Regulation of the Na⁺2Cl^–K⁺ cotransporter in isolated rat colon crypts

Abstract

The Na⁺2Cl^–K⁺ cotransporter accepts NH₄ ⁺ at its K⁺-binding site. Therefore, the rate of cytosolic acidification after NH₄ ⁺ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH₄ ⁺ led to an initial alkalinization, corresponding to NH₃ uptake. This was followed by an acidification, corresponding to NH₄ ⁺ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δ fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na⁺2Cl^–K⁺ cotransporter could be amplified if the experiments were carried out in the presence of bath Ba²⁺ to inhibit NH₄ ⁺ uptake via K⁺ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba²⁺. In the absence of any secretagogue, preincubation of RCC in a low-Cl^– solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70±0.11 to 2.54±0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93±0.17 to 2.84±0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 µmol/l carbachol (CCH) led to a small but significant increase in NH₄ ⁺ uptake rate from 2.06±0.34 to 2.40±0.30 U/1000 s (n=11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 µmol/l and 5 µmol/l, respectively) was from 2.39±0.24 to 3.06±0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH₄ ⁺ uptake rate, azosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na⁺2Cl^–K⁺ cotransporter: (1) lowering of cytosolic Cl^– concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.