Rapid activation of the Na⁺-H⁺ exchanger, in response to steroid hormones, has been demonstrated in a wide variety of tissues including epithelia, vascular smooth muscle and lymphocytes. Non-genomic stimulation of the Na⁺-H⁺ exchanger can influence the activity of other ionic transporters, which in turn determine cell volume, secretion and absorption. Previous studies from our laboratory have shown that 17β-oestradiol (E2) activates the Na⁺-H⁺ exchanger in female rat distal colon.

Here, we investigated the signalling pathways involved in rapid E2 effects on the Na⁺-H⁺ exchanger. The acute response of the Na⁺-H⁺ exchanger to E2 was measured on pH_i recovery rate following an ammonium chloride acid load in female Sprague-Dawley rats (killed by cervical dislocation). The distal colon was removed, crypts were isolated, loaded with the pH-sensitive fluorescent dye, 2Ô,7Ô-bis(carboxyethyl)carboxyfluorescein (BCECF) and finally pH_i was recorded in the absence of bicarbonate. Data represent means ± S.E.M. of six independent experiments.

We have previously shown (unpublished data) that E2 induces a rapid (< 10 min), non-concentration-dependent activation of the pH_i recovery rate basal = 0.46 ± 0.05 ΔpH units min^-1; E2 (10 nM) = 0.96 ± 0.04 ΔpH units min^-1 (P < 0.05, ANOVA test). The rapidity of the E2 action and its insensitivity to the classical steroid receptor antagonist, ICI 182,780, indicate a non-genomic action of E2 on the Na⁺-H⁺ exchanger. Evidence from other studies supports the involvement of a membrane receptor in non-genomic responses. To test the involvement of a membrane receptor in our system, we used an impeded form of E2 linked to BSA (E2-CMO-BSA). Both E2 (10 nM) and E2-CMO-BSA (10 nM) induced the same activation of the pH_i recovery rate (E2 (10 nM) = 0.96 ± 0.04 ΔpH units min^-1, E2-CMO-BSA (10 nM) = 0.89 ± 0.06 ΔpH units min^-1; P > 0.05). These results are consistent with the presence of a membrane receptor. We subsequently investigated G protein-coupled receptor involvement in our rapid E2 response. The involvement of two G protein subtypes, G_αi and G_αs, were assessed using pertussis toxin (100 ng ml^-1) and cholera toxin (100 ng ml^-1), respectively. Inhibition of G_αs led to a significant reduction of the E2-induced pH_i recovery rate (E2 = 0.98 ΔpH units min^-1, E2 + cholera toxin = 0.60 ΔpH units min^-1; P < 0.05). Inhibition of G_αi was without effect. Phospholipase C (PLC) involvement in E2 action was assessed using the specific inhibitor, U73122 (800 nM). Pre-incubation with U73122 significantly reduced the E2-induced activation of pH_i recovery rate by 30 %. The role of intracellular calcium was demonstrated using the cell-permeant calcium chelator, BAPTA AM (50 mM). Pretreatment with BAPTA AM inhibited the E2-induced activation of pH_i recovery rate by 40 % (E2 = 0.98 ± 0.04 ΔpH units min^-1, E2 + BAPTA AM = 0.59 ± 0.04 ΔpH units min^-1; P < 0.05).

This study demonstrates a rapid non-genomic activation of the Na⁺-H⁺ exchanger in isolated female rat distal colonic crypts by E2. This early response to E2 appears to involve a cholera toxin-sensitive G protein-coupled membrane receptor, the identity of which is unknown. Phospholipase C and intracellular calcium are also involved in the rapid E2 effect on the Na⁺-H⁺ exchanger. This early response to E2 may result in an increase in the NaCl absorptive capacity of the colon and/or modulation of pH_i-sensitive ion channels.

This work was funded by the Higher Education Authority of Ireland.

All procedures accord with current National guidelines.