Liddle-Mutation of the ß-Subunit, but not the γ-Subunit, Attenuates Protein Kinase C-Mediated Inhibition of Human Epithelial Sodium Channels (hENaC).

Mammalian distal nephron and distal colon, prime sites for Na(+) homeostasis, contain amiloride-sensitive epithelial sodium channels (ENaC). Protein kinase C (PKC) inhibits ENaC by phosphorylating serine and threonine residues within COOH termini of the ß- and/or γ-subunits. Although some of these phosphorylation sites are close to the PY motifs, it is unclear whether they remain susceptible to PKC in Liddle-mutated ENaC ß- and/or γ-subunits, where PY motifs are truncated, resulting in increased apical ENaC expression. We therefore studied the effects of PKC in wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. PKC activation using 500 nmol/l phorbol 12-myristate 13-acetate (PMA) decreased amiloride-sensitive Na(+) currents by 80 % in oocytes expressing wild-type hENaC, an effect largely prevented by co-exposure to 50 µmol/l calphostin C (a specific inhibitor of PKC), whereas 500 nmol/l phorbol didecanoate (PDD), an inactive phorbol ester which does not stimulate PKC, had no effect. In oocytes expressing hENaC containing the Liddle-mutated ß-subunit, PMA elicited a 54 % decrease in amiloride-sensitive Na(+) currents, significantly (P < 0.0025) less than that in oocytes expressing wild-type hENaC. By contrast, in oocytes expressing hENaC containing the Liddle-mutated γ-subunit, PMA elicited a 68 % decrease in amiloride-sensitive Na(+) current, similar (P = 0.10) to that in oocytes expressing wild-type hENaC. We conclude that hENaC incorporating the Liddle-mutated ß-subunit lacks one or more PKC phosphorylation sites, thereby significantly reducing the inhibitory effect of PKC on Na(+) channel activity, whereas hENaC incorporating Liddle-mutated γ-subunits remains as susceptible to PKC as wild-type hENaC.

Calcium rapidly down-regulates human renal epithelial sodium channels via a W-7-sensitive mechanism.

Increases in intracellular calcium (Ca(2+)) inhibit renal sodium (Na(+)) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca(2+) (using 10 µmol/L A23187, a Ca(2+) ionophore) on wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na(+) current by 55% in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 µmol/L W-7 (to inhibit the Ca(2+)/calmodulin complex). By contrast, co-exposure to 50 µmol/L calphostin (to inhibit protein kinase C) or 5 µmol/L KN-62 (to inhibit Ca(2+)/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na(+) current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na(+) current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na(+) channels in situ was unchanged by the rise in intracellular Ca(2+). These data suggest that the A23187-induced rise in intracellular Ca(2+) inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na(+) channels from the cell membrane by endocytosis. We, therefore, propose that Na(+) absorption in cortical collecting duct cells is inhibited by Ca(2+), possibly when complexed with calmodulin.

Increased colorectal epithelial cell proliferation and crypt fission associated with obesity and roux-en-Y gastric bypass.

BACKGROUND AND AIMS: The relationship between obesity, weight reduction, and future risk of colorectal cancer is not well understood. Therefore, we compared mucosal biomarkers in normal weight individuals [body mass index (BMI), 18.5-24.9 kg/m(2)] with those in morbidly obese patients (BMI >40 kg/m(2)) before and 6 months after Roux-en-Y gastric bypass (RYGB). METHODS: Rectal epithelial cell mitosis, crypt area, and crypt branching were measured following whole crypt microdissection. Apoptosis was measured by immunohistochemistry for neo-cytokeratin 18 on fixed tissue sections. Serum levels of C-reactive protein and cytokines were assayed in combination with quantification of mucosal proinflammatory gene expression by real-time RT-PCR. RESULTS: Twenty-six morbidly obese patients (mean BMI, 54.4 kg/m(2)) had significantly increased mitosis, crypt area, and crypt branching (all P < 0.01) compared with 21 age- and sex-matched normal weight individuals (mean BMI, 22.5 kg/m(2)). Morbidly obese patients underwent a mean excess weight loss of 41.7% at a mean of 26 weeks after RYGB. Surprisingly, this was associated with a further increase in mitosis and decreased apoptosis of epithelial cells. At the same time, lower levels of serum C-reactive protein and interleukin-6 following RYGB were accompanied by a reduction in mucosal IL-6 protein content but elevated mucosal expression of other proinflammatory genes such as cyclooxygenase-1 and cyclooxygenase-2. CONCLUSIONS: Mucosal biomarkers, accepted as indicators of future colorectal cancer risk, are increased in morbidly obese patients compared with normal weight controls. The hyperproliferative state that exists 6 months after RYGB may have important implications for long-term colorectal cancer risk in bariatric surgery patients.