Korean J Physiol Pharmacol.  2010 Apr;14(2):105-111. 10.4196/kjpp.2010.14.2.105.

Caffeine and 2-Aminoethoxydiphenyl Borate (2-APB) Have Different Ability to Inhibit Intracellular Calcium Mobilization in Pancreatic Acinar Cell

• Choi KJ
• Kim KS
• Kim SH
• Kim DK
• Park HS

• KMID: 1457676
• DOI: http://doi.org/10.4196/kjpp.2010.14.2.105

Abstract

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) modulate Ca2+ release from intracellular Ca2+ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP3Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca2+ release from intracellular Ca2+ stores and Ca2+ entry through store-operated Ca2+ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca2+ entry, but significantly inhibited carbamylcholine (CCh)-induced Ca2+ release. In contrast, 2-APB did not block CCh-induced Ca2+ release, but remarkably blocked SOC-mediated Ca2+ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced Ca2+ release, but 2-APB at lower concentration, which effectively blocked Ca2+ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP3-induced Ca2+ release and direct stimulation of Ca2+ release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP3R, and 2-APB at lower concentration is considered a blocker of Ca2+ entry through SOC channels in the pancreatic acinar cell.

Keyword

Caffeine; 2-APB; InsP3R; SOC; Acinar

MeSH Terms

Acinar Cells
Animals
Boron Compounds
Caffeine
Calcium
Carbachol
Inositol 1,4,5-Trisphosphate Receptors
Membranes
Mice
Reticulum
Boron Compounds
Caffeine
Calcium
Carbachol
Inositol 1,4,5-Trisphosphate Receptors

Figure

• 

  Fig. 1. Both caffeine and 2-aminoethoxydiphenyl borate (2-APB) dose-dependently inhibit lower carbamylcholine (CCh)-induced [Ca2+]i oscillation in pancreatic acinar cell clusters. (A) Representative trace shows the effects of caffeine and 2-APB on CCh-induced [Ca2+]i oscillation. The data were obtained from 5 separate experiments. The physiological concentration of CCh (300 nM) evoked [Ca2+]i oscillation in the presence of 1.28 mM Ca2+. Addition of 20 mM caffeine or 30 μM 2-APB abolished [Ca2+]i oscillation completely. Representative traces of dose responses to caffeine (B) and 2-APB (C) in CCh-induced [Ca2+]i oscillations obtained from at least 4 separate experiments. A gradual increase in caffeine (1∼30 mM) or 2-APB (3∼100 μM) effectively blocked the [Ca2+]i oscillation in a dose-dependent manner. The oscillation did not fully recover after washout of 2-APB, but it completely recovered after caffeine removal.

• 

  Fig. 2. Caffeine and 2-APB have different effects on higher carbamylcholine (CCh)-induced intracellular Ca2+ mobilization in pancreatic acinar cell clusters. (A) Representative trace shows the effect of caffeine on CCh-induced [Ca2+]i rise. The data were obtained from 5 separate experiments. Caffeine was added 100 sec prior to CCh perfusion. CCh (10 μM) evoked initial [Ca2+]i peak followed by sustained plateau in the presence of 1.28 mM Ca2+. Caffeine (30 mM) blocked [Ca2+]i rise completely. (B) Representative trace shows the effect of 2-APB on CCh-induced [Ca2+]i rise. The data were obtained from 5 separate experiments. 2-APB had no effect on initial [Ca2+]i peak, but it markedly blocked the sustained plateau. (C) Summarized dose responses to caffeine and 2-APB in CCh-induced initial [Ca2+]i peak. Data were represented as % of initial levels, and expressed as mean±S.E. Asterisks indicate the value is significantly different from the corresponding value of control (p<0.05). Only caffeine inhibited CCh-induced initial [Ca2+]i peak, dose dependently.

• 

  Fig. 3. 2-APB blocks thapsigargin (TG)-induced Ca2+ entry in a dose-dependent manner in pancreatic acinar cell clusters. Representative traces show the effects of caffeine (A) and 2-APB (B) on TG-induced Ca2+ entry. The data were obtained from at least 6 separate experiments. TG raised [Ca2+]i transiently in the absence of [Ca2+]o, and reintroduction of 1.28 mM Ca2+ resulted in a marked increase of Ca2+ entry. The Ca2+ entry was not reduced by caffeine, but it was blocked by 2-APB dose-dependently. (C) Summarized dose responses to caffeine and 2-APB in TG-induced Ca2+ entry. Data were represented as % of maximal levels. Asterisks indicate the value is significantly different from the corresponding values of control (p<0.05). Only 2-APB inhibited SOC-mediated Ca2+ entry, dose-dependently.

