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

• Park IK
• Kim JH
• Park CG
• Kim MY
• Parajuli S
• Hong CS
• Choi S
• Jun JY

• KMID: 2458587
• DOI: http://doi.org/10.4068/cmj.2018.54.1.63

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.

Reference

1. Szurszewski JH. Electrical basis for gastrointestinal motility. In : Johnson LR, editor. Physiology of the Gastrointestinal Tract. 2nd ed. New York: Ravin Press;1987. p. 384–422.

2. Olsson C, Holmgren S. The control of gut motility. Comp Biochem Physiol A Mol Integr Physiol. 2001; 128:481–503.

3. Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology. 1996; 111:492–515.

4. Thomsen L, Robinson TL, Lee JC, Farraway LA, Hughes MJ, Andrews DW, et al. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med. 1998; 4:848–851.

5. Koh SD, Sanders KM, Ward SM. Spontaneous electrical rhythmicity in cultured interstitial cells of cajal from the murine small intestine. J Physiol. 1998; 513:203–213.

6. Hanani M, Farrugia G, Komuro T. Intercellular coupling of interstitial cells of cajal in the digestive tract. Int Rev Cytol. 2005; 242:249–282.

7. Sanders KM, Hwang SJ, Ward SM. Neuroeffector apparatus in gastrointestinal smooth muscle organs. J Physiol. 2010; 588:4621–4639.

8. Won KJ, Sanders KM, Ward SM. Interstitial cells of Cajal mediate mechanosensitive responses in the stomach. Proc Natl Acad Sci U S A. 2005; 102:14913–14918.

9. Vanderwinden JM, Rumessen JJ. Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech. 1999; 47:344–360.

10. Jain D, Moussa K, Tandon M, Culpepper-Morgan J, Proctor DD. Role of interstitial cells of Cajal in motility disorders of the bowel. Am J Gastroenterol. 2003; 98:618–624.

11. Burnstock G, Kennedy C. P2X receptors in health and disease. Adv Pharmacol. 2011; 61:333–372.

12. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998; 50:413–492.

13. Giaroni C, Knight GE, Ruan HZ, Glass R, Bardini M, Lecchini S, et al. P2 receptors in the murine gastrointestinal tract. Neuropharmacology. 2002; 43:1313–1323.

14. Bornstein JC. Purinergic mechanisms in the control of gastrointestinal motility. Purinergic Signal. 2008; 4:197–212.

15. Christofi FL. Purinergic receptors and gastrointestinal secretomotor function. Purinergic Signal. 2008; 4:213–236.

16. Burnstock G. The journey to establish purinergic signalling in the gut. Neurogastroenterol Motil. 2008; 20:Suppl 1. 8–19.

17. Burnstock G, Lavin S. Interstitial cells of Cajal and purinergic signalling. Auton Neurosci. 2002; 97:68–72.

18. Vanderwinden JM, Timmermans JP, Schiffmann SN. Glial cells, but not interstitial cells, express P2X7, an ionotropic purinergic receptor, in rat gastrointestinal musculature. Cell Tissue Res. 2003; 312:149–154.

19. Van Nassauw L, Costagliola A, Van Op den Bosch J, Cecio A, Vanderwinden JM, Burnstock G, et al. Region-specific distribution of the P2Y4 receptor in enteric glial cells and interstitial cells of Cajal within the guinea-pig gastrointestinal tract. Auton Neurosci. 2006; 126-127:299–306.

20. Chen H, Redelman D, Ro S, Ward SM, Ordög T, Sanders KM. Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine. Am J Physiol Cell Physiol. 2007; 292:C497–C507.

21. Jiang R, Taly A, Grutter T. Moving through the gate in ATP-activated P2X receptors. Trends Biochem Sci. 2013; 38:20–29.

22. Ward SM, Ordog T, Koh SD, Baker SA, Jun JY, Amberg G, et al. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol. 2000; 525:355–361.

