Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H+-ATPase and on cytosolic free calcium concentration ([Ca2+]i) were investigated in isolated proximal S3 segments of rats during superfusion with an Na+-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH4Cl pulse, was 0.064 ± 0.003 pH units/min ( n = 17/74) and was abolished with concanamycin. Aldosterone (10−12, 10−10, 10−8, or 10−6 M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca2+]i was 103 ± 2 nM ( n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca2+]i and after 6-min preincubation there was a new increase in [Ca2+]i that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca2+]i but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca2+ chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H+-ATPase and on [Ca2+]i. The results are compatible with stimulation of the H+-ATPase by increases in [Ca2+]i (at 10−12-10−6 M aldosterone) and inhibition of the H+-ATPase by decreases in [Ca2+]i (at 10−12 or 10−6 M aldosterone plus RU 486).

CFTR inhibition augments NHE3 activity during luminal high CO2exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H+absorption from the lumen. We thus examined the effect of inhibition of Na+/H+exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO2diffusion and HCO3−secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO2solution or pH 2.2 low CO2solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTRinh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO2]). Luminal high CO2solution increased CO2absorption and HCO3−secretion, accompanied by PV acidification and PV Pco2increase. During CO2challenge, CFTRinh-172 induced HCO3−absorption, while inhibiting PV acidification. S3226 reversed CFTRinh-associated HCO3−absorption. Luminal pH 2.2 challenge increased H+and CO2absorption and acidified the PV, inhibited by CFTRinh-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO3−secretion to absorption and inhibited PV acidification during CO2challenge, suggesting that HCO3−secretion helps facilitate CO2/H+absorption. Furthermore, CFTR inhibition prevented CO2-induced cellular acidification reversed by S3226. Reversal of increased HCO3−loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H+available to neutralize luminal HCO3−with consequent CO2absorption. NHE3, by secreting H+into the luminal microclimate, facilitates net transmucosal HCO3−absorption with a mechanism similar to proximal tubular HCO3−absorption.

Ammonium transport and pH regulation by K+-Cl- cotransporters

The Na+-K+-Cl- cotransporters (NKCCs), which belong to the cation-Cl- cotransporter (CCC) family, are able to translocate [Formula: see text] across cell membranes. In this study, we have used the oocyte expression system to determine whether the K+-Cl- cotransporters (KCCs) can also transport [Formula: see text] and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote [Formula: see text] translocation, presumably through binding of the ion at the K+ site. Moreover, kinetic studies for both NKCCs and KCCs suggest that [Formula: see text] is an excellent surrogate of Rb+ or K+ and that [Formula: see text] transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [[Formula: see text]]). Indeed, NKCC2, KCC1, KCC2, and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated [Formula: see text] transport could bear great physiological implication given the ubiquitous distribution of these carriers.