Tissue pharmacokinetics, inhibition of DNA synthesis and tumor cell kill after high-dose methotrexate in murine tumor models.
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In Sarcoma 180 and L1210 ascites tumor models, the initial rate of methotrexate accumulation in tumor cells in the peritoneal cavity and in small intestine (intracellularly) after s.c. doses up to 800 mg/kg, showed saturation kinetics. These results and the fact that initial uptake in these tissues within this dosage range was inhibited to the expected relative extent by the simultaneous administration of leucovorin suggest that carrier mediation and not passive diffusion is the major route of drug entry at these extremely high doses. Maximum accumulation of intracellular drug occurred within 2 hr and reached much higher levels in small intestine than in tumor cells at the higher dosages. At a 3-mg/kg dose of methotrexate s.c., intracellular exchangeable drug levels persisted more than four times longer in L1210 cells than in small intestine, but differences in persistence (L1210 cell versus gut) diminished markedly with increasing dosage. At 96 mg/kg, the difference in persistence was less than 2-fold. In small intestine and L1210 cells, theduration of inhibition of DNA synthesis at different dosages correlated with the extent to which exchangeable drug was retained. Toxic deaths occurred when inhibition in small intestine lasted longer than 25 to 30 hr. Recovery of synthesis in small intestine and L1210 cells occurred synchronously and only below dosages of 400 mg/kg. Within 24 hr after dosages of greater than 24 mg/kg, the rate of tumor cell loss increased to a point characterized by a single exponential (t1/2=8.5 hr). The total cell loss, but not the rate of cell loss, was dose dependent. (+info)
Analysis of the effects of food and of digestive secretions on the small intestine of the rat. 1. Mucosal morphology and epithelial replacement.
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A modified Roux-en-Y repositioning of rat small intestine was performed so that the proximal segment of bowel (A) received only bile and pancreastic secretions, the second (B) received food direct from the stomach, and these two segments drained into a third (C). Four to five weeks after operation, cell production was assessed by injection of vincristine into operated, sham-operated and unoperated rats, and counts of blocked metaphases were made on isolated microdissected crypts. Villus height, crypt depth, and the number of crypts per villus (crypt/villus ratio) were also measured. Most of segment A showed no significant differences from sham-operated intestine, although the normal proximo-distal gradient of villus height was abolished. At the distal end (near the anastomosis with segments B and C), crypt depth and cell production were increased. The villus height gradient in segment B was also abolished, although crypt depth and cell production were significantly increased, especially at the proximal end. Crypt/villus ratio was also increased. Segment C showed all the characteristics of small bowel promoted to a more proximal position: increased villus height, crypt depth and cell production. Increased crypt/villus ratio was also observed. These results are discussed in terms of the role of food and of digestive secretions in the control of mucosal morphology and epithelial replacement. (+info)
Energy depletion differently affects membrane transport and intracellular metabolism of riboflavin taken up by isolated rat enterocytes.
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Isolated rat enterocytes, both normal and those de-energized with rotenone, were used to study the energy dependence of membrane and intracellular intestinal riboflavin transport in vitro. Membrane and intracellular transport were investigated by using short (3 min) and long (20 min) incubation times, respectively. For both types of cells and incubation times, [3H]-riboflavin uptake presented a saturable component prevailing at physiologic intraluminal concentrations. At 3 min incubation, saturable [3H]-riboflavin transport was apparently an energy-independent process with high affinity and low capacity. Values of the saturable component and its apparent constants, Km and Jmax, did not differ in normal and de-energized enterocytes. At 20 min incubation, saturable [3H]-riboflavin transport was a strictly energy-dependent process in which values of the saturable component were significantly greater in normal than in de-energized enterocytes. Km values did not differ in the two types of cells and were unmodified over 3 min, whereas in normal enterocytes, Jmax at 20 min [6.25 +/- 0.2 pmol/(mg protein. 20 min)] was significantly greater than at 3 min [2.67 +/- 0.33 pmol/(mg protein. 3 min)] and compared with de-energized enterocytes at 20 min [2.54 +/- 0.16 pmol/(mg protein. 20 min)]. Both membrane and intracellular events were inhibited by unlabeled riboflavin and analogs, which are good substrates for flavokinase, thus demonstrating the paramount role of this enzyme in riboflavin intestinal transport. (+info)
Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules.
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Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1-10 micrograms/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1alpha and IL-1beta, and IL-6, IL-8 and TNF-alpha as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria. (+info)
Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines.
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Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent beta-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted beta-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa. (+info)
Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine.
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1. Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. 2. Initially, 1-1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2-3 h (old tissues). 3. Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. 4. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+. 5. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues. 6. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. 7. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. 8. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels. (+info)
Expression of arginase II and related enzymes in the rat small intestine and kidney.
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Arginase, which catalyzes the conversion of arginine to urea and ornithine, and consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Arginine is also used for the synthesis of nitric oxide and creatine phosphate, while ornithine is used for the synthesis of polyamines and proline, and thus collagen. Arginase II mRNA and protein are abundant in the intestine (most abundant in the jejunum and less abundant in the ileum, duodenum, and colon) and kidney of the rat. In the kidney, the levels of arginase II mRNA do not change appreciably from 0 to 8 weeks of age. In contrast, arginase II mRNA and protein in the small intestine are not detectable at birth, appear at 3 weeks of age, the weaning period, and their levels increase up to 8 weeks. On the other hand, mRNAs for ornithine aminotransferase (OAT), ornithine decarboxylase, and ornithine carbamoyltransferase (OCT) are present at birth and their levels do not change much during development. Arginase II is elevated in response to a combination of bacterial lipopolysaccharide, dibutyryl cAMP, and dexamethasone in the kidney, but is not affected by these treatments in the small intestine. Immunohistochemical analysis of arginase II, OAT, and OCT in the jejunum revealed their co-localization in absorptive epithelial cells. These results show that the arginase II gene is regulated differentially in the small intestine and kidney, and suggest different roles of the enzyme in these two tissues. The co-localization of arginase II and the three ornithine-utilizing enzymes in the small intestine suggests that the enzyme is involved in the synthesis of proline, polyamines, and/or citrulline in this tissue. (+info)
Cellular uptake of biotin: mechanisms and regulation.
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This review describes our knowledge of biotin transport in the small intestine of humans and other mammals and presents recent findings in the area. Previous studies have shown that biotin transport across the brush border membrane of the small intestinal absorptive cells occurs via a carrier-mediated, Na+ gradient-dependent, electroneutral mechanism. Exit of biotin out of the enterocyte, i.e., transport across the basolateral membrane, also occurs via a carrier-mediated process, but the process is Na+ independent and electrogenic. Recent studies from our laboratory have shown that the uptake process of biotin in Caco-2 cells, a human-derived cultured intestinal epithelial cell line, are under the cellular regulation of both a protein kinase C- and a Ca/calmodulin-mediated pathway. In addition, the uptake process is shared by another water-soluble vitamin, pantothenic acid. For the first time, other recent studies have detected the existence of a Na+-dependent, carrier-mediated mechanism for biotin uptake at the apical membrane of colonocytes, which could theoretically mediate absorption of the biotin synthesized by colonic microflora. This system was again found to be shared by pantothenic acid, which is also synthesized by the normal microflora of the large intestine. (+info)