Local Anesthetics Inhibit Transient Receptor Potential Vanilloid Subtype 3 Channel Function in Xenopus Oocytes.

BACKGROUND: The transient receptor potential vanilloid subtype 3 (TRPV3) channel is activated by innocuous temperature and several chemical stimuli. It is proposed to be involved in pathological pain development and is therefore considered a potential target for treating pain. Local anesthetics have been used for patients with both acute and chronic pain. Although blockage of the voltage-gated sodium channel is the primary mechanism by which local anesthetics exert their effects, they cannot be explained by this mechanism alone, especially in pathologic states such as chronic pain. Indeed, the effects of local anesthetics on multiple targets involved in the pain pathway have been reported. It has also been suggested that modulating the function of transient receptor potential (TRP) channels (eg, TRPV1 and transient receptor potential ankyrin 1 [TRPA1]) is one of the mechanisms of action of local anesthetics. However, the effects of local anesthetics on TRPV3 have not been reported. METHODS: We expressed TRPV3 in Xenopus oocytes and investigated the effects of local anesthetics on 2-aminoethoxydiphenyl borate (2APB)-induced currents using 2-electrode voltage-clamp techniques. RESULTS: Clinically used local anesthetics inhibited the 2APB-activated currents from the TRPV3 channel in a concentration-dependent manner at pharmacologically relevant concentrations with half maximal inhibitory concentration (IC50) values of 2.5 (lidocaine), 1.4 (mepivacaine), 0.28 (ropivacaine), and 0.17 (bupivacaine) mmol/L, respectively. Conversely, these local anesthetics also directly induced currents at higher concentrations, although these currents were quite small compared to the 2APB-induced currents. We found that the inhibition of TRPV3 by lidocaine is noncompetitive and independent of intracellular signaling cascades. 2APB-induced TRPV3 currents were reduced by extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) but not by intracellular QX-314 nor benzocaine. Moreover, lidocaine showed a use-dependent block in TRPV3 inhibition. Finally, QX-314 appeared to slightly permeate the activated TRPV3 channel pore based on examination of oocytes coexpressing TRPV3 and a sodium channel. These results suggest that local anesthetics could inhibit TRPV3 channel function by extracellular interactions of their charged forms with the channel pore. CONCLUSIONS: Local anesthetics inhibited TRPV3 2APB-induced currents at pharmacologically relevant concentrations when TRPV3 was expressed in Xenopus oocytes. These effects seem to occur via an extracellular interaction between the charged form of the anesthetic with the TRPV3 channel pore. These results help to elucidate the mechanisms of action of local anesthetics.

Carvacrol inhibits the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.3, Nav1.7, and Nav1.8 expressed in Xenopus oocytes with different potencies.

Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Nav) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Nav. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 were 233, 526, 215, 367, and 824 µmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Nav1.2 and Nav1.6 than Nav1.3, Nav1.7, and Nav1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Nav subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol.

The neurosteroid allopregnanolone sulfate inhibits Nav1.3 α subunit-containing voltage-gated sodium channels, expressed in Xenopus oocytes.

The neurosteroid allopregnanolone has potent analgesic effects, and its potential use for neuropathic pain is supported by recent reports. However, the analgesic mechanisms are obscure. The voltage-gated sodium channels (Nav) α subunit Nav1.3 is thought to play an essential role in neuropathic pain. Here, we report the effects of allopregnanolone sulfate (APAS) on sodium currents (INa) in Xenopus oocytes expressing Nav1.3 with ß1 or ß3 subunits. APAS suppressed INa of Nav1.3 with ß1 and ß3 in a concentration-dependent manner (IC50 values; 75 and 26 µmol/L). These results suggest the possible importance of Nav1.3 inhibition for the analgesic mechanisms of allopregnanolone.

Cancer Risks of Hexavalent Chromium in the Respiratory Tract.

