Chemotherapy-induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity

The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration-dependent manner. Comparison of the EC50 values for cerebellar neurons and astrocytes with the median EC50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100-fold higher sensitivity of cerebellar neurons towards vincristine and 10-fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt-dependent expression of phosphorylated BAD. zVAD-fmk, a caspase inhibitor and brain-derived neurotrophic factor (BDNF), but not MK-801, a non-competitive NMDA receptor antagonist, significantly reduced vincristine- or topotecan-induced cell death.     

Characterization of HSP27 phosphorylation induced by microtubule interfering agents: implication of p38 signalling pathway

Vincristine and paclitaxel are widely used antitumoral drugs that interfere with microtubule dynamics. We have previously demonstrated that vincristine induces phosphorylation of HSP27 at serine 82 in MCF-7 cells. In this report, we show that vincristine also causes phosphorylation of serines 78 and 15. Moreover, we demonstrate that phosphorylation of this chaperone is induced by the p38 signalling pathway while the JNK pathway is not implicated. Differences between vincristine and paclitaxel treatments are also appreciated. Thus, while vincristine induces a strong phosphorylation of the three serines, paclitaxel induces a weak phosphorylation of serine 78 and has no effect over serines 82 and 15 phosphorylation. Interestingly, pre-treatment of cells with a ten-fold excess of paclitaxel abolishes vincristine-induced phosphorylation of HSP27.     

Aging and neural control of the GI tract. II. Neural control of the aging gut: can an old dog learn new tricks?

There has been a dramatic increase in funding available for aging research, primarily due to the fact that answers to questions on aging are likely to have a major impact on the well-being and healthy aging of the world's population for decades to come. The incidence of certain gastrointestinal problems, such as dysphagia and constipation, increases dramatically with age. Changes in gastrointestinal neuromuscular function with aging have been demonstrated in both human and animal models of aging. This article focuses on recent advances in our knowledge of the effects of aging on gastrointestinal function, treatment options, and future opportunities for research.     

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.     

Orally administered kappa as well as mu opiate agonists delay gastrointestinal transit time in the guinea pig

When an orally administered opiate agonist is systemically bioavailable, the relative activity of that opioid in delaying gastrointestinal transit (GIT) depends on its relative action at central and peripheral sites. This in turn depends on the density of opioid receptor specific subtypes at those sites of action in the species under study. In rats the kappa selective agonist U-50,488H has no effect on GIT. We have found that this same agonist is equipotent to mu agonists morphine and 1-methadone in delaying the orocecal transit of a charcoal meal when administered orally to guinea pigs. Thus, both kappa as well as mu receptor subtypes are involved in the mechanisms of opiate induced slowing of GIT in the guinea pig in contrast to the rat. Interspecies differences must be considered when determining the contribution of opiate receptor subtypes to the mechanisms of opiate-induced constipation.     

Iron-loaded cardiac myocytes stimulate cardiac myofibroblast DNA synthesis

Zinc is an essential nutrient with a wide range of functions and closely involved in a variety of enzymatic processes of importance in glucose, protein and lipid metabolism. Ghrelin is the endogenous ligand of the G protein coupled growth hormone secretagogue receptor. The regulatory mechanism that explain the biosynthesis and secretion of ghrelin in the gastrointestinal tract has not been clarified. This study was undertaken to examine the effect of zinc supplementation on the streptozotocin (STZ)-induced diabetic rats, which exhibits ghrelin production and secretion, and lipid metabolism on the gastrointestinal tract. The animals were divided into four groups. Group I: Non-diabetic untreated animals. Group II: Zinc-treated non-diabetic rats. Group III: STZ-induced diabetic untreated animals. Group IV: Zinc-treated diabetic animals. Zinc sulfate was given to some of the experimental animals by gavage at a dose of 100 mg/kg body weight every day for 60 days. In the zinc-treated diabetic group, the blood glucose levels decreased and body weight increased as compared to the diabetic untreated group. Zinc supplementation to STZ-diabetic rats revealed the protective effect of zinc on lipids parameters such as total lipid, cholesterol, HDL-cholesterol and atherogenic index. There is no statistically change in ghrelin-immunoreactive cells in gastrointestinal tissue. But, it has found that zinc supplementation caused a significant reduction in densities of ghrelin-producing cells of fundic mucosa of zinc-treated diabetic animals as compared to untreated, non-diabetic controls. Zinc supplementation may contribute to prevent some complications of diabetic rats, biochemically.     

