The photoprotein aequorin (aeq) has been used as an indicator of intracellular calcium levels ([Ca]i) in platelets (pits). Aeq is believed to monitor aspects of [Ca]i somewhat different from those reported by quin-2, since it reports a higher basal [Ca]i than quin-2 and the presence, rather than the absence, of increased [Ca]i following epinephrine (epi) stimulation. To characterize further aeq-indicated pit Ca signals we measured their time dependence in both stimulated and untimulated pits. Aeq was loaded into pits by membrane permeabilization and resealing at 0°C. The maximum aeq signal (Lmax), obtained by Triton X-100 lysis of pits in the presence of 1 mM Ca, decreased over 40-60 min in a non-linear manner to about 60% of its initial value. Basal [Ca]i also decreased in a similar manner, even after correction for the changes in Lmax. The decreases in both responses were greater in pits isolated in the presence of 1 mM Ca than in the absence of Ca, indicating a sensitivity to external Ca. Epi (1-50 uM) induced an aeq signal in pits of most, but not all, subjects initially, but this response disappeared completely over 40-60 min in most subjects, without a concomitant loss of aggregation response. In contrast, the endoperoxide analog U44069 (10 uM) induced aeq signals in pits from all subjects, which decreased only slightly with time. These results suggest 1) that a portion of pit aeq may be localized in a region of high [Ca]i which may be partially accessible to external Ca, and this may account, in part, for the higher basal [Ca]i reported by aeq than by quin-2; and 2) that the epi-induced aeq signal, which is not prerequisite for aggregation, may be derived primarily from this labile aeq pool since the time courses for the decay of Lmax and basal [Ca]i and the loss of the epi-induced aeq signal are similar.
Methodology for correcting the nonlinear response of radionuclide calibrators
