Nitric oxide synthase stimulates prostaglandin synthesis and barrier function inC. parvum-infected porcine ileum

Abstract

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.

NF-κB-mediated expression of iNOS promotes epithelial defense against infection byCryptosporidium parvumin neonatal piglets

“…Similarly, expression of inducible NO synthase (iNOS) and synthesis of NO are significantly increased in intestinal mucosa from Cryptosporidium-infected piglets and mice (14,26,30). Induction of iNOS by or exposure of cell monolayers to high concentrations of NO promotes cytotoxicity and barrier disruption (4,36,43,44,47).…”

Neutrophils Do Not Mediate the Pathophysiological Sequelae of Cryptosporidium parvum Infection in Neonatal Piglets

“…In infected piglets, neutrophil depletion had no effect on severity of diarrhea, maintenance of body weight (a parameter indicative of diarrheal severity in this model [11]) (Table 1) Mucosal neutrophil influx promotes barrier function in C. parvum infection. We have shown previously that C. parvum infection is associated with a decrease in paracellular barrier function (12). To determine whether neutrophils mediate physical or biochemical effects on barrier function in C. parvum infection, infected piglets were treated in vivo with isotype control or anti-CD18 antibody daily, beginning at the time of infection and continuing until peak infection severity (day 4), at which time mucosa from each piglet was mounted in Ussing chambers for measurement of TER and flux of 22 Na ϩ and [ 3 H]mannitol.…”

“…Neutrophils mediate barrier function independently of effects on mucosal prostaglandin synthesis. An increase in endogenous prostaglandin synthesis has been demonstrated previously to promote paracellular barrier function in C. parvum infection (2,12). Therefore, we sought to determine whether neutrophils mediate barrier function of C. parvum-infected mucosa by serving as a source or stimulus of mucosal prostaglandin synthesis.…”

“…Diarrhea has been attributed both to villus atrophy-associated malabsorption and to mucosal inflammation-induced alteration in intestinal water and electrolyte transport. Neutrophils are a source of mediators, including reactive oxygen metabolites (4), TNF-␣ (17), 5Ј-AMP (24), and prostaglandins (1,2,4,12), that may directly or indirectly promote epithelial secretion and diarrhea in C. parvum infection. Neutrophil depletion of C. parvum-infected piglets had no effect on clinical severity of diarrhea or epithelial Cl Ϫ secretion, suggesting that mediators derived from or stimulated by neutrophils do not play a significant role in the genesis of diarrhea in the infection.…”

“…We have shown previously that C. parvum infection results in a loss of barrier function that is compensated for by an increase in mucosal synthesis of prostaglandins (12). To determine if neutrophils are the cause of this defect in barrier function, transepithelial electrical resistance and flux of 22 Na ϩ and [ 3 H]mannitol were measured using ileal mucosae from neutrophil-depleted and isotype control-treated piglets infected with C. parvum.…”