REGULATION OF THE NA(-K+ COTRANSPORTER IN IN-VITRO PERFUSED RECTAL GLAND TUBULES OF SQUALUS-ACANTHIAS()2CL)

Previously it has been shown that the Na(+)2Cl(-)K(+) cotransporter ac cepts NH4+ at its K+ binding site. This property can be used to estima te its transport rates by adding NH4+ to the bath and measuring the in itial furosemide-dependent rates of change in BCECF fluorescence. We h ave utilized this technique to determine the regulation of the furosem ide-inhibitable Na(+)2Cl(-)K(+) cotransporter in in vitro perfused rec tal gland tubules (RGT) of Squalus acanthias. Addition of NH4+ to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH , uptake. This was followed by an acidification, corresponding to NH4 uptake. The rate of this uptake was quantified by exponential curve f itting and is given in arbitrary units (Delta fluorescence/time). This acidification could be completely inhibited by furosemide. In the abs ence of any secretagogue preincubation of RGT in a low Cl- solution (6 mmol/l, low Cl-) for 10 min enhanced the uptake rate significantly fr om 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 man nitol) 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 cyto solic cAMP concentration (Stim) led to a significant increase in NH4uptake rate from 5.00+/-1.33 to 13.3+/-1.54 (n=6). Similar results wer e obtained in the additional presence of Ba2+ (1 mmol/l): the uptake r ate 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 upta ke rate: 15.1+/-3.1 versus 15.2+/-2.8 in high Cl- (n=6). The uptake ra te in Stim containing additional +300 mannitol (22.3+/-4.0, n=5) was n ot significantly different from that obtained with Stim or +300 mannit ol alone. By whatever mechanism the NH4+ uptake rate was increased fur osemide (500 mu mol/l) always reduced this rate to control values. Hen ce three manoeuvres enhanced furosemide-inhibitable uptake rates of th e Na(+)2Cl(-)K(+) cotransporter probably independently: (1) lowering o f cytosolic Cl- concentration; (2) cell shrinkage; and (3) activation by cAMP.