Deoxycholic acid inhibits pacemaker currents by activating ATP-dependent K+ channels through prostaglandin E2 in interstitial cells of Cajal from the murine small intestine

Abstract

1. We investigated the role of deoxycholic acid in pacemaker currents using whole-cell patch-clamp techniques at 30 degrees C in cultured interstitial cells of Cajal (ICC) from murine small intestine. 2. The treatment of ICC with deoxycholic acid resulted in a decrease in the frequency and amplitude of pacemaker currents and increases in resting outward currents. Also, under current clamping, deoxycholic acid produced the hyperpolarization of membrane potential and decreased the amplitude of the pacemaker potentials. 3. These observed effects of deoxycholic acid on pacemaker currents and pacemaker potentials were completely suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. 4. NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, significantly inhibited the deoxycholic acid-induced effects. The treatment with prostaglandin E(2) (PGE(2)) led to a decrease in the amplitude and frequency of pacemaker currents and to an increase in resting outward currents, and these observed effects of PGE(2) were blocked by glibenclamide. 5. We next examined the role of deoxycholic acid in the production of PGE(2) in ICC, and found that deoxycholic acid increased PGE(2) production through the induction of COX-2 enzyme activity and its gene expression. 6. The results suggest that deoxycholic acid inhibits the pacemaker currents of ICC by activating ATP-sensitive K(+) channels through the production of PGE(2).

Figure 1

Cultured ICC from the murine small intestine. The tunica muscularis of the small bowel was digested with collagenase, and the dispersed cells were cultured for 2 days. (a) Light microscope image of a small ICC network and (b) confocal microscope image of Kit-immunopositive ICC network in culture (a and b are of same field image).

Figure 2

Effects of deoxycholic acid on pacemaker currents in cultured ICC of the murine small intestine. (a) Pacemaker currents from ICC exposed to deoxycholic acid (1–50 μM) at a holding potential of −70 mV. Deoxycholic acid caused a concentration-dependent decrease in the frequency, and amplitude of pacemaker currents, and increased basal outward currents. The responses to deoxycholic acid are summarized in (b), (c), and (d). Bars represent mean values±s.e. ^*(P<0.05), ^**(P<0.01) Significantly different from the untreated control. The dot lines indicate the zero current levels.

Figure 3

Effects of pinacidil and deoxycholic acid on pacemaker currents in cultured ICC of the murine small intestine. (a) Pacemaker currents of ICC exposed to pinacidil (10 μM) at a holding potential of −70 mV. Pinacidil decreased the frequency and amplitude of the pacemaker currents, and increased the basal outward currents, and these effects were reversed by adding glibenclamide (10 μM). (b) The effects of pinacidil (10 μM) on pacemaker currents after pretreatment with glibenclamide. GBC: glibenclamide (10 μM). (c) Pacemaker currents exposed to deoxycholic acid (50 μM) at a holding potential of −70 mV. The effects of deoxycholic acid were qualitatively the same as the effects of pinacidil on pacemaker currents. Also, these effects were reversed adding glibenclamide (10 μM). (d) The effect of deoxycholic acid (10 μM) on pacemaker currents after pretreating cells with glibenclamide (10 μM). (e, f, and g) Bar graphic representation of the blocking response to glibenclamide on effects of deoxycholic acid. Bars represent mean values±s.e. ^**(P<0.01) Significantly different from the untreated control. The dot lines indicate the zero current levels. DCA: deoxycholic acid, GBC: glibenclamide.

Figure 4

Effects of deoxycholic acid on pacemaker potentials in cultured ICC of murine small intestine. (a) Pacemaker potentials of ICC exposed to deoxycholic acid (10 μM) in the current clamping mode (I=0). The deoxycholic acid induced membrane hyperpolarization and the decreased amplitude of pacemaker potentials was reversed by glibenclamide. Responses to deoxycholic acid are summarized in (b) and (c). Bars represent mean values±s.e. ^*(P<0.05), ^**(P<0.01) Significantly different from the untreated control. DCA: deoxycholic acid, GBC: glibenclamide.

Figure 5

Effect of NS-398 upon deoxycholic acid-induced responses on pacemaker currents in cultured murine small intestine ICC. (a) NS-398 (5 μM) blocked the effect of deoxycholic acid (20 μM) on pacemaker currents. The blocking effect of NS-398 is summarized in (b), (c) ,and (d). Bars represent mean values±s.e. DCA: deoxycholic acid. The dot lines indicate the zero current levels.

Figure 6

Effects of PGE₂ on pacemaker currents in cultured murine small intestine ICC. (a) Pacemaker currents exposed to PGE₂ (0.5 μM) at a holding potential of −70 mV. PGE₂ decreased the frequency and amplitude of the pacemaker currents, and increased the basal outward currents. These effects were reversed by adding glibenclamide (10 μM). The responses to PGE₂ are summarized in (b), (c), and (d). Bars represent mean values±s.e. ^**(P<0.01) Significantly different from the untreated control. (e) PGE₂ effects on pacemaker currents in the pretreatment of glibenclamide. The dot lines indicate the zero current levels. GBC: glibenclamide.

Figure 7

(a) Deoxycholic acid increased PGE₂ in a time-dependent manner. ICC were incubated with indicated doses of deoxycholic acid for 5, 15, and 30 min. The results are means±s.e. ^**(P<0.01). DCA: deoxycholic acid. (b) Deoxycholic acid upregulated COX-2 mRNA expression. ICC were treated with different quantities of deoxycholic acid for 3 h. Total RNA was isolated and subjected to RT–PCR analysis with COX-1 and COX-2 primers. The result of a quantitative analysis of COX-2 mRNA expression from three independent experiments is shown in the right panel. The results are expressed as ratios of COX-2 to GAPDH mRNA, and bars represent mean values±s.e. ^**(P<0.01) Significantly different from the untreated control. (c) Deoxycholic acid upregulated COX-2 protein expression. ICC were treated with 1–20 μM deoxycholic acid for 24 h. Protein was extracted and subjected to Western blot analysis with a polyclonal anti-COX-2 antibody. The levels of COX-2 expression, which were quantified by densitometry from three independent experiments, are shown in the lower panel. The results are means±s.e. ^**(P<0.01) Significantly different from the untreated control. (d) ICC were incubated with indicated doses of deoxycholic acid for 24 h in the presence or absence 1 μM NS-398. The results are means±s.e. ^**(P<0.01) Significantly different from the untreated control.