Unconjugated bilirubin promotes intestinal secretion without affecting nutrient digestion or absorption. In the current study, the effects of unconjugated bilirubin (UCB) on the barrier function of the intestinal epithelium were investigated. The apical side of human intestinal cell line Caco-2 monolayers was challenged with purified UCB. Transepithelial electrical resistance and paracellular fluxes of 10 kD Cascade blue conjugate dextran were measured. Cell monolayer viability was studied using LDH release and trypan blue exclusion tests. Redistribution of enterocyte tight junction occludin was studied by confocal microscopy. Bilirubin induced a dose-dependent decrease of transepithelial electrical resistance (TEER). This effect was maximal at 6 h and tended to be reversed at 48 h. Oxidated bilirubin was ineffective. Bilirubin significantly increased fluorescent dextran paracellular passage. Cell viability was not affected by UCB over the 5-200 nmol/L concentration range. H yperbilirubinemia is a common finding in both term and preterm neonates (1). Because bilirubin can cause severe damage to the central nervous system, most investigators have focused their research on its effects on neural cells (2). The interaction of bilirubin with other organs has also been explored, although to a lesser extent. In the intestine, for example, bilirubin is excreted with bile in the gut lumen mainly as mono or diglicuronides. However, the enzyme glycuronidase, of both endogenous and microbial origin, partially converts it to UCB (3). Previous reports have shown UCB concentrations in the intestinal lumen of hyperbilirubinemic newborns to be in the micromolar range (4,5). Investigators have also documented that UCB in the gut does not affect cell viability, nutrient absorption, or brush border enzyme activities, although it can induce watery diarrhea by direct stimulation of ion secretion (6,7). Jaehrig et al. (8) found that UCB transit in the intestinal lumen of the human newborn was associated with a decreased transmural potential difference. As the latter is an indirect measurement of the tightness of the intestinal barrier, they suggested that bilirubin could cause "some injury" to the intestinal fence by increasing its permeability. This is of potential clinical interest because a decrease in the gut barrier function has been linked to the pathogenesis of intestinal (i.e. necrotizing enterocolitis) diseases (9) and extraintestinal, particularly allergic and autoimmune, disorders (10,11). The aim of this study was to further investigate in vitro the effect of bilirubin on intestinal epithelial permeability. For this purpose, we have used monolayers of the well-established human transformed cell line Caco-2 that displays many biologic features of the primary intestinal epithelium (12,13). The study was approved by the Institutional Review Board of the "Facoltà di Medicina-Federico II." MATERIALS AND METHODSReagents. Cell culture chemicals were obtained from GIBCO-Life Technologies (Milan, Italy). 10 kD Cascade blu...

SUMMARY1. Embryos of Xenopus laevis were selected prior to the onset of innervation and were raised for 2 days in the anaesthetic tricaine methanesulphonate (200 ,ug/ml). The gross development of these tricaine-reared animals appeared normal despite the absence of spontaneous motor activity and the lack of motor responses to prodding with a pin. Motor activity quickly appeared when the anaesthetic was withdrawn.2. Intracellular recording from the myotomes of intact, tricaine-maintained animals failed to reveal any spontaneous muscle action potentials. Synaptic potentials increased in frequency and amplitude upon withdrawing tricaine, but resting potentials remained unchanged.3. Cholinesterase activity, detected histochemically, was observed at the ends of the myotomes, the main site of innervation. The intensity of the histochemical reaction product at these sites appeared to be about as great in the myotomes of tricaine-reared animals as in control myotomes.4. Miniature end-plate currents (m.e.p.c.s), examined by focal external recording, declined with a time constant of 2-9 + 0-2 ms (mean+ S.E. of mean) in the myotomes of tricaine-reared animals (stages 40-41). The time constants in the myotomes of control animals were 1'8 + 0-1 ms at stages 40-41 and 8-7 + 0-7 ms at stages 24-26 (shortly after the onset of innervation).5. The anticholinesterase neostigmine doubled m.e.p.c. time constants in the myotomes of tricaine-reared animals as well as in control myotomes at stages 40-41.6. It is concluded that motor activity is not required for the in vivo development of physiological levels of synaptic cholinesterase in Xenopus myotomal muscle.

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