The IPEC-J2 cells were grown in Dulbeccos modified eagle medium with high D-glucose (4
The IPEC-J2 cells were grown in Dulbeccos modified eagle medium with high D-glucose (4.5 g/l) (Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum, 0.1 million units/l penicillin, and 100 mg/l streptomycin (Atlanta Bio, Lawrenceville, GA), and maintained in an atmosphere of 5% CO2 at 37 C. Cit were protective against HX-related damage. At the final time point (14 h), the mean TEER ratio (TEER as compared with baseline) for Arg + HX and Cit + HX was significantly higher than that for HX alone. Both Arg and Cit were associated with decreased inulin flux across hypoxic monolayers and qualitatively preserved TJ proteins. Irreversible inhibition of NOS blocked this protective effect. Lipid peroxidation assay showed that our model did not produce oxidant injury. Conclusion Arg AKBA and Cit, via a mechanism dependent on NO donation, protected intestinal epithelial integrity. Intestinal injury and inflammation resulting from ischemia is integral to the pathogenesis of multiple disease states affecting diverse populations from necrotizing enterocolitis (NEC), spontaneous perforation, and hypoxic injury in the context of cardiac or lung disease in infants, to atherosclerotic mesenteric ischemia and hypoxic injury related to venous thrombosis, autoimmune disease, or chronic cardiopulmonary disease in adult and geriatric populations. NEC, as one example, is the AKBA most common gastrointestinal disorder in low birth weight infants, and the total annual cost of caring for affected infants in the United States is estimated to be around $5 billion (1,2). NEC has been described as an aberrant reaction of the immature intestinal immune system that occurs in the context of enteral nutrition and is associated with mucosal injury, barrier compromise, and systemic immune response (3,4). Nitric oxide (NO) production by inducible NO synthetase (iNOS) increases in the presence of acute intestinal injury (5C7). However, there are conflicting data about the effect of this increase. Excessive production of NO can be destructive to intestinal tissues after ischemia/reperfusion injury (8). Conversely, it has been observed that NO and NOS activation do not have deleterious effects on epithelial barrier function (9). In addition, inhibition of iNOS has been shown to exacerbate inflammation during acute intestinal injury and to delay repair (10). Arginine (Arg) is the physiological substrate for NO synthesis and has been recognized as an enhancer of protein synthesis and wound healing (11). Arg is deficient in preterm neonates due to inadequate availability in the diet and the underdevelopment of its synthetic pathways in the small intestine (12). Serum levels of Arg have been shown to be low in patients with NEC, drifting downward 1 wk before the onset of NEC, suggesting that Arg may be essential to a process that is protective against NEC (13,14). Arg has been shown to stimulate intestinal cell migration and recovery of intestinal monolayer transepithelial resistance in a NO-dependent manner (15C17). Arg is synthesized from citrulline (Cit) by the sequential action of the cytosolic enzymes argininosuccinate synthetase and argininosuccinate lyase. Cit is potentially a key precursor of Arg that can then Mouse monoclonal to PRAK serve as a substrate for the production of NO and polyamines. Oral supplementation of L-Cit has AKBA been shown to increase plasma L-Arg concentration and augment NO-dependent signaling in a dose-dependent manner (18). In addition, similar to Arg, serum levels of Cit have also been shown to be low in premature infants and so may play a role in the pathophysiology of NEC (19). We hypothesized that either Arg or Cit may protect intestinal monolayers from hypoxia (HX)-mediated damage and that the NO synthetic pathway may be involved. RESULTS Transepithelial electrical resistance (TEER) was maintained in IPEC-J2 monolayers treated with Arg and Cit. IPEC-J2 cell monolayers were exposed to HX, TEER measurements were made hourly with brief reoxygenation for 10 min, and the percentage of each value to the value of the resistance before beginning HX was identified. Monolayers that were not treated and were not exposed to HX were measured as settings. TEER for these monolayers remained constant throughout the period of HX exposure. For monolayers that were exposed to HX but untreated, TEER started to fall, normally, after hour 12 and continued to fall through the period of exposure (Number 1a). Monolayers treated with Arg and Cit but not exposed to HX were not found to be significantly different from controls at any time point. Open in a separate window Number 1 Transepithelial electrical resistance (TEER) of IPEC-J2 monolayers during exposure to hypoxia (HX). (a) TEER of control monolayers not exposed to HX (solid black collection) remained constant (= 8). TEER of monolayers exposed to HX but untreated (solid dark gray collection) started to fall after hour 12 (= 12). TEER of monolayers not exposed to AKBA HX and treated with arginine (Arg) (short-dashed collection) (= 8) or citrulline (Cit) (long-dashed collection) (= 8) were not significantly different from controls at any time point. (b) TEER of monolayers exposed to HX and treated with Arg (short-dashed collection) (= 16) or Cit (long-dashed collection) (= 16) remained constant and were not significantly different from controls (monolayers not exposed to HX, solid black collection; monolayers exposed to HX but untreated, solid dark gray collection) at any time point,.