The objective of our proposal is to define the mechanism of action of cholera toxin (CT), and our hypothesis is that arachidonic acid (AA) metabolites (e.g., prostaglandin E2 [PGE2] and leukotriene C4 [LTC4] exert a significant impact on the physiological functions of the small intestine. While CT can directly upregulate adenylate cyclase activity by catalyzing the ADP-ribosylation of Gs-alpha and increase 3, 5 adenosine monophosphate (cAMP) levels, equally important are stimulatory effects on AA metabolism leading to increased production of eicosanoids (e.g, PGE2 and LTC4). PGE2 can stimulate adenylate cyclase and intestinal ion transport, while LTC4 is known to stimulate Ca++ mobilization. Further, Ca++ channel blockers reduce CT-induced fluid transport. Importantly, our Preliminary Studies show that CT stimulates AA metabolism independent of ADP-ribosylation of Gs-alpha by signaling the expression of the plaa gene, which encodes phospholipase A2-activating protein (PLAA). Immunoelectron microscopy and plaa antisense oligonucleotide experiments show that PLAA is an important nucleoprotein that upregulates PLA2 activity. After cloning human plaa cDNA, we overexpressed the gene in several prokaryotic and eucaryotic systems, We have also observed the celecoxib, a highly specific COX-2 inhibitor, blocks synthesis of PGE2 and CT-induced fluid transport in murine intestinal segments. We discovered novel imidazole covalent analogs of PGE2 that reduce cAMP levels and fluid accumulation in murine intestinal loops challenged with CT.
In Aim1, intestinal cells (e.g., Paneth, epiteilial) that synthesize PLAA in response to CT will be identified using in situ hybridization and immunohistochemistry, and the role of PLAA and Paneth cells will be established with PLAA and Paneth cell knockout mice. The role of the major constituents of the AA pathway (e.g., PLA2, COX-2, PGE2 synthase [PGES], and PGE2 receptors) will be evaluated in Aim 2 using specific inhibitors and knockout mice.
In Aim 3, we propose to define the regulatory mechanism by which CT signals the transcription of the plaa gene and PLAA synthesis.
Aim 4 consists of using plaa cDNA expressed in eucaryotic cells and PLAA protein to evaluate stimulatory effects on PLA2 activity. Although CTs action is a multifaceted mechanism, therapeutic control of CT- induced hypersecretion of water and electrolytes in cholera may be possible by modulating AA metabolism.
| Zhang, Fan; Suarez, Giovanni; Sha, Jian et al. (2009) Phospholipase A2-activating protein (PLAA) enhances cisplatin-induced apoptosis in HeLa cells. Cell Signal 21:1085-99 |
| Zhang, Fan; Sha, Jian; Wood, Thomas G et al. (2008) Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA). Cell Signal 20:844-61 |
| Lomada, Dakshayani; Gambhira, Ratish; Nehete, Pramod N et al. (2004) A two-codon mutant of cholera toxin lacking ADP-ribosylating activity functions as an effective adjuvant for eliciting mucosal and systemic cellular immune responses to peptide antigens. Vaccine 23:555-65 |
| Saini, Shamsher S; Gessell-Lee, Deborah L; Peterson, Johnny W (2003) The cox-2-specific inhibitor celecoxib inhibits adenylyl cyclase. Inflammation 27:79-88 |
| Gessell-Lee, Deborah L; Popov, Vsevolod L; Boldogh, Istvan et al. (2003) Role of cyclooxygenase enzymes in a murine model of experimental cholera. Infect Immun 71:6234-42 |
