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Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual-wavelengths studies with Mn2+.

ABSTRACT: 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.

SUBMITTER: Sage SO

PROVIDER: S-EPMC1138454 | biostudies-other | 1989 Mar

REPOSITORIES: biostudies-other

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Project description:Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

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Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual-wavelengths studies with Mn2+.

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