Action of ANG II and ANP on colon epithelial cells

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH(i)) and calcium ([Ca2+](i)) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH(i) recovery rate mediated by the Na(+)/H+ exchanger (NHE) was examined following an NH4Cl pulse. Under control conditions pH(i) recovered at 0.114+/-0.005 pH units/min (n=35). ANG II (10(-12) or 10(-9) M) increased this value, whilst ANG II (10(-7) M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 microM or 25 microM HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH(i) recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca2+]i concentration-dependently. ANP (10(-6) M) or dimethyl-BAPTA/AM (50 microM) blocked the effects of ANG II on [Ca2+]i and on the rate of pH(i) recovery. Thapsigargin (10(-5) M) enhanced the effect of ANG II on [Ca2+]i and reversed its stimulatory effect on the rate of pH(i) recovery to an inhibitory one. External Ca(2+)-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca2+]i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na(+)/H+ exchanger, one stimulating Na(+)/H+ activity by increases of [Ca2+]i in the lower range (at 10(-12) or 10(-9) M ANG II) and the other inhibiting this activity at high [Ca2+]i levels (at 10(-7) M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca2+]i, block both the stimulatory and inhibitory effects of ANG II.