Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas.

Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in gliomas, myeloid leukemias, chondrosarcomas and thyroid cancer. We discovered IDH1 and IDH2 mutations in 34 of 326 (10%) intrahepatic cholangiocarcinomas. Tumor with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3 lysine 79 (H3K79). Mutations in IDH1 or IDH2 were associated with longer overall survival (P=0.028) and were independently associated with a longer time to tumor recurrence after intrahepatic cholangiocarcinoma resection in multivariate analysis (P=0.021). IDH1 and IDH2 mutations were significantly associated with increased levels of p53 in intrahepatic cholangiocarcinomas, but no mutations in the p53 gene were found, suggesting that mutations in IDH1 and IDH2 may cause a stress that leads to p53 activation. We identified 2309 genes that were significantly hypermethylated in 19 cholangiocarcinomas with mutations in IDH1 or IDH2, compared with cholangiocarcinomas without these mutations. Hypermethylated CpG sites were significantly enriched in CpG shores and upstream of transcription start sites, suggesting a global regulation of transcriptional potential. Half of the hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant gliobastomas, suggesting the existence of a common set of genes whose expression may be affected by mutations in IDH1 or IDH2 in different types of tumors.

Beta-adrenoceptor control of G protein function in the neonate: determinant of desensitization or sensitization.

Neonatal beta-adrenoceptors (beta-ARs) are resistant to agonist-induced desensitization. We examined the functioning of G(i) and G(s) after repeated administration of beta-AR agonists to newborn rats. Isoproterenol (beta(1)/beta(2) agonist) obtunded G(i) function in the heart but not the liver; in contrast, terbutaline, a beta(2)-selective agonist, enhanced G(i) function. Isoproterenol, but not terbutaline, increased membrane-associated G((s)alpha), which would enhance receptor function. In addition, isoproterenol increased and terbutaline maintained the proportion of the short-splice (S) variant of G((s)alpha) in the membrane fraction; G((s)alpha)S is functionally more active than the long-splice variant. Either isoproterenol or terbutaline treatment increased G((s)alpha) in the cytosolic fraction, a characteristic usually associated with desensitization in the adult. Decreased G(i) activity, coupled with increased membrane-associated G((s)alpha) concentrations and maintenance or increases in membrane G((s)alpha)S, provide strong evidence that unique effects on G protein function underlie the ability of the immature organism to sustain beta-AR cell signaling in the face of excessive or prolonged stimulation; these mechanisms also contribute to tissue selectivity of the effects of beta-agonists with divergent potencies toward different beta-AR subtypes.

Are developing beta-adrenoceptors able to desensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver.

During fetal and neonatal development, beta-adrenergic receptors (beta-ARs) appear to be resistant to desensitization by beta-agonist drugs. To determine the mechanisms underlying the regulatory differences between adults and neonates, we administered isoproterenol, a mixed beta(1)/beta(2)-AR agonist, and terbutaline, a beta(2)-selective agonist. Effects were examined in the ensuing 4 h after a single injection, or after the last of four daily injections. We prepared cell membranes from heart (predominantly beta(1)-ARs) and liver (predominantly beta(2)-ARs) and assessed signal transduction in the adenylyl cyclase (AC) pathway. In the first few hours after a single administration of isoproterenol to adult rats, cardiac beta-ARs showed activation of G proteins (elevated AC response to forskolin) and desensitization of beta-AR-mediated responses; after the fourth injection, heterologous desensitization emerged, characterized by a loss of signaling mediated either through beta-ARs or glucagon receptors. Terbutaline evoked an increase in the forskolin response but no desensitization of receptor-mediated responses. When we gave the same treatments to neonatal rats, we observed cardiac G protein activation, but there was neither homologous nor heterologous desensitization of beta-ARs or glucagon receptors. In the adult liver, isoproterenol and terbutaline both failed to evoke desensitization, regardless of whether the drugs were given once or for 4 days. In neonates, however, acute or chronic treatment elicited homologous desensitization of beta-AR-mediated AC signaling, while sensitizing the response to glucagon. These results show that neonatal beta-ARs are inherently capable of desensitization in some, but not all, cell types; cellular responses can be maintained through heterologous sensitization of signaling proteins downstream from the receptor. Differences from adult patterns of response are highly tissue selective and are likely to depend on ontogenetic differences in subtypes of beta-ARs and AC.

beta-Adrenergic modulation of muscarinic cholinergic receptor expression and function in developing heart.