• 

  Fig. 4. Caffeine inhibits inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from internal store in the permeabilized pancreatic acinar cell. The effects of caffeine (A) and 2-APB (B) on IP3-induced Ca2+ release were obtained from at least 5 experiments. Arrows indicate the starting point of IP3 perfusion. The fluorescence ratio was normalized to an initial 10 sec period prior to IP3 perfusion. Caffeine (20 mM) inhibited submaximal (0.5 μM) and maximal (3 μM) IP3-induced Ca2+ release, whereas 2-APB (30 μM) failed to inhibit both concentrations of IP3-induced Ca2+ release. (C) Summarized data show the effects of caffeine and 2-APB on Ca2+ release rates in the permeabilized cell. Rates of Ca2+ release were fitted by a single exponential function. Asterisks indicate the value is significantly different from the corresponding values for control (p < 0.05). Only caffeine had an inhibitory effect on submaximal and maximal IP3-induced Ca2+ release.

• 

  Fig. 5. Higher 2-APB has multiple effects on Ca2+ mobilization. (A) Representative trace shows the effects of higher 2-APB on Ca2+ mobilization in intact pancreatic acinar cell clusters. The data were obtained from 4 separate experiments. Gradual increase of 2-APB (30 μM∼1 mM) enhanced [Ca2+]i dose-dependently. However, patterns of [Ca2+]i rise were variable according to dosage used. (B) Dual effects of 2-APB on Ca2+ release in the permeabilized cells were obtained from 4 separate experiments. 2-APB was perfused (arrow head) 30 sec prior to IP3 perfusion (arrow). The fluorescence ratio was normalized to initial 10 sec period prior to 2-APB perfusion. 2-APB (100 μM) itself elicited Ca2+ release, but reduced the submaximal (0.5 μM) IP3-induced Ca2+ release. (C) Summarized data of 2-APB effect on Ca2+ release rates in permeabilized cell. Asterisks indicate the value is significantly different from the corresponding values of control (p<0.05). 2-APB (100 μM) markedly inhibited submaximal (0.5 μM) IP3-induced Ca2+ release.

Reference

References

1. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003; 4:517–529.

2. Petersen OH, Michalak M, Verkhratsky A. Calcium signalling: past, present and future. Cell Calcium. 2005; 38:161–169.

3. Williams JA, Yule DI. Stimulus-secretion coupling in pancreatic acinar cell. Johnson LR, editor. ed.Physiology of the gastrointestinal tract. 4th ed.New York: Elsevier Academic Press;2006. p. 1337–1369.

4. Berridge MJ. Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophys Acta. 2009; 1793:933–940.

5. Putney JW, Bird GS. Cytoplasmic calcium oscillations and store-operated calcium influx. J Physiol. 2008; 586:3055–2059.

6. Taylor CW, Prole DL, Rahman T. Ca²⁺ channels on the move. Biochemistry. 2009; 48:12062–12080.

7. Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY. Cell-permeant caged InsP3 ester shows that Ca²⁺ spike frequency can optimize gene expression. Nature. 1998; 392:936–941.

8. Ehrlich BE, Kaftan E, Bezprozvannaya S, Bezprozvanny I. The pharmacology of intracellular Ca²⁺-release channels. Trends Pharmacol Sci. 1994; 15:145–149.

9. Solovyova N, Fernyhough P, Glazner G, Verkhratsky A. Xestospongin C empties the ER calcium store but does not inhibit InsP₃-induced Ca²⁺ release in cultured dorsal root ganglia neurons. Cell Calcium. 2002; 32:49–52.

10. Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P₃-induced Ca²⁺ release. J Biochem. 1997; 122:498–505.

11. Missiaen L, Callewaert G, De Smedt H, Parys JB. 2-Amino-ethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca²⁺ pump and the non-specific Ca²⁺ leak from the non-mitochondrial Ca²⁺ stores in permeabilized A7r5 cells. Cell Calcium. 2001; 29:111–116.

12. Bilmen JG, Michelangeli F. Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate. Cell Signal. 2002; 14:955–960.

13. Soulsby MD, Wojcikiewicz RJ. 2-Aminoethoxydiphenyl borate inhibits inositol 1,4,5-trisphosphate receptor function, ubiquitination and downregulation, but acts with variable characteristics in different cell types. Cell Calcium. 2002; 32:175–181.

