Isolation of retinoic acid-resistant clones from human breast cancer cell line MCF-7 with altered activity of cellular retinoic acid-binding protein.

After ethyl methane sulfonate mutagenesis of the mammary carcinoma cell line, MCF-7, we have isolated three clones, U-2, U-3, and U-9, resistant to retinoic acid. These three clones showed more than a 1000-fold higher level of resistance to retinoic acid than the parental MCF-7 cells when assayed by colony formation in monolayer culture system or by growth curves. The three resistant clones showed a 200-fold higher resistance to 13-cis-retinoic acid, about 10-fold higher resistance to retinol, and about 2-fold higher resistance to retinyl acetate, respectively, than MCF-7. Binding of [3H]retinoic acid or [3H]-retinol to a cellular fraction in situ showed apparent decrease of the specific binding of retinoic acid in U-2, but there was no such specific fraction bound to retinol in U-2 and MCF-7. Sucrose gradient analysis with cytoplasmic fraction showed little, if any, cellular retinoic acid-binding protein in U-2, but a significant amount of the cellular retinoic acid-binding protein could be found in MCF-7. By contrast, there was no activity of cellular retinol-binding protein in both MCF-7 and U-2. The sensitivity or resistance of mammalian cells in culture to retinoic acid is discussed in relation with cellular binding activity for vitamin A.

Potentiation by a biscoclaurine alkaloid, cepharanthine, of the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin in HeLa cells.

Cepharanthine, a biscoclaurine alkaloid, causes an 8-fold enhancement of the cytotoxic effect of a conjugate of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin in HeLa cells. Cepharanthine also potentiates the effect of Pseudomonas exotoxin. Cepharanthine does not affect the binding and uptake of 125I-EGF by HeLa cells, but it delays the release of radioactivity associated with 125I-EGF into the medium. Analysis by colloidal silica gradients using cell homogenates suggests that 125I-EGF accumulates in the lysosomes of cells treated with cepharanthine and that [3H]cepharanthine accumulates in lysosomes. The pH in HeLa cell lysosomes is 5.2, and cepharanthine does not significantly increase the pH. Electron microscopy shows an increased number of electron-dense bodies and dilated Golgi apparatus after cepharanthine treatment. Cepharanthine appears to accumulate in lysosomes, and it may delay degradation of EGF-Pseudomonas exotoxin in the cells as does 125I-EGF.

Effects of exogenous phospholipase enzymes, arachidonic acid and 1-oleoyl-2-acetyl-sn-glycerol on ketogenesis in isolated rat hepatocytes.

Studies were conducted to see whether exogenous phospholipase C from Clostridium perfringens, phospholipase A2 from Crotalus adamanteus venom, arachidonic acid and 1-oleoyl-2-acetyl-sn-glycerol (OAG) mimic the anti-ketogenic action of vasopressin in isolated rat hepatocytes. Exogenous phospholipase C inhibited ketogenesis in the presence of 0.5 mM oleate. Experiments employing [1-14C]oleate, however, indicated that the mechanism involved in the anti-ketogenic action of exogenous phospholipase C is distinct from that of vasopressin. The decreased rate of the production of acid-soluble products from [1-14C]oleate in response to vasopressin could be explained by the sum of the increased rates of 14CO2 formation and [1-14C]oleate esterification. By contrast, exogenous phospholipase C suppressed not only the formation of acid-soluble products but also 14CO2 production and [1-14C]oleate esterification. Indeed, phospholipase C greatly inhibited [1-14C]oleate uptake into hepatocytes. It is suggested that the alteration of the architecture of plasma membrane by exogenous phospholipase C may lead to the disturbance of oleate uptake and consequent general suppression of oleate metabolism. Exogenous phospholipase A2, arachidonic acid and OAG increased ketogenesis regardless of the presence of oleate. The ketogenic effects may be attributed to the supply of fatty acids by these agents to hepatocytes.

Alpha 1-adrenergic regulation of ketogenesis in isolated rat hepatocytes.

