Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides.
In aspirin-treated platelets the thrombin-induced increase of cytosolic Ca2+ ([Ca2+]i) associated with the release from the intracellular stores is followed by a decrease to the baseline which is largely dependent on the re-uptake into the stores. This is shown by the further increase of [Ca2+]i upon inhibition of the endomembrane Ca(2+)-ATPase with thapsigargin. The re-uptake of Ca2+ into the stores is accelerated by sodium nitroprusside (SNP) or prostacyclin (PGI2). In all cases, after store depletion with thapsigargin the influx of external Ca2+ is maximal. After a thrombin-induced cycle of Ca(2+)-release re-uptake the stores are partly full: in these conditions the addition of external Ca2+ elicits a significant increment of [Ca2+]i and a further filling of the stores. Both are strongly reduced if Ca2+ addition is preceded by SNP or PGI2. Similar results are obtained also if (by supplementing and then cheleting Ca2+) the stores are as full as in native platelets at the moment of adding Ca2+. The thrombin-activated Ca2+ influx is reversed by hirudin. A PGI2- and SNP-sensitive Mn2+ influx is observed if Mn2+ is added in place of Ca2+. It is concluded that thrombin activates a cyclic nucleotide-sensitive Ca2+ (and Mn2+) influx pathway dependent on the occupancy of the thrombin receptor and independent of the filling state of the stores. In the absence of thrombin, thapsigargin releases Ca2+ relatively rapidly from a fraction of the stores; the remaining deposits are discharged much more slowly. This may indicate that platelets contain two distinct classes of agonist-sensitive stores. The addition of external Ca2+ (or Mn2+) at short or long incubation times with thapsigargin monitors the influx of Ca2+ activated by the depletion of one or both types of stores. The depletion of each type of store activates Ca2+ (Mn2+) influx. This type of cation influx is not inhibited by the cyclic nucleotides.
Doni
M G
MG
Department of Biological Chemistry, University of Padova, Italy.
Cavallini
L
L
Alexandre
A
A
D001241:Aspirin / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004347:Drug Interactions; D004533:Egtazic Acid / Q000494:pharmacology; D011464:Epoprostenol / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D009712:Nucleotides, Cyclic / Q000037:antagonists & inhibitors / Q000502:physiology*; D015539:Platelet Activation / Q000187:drug effects*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology*
D006629:Hirudins; D009712:Nucleotides, Cyclic; D013729:Terpenes; D009599:Nitroprusside; D008345:Manganese; D004533:Egtazic Acid; D015759:Ionomycin; D019284:Thapsigargin; D011464:Epoprostenol; D013917:Thrombin; D000252:Calcium-Transporting ATPases; D001241:Aspirin; D002118:Calcium