Imbalances of beta-adrenoceptor (beta-AR) and muscarinic ACh receptor (mAChR) input are thought to underlie perinatal cardiovascular abnormalities in conditions such as sudden infant death syndrome. Administration of isoproterenol, a beta(1)/beta(2)-AR agonist, to neonatal rats on postnatal days (PN) 2-5 caused downregulation of cardiac m(2)AChRs and a corresponding decrement in their control of adenylyl cyclase activity. Terbutaline, a beta(2)-selective agonist that crosses the placenta and the blood-brain barrier, was also effective when given either on PN 2-5 or during gestational days 17-20. Terbutaline failed to downregulate brain m(2)AChRs, even though it downregulated beta-ARs; beta-ARs and m(2)AChRs are located on different cell populations in the brain, but they are on the same cells in the heart. Destruction of catecholaminergic neurons with neonatal 6-hydroxydopamine upregulated cardiac but not brain m(2)AChRs. These results suggest that perinatal beta-AR stimulation shifts cardiac receptor production away from the generation of m(2)AChRs so that the development of sympathetic innervation acts as a negative modulator of cholinergic function. Accordingly, tocolytic therapy with beta-AR agonists may compromise the perinatal balance of adrenergic and cholinergic inputs.

Perinatal exposure to environmental tobacco smoke upregulates nicotinic cholinergic receptors in monkey brain.

In humans, perinatal exposure to environmental tobacco smoke (ETS) is associated with neurobehavioral deficits. In the current study, we exposed Rhesus monkeys to ETS in late gestation and in the early neonatal period, and examined changes in neurotransmitter receptors in the brainstem and caudal portion of the cerebral cortex. Nicotinic acetylcholine receptors were markedly upregulated and the effect was selective in that there were no changes in m(2)-muscarinic acetylcholine receptors or in beta-adrenergic receptors. Nicotinic receptor upregulation is indicative of chronic cell stimulation by nicotine, and is a hallmark of nicotine-induced neuroteratogenesis. These results indicate that perinatal ETS exposes the fetus and neonate to quantities of nicotine that are sufficient to alter brain development.

Beta-adrenoceptor-mediated cell signaling in the neonatal heart and liver: responses to terbutaline.

Terbutaline, a beta(2)-adrenoceptor (beta(2)-AR) agonist, is a widely used tocolytic that also crosses the placenta to stimulate fetal beta-ARs. The current study examines the effects of terbutaline administered to neonatal rats. Terbutaline (10 mg/kg sc) given on postnatal day (PN) 2-5 or PN 11-14 elicited significant downregulation of both cardiac and hepatic beta-ARs, with a much greater effect in the liver. Despite the reduction in cardiac beta-ARs, receptor desensitization was absent as evidenced by the maintained ability of isoproterenol to stimulate adenylyl cyclase (AC) in membrane preparations. The underlying mechanism was dissected by using stimulants that operate at different points in the AC signaling pathway, NaF, forskolin, and Mn(2+). When administered in the early neonatal period, terbutaline failed to evoke any changes in cardiac AC activity; however, treatment on PN 11-14 evoked heterologous sensitization downstream from the receptor, evidenced by increases in the response to NaF and forskolin. In the liver, neonatal terbutaline administration elicited a small (approximately equal to 10%) decrease in the AC response to isoproterenol, an effect much smaller than the downregulation of beta-ARs (>40%). In this tissue, desensitization was again offset by heterologous sensitization of AC signaling. These results indicate that, in the developing organism, beta-AR-mediated cell signaling responses are maintained in the face of receptor downregulation through heterologous induction of downstream signaling elements. These unique responses serve to sustain beta-AR signaling in the perinatal period.

Regulation of fetal cardiac and hepatic beta-adrenoceptors and adenylyl cyclase signaling: terbutaline effects.

Terbutaline (Ter), a beta(2)-adrenergic agonist used in preterm labor, stimulates fetal beta-adrenoceptors (beta-ARs). We administered Ter to pregnant rats on gestational days 17-20 and examined beta-ARs and adenylyl cyclase (AC) signaling in heart and liver. Ter produced less downregulation of cardiac beta-ARs than in adults, despite a higher proportion of the beta(2)-subtype, and failed to elicit desensitization of the receptor-mediated AC response. AC stimulants acting at different points indicated an offsetting of homologous desensitization at the level of the beta-AR by heterologous sensitization at the level of AC: induction of total AC catalytic activity and a shift in the catalytic profile or AC isoform. In fetal liver, Ter produced downregulation of beta-ARs, in keeping with the predominance of the beta(2)-subtype; hepatic receptor downregulation was equivalent in fetus and adult. Nevertheless, there was still no desensitization of beta-AR-mediated AC responses and again AC was induced. Our results indicate that, unlike in the adult, fetal beta-AR signaling is not desensitized by beta-agonists and, in fact, displays heterologous sensitization, thus sustaining responses during parturition. At the same time, the inability to desensitize beta-AR AC responses may lead to disruption of cardiac, hepatic, or neural cell development as a consequence of tocolytic therapy with beta-agonists.

Perinatal exposure to environmental tobacco smoke induces adenylyl cyclase and alters receptor-mediated cell signaling in brain and heart of neonatal rats.

