Structure-activity relationship studies of pyrogallol as a calcineurin/NFAT signaling suppressor.

Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC50 value of 82.6 µM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.

Pharmacology of Na+/Ca2+ exchanger.

KB-R7943 inhibits the Na(+)/Ca(2+) exchanger in an independent manner or in a manner dependent on the direction of the current. This effect may be due to the experimental protocols bawed on the competition between the drug and external substrate ions. Some antiarrhythmic drugs inhibit NCX. A new column of NCX was added in Sicilian Gambit.

Inhibitory effects of metals on ATP-induced current through P2X7 receptor in NG108-15 cells.

We investigated the effects of heavy metal ions on the ATP-induced nonselective cation current through P2X7 receptor (I(NS x P2X7)) in NG108-15 cells using the whole-cell patch-clamp technique. Cu2+ inhibited the I(NS x P2X7) most potently among the metal ions investigated. Other metals such as Ni2+, Cd2+, Zn2+ and Co2+ also inhibited the I(NS x P2X7) in concentration-dependent manners. The order of potency was Cu2+ >> Ni2+ > Cd2+ > Zn2+ > Co2+ with IC50 values of 16 nM, 0.79 microM, 1.2 microM, 3.0 microM and 4.6 microM, respectively. Fe3+ (10 and 100 microM) and Mn2+ (10 microM) did not affect the INS P2X7. A high concentration of Mn2+ (100 microM) slightly inhibited the I(NS x P2X7). When the concentration-response curve of ATP was obtained in the presence of 3 and 10 nM Cu2+, the maximal response but not the EC50 value appeared to be reduced, suggesting that the inhibition is not competitive. These results suggest that under physiological and toxicological conditions, metal ions, such as Cu2+, Ni2+, Cd2+, Zn2+ and Co2+, may modulate P2X7 receptor channels as inhibitors.

Characteristics of ATP-induced current through P2X7 receptor in NG108-15 cells: unique antagonist sensitivity and lack of pore formation.

ATP activates the mouse P2X7 receptor and induces a nonselective-cation current in NG108-15 cells. We investigated the effects of five receptor antagonists on the ATP-induced nonselective-cation current through P2X7 receptor (I(NS.P2X7)) in NG108-15 cells. Nonselective P2 receptor antagonists, RB-2, PPADS and suramin inhibited the I(NS.P2X7) with IC50 values of 4.3, 53 and 40 microM, respectively. However, KN-04, which is a potent antagonist of human P2X7 receptors but is not that of rat P2X7 receptors, had only a weak blocking effect. Furthermore, oxidized-ATP (300 microM), an antagonist of the P2X7 receptor-mediated pore-formation, did not affect the I(NS.P2X7). Prolonged ATP application did not increase the membrane permeability to large molecules, N-methyl-D-glucamine or Yo-Pro-1, indicating that pore-formation was not promoted by the P2X7 receptor activation in NG108-15 cells. These results suggest that antagonist sensitivities and pore-forming properties of the P2X7 receptors in NG108-15 cells are different from those of other cells types.

Effects of anions on ATP-induced [Ca2+], increase in NG108-15 cells.

We investigated the effects of anions on different P2 receptors by measuring ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded NG108-15 and PC12 cells. In NG108-15 cells, ATP at 100 microM and 1 mM induced a transient and a sustained [Ca2+]i increase, respectively. The former, but not the latter, was inhibited by U-73122, indicating that the former was via the P2Y2 receptor and the latter via the P2X7 receptor. When external Cl- was replaced by other anions, the [Ca2+]i increase mediated by the P2Y2 receptor was not changed, but that mediated by the P2X7 receptor varied in the order of aspartate- > methanesulfonate > Cl- > Br > or = I-. In PC12 cells, transient [Ca2+]i increases mediated by the P2Y2 and P2X2 receptors were not affected by various anions. These results suggest that modulation by anions is unique to the P2X7 receptor and does not occur in P2Y2 and P2X2 receptors. This may be because the mechanism of ATP binding to the P2X7 receptor may be different than that to other P2 receptors.

P2X receptor subtype-specific modulation of excitatory and inhibitory synaptic inputs in the rat brainstem.

The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole-cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPgammaS (100 microm) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 microm). alpha,beta-meATP also facilitated sEPSC and sIPSC frequency, while l-beta,gamma-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPgammaS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 microm) and the selective P2X(3) receptor antagonist A-317491 (3 microm), suggesting that modulation of sEPSCs involves P2X(3) receptor subunits. alpha,beta-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X(1) receptor-deficient mice demonstrating a functional role for P2X(1) receptors in the CNS.

Stoichiometry of Na+-Ca2+ exchange is 3:1 in guinea-pig ventricular myocytes.

In single guinea-pig ventricular myocytes, we examined the stoichiometry of Na(+)-Ca(2+) exchange (NCX) by measuring the reversal potential (E(NCX)) of NCX current (I(NCX)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) with the whole-cell voltage-clamp technique and confocal microscopy, respectively. With given ionic concentrations in the external and pipette solutions, the predicted E(NCX) were -73 and -11 mV at 3:1 and 4:1 stoichiometries, respectively. E(NCX) measured were -69 +/- 2 mV (n = 11), -47 +/- 1 mV (n = 14) and -15 +/- 1 mV (n = 15) at holding potentials (HP) of -73, -42 and -11 mV, respectively. Thus, E(NCX) almost coincided with HP, indicating that [Ca(2+)](i) and/or [Na(+)](i) changed due to I(NCX) flow. Shifts of E(NCX) (deltaE(NCX)) were measured by changing [Ca(2+)](o) or [Na(+)](o). The measured values of deltaE(NCX) were almost always smaller than those expected theoretically at a stoichiometry of either 3:1 or 4:1. Using indo-1 fluorescence, [Ca(2+)](i) measured under the whole-cell voltage-clamp supported a 3:1 but not 4:1 stoichiometry. To prevent Ca(2+) accumulation, we inhibited I(NCX) with Ni(2+) and re-examined E(NCX) during washing out Ni(2+). With HP at predicted E(NCX) at a 3:1 stoichiometry, E(NCX) developed was close to predicted E(NCX) and did not change with time. However, with HP at predicted E(NCX) for a 4:1 stoichiometry, E(NCX) developed initially near a predicted E(NCX) for a 3:1 stoichiometry and shifted toward E(NCX) for a 4:1 stoichiometry with time. We conclude that the stoichiometry of cardiac NCX is 3:1.