The 2-aminoethoxydiphenyl borate analog, DPB161 blocks store-operated Ca2+ entry in acutely dissociated rat submandibular cells

Abstract

Cellular Ca²⁺ signals play a critical role in cell physiology and pathology. In most non-excitable cells, store-operated Ca²⁺ entry (SOCE) is an important mechanism by which intracellular Ca²⁺ signaling is regulated. However, few drugs can selectively modulate SOCE. 2-Aminoethoxydiphenyl borate (2APB) and its analogs (DPB162 and DPB163) have been reported to inhibit SOCE. Here, we examined the effects of another 2-APB analog, DPB161 on SOCE in acutely-isolated rat submandibular cells. Both patch-clamp recordings and Ca²⁺ imaging showed that upon removal of extracellular Ca²⁺ ([Ca²⁺]_o=0), rat submandibular cells were unable to maintain ACh-induced Ca²⁺ oscillations, but restoration of [Ca²⁺]_o to refill Ca²⁺ stores enable recovery of these Ca²⁺ oscillations. However, addition of 50 μM DPB161 with [Ca²⁺]_o to extracellular solution prevented the refilling of Ca²⁺ store. Fura-2 Ca²⁺ imaging showed that DPB161 inhibited SOCE in a concentration-dependent manner. After depleting Ca²⁺ stores by thapsigargin treatment, bath perfusion of 1 mM Ca²⁺ induced [Ca²⁺]_i elevation in a manner that was prevented by DPB161. Collectively, these results show that the 2-APB analog DPB161 blocks SOCE in rat submandibular cells, suggesting that this compound can be developed as a pharmacological tool for the study of SOCE function and as a new therapeutic agent for treating SOCE-associated disorders.

Keywords: 2-aminoethoxydiphenyl borate; Ca2+ oscillations; DPB161; rat submandibular cells; store-operated Ca2+ entry (SOCE).

Figure 1. Chemical structure of DPB-161

Figure 2. Comparison of ACh-induced Ca2+ oscillations between pancreatic acinar cells and submandibular cells using patch-clamp recordings

Under [Ca²⁺]_o = 0 condition, ACh (20 nM) induced a long-lasting Ca²⁺ oscillatory response in mouse pancreatic acinar cells (A) but induced a short-lasting Ca²⁺ oscillatory response in rat submandibular cells (B). Typical traces from 6 cells tested are presented in A (mouse pancreatic acinar cells) and B (rat submandibular cells), respectively.

Figure 3. Effects of DPB-161 on [Ca²⁺]_i responses to ACh measured by patch-clamp recordings in rat submandibular cells

(A) Under [Ca²⁺]_o = 0 conditions, bath-application of 20 nM ACh induced Ca²⁺oscillatory response. Bath-application of 1 mM Ca²⁺ refills the Ca²⁺stores through opened SOC channels. (B) Using the same experimental protocol but refilling Ca²⁺ stores with 1 mM Ca²⁺ and 50 μM DBP161 prevented the refilling of Ca²⁺ stores, suggesting that DPB161 blocks SOCE. Typical traces in A and B are presented from 6 submandibular cells tested, respectively.

Figure 4. Effects of DPB-161 on [Ca²⁺]_i responses to ACh measured by Ca²⁺ imaging in rat submandibular cells

(A) In the presence of extracellular Ca²⁺, the cell was stimulated with 1 μM ACh (4 min). (B) ACh was applied to the cell in a Ca²⁺-free solution. (C) Before (1 min) and during stimulation with ACh in the Ca²⁺-free solution, 50 μM DPB-161 was applied. (D) Before (1 min) and during stimulation with ACh in the Ca²⁺-containing solution, 50 μM DPB-161 was applied. The representative trace of 11-16 experiments is shown in each panel.

Figure 5. Effects of DPB-161 on Ca²⁺ store refilling following ACh-induced Ca²⁺ store depletion in rat submandibular cells

(A) In Ca²⁺-free solution, the cell was stimulated by 1 μM ACh (4 min). After the first challenge of ACh, a second ACh challenge (4 min) was performed. (B) Between the two ACh challenges, Ca²⁺-containing solution was applied for 2 min. (C) Before (1 min) and during addition of extracellular Ca²⁺, 5 μM DPB-161 was applied. (D) Before (1 min) and during addition of extracellular Ca²⁺, 50 μM DPB-161 was applied. The representative trace of 5-19 experiments is shown in each panel. (E) Concentration-response relationships for DPB-161 inhibition of store-operated Ca²⁺ entry. Relative sizes of the second ACh responses to the first ones are shown, measured by the area under the curve. Values are means of 5, 8, 8, and 15 cells tested for 0, 5, 20 and 50 μM DPB-161, respectively. Vertical bars indicate SE. Statistical significance was obtained by one-way ANOVA. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 6. Effects of DPB-161 on the Ca²⁺ entry following thapsigargin-induced Ca²⁺ store depletion in rat submandibular gland acinar cells

(A) In Ca²⁺-free solution, 1 μM thapsigargin (TG) was first applied (4 min), followed by 1 μM ACh. (B) After TG treatment (4 min), the Ca²⁺-containing solution was applied (2 min). (C) Before (1 min) and during addition of extracellular Ca²⁺, 5 μM DPB-161 was applied. (D) Before (1 min) and during addition of extracellular Ca²⁺, 50 μM DPB-161 was applied. (E) Statistical analysis of 14-21 experiments are shown in panels B, C, D.