Cyclic Nucleotides and Inducible Nitric Oxide Synthesis in Pulmonary Artery Smooth Muscle

Background.Nitric oxide (NO), cGMP, and cAMP affect the synthesis, metabolism, and cellular effects each other. We wanted to study how cGMP and cAMP interact to affect the induced synthesis of NO in response to interleukin-1β (IL-1β) in rat pulmonary artery smooth muscle cells. To further dissect the relative contributions of each cyclic nucleotide, and to detect any possible “crossover” effect of one cyclic nucleotide activating the other protein kinase, we tested how pharmacological inhibition of cGMP-dependent and cAMP-dependent protein kinases (PKG and PKA, respectively) affected responses. Materials and Methods.We tested the effects of IL-1β, dibutyryl (db)-cAMP (1–100 μM), and 8-bromo (Br)-cGMP (1 μM–1 mM) on NO synthesis in cultured rat pulmonary artery smooth muscle cells. Positive effects were then tested in the presence of KT5720 (10−9–10−5M), the pharmacological inhibitor of PKA, and KT5823 (10−9–10−5M), the pharmacological inhibitor of PKG. NO production was measured using the Greiss reaction, and mRNA abundance of the inducible NO synthase (iNOS), using semiquantitative RT-PCR. Results.IL-1β caused nitrite levels to increase nearly 10-fold over basal levels at 24 h (P< 0.05). Nitrite levels increased with the addition of either db-cAMP (100 μM, an 8-fold increase) or 8-Br-cGMP (100 μM, a 3-fold increase) to IL-1β (P< 0.05). PKA inhibition with KT5720 (10−5M) completely inhibited NO synthesis in response to the combination of IL-1β and cAMP, while KT5823 had less effect at all doses tested. NO synthesis in response to IL-1β plus cGMP also decreased to PKA inhibition, but not PKG inhibition, indicating that cGMP responses are a crossover effect. Both cAMP and cGMP in combination with IL-1β increased iNOS mRNA abundance above basal levels on reverse transcription polymerase chain reaction. KT5720, but not KT5823, decreased iNOS mRNA to basal levels. Conclusion.Both cAMP and cGMP augment cytokine induction of NO synthesis through activation of PKA: cAMP does so directly; cGMP, through a crossover stimulation of PKA.