| Abstrakt: |
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. |
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