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Sci Rep
2016 Dec 22;6:39683. doi: 10.1038/srep39683.
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Glycosides from edible sea cucumbers stimulate macrophages via purinergic receptors.
Aminin D
,
Pislyagin E
,
Astashev M
,
Es'kov A
,
Kozhemyako V
,
Avilov S
,
Zelepuga E
,
Yurchenko E
,
Kaluzhskiy L
,
Kozlovskaya E
,
Ivanov A
,
Stonik V
.
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Since ancient times, edible sea cucumbers have been considered a jewel of the seabed and used in Asian folk medicine for stimulation of resistance against different diseases. However, the power of this sea food has not been established on a molecular level. A particular group of triterpene glycosides was found to be characteristic metabolites of the animals, responsible for this biological action. Using one of them, cucumarioside A2-2 (CA2-2) from the edible Cucumaria japonica species as an example as well as inhibitory analysis, patch-clamp on single macrophages, small interfering RNA technique, immunoblotting, SPR analysis, computer modeling and other methods, we demonstrate low doses of CA2-2 specifically to interact with P2X receptors (predominantly P2X4) on membranes of mature macrophages, enhancing the reversible ATP-dependent Ca2+ intake and recovering Ca2+ transport at inactivation of these receptors. As result, interaction of glycosides of this type with P2X receptors leads to activation of cellular immunity.
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/Ñ 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.
Figure 2. P2X receptors, the main molecular targets of CA2-2, are localized on membranes of large granular macrophages (mature macrophages).(a) Percentage of F4/80+ cells in a total fraction of peritoneal BALB/c mouse macrophages (mature macrophages) determined by flow cytometry (1, 2) and confocal microscopy (3). Mature macrophages were dyed green on a dotogram (2) and in the confocal representation (3). (b) Localization of P2X receptors on the surface of peritoneal macrophages from BALB/c mice by the immunocytochemical method followed by confocal microscopy. Secondary antibodies were conjugated with TRITC (red staining). (c) Colocalization of Ð 2Ð¥+ and F4/80+ cells in a population of peritoneal mouse macrophages, indicated by flow cytometry (1, 3) and confocal microscopy (2, 4). Immunochemical staining of the cells using antibodies to F4/80 and FITC (green staining) and to Ð 2Ð¥1, P2X4, and TRITC (red staining).
Figure 3. CA2-2 increases the Ca2+ current in single macrophages as a result of action on P2X family receptors and partly recovers the inactivation of P2X receptors caused by repeated application of ATP.(a), Patch clamp in configuration of “whole-cellâ€. Registration of Са2+ current in single macrophage at application of CA2-2 (100 nM) before and after pre-treatment with BBG (100 μM). (b) Registration of Са2+ current evoked by ATP, and ATP after pre-treatment with CA2-2. Left, membrane potential −40 mV, ATP, 0.03 μМ, 2 sec, on the right, ATP, 0.03 μМ, 2 sec after 2 min pre-incubation with CA2-2, 100 nM, below a concentration–response relationship for ATP, EC50 = 0.16 μМ. (c) The clearing of inactivation of single macrophage by CÐ2-2, 1-3 – consequent answers of the cell to action of repeated portions of 0.3 μМ of ATP, and 4 – after 0.3 μМ ATP + 100 nM CA2-2 with 2 minutes headway. Cells that were not pretreated with apyrase (a) and pretreated with apyrase (b,c).
Figure 4. The interaction of CA2-2 with P2X4 receptor.(a) (side view), (b) (top view), The modeling of the mP2X4 receptor (30-354 amino acid residues) with ATP and CA2-2 interaction is shown. Membrane surface is grey, different subunits of the receptor are red, blue and grey, ATP is presented as sphere packing structure, CA2-2 as ball-and-stick diagram by magenta with oxygen atoms marked by red, hydrogen atoms marked by white and sulfur atoms marked by yellow. Binding sites of ATP and CA2-2 are shown by arrows. (c,d) Fragments of mP2X4 receptors, containing β-sheets with localized ATP and CA2-2 molecules in initial and modified by CA2-2 states, respectively. ATP is shown by ball-and-sticks, CA2-2 by magenta sticks, amino acids residues, interacting with the ligands by grey and blue in dependence on the belonging to some or other receptor. Green arrow indicates the changes in structure of mP2X4 receptor after interaction with CA2-2. (e) Sensograms of CA2-2 (100-150-250 and 400 μM) interaction with immobilized Р2Х4 receptor. The arrows indicate start of the CA2-2 injections and the end of CA2-2 injections, and start of complex dissociation during washing with the running buffer. (f), P2X4 protein expression in mouse peritoneal macrophages in control cells and after stimulation of mouse peritoneal macrophages with CA2-2 (10 nM) for 48 h. Western blot of total P2X4 receptors and β-actin from macrophages are shown.
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