Seiji Ichida

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.

Occurrence of a Ca2+- and modulator protein-activatable ATPase in the synaptic plasma membranes of brain.

Ca2*-dependent modulator protein was discovered as an activator of phosphodiesterase [ 1,2], or a protein factor required for the Ca*+-dependent activation of phosphodiesterase [3,4]. Later, this protein was shown to be structurally similar to troponin-C [5,6] and to cause Ca2+-dependent activation of several enzymes including phosphodiesterase, brain adenylate cyclase [7], myosin light chain kinases from skeletal muscles [g-10] and chicken gizzard muscle [ll], and actomyosin ATPase [ 12,131. Recently, two groups [14,15] have demonstrated that the activator protein [16,17] for the erythrocyte membrane ATPase is identical to this modulator protein. (Ca” t Mg2+)-ATPase activity was also found in brain tissue [ 18,191. However, requirement of brain enzyme for an activator has not been reported. In the present study, we are able to show the dependency of the activity of brain enzyme upon the modulator protein. Although brain (Ca2’ + Mg*‘)-ATPase activity was detected in all particulate fractions upon subcellular fractionation, only enzyme in the synaptic plasma membrane fraction was responsive to modulator protein.

Regulation of ATP-dependent Ca-uptake of synaptic plasma membranes by Ca-dependent modulator protein.

Abstract 1. 1)Treatment of synaptic plasma membranes (S.P.M.) with 1 mM EGTA decreased their MgCaATPase activity about 50% without significantly affecting their MgATPase or NaKATPase activity. 2. 2)EGTA-treatment of S.P.M. did not affect the maximum binding of Cd-uptake but decreased its initial velocity about 50%. This treatment also decreased Ca-accumulation in the presence of oxalate about 60%. 3. 3)Addition of Ca-dependent modulator protein (M.P.) purified from bovine brain to EGTA-treated S.P.M. completely restored that MgCaATPase activity and Ca-uptake to the control levels and partially restored their Ca-accumulation. The half-maximum activations of MgCaATPase, Ca-uptake and Ca-accumulation were observed with about 3 μg of M.P. per mg protein of S.P.M. These results suggest that M.P. regulates MgCaATPase and the translocation of Ca ion at the S.P.M.

Study of metallothionein using capillary zone electrophoresis

Metallothioneins (MTs) have many different functions in tissues, but the roles of individual isoforms are still not entirely clear. Capillary zone electrophoresis (CZE) is a powerful method for the separation of substances because of its small sample requirement, rapid analysis, high sensitivity and high resolution. The separation and identification of mammalian MT-1, MT-2, and MT-3 and class III MTs by CZE has been reported. Uncoated and polyacrylamide-coated capillary tubes were recently used for the separation of MTs, and a UV detector is usually employed for observations of peaks of MTs. Small changes to the structure and metal components of MTs are reflected in the migration times of the peaks. N-acetylated and non-acetylated MTs can be separated and identified by CZE-mass spectrometry (MS). In addition, metal complexes with MTs can be characterized by CZE-proton-induced X-ray emission (PIXE) detector and CZE-inductively coupled plasma (ICP)-MS. For the quantification of an MT isoform, the peak area of UV absorption is used, but the technique has problems. One is lack of a purified isoform standard. The other is the need for a suitable internal standard substance. CZE-ICP-isotope dilution (ID)-MS is also reported to be able to quantify MT isoforms. CZE combined with other techniques is very effective for separation and quantitative and qualitative analyses of MT isoforms in biological materials.

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels

We investigated if stimulation of T‐type Ca2+ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2S), could cause neuronal differentiation of NG108‐15 cells. Like dibutyryl cyclic AMP (db‐cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration‐dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db‐cAMP at subeffective concentrations. Exposure to NaHS or db‐cAMP for 2 days resulted in enhancement of expression of high‐voltage‐activated currents consisting of N‐, P/Q‐, L‐ and also other types, but not of T‐type currents. Mibefradil, a pan‐T‐type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db‐cAMP. The NaHS‐evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca2+, and by zinc chloride at a concentration known to selectively inhibit Cav3.2 isoform of T‐type Ca2+ channels, but not Cav3.1 or Cav3.3. Further, l‐ascorbate, recently proven to selectively inhibit Cav3.2, abolished the neuritogenic effect of NaHS, but not db‐cAMP. Our data thus demonstrate that NaHS/H2S is a novel inducer of neuronal differentiation in NG108‐15 cells, as characterized by neuritogenesis and expression of high‐voltage‐activated currents, and suggest the involvement of T‐type Ca2+ channels, especially Cav3.2.

Selective inhibition by ketanserin and spiroperidol of 5-HT-induced myometrial contraction

The inhibitory effects of ketanserin and spiroperidol (a neuroleptic drug) on the contractile response of isolated rat uterus to serotonin (5-HT) were investigated. Ketanserin caused non-competitive inhibition of the contractile response to 5-HT and showed more selective inhibition than the other 5-HT antagonists tested. The inhibitory effect of spiroperidol was comparable with the effects of classical 5-HT antagonists. These results suggest that ketanserin and spiroperidol selectively inhibit the contractile response of isolated rat uterus to 5-HT.

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.

Determination of metallothionein-1/metallothionein-2 ratios in the mouse liver and pancreas by capillary zone electrophoresis using a polyacrylamide-coated capillary at neutral pH.

Metallothionein (MT) isoforms, MT-1 and MT-2, in biological specimens are clearly separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated capillary. The effectiveness of CZE analysis in the study of MT isoforms in biological specimens is discussed. We did two experiments to determine the MT-1/MT-2 ratio in biological specimens. The ratio of MT-1/MT-2 can be determined by CZE under a neutral pH without any detergents. One of these studies is time-dependent changes of the MT-1/MT-2 ratio in the cytosol of the pancreas and liver in mice after Zn or Cd injection. In the pancreas, both isoforms were detected in the control mice and the ratio of MT-1/MT-2 was below 1.0. When Zn was injected, the maximum peak areas of both isoforms were obtained at 24 h, and the ratios increased over a value of 1.0 at 3 h and peaked at 10 h. However, in the Cd-injected mice, the peak areas of both isoforms increased up to 72 h, and the ratios were below 1.0 up to 72 h. On the contrary, neither isoform was detected in the livers of control mice. The ratios of Zn-injected mice liver were near the value 1.0 between 6 and 72 h, although the areas of both isoforms showed peaks at 48 h. The ratios of Cd-injected mice livers were detected to be over 1.0 from 10 h, but there were no significant difference between 10 and 72 h, and the areas of both isoforms showed peaks at 24 h. The other experiment investigated the ratio in each fraction of cell fractionation. Cell fractionation was done in the livers of Zn-treated mice. Twenty-four hours after the injection, the ratio of MT-1/MT-2 was 0.80+/-0.12 and 1.19+/-0.21 (mean+/-SD) in nuclear and cytosol fractions, respectively. Neither isoform was detected in mitochondrial or microsomal fraction. From the present results, CZE analysis is a suitable method for observation of the ratio of MT-1/MT-2 in biological specimens, and dynamic changes in both isoforms can be detected.

Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites

Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 microM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.