Tetsuyuki Wada

Hydrogen sulfide as a novel nociceptive messenger

Abstract Hydrogen sulfide (H2S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H2S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H2S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H2S‐evoked hyperalgesia was blocked by 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T‐type Ca2+ channel inhibitors. l‐Cysteine, an endogenous source for H2S, given i.pl., also elicited hyperalgesia, an effect being abolished by dl‐propargylglycine (PPG) and β‐cyanoalanine (BCA), inhibitors of cystathionine‐γ‐lyase, a H2S synthesizing enzyme. PPG and/or BCA partially inhibited the hyperalgesia induced by i.pl. lipopolysaccharide, an effect being reversed by i.pl. NaHS. In the patch‐clamp study using undifferentiated NG108‐15 cells that express T‐type, but not other types, of Ca2+ channels, NaHS enhanced the currents through the T‐type channels, an effect being blocked by DTNB. Thus, H2S appears to function as a novel nociceptive messenger through sensitization of T‐type Ca2+ channels in the peripheral tissues, particularly during inflammation.

Transfollicular Drug Delivery: Penetration of Drugs Through Human Scalp Skin and Comparison of Penetration Between Scalp and Abdominal Skins In Vitro

In order to quantitatively investigate the importance of transfollicular pathway for drug delivery, drug penetration through human scalp skin was investigated using liquid formulations containing lipophilic and hydrophilic drugs in vitro. The penetration pathway for drugs through the scalp skin was examined using fluorescent probes. Additionally, the drug penetration through the scalp skin was compared with that via human abdominal skin to clarify the usefulness of intrafollicular delivery. Lipophilic melatonin (MT) and ketoprofen (KP) showed high permeabilities through the scalp skin, although the flux of KP was much higher. Absorption enhancers, N -methyl-2-pyrrolidone and isopropylmyristate, only slightly increased the fluxes. Hydrophilic fluorouracil (5FU) and acyclovir (ACV) penetrated through the scalp skin with relatively large fluxes. However, there was large variability in the fluxes of these drugs across scalp skin from different sources. When the relationship between the flux and hair follicle density was estimated, there was good correlation between the two (r =0.651 for MT and r =0.666 for ACV, P <0.05) . The histologic examination of the scalp skin, following application of the formulation with nile red or sodium fluorescein, indicated that the probes permeated into the junction of the internal and external root sheath of follicles and diffused into the dermis via the outer root sheath at the initial times. The penetration of nile red, a lipophilic probe, via the stratum corneum of scalp skin was later than that via the follicles. The permeation of MT and 5FU through the scalp skin was much higher than that via the abdominal skin, being 27 and 48 times as high as the abdominal skin, respectively. These results indicate that the drug delivery through the scalp skin will offer an available delivery means for drugs, particularly for hydrophilic drugs.

Bleaching of alkali-refined vegetable oils with clay minerals

Bleaching efficiencies of bentonites, montmorillonites and sepiolites for alkali-refined rapesseed, soybean, wheatgerm, safflower, corn, cottonseed and sunflower oils were investigated by a batch method at 110°C. The sepiolites with more acid sites at −5.6 < Ho ≥ −3.0 were the most effective in bleaching of each alkali-refined oil. Surface area and acidity at −5.6>Ho ≥ −3.0 were highly significant with bleaching efficiency. The sepiolites (numbers 2 and 3) were more suitable than standard activated clay because they were more effective both in retaining tocopherols and in reducing free fatty acids after bleaching.

Characteristics of45Ca uptake stimulated by high KCl of differentiated and undifferentiated NG108-15 and PC12h cells

The characteristics of KCl-stimulated45Ca uptake by neuroblastoma x glioma hybrid NG108-15 cells induced to differentiate with dibutyryl cAMP (Bt2cAMP) and of PC12h pheochromocytoma cells induced to differentiate with nerve growth factor (NGF) were studied. The extent and rate of KCl-stimulated45Ca uptake by differentiated NG108-15 cells induced with Bt2cAMP were significantly higher than those of the undifferentiated cells. However, differentiation of PC12h cells induced with NGF did not enhance their extent or rate of KCl-stimulated45Ca uptake. The effects of Ca agonist and antagonists indicated that the characteristics of KCl-stimulated45Ca uptake by Bt2cAMP-treated NG108-15 cells and NGF-treated PC12h cells mainly reflected those of peripheral L-type voltage-sensitive calcium channels activated by high KCl. These results suggest that differentiated neural cells did not all show an enhanced capacity for KCl-stimulated45Ca uptake, although the characteristic patterns of differentiation (extension of neurite-like processes, etc.) and that of effect by Ca agonist or antagonists on NG108-15 cells and PC12h cells were similar.

Characteristics of specific125I-ω-conotoxin GVIA binding in rat whole brain

Characteristics of specific125I-omega-conotoxin (ω-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1–13), in particular, specifically inhibited125I-ω-CgTX binding, but not that of [3H](+)PN200-110. Spider venom fromPlectreurys tristes did not specifically inhibit specific binding of125I-ω-CgTX, because the venom also inhibited the binding of [3H](+)PN200-110 to a similar degree. The amount of specific binding of125I-ω-CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with125I-ω-CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1–13) was a selective blocker of ω-CgTX-sensitive Ca channels in crude membranes from rat whole brain and that ω-CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.

