Regulation of type 1 IP3 receptor expression by dopamine D2-like receptors via AP-1 and NFATc4 activation.

Type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca²âº concentration. Although our recent report demonstrates the essential involvement of IP3R-1 up-regulation induced by dopamine D1-like and D2-like receptor (D1 and D2R) stimulation in psychological dependence, exact regulatory mechanisms of IP3R-1 expression by D2Rs have not yet been clarified. Mouse cerebral cortical neurons were treated with inhibitor of Ca²âº-related signal transduction pathways coupling to D2Rs and used to analyze the mechanisms of IP3R-1 expression regulated by transcriptional factor. A selective D2R agonist, quinpirole, up-regulated IP3R-1 protein following its mRNA increase, which was significantly inhibited by gallein (a Gßγ modulator), U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor). Immunocytochemical assessment showed that quinpirole increased expression of both cFos and phosphorylated-cJun in nucleus and enhanced translocation of NFATc4 complex to nucleus from cytoplasm. In addition, quinpirole directly recruited bindings between AP-1 and IP3R-1 promoter region and between NFATc4 and IP3R-1 promoter region. These results indicate that D2Rs enhance IP3R-1 gene transcription via increased bindings of AP-1 and NFATc4 to IP3R-1 promoter region after Gßγ activation.

Possible involvement of type 1 inositol 1,4,5-trisphosphate receptors up-regulated by dopamine D1 and D2 receptors in mouse nucleus accumbens neurons in the development of methamphetamine-induced place preference.

Little is known about regulatory mechanisms of type 1 inositol-1,4,5-triphosphate receptor (IP(3)R-1) expression in conditioned place preference by methamphetamine (METH), though significant enhancement of IP(3)R-1 expression in the mouse frontal cortex and limbic forebrain by intermittent administration of cocaine is reported. The present study investigated the role and regulation of IP(3)R-1 in mice with METH-induced place preference. Injection of IP(3)R antagonists with different chemical structures, 2-aminophenoxyethane-borate and xestospongin C, into the mouse nucleus accumbens (NAcc) dose-dependently inhibited METH-induced place preference. The levels of IP(3)R-1 protein in the NAcc of METH-conditioned mice significantly increased, which was completely abolished by microinjection of SCH23390 and raclopride, selective dopamine D1-like and D2-like receptor (D1 and D2DR) antagonists respectively, into the mouse NAcc. Immunohistochemical assessment revealed co-localization of immunoreactivity for IP(3)R-1 and those for D1 and D2DRs in the NAcc. These findings suggest that IP(3)R-1 could be involved in the development of METH-induced place preference and that D1 and D2DRs in the NAcc of mice showing METH-induced place preference play possible regulatory roles in IP(3)R-1 expression.

Gabapentin blocks methamphetamine-induced sensitization and conditioned place preference via inhibition of α2/δ-1 subunits of the voltage-gated calcium channels.

Our previous investigation demonstrated that repeated administration of morphine significantly enhanced α(2)/δ-1 subunit expression in the frontal cortex and limbic forebrain of mice as well as morphine-induced place preference. However, little is known about regulatory mechanisms of α(2)/δ-1 subunit expression in conditioned place preference by methamphetamine (METH). In the present study, we investigated the role of α(2)/δ-1 subunit of voltage-gated calcium channels (VGCCs) in the mouse brain under repeated treatment with METH. The level of α(2)/δ-1 subunit increased significantly in the limbic forebrain including the nucleus accumbens and the frontal cortex of mice showing METH-induced sensitization. Under these conditions, the development of behavioral sensitization induced by the intermittent administration of METH was significantly suppressed by the co-administration of gabapentin (GBP) with binding activity to an exofacial epitope of α(2)/δ-1 subunit. Furthermore, GBP administered i.c.v. caused a dose-dependent inhibition of the METH-induced place preference. Chronic GBP treatment at the dose alleviating sensitization and place preference significantly reduced the elevation of α(2)/δ-1 subunit of VGCC induced by the repeated administration of METH in the limbic forebrain and frontal cortex, whereas there were no changes in the increase of α(2)/δ-1 subunit mRNA. These findings indicate that α(2)/δ-1 subunit plays a critical role in the development of METH-induced place preference following neuronal plasticity, and that GBP, which significantly suppressed METH-induced place preference by its possible inhibitory action of α(2)/δ subunit to neuronal membrane, may possibly be used as an alternative drug to treat or prevent drug dependence.

Role of alpha2/delta subunit in the development of morphine-induced rewarding effect and behavioral sensitization.

