AbstractThis study concerns the production and action of nitric oxide (NO) in the rat gastric mucosa. The major objectives were to: (i) validate the use of a novel nitric oxide sensitive fluorescent indicatior 4,5-diaminfluorescein (DAF-2), for real time measurement of NO, (ii) identify the intracellular pathway(s) regulating NO production in gastric epithelial cell suspensions and (iii) examine the release of NO from a novel class of non-steroidal antiinflammatory drugs (NO-NSAIDS) and its subsequent action on the rat gastric mucosa.
Rat gastric epithelial cells were isolated by pronase digestion coupled with intermittent calcium chelation. Cellular viability and toxicity were assessed by trypan blue dye exclusion and acid phosphatase release respectively. The production or release of NO was detected by spectrofluorimetry in the presence of 4,5-diaminfluorescein (DAF-2;10μM) at excitation and emission wavelengths of 495 nm and 515 nm respectively.
NO donating compounds produced a concentration dependent increase in DAF-2 fluorescence with a detection limit of approximately 2nM. The increase in fluorescence was blocked by the NO-scavenger 2-(4-carboxyphenyl1)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO). Futhermore, oxidation products of NO were unable to increase the fluorescence signal of DAF-2.
Incubation of isolated gastric epithelial cells with the cholinergic agonists acetycholine (ACh) and carbachol, resulted in a concentration-dependent increase in NO production, which was inhibited by NG-nitro-L-argininev methyl ester (L-NAME), atropine, inhibitors of protein kinase A and C (PKA; PKC), a calcium/calmodulin dependent protein kinase II inhibitor and a microsomal Ca2+-ATPase inhibitor. Actvators of PKA and PKC mimicked the effect of ACh. Furthermore, the calcium ionophore ionomycin also stimulated NO production, which was blocked by intracelluar modulators of calcium function.The release of NO from two NO-releasing NSAIDs (NO-NSAIDs: NCX 4016 and HCT 1026) was concentration dependent and occurred in both the presence and absence of gastricepithial cells. Furthermore, both NCX 4016 and HCT 1026 (10 μM-1000 μM) stimulated mucus secretion in rat gastric epithelial cells which was associated with an increase in intracellular cGMP concentrations. I addition, intra-gastric administration of both NO-NSAIDs resulted in a concentration-related increase in mucus gel thickness.In summary, these findings suggest that DAF-2 is a suitably sensitive indicator for detecting NO production in gastric epithelial cell suspensions. Cholinergic mediated NO prduction by these cells is likely to involve PKC, PKA, calcium mobilisation and calmodulin. Finally, No-SAIDs appear to act as NO-donating compounds which stimulate both the in vitro and in vivo release of gastric mucus, via a cGMP-dependent pathway, a mechanism which may account for their apparent gastric sparing properties.
|Date of Award||1 May 2002|
|Supervisor||James Brown (Supervisor) & Matt Guille (Supervisor)|