A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

Abstract

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA in vitro and in vivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC. (Am J Pathol 2017, 187: 401e417; http://dx

β-arrestin-2 enhances intestinal epithelial apoptosis in necrotizing enterocolitis

“…In our study, salmeterol may also have induced cyclic adenosine monophosphate (cAMP), the typical intracellular second messenger of β2ARs. Previously, the cAMP/protein kinase A (PKA) pathway was reported to promote apoptosis in NEC [38]. Whether both G-protein-biased (β2ARs/cAMP/PKA) and β-arrestin-2-biased (β2ARs/β-arrestin-2/BiP) signaling pathways induce apoptosis may be worthy of future study.…”

The science and necessity of using animal models in the study of necrotizing enterocolitis

“…correlation on intestinal fatty acid binding protein with timing of ischemia and severity of tissue injury(23)). Currently, the administration of a protein kinase A inhibitor(24) and milk-derived exosomes(25) are being studied in a rat model with promising results as potential treatments for NEC.…”

H89 Treatment Reduces Intestinal Inflammation and Candida albicans Overgrowth in Mice

“…Although the exact aetiology of IBD is not well known, it is believed that genetic susceptibility, environmental factors, a dysfunctional immune system and changes to the gut microbiota are involved in the onset and progression of this disease [ 33 ]. Different studies have implicated cAMP in inflammatory disorders [ 12 , 24 , 34 ]. Zimmerman et al showed that cAMP dysregulates intestinal epithelial cell restitution through PKA indicating that increased levels of cAMP in intestinal epithelial cells inhibit the migratory and restitutive potential in the gut [ 12 ].…”

“…Zimmerman et al showed that cAMP dysregulates intestinal epithelial cell restitution through PKA indicating that increased levels of cAMP in intestinal epithelial cells inhibit the migratory and restitutive potential in the gut [ 12 ]. Additionally, PKA inhibition appears to protect against gut injury in the rat pup model [ 24 ]. H89 is an isoquinoline derivative that inhibits PKA by acting as competitive antagonists of ATP at its binding site on the PKA catalytic subunit [ 35 ].…”

“…For H89 treatment, mice received 5 mg H89/kg/mouse (Sigma-Aldrich, France) in 200 µL PBS daily for 5 days by oral gavage. The dose of 5 mg H89/kg/mouse was established in this in vivo model based on previous publications and on our in vitro migration assay [ 24 , 25 ]. The mice were then sacrificed on Day 14 by cervical dislocation and the colon was collected to determine the fungal load and to carry out PCR and histological examination.…”