Previous data demonstrate that L-type voltage-gated calcium channel (VGCC) blockers, which bind to alpha(1) subunits of VGCC to suppress Ca(2+) entry into cells, inhibit the development of psychological dependence on drugs of abuse, suggesting the upregulation of L-type VGCC in the development of psychological dependence. However, there are few available data on changes of the auxiliary subunit alpha(2)/delta modifying L-type VGCC under such conditions. We therefore investigated here the role of alpha(2)/delta subunits of VGCCs in the brain of mouse after repeated treatment with morphine. The treatment with morphine increased alpha(2)/delta subunit expression in the frontal cortex and the limbic forebrain of mice showing rewarding effect and sensitization to hyperlocomotion by morphine. The morphine-induced behavioral sensitization and place preference were also suppressed by gabapentin, which binds to an exofacial epitope of the alpha(2)/delta auxiliary subunits of VGCCs. These findings indicate that the upregulation of alpha(2)/delta subunit as well as alpha(1) subunits of VGCC in the frontal cortex and the limbic forebrain plays a critical role in development of morphine-induced rewarding effect and behavioral sensitization following neuronal plasticity.

Chemotherapeutic choice of ranimustine or nimustine on the basis of regional polyamine levels in rat brain.

The aim of the present study was to improve the chemotherapeutic efficacy of anticancer drugs by choosing on the basis of the polyamine level induced by the drug in each host cancer-bearing tissue. We propose an "organ-specific therapy" in the article. The polyamines, putrescine, spermidine and spermine are strongly associated with tumor cell growth. The effects of ranimustine (MCNU) and nimustine (ACNU) on body weight, regional brain weights and concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, combined thalamus and hypothalamus and diencephalon of the brain were examined in rats. MCNU and ACNU reduced spermidine and spermine in the corpus striatum, and spermine in the diencephalon, but increased putrescine in the corpus striatum and combined thalamus and hypothalamus. These results indicate that both MCNU and ACNU are suitable for the treatment of cancers of the corpus striatum, but ACNU is not suitable for cancers of the corpus striatum, thalamus and hypothalamus.

Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation.

During endochondral ossification, the production of hyaluronan (HA) is strictly and selectively regulated by chondrocytes, with a temporal peak at the hypertrophic stage. This study was conducted to clarify the effects of HA on expression and activity of runt-related gene 2 (Runx2), a potent transcription factor for chondrocyte differentiation in hypertrophic chondrocytes. Immature chondrocytes from an ATDC5 cell line were cultured and differentiated in DMEM/Ham's F12 with pre-defined supplements. Using real-time PCR, the gene expressions of type II collagen, MMP-13, HAS2, and Runx2 in cultured chondrocytes were analysed from days 0 to 18 of cell differentiation. The activity and expression of Runx2 in hypertrophic chondrocytes were analysed after the treatment with HA oligosaccharide (HAoligo) using AML-3/Runx2 binding, real-time PCR and Western blot analysis. The effects of pre-incubation of anti-CD44 antibody on Runx2 expression were also examined. Expression of type X collagen and Runx2 mRNAs reached a maximum at the terminal differentiation of chondrocytes. The activity and expression of Runx2 was significantly inhibited in hypertrophic chondrocytes treated with HAoligo compared to the untreated controls. High molecular weight-HA did not affect the expression or activity of Runx2. The expression of Runx2 mRNA was significantly decreased in hypertrophic chondrocytes treated with anti-CD44 antibody. These results suggest that HAoligo may affect the terminal differentiation of chondrocytes during the endochondral ossification by inhibiting the expression and activity of Runx2.

Regression of papular elastolytic giant cell granuloma using narrow-band UVB irradiation.

A 71-year-old Japanese man presented with asymptomatic, firm, brown-red papules on the dorsum of his left hand and back. A biopsy specimen revealed granulomatous infiltration consisting of multinucleated giant cells and histiocytes in the upper dermis. Elastic fibers were absent in the upper dermis, having been phagocytosed by giant cells. These papular elastolytic giant cell granuloma lesions regressed very well with narrow-band ul traviolet B irradiation, with only residual brown pigmentation left after treatment.

Influence of lipophilicity and lysosomal accumulation on tissue distribution kinetics of basic drugs: a physiologically based pharmacokinetic model.

