Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual-wavelengths studies with Mn2+.
Previous studies of the early kinetics of rises in cytosolic free [Ca2+] in fura-2-loaded human platelets suggested that: (1) Ca2+ entry slightly preceded internal discharge with thrombin and other agonists known to promote inositol lipid hydrolysis; (2) with ADP, Ca2+ entry occurred without measurable delay and clearly preceded internal Ca2+ discharge. In the present work, Mn2+ added to the external medium was used as a marker for Ca2+ entry. By using an excitation wavelength of 360 nm, a quench of fura-2 can be followed to report Mn2+ entry without 'contamination' of the signal by changes in [Ca2+], because at this isosbestic wavelength Ca2+ does not alter fura-2 fluorescence. The present results show that, with thrombin stimulation, readily discernible Mn2+ entry starts after discharge of internal Ca2+ and is maintained for many minutes. With ADP, Mn2+ entry starts without measurable delay (less than 20 ms) and clearly precedes internal Ca2+ discharge. However, the enhanced Mn2+ permeability is only short-lived. These results, considered alongside previous data, point to the possible presence of at least three different receptor-mediated Ca2+-entry mechanisms in human platelets, one of which may include regulation by the 'state of filling' of this dischargeable Ca2+ store.
Sage
S O
SO
Physiological Laboratory, University of Cambridge, U.K.
Merritt
J E
JE
Hallam
T J
TJ
Rink
T J
TJ
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001572:Benzofurans; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D008345:Manganese / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*
D001572:Benzofurans; D005456:Fluorescent Dyes; D008345:Manganese; D000244:Adenosine Diphosphate; D013917:Thrombin; D002118:Calcium; D016257:Fura-2