Role of epithelial ion transports in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with a complex pathogenesis. Diarrhea is a highly prevalent and often debilitating symptom of IBD patients that results, at least in part, from an intestinal hydroelectrolytic imbalance. Evidence suggests that reduced electrolyte absorption is more relevant than increased secretion to this disequilibrium. This systematic review analyses and integrates the current evidence on the roles of epithelial Na(+)-K(+)-ATPase (NKA), Na(+)/H(+) exchangers (NHEs), epithelial Na(+) channels (ENaC), and K(+) channels (KC) in IBD-associated diarrhea. NKA is the key driving force of the transepithelial ionic transport and its activity is decreased in IBD. In addition, the downregulation of apical NHE and ENaC and the upregulation of apical large-conductance KC all contribute to the IBD-associated diarrhea by lowering sodium absorption and/or increasing potassium secretion.

Amine neurotransmitters, inflammation and epithelial sodium transport.

NEW FINDINGS: What is the topic of this review? The present work reviews the roles of renal and intestinal dopamine and 5-HT in the maintenance of fluid and electrolyte homeostasis. The role of inflammatory agents at the intestinal level that affect fluid and electrolyte homeostasis is also addressed. What advances does it highlight? General mechanisms of epithelial cell ion transport in the gastrointestinal tract and kidney share considerable similarities, particularly with regard to basolateral Na(+) ,K(+-) ATPase as a driving force for the movement of numerous substrates across the cell membrane. The physiological importance of the renal actions of monoamines (dopamine, noradrenaline and 5-HT) mainly depends on the sources of the amines in the kidney and on their availability to activate the amine-specific receptors. Dopamine and 5-HT are also relatively abundant in the mucosal cell layer of the intestine, and recent evidence suggests their physiological relevance in regulating electrolyte transport. The gastrointestinal tract can be an important site for the loss of water and electrolytes, in the presence of intestinal inflammation. General mechanisms of epithelial cell ion transport in the gastrointestinal tract and kidney share considerable similarities with regard to basolateral Na(+) ,K(+) -ATPase as a driving force for the movement of numerous substrates across the cell membrane. The present work reviews the roles of renal and intestinal dopamine and 5-HT in the maintenance of fluid and electrolyte homeostasis. The role of inflammatory agents at the intestinal level that affect fluid and electrolyte homeostasis is also addressed.

Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.

Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 µg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Short- and long-term regulation of intestinal Na+/H+ exchange activity associated with TLR2 receptor activation is independent of nuclear factor-κB signaling.

Type 2 Toll-like receptors (TLR2s) are expressed in cell membranes and recognize a wide range of pathogen-associated molecular patterns derived from bacteria, such as lipoteichoic acid (LTA). The aim of this study was to evaluate the effect of TLR2 activation by LTA on the activity of type 1 Na(+)/H(+) exchanger (NHE) in T84 intestinal epithelial cells. Short-term (0.5 hour) and long-term (18 hours) TLR2 activation significantly inhibited NHE1 activity in a concentration-dependent manner (0.01-100 µg/ml; -7 ± 3 to -21 ± 3% and 3 ± 3 to -21 ± 3% of control values, respectively). S3226 [3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride], an NHE3-selective inhibitor, did not affect the inhibitory effect on NHE activity. LTA-induced NHE inhibition did not occur in the presence ofethylisopropylamiloride (an NHE1 inhibitor). Long-term TLR2 activation decreased NHE1 affinity for Na(+) (Km= 64.98 ± 1.67 mM) compared with control (Km= 20.44 ± 0.54 mM) without changes in Vmax values. After TLR2 activation, we observed tyrosine-protein kinase (SRC) activation, phosphatidylinositol 3-kinase (PI3K) recruitment, and adenylyl cyclase (AC3) phosphorylation. The total amount of AC3 increased (23 ± 8% of control) after long-term treatment with LTA. Anti-AC3 small interfering RNA prevented LTA-induced NHE1 inhibition, similar to that observed with the AC3 inhibitor KH7 [(±)-2-(1H-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide]. A significant increase in cAMP levels (32 ± 3% and 14 ± 2% after short- and long-term stimulation, respectively) was detected, and inhibition of protein kinase A (PKA), phospholipase C (PLC), and downregulation of protein kinase C (PKC) prevented NHE1 inhibition. Inhibition of nuclear factor-κΒ (NF-κB) failed to revert NHE1 inhibition. We concluded that activation of TLR2 reduces NHE1 activity in epithelial cells through an alternative pathway that is unrelated to NF-κB, which involves SCR, PI3K, AC3, PKA, PLC, and PKC.

Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats.

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

LAT1 overexpression and function compensates downregulation of ASCT2 in an in vitro model of renal proximal tubule cell ageing.

Amino acid transporters provide cells neutral amino acids indispensable for growth and proliferation-dependent protein synthesis. This study evaluates whether prolonged serial cell passaging during 6 months (over 50 passages) may induce changes in amino acid transporters properties in Opossum kidney (OK) proximal tubular cells. High passage OK cells exhibit polyploidy, but no difference in the proliferation potential was observed when compared to low passage OK cells. Increased time in culture was accompanied by an increased total, membrane and cytosol protein content. The Na(+)-insensitive [(14)C]-L-leucine uptake was promoted almost exclusively thought LAT1 (~ 90 vs 80%, high versus low passage OK cells). The increased LAT1 protein abundance in high passage OK cells correlated positively with enhanced ability to take up [(14)C]-L-leucine, despite a 4.3-fold decrease in affinity for the substrate. The Na(+)-sensitive [(14)C]-L-alanine transport was decreased by 2.5-fold in high passage OK cells. However, no differences in ASCT2 expression were observed between high and low passage OK cells. It is concluded that OK cells show functional differences in both L-leucine and L-alanine uptake as a function of passage time in culture. The increased expression and activity of LAT1 in high passage OK cells may correspond to a mechanism enabling the cell to develop the hypertrophy response to prolonged cell passaging, when the function of ASCT2 is markedly depressed.