This paper examines the role of lipophilicity in the tissue distribution kinetics of basic drugs. Basic drugs have a large distribution volume and are distributed widely in various tissues in the following order: lung, fat, heart, kidney, brain, gut, muscle and bone. The fat volume in the whole body influences the disposition kinetics. There is a good correlation in various tissues between the tissue-plasma concentration ratio and the octanol-water partition coefficient among various drugs. We constructed a physiologically-based pharmacokinetic model on the basis of drug lipophilicity and found that drug distribution decreased when NH4Cl was administered concomitantly. In regards to subcellular distribution, the relative specific contents of chlorpromazine, imipramine and biperiden with respect to the protein in lysosomes were 7.3, 9.6 and 4.2, respectively, while those in other subcellular organella, including mitochondria, were only 0.4-1.7, indicating preferential accumulation of these drugs in lysosomes. The uptake of basic drugs into lysosomes depended on both intralysosomal pH and drug lipophilicity. As the lipophilicity of the basic drugs increased, they accumulated more than would have been predicted from the pH-partition theory and raised the intralysosomal pH more potently, probably owing to their binding with lysosomal membranes, with or without intralysosomal aggregation. We conclude that the distribution kinetics of basic drugs is driven by drug lipophilicity and uptake into lysosomes, and these phenomena provide a possible basis for drug interaction in clinical treatments.

Withdrawal from nicotine facilitates diazepam binding inhibitor mRNA expression in mouse cerebral cortex.

Changes in diazepam binding inhibitor (DBI) mRNA expression after withdrawal from nicotine were examined. Withdrawal from nicotine Increased DBI mRNA expression in cerebral cortices derived from nicotine-dependent mice and in the neurons continuously exposed to nicotine (0.1 microM). These results indicate that withdrawal from nicotine after its long-term exposure induces steep increase of DBI mRNA expression as reported previously in ethanol- and morphine-dependent animals.

A simple estimation of peroxisomal degradation with green fluorescent protein - an application for cell cycle analysis.

When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO-K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient-starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3-methyladenine suppressed the decay of GFP fluorescence (13-60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.

[Studies on the mechanism of subcellular distribution of basic drugs based on their lipophilicity].

This paper described the studies on the mechanism of subcellular distribution of lipophilic weak bases. Although the tissue distribution of basic drugs appeared to decrease with time simply in parallel with their plasma concentration, their subcellular distribution in various tissues exhibited a variety of patterns. Basic drugs were distributed widely in various tissues, but were concentrated in lung granule fraction, where their accumulation was dependent on their lipophilicity and lysosomal uptake. As the plasma concentration of drugs decreased after maximum level, the contribution of lysosomes to their subcellular distribution increased. The uptake of the basic drugs into lysosomes depended both on their intralysosomal pH and on the drug lipophilicity. As the lipophilicity of the basic drugs increased, they accumulated more than the values predicted from the pH-partition theory and raised the intralysosomal pH more potently, probably owing to their binding with lysosomal membranes with or without additional intralysosomal aggregation. These phenomena should be considered as a basis of drug interaction in clinical treatments.

Suppression of Ca2+ influx through L-type voltage-dependent calcium channels by hydroxyl radical in mouse cerebral cortical neurons.

In the present study, we investigated the effect of hydroxyl radical (.OH) produced by the Fenton reaction with FeSO(4) to H(2)O(2) on Ca2+ influx by measuring [(45)Ca2+] influx into mouse cerebral cortical neurons in primary culture.OH formed from 3 microM FeSO(4) and 0.01 microM H(2)O(2) significantly reduced 30 mM KCl-induced [(45)Ca2+] influx and this reduction was abolished by .OH scavengers such as N,N'-dimethylthiourea and mannitol. Nifedipine (1 microM), an inhibitor for L-type voltage-dependent Ca2+ channels (VDCCs) showed no additive effect on the reduction of the 30 mM KCl-induced [(45)Ca2+] influx, while the inhibitors for P/Q- and N-type VDCCs showed further suppression of the KCl-induced [(45)Ca2+] influx even in the presence of .OH. Bay k 8644, an activator of L-type VDCCs, dose-dependently stimulated [(45)Ca2+] influx, and this stimulation disappeared in the presence of nifedipine. Similarly, .OH also suppressed significantly [(45)Ca2+] influx induced by Bay k 8644. These inhibitory actions of .OH on the KCl- and Bay k 8644-induced [(45)Ca2+] influx were completely abolished by .OH scavengers. These results indicate that .OH has the activity to suppress Ca2+ influx through L-type VDCCs.

