Aluminum is a weak agonist for the calcium-sensing receptor Robert F. Spurney, Min Pi, Patrick Flannery, L. Darryl Quarles  Kidney International  Volume 55, Issue 5, Pages 1750-1758 (May 1999) DOI: 10.1046/j.1523-1755.1999.00432.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Western blot analysis of calcium sensing receptor (CaSR) expression in human embryonic kidney (HEK) 293 cells. Expression of rat CaSR was assessed by Western blotting, as described in the Methods section, in human parathyroid tissue (lane 1), in HEK 293 cells stably transfected with the CaSR cDNA (lane 2), and in nontransfected HEK 293 cells (lane 3). We detected three major bands of apparent molecular mass of 140, 165, and 250kDa in membrane protein preparations from HEK 293 transfected with CaSR cDNA. These bands were not observed in nontransfected HEK 293 cells. The 250 and 165kDa bands predominated in human parathyroid tissue. Kidney International 1999 55, 1750-1758DOI: (10.1046/j.1523-1755.1999.00432.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Effects of extracellular calcium on [Ca2+]i levels in HEK 293 cells. Calcium-induced activation of rat CaSR was assessed by monitoring [Ca2+]i levels using fura 2-loaded cells as described in the Methods section. (A) Results for HEK 293 cells stably expressing the CaSR. (B) Results in nontransfected HEK 293 cells. Extracellular calcium concentrations above 2mm were required to elicit an increase in [Ca2+]i levels in cells transfected with CaSR. A maximal response was obtained at extracellular calcium concentrations of 10mm. In contrast, increasing the extracellular calcium concentration did not enhance [Ca2+]i levels in nontransfected cells, although the addition of the calcium ionophore ionomycin caused a rapid increase in [Ca2+]i levels. Results are representative of three to six separate experiments. Kidney International 1999 55, 1750-1758DOI: (10.1046/j.1523-1755.1999.00432.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Effects of CaSR agonists on [Ca2+]i levels in HEK 293 transfected with rat CaSR. The ability of CaSR agonists to activate rat CaSR was assessed by monitoring [Ca2+]i levels following the addition of gadolinium (A), neomycin (B), or magnesium (C) to the extracellular fluid using fura 2-loaded cells, as described in the Methods section. Additions of gadolinium, neomycin, and magnesium at the indicated concentrations caused similar increments in [Ca2+]i levels in HEK 293 cells transfected with rat CaSR. Results are representative of four to six separate experiments per compound. Kidney International 1999 55, 1750-1758DOI: (10.1046/j.1523-1755.1999.00432.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Effects of cations on inositol monophosphate (IP1) generation in HEK 293 cells transfected with rat CaSR. IP1 generation was measured as described in the Methods section 30minutes after the addition of calcium (A) or aluminum (B) to the extracellular fluid. PI hydrolysis was enhanced, in a dose-dependent fashion, by additions of calcium between 3 and 20mm. Aluminum also caused a modest increase in IP1 generation at the 0.5 and 1mm concentrations. No significant increase in IP1 generation was detected at the 10, 25, or 100 μm concentrations of aluminum. Values are the mean ± sem of three separate determinations. The asterisk indicates a significant difference at P < 0.05 vs. 2mm calcium (A) or vs. 10 μm aluminum (B). Kidney International 1999 55, 1750-1758DOI: (10.1046/j.1523-1755.1999.00432.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Comparison of aluminum and gadolinium effects on [Ca2+]i levels in HEK 293 cells transfected with rat CaSR. The ability of aluminum (A) and gadolinium (B) to increase [Ca2+]i was assessed by monitoring [Ca2+]i levels following the addition of the trivalent cations to the extracellular fluid using fura 2-loaded cells as described in the Methods section. Additions of 10 or 100 μm aluminum did not cause an increase in [Ca2+]i levels. At 1mm aluminum, a small increment in [Ca2+]i was observed that did not prevent the subsequent activation of CaSR by extracellular calcium. In contrast, the addition of 50 μm gadolinium to the extracellular fluid caused an increase in [Ca2+]i levels and blocked subsequent calcium-induced increases in [Ca2+]i levels. Results are representative of four to six separate experiments per compound. Kidney International 1999 55, 1750-1758DOI: (10.1046/j.1523-1755.1999.00432.x) Copyright © 1999 International Society of Nephrology Terms and Conditions