PKG II-dependent inhibition of an Fe2+-evoked electrogenic transport pathway by cGMP in human intestinal Caco-2 epithelia

Apical exposure to Fe2+ stimulates a pH-dependent inward short circuit current (ISC) in Caco-2 epithelia (Scott et al. 2002). This transport process is consistent with H+-coupled apical entry of Fe2+ via DMT-1 (Gunshin et al. 1997). The present study set out to investigate intracellular regulation of Fe2+-evoked electrogenic transport by cGMP. ISC determinations were made on voltage-clamped Caco-2 epithelia, grown on permeable supports (Anotec, Nunc). Cells were bathed with isotonic mannitol-Hepes buffer (37°C; pH 7.4). At the onset of the experiment, apical pH was adjusted to 6.0. Iron ascorbate (1:10 molar ratio) was applied apically. The cGMP analogue 8-Br cGMP (100 µM) was applied to the apical bathing medium for 20 min prior to Fe2+ exposure. In experiments investigating the effects of staurosporine (0.5 µM), The PKA inhibitor H-89 (50 µM) and the PKG II inhibitor Rp-8-pCPT-cGMPs (20 µM), these were applied 30 min prior to 8-Br cGMP. Exposure to 8-Br cGMP inhibited Fe2+-induced inward ISC to levels not distinguishable from zero at all Fe2+ concentrations tested (25-1000 µM). Pre-incubation with staurosporine reversed cGMP-induced inhibition of Fe2+-evoked ISC and stimulated (P < 0.05, ANOVA) an additional inward ISC (control Vmax = 1.06 ± 0.05 µA cm-2 (9); stauro + cGMP Vmax = 1.71 ± 0.12 µA cm-2 (5) (means ± S.E.M. (n)). Staurosporine alone stimulated the Fe2+-induced ISC with Vmax rising (P < 0.001 vs. control) to 2.45 ± 0.40 µA cm-2 (4). Rp-8-pCPT-cGMPs also abolished the inhibitory action of 8-Br cGMP on Fe2+-induced ISC (control Vmax = 1.05 ± 0.15 µA cm-2 (4); Rp-8-pCPT-cGMPs + cGMP Vmax = 0.99 ± 0.04 µA cm-2 (4)). As with staurosporine, Rp-8-pCPT-cGMPs alone stimulated the Fe2+-induced ISC, Vmax rising (P < 0.01) to 1.99 ± 0.21 µA cm-2 (4). H89 had no effect on the actions of 8Br-cGMP or on the Fe2+-evoked ISC. These data identify an intracellular regulatory mechanism for an Fe2+-induced electrogenic transport pathway in Caco-2 epithelia. Regulation by cGMP was inhibitory and was dependent upon PKG II with no discernible involvement of PKA. The stimulatory actions of both staurosporine and Rp-8-pCPT-cGMPs when applied alone suggest that there was baseline inhibitory PKG II activity in these cells. To our knowledge, this is the first reported involvement of cGMP/PKG II-dependent regulation of an Fe2+-evoked transport pathway.