Background: Previous studies have suggested that nitric oxide (NO) produced within cells of the macula densa (MD) modulates tubuloglomerular feedback (TGF). We tested the hypothesis that NO produced in the MD acts locally as an autacoid to activate soluble guanylate cyclase and cGMP-dependent protein kinase in the MD itself.
Methods: Rabbit afferent arterioles (Af-Arts) and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring the Af-Art diameter before and after increasing NaCl in the MD perfusate (from 17 mmol/L of Na and 2 of Cl to 65 mmol/L of Na and 50 of Cl). TGF was studied before (control TGF) and after inhibiting components of the NO-cGMP-dependent cascade in the tubular or vascular compartment.
Results: Increasing NaCl concentration in the MD perfusate decreased the Af-Art diameter by 3.2 +/- 0.5 microm (from 18.5 +/- 1.3 to 15.4 +/- 1.3 microm, P < 0.001). Adding a soluble guanylate cyclase inhibitor (LY83583) to the MD increased TGF response to 6.3 +/- 1.1 microm (P < 0.031 vs. control TGF). Similarly, when cGMP-dependent protein kinase was inhibited with KT5823, TGF was augmented from 2.6 +/- 0.3 to 4.0 +/- 0.7 microm (P < 0.023). An analogue of cGMP in the MD reversed the TGF-potentiating effect of both 7-nitroindazole (7NI; an nNOS inhibitor) and LY83583. Inhibition of MD guanylate cyclase did not alter the effect of acetylcholine (a NO-cGMP-dependent vasodilator) on the Af-Art. Perfusing the Af-Art with the guanylate cyclase inhibitor did not potentiate TGF, suggesting that the effect of NO occurred at the MD via a cGMP-dependent mechanism. To determine whether the effect of NO in the MD was entirely mediated by cGMP, TGF was studied after giving (1) LY83583 or (2) LY83583 plus 7NI. Adding the nNOS inhibitor to the MD did not potentiate the TGF response further.
Conclusions: We concluded the following: (1) NO produced by the MD inhibits TGF via stimulation of soluble guanylate cyclase, generating cGMP and activating cGMP-dependent protein kinase; (2) NO acts on the MD itself rather than by diffusing to the Af-Art; and (3) most, if not all, of the effect of NO in the MD is due to a cGMP-dependent mechanism rather than to other NO mediators.