An evolutionary scenario for gonadotrophin-inhibitory hormone in chordates.

In 2000, we discovered a novel hypothalamic neuropeptide that actively inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH peptides have subsequently been identified in most representative species of gnathostomes. They all share a C-terminal LPXRFamide (X = L or Q) motif. GnIH can inhibit gonadotrophin synthesis and release by decreasing the activity of GnRH neuroes, as well as by directly inhibiting pituitary gonadotrophin secretion in birds and mammals. To investigate the evolutionary origin of GnIH and its ancestral function, we identified a GnIH precursor gene encoding GnIHs from the brain of sea lamprey, the most ancient lineage of vertebrates. Lamprey GnIHs possess a C-terminal PQRFamide motif. In vivo administration of one of lamprey GnIHs stimulated the expression of lamprey GnRH in the hypothalamus and gonadotophin ß mRNA in the pituitary. Thus, GnIH may have emerged in agnathans as a stimulatory neuropeptide that subsequently diverged to an inhibitory neuropeptide during the course of evolution from basal vertebrates to later-evolved vertebrates, such as birds and mammals. From a structural point of view, pain modulatory neuropeptides, such as neuropeptide FF (NPFF) and neuropeptide AF, share a C-terminal PQRFamide motif. Because agnathans possess both GnIH and NPFF genes, the origin of GnIH and NPFF genes may date back before the emergence of agnathans. More recently, we identified a novel gene encoding RFamide peptides in the amphioxus. Molecular phylogenetic analysis and synteny analysis indicated that this gene is closely related to the genes of GnIH and NPFF of vertebrates. The results suggest that the identified protochordate gene is similar to the common ancestor of GnIH and NPFF genes, indicating that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. The GnIH and NPFF genes may have diverged by whole-genome duplication during the course of vertebrate evolution.

Chair rising exercise is more effective than one-leg standing exercise in improving dynamic body balance: a randomized controlled trial.

A randomized controlled trial was conducted to compare the effect of a one-leg standing exercise and a chair-rising exercise on body balance in patients with locomotive disorders. Thirty ambulatory patients (mean age: 66.6 years) were randomly divided into two groups (n=15 in each group): a one-leg standing exercise group and a chair-rising exercise group. All the participants performed calisthenics of the major muscles, a tandem gait exercise, and a stepping exercise. The exercises were performed 3 days per week, and the study period was 5 months. Physical function was evaluated at baseline and at one-month intervals. No significant differences in the baseline characteristics were observed between the two groups. After the 5-month exercise program, the timed up and go, one-leg standing time, and tandem gait time improved significantly in the one-leg standing exercise group, while the walking time and chair-rising time in addition to above parameters improved significantly in the chair-rising exercise group. The improvements in the walking time, chair-rising time, and tandem gait time were significantly greater in the chair-rising exercise group than in the one-leg standing exercise group. The present study showed that the chair-rising exercise was more effective than the one-leg standing exercise for improving walking velocity and dynamic body balance.

Identification, localisation and functional implication of 26RFa orthologue peptide in the brain of zebra finch (Taeniopygia guttata).

Several neuropeptides with the C-terminal Arg-Phe-NH(2) (RFa) sequence have been identified in the hypothalamus of a variety of vertebrates. The present study was conducted to isolate novel RFa peptides from the zebra finch brain. Peptides were isolated by immunoaffinity purification using an antibody that recognises avian RFa peptides. The isolated peptide consisted of 25 amino acids with RFa at its C-terminus. The sequence was SGTLGNLAEEINGYNRRKGGFTFRFa. Alignment of the peptide with vertebrate 26RFa has revealed that the identified peptide is the zebra finch 26RFa. We also cloned the precursor cDNA encoding this peptide. Synteny analysis of the gene showed a high conservation of this gene among vertebrates. In addition, we cloned the cDNA encoding a putative 26RFa receptor, G protein-coupled receptor 103 (GPR103) in the zebra finch brain. GPR103 cDNA encoded a 432 amino acid protein that has seven transmembrane domains. In situ hybridisation analysis in the brain showed that the expression of 26RFa mRNA is confined to the anterior-medial hypothalamic area, ventromedial nucleus of the hypothalamus and the lateral hypothalamic area, the brain regions that are involved in the regulation of feeding behaviour, whereas GPR103 mRNA is distributed throughout the brain in addition to the hypothalamic nuclei. When administered centrally in free-feeding male zebra finches, 26RFa increased food intake 24 h after injection without body mass change. Diencephalic GPR103 mRNA expression was up-regulated by fasting for 10 h. Our data suggest that the hypothalamic 26RFa-its receptor system plays an important role in the central control of food intake and energy homeostasis in the zebra finch.

