Sistema FMUSP-HC: Faculdade de Medicina da Universidade de São Paulo (FMUSP) e Hospital das Clínicas da FMUSPROSSETTO, Silmara LarissaQUEIROZ-LEITE, Gabriella DuarteCRAJOINAS, Renato OliveiraOMAE, Samantha V.MALNIC, GerhardGIRARDI, Adriana C.2013-10-112013-10-112012FASEB JOURNAL, v.26, 20120892-6638https://observatorio.fm.usp.br/handle/OPI/3144NHE3 transport function is inhibited by PKA. Binding of angiotensin II (ANG II) to AT1 receptor has been reported to activate Gi-protein signaling pathway, resulting in the inhibition of adenylyl cyclase, and consequently, in decreased PKA activation. We therefore tested the hypothesis that decrement of PKA-mediated NHE3 phosphorylation is one of the mechanisms by which ANG II acutely stimulates NHE3 activity in renal proximal tubule. To this end, OKP cells were treated with 100 μM Dopamine (DOP) for 30 min in the presence or absence of 10–10 M ANG II for 5, 15, and 30 min; or only with ANG II for 30 min. Extrusion of H+ from OKP cells was measured. DOP significantly inhibited NHE3 activity (0.2013 ± 0.009 vs. 0.3036 ± 0.0019 pH units/min in control). Simultaneous incubation of DOP with ANG II for 15 (0.2347 ± 0.044 pH units/min) and 30 min (0.3295 ± 0.032 pH units/min) completely blocked this inhibitory effect. As expected, ANG II stimulated NHE3 activity. DOP increased the levels of NHE3 phosphorylated at the PKA consensus phosphorylation sites [serines 552 and 605] by 64 ± 8% and 288 ± 37% respectively vs. control. ANG II treatment for 15 and 30 min prevented this increase. PKA activity was also measured in these groups of cells. We found that PKA activity was significantly increased in cells treated with DOP for 30 min and in cells treated with DOP plus ANG II for 5 min. Longer incubation times with ANG II (15 and 30 min) in the presence of DOP restored PKA activity near to control levels. Losartan blocked all the effects of ANG II here reported. Collectively, these data suggest that inhibition of PKA activity leading to lower levels of endogenous phosphorylation of NHE3 is one of the mechanisms by which ANG II stimulates NHE3 activity in the proximal tubule.engrestrictedAccessconferenceObjectCopyright FEDERATION AMER SOC EXP BIOLBiochemistry & Molecular BiologyBiologyCell Biology