Angiotensin II modulates thick ascending limb ADH–stimulated transport: Role of AT1 receptor, cAMP and O2-.

GUILLERMO B. SILVA; LUIS I. JUNCOS; NESTOR H. GARCIA

Orlando

Congreso; High Blood Pressure Research 2011 Scientific Sessions Abstracts; 2011

Council of High Blood Pressure

Na transport at the thick ascending limb regulates water excretion and thus plasma volume. While ADH increases transport in this segment vía cAMP stimulation, Ang II increases superoxide (O2 -) production, modulating Na reabsorption. However, the mechanism of interaction of these hormones in this segment has not been assessed. We hypothesized that Ang II and ADH potentiate each other via their second messengers. To test this, we measured oxygen consumption (QO2) in thick ascending limb suspensions from Wistar rats as an indirect transport measurement. We found that basal QO2 was 127.8
17.6 nmol O2/min/mg protein. Addition of ADH (1 nM) increased thick ascending limbs QO2 to 301
25.8 O2/min/mg protein (p 0.01, n5). This effect was maintained throughout the experimental period. Then we evaluated the effect of Ang II on ADH-stimulated transport. After addition of Ang II (1 nM) QO2 decreased from 295
29 to 117
11 nmol O2/min/mg protein and this inhibition was maintained during the first 3 minutes (p 0.01vs ADH). However, after this transient inhibitory effect QO2 was markedly stimulated to 226
29 nmol nmol O2/min/mg protein (p 0.01 vs basal). To test receptor‘s role, we used AT1 (Losartan) and AT2 (PD123319) receptor antagonists. Losartan completely abolished the effects of Ang II (p0.01 vs control). In contrast, PD123319 failed to block the Ang II-derived response (p  N.S vs control; n5). In the presence of the O2 - scavenger Tempol, (100 M), ADH increased QO2 from 103
8 to 226
10 nmol O2/min/mg protein. Addition of Ang II decreased QO2 to 90
15 nmol O2/min/mg protein (p 0.01 vs ADH; n5) and remained at 94
18 nmol O2/min/mg protein during all the experimental period. To study the second messengers involved, we measured cAMP and O2 -. ADH increased cAMP by 296% (p 0.02 vs basal; n5) and co-incubation of ADHAng II for 3 minutes blocked ADH-stimulated cAMP (p0.03 vs ADH). ADH produced no effect on O2 - production (p  N.S vs basal; n6), however increased by 206% at 5 minutes after addition of Ang II (p0.01 vs basal). We conclude that 1) Ang II has a time-dependent biphasic effect on ADH-stimulated transport. 2) This effect is mediated by AT1 receptors and 3) that the Ang II inhibitory phase is mediated by decreased cAMP while 4) the stimulatory phase is mediated by O2 -.