PKA-dependent Akt/nNOS pathway mediates baroreflex enhancement by adrenomedullin in the nucleus tractus solitarii of rats

JC Yen1, YC Chiu1, FCH Li1,2, AYW Chang2. 1National Yang-Ming University, Institute of Pharmacology, 11221, Taiwan, 2Kaohsiung Chang Gung Memorial Hospital, Center for Translational Research in Biomedical Sciences, 88301, Taiwan

Adrenomedullin (ADM), a 52-amino acid vasoactive peptide, contributes to the modulation of cardiovascular functions through both central and peripheral mechanisms. We have previously demonstrated that ADM in the nucleus tractus solitarii (NTS) enhances baroreceptor reflex (BRR) response through the mechanism mediated by protein kinase A (PKA)-dependent activation of neuronal nitric oxide synthase (nNOS) as indicated by phosphorylation at Ser1416, a well-known phosphorylation site for Akt. In NTS, phosphatidylinositol 3-kinase (PI3K)/Akt/nNOS pathway is reported to play an important role in central cardiovascular regulation. Therefore, this study was undertaken to examine the hypothesis that Akt may mediate the PKA-dependent nNOS phosphorylation induced by ADM in the NTS. Male, adult Sprague-Dawley rats (250-300 g) used in this study were anesthetized with pentobarbital sodium. All pharmacological agents were prepared with artificial cerebrospinal fluid and were bilaterally microinjected into the NTS at a total volume of 20 nl per site. Results were analyzed by one-way or two-way analysis of variance with repeated measures for group means (n = 6-8 for each group), as appropriate, followed by the Scheffe’s post hoc test for individual means; p < 0.05 was taken as statistically significant. We found that ADM-induced BRR enhancement was significantly reduced after inhibition of PI3K/Akt signaling by LY294002, wortmannin, or Akt inhibitor API-2. Furthermore, ADM administration caused an increase of phospho-nNOS (Ser1416) which was attenuated by administration of PI3K, Akt, or PKA inhibitors. ADM-induced elevation of the protein level of phospho-nNOS (Ser1416) was also inhibited by the PKA inhibitor Rp-cAMP, but not by NPLA (a selective nNOS inhibitor). These results suggested that a PKA-dependent PI3K/Akt signaling pathway participates in ADM-induced enhancement of BRR response and activation of nNOS. This novel signaling pathway activated by ADM was substantiated by the co-localization of Akt and nNOS proteins in NTS neurons. In conclusion, these results suggested that ADM may enhance BRR response though PKA-dependent PI3K/Akt/nNOS signaling pathway in the NTS of rats.