Fig. 1. (A) HEK CaR cells can oscillate for up to 10 minutes in the absence of external Ca²⁺ (10-20 µM BAPTA free acid in bath) following stimulation with the CaR agonist spermine (1 mM). Traces from several representative cells are shown. Time bar indicates three minutes. (B) Increasing the BAPTA concentration from 10 µM to 1 mM reversibly interrupts cytosolic Ca²⁺ signals (oscillations) stimulated by 1 mM spermine. (C) Comparison of oscillations induced by 1 mM spermine in nominally Ca²⁺-free Ringer's containing 10 µM BAPTA vs a solution containing 1 mM BAPTA + 0.49 mM CaCl₂. The free Ca²⁺ in the 10 µM BAPTA solution (approx. 150 nM) was directly measured to be slightly lower than that in the 1 mM BAPTA/0.49 mM CaCl₂ solution (approx. 200 nM; see Materials and Methods for details). (D) Oscillations elicited by spermine (1 mM) in the presence of 1 mM Ca²⁺ were attenuated in a buffer containing 20 mM citrate (added to buffer extracellular Ca²⁺ changes) plus approx. 10 mM CaCl₂. Free Ca²⁺ was the same in control and buffered solutions, and was titrated using a calcium-selective electrode as described in Materials and Methods. (E) Frequency and amplitude of oscillatory signals elicited by spermine in the presence of 0.65 mM added Ca²⁺ were reversibly altered by citrate-buffered solution. As in panel D, the solution containing 20 mM citrate plus approx. 8 mM CaCl₂, contained the same free [Ca²⁺] as the control solution, matched using a Ca²⁺ electrode. (F) Oscillations in HEK CaR cells stimulated by 100 µM carbachol (CCh) and 100 µM ATP were not affected by the same citrate buffer used in Fig. 1E. (G) Plot of Ca²⁺ buffering capacity of citrate solution used in Fig. 1D. Top trace, unbuffered (control) solution showing free [Ca²⁺] change as measured with a Ca²⁺-electrode with the addition of 600 µM increments of added CaCl₂; bottom trace, parallel measurement with citrate-containing solution. The buffering characteristics of the solutions used in Fig. 1E containing 0.65 mM Ca²⁺ and 20 mM citrate plus CaCl₂ were similar (not shown).

Return to article