Epoxyeicosatrienoic acid activates BK channels in the cortical collecting duct

Abstract

The cortical collecting duct (CCD), which is involved in renal potassium (K) excretion, expresses cytochrome P450 (CYP)-epoxygenase. Here, we examined the effect of high dietary K on renal expression of CYP2C23 and CYP2J2 in the rat, as well as the role of CYP-epoxygenase-dependent metabolism of arachidonic acid in the regulation of Ca(2+)-activated big-conductance K (BK) channels. By Western blot analysis, high dietary K stimulated the expression of CYP2C23 but not CYP2J2 and increased 11,12-epoxyeicosatrienoic acid (11,12-EET) levels in isolated rat CCD tubules. Application of arachidonic acid increased BK channel activity, and this occurred to a greater extent in rats on a high-K diet compared with a normal-K diet. This effect was unlikely due to arachidonic acid-induced changes in membrane fluidity, because 11,14,17-eicosatrienoic acid did not alter BK channel activity. Inhibiting CYP-epoxygenase but not cyclooxygenase- or CYP-omega-hydroxylase-dependent pathways completely abolished the stimulatory effect of arachidonic acid on BK channel activity. In addition, application of 11,12-EET mimicked the effect of arachidonic acid on BK channel activity, even in the presence of CYP-epoxygenase inhibition. This effect seemed specific to 11,12-EET, because both 8,9- and 14,15-EET failed to stimulate BK channels. Finally, inhibition of CYP-epoxygenase abolished iberiotoxin-sensitive and flow-stimulated but not basal net K secretion in isolated microperfused CCD. In conclusion, high dietary K stimulates the renal CYP-epoxygenase pathway, which plays an important role in activating BK channels and flow-stimulated K secretion in the CCD.

Figure 1.

(A) Western blot shows the effect of K intake on the expression of CYP2C23 and CYP2J2 in renal cortex and outer medulla (mixture) from rats on an NK or HK diet. (B) Bar graph summarizes the changes in CYP2C23 and CYP2J2 expression (data are normalized in comparison with actin level). *Significant difference (P < 0.05). (C) Effect of HK on 11,12-EET level in isolated CCDs. *Significant difference.

Figure 2.

(A) A channel recording showing the effect of AA on BK channels in the CCD. The experiments were performed in a cell-attached patch, and the holding potential was 0 mV. The channel closed level is indicated by C and a dotted line. The top trace shows the time course of the experiment, and two parts of the recording indicated by numbers and a bar are extended to show the fast time resolution. (B). A dose-response curve of AA effect on BK channels. Each point represents a mean value from five to six patches. (C) A recording showing the effect of 10 μM 11,14,17-EA on BK channels in the CCD. (D) The effect of 10 μM 11,14,17-EA and AA on BK channel activity. The experiments were performed in cell-attached patches of the CCD from rats on a HK diet. *Significant difference (P < 0.05). The pipette solution was composed of 140 mM KCl, 1.8 mM MgCl₂, and 5 mM HEPES (pH 7.4).

Figure 3.

(A) A channel recording showing the effect of 10 μM AA on BK channels in the CCD from rats on NK diet. The top trace is the time course of the experiment, and two parts of the recording indicated by numbers are extended to show the fast time resolution. The channel closed level is indicated by C, and the holding potential was 0 mV. An arrow indicates the addition of AA. (B) A bar graph summarizes the effect of AA on BK channels in the CCD from rats on NK or HK diet, respectively. *Significant difference between AA and corresponding control group; #significance between HK and NK groups.

Figure 4.

(A) A channel recording showing the effect of AA on BK channels in the CCD in the presence of 5 μM indomethacin (top), DDMS (middle), or 10 μM MS-PPOH (bottom). The experiments were performed in a cell-attached patch in the CCD from rats on an HK diet. The holding potential was 0 mV, and the channel closed level is indicated by C. (B) The effect of 10 μM AA on BK channels in the presence of 5 μM indomethacin (Indo), 5 μM DDMS, and 10 μM MS-PPOH. *Significantly different from the corresponding control value.

Figure 5.

A channel recording showing the effect of 100 nM 11,12-EET on BK channels in the CCD from rats on an HK diet. The experiments were performed in a cell-attached patch, and the holding potential was 0 mV. The channel closed level is indicated by C and a dotted line. The top trace shows the time course of the experiment, and two parts of the recording indicated by numbers and a bar are extended to show the fast time resolution.

Figure 6.

A channel recording showing the effect of 100 nM 8,9-EET and 11,12-EET (A) and 14,15-EET (B) on BK channels in the CCD from rats on an HK diet. The experiments were performed in a cell-attached patch, and the holding potential was 0 mV. The channel closed level is indicated by C and a dotted line. (C) The effect of 100 nM 8,9-, 14,15-, and 11,12-EET on BK channels in the CCD. Experiments were performed in cell-attached patches. *Significant difference (n = 5 to 8) between the experimental and corresponding control values.

Figure 7.

A channel recording showing the effect of AA (10 μM) and 11,12-EET (100 nM) on BK channels in the CCD treated with MS-PPOH. The experiments were performed in a cell-attached patch in the CCD from rats on an HK diet, and the holding potential was 0 mV. The channel closed level is indicated by C and a dotted line. The top trace shows the time course of the experiment, and three parts of the recording indicated by numbers and a bar are extended to show the fast time resolution.

Figure 8.

(A) Effect of tubular fluid–flow rate on J_Na and J_K in the presence or absence of 10 μM MS-PPOH. The rabbit CCD was treated with MS-PPOH (n = 3) or vehicle (n = 3) for 10 min before experiments, and 10 μM MS-PPOH was present throughout the entire experiments. (B) Effect of MS-PPOH (10 μM) and luminal IBX (50 nM) + MS-PPOH on J_Na (left) and J_K (right) in CCDs perfused at 5 nl/mm per min. Control measurements were determined in the absence of the inhibitors. *Significant difference (P < 0.05).