Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration (Ca²⁺]_i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10^?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10^?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10^?6 M) or BAPTA (5 × 10^?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal Ca²⁺]_i was 104 ± 3 nM (15), and ALDO (10^?12 or 10^?6 M) increased the basal Ca²⁺]_i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the Ca²⁺]_i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for Ca²⁺]_i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.