Hexavalent chromium (Cr(VI)) compounds are recognized as carcinogens in the respiratory tract, giving rise to cancers of the lung, nose and nasal sinuses, especially in certain occupational environments. Inhalation exposure of Cr(VI)-containing particles, dusts and fumes commonly occurs in chromium-related occupational environments, such as chromium production, plating, welding of chromium-containing metals and alloys, electroplating, chromium-containing pigments and paints. Epidemiological surveys of chromium compounds have shown strong associations between exposure to Cr(VI) and mortality due to lung cancer, as well as positive associations with cancers of the nose and nasal cavity. Nasal symptoms, such as nasal irritation, ulceration and perforation of the nasal septum, nasal turbinate engorgement and hypertrophy, are important signs for the early diagnosis of lung cancer and cancers of the nose and nasal cavity in those with an occupational history of Cr(VI) exposure. Cr(VI) exposure in the workplace remains a serious problem as a cause of lung cancer and cancers of nose and nasal cavity, especially in relatively small enterprises that use chromium compounds. Appropriate protection for workers should be considered in occupations that involve exposure to chromium compounds.

Lidocaine preferentially inhibits the function of purinergic P2X7 receptors expressed in Xenopus oocytes.

BACKGROUND: Lidocaine has been widely used to relieve acute pain and chronic refractory pain effectively by both systemic and local administration. Numerous studies reported that lidocaine affects several pain signaling pathways as well as voltage-gated sodium channels, suggesting the existence of multiple mechanisms underlying pain relief by lidocaine. Some extracellular adenosine triphosphate (ATP) receptor subunits are thought to play a role in chronic pain mechanisms, but there have been few studies on the effects of lidocaine on ATP receptors. We studied the effects of lidocaine on purinergic P2X3, P2X4, and P2X7 receptors to explore the mechanisms underlying pain-relieving effects of lidocaine. METHODS: We investigated the effects of lidocaine on ATP-induced currents in ATP receptor subunits, P2X3, P2X4, and P2X7 expressed in Xenopus oocytes, by using whole-cell, two-electrode, voltage-clamp techniques. RESULTS: Lidocaine inhibited ATP-induced currents in P2X7, but not in P2X3 or P2X4 subunits, in a concentration-dependent manner. The half maximal inhibitory concentration for lidocaine inhibition was 282 ± 45 µmol/L. By contrast, mepivacaine, ropivacaine, and bupivacaine exerted only limited effects on the P2X7 receptor. Lidocaine inhibited the ATP concentration-response curve for the P2X7 receptor via noncompetitive inhibition. Intracellular and extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) and benzocaine suppressed ATP-induced currents in the P2X7 receptor in a concentration-dependent manner. In addition, repetitive ATP treatments at 5-minute intervals in the continuous presence of lidocaine revealed that lidocaine inhibition was use-dependent. Finally, the selective P2X7 receptor antagonists Brilliant Blue G and AZ11645373 did not affect the inhibitory actions of lidocaine on the P2X7 receptor. CONCLUSIONS: Lidocaine selectively inhibited the function of the P2X7 receptor expressed in Xenopus oocytes. This effect may be caused by acting on sites in the ion channel pore both extracellularly and intracellularly. These results help to understand the mechanisms underlying the analgesic effects of lidocaine when it is administered locally at least.

Prenatal Exposure to 1-Bromopropane Suppresses Kainate-Induced Wet Dog Shakes in Immature Rats.

1-Bromopropane (1-BP) is used in degreasing solvents and spray adhesives. The adverse effects of 1-BP have been reported in human cases and adult animal models, and its developmental toxicity has also been reported, but its effects on developmental neurotoxicity have not been investigated in detail. We evaluated the effects in rat pups of prenatal exposure to 1-BP on behaviors such as scratching and wet dog shakes (WDS), which were induced by injection of kainate (KA). Pregnant Wistar rats were exposed to vaporized 1-BP with 700 ppm from gestation day 1 to day 20 (6 h/day). KA at doses of 0.1, 0.5, and 2.0 mg/kg were intraperitoneally injected into a control group and a 1-BP-exposed group of pups on postnatal day 14. There was no significant difference in scratching between the control and the prenatally 1-BP-exposed groups, while suppression of the occurrence ratio of WDS was observed at the low dose of 0.1 mg/kg of KA in the prenatally 1-BP-exposed pups. Our results suggest that prenatal exposure to 1-BP affects neurobehavioral responses in the juvenile period.

