Release of leukotrienes into the perfusate of calcium-ionophore stimulated rabbit lungs: Influence of 5-lipoxygenase inhibitors

Abstract

Arachidonic acid and the calcium ionophore A23187 are known to provoke a pulmonary artery pressor response, edema formation and release of thromboxane B2 (TxB2) and 6-keto prostaglandin-F1 α (6-keto PGFlα) into the recirculating perfusion fluid of isolated blood-free perfused rabbit lungs. Here we investigated the release of leukotrienes (LTs) by repetitive 0.1 μM A23187 challenge in the presence or absence of cyclooxygenase and 5-lipoxygenase inhibitors. RP-HPLC analysis of perfusion fluid extracts persistently showed peaks with retention times of authentic LTC4, -D4, -E4 and -B4. Fractionated RPHPLC eluate subjected to radioimmunoassay (RIA) with LTC4 and LTB4 antibodies showed two major peaks of immunoreactivity corresponding to those compounds and minor immunoreactivity with LTD4 and LTE4 in accordance with the stated cross-reactivities of the LTC4 antibody. Good correlation for both LTB4 and LTC4 levels measured by RP-HPLC versus RIA of collected HPLC peaks was found. Five to ten min after A23187 challenge, LTC4, -D4 and -B4 levels ranged from 800 to 1600 pg/ml perfusate. LTC4 reached a maximum level at 20 min whereas LTB4 slightly increased over a 35 min period. Upon repeated A23187 challenge, interrupted by rinsing phases with fresh perfusion fluid, the LT release was reproducible several times with increasing reaction strength. This performed in presence of increasing concentration of the 5-lipoxygenase inhibitors AA-861 or U-60,257 caused a dose-dependent inhibition of the release of all LTs with an ic 50 of approximately 10 −8 to 10 −7 M and 10 −6 M, respectively. Cyclooxygenase inhibition with acetylsalicylic acid at doses completely suppressing the A23187 induced pressor response did not inhibit the peptidoleukotriene release and only slightly depressed LTB4 release. Conclusion: using a rapid and sensitive extraction and RP-HPLC method isolated lungs are found to release nanomolar amounts of LTs into the perfusate upon repetitive A23187 challenge, suppressed by 5-lipoxygenase inhibition.