Contribution of the cAMP second messenger pathway to the control of cation channels in human syncytiotrophoblast.

MONTALBETTI, N., CANTERO, M.DEL R., AND H.F. CANTIELLO

Santiago, Chile

Simposio; 2nd Latino-American Symposium on Placenta & Materno-Fetal Interaction: From Basic to Clinical Research; 2005

The cAMP second messenger pathway plays an important role in placental development and physiology, including a contribution to cell differentiation, the release of important metabolites, and the activation of transcription factors. The cAMP pathway is the concerted effort of G protein-dependent receptor induced production of cAMP, cAMPdependent kinase (PKA) activation, and phosphatase-mediated cAMP degradation. Here, we explored the role of the cAMP pathway on channel function in apical membranes of the term human syncytiotrophoblast (hST). Methods: hST apical membranes were prepared by ultra-centrifugation, and reconstituted in lipid bilayer chamber to assess cation channel activity. Addition of the purified enzyme (10 ìg/ml) and MgATP (1 mM) assessed the effect of PKA in the hST membranes. Two other maneuvers were also tested. The cAMP analog, 8-Br-cAMP was added to mimic cAMP increase, and vanadate was added to inhibit phosphatase activity. Channel activity was followed with a patch-clamp amplifier and signals were processed for single channel analysis. Results: Addition of PKA and ATP, but not ATP alone, induced an increase in K+ currents. This effect was mimicked in purified polycystin-2 (PC2), the TRP channel associated with this channel activity in hST. Interestingly, direct addition of either 8-Br-cAMP or the phosphatase inhibitor vanadate to the cis chamber, also increased PC2 channel activity in hST vesicles. Conclusions: The data indicate that PKA phosphorylation directly activates PC2 channel function in apical membranes from hST. The data further suggest that local cAMP production and hydrolysis both help regulate PC2-mediated cation transport and electrical parameters in the hST.