Development of an experimentally useful model of acute myocardial infarction: 2/3 nephrectomized triple nitric oxide synthases-deficient mouse.

We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy (NX) was performed on male triple NOSs(-/-) mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs(-/-) mice as early as 4months after the surgery. The 2/3NX triple NOSs(-/-) mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs(-/-) mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin-angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs(-/-) mouse is a new experimentally useful model of AMI. Renin-angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.

Neurosteroids allopregnanolone sulfate and pregnanolone sulfate have diverse effect on the α subunit of the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 expressed in xenopus oocytes.

BACKGROUND: The neurosteroids allopregnanolone and pregnanolone are potent positive modulators of γ-aminobutyric acid type A receptors. Antinociceptive effects of allopregnanolone have attracted much attention because recent reports have indicated the potential of allopregnanolone as a therapeutic agent for refractory pain. However, the analgesic mechanisms of allopregnanolone are still unclear. Voltage-gated sodium channels (Nav) are thought to play important roles in inflammatory and neuropathic pain, but there have been few investigations on the effects of allopregnanolone on sodium channels. METHODS: Using voltage-clamp techniques, the effects of allopregnanolone sulfate (APAS) and pregnanolone sulfate (PAS) on sodium current were examined in Xenopus oocytes expressing Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits. RESULTS: APAS suppressed sodium currents of Nav1.2, Nav1.6, and Nav1.7 at a holding potential causing half-maximal current in a concentration-dependent manner, whereas it markedly enhanced sodium current of Nav1.8 at a holding potential causing maximal current. Half-maximal inhibitory concentration values for Nav1.2, Nav1.6, and Nav1.7 were 12 ± 4 (n = 6), 41 ± 2 (n = 7), and 131 ± 15 (n = 5) µmol/l (mean ± SEM), respectively. The effects of PAS were lower than those of APAS. From gating analysis, two compounds increased inactivation of all α subunits, while they showed different actions on activation of each α subunit. Moreover, two compounds showed a use-dependent block on Nav1.2, Nav1.6, and Nav1.7. CONCLUSION: APAS and PAS have diverse effects on sodium currents in oocytes expressing four α subunits. APAS inhibited the sodium currents of Nav1.2 most strongly.

New insights into the pharmacological potential of plant flavonoids in the catecholamine system.

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 - 1.0 µM), a soy isoflavone, stimulated (14)C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 - 100 µM), a citrus polymethoxy flavone, enhanced (14)C-catecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with (45)Ca(2+) influx and catecholamine secretion. Treatment with genistein (0.01 - 10 µM), another isoflavone, but not daidzein, enhanced [(3)H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 µM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons.

Effects of selective estrogen receptor modulators on plasma membrane estrogen receptors and catecholamine synthesis and secretion in cultured bovine adrenal medullary cells.

