Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.
Previously it has been shown that the Na+2Cl–K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl–K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) 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 (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl– solution (6 mmol/l, low Cl–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na+2Cl–K+ cotransporter probably independently: (1) lowering of cytosolic Cl– concentration; (2) cell shrinkage; and (3) activation by cAMP.
Warth
R
R
Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Bleich
M
M
Thiele
I
I
Lang
F
F
Greger
R
R
D000818:Animals; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D004594:Electrophysiology; D005665:Furosemide / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012491:Salt Gland / Q000033:anatomy & histology* / Q000187:drug effects / Q000378:metabolism*; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D048251:Squalus acanthias / Q000033:anatomy & histology* / Q000378:metabolism*
D002712:Chlorides; D004232:Diuretics; D000644:Quaternary Ammonium Compounds; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D005665:Furosemide; D000242:Cyclic AMP