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. 2004 Dec;124(6):719-27.
doi: 10.1085/jgp.200409140. Epub 2004 Nov 15.

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways

Yuan Wei  1 , Dao-Hong Lin, Rowena Kemp, Ganesh S S Yaddanapudi, Alberto Nasjletti, John R Falck, Wen-Hui Wang
Affiliations

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways

Yuan Wei et al. J Gen Physiol. 2004 Dec.

Abstract

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.

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Figures

F<sc>igure</sc> 1.
Figure 1.
A channel recording showing the channel activity of ENaC under control conditions (without amiloride) (A) and in the presence of 0.5 μM amiloride (B). The channel closed level is indicated by “C” and the holding potential was indicated on the top of the corresponding trace.
F<sc>igure</sc> 2.
Figure 2.
A channel recording showing the effect of AA on ENaC in a cell-attached patch. The channel closed level is indicated by “C” and a dotted line. The holding potential was −40 mV (hyperpolarization).
F<sc>igure</sc> 3.
Figure 3.
A dose–response curve of AA effect on ENaC. Each point represents a mean value from 3–10 patches.
F<sc>igure</sc> 4.
Figure 4.
Effect of 10 μM ETYA, 11,14,17-EA, and AA on ENaC activity. The experiments were performed in cell-attached patches.
F<sc>igure</sc> 5.
Figure 5.
The effect of 10 μM AA in the presence 5 μM indomethacin (Indo), 15 μM MS-PPOH (PPOH), and 5 μM DDMS. The experiments were performed in cell-attached patches and asterisk indicates that the data is significantly different from the corresponding control value.
F<sc>igure</sc> 6.
Figure 6.
A channel recording showing the effect of 10 μM AA on ENaC in the presence DDMS. The channel closed level is indicated by a dotted line.
F<sc>igure</sc> 7.
Figure 7.
A channel recording showing the effect of 10 μM AA on ENaC in the presence of MS-PPOH. The channel closed level is indicated by a dotted line.
F<sc>igure</sc> 8.
Figure 8.
A channel recording showing the effect of 200 nM 11,12 EET on ENaC. The experiment was performed in a cell-attached patch and the channel closed level is indicated by a dotted line.
F<sc>igure</sc> 9.
Figure 9.
The effect of 200 nM 5,6-, 8,9-, 11,12-, 14,15-EET and 20-HETE on ENaC. The asterisk indicates that the difference is significant from the corresponding control value. Experiments were performed in cell-attached patches.
F<sc>igure</sc> 10.
Figure 10.
A channel recording showing the effect of 200 nM 11,12-EET on ENaC in the presence MS-PPOH. The channel closed level is indicated by a dotted line and the experiment was conducted in a cell-attached patch.
F<sc>igure</sc> 11.
Figure 11.
A histogram showing the presence of 11,12-EET in the isolated CCD (top). The standard of 11,12-EET is shown on the bottom of the figure.
F<sc>igure</sc> 12.
Figure 12.
Confocal images showing the CYP2C23 (green) and AQP2 (red) staining in the renal cortex. The bar represents 10 μM.
F<sc>igure</sc> 13.
Figure 13.
A schema illustrating the mechanism by which high Na intake suppresses ENaC activity.
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