Lactobacillus acidophilus attenuates downregulation of DRA function and expression in inflammatory models.

Probiotics, including Lactobacilli, are commensal bacteria that have been used in clinical trials and experimental models for the prevention and treatment of diarrheal disorders. Our previous studies have shown that Lactobacillus acidophilus (LA) and its culture supernatant (CS) stimulated Cl(-)/HCO3 (-) exchange activity, acutely via an increase in the surface levels of downregulated in adenoma (DRA, SLC26A3) and in long-term treatments via increasing its expression involving transcriptional mechanisms. However, the role of LA in modulating DRA activity under inflammatory conditions is not known. Current in vitro studies using human intestinal epithelial Caco-2 cells examined the efficacy of LA or its CS in counteracting the inhibitory effects of interferon-γ (IFN-γ) on Cl(-)/HCO3 (-) exchange activity. Pretreatment of cells with LA or LA-CS for 1 h followed by coincubation with IFN-γ significantly alleviated the inhibitory effects of IFN-γ on Cl(-)/HCO3 (-) exchange activity. In the in vivo model of dextran sulfate sodium-induced experimental colitis (3% in drinking water for 7 days) in C57BL/6J mice, administration of live LA (3 × 10(9) colony-forming units) via oral gavage attenuated colonic inflammation. LA administration also counteracted the colitis-induced decrease in DRA mRNA and protein levels. Efficacy of LA or its secreted soluble factors in alleviating inflammation and inflammation-associated dysregulation of DRA activity could justify their therapeutic potential in inflammatory diarrheal diseases.

Lactobacillus acidophilus upregulates intestinal NHE3 expression and function.

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice.

P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [(3)H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (∼3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (∼2-fold, P < 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 × 10(9) colony-forming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2- to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders.

The probiotic Lactobacillus plantarum counteracts TNF-{alpha}-induced downregulation of SMCT1 expression and function.

The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na(+)-coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na(+)-dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na(+)-dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value.

Lactobacillus acidophilus stimulates the expression of SLC26A3 via a transcriptional mechanism.

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, Na(+) and Cl(-), occurs mainly through the coupled operation of Na(+)/H(+) exchangers and Cl(-)/OH(-) exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated Cl(-)/OH(-) exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6-24 h resulted in a significant increase in Cl(-)/OH(-) exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA ( approximately 4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA.

Role of muscarinic signal transduction and CREB phosphorylation in dichlorvos-induced memory deficits in rats: an acetylcholine independent mechanism.

The present study was designed to explore the alternative mechanism (other than AChE inhibition) for chronic, low-level exposure to dichlorvos, an organophosphate, in vivo. Dichlorvos, at a dose of 1.0 and 6.0 mg/kg body weight (b.wt.) for 12 weeks, showed impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests. Though high dose of dichlorvos had a detrimental effect on acetylcholinesterase activity, no significant inhibition was seen with low dose of dichlorvos. Western blot analysis and immunofluorescence studies showed a significant reduction in the expression of M(1), M(2) and M(3) muscarinic receptor subtypes in high dose group animals, whereas in low dose group animals only the M(2) receptor subtype was reduced significantly. Further, the signal transduction cascade linked to these receptor subtypes was affected in high dose group animals whereas in low dose group only adenylyl cyclase-linked signaling pathway was impaired. Finally, the phosphorylation of CREB, a memory enhancing transcription factor, was significantly reduced in both low dose and high dose group animals. Thus, the present study reveals the significance of M(2) muscarinic receptor linked adenylyl cyclase signaling pathway and phosphorylation of CREB in the development of neurobehavioral impairments after chronic low-level exposure to dichlorvos.

Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain.

