Diluting power of thick limbs of Henle. II. Bumetanide-sensitive ²²Na⁺ influx in medullary vesicles

We evaluated the effects of osmotic gradients on ²²Na⁺ influx in vesicles prepared from rat outer renal medulla. ²²Na⁺ influx driven in a coflow mode by an inwardly directed 100 mM KCl gradient was measured at 20 and 60 s; 1 mM bumetanide inhibited ~ 30% of ²²Na⁺ influx. The bumetanide-sensitive ²²Na⁺ influx was reduced by ~ 65% when either K⁺ or Cl^- was omitted from the aqueous phases. We found that an osmotic gradient for vesicle shrinkage, that is, 600 mM urea in the extravesicular medium, enhanced the bumetanide-sensitive ²²Na⁺ influx twofold. Conversely, an osmotic gradient for vesicle swelling, that is, with vesicles but not extravesicular media loaded with 600 mM urea, produced a 50% suppression of bumetanide-sensitive ²²Na⁺ influx. Moreover, 600 mM extravesicular urea, an osmotic gradient for vesicle shrinkage, also reduced uptake of the nonspecific marker [¹⁴C]mannitol. These effects of osmotic gradients were not due to alterations in ionic driving forces, since bumetanide-sensitive ²²Na⁺ influx driven in a counterflow mode by loading the vesicles with 100 mM NaCl also was activated or suppressed by osmotic gradients for vesicles shrinkage or swelling, respectively. We conclude that osmotic gradients, and/or vesicle volume changes, modulate bumetanide-sensitive Na⁺:K⁺:2Cl^- activity.