Figure 1

CA₂-2 at nanomolar concentrations induces a reversible [Ca²⁺]_i increase in macrophages by binding with purinergic receptors of the P2X family.

(a,b) Influence of CA₂-2 (0.02 μM) or ATP (30 μM) on duration and (c,d) amplitude of Ca²⁺ response in murine peritoneal macrophages, loaded with Ca²⁺-sensitive fluorescent probe Fura-2/AM. Arrows show the moment of addition of inductors to macrophages. (e) Pre-incubation with different calcium channel blockers and apyrase followed by Ca⁺² influx at application of CA₂-2: 1 control; 2 suramin, 100 μM; 3 PPADS, 10 μM; 4 PhR 10 μM; 5 Phph, 10 μM; 6 BBG 100 μM; 7 KN-62, 10 μM; apyrase, 2.0 U. (f) Effect of 30 min pre-incubation with receptor antibodies (ab81122, rabbit polyclonal to P2X1, Abcam,10 μg/mL, orb100036, rabbit polyclonal to P2X4, Biorbyt, 10 μg/mL) and normal rabbit immunoglobulin G (IgG, 10 μg/mL) on Са²⁺ influx into peritoneal macrophages (mice, Balb/с line), caused by CA₂-2 (100 nM); cells alone were used as a control. (g) Effect of transient knockdown approach, using P2X4 small interfering RNA, confirms a significant role for the P2X4 receptor in conferring CA₂-2 induced Ca²⁺ entry. (h) Expression of P2X4 mRNA in mouse macrophages and reduction of P2X4 mRNA in macrophages by P2X4 siRNA treatment in comparison with macrophages treated with scrambled RNA (PCR data). In addition, control reactions for loading were performed for each sample using β-actin specific primers generating the expected product. DNA molecular weight markers (M) are indicated in base pairs (bp). (i) chemical structure of CA₂-2. All data are presented as m ± sd, *p < 0.05.