Escherichia coli Heat-Stable Enterotoxin Mediates Na(+)/H(+) Exchanger 4 Inhibition Involving cAMP in T(84) 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. pH(i) was assayed by the NH(4)Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pH(i) recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) 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) (+) (~63%), without altering basal pH(i) (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pH(i) units. The dpHi/dt and J (H) (+) was almost abolished (~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.