Nitric Oxide Links the Apical Na⁺ Transport to the Basolateral K⁺ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP_o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.