Regulation of the Na(+)2Cl(-)K(+) cotransporter in isolated rat colon crypts

The Na(+)2Cl(-)K(+) cotransporter accepts NH4+ at its K+-binding site. Ther efore, 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 upt ake. This was followed by an acidification, corresponding to NH4+ uptake. T he rate of this uptake was quantified by exponential curve fitting and is g iven in arbitrary units (Delta fluorescence ratio units/1000 s). In pilot e xperiments it was shown that the pH signal caused by the Na(+)2Cl(-)K(+) co transporter could be amplified if the experiments were carried out in the p resence 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 t he 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 (hyp ertonic 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 containi ng 100 mu mol/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 incre ase in uptake rate observed with Stimulation of the cAMP pathway by iso but ylmethylxanthine (IBMX) and forskolin (100 mu mol/l and 5 mu mol/l, respect ively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the me chanism used to increase the NH4+ uptake rate. azosemide (500 mu mol/l) alw ays reduced this rate to control values. Hence three manoeuvres enhanced lo op-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K(+) cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activa tion of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a tran sient fall in cell volume.