Perinatal exposure to environmental tobacco smoke (ETS) has adverse effects on neurobehavioral development. In the current study, rats were exposed to ETS during gestation, during the early neonatal period, or both. Brains and hearts were examined for alterations in adenylyl cyclase (AC) activity and for changes in beta-adrenergic and m2-muscarinic cholinergic receptors and their linkage to AC. ETS exposure elicited induction of total AC activity as monitored with the direct enzymatic stimulant, forskolin. In the brain, the specific coupling of beta-adrenergic receptors to AC was inhibited in the ETS groups, despite a normal complement of beta-receptor binding sites. In the heart, ETS evoked a decrease in m2-receptor expression. In both tissues, the effects of postnatal ETS, mimicking passive smoking, were equivalent to (AC) or greater than (m2-receptors) those seen with prenatal ETS mimicking active smoking; the effects of combined prenatal and postnatal exposure were equivalent to those seen with postnatal exposure alone. These data indicate that ETS exposure evokes changes in cell signaling that recapitulate those caused by developmental nicotine treatment. Since alterations in AC signaling are known to affect cardiorespiratory function, the present results provide a mechanistic link reinforcing the participation of ETS exposure, including postnatal ETS, in disturbances culminating in events like Sudden Infant Death Syndrome.

Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity.

Chlorpyrifos has been hypothesized to interact with receptors and transduction proteins involved in the production of cyclic AMP, contributing to adverse effects on cell replication and differentiation. We studied the effects of neonatal chlorpyrifos exposure on hepatic adenylyl cyclase (AC) activity, as the liver accumulates the highest concentrations of chlorpyrifos and is the site for generation of its active metabolite, chlorpyrifos oxon. Newborn rats were given 1 mg/kg of chlorpyrifos s.c. on PN1-4. On PN5, 24 h after the last dose, AC catalytic activity was induced as assessed by the response to the direct AC stimulant, Mn(2+). In contrast, AC activation dependent upon interaction of the enzyme with G-proteins (forskolin) did not show any enhancement, suggesting impairment of G-protein function. This conclusion was confirmed by impaired responsiveness to fluoride, which directly activates G-proteins. In addition, the response of AC to hormonal signals was altered in a receptor-selective manner, with an enhanced response to glucagon but not to the beta-adrenoceptor agonist, isoproterenol. The effects of chlorpyrifos on AC signaling displayed a critical developmental period of vulnerability, as treatment of older rats (PN11-14) failed to cause substantial induction of AC or interference with G-protein signaling, although it did still enhance the glucagon response. In all cases, the effects of chlorpyrifos disappeared within a few days of discontinuing treatment. These results stand in contrast to the delayed deterioration of AC signaling seen in the brain after the same chlorpyrifos treatment. The temporal and organ selectivity of chlorpyrifos' effects on the AC cascade suggest that disruption of membrane signaling occurs consequent to selective effects on cell development, rather than representing a direct interaction between chlorpyrifos and signaling proteins.

Working memory performance and cholinergic effects in the ventral tegmental area and substantia nigra.

The nicotinic antagonist mecamylamine has been found to impair working memory performance in the radial-arm maze (RAM) after s.c. or i.c.v. administration. Mecamylamine has important interactions with dopaminergic (DA) systems. Mecamylamine-induced memory deficits in the RAM are potentiated by the D2 antagonist raclopride and reversed by the D2 agonist quinpirole. The nicotinic agonist nicotine has been found to improve working memory performance in the RAM after s.c. or i.c.v. administration. Nicotine-induced memory improvement in the RAM is potentiated by the D2 agonist quinpirole. The midbrain DA nuclei, the substantia nigra (SN) and the ventral tegmental area (VTA) have relatively dense concentrations of nicotinic receptors which may be critical sites of action for mecamylamine and nicotine. In the current study, the effects of mecamylamine (1, 3.3 and 10 micrograms/side) infusions into the SN (n = 12) and VTA (n = 13) on working memory in the radial-arm maze were examined in adult female Sprague-Dawley rats. The 10-micrograms/side dose of mecamylamine significantly impaired radial-arm maze working memory performance when infused into either the SN or VTA. No significant effects of mecamylamine on response latency were seen. The nicotinic agonists cytisine (0.1, 0.33 and 1.0 microgram/side) and nicotine (0.3, 1.0 and 3.3 micrograms/side) were administered in a counterbalanced order. The high dose of cytisine (1 microgram/side) nearly caused a significant deficit in choice accuracy. Nicotine slightly depressed choice accuracy but not significantly in this study. The interaction of nicotine and mecamylamine was then studied. A dose of 1.0 microgram/side of nicotine caused a significant decrease in choice accuracy. Interestingly, this was significantly reversed by a 3.3-micrograms/side dose of mecamylamine.(ABSTRACT TRUNCATED AT 250 WORDS)