Studies were conducted to clarify the effects of noradrenaline on oleate metabolism in isolated hepatocytes from fed rats. Noradrenaline caused an inhibition of ketogenesis from oleate along with a stimulation of glucose release through alpha 1-adrenergic receptors. Anti-ketogenic action of noradrenaline was confirmed by the suppression of the formation of radioactive acid-soluble products from [1-14C]oleate in response to this agent. Noradrenaline increased the conversion of [1-14C]oleate into 14CO2 but failed to affect [1-14C]oleate esterification. When hepatocytes were incubated in a medium containing 1 mM EGTA but no Ca2+, the effects of noradrenaline on oleate oxidation were negated. On the other hand, noradrenaline-induced increase in glucose release remained unchanged even in the absence of Ca2+ in the incubation medium. Decrease in ketogenesis and increase in glucose release produced by vasopressin was completely abolished by calcium depletion. Noradrenaline caused a significant increase in cAMP levels in both the presence and absence of Ca2+, although the effect was more marked in the latter. Vasopressin did not affect it. The noradrenaline-induced increase in cAMP and glucose release in the absence of Ca2+ was also mediated by alpha 1-adrenergic receptors. These data are discussed and it is suggested that alpha 1-adrenergic agonists may control hepatic ketogenesis and glycogenolysis through two separate signal transduction mechanisms, i.e., a calcium-mobilizing system which is common with vasopressin, and a cAMP generation system which vasopressin lacks.

Usefulness of transanal endoscopic surgery for carcinoid tumor in the upper and middle rectum.

BACKGROUND: This study evaluated the indications and outcome for transanal endoscopic surgery (TES) used to manage rectal carcinoid tumor as compared with those of conventional transanal local resection (TAR). METHODS: The retrospective study subjects were 28 patients with rectal carcinoid tumor treated by TES (n = 17) or TAR (n = 11) between January 1995 and December 2001. Patient and tumor characteristics, operative results, and postoperative outcomes were compared between the two groups. RESULTS: The distance from the anal verge to the distal tumor margin in the TES group (range, 4-12 cm; median, 6.8 cm) was significantly greater than in the TAR group (range, 3-6 cm; median, 4.5 cm) (p = 0.001). The median tumor diameter was 5.5 mm (range, 3-11 mm) in the TES group and 5.0 mm (range, 3-8 mm) in the TAR group, showing no statistical difference. Microscopically, resected specimens in both groups were typical carcinoid tumors restricted to the submucosal layer. No recurrence was noted in either group. CONCLUSION: Whereas TES is useful for patients with small rectal carcinoid tumor of typical histology within the submucosal layer in the upper and middle rectum, TAR is effective for accessing the lower rectum.

Specific Roles of NMDA Receptor Subunits in Mental Disorders.

N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed.

Improvement of learning and increase in dopamine level in the frontal cortex by methylphenidate in mice lacking dopamine transporter.

The symptoms of attention-deficit/hyperactivity disorder (ADHD) are characterized by inattention and hyperactivity-impulsivity. It is a common childhood neurodevelopmental disorder that often persists into adulthood. Improvements in ADHD symptoms using psychostimulants have been recognized as a paradoxical calming effect. The psychostimulant methylphenidate (MPH) is currently used as the first-line medication for the management of ADHD. Recent studies have drawn attention to altered dopamine-mediated neurotransmission in ADHD, particularly reuptake by the dopamine transporter (DAT). This hypothesis is supported by the observation that DAT knockout mice exhibit marked hyperactivity that is responsive to acute MPH treatment. However, other behaviors relevant to ADHD have not been fully clarified. In the present study, we observed learning impairment in shuttle-box avoidance behavior together with hyperactivity in a novel environment in DAT knockout mice. Methylphenidate normalized these behaviors and enhanced escape activity in the tail suspension test. Interestingly, the effective dose of MPH increased extracellular dopamine in the prefrontal cortex but not striatum, suggesting an important role for changes in prefrontal dopamine in ADHD. Research that uses rodent models such as DAT knockout mice may be useful for elucidating the pathophysiology of ADHD.

Molecular cloning of the human Nurr1 gene: characterization of the human gene and cDNAs.

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing dopaminergic neurons. Recently, it was demonstrated that Nurr1 is critical for midbrain dopaminergic cell differentiation. In order to investigate a possible relation of Nurr1 with the pathogenesis of Parkinson's disease or other neuropsychiatric disorders, we have cloned and characterized the human Nurr1 gene. The gene exists as a single copy in the human genome and comprises eight exons spanning 8kb. We determined the complete nucleotide sequence and flanking regions of the gene. Potential regulatory regions included consensus binding sites for NF-kappaB, CREB, and Sp1. Isolation of human Nurr1 cDNAs from fetal brain suggested the presence of a new splicing variant of Nurr1 in the human brain.

