Mesenchymal stem cells and immunomodulation: current status and future prospects.

The unique immunomodulatory properties of mesenchymal stem cells (MSCs) make them an invaluable cell type for the repair of tissue/ organ damage caused by chronic inflammation or autoimmune disorders. Although they hold great promise in the treatment of immune disorders such as graft versus host disease (GvHD) and allergic disorders, there remain many challenges to overcome before their widespread clinical application. An understanding of the biological properties of MSCs will clarify the mechanisms of MSC-based transplantation for immunomodulation. In this review, we summarize the preclinical and clinical studies of MSCs from different adult tissues, discuss the current hurdles to their use and propose the future development of pluripotent stem cell-derived MSCs as an approach to immunomodulation therapy.

Effects of metformin on survival outcomes of lung cancer patients with type 2 diabetes mellitus: a meta-analysis.

BACKGROUND: Anti-cancer effect of metformin on different kinds of lung cancer has been studied frequently. However, the association between metformin and the prognosis of lung cancer in type 2 diabetes patients is still controversial. METHODS: An electronic search was conducted using PubMed/Medicine, EMBASE and Cochrane library databases. Statistical analyses were carried out using either random-effects or fixed-effects models according to the heterogeneity examined by I (2) statistics. RESULTS: Six studies involving 2350 patients were included in the current meta-analysis. In all, the pooled HR of overall survival (OS) was 0.90 (95 % CI 0.84-0.96; P = 0.003). Sub-group analysis showed that when stratified by region the HR of OS was 0.52 (95 % CI 0.31-0.87; P = 0.012) and 0.86 (95 % CI 0.67-1.11, P = 0.361) for Asian and Western countries. When stratified by study design, the HR of OS was 0.78 (95 % CI 0.52-1.15, P = 0.206) and 0.82 (95 % CI 0.59-1.16, P = 0.264) for cohort and medical data-based studies. When stratified by lung cancer subtype, HR of OS was 0.52 (95 % CI 0.31-0.87; P = 0.012), 1.06 (95 % CI 0.51-2.19; P = 0.878) and 0.82 (95 % CI 0.59-1.16; P = 0.264) for SCLS, NSCLC and non-divided subtypes, respectively. CONCLUSION: Metformin use may associate with a good prognosis in lung cancer patients with type 2 diabetes but the effect was modest. However, it could achieve benefits in a selective sub-group of lung cancer patients especially in SCLC patients from Asian. Further studies are warranted to confirm this efficacy.

Estrogen regulates the expression and activity of epithelial sodium channel in mouse osteoblasts.

Estrogen plays an important role in bone metabolism and only high dose can stimulate osteoblast bone formation. However, the underlying mechanisms have not been elucidated. The epithelial sodium channel (ENaC) is a key pathway for sodium transport in epithelia, vascular endothelium, and other tissues; although the expressions of α and γ ENaC mRNA were found in osteoblasts, the regulation of ENaC by estrogen in osteoblasts has not been studied. Our recent data confirmed the ENaC expression in mouse primary osteoblasts by immunocytofluorescence, RT-PCR, western blot, and patch clamp. Furthermore, we found estrogen (10(-5)M) increased the expression of α and γ ENaC subunits at both the mRNA and protein levels in osteoblasts. On the other hand, 17ß estradiol (20 nM) increased inward Na+ currents which were inhibited by amiloride. The estrogen dose used in patch clamp is much lower than those of mRNA and protein analysis, which means single cell ENaC electrophoretic mobility is much more sensitive to estrogen than the mRNA and protein production by estrogen stimulation. Our results suggest that estrogen regulates expression and function of ENaC in osteoblasts may provide a new clue that the mechanism of high dose of estrogen influence osteoblast bone formation via ENaC activity.

Surface nanomechanical behavior of ZrN and ZrCN films deposited on NiTi shape memory alloy by magnetron sputtering.

Surface nanomechanical behavior under nanoindentation of ZrN and ZrCN film on NiTi substrate was studied. The surface hardness and modulus of the films increase initially with larger nanoindentation depths and then reach their maximum values. Afterwards, they diminish gradually and finally reaching plateau values which are the composite modulus and composite hardness derived from the ZrN/ZrCN film and NiTi substrate. They are higher than those of electropolished NiTi SMA due to the properties of ZrN and ZrCN. In comparison, the surface nanomechanical properties of electropolished NiTi exhibit a different change with depths.

Thermal diversities of two Na+/H+ exchanges in guinea pig red cells.

OBJECTIVE: To test the effect of hypothermia on Na+/H+ exchange, activated by shrinkage and cytoplasmic acidosis. METHOD: Amiloride-sensitive Na+ influx in guinea pig red cells was traced with isotope 22Na and intracellular Na+ concentration was measured by emission flame photometry. RESULT: Amiloride-sensitive Na+ influx decreased linearly as a function of temperatures (about 37 degrees C) in shrunken cells, but increased in acidified cells. The up-regulation of acid-induced Na+/H+ exchange by elevated temperature was enhanced by hypo-osmolarity. Less sensitivity of intracellular H+ site at 41 degrees C may be the mechanism for the inhibition of shrinkage-induced Na+/H+ exchange by elevated temperature. Heating-mediated explosive increase in the activity of acid-induced Na+/H+ exchange may be due to enhanced extracellular Na+ sensitivity and lower intracellular pH caused by acidic metabolites. Acid-induced Na+/H+ exchange contributes to cytoplasmic Na+ accumulation. CONCLUSION: These two modes of Na+/H+ exchange with different response to elevated temperature may play different roles in the cellular pathogenesis of heatstroke.

