Intracellular Ca²⁺ and PKC activation do not inhibit Na⁺ and water transport in rat CCD

Experiments examined the effects of elevation of intracellular calcium concentration ([Ca²⁺]_i) or activation of protein kinase C (PKC) on Na⁺ and water transport in the rat cortical collecting duct (CCD). We measured the lumen-to-bath ²²Na⁺ flux (J_1→b), transepithelial voltage (V_T), and water permeability (P_f) in CCD from deoxycorticosterone (DOC)-treated rats. lonomycin (0.5 and 1 μM) and thapsigargin (1 and 2 μM) were used to increase [Ca²⁺]_i. Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 μM) and oleoyl-acetyl-glycerol (OAG; 100 μM) were used as activators of PKC. [Ca²⁺]_i was measured in isolated perfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin (AVP) failed to affect [Ca²⁺]_i, whereas 1 μM ionomycin increased [Ca²⁺]_i by 103 ± 15% and 2 μM thapsigargin increased [Ca²⁺]_i by 24 ± 4%. In flux studies, neither ionomycin nor thapsigargin affected J_l→b or P_f, although ionomycin caused marked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100 μM OAG nor 1 μM PMA affected J_l→b or P_f. OAG at 50 μM had no effect on V_T or transepithelial resistance, indicating no inhibition of conductive Na⁺ transport. We conclude that increased [Ca²⁺]_i and PKC activation do not affect J_l→b or P_f in the rat CCD. These findings may account for the sustained increase in J_l→b produced in the rat CCD by AVP.