Recent research on alcohol tolerance and dependence.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hiroshi Suwaki and Harold Kalant. The presentations were (1) Influence of ADH genotypes on acute alcohol withdrawal syndrome in Japanese, by Susumu Higuchi; (2) Use of genetic analyses to refine phenotypes related to alcohol tolerance and dependence, by John C. Crabbe; (3) Neurochemical basis for alcohol dependence, by Seitaro Ohkuma and Masashi Katsura; (4) Adenylyl cyclase and development of tolerance to addictive drugs, by Masami Yoshimura; (5) Tolerance in rat lines selectively bred for alcohol preference, by Robert C. Stewart and Ting-Kai Li; and (6) Ethanol reinforcement, dependence, and vulnerability to relapse: Interactions between neuroadaptive and conditioning factors, by Friedbert Weiss.

NMDA receptor activation enhances diazepam binding inhibitor and its mRNA expressions in mouse cerebral cortical neurons.

Effects of N-methyl-D-aspartate (NMDA) on diazepam binding inhibitor (DBI) and its mRNA expression in mouse cerebral cortical neurons were examined. A significant increase in DBI mRNA expression was observed 1 day after the exposure to 0.1 microM NMDA and the maximal expression occurred 2 days after the exposure, whereas transient exposure to 0.1 microM NMDA for 15 min, 1 and 3 h produced no changes in the expression. Similarly, no changes in the expression were found by the concomitant exposure to NMDA and MK-801, a NMDA receptor antagonist, for 72 h subsequent to the incubation with NMDA alone for 3 h. Such NMDA-induced increases in DBI mRNA expression were dose-dependently inhibited by MK-801. Moreover, neuronal DBI content significantly increased by treatment with NMDA, which was completely abolished by MK-801. These results indicate that continuous activation of NMDA receptors is an essential factor for increasing DBI expression in the neurons.

Alterations in cerebral diazepam binding inhibitor expression in drug dependence: a possible biochemical alteration common to drug dependence.

Mechanisms for formation of drug dependence and expression of withdrawal syndrome have not fully clarified despite of huge accumulation of experimental and clinical data at present. Several clinical features of withdrawal syndrome are considered to be common among patients with drug dependence induced by different drugs of abuse. One of them is anxiety. Recent investigations have revealed that diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids with molecular weight of about 10 kDa, serves as an inverse agonist for benzodiazepine (BZD) receptors with endogenously anxiogenic potential. These lines of data suggest that cerebral DBI expression in brain may participates in formation of drug dependence and/or emergence of withdrawal syndrome. Based on this working hypothesis, we have examined DBI expression in the brain derived from mice depended on alcohol (ethanol), nicotine, and morphine to investigate functional relationship between cerebral DBI expression and drug dependence. Cerebral DBI expression significantly increases in animals with drug dependence induced by these drugs, and in the cases of nicotine- and morphine-dependent mice concomitant administration of antagonists for nicotinic acetylcholine and opioid receptors, respectively, abolished the increase. Abrupt cessation of administration of drugs facilitated further increase in DBI expression. Therefore, these alterations in DBI expression have close relationship with formation of drug dependence and/or emergence of withdrawal syndrome, and are considered to be a common biochemical process in drug dependence induced by different drugs of abuse. Finding and elucidation of mechanisms for common biochemical alterations among drug dependence may provide a clue to clarify mechanisms for formation of drug dependence and/or emergence of withdrawal syndrome.

Nitric oxide and peroxynitrite as factors to stimulate neurotransmitter release in the CNS.

This review summarizes the stimulatory potentials of NO and peroxynitrite (OONO-) on neurotransmitter release in the central nervous system. Exogenous and endogenous NO stimulates to release neurotransmitter. NO synthesized intracellularly diffuses out through neuronal membrane and acts on the outer side of membrane to depolarize neuronal membrane, which triggers neurotransmitter release. NO-induced release of neurotransmitters is mediated by Ca2+-dependent and -independent processes. The latter process is operated by reverse process of the Na+-dependent carrier-mediated neurotransmitter uptake system or by unknown mechanisms. Ca2+-dependent release of neurotransmitter occurs in part subsequent to increase in Ca2+ influx via VDCCs, although N-type VDCCs may not involve in this action of NO because of suppression of Ca2+ influx through N-type VDCCs by NO. Participation of cGMP formation by NO on neurotransmitter release is controversial. A superoxide scavenger, Ca2+, Zn(2+)-superoxide dismutase, abolishes NO-induced neurotransmitter release and synthesized OONO- induces neurotransmitter release, indicating that OONO- participates in NO-evoked neurotransmitter release.

Peroxynitrite affects Ca2+ influx through voltage-dependent calcium channels.