Characteristics of specific 125I-ω-conotoxin GVIA binding and 125I-ω-conotoxin GVIA labeling using bifunctional crosslinkers in crude membranes from chick whole brain

Abstract Characteristics of specific 125 I-ω-conotoxin GVIA ( 125 I-ω-CgTX) binding and 125 I-ω-CgTX labeling using bifunctional crosslinkers were systematically investigated in crude membranes from chick whole brain. Aminoglycosides and dynorphine A (1–13) inhibited the specific binding of 125 I-ω-CgTX, but not that of the L-type calcium ion channel antagonist [ 3 H](+)PN200-110. It seems likely that the inhibitory effect of dynorphine A (1–13) does not involve κ-opiate receptors, based on results with the opiate receptor antagonist naloxone and the κ-opiate receptor agonist U50488H. Spider venom, Cd 2+ and La 3+ inhibited the specific binding of 125 I-ω-CgTX, as well as that of [ 3 H](+)PN200-110. Various L-type Ca 2+ channel antagonists did not affect the specific binding of 125 I-ω-CgTX. 125 I-ω-CgTX specifically labeled 135 kDa and 215 kDa bands in crude membranes under reduced and non-reduced conditions, respectively. The crosslinker disuccinimidyl suberate (DSS) yielded better 125 I-ω-CgTX labeling than the other two crosslinkers tested. We investigated the effect of various Ca 2+ channel antagonists on 125 I-ω-CgTX labeling with DSS in detail, and found that there is a strong correlation between the effects of Ca 2+ channel antagonists on 125 I-ω-CgTX labeling of the 135 kDa band and specific 125 I-ω-CgTX binding. These results suggest that aminoglycosides and dynorphine A (1–13) are specific inhibitors of specific 125 I-ω-CgTX binding, and that labeling of the 135 kDa band with 125 I-ω-CgTX using DSS involves the specific binding sites of 125 I-ω-CgTX, perhaps including one of the neuronal N-type Ca 2+ channel subunits in the crude membranes.

Effects of filtration through activated carbons on peroxide, thiobarbituric acid and carbonyl values of autoxidized soybean oil

Effects of filtration bleaching on peroxide value (PV), thiobarbituric acid value (TAV) and carbonyl value (CV) of autoxidized soybean oil were investigated by using twenty-three kinds of activated carbon in order to improve oil quality. From the decreases in PV, TAV and CV and from the physical and chemical properties of activated carbons, it was suggested that hydroperoxides, aldehydes and ketones were adsorbed on the acid sites distributed over the surface or within the pores of the activated carbons while the autoxidized soybean oil flowed through the packed column. The residual tocopherols in autoxidized soybean oil and treated soybean oil were determined during storage. The decrease in oxidative stability of treated soybean oil seemed to be caused by elimination ofα-,β-andγ-tocopherols.δ-Tocopherol was chemically more stable thanα-,β- andγ-tocopherols in autoxidized soybean oil.

Increased Response to High KCl-Induced Elevation in the Intracellular-Ca2+ Concentration in Differentiated NG108-15 Cell and the Inhibitory Effect of the L-Type Ca2+ Channel Blocker, Calciseptine

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca2+]i) by high-KCl application were investigated in the neuroblastoma×glioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca2+]i by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt2cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca2+ channel blocker, but not by N-, P- and R-type Ca2+ channel blockers. The IC50 values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca2+]i by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca2+]i by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl2). Together, these results suggest that the enhancement of the response effect of [Ca2+]i by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.

Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [3H](+)PN200-110 (PN: L-type Ca channel antagonist) and [125I]ω-conotoxin G VI A (ω-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt2cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated45Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt2cAMP (unpublished results). The specific binding of [3H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [3H](+)PN. Scatchard analysis showed that the specific binding of [3H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt2cAMP, but that the apparent Kd value for the specific binding of [3H](+)PN was not influenced by treatment with Bt2cAMP. The specific binding of [3H](+)PN to crude membranes from Bt2cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(−)PN≧Bay K 8644≫diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [3H(+)PN. On the other hand, [125I]ω-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated45Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [125I]ω-CgTX on Bt2cAMP-treated or untreated NG108-15 cells.

Characteristics of the inhibitory effect of calmodulin on specific [125i]omega-conotoxin GVIA binding to crude membranes from chick brain.

The characteristics of the inhibitory effect of calcium ion (Ca2+)/calmodulin (CaM) on specific [125I]-omega-conotoxin GVIA (125I-ω-CTX) binding and on the labeling of 125I-ω-CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca2+/CaM depended on the concentrations of free Ca2+ and CaM. The IC50 values for free Ca2+ and CaM were about 2.0 × 10−8 M and 3.0 μg protein/ml, respectively. The inhibitory effect of Ca2+/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca2+/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca2+ channel α1 subunits) with 125I-ω-CTX using a cross-linker. These results suggest that Ca2+/CaM affects specific 125I-ω-CTX binding sites, probably N-type Ca2+ channel α1 subunits, in crude membranes from chick whole brain.