We previously reported the occurrence and function of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of raloxifene and tamoxifen, selective estrogen receptor modulators, on plasma membrane estrogen receptors and catecholamine synthesis and secretion in these cells. Raloxifene caused dual effects on the specific binding of [(3)H]17ß-estradiol to the plasma membranes isolated from bovine adrenal medulla; that is, it had a stimulatory effect at 1.0 - 10 nM but an inhibitory effect at 1.0 - 10 µM, whereas tamoxifen (1.0 nM - 10 µM) increased binding at all concentrations (except for 100 nM). Tamoxifen at 100 nM caused a significant increase in basal (14)C-catecholamine synthesis from [(14)C]tyrosine, whereas tamoxifen and raloxifene at higher concentrations attenuated basal and acetylcholine-induced (14)C-catecholamine synthesis. Raloxifene (0.3, 1.0, and 3 - 100 µM) and tamoxifen (10 - 100 µM) also suppressed catecholamine secretion and (45)Ca(2+) and (22)Na(+) influx, respectively, induced by acetylcholine. Raloxifene (1.0 µM) inhibited Na(+) current evoked by acetylcholine in Xenopus oocytes expressing α4ß2 neuronal nicotinic acetylcholine receptors. The present findings suggest that raloxifene and tamoxifen at low concentrations allosterically modulate plasma membrane estrogen receptors and at high concentrations inhibit acetylcholine-induced catecholamine synthesis and secretion by inhibiting Na(+) and Ca(2+) influx in bovine adrenal medulla.

The endocannabinoid anandamide inhibits voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8 in Xenopus oocytes.

BACKGROUND: Anandamide is an endocannabinoid that regulates multiple physiological functions by pharmacological actions, in a manner similar to marijuana. Recently, much attention has been paid to the analgesic effect of endocannabinoids in terms of identifying new pharmacotherapies for refractory pain management, but the mechanisms of the analgesic effects of anandamide are still obscure. Voltage-gated sodium channels are believed to play important roles in inflammatory and neuropathic pain. We investigated the effects of anandamide on 4 neuronal sodium channel α subunits, Nav1.2, Nav1.6, Nav1.7, and Nav1.8, to explore the mechanisms underlying the antinociceptive effects of anandamide. METHODS: We studied the effects of anandamide on Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits with ß1 subunits by using whole-cell, 2-electrode, voltage-clamp techniques in Xenopus oocytes. RESULTS: Anandamide inhibited sodium currents of all subunits at a holding potential causing half-maximal current (V1/2) in a concentration-dependent manner. The half-maximal inhibitory concentration values for Nav1.2, Nav1.6, Nav1.7, and Nav1.8 were 17, 12, 27, and 40 µmol/L, respectively, indicating an inhibitory effect on Nav1.6, which showed the highest potency. Anandamide raised the depolarizing shift of the activation curve as well as the hyperpolarizing shift of the inactivation curve in all α subunits, suggesting that sodium current inhibition was due to decreased activation and increased inactivation. Moreover, anandamide showed a use-dependent block in Nav1.2, Nav1.6, and Nav1.7 but not Nav1.8. CONCLUSION: Anandamide inhibited the function of α subunits in neuronal sodium channels Nav1.2, Nav1.6, Nav1.7, and Nav1.8. These results help clarify the mechanisms of the analgesic effects of anandamide.

Pentazocine inhibits norepinephrine transporter function by reducing its surface expression in bovine adrenal medullary cells.

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [(3)H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (-)-PTZ and (+)-PTZ inhibited [(3)H]NE uptake by adrenal medullary cells in a concentration-dependent (3-100 µM) manner. Eadie-Hofstee analysis of [(3)H]NE uptake showed that both PTZs caused a significant decrease in the V(max) with little change in the apparent K(m), suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [(3)H]NE uptake induced by (-)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [(3)H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [(3)H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the B(max) without changing the apparent K(d). Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (-)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor-independent pathway.

[Toxicology of chemical substances (metals and organic solvents): management as an occupational physician].

Even in Japan, there was a time when cases of occupational poisoning had frequently occurred, which led to the enactment of the Industrial Safety and Health Act in 1972. Currently, the use of only a part of chemical substances utilized in the workplace is regulated according to their designated hazardous level, but there are many other substances whose toxicities have not been elucidated. Risk assessment is now required of entrepreneurs in all categories of industry by the recently-revised Industrial Safety and Health Act. This article will focus on the toxicology of metals and organic solvents, and it will discuss how occupational physicians should manage chemicals, including the ones whose toxicities have not been clarified.