23. Gallego D, Vanden Berghe P, Farré R, Tack J, Jiménez M. P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine. Neurogastroenterol Motil. 2008; 20:159–168.

24. Hwang SJ, Blair PJ, Durnin L, Mutafova-Yambolieva V, Sanders KM, Ward SM. P2Y1 purinoreceptors are fundamental to inhibitory motor control of murine colonic excitability and transit. J Physiol. 2012; 590:1957–1972.

25. Hedlund H, Fändriks L, Delbro D, Fasth S. Effect of alpha, beta-methylene ATP on distal colonic and rectal motility--a possible involvement of P2-purinoceptors in pelvic nerve mediated non-adrenergic, non-cholinergic contraction. Acta Physiol Scand. 1986; 127:425–432.

26. Martínez-Cutillas M, Gil V, Gallego D, Mañé N, Clavé P, Martín MT, et al. α,β-meATP mimics the effects of the purinergic neurotransmitter in the human and rat colon. Eur J Pharmacol. 2014; 740:442–454.

27. Bian X, Ren J, DeVries M, Schnegelsberg B, Cockayne DA, Ford AP, et al. Peristalsis is impaired in the small intestine of mice lacking the P2X3 subunit. J Physiol. 2003; 551:309–322.

28. Matsuo K, Katsuragi T, Fujiki S, Sato C, Furukawa T. ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle. Br J Pharmacol. 1997; 121:1744–1748.

29. Furuzono S, Nakayama S, Imaizumi Y. Purinergic modulation of pacemaker Ca2+ activity in interstitial cells of Cajal. Neuropharmacology. 2005; 48:264–273.

30. Kurahashi M, Mutafova-Yambolieva V, Koh SD, Sanders KM. Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles. Am J Physiol Cell Physiol. 2014; 307:C561–C570.

31. Peri LE, Sanders KM, Mutafova-Yambolieva VN. Differential expression of genes related to purinergic signaling in smooth muscle cells, PDGFRα-positive cells, and interstitial cells of Cajal in the murine colon. Neurogastroenterol Motil. 2013; 25:e609–e620.

32. Bradley E, Kadima S, Kyle B, Hollywood MA, Thornbury KD, McHale NG, et al. P2X receptor currents in smooth muscle cells contribute to nerve mediated contractions of rabbit urethral smooth muscle. J Urol. 2011; 186:745–752.

33. Kim BJ, Lim HH, Yang DK, Jun JY, Chang IY, Park CS, et al. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity. Gastroenterology. 2005; 129:1504–1517.

34. Nakayama S, Kajioka S, Goto K, Takaki M, Liu HN. Calcium-associated mechanisms in gut pacemaker activity. J Cell Mol Med. 2007; 11:958–968.

35. Hwang SJ, Blair PJ, Britton FC, O'Driscoll KE, Hennig G, Bayguinov YR, et al. Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles. J Physiol. 2009; 587:4887–4904.

36. Alvarez J, Coulombe A, Cazorla O, Ugur M, Rauzier JM, Magyar J, et al. ATP/UTP activate cation-permeable channels with TRPC3/7 properties in rat cardiomyocytes. Am J Physiol Heart Circ Physiol. 2008; 295:H21–H28.

37. Koshimizu TA, Van Goor F, Tomić M, Wong AO, Tanoue A, Tsujimoto G, et al. Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells. Mol Pharmacol. 2000; 58:936–945.

38. Monaghan KP, Koh SD, Ro S, Yeom J, Horowitz B, Sanders KM. Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes. Am J Physiol Cell Physiol. 2006; 291:C985–C994.

39. So KY, Kim SH, Sohn HM, Choi SJ, Parajuli SP, Choi S, et al. Carbachol regulates pacemaker activities in cultured interstitial cells of Cajal from the mouse small intestine. Mol Cells. 2009; 27:525–531.

40. Povstyan OV, Harhun MI, Gordienko DV. Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries. Br J Pharmacol. 2011; 162:1618–1638.