Human mammary cancer cell mutants with altered hormone receptor activity.

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.

Changes in expression of the mouse homologues of KIAA genes after subchronic methamphetamine treatment.

Amphetamine abuse may be associated with adaptive changes in gene expression in the brain. In the present study, a newly developed cDNA array system comprising mouse KIAA (mKIAA) cDNA clones was used to examine the gene expression affected by chronic methamphetamine treatment. Approximately 800 mKIAA clones were blotted onto a nylon membrane and hybridized with 33P-labeled cDNA derived from mRNAs isolated from the whole brains of mice that had been treated daily with saline or methamphetamine (2 mg/kg, i.p.) for 2 weeks. The arrays displayed robust hybridization for almost all transcripts. The results obtained from five experiments were averaged, each performed with triplicate samples. Several clones were chosen as positive candidates for methamphetamine-induced changes; however, only Per2 and mKIAA0099 genes showed a significantly increased expression (P < .05). Subsequently, with the focus on the period-related proteins, the expression of these proteins in various parts of the rat brain were assessed by immunoblot analysis. Chronic administration of methamphetamine (8 mg/kg, i.p., for 10 days) caused increased Per2 protein expression in the hippocampus. Interestingly, chronic methamphetamine treatment at a lower dose (4 mg/kg, i.p., for 10 days) induced an increase in SCN circadian oscillatory protein (SCOP) expression, also in the hippocampus. These data suggest that long-lasting alterations of the period-related gene expressions in the hippocampus might play an important role in methamphetamine addiction.

Effects of prenatal methylazoxymethanol treatment on striatal dopaminergic systems in rat brain.

To further examine the effects of prenatal methylazoxymethanol (MAM) treatment on striatal dopaminergic systems, the status of presynaptic dopamine transporters was examined by quantitative autoradiography of [3H]GBR 12935 binding. Significantly higher [3H]GBR 12935 binding was seen in MAM-lesioned striatum in comparison to the controls, indicating relative dopaminergic hyperinnervation in MAM-induced hypoplastic striatum. The effect of prenatal MAM treatment on extracellular levels of dopamine and its metabolites in the striatum was also examined using in vivo microdialysis. As measured in conscious freely-moving rats, prenatal MAM treatment significantly increased basal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the striatum in comparison with control rats. These data suggest that in accordance with morphological dopaminergic hyperinnervation, dopaminergic functions are significantly augmented in MAM-lesioned brains. Thus, it is suggested that MAM-induced microencephalic rats should serve as a good animal model for the study of augmented dopaminergic functions in the striatum.

Bilateral subdiaphragmatic vagotomy does not prevent the behavioral effects of systematically administered ceruletide in mice.

To clarify the vagal mediation of behavioral changes following systematically administered CCK-like peptides, we examined the effects of subcutaneously injected ceruletide on several behavioral parameters. Ceruletide at a dose of 100 micrograms/kg reduced the rates of spontaneous locomotor activity and rearing, and also inhibited methylphenidate- and methamphetamine-induced hyperactivity in both sham-operated and vagotomized mice to same extent, whereas bilateral subdiaphragmatic vagotomy attenuated these behavioral parameters. These results indicate that the ascending sensory pathway mediating a peripheral CCK-elicited signal may not be responsible for producing the behavioral effects of systematically administered CCK-like peptides.

A behavioral pharmacological study on CCK-8 related peptides in mice.

The effects of CCK-8, ceruletide, their non-sulfated forms and CCK-4 on locomotor activity and rearing in mice were examined. CCK-8 and ceruletide, but not their non-sulfated forms and CCK-4, significantly inhibited the behavioral parameters dose-dependently. The inhibitory effects of CCK-8 and ceruletide were similar, but ceruletide acted more slowly and its inhibitory effect continued much longer than CCK-8, suggesting a difference in stability with the chemical structure. The antagonistic effects of ceruletide on behavioral changes induced by DA agonists were also examined. Peripherally injected ceruletide antagonized both methylphenidate- and methamphetamine-induced hyperactivity in mice dose-dependently, whereas it had no dose-related antagonistic effect on methylphenidate-induced stereotyped behavior. Ceruletide also significantly inhibited apomorphine-induced hyperactivity when injected peripherally. However, no obvious dose-response relationship was observed in either intensity or duration of inhibitory action of ceruletide. These findings suggest that ceruletide does not interfere with dopaminergic transmission due to the blockade of postsynaptic DA receptors in the brain. In conclusion, peripheral injection of CCK-8 and ceruletide may affect directly or indirectly dopamine function producing behavioral changes that resemble those of neuroleptics in some respects.

