Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.

In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophilderived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum.