Chonnam Med J.  2018 Jan;54(1):63-71. 10.4068/cmj.2018.54.1.63.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine

Affiliations
  • 1Department of Internal Medicine, College of Medicine, Chosun University, Gwangju, Korea.
  • 2Department of Neurology, College of Medicine, Chosun University, Gwangju, Korea.
  • 3Department of Physiology, College of Medicine, Chosun University, Gwangju, Korea. jyjun@chosun.ac.kr

Abstract

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5"²-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca²âº ([Ca²âº]áµ¢) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na⁺-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca²âº or treatment with thapsigargin (inhibitor of Ca²âº uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca²âº]áµ¢ oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca²âº influx and [Ca²âº]áµ¢ release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Keyword

Adenosine Triphosphate; Receptors, Purinergic; Interstitial Cells of Cajal; Pacemaker, Artificial; Intestine, Small

MeSH Terms

Adenosine
Adenosine Triphosphate*
Animals
Endoplasmic Reticulum
Flufenamic Acid
Interstitial Cells of Cajal*
Intestine, Small*
Membranes
Mice*
Muscle, Smooth
Niflumic Acid
Pacemaker, Artificial
Receptors, Purinergic
Receptors, Purinergic P2
Suramin
Thapsigargin
Adenosine
Adenosine Triphosphate
Flufenamic Acid
Niflumic Acid
Receptors, Purinergic
Receptors, Purinergic P2
Suramin
Thapsigargin

Figure

  • FIG. 1 Effects of external ATP on pacemaker potentials in cultured ICCs from mouse small intestine. (A) The treatment with 200 µM ATP depolarized the membranes and decreased the amplitude of pacemaker potentials. The effects of ATP on pacemaker potentials are summarized in (B) and (C). Bars represent mean±SE values. *p<0.05: significantly different from control. RMP: resting membrane potential.

  • FIG. 2 Concentration-dependent effects of ATP on pacemaker currents in cultured ICCs from mouse small intestine. Pacemaker currents of ICCs recorded at a holding potential of −70 mV and exposed to various concentrations of ATP (50–200 µM). (A-C) External ATP produced tonic inward currents and reduced the amplitude of pacemaker currents in a concentration-dependent manner. Responses to ATP are summarized in (D) and (E). Bars represent mean±SE values. *p<0.05: significantly different from control.

  • FIG. 3 Effects of purinergic P2 receptor antagonism on ATP-induced tonic inward currents of ICCs from mouse small intestine. (A) Pretreatment with a purinergic P2 receptor antagonist, suramin (200 µM) alone, had no effect on pacemaker currents. In the presence of suramin, ATP-induced tonic inward currents were essentially blocked. Responses to ATP in the presence of suramin are summarized in (B) and (C). Bars represent mean±SE values. *p<0.05: significantly different from control.

  • FIG. 4 The effects of external Na+-free solution on the ATP-induced tonic inward currents of ICCs from mouse small intestine. (A) Application of an external Na+-free solution abolished the generation of pacemaker currents. The tonic inward currents induced by ATP (200 µM) were markedly suppressed. Responses to ATP in the external Na+-free solution are summarized in (B). Bars represent mean±SE values. *p<0.05: significantly different from control.

  • FIG. 5 The effects of nonselective cation channel (NSCC) blockage on the ATP-induced tonic inward currents of ICCs from mouse small intestine. (A) NSCC inhibitors, flufenamic acid (50 µM) and (B) niflumic acid (50 µM), abolished the generation of pacemaker currents. The tonic inward currents induced by ATP (200 µM) were essentially blocked. Responses to ATP in the presence of flufenamic acid and niflumic acid are summarized in (C) and (D). Bars represent mean±SE values. *p<0.05: significantly different from control.

  • FIG. 6 The effects of external Ca2+-free solution and thapsigargin treatment on the ATP-induced tonic inward currents of ICCs from mouse small intestine. (A) Application of an external Ca2+-free solution or (B) thapsigargin, (10 µM), abolished the generation of pacemaker currents. The tonic inward currents induced by ATP (200 µM) were markedly suppressed. Responses to ATP in the external Ca2+-free solution and in the presence of thapsigargin are summarized in (C) and (D). Bars represent mean±SE values. *p<0.05: significantly different from control.

  • FIG. 7 The effects of ATP on intracellular Ca2+ [Ca2+]i in ICCs from mouse small intestine. ATP (200 µM) increased the basal level of [Ca2+]i intensity and increased the frequency of Ca2+ oscillations (A). The percentage increasing of frequency by ATP is shown in Fig.7B.


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