Inhibition of Citric Acid-Induced Dentin Erosion by an Acidulated Phosphate Sodium Monofluorophosphate Solution.

Sodium monofluorophosphate (Na2FPO3, MFP) is mainly used as an ingredient in fluoride-based dentifrices as it has a high safety profile, with one-third of the toxicity of sodium fluoride (NaF), as well as the ability to reach deep into the dentin. The purpose of this study was to assess the prevention of dentin erosion by MFP upon exposure to citric acid, which has a chelating effect, and to compare the effects to those of the conventional acidulated phosphate fluoride (APF) application method. Bovine dentin was used, and four groups were created: (i) APF (9000 ppmF, pH 3.6) 4 min group; (ii) acidulated phosphate MFP (AP-MFP, 9000 ppmF, pH 3.6) 4 min group; (iii) AP-MFP 2 min + APF 2 min (dual) group; and (iv) no fluoride application (control) group. Compared with the conventional APF application method, the application of AP-MFP was shown to significantly reduce substantial defects, mineral loss, and lesion depth; better maintain Vickers hardness; and promote the homogenous aggregation of fine CaF2 particles to seal the dentin tubules, enhancing acid resistance in their vicinity. The ΔZ value of the AP-MFP group was 2679 ± 290.2 vol% µm, significantly smaller than the APF group's 3806 ± 257.5 vol% µm (p < 0.01). Thus, AP-MFP-based fluoride application could effectively suppress citric acid-induced demineralization and could become a new, more powerful, and biologically safer professional-care method for preventing acid-induced dentin erosion than the conventional method.

The effect of mask fit test on the association between the concentration of metals in biological samples and the results of time-weighted average personal exposure: A study on Japanese male welders.

OBJECTIVES: The mask fit test confirms whether the wearing condition of the wearer's face and the facepiece of the respirators are used appropriately. This study aimed to examine whether the results of the mask fit test affect the association between the concentration of metals related to welding fumes in biological samples and the results of time-weighted average (TWA) personal exposures. METHODS: A total of 94 male welders were recruited. Blood and urine samples were obtained from all participants to measure the metal exposure levels. Using personal exposure measurements, the 8-h TWA (8 h-TWA) of respirable dust, TWA of respirable Mn, and 8-h TWA of respirable Mn were calculated. The mask fit test was performed using the quantitative method specified in the Japanese Industrial Standard T8150:2021. RESULTS: Fifty-four participants (57%) passed the mask fit test. Only in the Fail group of the mask fit test, it was observed that blood Mn concentrations be positively associated with the results of TWA personal exposure after adjusting for multivariate factors (8-h TWA of respirable dust; coefficient, 0.066; standard error (SE), 0.028; P = 0.018, TWA of respirable Mn: coefficient, 0.048; SE, 0.020; P = 0.019, 8 h-TWA of respirable Mn: coefficient, 0.041; SE, 0.020; P = 0.041). CONCLUSIONS: The results clarify that welders with high concentrations of welding fumes in their breathing air zone are exposed to dust and Mn if there is leaking air owing to the lack of fitness between respirators and the wearer's face when using human samples in Japan.

Associations between welding fume exposure and neurological function in Japanese male welders and non-welders.

OBJECTIVES: There are some studies reporting the association between (manganese [Mn]) exposure to welding fume and neurological dysfunction. This study examined the relationship between Mn exposure and neurological behavior in Japanese male welders and non-welders using biological samples, which to date has not been assessed in Japan. METHODS: A total of 94 male welders and 95 male non-welders who worked in the same factories were recruited. The blood and urine samples were obtained from all the participants to measure Mn exposure levels. Neurological function tests were also conducted with all participants. The sampling of the breathing air zone using a personal sampler was measured for welders only. RESULTS: The odds ratios (ORs) for the Working Memory Index (WMI) scores were significantly higher among all participants in the low blood Mn concentration group than those in the high blood Mn concentration group (OR, 2.77; 95% confidence interval [CI], 1.24, 6.19; P = .013). The association of WMI scores and blood Mn levels in welders had the highest OR (OR, 3.73; 95% CI, 1.04, 13.38; P = .043). Although not statistically significant, a mild relationship between WMI scores and blood Mn levels was observed in non-welders (OR, 2.09; 95% CI, 0.63, 6.94; P = .227). CONCLUSIONS: The results revealed a significant positive relationship between blood Mn and neurological dysfunction in welders. Furthermore, non-welders at the same factories may be secondarily exposed to welding fumes. Further research is needed to clarify this possibility.

