cGMP/PKG II-dependent inhibition of a pH-dependent Zn2+-evoked electrogenic transport pathway in human intestinal Caco-2 epithelia

Apical exposure to Zn2+ stimulates an inward short circuit current (ISC) in Caco-2 epithelia (Scott et al. 2003). This transport process is pH dependent, consistent with H+-coupled uptake of Zn2+ across the apical membrane (Fleet et al. 1993). The present study set out to investigate intracellular regulation of Zn2+-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. Zinc-histidine (1:5 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 Zn2+ exposure. In experiments investigating the effects of staurosporine (0.5 µM), H-89 (50 µM), chelerthryne chloride (20 µM) and Rp-8-pCPT-cGMPs (20 µM), these were applied 30 min prior to 8-Br-cGMP. Exposure to 8-Br-cGMP reversed Zn2+-induced inward ISC to a small, but significant (P < 0.05, Student's unpaired t test), outward ISC at all Zn2+ concentrations tested (25-1000 µM). Staurosporine reversed cGMP-induced inhibition of Zn2+-evoked ISC, resulting in an inward ISC (control Vmax = 0.73 ± 0.08 µA cm-2 (5); stauro + 8-Br-cGMP Vmax = 0.37 ± 0.03 µA cm-2 (4) (means ± S.E.M. (n)). Staurosporine alone stimulated the Zn2+-induced ISC with Vmax rising (P < 0.05 vs. control) to 1.79 ± 0.10 µA cm-2 (8). The PKG II inhibitor, Rp-8-pCPT-cGMPs, also abolished the inhibitory action of 8-Br-cGMP on Zn2+-induced ISC (control Vmax = 0.61 ± 0.09 µA cm-2 (5); Rp-8-pCPT-cGMPs + 8-Br-cGMP Vmax = 1.20 ± 0.08 µA cm-2 (4)). Rp-8-pCPT-cGMPs alone stimulated the Zn2+-induced ISC, Vmax rising (P < 0.01) to 1.39 ± 0.19 µA cm-2 (4). Neither the PKA inhibitor, H89, nor the PKC inhibitor, chelerthryne chloride, had any effect on the actions of 8Br-cGMP or on the Zn2+-evoked ISC. These data demonstrate that the pH-dependent inward ISC, induced by apical Zn2+ exposure in Caco-2 cells, was inhibited by intracellular cGMP. This inhibition was dependent on PKG II but not PKA or PKC. It has yet to be established whether the Zn2+-evoked ISC reflects Zn2+ transport per se. However, these results may suggest an important regulatory pathway for Zn2+ absorption in the small intestine.