Effect of ion transport inhibitors on dense cell formation by O-D cycling. SS ND cells were O-D cycled for 22 hours at pH 6.8 and density separated into LD, ND, ID, and HD fractions, and the percentage of cells in each fraction was quantitated as described and defined in Fig 1. All experiments contained ouabain (100 μmol/L final concentration) and bumetanide (10 μmol/L final concentration) to inhibit the Na⁺-K⁺-ATPase and Na⁺-K⁺-2Cl⁻ cotransporter, respectively. (A) Inhibitors of K:Cl cotransport, DIOA (100 μmol/L final concentration), and okadaic acid (OKA; 0.5 μmol/L final concentration) primarily inhibited ID cell formation (1.090 to 1.114 g/mL). (B) Inhibitors of K(Ca²⁺), charybdotoxin (CTX; 0.1 μmol/L final concentration), and clotrimazole (CLT; 10 μmol/L final concentration) inhibited primarily HD cell formation (>1.114 g/mL). (C) Combined use of K:Cl cotransport inhibitor (DIOA) and K(Ca²⁺) inhibitor (CLT) in the same SS patient on ID and HD cell formation. Inhibitor concentrations are as described above. A manifold was used to allow for the simultaneous O-D cycling of all four test conditions.