ca²⁺-pump activity and may be accomplished via Na⁺/Ca²⁺ exchange. Accepted: 29 June 1999