title: Inhibition of Ca2+ entry by Ca2+ overloading of intracellular Ca2+ stores in human platelets.
abstract: 1. This study examined the effect of overloading human platelet intracellular Ca2+ stores on the rate of agonist-evoked external Ca2+ entry. To overload the Ca2+ stores (presumably dense tubules), external Na(+)-dependent Ca2+ efflux via Na(+)-Ca2+ exchange was inhibited by pretreating the cells with ouabain or Na(+)-free medium. Ca2+ regulation was then examined after exposure to thrombin, ADP and thapsigargin. Cytosolic free Ca2+ levels were monitored using the fluorescent probe fura-2 and external Ca2+ influx was assessed by the rates of extracellular Mn2+ or 45Ca2+ uptake. 2. Both ouabain and Na(+)-free pretreatments caused a slight increase in the resting cytosolic free Ca2+. 3. In 1 mM Ca(2+)-containing medium, Ca(2+)-overloaded platelets showed similar thrombin-evoked cytosolic free Ca2+ responses to those of control platelets. However, in Ca(2+)-free medium, they showed substantially greater thrombin-evoked cytosolic free Ca2+ responses than control platelets. Moreover, increased thrombin-evoked Ca2+ mobilization from Ca2+ storage sites was accompanied by a diminished rate of thrombin-evoked external Ca2+ entry. 4. Similar reductions in the rate of external Ca2+ entry were observed after treatment with ADP and thapsigargin. 5. Protein kinase C inhibitors (calphostin C and staurosporine) failed to reverse the effect of ouabain pretreatment on thrombin-induced changes in the cytosolic free Ca2+ response. 6. Inositol 1,4,5-trisphosphate profiles in ouabain-treated and non-treated platelets were not significantly different. 7. These data indicate that increased Ca2+ in the dense tubules is associated with diminished agonist-evoked external Ca2+ influx.
authors.affiliation: Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103-2714.
authors.forename: M
authors.initials: M
authors.lastname: Kimura
authors.forename: J H
authors.initials: JH
authors.lastname: Cho
authors.forename: J P
authors.initials: JP
authors.lastname: Reeves
authors.forename: A
authors.initials: A
authors.lastname: Aviv
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000032:analysis* / Q000378:metabolism / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000502:physiology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D006224:Cricetinae; D016257:Fura-2; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D009281:Naphthalenes; D010042:Ouabain / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012964:Sodium / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger; D019311:Staurosporine; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D015220:Calcium Channels; D002352:Carrier Proteins; D009281:Naphthalenes; D011083:Polycyclic Compounds; D019831:Sodium-Calcium Exchanger; D013729:Terpenes; D010042:Ouabain; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D012964:Sodium; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; C058819:calphostin C; D002118:Calcium; D016257:Fura-2