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First published online 25 February 2003
doi: 10.1242/jcs.00368

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Evidence that Ca²⁺ cycling by the plasma membrane Ca²⁺-ATPase increases the `excitability' of the extracellular Ca²⁺-sensing receptor

¹ Dipartimento di Fisiologia Generale ed Ambientale, Universitá di Bari, Via Amendola 165/A, I-70126 Bari, Italy
² West Roxbury VAMC and the Department of Surgery, Harvard Medical School, West Roxbury, MA 02132, USA

View larger version (41K): [in a new window]   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).

View larger version (49K): [in a new window]   Fig. 2. (A) HEK CaR cells were stimulated with 1 mM spermine in the absence of extracellular Ca2+. Addition of 100 nM HgCl2 produced a rapid and reversible block of Ca2+ spiking. (B) Pretreatment with 100 nM HgCl2 did not stimulate HEK CaR cells by itself or prevent the release of Ca2+ from internal stores induced by 1 mM spermine. (C) Oscillations resulting from stimulation with carbachol (CCh; 100 µM) and ATP (100 µM) were not blocked by 100 nM HgCl2 in HEK CaR cells. These records also illustrate that Hg2+ is an effective blocker of the PMCA in HEK CaR cells, since the time to return to baseline following Ca2+ removal (in the presence of agonists) was significantly attenuated by HgCl2. (D) Ca2+ spiking was not abolished by 100 nM HgCl2 in HEK WT cells stimulated with 10 µM carbachol. (E) Spermine-induced oscillations were attenuated by 1 mM orthovanadate in HEK CaR cells. (F) Vanadate produced one of two effects on carbachol-stimulated oscillations in HEK WT cells. Shown here are cells in which 1 mM vanadate had little or no action on oscillations (42% of cells). (G) Vanadate (1 mM) sometimes produced an elevated plateau of Ca2+ when added acutely to carbachol-stimulated HEK WT cells (58% of cells). (H) 2 mM Caloxin 2A1, a peptide inhibitor of the PMCA, blocked oscillations elicited by NPS R-467 (5 µM).

View larger version (45K): [in a new window]   Fig. 3. (A) Top, confocal xy-section through center of a cluster of living HEK CaR cells incubated with an extracellular fluorescent marker (fluorescein-dextran; 10,000 MW) illustrating limited aqueous spaces between cells. Bottom, `side-view' profile (yz) reconstructed from stack of confocal z-sections (each 1 µm thick) through the region indicated by the line shown in the top image (line width=3 pixels or 1.5 µm). (B) Co-immunostaining of CaR and PMCA in HEK CaR cell clusters. Top left, Alexa 488 staining of CaR (red pseudocolor); middle, CY5 labeling of PMCA (green pseudocolor); top right (merged image), yellow color depicts areas of overlap. Bottom panels show control staining for Alexa 488 + CaR peptide block (see Materials and Methods for details) and Cy5 secondary antibody alone.

View larger version (58K): [in a new window]   Fig. 4. Measurement of extracellular near-membrane [Ca2+] with fura-C18. (A) Images a-c depict ratio images of fura-C18-loaded HEK CaR cells taken at times corresponding to points marked with arrows on the trace in panel B in four regions (indicated by black circles). Image d shows fluorescence at 340 nm excitation (510 nm emission) of the same fura-C18 loaded cells. (B) Fura-C18 ratio change of HEK CaR cells from regions shown in panel A in response to 1 mM spermine, followed by superfusion with 1 mM BAPTA and then 1 mM Ca2+. Inset shows enlarged time-scale of fura-C18 ratio from a selected region in the same experiment. The intracellular [Ca2+] signal as measured by fura-2 from a typical trace in a separate experiment is overlaid to illustrate the differences in time course of the two responses. (C) Example of an experiment in which extracellular [Ca2+] oscillations were detected following stimulation with 1 mM spermine in HEK CaR cells. (D) Significantly larger extracellular transients were detected in HEK CaR cells in response to stimulation with carbachol (CCh; 100 µM) and ATP (100 µM).

View larger version (74K): [in a new window]   Fig. 5. Model showing proposed mechanism by which the PMCA might provide positive feedback on CaR of the same cell or neighboring cells, reinforcing signaling via CaR.