IP3R/Inositol triphosphate receptor
2-APB is a functional and membrane-permeable antagonist of the D-myo-inositol 1,4,5-trisphosphate (IP3) receptor with an IC50 of approximately 42 μM. It inhibits IP3-induced calcium release from intracellular stores by blocking the IP3 receptor channel. At low concentrations (<10 μM), 2-APB inhibits store-operated calcium entry (SOCE), while at higher concentrations (up to 50 μM) it also inhibits TRP channels. 2-APB activates some TRP channels (TRPV1, V2, and V3) at certain concentrations. It is also known to inhibit the SOC channel activity.

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca(2+)]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca(2+) influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca(2+)]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca(2+)]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca(2+)]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca(2+) and SOC channel blockers such as SK&F96365, La(3+) and Gd(3+). PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca(2+)]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca(2+)]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling[3].
2-APB is an IP3 receptor inhibitor that regulates IP3-induced calcium release. It inhibits the SOC channel activity and, at higher concentrations, inhibits TRP channels. The compound's IC50 for IP3 receptor inhibition is approximately 42 μM. 2-APB inhibits store-operated Ca2+ release at low concentrations (<10 μM) and inhibits it at higher concentrations (up to 50 μM). The compound is a cell-permeable inhibitor of IP3R. It also activates some TRP channels (V1, V2 and V3).

Autologous arterial blood injection into the cisterna magna caused vasospasm in rats. 2-APB (2-Aminoethyl diphenylborate) treatment increased the BA wall thickness and reduced the BA lumen diameter, inducing significant vascular changes. 2-APB also alleviated cell apoptosis at 24 hours after SAH.
Conclusion: In experimental SAH in rats, 2-APB treatment increased the BA wall thickness and reduced the BA lumen diameter, inducing significant vascular changes. 2-APB also alleviated cell apoptosis at 24 hours after SAH.
Keywords: 2-Aminoethyl diphenylborate/2-APB; Ca(2+) channels; Cerebral vasospasm; Orai channels; Subarachnoid hemorrhage; TRP channels[2].

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Mean superoxide dismutase, total antioxidant capacity and glutathione were significantly higher in the sham treated group than in the ischemia-perfusion group (p <0.05). Mean malondialdehyde and DNA fragmentation levels were significantly lower in the sham treated group than in the ischemia-reperfusion group (p <0.05). After 2-APB treatment superoxide dismutase, total antioxidant capacity and glutathione were significantly increased but malondialdehyde and DNA fragmentation levels were significantly decreased compared to the ischemia-reperfusion group (p <0.05). The number of TUNEL positive cells was significantly lower in the 2-APB treatment groups than in the ischemia-reperfusion group (p <0.05). Conclusions: In rats 2-APB reduced the oxidative stress and apoptosis caused by testicular ischemia-reperfusion injury. The testicular protective effect of 2-APB appears to be mediated through its antiapoptotic and antioxidative effects. Keywords: 2-aminoethoxydiphenyl borate; apoptosis; oxidative stress; reperfusion injury; testis. https://pubmed.ncbi.nlm.nih.gov/25444954/
In vivo, 2-APB has been used in various animal models to study the role of IP3 receptors and calcium signaling in physiological and pathological processes. Its effects on calcium signaling pathways have implications for understanding neuronal function, muscle contraction, immune cell activation, and other calcium-dependent processes. However, detailed in vivo efficacy data are limited as 2-APB is primarily a research tool used in vitro.

IP3 receptor binding and functional assays are performed using membrane preparations from tissues or cells expressing IP3 receptors (e.g., cerebellar microsomes). Radioligand binding studies utilize [3H]-IP3 as the tracer. Membranes are incubated with varying concentrations of 2-APB and fixed [3H]-IP3 in binding buffer (25 mM Tris-HCl, pH 8.3, containing 1 mM EDTA) for 10-30 minutes on ice. Non-specific binding is determined in the presence of excess unlabeled IP3. Bound radioligand is separated by rapid filtration and counted. Functional assays measure IP3-induced calcium release in permeabilized cells or microsomes using fluorescent calcium indicators.

Dopamine-release experiments[3]
PC12 cells (1×10~5) were seeded in 24-well plates and cultured for 48 h. The medium was then removed, and the cells were washed with HEPES-buffered solution twice and preincubated in HEPES-buffered solution at 37 °C for 20 min. Then they were washed and treated with or without 2-APB in HEPES-buffered solution under each pH condition. Incubation was performed at room temperature for 2 min and then was stopped on ice. Sample solution was collected in a 1.5 mL tube and centrifuged. The supernatant was collected and prepared as supernatant assay solution containing 0.4 N perchloric acid (PCA) (Sup sol.). The cells remaining on the dish were extracted with 0.4 N PCA and prepared as a cell assay solution (Cell sol.). These preparations were performed on ice.
Cellular assays for 2-APB activity typically employ cell lines with functional IP3 receptor and calcium signaling pathways, such as HEK-293 cells, neurons, or smooth muscle cells. Cells are loaded with calcium-sensitive fluorescent dyes (e.g., Fura-2 AM or Fluo-4 AM). IP3-induced calcium release is triggered by stimulation with agonists that activate phospholipase C (e.g., ATP, carbachol, or histamine) or by direct application of caged IP3. 2-APB is pre-incubated for 10-30 minutes before stimulation, and the inhibition of the calcium response is measured. Store-operated calcium entry is assessed by depleting intracellular stores with thapsigargin followed by calcium readdition.

