2-APB

2-APB is an antagonist of Ins(1,4,5) P3-induced Ca2+ release with IC50 value of 42 μM [1].

Myo-Ins(1,4,5) P3 receptors (IP3R) mediates the mobilization of internal Ca2+. IP3R is involved in Ca2+ waves and Ca2+ oscillations.

2-APB is an antagonist of Ins(1,4,5) P3-induced Ca2+ release. 2APB inhibited Ins(1,4,5)-P3-induced Ca2+ release from rat cerebellar microsomal with IC50 value of 42 μM. Addition of 2APB to the extracellular inhibited the cytosolic Ca2+ rise in human platelets and neutrophils stimulated by thrombin. Also, 2APB inhibited the contraction of thoracic aorta induced by angiotensin II (AII) [1]. In HEK-293 cells, 2-APB in the extracellular blocked human TRPC5 channels with IC50 value of 20 μM. Also, 2-APB blocked TRPC6 and TRPM3. In cells overexpressing TRPC5, 2-APB inhibited cell proliferation [2]. In the mouse pancreatic acinar cell, 2-APB significantly inhibited store-operated Ca2+ (SOC)-mediated Ca2+ entry at low concentrations. In permeabilized acinar cell, 2-APB inhibited direct stimulation of Ca2+ release and InsP3-induced Ca2+ release at high concentrations [3].

In rats with I/R-induced testicular injury, 2-APB significantly increased superoxide dismutase (SOD), total antioxidant capacity (TAC) and glutathione (GSH) and reduced malondialdehyde (MDA) and DNA fragmentation, which suggested the antiapoptotic and antioxidative effects of 2-APB [4].

References:
[1].  Maruyama T, Kanaji T, Nakade S, et al. 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem, 1997, 122(3): 498-505.
[2].  Xu SZ, Zeng F, Boulay G, et al. Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect. Br J Pharmacol, 2005, 145(4): 405-414.
[3].  Choi KJ, Kim KS, Kim SH, et al. Caffeine and 2-Aminoethoxydiphenyl Borate (2-APB) Have Different Ability to Inhibit Intracellular Calcium Mobilization in Pancreatic Acinar Cell. Korean J Physiol Pharmacol, 2010, 14(2): 105-111.
[4].  Sari E, Aksit H, Erken HA, et al. Protective effect of 2-APB on testicular ischemia-reperfusion injury in rats. J Urol, 2015, 193(3): 1036-1041.

A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in O. tsutsugamushi-Infected Human Macrophages.[Pubmed:27472555]

PLoS One. 2016 Jul 29;11(7):e0159299.

Orientia (O.) tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF)-alpha reduced cell injury from a cytokine storm. Since TNF-alpha production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE) inhibitors on TNF-alpha production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB), but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-alpha and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70) expression associated with ERK activation; upregulated TNF-alpha production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-alpha production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response.

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak.[Pubmed:28179072]

Cell Calcium. 2017 Jul;65:91-101.

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca(2+) leak using synthetic Ca(2+) indicators that report changes in both the cytoplasmic ([Ca(2+)]i) and the luminal ER ([Ca(2+)]ER) Ca(2+) concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca(2+) leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca(2+)]ER but did not decrease the ER Ca(2+) leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca(2+) leak, while Orai1 and Orai2 inhibited this type of ER Ca(2+) leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca(2+) leak but did not modify the ER Ca(2+) leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca(2+)]i response after TG but only when the ER store had been overloaded with Ca(2+) by eliminating the acidic internal Ca(2+) store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca(2+) leak but forms an ER Ca(2+) leak channel that is limiting the overloading with Ca(2+) of the ER store.

Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration.[Pubmed:28249687]

Cell Calcium. 2017 Jul;65:80-90.

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca(2+) level ([Ca(2+)]ER) and we have carried out simultaneous recordings of both [Ca(2+)]i and the [Ca(2+)]ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca(2+)]ER by activating an ER-resident Ca(2+) permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca(2+)]ER is auto-limited because depends on a replete ER Ca(2+) store; a condition that thapsigargin does not require to decrease the [Ca(2+)]ER. Additionally, our data indicate that the ER Ca(2+) permeable channel activated by 2-APB does not seem to participate in the ER Ca(2+) leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5muM) would lead to the reduction in the [Ca(2+)]ER that might explain the inhibitory effect of this compound on those signals that require Ca(2+) release from the ER store.

Selective potentiation of 2-APB-induced activation of TRPV1-3 channels by acid.[Pubmed:26876731]

Sci Rep. 2016 Feb 15;6:20791.

Temperature-sensitive TRP channels are important for responses to pain and inflammation, to both of which tissue acidosis is a major contributing factor. However, except for TRPV1, acid-sensing by other ThermoTRP channels remains mysterious. We show here that unique among TRPV1-3 channels, TRPV3 is directly activated by protons from cytoplasmic side. This effect is very weak and involves key cytoplasmic residues L508, D512, S518, or A520. However, mutations of these residues did not affect a strong proton induced potentiation of TRPV3 currents elicited by the TRPV1-3 common agonist, 2-aminoethoxydiphenyl borate (2-APB), no matter if the ligand was applied from extracellular or cytoplasmic side. The acid potentiation was common among TRPV1-3 and only seen with 2-APB-related ligands. Using (1)H-nuclear magnetic resonance to examine the solution structures of 2-APB and its analogs, we observed striking structural differences of the boron-containing compounds at neutral/basic as compared to acidic pH, suggesting that a pH-dependent configuration switch of 2-APB-based drugs may underlie their functionality. Supporting this notion, protons also enhanced the inhibitory action of 2-APB on TRPM8. Collectively, our findings reveal novel insights into 2-APB action on TRP channels, which should facilitate the design of new drugs for these channels.