Synchronised expressions of LPXRFamide peptide and its receptor genes: seasonal, diurnal and circadian changes during spawning period in grass puffer.

Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa-R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real-time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa-R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle-stimulating hormone ß- and luteinising hormone ß-subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa-R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa-R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa-R system in the regulation of lunar-synchronised spawning of grass puffer.

Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction.

Gonadotrophin-releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin-inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic-pituitary-gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS-1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS-1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS-1, designated KiSS-2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.

Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

A new key neurohormone controlling reproduction, gonadotrophin-inhibitory hormone in birds: discovery, progress and prospects.

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.

Gonadotropin-inhibitory hormone in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments.

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.

Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.

BACKGROUND: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). METHODS AND RESULTS: The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. CONCLUSIONS: The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.

Bone morphogenetic protein-2 inhibits serum deprivation-induced apoptosis of neonatal cardiac myocytes through activation of the Smad1 pathway.

Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-x(L). Antisense oligonucleotides to bcl-x(L) attenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-x(L) via a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes.

gp130-Dependent signalling pathway is not enhanced in gp130 transgenic heart after LIF stimulation.

Activation of gp130 transduces a hypertrophic signal in the heart, but it is not clear whether signalling through gp130 is enhanced when gp130 is overexpressed in vivo. We generated gp130 transgenic mice (TG) and examined the activation of signalling pathways downstream of gp130 in the hearts. The tyrosine phosphorylation of gp130 was enhanced, the phosphorylation of STAT3 and ERK (extracellular signal regulated kinase) 1/2 was increased and induction of the beta-myosin heavy chain (MHC) gene was observed in TG hearts without significant phenotypic changes. Intravenous administration of leukaemia inhibitory factor (LIF) induced tyrosine phosphorylation of STAT3 and ERK 1/2 and expression of c-fos and beta-MHC mRNAs in wild-type littermates' (WT) hearts. However, enhancement of STAT3 and ERK 1/2 phosphorylation or augmented mRNA expressions was not observed in TG hearts after LIF stimulation. Next, STAT-induced STAT inhibitor (SSI) mRNA expression was examined. The expression of SSI-1, SSI-2, and SSI-3 mRNAs was significantly augmented in TG hearts after LIF stimulation. These results indicate that overexpressed gp130 does not always enhance downstream signals in the hearts and suggest that the SSI family plays a role in the regulation of the gp130-dependent signalling pathway in the hearts.

Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

Activation of glycoprotein (gp) 130 transduces hypertrophic and cytoprotective signals in cardiac myocytes. In the present study, we have demonstrated that signals through gp130 increase the expression of vascular endothelial growth factor (VEGF) in cardiac myocytes via the signal transducer and activator of transcription (STAT) 3 pathway. After activation of gp130 with leukemia inhibitory factor (LIF), expression of VEGF mRNA rapidly increased with a peak at 3 h in cultured cardiac myocytes. Cardiotrophin-1 also enhanced VEGF mRNA expression in a dose-dependent manner. VEGF protein production and secretion to the medium were also enhanced by LIF and cardiotrophin-1 but not by interleukin-6. Adenovirus transfer of the dominant-negative form of STAT3 to cultured cardiac myocytes inhibited induction of VEGF expression induced by LIF, but neither PD98059 nor wortmannin was affected. In murine hearts, intravenous administration of LIF augmented expression of VEGF mRNA; however, the hearts of transgenic mice overexpressing dominant-negative STAT3 showed reduced expression of VEGF mRNA that was not induced after LIF stimulation. These data provide the first evidence that a STAT family protein functions as a regulator of angiogenic growth factors and suggest that gp130/STAT signaling in cardiac myocytes can control vessel growth during cardiac remodeling.

Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy.

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3 in vivo, transgenic mice with cardiac-specific overexpression of the Stat3 gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of the atrial natriuretic factor (ANF), beta-myosin heavy chain (MHC), and cardiotrophin (CT)-1 genes. The animals were injected i.p. with 15 mg/kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5/20) for control littermates (WT), but 80% (16/20) for STAT3-TG (P < 0.01). WT showed increased expression of beta-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of the cardiac alpha-actin and Stat3 genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors.

[Antiretroviral agent nevirapine: its pharmacological action and potential for clinical use].

Nevirapine (NVP) is a potent noncompetitive inhibitor of the reverse transcriptase enzyme, which is necessary for HIV replication. NVP selectively inhibits HIV-1 but not HIV-2 and any of the human DNA polymerases. NVP is active against ZDV-resistant HIV-1 and synergistic with nonnucleoside reverse transcriptase inhibitors. NVP has a favorable pharmacokinetic profile, becomes widely distributed throughout body tissues including the central nervous system, and is active in the adult at an oral dose of 200 mg administered twice daily after a 2 week lead-in dose of 200 mg/day due to its long elimination half life. Although the currently used protease inhibitors (PIs) may undergo more rapid rates of metabolism because NVP induces CYP3A, No dosage adjustments are required when NVP is taken in combination with PIs so far. When administered in triple combinations with antiretroviral agents, the antiviral effect of NVP has been profound and sustained in HIV-infected patients, particularly in naive patients to antiretroviral therapy. Resistance to NVP is rapid when given as monotherapy, but this is altered and made less clinically relevant when NVP is administered as a triple combination. NVP has a safety profile that does not overlap with other antiretroviral therapies, the most common treatment-limiting reaction being rash. It seems that NVP would be a very useful option in combination with antiretroviral agents.

Characterization of a nuclear factor that enhances DNA binding activity of SSCRE-BP/PUR alpha, a single-stranded DNA binding protein.

Pur alpha has been identified as a single-stranded DNA binding protein that specifically binds to the purine-rich strand present in the DNA replication initiation zone of the human c-myc gene. We have previously demonstrated that chronic morphine treatment decreases the DNA binding activity of ssCRE-BP (single-stranded cyclic AMP response element-binding protein), which has been shown to be identical to pur alpha by cDNA cloning, and is abundant in the brain. In this report we identified an activator of ssCRE-BP/pur alpha in the brain and characterized it. Although purified ssCRE-BP/pur alpha or its GST-fusion protein exhibited very low DNA binding activities, they were markedly enhanced by including nuclear extract in the binding assay. The enhanced binding activity is trypsin-sensitive, heat-stable and has a molecular weight of approximately 66 kDa. Casein could substitute for the activator and increased the DNA binding activity of ssCRE-BP/pur alpha by one order. A series of deletion mutants were prepared in order to determine the DNA binding and activator interacting domains, and both of them were found to reside in AA 50-215 of ssCRE-BP/pur alpha. These data suggest that the DNA binding activity of ssCRE-BP/pur alpha is augmented by a nuclear protein, which may modulate the ssCRE-BP/pur alpha activity to develop morphine dependence and tolerance.

Characterization of single-stranded cAMP response element binding protein (ssCRE-BP) from mouse cerebellum.