Effect of ceruletide on discriminated avoidance behavior in rats.

To clarify the central action of peripherally administered ceruletide, we examined the effects of ceruletide and four different classes of neuroleptics, i.e. haloperidol, chlorpromazine, oxypertine and sulpiride, on the discriminated avoidance response (DAR) in rats. Ceruletide and sulpiride did not suppress the DAR over a broad range of doses, whereas other three neuroleptics caused a dose-related decrease in both avoidance and response rates. In addition, the combined administration of ceruletide (100 micrograms/kg s.c.) and neuroleptics at critical doses that suppress the DAR caused a significant reduction in the avoidance rate without affecting the response rate, compared with neuroleptics alone. These findings suggest that ceruletide influences the central dopaminergic system, potentiating the central effects of neuroleptics and producing the favorable therapeutic effects observed in the clinical trials.

Effects of ceruletide and haloperidol on the hypothalamo-pituitary beta-endorphin system and brain beta-endorphin contents in the rat: with special reference to effects of ceruletide in chronically haloperidol-treated rats.

Subcutaneous (sc) administration of 200 micrograms/kg ceruletide (CER), a decapeptide chemically related CCK-8, and 5 mg/kg haloperidol (HLP) to rats increased the plasma immunoreactive beta-endorphin (ir-beta-END) level. The combined injection of CER and haloperidol caused higher plasma ir-beta-END levels than either drug alone. High plasma ir-beta-END levels returned to control levels on the 2nd day. Prior intraperitoneal (ip) administration of a CCK receptor antagonist, L-364,718 (3 mg/kg), but not proglumide (400 mg/kg, ip), inhibited CER-induced, but not HLP-induced, elevation in plasma ir-beta-END levels. The dopamine agonist, bromocriptine (1 mg/kg, ip) decreased plasma ir-beta-END levels, but had not effect on CER-induced elevation in plasma ir-beta-END levels, whereas bromocriptine-induced reduction in plasma ir-beta-END levels was antagonised by HLP. CER injection to chronically HLP-treated rats caused a greater elevation of plasma ir-beta-END levels compared to saline-injected rats. In contrast to the acute experiment, plasma ir-beta-END levels remained elevated over a period of 24 h. In the acute experiment, CER, HLP or the combined treatment with these two drugs had no effect on ir-beta-END contents in the pituitary gland and brain. In the chronic experiment, HLP increased the adenohypophyseal and septal ir-beta-END contents and decreased the hippocampal ir-beta-END contents 24 h after the final HLP injection. CER caused a small reduction only in the hippocampal ir-beta-END contents of CER-injected rats 15 min after injection. When determined on the 2nd day, however, the increases in the adenohypophyseal and septal ir-beta-END contents and the decrease in the hippocampal ir-beta-END contents observed in CER-injected rats were of the same magnitude as those of rats not given the CER injection. These findings indicate that CER stimulates the release of ir-beta-END from the adenohypophysis through CCK-A receptors and that elevated plasma ir-beta-END levels is partly involved in some behavioural effects induced by CER. Furthermore, sustained elevation of plasma ir-beta-END levels after a single injection of CER to chronically HLP-treated rats may explain its long-lasting therapeutic and behavioural effects.

A behavioural pharmacological study on intracerebroventricularly administered CCK-8 related peptides in mice.

The sulfated form of cholecystokinin octapeptide (CCK-8) and ceruletide (CER), but not their non-sulfated forms of CCK-4, significantly decreased the rates of locomotor activity and rearing during the first 10 min test session 10 min after intracerebroventricular (ICV) administration at doses more than 25 and 3.125 mg, respectively. CER-S antagonized methylphenidate-induced hypermotility after ICV administration at a dose of 800 ng. Plasma levels of CER-like immunoreactivity (CER-LI) measured at 120 min after subcutaneous injection, when the locomotor suppressive activity induced by 100 and 200 micrograms was no longer observed, were similar to or much higher than that 30 min after ICV administration at a dose of 800ng, suggesting that the effects of ICV CER-S are not mediated by a peripheral redistribution. These findings indicate that (1) the structural requirement for the locomotor suppressive activity is sulfated tyrosine residue; (2) the behavioural effects of ICV-administered CCK-8-S and CER-S are due to their central actions and mediated by the/inhibition of the central dopamine (DA) function; and (3) CCK-8-S in the brain is functionally associated with the central DA system.