A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.

1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

Regions in the carboxy terminus of alpha-bENaC involved in gating and functional effects of actin.

Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.

Ca(2+)-activated Cl(-) channels: a newly emerging anion transport family.

A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca(2+)-sensitive Cl(-) channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca(2+)-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca(2+)-mediated Cl(-) conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.

Charged residues in the M2 region of alpha-hENaC play a role in channel conductance.

The epithelial Na(+) channel (ENaC) is a low-conductance channel that is highly selective for Na(+) and Li(+) over K(+) and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of alpha-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in alpha-subunit human ENaC (alpha-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na(+):K(+) permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 alpha-hENaC mutants along with wild-type (wt) beta- and gamma-hENaC was low (46-93 nA) compared with the wt channel (1-3 microA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant alpha-hENaC subunits with green fluorescent protein-tagged beta- and gamma-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of alpha-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes.

The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic.

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system.

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland.

The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins.

We have cloned and molecularly and functionally characterized the first human member of the family of Ca2+-activated Cl- channels, human (h) CLCA1. The 31,902-bp gene is located on chromosome 1p22-31 and is preceded by a canonic promoter region that contains an L1 transposable element. In contrast to all previously known homologs in other species, hCLCA1 is exclusively expressed in intestinal basal crypt epithelia and goblet cells, suggesting that it does not represent the human counterpart of any of them. Expression of the 914-amino-acid hCLCA1 protein in HEK 293 cells yielded a 125-kDa precursor that was processed to yield two cell-surface-associated subunits, a 90-kDa protein and a group of 37- to 41-kDa proteins. Four transmembrane domains were established within the 90-kDa subunit. HEK 293 cells transfected with CLCA1 exhibited an increase in whole-cell Ca2+-sensitive Cl- currents that were outwardly rectified and inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells revealed single channels with a slope conductance of 13.4 pS. These findings suggest that human CLCA1 mediates a Ca2+-activated Cl- conductance in the human intestine and make it an interesting candidate as a modulating factor in the pathogenesis of cystic fibrosis.

Osmotic pressure regulates alpha beta gamma-rENaC expressed in Xenopus oocytes.

The hypothesis that amiloride-sensitive Na+ channels (ENaC) are involved in cell volume regulation was tested. Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to superfuse Xenopus oocytes expressing alpha beta gamma-rat ENaC (alpha beta gamma-rENaC). Whole cell currents were reversibly dependent on external osmolarity. Under conditions of swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased and increased, respectively. In contrast, there was no change in current amplitude of H2O-injected oocytes to the above osmotic insults. Currents recorded from alpha beta gamma-rENaC-injected oocytes were not sensitive to external Cl- concentration or to the K+ channel inhibitor BaCl2. They were sensitive to amiloride. The concentration of amiloride necessary to inhibit one-half of the maximal rENaC current expressed in oocytes (Ki; apparent dissociation constant) decreased in swollen cells and increased in shrunken oocytes. The osmotic pressure-induced Na+ currents showed properties similar to those of stretch-activated channels, including inhibition by Gd3+ and La3+, and decreased selectivity for Na+. alpha beta gamma-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first demonstration that alpha beta gamma-rENaC heterologously expressed in Xenopus oocytes may contribute to oocyte volume regulation following shrinkage.

Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

Functional expression of a truncated Ca(2+)-activated Cl- channel and activation by phorbol ester.

We have isolated a niflumic acid-insensitive, Ca(2+)-activated Cl- channel (CaCC) from bovine trachea that migrates at 38 kDa (p38) on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC isolated from a tracheal cDNA expression library by screening with an antibody raised against p38 has a primary cDNA transcript of 2712 base pairs that codes for a 100-kDa protein and is not susceptible to dithiothreitol reduction. To test the hypothesis that the functional channel may be a much smaller posttranslationally processed form of the 100-kDa protein, we generated a mutant construct (CaCCX, 42.5-kDa protein) truncated at the NH2 and COOH termini. The whole cell currents of wild-type (wt) CaCC and CaCCX expressed in Xenopus oocytes were 10-fold higher than those of water-injected oocytes and were further increased by ionomycin or A-23187 and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dithiothreitol. Whole cell currents in wtCaCC- and CaCCX-expressing oocytes could also be activated by phorbol 12-myristate 13-acetate and could be inhibited by chelerythrine chloride, suggesting that the cloned CaCC is regulated by protein kinase C. These results suggest that a smaller form of the full-length CaCC can form a functional channel.

Puerarin inhibits tetrodotoxin-resistant sodium current in rat dorsal root ganglion neurons.

AIM: To test if puerarin (Pue) affects slow sodium current in dorsal root ganglion (DRG) neurons. METHODS: Tetrodotoxin resistant (TTXr) sodium current was recorded with whole cell patch clamp technique on DRG neurons of young adult rats. RESULTS: Pue 0.01-2 mmol.L-1 inhibited TTXr sodium current by 9.5%-83.2%. The inhibition was concentration-dependent and partially reversible, but was not use-dependent nor voltage-dependent. Pue did not affect the inactivation but changed the potential for half maximal conductance from -26 mV to -16 mV, suggesting the activation process was inhibited. CONCLUSION: Pue moderately inhibits TTXr sodium current of rat DRG neurons.