It has been speculated that opiate tolerance and dependence may occur at the level of gene expression. Our previous studies have shown that the binding activity of a nuclear factor (ssCRE-BP) to single-stranded CRE of somatostatin gene is altered by long-term treatment with morphine in the mouse cerebellum. ssCRE-BP was purified from the mouse cerebellum by a combination of chromatography on DNA affinity agarose and Mono Q HR. The native protein exhibited a molecular size of 110-150 kDa by gel filtration, and two polypeptides of about 35-40 kDa were observed on SDS-PAGE. The cloning and sequencing of a cDNA encoding ssCRE-BP showed that the protein possesses a glycine-rich domain and a glutamine-rich domain in the amino terminus and the carboxyl terminus, respectively. To investigate the function of ssCRE-BP in the brain, recombinant glutathione-S-transferase (GST) fusion proteins containing ssCRE-BP were expressed in bacterial systems. Rabbit anti-ssCRE-BP antibodies were raised against a GST-ssCRE-BP fusion protein. Using the antibodies in western blot analysis, a polypeptide of approximately 66 kDa was detected in the brain. These findings indicate that ssCRE-BP is involved in opiate tolerance and dependence.

Involvement of a single-stranded DNA binding protein, ssCRE-BP/Pur alpha, in morphine dependence.

We have purified a nuclear protein from mouse cerebella that binds to single-stranded oligo-DNA of cAMP response element and is modulated by morphine treatment. Isolation of the cDNA clone showed that the nuclear protein (ssCRE-BP) was identical to Pur alpha, a DNA binding protein for single-stranded purine-rich sequences that was originally isolated as a replication factor. ssCRE-BP/Pur alpha and mRNA were abundant in the brain. The levels of ssCRE-BP/Pur alpha and the transcript were not changed by chronic morphine treatment, however, the levels of an activator of ssCRE-BP/Pur alpha, which is necessary for the DNA binding, may be modulated by the treatment.

Decrease in CRE binding activity by chronic morphine administration in mouse brain.

Recent studies have suggested that opiate addiction is associated with transcriptional changes. We developed a novel method, in situ DNA-protein binding (ISDB), for investigating the distribution and changes of DNA binding activity of transcription factors in the brain. Using this method, we found that cAMP response element (CRE) binding activity was decreased by chronic morphine treatment in specific regions including the amygdala complex, thalamus, cerebral cortex and hypothalamus in mouse brain. This effect persisted for at least 14 days after the cessation of morphine. These data suggest that chronic morphine treatment elicits a long-term change in cAMP-mediated gene expression in the brain.

[Involvement of gene expression in drug tolerance and dependence].

The development of drug tolerance and dependence are thought to be associated with gene expression. Our studies showed that the binding activity of nuclear factors to several DNA sequences is altered by long-term treatment with methamphetamine, cocaine and morphine: 1) the binding activity of AP-1 increased markedly in the mouse brain after administration of methamphetamine and cocaine, 2) CRE-binding activity was decreased by chronic morphine treatment in the amygdala complex, cerebral cortex and hypothalamus of the mouse brain, and 3) the binding activity of single-stranded CRE binding proteins was decreased by chronic morphine treatment in the mouse cerebellum. These data suggest that the changes of DNA binding proteins can be involved in the development of drug tolerance and dependence.

Tyrosine phosphorylation of a 58 kDa protein induced by morphine in SK-N-SH cells.

A 58 kDa protein which was phosphorylated on tyrosine residues with morphine was found in human neuroblastoma cells (SK-N-SH cells) by immunoblot with monoclonal anti-phosphotyrosine antibody. The tyrosine phosphorylation was induced by morphine in 5 min in a dose-dependent manner and the increment was completely inhibited by naloxone. A Delta (d) agonist, [D-Pen2,Pen5]-enkephalin (DPDPE), but not a m agonist, [D-Ala2,N-Me-Phe,Gly5-ol]-enkephalin (DAGO), stimulated the phosphorylation and treatment of the cells with pertussis toxin inhibited the phosphorylation by morphine. These data suggest that d receptor-stimulation increases tyrosine phosphorylation of the 58 kDa protein through Gi protein in SK-N-SH cells.