Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts.
The Na(+2)Cl(-)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 (delta fluorescence ratio units/1000 s). In pilot experiments 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 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 micromol/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 micromol/l and 5 micromol/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 micromol/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.
Heitzmann
D
D
Physiologisches Institut, Albert-Ludwigs Universität, Freiburg, Germany.
Warth
R
R
Bleich
M
M
Henger
A
A
Nitschke
R
R
Greger
R
R
D000465:Algorithms; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D015854:Up-Regulation / Q000502:physiology
D002352:Carrier Proteins; D002712:Chlorides; D006982:Hypertonic Solutions; D018721:Muscarinic Agonists; D000644:Quaternary Ammonium Compounds; D028021:Sodium-Potassium-Chloride Symporters; D002217:Carbachol; D012964:Sodium; D000242:Cyclic AMP; D011188:Potassium