Cell volume regulation in proximal renal tubules from trout (Salmo trutta)

Regulatory volume decrease (RVD) following hypoosmotic stimulation was studied using videometric methods in isolated proximal renal tubules from trout (Salmo trutta), The relative tubule diameter increased by 132.0+/-4.8% (maximum swelling within 1 min at 15 and 25 degrees C and within 4 min at 10 degrees C) following a change from iso-osmotic (290 mosmol kg(-1)) to hypo-osmotic (160 mosmol kg(-1)) Ringer's solution. The tubule diameter subsequently decreased to approximately one-quarter of the maximal value. Ouabain (1 mmol l(-1)) reduced cell swelling and inhibited the RVD response by 28.0+/-10.5%. Furthermore, increasing the bath K+ concentration by 30 mmol l(-1) inhibited RVD by 76.5+/-3.6%. The K+ channel blocker quinine, but not Ba2+ (1 and 2 mmol l(-1)), significantly decreased the RVD response (by 25.0+/-5.4 and 72.3+/-5.1% at 0.1 and 0.5 mmol l(-1), respectively), Similarly, increasing the Cl- concentration in the bath from 47 to 102 mmol l(-1) induced a significant reduction (45.2+/-7.9%) in RVD. The RVD response was also markedly reduced (by 54.7+/-5.3%) by the Cl- channel blocker indacrinone (MK-196; 0.5 mmol l(-1)), but only marginally by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1, 5, 8 and 10 mu mol l(-1)). Addition of the K+/Cl- symport inhibitor furosemide (0.1 mmol l(-1)) resulted in a 39.8+/-3.9% inhibition of RVD. This inhibition could be completely overcome by simultaneous administration of 1 mu mol l(-1) tributyltin (anion exchanger) and furosemide. Chelation of either extracellular (1 mmol l(-1) EGTA) or both extra- and intracellular Ca2+ (1 mmol l(-1) EGTA, 10 mu mol l(-1) A23187) had no effect on this RVD process. Furthermore, as measured using the fluorescent dye Fura-2/AM, there was no increase in the intracellular free Ca2+ concentration upon hypo-osmotic stimulation. Administration of the 5-lipoxygenase antagonist ETH 615-139 (20 mu mol l(-1)), however, induced a 60% inhibition of RVD. Simultaneous addition of ETH-615 and either the K+ ionophore gramicidin (0.5 mmol l(-1)) or the anion exchanger tributyltin (1 mu mol l(-1)) could not reverse the ETH 615-139 inhibition, Finally, administration of the cycloxygenase inhibitor indomethacin had only a small, but significant, effect on RVD. We conclude that RVD following hypo-osmotic swelling is in these cells a temperature- and ouabain-sensitive process that appears to be the result of K+ efflux through quinine-sensitive, Ba2+-insensitive K+ channels and Cl- efflux through an MK-196- and furosemide-sensitive Cl- conductance that is relatively unaffected by NPPB. This KCl efflux seems to be regulated by eicosanoids produced by the 5-lipoxygenase. Arachidonic acid metabolites from the cycloxygenase pathway are not involved in this process. Similarly, neither extra-nor intracellular Ca2+ appears to be important for the signalling of RVD.