Analysis of spatiotemporal pattern and quantification of gastrointestinal slow waves caused by anticholinergic drugs

Anticholinergic drugs are well-known to cause adverse effects, such as constipation, but their effects on baseline contractile activity in the gut driven by slow waves is not well established. In a video-based gastrointestinal motility monitoring (GIMM) system, a mouse's small intestine was placed in Krebs solution and recorded using a high definition camera. Untreated controls were recorded for each specimen, then treated with a therapeutic concentration of the drug, and finally, treated with a supratherapeutic dose of the drug. Next, the video clips showing gastrointestinal motility were processed, giving us the segmentation motions of the intestine, which were then converted via Fast Fourier Transform (FFT) into their respective frequency spectrums. These contraction quantifications were analyzed from the video recordings under standardised conditions to evaluate the effect of drugs. Six experimental trials were included with benztropine and promethazine treatments. Only the supratherapeutic dose of benztropine was shown to significantly decrease the amplitude of contractions; at therapeutic doses of both drugs, neither frequency nor amplitude was significantly affected. We have demonstrated that intestinal slow waves can be analyzed based on the colonic frequency or amplitude at a supratherapeutic dose of the anticholinergic medications. More research is required on the effects of anticholinergic drugs on these slow waves to ascertain the true role of ICC in neurologic control of gastrointestinal motility.     

Mast cell heterogeneity: effects of neuroenteric peptides on histamine release

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.     

Profound destructive effects of adolescent exposure to vincristine accompanied with some sex differences in motor and memory performance

Vincristine, an anticancer drug, is known to induce neuronal cell damage. We have elucidated the alteration in performance of the hippocampus and cerebellum following chronic vincristine treatment (0.2?mg·(kg body mass)(-1)·week(-1)) in male and female rats. Intraperitoneal injection of vincristine in adolescent rats caused impairment of motor and cognitive behavior. In the probe test, the length of path traveled and percent swimming time for vincristine-treated rats in the correct quadrant was significantly less than for the saline-treated (control) groups. The path length and time latency at the 2nd and 3rd blocks of trials for the male vincristine-treated group was significantly higher than that for the female saline- and the vincristine-treated rats. In the rod test, vincristine exposure impaired the motor coordination in both male and female rats. Exposure to vincristine caused a significant decrease in hanging time in male rats, compared with the saline- and the vincristine-treated female rats, while there were no differences between the female vincristine-treated rats and the saline-treated rats of both sexes. The rearing frequency, total distance moved, and velocity for both male and female rats were dramatically affected by exposure to vincristine. We have observed that the hippocampal and cerebellar functions of male and female rats were profoundly affected by exposure to vincristine, especially the male rats, suggesting a sexual dimorphism in the developing central nervous system that is affected by chemicals such as anticancer drugs.     

Quantitative characteristics of the endocrine cells of the duodenal glands of Carnivora

In the duodenal glands of the Carnivora investigated endocrine elements have been revealed, a part of them is presented as serotonin-producing EC-cells. Endocrine cells are situated in terminal parts and in glandular ducts, among them elements of open and close types are distinguished. Distribution of these cells in the glandular lobules is subjected to the distal gradient regularity, specific for the gastrointestinal tract mucosal membrane. Amount of endocrinocytes in the glands is much less than in the gut crypts. There is no correlation between distribution of the endocrine cells in the glands and in the crypts. The results of unifactor analysis of variance demonstrate a slight effect of the taxonomic position of the species on the number of endocrine cells in the duodenal glands. The proper endocrine apparatus of the duodenal glands is supposed to produce a local regulatory influence on the secretory activity of exogenic glandulocytes, as well as ensure humoral connections of the duodenal glands with other parts of the gastrointestinal tract.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

A reduction in energy-dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady-state intracellular α-aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α-aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α-aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α-aminoisobutyric acid. Inhibition of net α-aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady-state α-aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine. The data indicate that these agents inhibit a small component of the uphill transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy-dependent transport of α-aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy-metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.     

LF 15-0195 treatment protects against central nervous system autoimmunity by favoring the development of Foxp3-expressing regulatory CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vbeta8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-beta. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.     

In vivo and in vitro effects of amylin and amylin-amide on calcium metabolism in the rat and rabbit

Amylin is a new member of the calcitonin/CGRP family: it is a 37 amino acid polypeptide which was recently isolated from amyloid deposits in pancreatic islets obtained from type II diabetics. In the present study we investigated the effect of amylin and amylin-amide on calcium metabolism in the rat and rabbit. Two main methods were used: in vivo hypocalcaemic activity was assessed by measuring plasma calcium levels after injection of the peptide in 50 g rats; and in vitro resorption of cortical bone by disaggregated rat osteoclasts was quantified by scanning electron microscopy together with image analysis. We demonstrate that amylin and amylin-amide have calcitonin-like effects: both are powerful inhibitors of osteoclastic resorption and as a consequence lower plasma calcium in both rats and rabbits. We speculate that the peptide may exert systemic or local regulatory effects on bone cells.     

Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ketoprofen, are widely used in clinical medicine. However, these drugs may damage the gastrointestinal mucosa. Some reports have suggested that intestinal diseases, such as ulcers, are associated with lipid peroxidation and oxidative damage in the mucosa. Phytochemicals, such as polyphenols, are common dietary antioxidants that possess many beneficial characteristics, such as antioxidant and anti-inflammatory capabilities. The objective of this study was to investigate the protective effects of polyphenols on ketoprofen-induced oxidative damage in the gastrointestinal mucosa. We evaluated the effects of catechin, theaflavin, malvidin, cyanidin and apigenin on the activity of antioxidant enzymes in human intestinal-407 (Int-407) cells and rat primary gastric cells treated with ketoprofen. The results indicated that catechin significantly (P<.05) decreased the levels of lipid peroxidation (40.5%) and reactive oxygen species (30.0%), and increased the activity of intracellular antioxidant enzymes glutathione peroxidase, glutathione reductase and total sulfhydryl groups. More importantly, the treatment of Sprague–Dawley rats with catechin (35 mg/kg/day) prior to the administration of ketoprofen (50 mg/kg/day) successfully inhibited oxidative damage and reversed the impairment of the antioxidant system in the intestinal mucosa. Western blot analysis revealed that catechin stimulated a time-dependent increase in both the nuclear factor erythroid 2-related factor 2 and total heme oxygenase-1 protein expression in Int-407 cells. These results suggest that catechin may have a protective effect on gastrointestinal ulcers.     

后生元缓解胃肠道疾病的研究进展及其潜在机制

胃肠道是全身代谢最活跃的器官之一，也是人体内最大的细菌库。人体胃肠道中含有丰富的微生物群，其与宿主健康存在着错综复杂的关系。肠道菌群处于一种动态平衡的状态，当这种平衡被打破时会引起便秘、腹泻、肠易激综合征、炎症性肠病和结直肠癌等胃肠道疾病的发生。近年来，关于后生元的研究越来越多，其对肠道屏障的保护作用与益生菌类似甚至效果更佳。本文重点介绍了当前后生元在动物实验和临床中改善胃肠道疾病的相关研究，探讨了后生元在胃肠道中的作用及其在增强上皮屏障、调节免疫系统、肠道菌群和神经系统4个方面的潜在作用机制。     

Neurohumoral control of gastrointestinal motility

Neurohumoral substances and their receptors play a major part in the complex regulation of gastrointestinal motility and have therefore been the predominant targets for drug development. The numerous receptors involved in motility are located mainly on smooth muscle cells and neuronal structures in the extrinsic and intrinsic parts of the enteric nervous system. Within this system, receptor agonists and antagonists interacts directly to modify excitatory or inhibitory signals. In view of this complexity it is not surprising that our knowledge about the mechanisms of actions of the various neurohormones and drugs affecting gut motility has been rather fragmented and incomplete. However, recently substantial progress has been achieved, and drug therapy for gut dysmotility is emerging, based primarily on neurohumoral receptors. This paper presents a selective review of the neurohumoral regulatory mechanisms of gastrointestinal motility. In this context, the physiology and pharmacology of the smooth muscle cells, gastrointestinal motility and dysmotility, the enteric nervous system, gastrointestinal reflexes, and serotonin is presented. Further investigation and understanding of the transmitters and receptors involved in especially the reflex activation of peristalsis is crucial for the development of novel therapies for motility disorders.     

Fasting and postprandial concentrations of GLP-1 in intestinal lymph and portal plasma: evidence for selective release of GLP-1 in the lymph system

Glucagon like peptide 1 (GLP-1) is an intestinal hormone that plays an important role in glucose metabolism. GLP-1 is released from mucosal L cells following nutrient ingestion and contributes to the incretin effect, with the enhancement of insulin secretion occurring with enteral compared with intravenous glucose administration. The mechanisms linking nutrient absorption and GLP-1 secretion are unknown, and studies addressing this topic, particularly in small animal models, have been hampered by the relatively low concentrations of GLP-1 in the circulation. We hypothesized that GLP-1 levels would be higher in samples of intestinal lymph compared with plasma and could provide a novel system in which to study meal-induced hormone secretion. We addressed this hypothesis in conscious rats with indwelling catheters in the portal vein and distal intestinal lymph duct. These animals had plasma and lymph sampled before and for 240 min after instillation of a liquid meal in the gastrointestinal tract. Lymph contained detectable concentrations of glucose, insulin, and GLP-1 that were reliably measured using our assays. Before and after the Ensure feeding, plasma insulin levels were approximately two times as high in portal plasma as intestinal lymph. In marked contrast, GLP-1 levels were five to six times higher in lymph relative to portal plasma following nutrient administration. This relative difference in GLP-1 levels was even greater when lymph was compared with peripheral plasma and dramatically exceeded the ratio of lymph to plasma peptide tyrosine-tyrosine concentrations. This is the first observation of a gastrointestinal hormone being disproportionately transported in lymph. The remarkable levels of GLP-1 in intestinal lymph demonstrate the potential for lymphatic sampling as a more sensitive means of studying the secretory physiology of this hormone in vivo. In addition, these data raise the possibility that intestinal lymph may serve as a specialized signaling conduit for regulatory peptides secreted by gastrointestinal endocrine cells.