Involvement of thromboxane a(2) in the modulation of pacemaker activity of interstitial cells of cajal of mouse intestine

Abstract

Although many studies show that thromboxane A(2) (TXA(2)) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA(2) on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA(2) in ICC with whole cell patch-clamp technique. Externally applied TXA(2) (5microM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA(2) were inhibited by intracellular application of GDP-beta-S. The pretreatment of ICC with Ca(2+) free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA(2)-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA(2)-induced effects on pacemaker currents. These results suggest that TXA(2) can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA(2) may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca(2+) modulation.

Keywords: Interstitial cells of Cajal (ICC); Intestinal motility; Pacemaker currents; Thromboxane A2 (TXA2).

Fig. 1

The effects of TXA₂ on pacemaker potentials and pacemaker currents recorded in cultured ICC from mouse small intestine (A) Pacemaker potentials of ICC which were exposed to TXA₂ (5µM) in the current-clamping mode (I=0). Vertical solid line scales amplitude of pacemaker potential and horizontal solid line scales for duration of recording (s) pacemaker potentials. (B), (C), and (D) Pacemaker currents of ICC recorded at a holding potential of -70 mV, when exposed to various concentrations of TXA₂ (1, 3, and 5µM). The dotted lines indicate zero current levels. Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker currents. The responses to TXA₂ are summarized in (E), (F) and (G). The bars represent mean values±SE. ^**Significantly different from the untreated control (Con) (p < 0.01).

Fig. 2

The effects of GDP-β-S in response to TXA₂ induced pacemaker currents from ICC of mouse small intestine (A) Pacemaker currents from ICC exposed to TXA₂ (5µM) in the presence of GDP-β-S (1 mM) in the pipette. The tonic inward currents with suppressed amplitudes and frequency induced by TXA₂ were blocked by internally applied GDP-β-S (1 mM). The dotted lines indicate the zero current levels. The effects of TXA₂ in the presence of GDP-β-S are summarized in (B). Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales for duration of recording (s) pacemaker currents. Bars represent mean values ± SE. The effects of GDP-β-S on TXA₂-induced pacemaker currents were significantly different from the TXA₂-induced pacemaker currents (p < 0.01). The bars represent mean values±SE. ^**Significantly different from the untreated control (Con) (p < 0.01).

Fig. 3

The effects of an external Ca²⁺-free solution or thapsigargin on the TXA₂-induced response on pacemaker currents in cultured ICC from mouse small intestine (A) External Ca²⁺-free solution abolished the generation of pacemaker currents. Under this condition, the TXA₂ (5µM)-induced tonic inward currents were blocked. (C) Thapsigargin (5µM) abolished the generation of pacemaker currents. Thapsigargin also blocked the TXA₂ (5µM)-induced tonic inward currents. The dotted lines indicate the zero current levels. Responses to the TXA₂ in the external Ca²⁺-free solution and in the presence of thapsigargin are summarized in (B) and (D). Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker current. The bars represent mean values ± SE. ^**Significantly different from the untreated Control (Con) (p < 0.01).

Fig. 4

The effects of chelerythrine or calphostin C on the TXA₂-induced response on pacemaker currents in cultured ICC from mouse small intestine (A), (B) Pacemaker currents of ICC exposed to TXA₂ (10µM) in the presence of chelerythrine (1µM) or calphostin C (0.1µM). In this condition, the TXA₂ caused tonic inward currents. The dotted lines indicate the zero current levels. Responses to the TXA₂ in the presence of chelerythrine or calphostin C are summarized in (C). Vertical solid line scales amplitude of pacemaker current and horizontal solid line scales duration of recording (s) pacemaker current. The bars represent mean values ± SE.