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Activation of Adenylyl Cyclase in Pulmonary Microvascular Endothelial Cells Increases Microparticle cAMP Content
Author(s) -
Sayner Sarah L,
Hargett Leslie,
Scruggs April,
Ramila KC,
Yarbrough Thomas,
Bauer Natalie
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.980.4
Subject(s) - adenylyl cyclase , microvesicles , cyclic adenosine monophosphate , phosphodiesterase , microbiology and biotechnology , chemistry , stimulation , forskolin , endothelial stem cell , cell type , cell , biochemistry , endocrinology , signal transduction , enzyme , in vitro , biology , receptor , microrna , gene
Rationale Microparticles (MPs) are submicron intact vesicles released from many cell types constitutively or following activation. MPs play a role in cellular homeostasis, injury and act as biomarkers. These submicron vesicles can encapsulate a variety of functional proteins and RNAs/miRNAs; however, little is known about whether they encapsulate signaling molecules, such as cyclic adenosine monophosphate (cAMP). Thus, we investigated the hypothesis that MPs from endothelial cells contain cAMP and that pulmonary microvascular endothelial cells (PMVEC) can be stimulated to increase MP‐cAMP content. Methods MPs were collected from the media of unstimulated pulmonary and aortic endothelial cells as well as pulmonary artery smooth muscle cells (PASMCs) by ultracentrifugation. MPs were also collected from (PMVECs) following treatment with agonists to stimulate endogenous cAMP production from the transmembrane adenylyl cyclase (AC) or bicarbonate‐stimulation of the soluble AC (AC10) in the presence or absence of the phosphodiesterase (PDE) 4 inhibitor, rolipram. Cyclic AMP in the MPs as well as cellular lysates was measured by immunoassay or mass spectrometry and normalized to protein concentration. Results While cAMP was detected in MPs derived from various endothelial cell types as well as PASMCs, the cAMP content was highest in PMVECs compared to the other cell types. Further, stimulation of PMVECs with agents that increase the near membrane cAMP pool led to an increase in cAMP content in MPs. Conclusion Microparticles released from the vasculature contain cAMP while stimulation of transmembrane ACs in PMVECs increases MP‐cAMP content. Support or Funding Information This work was supported by the Parker B. Francis Fellowship Foundation Award (SLS) and HL121513 (SLS), AHA 11SDG7390037 (NB) and T32 HL076125 (LH, AS, TY)