Inhibition by pregnenolone sulphate, a metabolite of the neurosteroid pregnenolone, of voltage-gated sodium channels expressed in Xenopus oocytes.

Neurosteroids are known as allosteric modulators of the ligand-gated ion channel superfamily. Voltage-gated sodium channels (Na(v)) play an important role in mediating excitotoxic damages. Here we report the effects of neurosteroids on the function of Na(v), using voltage-clamp techniques in Xenopus oocytes expressed with the Na(v)1.2 α subunit. Pregnenolone sulphate, but not pregnenolone, inhibited sodium currents (I(Na)) at 3 - 100 µmol/L. The suppression of I(Na) by pregnenolone sulphate was due to increased inactivation with little change in activation. These findings suggest that pregnenolone sulphate, a metabolite of pregnenolone, suppresses the function of Na(v) via increased inactivation, which may contribute to the neuroprotection.

Binding of titanium dioxide nanoparticles to lactate dehydrogenase.

OBJECTIVE: Measurement of released lactate dehydrogenase (LDH) activity, a commonly used marker of lethal cell injury in both in vitro and in vivo screenings, has been used to assess the cytotoxicity of nanoparticles (NPs), chemical compounds, and environmental factors. We have recently demonstrated that titanium dioxide (TiO2) particles bind to several serum proteins. In the present study we investigated the binding of TiO2 NPs to LDH. METHODS: Purified LDH was incubated with TiO2 NPs at 37°C for 1 h. The particles were then sedimented by centrifugation, and the activity and quantity of LDH in the supernatant and precipitated fraction were analyzed. RESULTS: Incubation with TiO2 reduced the LDH activity in the supernatant in a dose-dependent manner, while LDH activity in the precipitated fraction increased in a dose-dependent manner. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a TiO2 dose-dependent reduction in the quantity of LDH protein in the supernatant and an increase of LDH in particulate re-suspensions. CONCLUSIONS: These findings, although based on a purified form of LDH, suggest that TiO2 NPs bind to LDH, and consequently, TiO2 NP-induced toxicity could be underestimated by the LDH activity assay.

Nuclear factor-kappa B is not involved in titanium dioxide-induced inflammation.

Research over recent years have shown that titanium dioxide (TiO2) nanoparticles (NPs) induce inflammation in various lung, kidney, liver and brain cells. Although the mechanism of inflammation is unclear, existing literature suggests the underlying role of oxidative stress. On the other hand, it has also been shown that nuclear factor-kappa B (NF-kappaB) is activated in response to pro-inflammatory cytokines. In this study we investigated the involvement of NF-kappaB in TiO2-induced inflammation in human lung adenocarcinomic epithelial cells (A549 cells). After 24h of treatment, IL-8 protein release from A549 cells, induced by 10, 50 and 250 microg/ml of P25 TiO2 NPs, were statistically significantly raised, compared to that of the control. This finding corroborates existing literature in that TiO2 NPs induce a dose-dependent increase in the release of IL-8 protein when exposed to A549 cells. However, the binding of NF-kappaB DNA was not affected after 6 h of incubation with P25. Therefore, NF-kappaB DNA binding is not the likely transcription pathway that leads to TiO2-induced inflammation.

Crucial vasculoprotective role of the whole nitric oxide synthase system in vascular lesion formation in mice: Involvement of bone marrow-derived cells.

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism.