Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations

Abstract

P2X7 receptor (P2X7R), a purinergic receptor, is involved in pathophysiological events such as inflammation and cell death, and thus is an attractive target for therapeutic approaches. It is reported that divalent metal cations (DMCs) inhibit P2X7R activation and that there are species differences in their inhibitory effects. To extrapolate the findings in experimental animals to humans, these species differences have to be clarified, but species differences in the sensitivity of P2X7R to DMCs between man and mouse have not been demonstrated. Here we performed direct comparison of the inhibitory effects of DMCs on human and mouse P2X7R activation. Cell lines constitutively expressing human and mouse P2X7R were used, and their P2X7R activation was evaluated as means of YO-PRO-1 dye uptake. MgCl 2 , NiCl 2 , ZnCl 2 , CuCl 2 and CaCl 2 dose-dependently decreased agonist-induced YO-PRO-1 uptake via both human and mouse P2X7Rs. Apparent differences in the inhibitory profiles for NiCl 2 and CaCl 2 between them were found, and the IC 50 values of DMCs were in the order of CaCl 2 >MgCl 2 >NiCl 2 ≈ZnCl 2 >CuCl 2 for both human and mouse P2X7Rs. In this study, we demonstrate that human P2X7R exhibits different sensitivity to nickel and calcium compared with the case of the mouse one, while there is no species difference in the sensitivity of their P2X7Rs to magnesium, zinc and copper, suggesting that the effects of magnesium, zinc and copper on P2X7R-associated pathophysiological events in humans might be predicted from those in mice.Key words P2X7 receptor; divalent metal cation; species difference; IC 50 value ATP is an energy currency, and the finding of purinergic receptors (P2Rs) revealed another role of it as a signaling molecule. For instance, ATP acts as a neuro-and glio-transmitter in the neuronal system, and as a regulator of inflammatory response in immune system. 1-3) P2Rs are classified into two groups, ionotropic P2XRs and metabotropic P2YRs.4,5) Of P2XRs, P2X7R has unique characteristics, and its affinity for ATP is extremely low and its prolonged activation leads to formation of channels/pores. 6,7) In neuronal cells, extensive activation of P2X7Rs causes the death of neurons, 8,9) and induces microglial activation 10) and astrocytic engulfing activity. 11,12) In immune cells, ATP-induced P2X7R activation triggers Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, followed by generation/ release of inflammatory cytokines.13-15) Kurashima et al. demonstrated that activation of P2X7R expressed by resident mast cells in the colon induces initiation of inflammation through the release of chemokines and cytokines, and exacerbation of inflammation through the recruitment of neutrophils, and robust release of cytokines in inflammatory bowel disease model mice and Crohn's disease patients.16) These findings strongly indicate that P2X7Rs are an attractive target for the development of therapeutic approaches for various diseases such as neurodegenerative disorders, inflammatory dise...

Prophylactic Oral Administration of Magnesium Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice through a Decrease of Colonic Accumulation of P2X7 Receptor-Expressing Mast Cells

“…In our previous study, calcium exhibited an inhibitory effect on P2X7R activation, and its inhibitory effect was the weakest among the DMCs examined, its IC 50 value being 2 or more mM. 23) Thus, the concentration of calcium in RDSW is low to inhibit P2X7R activation, but interaction of magnesium and calcium such as an additive/synergistic effect might explain the difference between the MgCl 2 solution and RDSW. This might be supported by the findings that nevertheless the colonic magnesium concentration in RDSW-ingested mice was approximately half of that in MgCl 2 -p.o.…”

“…administration and ad libitum ingestion of magnesium had preventive effects on the development of experimental colitis and that an underlying mechanism was inhibition of colonic mast cell activation via P2X7R. As there is no species difference in the inhibitory effect of magnesium between humans and mice, 23) this prophylactic effect of magnesium on colitis might be obtained in IBD patients. …”

“…Recently, we reported that there is no species difference in the sensitivity of P2X7R to magnesium between humans and mice. 23) Overall, it is suggested that daily ingestion of magnesium might have a prophylactic effect in IBD patients through inhibition of inflammatory conditions including P2X7R-induced mast cell activation.…”

“…administration, and has a relatively greater inhibitory ability as to P2X7R activation (the IC 50 =0.3-0.4 mM as MgCl 2 ), compared to the case of calcium (IC 50 value=2 or more mM as CaCl 2 ). 23) In addition, magnesium at concentrations found in vivo inhibited agonist-induced P2X7R activation in vitro experiments, 19,21) while the other P2XRs, except for P2X1R, are known to be potentiated or unaffected by DMCs. [24][25][26][27][28][29][30] Therefore, there is the possibility that DMCs might exhibit specific antagonism as to P2X7R without any effect on other P2XRs.…”

“…[19][20][21][22] Recently, we revealed that the inhibitory effects of DMCs were greater in the order of copper > zinc ≈ nickel ≈ magnesium > calcium for both human and mouse P2X7Rs, and that there was species differences in the effects of nickel and calcium, but not of the others, between humans and mice. 23) Of these DMCs, we have focused on magnesium as a blocker for P2X7R activation because magnesium oxide, which gives magnesium chloride by reacting with gastric acid in the stomach, is used as a medicine for gastritis/gastric ulcers and constipation, guaranteeing safety and colonic delivery of magnesium via p.o. administration, and has a relatively greater inhibitory ability as to P2X7R activation (the IC 50 =0.3-0.4 mM as MgCl 2 ), compared to the case of calcium (IC 50 value=2 or more mM as CaCl 2 ).…”