We showed previously that angiotensin-(1-7) [Ang-(1-7)] reversed stimulation of proximal tubule Na+-ATPase promoted by angiotensin II (Ang II) through a D-ala(7)-Ang-(1-7) (A779)-sensitive receptor. Here we investigated the signaling pathway coupled to this receptor. According to our data, Ang-(1-7) produces a MAS-mediated reversal of Ang II-stimulated Na+-ATPase by a Gs/PKA pathway because: (1) the Ang-(1-7) effect is reversed by GDPbetaS, an inhibitor of trimeric G protein and Gs polyclonal antibody. Cholera toxin, an activator of Gs protein, mimicked it; (2) in the presence of Ang II, Ang-(1-7) increased the PKA activity 10-fold; (3) the peptide inhibitor of PKA blocked the Ang-(1-7) effect on Ang II-stimulated Na+-ATPase; (4) Ang-(1-7) reverses the Ang II-stimulated PKC activity; (5) cAMP mimicked the Ang-(1-7) effect on the Ang II-stimulated Na+-ATPase. Our results provide new understanding about the signaling mechanisms coupled to MAS receptor-mediated renal Ang-(1-7) effects.
The signaling pathway mediating modulation of Na(+)-ATPase of proximal tubule cells by atrial natriuretic peptides (ANP) and urodilatin through receptors located in luminal and basolateral membranes (BLM) is investigated. In isolated BLM, 10(-11)M ANP or 10(-11)M urodilatin inhibited the enzyme activity (50%). Immunodetection revealed the presence of NPR-A in BLM and LLC-PK1 cells. Both compounds increased protein kinase G (PKG) activity (80%) and this effect did not occur with 10(-6)M LY83583, a specific inhibitor of guanylyl cyclase. The inhibitory effect of these peptides on Na(+)-ATPase activity did not occur after addition of 10(-6)M KT5823, a specific inhibitor of PKG. LLC-PK1 cells were used to investigate if ANP and urodilatin change the activity of sodium pumps by luminal receptor interaction. ANP and urodilatin inhibited Na(+)-ATPase activity (50%), with maximal effect at 10(-10)M, similar to 10(-7)M db-cGMP, and did not occur with 10(-7)M LY83583, a guanylyl cyclase inhibitor. ANP and urodilatin specifically inhibit Na(+)-ATPase activity by activation of the cGMP/PKG pathway through NPR-A located in luminal membrane and BLM, increasing understanding of the mechanism of natriuretic peptides on renal sodium excretion, with proximal tubule Na(+)-ATPase one possible target.
This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.