Deliberate quin2 overload as a method forIn Situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet

Abstract

The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9 mM and (ii) are exposed to 2 mM external Ca2+ and 1.0 microM ionomycin to rapidly achieve cytoplasmic Ca2+ ([Ca2+]cyt) of ca. 1.5 microM. (iii) The external Ca2+ is removed by EGTA addition, and (iv) the active Ca2+ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca2+ efflux process during phases iii-iv and that the extrusion process is sensitive to metabolic inhibitors. The progress curves for the decline of quin2 fluorescence (resulting from active Ca2+ extrusion) were analyzed as a function of [Ca2+]cyt using a mathematical model involving the probability that an exported Ca2+ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca2+]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of its Km, Vm and Hill coefficient (n)), (ii) the intrinsic Ca2+ buffer capacity of the cytoplasm (a function of the total site concentration ([B]T) and its Kd) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration and Kd). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca2+]cyt. The Ca2+ binding data were verified by 45Ca2+ experimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of Intracellular Calcium

“…However, the absorption coefficient and the quantum yield of quin 2 are low and hence, at concentrations needed to measure observable fluorescence, quin 2 buffers cellular Ca 2ϩ . Therefore, quin 2 has most recently been used not as a simple indicator for measuring [ (176,393,410,411). Quin 2 has also been used as an intracellular Ca 2ϩ chelator (66,123,153,433).…”

“…Hence, if the amount of increase in total Ca 2ϩ can be calculated, the endogenous Ca 2ϩ -buffering capacity can be estimated based on the assumption that Ca 2ϩ that enters the cell diffuses throughout the cell immediately (before the pump or sequestration mechanism work) (108,272,274,410). In addition, sufficiently loaded Ca 2ϩ indicators dominate the intrinsic Ca 2ϩ buffers and allow accurate quantification of Ca 2ϩ efflux across the plasma membrane (176,272,274).…”

Regulation of Platelet Plasma Membrane Ca2+-ATPase by cAMP-dependent and Tyrosine Phosphorylation

“…The appropriate fractions were pooled, applied to a precycled polybrene-coated glass fiber disc, and sequenced on an Applied Biosystems 477A sequencer with pulsed liquid phase chemistry and on-line PTH-derivative analysis. (19) showed that the Na-Ca 2ϩ exchanger makes a negligible contribution to platelet calcium efflux at resting calcium concentrations, thus implying that the main effect of cAMP on platelet Ca 2ϩ efflux is on PMCA. Consequently, we attempted to demonstrate that elevated cAMP leads to direct phosphorylation of PMCA in the platelet.…”

Evidence against a functional ATP‐dependent calcium extrusion mechanism in bovine epididymal sperm

“…Finally, we used a technique, recently reported, that enabled the detection of calcium pump activity in platelets (Johansson and Haynes, 1988). This consists of overloading cells with Quin 2, increasing intracellular calcium with ionomycin, and followed by measurement of the rate of calcium extrusion after chelation of external calcium.…”