The Tyrosine Phosphatase Inhibitor Orthovanadate Inhibits Chloride Secretion in Rat Distal Colon
Abstracts
|
Objective : Vanadium-based tyrosine phosphatase inhibitors decrease blood glucose levels in diabetic rats but may interact with membrane ion transporters and protein tyrosine phosphatases in epithelial cells. Thus, our aim was to study the impact of orthovanadate on transepithelial electrical resistance and chloride secretion in rat distal colon. Material and methods: Unstripped rat distal colon segments were mounted in modified Ussing chambers and bathed in buffer solution. Potential difference across colon segments was monitored with a dual voltage/current clamp. Transepithelial resistance (TER, in O· cm2) and short-circuit current (Isc, in µA· cm-2) were calculated by Ohm´s law. Data are presented as mean ? SEM. Student´s t test was used for statistical analysis. Results: Neither apical nor basolateral application of orthovanadate (1-10 mM) for 30 minutes induced significant changes in transepithelial resistance (TER = 199 ? 12 O· cm2 for 1 mM orthovanadate vs. 213 ? 19.4 O· cm2 for control; n = 4 for each group; p = 0.56) and basal short-circuit current (i.e. basal Isc = 45 ? 11 µA· cm2 for control vs. 37 ? 10 µA· cm-2 for basolateral orthovanadate; n = 4 for each group; p = 0.6). Basolateral orthovanadate dose-dependently inhibited forskolin (peak. Isc = 147 ? 36 vs. 45 ? 11 µA· cm-2 for control vs. samples treated with 1 mM orthovanadate; n = 4 for each condition; p<0.05) and 8-Br-cAMP-activated currents. The same effect was seen after luminal exposure. Luminal ouabain did not inhibit the cAMP-stimulated rise in Isc, which discards a luminal ouabain-sensitive ATPase as the site for orthovanadate inhibition. In addition, the effect of orthovanadate remained unchanged by 10 µM indomethacin (peak Isc = 45 ? 11 µA· cm-2 for orthovanadate + indomethacin treated samples vs. 147 ? 36 µA· cm-2 for indomethacin alone; n=4 for each group; p < 0.05) and 5 µM genistein. The tyrosine phosphatase inhibitor diamide (1 mM) did not block the response to forskolin ( peak Isc = 147 ? 7 vs. 151 ? 16 µA· cm-2 for basolateral 1 mM diamide vs. control n = 3; p = 0.83). Conclusion: Orthovanadate blocks cAMP-stimulated chloride secretion in rat distal colon without interfering with either basal secretion or transepithelial resistance. Neither tyrosine phosphatase activity nor luminal ouabain-sensitive ATPases seems to be involved in the inhibitory effect of orthovanadate on chloride secretion. |