In this study, researchers performed an experimental study using 32 Sprague-Dawley rats divided into 4 groups: sham group (n = 8), SAH group (n = 8), 2-APB group (SAH rats intraperitoneally administered with 0.5 mg/kg 2-APB; n = 8), and 2-APB-2 group (SAH rats intraperitoneally administered with 2 mg/kg 2-APB; n = 8). The rats were sacrificed after 24 hours, and superoxide dismutase, glutathione peroxidase, malondialdehyde, tumor necrosis factor-α, and interleukin-1β in the brain tissue and serum were measured. The histopathological investigation of brain tissue included measurement of the luminal diameter and wall thickness of the basilar artery (BA), and apoptotic cells in the hippocampus were counted after caspase staining.[2]

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A total of 28 rats were randomly divided into 4 groups, including sham treated, ischemia-reperfusion, ischemia-reperfusion plus 2 mg/kg 2-APB and ischemia-reperfusion plus 4 mg/kg 2-APB. Testicular tissue superoxide dismutase, glutathione, malondialdehyde, total antioxidant capacity and DNA fragmentation levels were determined. Testicular tissue samples were examined by histopathology and TUNEL staining. https://pubmed.ncbi.nlm.nih.gov/25444954/
In vivo studies with 2-APB are typically conducted in rodent models to investigate the role of IP3 receptors and calcium signaling in various conditions. The compound is administered via intraperitoneal, intravenous, or intracerebroventricular routes depending on the target. Doses typically range from 1-10 mg/kg for systemic administration. Effects on calcium-dependent processes such as neuronal excitability, muscle contraction, immune responses, or pain sensation are measured. However, 2-APB's multiple targets and relatively low potency limit its utility for in vivo studies.

2-APB has a molecular weight of 225.09 and molecular formula C14H16BNO. The compound is a small, cell-permeable molecule that readily crosses cell membranes. It is soluble in DMSO and other organic solvents. Following administration, 2-APB is rapidly distributed and metabolized. The compound's pharmacokinetic properties support its use in in vitro and some in vivo applications, though its multiple targets and relatively low potency limit its utility as a selective pharmacological tool.

The intravenous LD50 in mice was 56 mg/kg. NIOSH Exchange Chemicals, Chemical Systems Laboratory, U.S. Army Armaments Research and Development Command, NX#01899
Toxicological data for 2-APB are limited as it is a research compound. At high concentrations, 2-APB may have off-target effects due to its activity at multiple ion channels and receptors. The compound should be handled with appropriate laboratory safety precautions. In cell culture, 2-APB is typically used at concentrations ranging from 10-100 μM, which are generally well-tolerated in short-term experiments. However, prolonged exposure or higher concentrations may cause cytotoxicity.

[1]. Effect of Wilforine on the Calcium Signaling Pathway in Mythimna separata Walker MyocytesUsing the Calcium Imaging Technique. J Agric Food Chem. 2019 Dec 11;67(49):13751-13757.

[2]. Effects of 2-Aminoethyl Diphenylborinate, a Modulator of Transient Receptor Potential and OraiChannels in Subarachnoid Hemorrhage: An Experimental Study.

[3]. Molecular mechanism of 2-APB-induced Ca²⁺ influx in external acidification in PC12. Exp Cell Res. 2014 May 1;323(2):337-45.

2-Aminoethoxydiphenylborane is an organoboron compound, consisting of diphenylborane in which the borane hydrogen is replaced by a 2-aminoethoxy group. It possesses multiple functions, including acting as an IP3 receptor antagonist, calcium channel blocker, and potassium channel opener. It is both an organoboron compound and a primary amino compound.
2-APB is a widely used research tool for studying calcium signaling pathways. Its ability to both inhibit IP3 receptors and modulate TRP channels makes it valuable for investigating the roles of these proteins in various cellular processes. The compound is available from multiple research chemical suppliers and is used in a wide range of studies including neurobiology, immunology, and cardiovascular research. However, its multiple targets require careful interpretation of experimental results.

C14H16BNO

225.09394

225.132

524-95-8

1598

White to off-white solid powder

1.0±0.1 g/cm3

325.3±34.0 °C at 760 mmHg

192-194 °C(lit.)

150.6±25.7 °C

0.0±0.7 mmHg at 25°C

1.560

3.48

1

2

5

17

182

0

BLZVCIGGICSWIG-UHFFFAOYSA-N

InChI=1S/C14H16BNO/c16-11-12-17-15(13-7-3-1-4-8-13)14-9-5-2-6-10-14/h1-10H,11-12,16H2

2-diphenylboranyloxyethanamine

2-Aminoethyl diphenylborinate; 524-95-8; 2-APB; (2-Aminoethoxy)diphenylborane; 2-Aminoethoxydiphenylborane; 2-((Diphenylboryl)oxy)ethanamine; 2-Aminoethoxydiphenylborate; 2-aminoethoxydiphenyl borate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~1110.67 mM)
H2O : ~1.61 mg/mL (~7.15 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (9.24 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (9.24 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)