Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T-84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pH(i)), and NHE1, NHE2, and NHE4 are expressed in T-84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 mu mol/L STa (30 minutes), 25 mu mol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 mu mol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 mu mol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 mu mol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (Bi) and H+ efflux (J(H)(+)) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J(H)(+) (similar to 63%), without altering basal pHi (range 7.144-7.172). STa did not alter Bi value in a range of 1.6 pHi units. The dpHi/dt and J(H)(+) was almost abolished (similar to 94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and J(H)(+) was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T-84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pH(i) recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.