[6]-gingerol and [6]-shogaol, active ingredients of the traditional Japanese medicine hangeshashinto, relief oral ulcerative mucositis-induced pain via action on Na+ channels.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch-clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30min after the swab application of HST despite the absence of anti-bacterial and anti-inflammatory actions in the OUM area. A swab application of a mixture of [6]-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUM-induced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain.

Mutation D384N alters recovery of the immobilized gating charge in rat brain IIA sodium channels.

Rat brain (rBIIA) sodium channel fast inactivation kinetics and the time course of recovery of the immobilized gating charge were compared for wild type (WT) and the pore mutant D384N heterologously expressed in Xenopus oocytes with or without the accessory beta1-subunit. In the absence of the beta1-subunit, WT and D384N showed characteristic bimodal inactivation kinetics, but with the fast gating mode significantly more pronounced in D384N. Both, for WT and D384N, coexpression of the beta1-subunit further shifted the time course of inactivation to the fast gating mode. However, the recovery of the immobilized gating charge (Qg) of D384N was clearly faster than in WT, irrespective of the presence of the beta1-subunit. This was also reflected by the kinetics of the slow Ig OFF tail. On the other hand, the voltage dependence of the Qg-recovery was not changed by the mutation. These data suggest a direct interaction between the selectivity filter and the immobilized voltage sensor S4D4 of rBIIA sodium channels.

Mechanisms of aldosterone's action on epithelial Na + transport.

Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.

Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes.

The epithelial sodium channel (ENaC) provides the rate-limiting step in the reabsorption of sodium by many epithelia. The number of channels at the cell surface is tightly regulated; most cells express only a few channels. We have examined the biosynthesis and cell surface expression of ENaC in Xenopus oocytes. The subunits of ENaC are readily synthesized in the endoplasmic reticulum, but most of them remain as immature proteins in pre-Golgi compartments, where they are degraded by the proteasomal pathway without apparent ubiquitination. Even when the three subunits, alpha, beta, and gamma, are expressed in the same cell, only a very small fraction of the total channel population leave the endoplasmic reticulum, acquire complex oligosaccharides, and reach the plasma membrane. Overexpression of subunits does not increase the number of channels in the plasma membrane but results in the appearance of cytoplasmic subunits in a form not membrane bound. The data indicate that maturation and assembly of the subunits are slow and inefficient processes, and constitute limiting steps for the expression of functional ENaC channels in the plasma membrane.

Disruption of cell volume regulation by mercuric chloride is mediated by an increase in sodium permeability and inhibition of an osmolyte channel in skate hepatocytes.

The mechanism by which mercury leads to cell swelling and impairs the normal regulatory volume decrease (RVD) in cells swollen in hypotonic media was examined in hepatocytes isolated from the little skate, Raja erinacea, an osmoconforming marine elasmobranch. Skate hepatocytes treated with 50 microM HgCl2 in isotonic medium swelled to volumes double those of control cells, and this was associated with an increase in Na+ and K+ permeability. The gain in intracellular Na+ exceeded the K+ loss by 0.27 microEq/mg protein, accounting in large part for the observed cell swelling. The effects of mercury were blunted when hepatocytes were incubated in medium in which the Na+ was replaced with K+, and were essentially absent when Na+ was replaced with choline+, indicating an important role of Na+ influx in mediating mercury's effects on cell volume regulation. The inhibition of RVD by mercury was prevented if the metal was administered as a mercaptide with dithiothreitol or glutathione. However, when these chelating agents were added after the mercury, only the membrane permeant dithiothreitol was able to reverse the inhibition of RVD, suggesting an intracellular site of action. Mercuric chloride also produced a concentration-dependent inhibition of the ATP-sensitive volume-regulatory osmolyte channel in skate hepatocytes, as assessed by inhibition of swelling-activated [14C]taurine efflux. [14C]Taurine efflux was inhibited at mercury concentrations (20-40 microM) that had no effect on intracellular ATP levels or ATP/ADP ratios, consistent with a direct interaction with the channel. These findings indicate that mercury impairs cell volume regulation in skate hepatocytes at multiple sites, including the volume-regulatory osmolyte channels, and Na+ and K+ permeability pathways. The combined effects of increased Na+ influx and the inability to extrude organic osmolytes may account for the inhibition of RVD.