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 Ca(2+) because removal of Ca(2+) from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca(2+), we observed that amiloride and benzamil significantly decreased intracellular Ca(2+) in the Ca(2+)-containing solution but had no effect in a Ca(2+)-free bath. Furthermore, raising intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-free bath solution. We conclude that Ca(2+)-dependent NO generation mediates the effect of inhibiting the apical Na(+) transport on the basolateral SK in the rat CCD.