Prostacyclin production in tryptase and thrombin stimulated human bladder endothelial cells: effect of pretreatment with phospholipase A2 and cyclooxygenase inhibitors.
OBJECTIVE
Thrombin and tryptase stimulation of human bladder microvascular endothelial cells (Cambrex Bioscience, Walkersville, Maryland) results in the production of multiple membrane phospholipid derived inflammatory mediators via the activation of a calcium independent phospholipase A2 that may have important implications in bladder inflammatory conditions, such as interstitial cystitis. We examined the effect of multiple phospholipase A2 and cyclooxygenase inhibitors on the immediate release of prostacyclin from human bladder microvascular endothelial cells.


METHODS
We stimulated confluent human bladder microvascular endothelial cell monolayers with thrombin or tryptase and measured the immediate release of prostacyclin. Human bladder microvascular endothelial cells were pretreated with several selective phospholipase A2 and cyclooxygenase inhibitors before thrombin or tryptase stimulation to determine which combination of phospholipase A2/cyclooxygenase isoforms was involved in this process. Phospholipase A2 activity was measured using (16:0, [3H]18:1) plasmenylcholine substrate in the absence of calcium. [3H] arachidonic acid release was measured in the surrounding medium from prelabeled human bladder microvascular endothelial cell monolayers. Prostacyclin release into the surrounding medium was measured using a commercially available immunoassay kit.


RESULTS
The immediate increase in prostacyclin release from thrombin or tryptase stimulated human bladder microvascular endothelial cells depended on the activation of membrane associated calcium independent phospholipase A2, resulting in an increase in arachidonic acid production. Constitutively active cyclooxygenase-1 was then responsible for further metabolism of free arachidonic acid to prostacyclin.


CONCLUSIONS
These results show that the search for a suitable anti-inflammatory agent that selectively target specific phospholipase A2 isoforms requires rigorous testing in several cell types in response to various stimuli.
Portell
Craig
C
Department of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Rickard
Alice
A
Vinson
Suzanne
S
McHowat
Jane
J
D001095:Arachidonic Acids / Q000494:pharmacology; D001798:Blood Proteins / Q000494:pharmacology*; D018929:Cell Culture Techniques; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D063065:Organophosphonates / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011753:Pyrones / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D053802:Tryptases; D001743:Urinary Bladder / Q000166:cytology*
C501048:3-(4-methylsulfonylphenyl)-4-phenyl-5-trifluoromethylisoxazole; D001095:Arachidonic Acids; D001798:Blood Proteins; D016861:Cyclooxygenase Inhibitors; D006490:Hemostatics; D007555:Isoxazoles; D009281:Naphthalenes; D063065:Organophosphonates; C400481:PLIalpha; D011720:Pyrazoles; D011753:Pyrones; C115461:SC 560; D013450:Sulfones; C104810:methyl arachidonylfluorophosphonate; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D011464:Epoprostenol; D012697:Serine Endopeptidases; D013917:Thrombin; D053802:Tryptases