Leukotriene D4 receptors are not negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma.
1. The possibility that receptors for the peptide-containing leukotrienes may be negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma was investigated by comparing the effect of leukotriene D4 (LTD4) on the intracellular cyclic nucleotide (cyclic AMP and cyclic GMP) content and on the activity of cyclic AMP-dependent protein kinase (PKA). In addition, the potential association between changes in the cyclic nucleotide content and the ability of LTD4 to increase lung parenchymal tone was also evaluated. 2. Non-cumulative challenge of parenchymal lung strips with LTD4 elicited concentration-dependent contractions (pD2 = 8.23) that were paralleled by concentration-related increases in the intracellular level of cyclic AMP and cyclic GMP, and in the activation state of PKA (Kact = 33 nM). Temporally, these biochemical effects of LTD4 were transient, peaking after approximately 5 min drug contact thereafter decaying, despite the continued generation of tone. Both the biochemical and mechanical effects of LTD4 were antagonized by the LTD4-receptor blocking drug, ICI 198,615 (1 microM for 60 min), indicating that they were receptor-mediated events. 3. Challenge of guinea-pig lung with LTD4 (200 nM; EC100 for tension generation) stimulated a 150 and 70 fold increase in the elaboration of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) respectively, relative to that generated spontaneously. 4. Pretreatment of lung strips for 60 min with an irreversible inhibitor of cyclo-oxygenase, flurbiprofen,at a concentration (8 microM) that abolished both basal and LTD4 (200 nM)-induced TXB2 and 6-keto-PGF1alpha release, relaxed rapidly the spontaneous tone of the tissues, reduced the cyclic AMP content by ~50%and lowered the PKA activity ratio from 29% to 17%. In addition, flurbiprofen abolished the ability of LTD4 (200 nM) to increase the cyclic AMP content and to activate PKA. Functionally, the magnitude of LTD4 (200 nM)-induced tone and the increase in cyclic GMP content were attenuated by approximately 20% and 50% respectively in flurbiprofen-treated tissues.5. In flurbiprofen-treated tissues, isoprenaline (10 microM for 10 min) increased the cyclic AMP content(from 4 to 27 pmol mg-1 protein) and activated PKA (from 15% to 26%). Preincubation (30 s or 5 min)of lung with LTD4 (200 nM) did not inhibit (or enhance) these isoprenaline-induced effects.6. Pretreatment of lung strips for 60 min with the thromboxane synthetase inhibitor, dazmegrel (10 microM),relaxed the spontaneous tone of the tissues, abolished the LTD4 (200 nM)-stimulated release of TXB2 and significantly enhanced (~two fold) the elaboration of 6-keto-PGF1alpha. In addition, dazmegrel attenuated (by ~50%) LTD4 (200 nM)-induced cyclic GMP accumulation but approximately doubled both the cyclic AMP content and PKA activity ratio. LTD4-induced contractions, in contrast, were not affected by dazmegrel.7. EP 092 (1 microM for 60 min), a selective TP-receptor blocking drug, had no effect on spontaneous tone,eicosanoid formation or on the cyclic GMP content of guinea-pig lung parenchymal strips. Likewise,EP 092 exerted no significant mechancial effect in lung challenged with LTD4 (200 nM) although it did potentiate, to a small extent, the ability of LTD4 (200 nM) to increase the cyclic AMP content.8. It is concluded that LTD4 can increase the intracellular level of cyclic AMP in guinea-pig parenchyma and activate PKA by a leukotriene-receptor-mediated mechanism sensitive to ICI 198,615. However,these biochemical actions of LTD4 are induced indirectly by an arachidonic acid-derived cyclo-oxygenase product(s) other than TXA2. Thus, contrary to reports of other investigators, no evidence was found to corroborate the finding that stimulation of leukotriene receptors on guinea-pig lung parenchyma results in a rapid lowering of the cyclic AMP content even in cyclo-oxygenase-blocked tissues. These data,therefore, do not support the hypothesis that leukotriene-induced tension generation is dependent upon a prior reduction in the cyclic AMP content.
Department of Thoracic Medicine, Royal Brompton National Heart and Lung Institute, London.
M A
MA
Giembycz
J
J
Diamond
I W
IW
Rodger
D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006168:Guinea Pigs; D006454:Hemoglobins / Q000378:metabolism; D066298:In Vitro Techniques; D008168:Lung / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013929:Thromboxane B2 / Q000037:antagonists & inhibitors / Q000378:metabolism
D006454:Hemoglobins; D011506:Proteins; D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D006152:Cyclic GMP