The effects of intraperitoneally administered phencyclidine on the central nervous system: behavioral and neurochemical studies.

The effects of intraperitoneally (IP) injected phencyclidine (phencyclohexyl piperidine; PCP) on the metabolism of dopamine (DA) and cholecystokinin-like immunoreactivity (CCK-LI) in the rat brain were investigated in connection with PCP-induced behavioral changes. The predominant behavior change elicited by 2.5 mg/kg PCP was locomotion, while with higher doses (5 and 10 mg/kg) sniffing, swaying and falling were observed in addition to the enhanced locomotor activity. Backpedaling and rotation were observed in 10 mg/kg PCP-treated rats. IP injection of PCP caused a dose-related increase in the levels of DA and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the medial frontal cortex (MFC) and anterior cingulate cortex (ant.CC) without any changes in the nucleus accumbens (NAc) or striatum. CCK-LI in the MFC, ant.CC and NAc was decreased in a dose-dependent manner following IP injection of PCP. These findings support the evidence that PCP selectively activates the mesocortical DA systems. Furthermore, our results indicate a functional relationship between the mesocortical DA neurons and intrinsic CCK containing cortical neurons, and the change in the activity of the intrinsic CCK-containing cortical neurons in these two areas, perhaps due to an alteration in DA transmission, might be involved in behavioral changes after PCP injection.

Effects of systemically administered ceruletide on the in vivo release and metabolism of dopamine in the medial prefrontal cortex of awake, freely moving rats: an in vivo microdialysis study.

The effects of the cholecystokinin octapeptide-related peptide, ceruletide (CER), on the in vivo release and metabolism of dopamine (DA) in the medial prefrontal cortex were examined in awake, freely moving rats, using in vivo microdialysis. Subcutaneously administered CER (200 micrograms/kg) increased extracellular levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), indicating that extracellular levels of DOPAC and HVA may reflect DA release in the medial prefrontal cortex. Bilateral subdiaphragmatic vagotomy markedly attenuated the CER-induced effect, but did not abolish it completely. CER (10(-7) and 10(-10) M), applied locally via the dialysis tube, had no effect on the extracellular levels of either DOPAC or HVA. The CCK-A receptor antagonist, L-364,718 (3 mg/kg, i.p.), completely prevented CER-induced increases in the extracellular levels of DOPAC and HVA. The CCK-B antagonist, L-365,260 (3 mg/kg, i.p.), however, given 1 h before the CER treatment, slightly attenuated the CER-induced increase in the extracellular levels of DOPAC, but not the CER-induced increase in HVA, 60-180 min after the treatment. These findings indicate that systemically administered CER modulates the in vivo release and metabolism of DA in the medial prefrontal cortex. We suggest that systemically administered CER exerts its action on both vagal afferent nerves and the area postrema via CCK-A receptors, thus enhancing the in vivo release and metabolism of DA in the medial prefrontal cortex.

Effect of chronic amitriptyline administration on serotonergic receptors in rats with methylazoxymethanol-induced microencephaly.

Methylazoxymethanol (MAM)-induced cortical hypoplasia resulted in a 20% decrease in the Bmax of 5-HT2A receptors in the frontal cortex with no change in the Bmax of 5-HT1A receptors. Chronic treatment with amitriptyline did not further decrease the Bmax of 5-HT2A receptors in the MAM-lesioned cortex, suggesting that the persistent down-regulation of cortical 5-HT2A receptors in MAM-lesioned rats was induced by serotonergic hyperinnervation.

The atypical antipsychotic sertindole enhances efflux of dopamine and its metabolites in the rat cortex and striatum.

Previous studies have shown that sertindole (1-[2-[4-[5-chloro-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl]ethyl ]-2 imidazolidinone), an atypical antipsychotic drug that is a potent 5-HT2A and dopamine D2 receptor antagonist, preferentially affects mesocorticolimbic rather than mesostriatal dopamine neurons. Using in vivo microdialysis in conscious rats, we investigated the effects of sertindole on dopamine release and metabolism in the striatum and the medial prefrontal cortex. Systemic administration of sertindole dose dependently enhanced dopamine release in the medial prefrontal cortex and the striatum to the same extent.