title: Thapsigargin-induced calcium influx in the absence of detectable tyrosine phosphorylation in human platelets.
abstract: Tyrosine phosphorylation is a potential mechanism for mediating store-operated calcium (SOC) influx in platelets and other nonexcitable cells. Thapsigargin induces calcium-dependent tyrosine phosphorylation and SOC influx in platelets. We prevented thapsigargin-induced tyrosine phosphorylation by buffering cytosolic calcium rise with the calcium chelator 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetomethoxyester (BAPTA-AM). Calcium influx, induced by thapsigargin and measured by 45Ca2+ accumulation, persisted in BAPTA-loaded platelets in the absence of tyrosine phosphorylation. This calcium influx was blocked by the SOC influx inhibitor SKF-96365. Tyrosine kinase inhibitors have been used to demonstrate a role for tyrosine phosphorylation in SOC influx. We compared the effects of four tyrosine kinase inhibitors genistein, methyl-2, 5-dihydroxycinnamate (erbstatin analog), tyrphostin A47, and lavendustin A, on thapsigargin-induced tyrosine phosphorylation in control platelets and on thapsigargin-induced SOC influx into BAPTA-loaded platelets in absence of tyrosine phosphorylation. Tyrphostin A47 prevented all measurable tyrosine phosphorylation in control platelets, but did not decrease calcium influx into BAPTA-loaded platelets. Genistein and the erbstatin analog were poor inhibitors of tyrosine phosphorylation but decreased SOC influx into BAPTA-loaded platelets to 55.8 +/- 3% and 51.9 +/- 7.5% of control, respectively. Lavendustin A did not decrease tyrosine phosphorylation or calcium influx. Thus, thapsigargin-induced SOC influx can occur without detectable tyrosine phosphorylation and the inhibition of SOC influx by tyrosine kinase inhibitors does not correlate with their ability to prevent tyrosine phosphorylation.
authors.affiliation: Laboratory of Cellular Hematology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Vostal
authors.forename: B
authors.initials: B
authors.lastname: Shafer
mesh_terms: D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002614:Chelating Agents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D013729:Terpenes; D014443:Tyrosine; D019284:Thapsigargin; D011505:Protein-Tyrosine Kinases; D000252:Calcium-Transporting ATPases; D002118:Calcium