Abstract
In this study, we examined 2-aminoethoxydiphenyl borate (2APB) as an inhibitor of Ca2+ influx in human platelets. 2APB was found to inhibit thrombin-mediated intracellular Ca2+mobilization rapidly in platelets incubated in the absence of extracellular Ca2+. This result supports an intracellular action of 2APB on inositol 1,4,5-trisphosphate (IP3)-receptor Ca2+ channels. 2APB was without effect on the ability of thapsigargin to mobilize intracellular Ca2+. This result suggests that the efflux of Ca2+ from the endoplasmic reticulum mediated by thapsigargin is not via IP3 Ca2+ channels. However, 2APB was able to prevent the entry of Ca2+ and Sr2+ through thapsigargin-activated, store-operated Ca2+ channels (SOCC). This result supports a direct inhibitory effect of 2APB on SOCC. 2APB was also able to block the entry of Sr2+, Ba2+, and Mn2+ entry into unstimulated platelets, which suggests that 2APB was inhibiting the Ca2+ influx channels directly. The capacity of 2APB to prevent Ca2+ influx and Sr2+ influx was rapid because it occurred immediately upon addition to the platelets. The inhibition of Ca2+ and Sr2+ influx by 2APB was similar to that seen with the cell-impermeable nonselective Ca2+-channel blocker La3+ or the Ca2+ chelator EGTA. Diphenylboronic anhydride and 2,2-diphenyltetrahydrofuran, two compounds that are structurally similar to 2APB, also inhibited Ca2+ influx. It was concluded that 2APB was a rapid and effective direct inhibitor of SOCC in human platelets; as such, it cannot be used to support the involvement of IP3 receptors in the activation of SOCC.
Footnotes
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The laboratory of P.F.B. was supported by grants from the Jeffress Memorial Trust; the American Heart Association, VA affiliate; and the Virginia Academy of Science. Some of the data presented in this article were presented at Experimental Biology; 2001 March 31 to April 4; in Orlando, FL (abstract 718.6).
- Abbreviations:
- 2APB
- 2-aminoethoxydiphenyl borate
- IP3
- inositol 1,4,5-trisphosphate
- SOCC
- store-operated Ca2+ channels
- hTrp
- human transient receptor potential
- fura-2
- 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid
- ER
- endoplasmic reticulum
- AM
- acetoxymethyl ester
- SERCA
- smooth endoplasmic reticulum Ca2+ ATPase
- DPBA
- diphenylborinic anhydride
- IP3R
- inositol 1,4,5-trisphosphate receptor
- DPTTF
- 2,2-diphenyltetrahydrofuran
- Received December 12, 2000.
- Accepted May 21, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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