14. Prakriya M, Lewis RS. Potentiation and inhibition of Ca²⁺ release-activated Ca²⁺ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP₃ receptors. J Physiol. 2001; 536:3–19.

15. Gregory RB, Rychkov G, Barritt GJ. Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca²⁺ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors. Biochem J. 2001; 354:285–290.

16. Park MK, Lee KK, Uhm DY. Slow depletion of endoplasmic reticulum Ca²⁺ stores and block of store-operated Ca²⁺ channels by 2-aminoethoxydiphenyl borate in mouse pancreatic acinar cells. Naunyn Schmiedebergs Arch Pharmacol. 2002; 365:399–405.

17. Bootman MD, Collins TJ, Mackenzie L, Roderick HL, Berridge MJ, Peppiatt CM. 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca²⁺ entry but an inconsistent inhibitor of InsP₃-induced Ca²⁺ release. FASEB J. 2002; 16:1145–1150.

18. DeHaven WI, Smyth JT, Boyles RR, Bird GS, Putney JW Jr. Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry. J Biol Chem. 2008; 283:19265–19273.

19. Park HS, Betzenhauser MJ, Won JH, Chen J, Yule DI. The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP₃-induced Ca²⁺ release to ATP in pancreatic acinar cells. J Biol Chem. 2008; 283:26081–26088.

20. Betzenhauser MJ, Wagner LE 2nd, Park HS, Yule DI. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs. J Biol Chem. 2009; 284:16156–16163.

21. Yule DI, Gallacher DV. Oscillations of cytosolic calcium in single pancreatic acinar cells stimulated by acetylcholine. FEBS Lett. 1988; 239:358–362.

22. Sei Y, Gallagher KL, Daly JW. Multiple effects of caffeine on Ca²⁺ release and influx in human B lymphocytes. Cell Calcium. 2001; 29:149–160.

23. Han MH, Kawasaki A, Wei JY, Barnstable CJ. Miniature postsynaptic currents depend on Ca²⁺ released from internal stores via PLC/IP₃ pathway. Neuroreport. 2001; 12:2203–2207.

24. Nakamura T, Nakamura K, Lasser-Ross N, Barbara JG, Sandler VM, Ross WN. Inositol 1,4,5-trisphosphate (IP3)-mediated Ca²⁺ release evoked by metabotropic agonists and backpropagating action potentials in hippocampal CA1 pyramidal neurons. J Neurosci. 2000; 20:8365–8376.

25. Sjödin L, Gylfe E. Caffeine inhibits a low affinity but not a high affinity mechanism for cholecystokinin-evoked Ca²⁺ signalling and amylase release from guinea pig pancreatic acini. Naunyn Schmiedebergs Arch Pharmacol. 2000; 361:113–119.

26. Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev. 2005; 85:757–810.

27. Putney JW. Physiological mechanisms of TRPC activation. Pflugers Arch. 2005; 451:29–34.

28. Smyth JT, Dehaven WI, Jones BF, Mercer JC, Trebak M, Vazquez G, Putney JW Jr. Emerging perspectives in store-operated Ca²⁺ entry: roles of Orai, Stim and TRP. Biochim Biophys Acta. 2006; 1763:1147–1160.

29. McCarron JG, Bradley KN, MacMillan D, Muir TC. Sarco-lemma agonist-induced interactions between InsP₃ and ryanodine receptors in Ca²⁺ oscillations and waves in smooth muscle. Biochem Soc Trans. 2003; 31:920–924.

30. Rousseau E, Ladine J, Liu QY, Meissner G. Activation of the Ca²⁺ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch Biochem Biophys. 1988; 267:75–86.

31. Toescu EC, O'Neill SC, Petersen OH, Eisner DA. Caffeine inhibits the agonist-evoked cytosolic Ca²⁺ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production. J Biol Chem. 1992; 267:23467–23470.

32. Ashby MC, Petersen OH, Tepikin AV. Spatial characterisation of ryanodine-induced calcium release in mouse pancreatic acinar cells. Biochem J. 2003; 369:441–445.

33. Straub SV, Giovannucci DR, Yule DI. Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen Physiol. 2000; 116:547–560.

34. Ascher-Landsberg J, Saunders T, Elovitz M, Phillippe M. The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4, 5-trisphosphate receptor modulator on myometrial contractions. Biochem Biophys Res Commun. 1999; 264:979–982.

35. Peppiatt CM, Collins TJ, Mackenzie L, Conway SJ, Holmes AB, Bootman MD, Berridge MJ, Seo JT, Roderick HL. 2-Aminoeth-oxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium. 2003; 34:97–108.