The effect of peroxynitrite (OONO-) on voltage-dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO- time- and dose-dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4-benzoic acid) porphyrin, a scavenger for OONO-. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO(-)-induced [45Ca2+] influx. OONO-, as well as 30 mm KCl, significantly increased fluorescence intensity of cell-associated bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose-dependently suppressed OONO(-)-induced [45Ca2+] influx. Although each of 1 microM nifedipine and 1 microM omega-agatoxin VIA (omega-ATX) significantly inhibited the OONO(-)-induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 microM omega-conotoxin GVIA (omega-CTX) showed no effect on the influx. On the other hand, OONO- itself reduced 30 mM KCl-induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO- alone, and the magnitude of this reduction was as same as that of KCl-induced [45Ca2+] influx by omega-CTX. These results indicate that OONO- increases [45Ca2+] influx into the neurones through opening P/Q- and L-type VDCCs subsequent to depolarization, and inhibits the influx through N-type VDCCs.

Presence of Vicia graminea or Vicia unijuga lectin-binding (Vgu) glycoproteins with and without Thomsen-Friedenreich (T) antigen in normal human seminal plasma.

Vgu glycoprotein (Vicia graminea lectin- or Vicia unijuga lectin-binding glycoprotein) has been reported as oncofetal antigen, which is found in many kind of tumor tissues, amniotic fluid and fetal membranes. In autoradiography with an 125I-labeled Vicia unijuga lectin (VUA) probe and an 125I-labeled Arachis hypogaea lectin (PNA, anti-T lectin) probe, seminal plasma samples of eight healthy men gave 2-7 Vgu glycoproteins without T antigen, 1-2 Vgu glycoproteins with Thomsen-Friedenreich antigen (T antigen) and 1-8 T-antigen glycoproteins, respectively. These results show that in addition to T-antigen glycoproteins, normal human seminal plasma contains Vgu glycoproteins and Vgu glycoproteins with T antigen as seminal plasma components as well as other tumor markers such as CA19-9, CA-125 and CEA.

Reversible and potent uncoupling of hog gastric (H(+)+K(+))-ATPase by prodigiosins.

Prodigiosin, prodigiosin 25-C, and metacycloprodigiosin all strongly inhibited the acidification activity of (H(+)+K(+))-ATPase on membrane vesicles from hog gastric mucosa (IC(50) = 32 to 103 pmol/mg protein). But, the prodigiosins, unlike omeprazole, showed little inhibitory effect on K(+)-dependent ATPase (K(+)-ATPase) activity, although at higher concentrations they inhibited K(+)-ATPase activity with an IC(50) of 1.5 to 3.0 microM. Furthermore, the inhibitory effect of the prodigiosins was rapid and completely reversible unlike that of omeprazole, and the mode of inhibition was non-competitive with respect to ATP. Hog gastric (H(+)+K(+))-ATPase itself showed an absolute requirement of halide (effectively, chloride) for acidification activity. Prodigiosins also showed a chloride requirement for inhibition of vesicular acidification, and quickly reversed the acidification of vesicular pH to neutrality even in the presence of N, N'-dicyclohexylcarbodiimide (DCCD), showing their ionophoric nature of acidification inhibitory activity. In fact, tributyltin chloride (TBT, an OH(-)/Cl(-) exchange ionophore) also inhibited vesicular acidification, but it inhibited K(+)-ATPase activity too. Finally, the prodigiosins inhibited the acid secretion from parietal cells isolated from rabbit gastric mucosa. These results suggest that prodigiosins are potent reversible uncouplers of (H(+)+K(+))-ATPase that inhibit gastric acid secretion.

Vacuolar H(+)-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts in cultured human saphenous vein.

BACKGROUND: The molecular mechanism of neointimal hyperplasia after vein graft surgery remains elusive. Vacuolar H(+)-ATPase (V-ATPase) is involved in intracellular trafficking and may play a crucial role in neointimal cell growth. METHODS AND RESULTS: Cultured human saphenous vein segments developed neointimal formation within 10 days. Neointimal cells were positive for vimentin and alpha-smooth muscle actin but negative for desmin, which is indicative of myofibroblasts. Those myofibroblasts were found to have originated from periadventitial fibroblasts, which upregulated the expression of 16-kDa proteolipid of V-ATPase before proliferation and phenotypic modulation. Neointimal myofibroblast growth and survival were highly sensitive to inhibition of V-ATPase by bafilomycin A(1) (BA(1)), because the incorporation of [(3)H]thymidine into the myofibroblasts was significantly inhibited by nanomolar concentrations of BA(1) and apoptotic cell death was induced by a similar concentration range of BA(1). In contrast, endothelial cells and differentiated smooth muscle cells were resistant to apoptosis by BA(1). CONCLUSIONS: These results suggest that V-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts that contributes to neointimal formation in cultured human saphenous vein.