In vivo monitoring of pH variations in the zebrafish gut lumen after ion transport(ers) inhibitors oral administration

In the gut, pH gradients sustain the activity of various transport systems involved in the trans-epithelial flux of ions, nutrients, xenobiotics and pharmacological compounds. The zebrafish (Danio rerio) has emerged as a model in vertebrate gut physiology, mainly for the ease with which it is possible to analyse basic functions at the gut level. Also, it is commonly used to test the fate of drugs, new pharmacological compounds and/or xenobiotics. Here, we describe – as a proof-of-principle – a simple, non-invasive method to monitor pH variations in vivo in the zebrafish gut lumen. As an agastric teleost, it is estimated that zebrafish gut luminal pH is alkaline (pH ≥ 7.0). Our protocol is based on the use of m-cresol purple, a pH indicator that easily detects colorimetric changes in the 6.5-9.5 pH range. Briefly, pH variations were monitored and visualized by color transition, and data quantitated by image analysis. We induced pH variations in the zebrafish gut lumen using inhibitors of bicarbonate transport systems {namely SITS; IUPAC Name: 5-acetamido-2-[(E)-2-(4-isothiocyanato-2-sulfophenyl)ethenyl]benzenesulfonic acid}, carbonic anhydrases {namely ACTZ; IUPAC Name: N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide} and Na+/H+ exchangers/Na+ channels {namely DMA; IUPAC Name: 3-amino-6-chloro-N-(diaminomethylidene)-5-(dimethylamino)pyrazine-2-carboxamide}. Administration of the inhibitors elicited dose- and time-dependent luminal pH changes. In particular, SITS and ACTZ promoted acidification of the gut lumen, while DMA induced limited pH alkalinization. Our results display the potential of the zebrafish to investigate pH changes in the gut and to study pH-dependent systems under physiological and/or pharmacological solicitations.