STIM and Orai: the long-awaited constituents of store-operated calcium entry.[Pubmed:19187978]

Trends Pharmacol Sci. 2009 Mar;30(3):118-28.

Rapid changes in cytosolic Ca(2+) concentrations [Ca(2+)](i) are the most commonly used signals in biology to regulate a whole host of cellular functions including contraction, secretion and gene activation. A widely utilized form of Ca(2+) influx is termed store-operated Ca(2+) entry (SOCE) owing to its control by the Ca(2+) content of the endoplasmic reticulum (ER). The underlying molecular mechanism of SOCE has eluded identification until recently when two groups of proteins, the ER Ca(2+) sensors stromal interaction molecule (STIM)1 and STIM2 and the plasma-membrane channels Orai1, Orai2 and Orai3, have been identified. These landmark discoveries have enabled impressive progress in clarifying how these proteins work in concert and what developmental and cellular processes require their participation most. As we begin to better understand the biology of the STIM and Orai proteins, the attention to the pharmacological tools to influence their functions quickly follow suit. Here, we briefly summarize recent developments in this exciting area of Ca(2+) signaling.

Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB).[Pubmed:18204483]

Br J Pharmacol. 2008 Mar;153(6):1324-30.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel and is known to be activated by adenosine 5'-diphosphoribose (ADP-ribose) and hydrogen peroxide. TRPM2 current responses are reported to be drastically potentiated by the combination of each of these ligands with heat. Furthermore, the combination of cyclic ADP-ribose with heat also activates TRPM2. Although flufenamic acid, antifungal agents (miconazole and clotrimazole), and a phospholipase A(2) inhibitor (N-(p-amylcinnamoyl)anthranilic acid) inhibit TRPM2, their inhibition was either gradual or irreversible. EXPERIMENTAL APPROACH: To facilitate future research on TRPM2, we screened several compounds to investigate their potential to activate or inhibit the TRPM2 channels using the patch-clamp technique in HEK293 cells, transfected with human TRPM2. KEY RESULTS: 2-aminoethoxydiphenyl borate (2-APB) exhibited a rapid and reversible inhibition of TRPM2 channels that had been activated by its ADP-ribose or cADP-ribose and heat in a dose-dependent manner (IC(50) about 1 microM). 2-APB also inhibited heat-evoked insulin release from pancreatic islets, isolated from rats. CONCLUSIONS AND IMPLICATIONS: 2-APB proved to be a powerful and effective tool for studying the function of TRPM2.

Block of specific gap junction channel subtypes by 2-aminoethoxydiphenyl borate (2-APB).[Pubmed:16985167]

J Pharmacol Exp Ther. 2006 Dec;319(3):1452-8.

2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC(50) of 3.7 microM, lower than that of most gap junction inhibitors. At a concentration of 20 microM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC(50), 3.0 microM), IC(50) values were higher for Cx43 (51.6 microM), Cx45 (18.1 microM), and Cx46 (29.4 microM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.

Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect.[Pubmed:15806115]

Br J Pharmacol. 2005 Jun;145(4):405-14.

1 2-aminoethoxydiphenyl borate (2-APB) has been widely used to examine the roles of inositol 1,4,5-trisphosphate receptors (IP3Rs) and store-operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+-indicator dye and patch-clamp recording we first examined the blocking effect of 2-APB on human TRPC5 channels expressed in HEK-293 cells. 3 The concentration-response curve has an IC50 of 20 microM and slope close to 1.0, suggesting one 2-APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N-propargylnitrendipine, or berberine. 4 In whole-cell and excised membrane patch recordings, 2-APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2-APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2-APB also blocks TRPC6 and TRPM3 expressed in HEK-293 cells, but not TRPM2. 7 Block of TRP channels by 2-APB may be relevant to cell proliferation because 2-APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2-APB. The site is only available via an extracellular route and the block shows mild voltage-dependence.

2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release.[Pubmed:9348075]

J Biochem. 1997 Sep;122(3):498-505.

The effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins(1,4,5)P3-induced Ca2+ release were examined. 2APB inhibited Ins(1,4,5)P3-induced Ca2+ release from rat cerebellar microsomal preparations without affecting [3H]Ins(1,4,5)P3 binding to its receptor. The IC50 value (concentration producing 50% inhibition) of 2APB for inhibition of Ins(1,4,5)P3 (100 nM) induced Ca2+ release was 42 microM. Further increase in the concentration of 2APB (more than 90 microM) caused a gradual release of Ca2+ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca2+ ([Ca2+]c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA2 or thrombin, and leukotriene B4 (LTB4) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angiotensin II (AII), STA2, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca2+ release from the ryanodine-sensitive Ca2+ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins(1,4,5)P3 receptor, and it should be a useful tool to investigate the physiological role of the Ins(1,4,5)P3 receptor in various cells.

2-Aminoethyl diphenylborinate (2-APB) is a cell-permeable inhibitor of IP3R. 2-Aminoethyl diphenylborinate also inhibits the store-operated Ca2+ (SOC) channel and activates some TRP channels (V1, V2 and V3).

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