Carbon Monoxide, a heme oxygenase metabolite, decreases ATP release by macula densa cells in vitro

Abstract

Tubuloglomerular feedback (TGF) is the mechanism by which macula densa (MD) cells sense changes in luminal NaCl concentration and trigger signals that alter the vascular tone of afferent arterioles. TGF is mediated by ATP. We have shown that heme oxygenase (HO) metabolites blunt TGF, but the mechanism is unknown. We hypothesize that carbon monoxide (CO), released by HO, reduces ATP release by MD cells in response to increased NaCl thus blunting TGF. We measured: 1) expression of HO‐1 and 2 in laser capture microdissected MD cells from Sprague‐Dawley rats by real‐time PCR; and 2) the effect of a CO releasing molecule (CORM‐3) on NaCl‐induced ATP release by MD cells in culture. ATP release was quantified by an ATP luciferin‐luciferase bioluminescence assay. We found that MD cells from Sprague‐Dawley rats express both HO‐1 and HO‐2, but HO‐2 expression was higher (33.5 ± 0.5 vs. 31.4 ± 0.6 cycles at inflection; n=3, p<0.05). In the presence of 10 mM NaCl, ATP release was 3.2 ± 1.01 pmol/mg protein. In the presence of 75 mM NaCl it was 38.4 ± 6.4 pmol/mg protein, 11‐fold greater (n=6, p<0.002). CORM‐3 (50μM) reduced ATP release in 75 mM NaCl by 33% (25.8 ± 4.3; n=6, p<0.002), but had no effect on ATP release in 10 mM NaCl. Increasing chloride without increasing sodium by adding choline chloride to the media, had no significant effect on ATP release. We conclude that CO released by HO blunts TGF in part by reducing ATP release by MD cells.