Chronic dichlorvos exposure (6 mg/kg b.wt/day) for a period of 8 weeks resulted in significant reduction in body weight gain of the male Wistar rats. However, the dietary intake remained unchanged in experimental animals following dichlorvos treatment. Activity of the synthesizing enzyme of acetylcholine (ACh) ie, choline acetyltransferase, was found to be significantly increased and the activity of hydrolyzing enzyme, acetyl cholinesterase (AChE), was inhibited in all the three brain regions studied. Chronic dichlorvos treatment also caused significant reduction in both high affinity (HA) and low affinity (LA) choline uptake (CU), with maximal effect being observed in the brain stem followed by cerebellum and cerebrum. Muscarinic receptor binding was significantly decreased in brain stem and cerebellum as reflected in the decreased receptor number (B(max)), without any change in the binding affinity (K(d)) of the receptors. Dichlorvos treatment caused marked inhibition in cAMP synthesis as indicated by decreased adenylate cyclase activity as well as cAMP levels in cerebrum, cerebellum and brain stem. Our study shows that organophosphates may interact with muscarinic receptor-linked second messenger system and this could be a potential mechanism for the neurotoxic effects observed after repeated exposure to low levels of organophosphates, which are unexplainable on the basis of cholinergic hyperactivity.

The L-type calcium channel blocker nimodipine mitigates cytoskeletal proteins phosphorylation in dichlorvos-induced delayed neurotoxicity in rats.

The present investigation was carried out to assess the protective efficacy of nimodipine against dichlorvos-induced organophosphate induced delayed neurotoxicity (OPIDN). Single subcutaneous dose of dichlorvos (200 mg/kg body weight) led to a consistent increase in the activity of both microtubule associated protein kinases viz. Ca2+/Calmodulin-dependent and cAMP dependent protein kinases, at all post exposure intervals (day 7, 15 and 21) as compared to that of controls. Autoradiography followed by microdensitometric studies demonstrated enhanced phosphorylation of 55 kDa and 280 kDa proteins in dichlorvos-exposed animals. These two proteins were confirmed to be tubulin and microtubule associated protein-2 (MAP-2) by western blotting. The hyperphosphorylation of these two proteins was shown to interfere with the assembly of neuronal microtubules as shown by electron microscopic studies that may eventually lead to possible disruption of neuronal cytoarchtecture resulting in axonal degeneration. Administration of nimodipine along with dichlorvos brought about a significant reduction in the activities of both the kinases as well as the extent of microtubule associated protein phosphorylation. This indicates that nimodipine, a centrally acting calcium channel blocker, may contribute to the amelioration of dichlorvos induced neurotoxicity by attenuation of calcium mediated disruption of cytoskeletal proteins and hence, calcium channel blockers like nimodipine have great future as new therapeutic agents for OPIDN.

Purification and characterization of neuropathy target esterase (NTE) from rat brain.

Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1-3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9'-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.

Possible involvement of dopaminergic neurotransmitter system in dichlorvos induced delayed neurotoxicity.

The present investigation was carried out to elucidate the possible involvement of the dopaminergic neurotransmitter system in the development of dichlorvos induced delayed neurotoxicity in the rat and to assess the protective efficacy of nimodipine against OPIDN (Organophosphate induced delayed neurotoxicity). Single subcutaneous dose of dichlorvos (200 mg/kg body weight) resulted in marked changes in the dopaminergic neurotransmitter system in terms of increased levels of both dopamine and norepinephrine along with significant increase in the activity of both the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase. This increase was accompanied with a concomitant decrease in the activity of the major degradative enzyme, monoamine oxidase. Scatchard plot analysis revealed a significant decrease in both K(d) and B(max) for dopamine D2 receptors. Administration of nimodipine, a centrally acting calcium channel blocker, along with dichlorvos restricted all these alterations to within control values and could also ameliorate certain behavioural deficits by maintaining the dopaminergic neurotransmitter system. The study underlines the importance of alterations in the dopamine system as a possible causative mechanism behind the behavioural and functional changes associated with delayed neurotoxicity.

Calcium homeostasis and dichlorvos induced neurotoxicity in rat brain.

The present study was designed to investigate the possible effects of chronic dichlorvos exposure on the various aspects of calcium homeostasis in rat brain. Chronic dichlorvos administration caused significant rise in the intrasynaptosomal calcium levels. The activity of major calcium expelling enzyme i.e. Ca2+ATPase was found to be declined. Also, the depolarization induced calcium uptake via voltage operated calcium channels increased significantly. Concomitant to the increase in intrasynaptosomal calcium, calpain activity was found to be increased. The results presented herein, indicate that the toxic effects of dichlorvos could be mediated through modifications in the intracellular calcium homeostasis which may lead to impaired neuronal function.