Resting and ADP-evoked changes in cytosolic free sodium concentration in human platelets loaded with the indicator SBFI.
1. Cytosolic free Na+ concentration, [Na+]i, was investigated in human platelets loaded with the fluorescent indicator SBFI (sodium-binding benzofuran isophthalate). 2. SBFI fluorescence from platelet suspensions was measured at excitation wavelengths of 340 and 385 nm and the 340/385 nm fluorescence ratio was calibrated in terms of [Na+]i in situ. [Na+]i was set to known values by resuspending cells in media with various [Na+], in the presence of the Na(+)-K+ ionophore, gramicidin. 3. Basal free [Na+]i was 5.5 +/- 0.3 mM (n = 50). This is considerably lower than estimates of total platelet Na+, suggesting that much intracellular Na+ is sequestered or bound. 4. ADP (40 microM) evoked a rise in [Na+]i from 6.4 +/- 0.7 to 18.3 +/- 1.1 mM (n = 8). The ADP-evoked rise in [Na+]i was abolished when external Na+ was replaced with N-methyl-D-glucamine. This indicates that the rise in [Na+]i was due to Na+ entry. 5. In platelets loaded with the fluorescent pH indicator, BCECF, 40 microM-ADP was shown to evoke a fall in cytosolic pH (pHi) from 7.21 +/- 0.03 to 7.12 +/- 0.03 (n = 10). Three minutes after ADP addition pHi had only recovered to 7.15 +/- 0.03. The recovery was dependent on external Na+, suggesting it was mediated by Na(+)-H+ exchange. However, this would only account for an increase in [Na+]i of approximately 0.5 mM, indicating most of the ADP-evoked Na+ entry occurred by other mechanisms. 6. Stopped-flow fluorimetry showed that the ADP-evoked rise in [Na+]i commenced without measurable delay and peaked within 1 s. The initial kinetics were thus similar to those reported for ADP-evoked rises in [Ca2+]i. 7. Cell-attached patch-clamp recordings showed that ADP evoked single-channel inward currents when included in the pipette-filling solution. The currents were similar whether Ca2+ was present or absent from the pipette. The slope conductance was 11 pS in the presence of external Ca2+ and 10 pS in its absence. Current-voltage relationships were similar and the reversal potentials were close to 0 mV under both conditions. 8. SK & F 96,365 (20 microM), a blocker of receptor-mediated Ca2+ entry in several non-excitable cells, blocked the ADP-evoked rise in [Na+]i. This compound has been shown to only partly block the biphasic ADP-evoked rise in [Ca2+]i, being selective for the fast, receptor-operated phase of entry. 9. These data suggest that ADP rapidly activates a channel in that platelet plasma membrane which is permeable to Na+ and divalent cations.
Sage
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Physiological Laboratory, University of Cambridge.
Rink
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TJ
Mahaut-Smith
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D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001572:Benzofurans; D001792:Blood Platelets / Q000187:drug effects*; D003600:Cytosol / Q000378:metabolism; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D012964:Sodium / Q000097:blood*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; C061665:sodium-binding benzofuran isophthalate; D000244:Adenosine Diphosphate; D012964:Sodium