Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression.

Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4(Chaos3) allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.

Hypersensitivity of primordial germ cells to compromised replication-associated DNA repair involves ATM-p53-p21 signaling.

Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression.

Accumulating evidence suggests that dormant DNA replication origins play an important role in the recovery of stalled forks. However, their functional interactions with other fork recovery mechanisms have not been tested. We previously reported intrinsic activation of the Fanconi anemia (FA) pathway in a tumor-prone mouse model (Mcm4chaos3) with a 60% loss of dormant origins. To understand this further, we introduced a null allele of Fancc (Fancc-), encoding a member of the FA core complex, into the Mcm4chaos3 background. Primary embryonic fibroblasts double homozygous for Mcm4chaos3 and Fancc- (Mcm4chaos3/chaos3;Fancc-/-) showed significantly increased levels of markers of stalled/collapsed forks compared to either single homozygote. Interestingly, a loss of dormant origins also increased the number of sites in which replication was delayed until prophase, regardless of FA pathway activation. These replication defects coincided with substantially elevated levels of genome instability in Mcm4chaos3/chaos3;Fancc-/- cells, resulting in a high rate of perinatal lethality of Mcm4chaos3/chaos3;Fancc-/- mice and the accelerated tumorigenesis of surviving mice. Together, these findings uncover a specialized role of dormant origins in replication completion while also identifying important functional overlaps between dormant origins and the FA pathway in maintaining fork progression, genome stability, normal development and tumor suppression.

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

Helq acts in parallel to Fancc to suppress replication-associated genome instability.

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive. Here, we investigated this question in mice using the Helq(gt) and Fancc(-) strains. Compared with Fancc(-)(/)(-) mice lacking FANCC, a component of the FA core complex, Helq(gt/gt) mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc(-)(/)(-) primary fibroblasts, Helq(gt/gt) cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helq(gt)(/gt);Fancc(-)(/)(-) double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.

Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ.

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease.

FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination.

Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNAK164R from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNAK164R cells. FANCD2 mono-ubiquitination is significantly reduced in PCNAK164R mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.

A reduction of licensed origins reveals strain-specific replication dynamics in mice.

Replication origin licensing builds a fundamental basis for DNA replication in all eukaryotes. This occurs during the late M to early G1 phases in which chromatin is licensed by loading of the MCM2-7 complex, an essential component of the replicative helicase. In the following S phase, only a minor fraction of chromatin-bound MCM2-7 complexes are activated to unwind the DNA. Therefore, it is proposed that the vast majority of MCM2-7 complexes license dormant origins that can be used as backups. Consistent with this idea, it has been repeatedly demonstrated that a reduction (~60%) in chromatin-bound MCM2-7 complexes has little effect on the density of active origins. In this study, however, we describe the first exception to this observation. A reduction of licensed origins due to Mcm4 ( chaos3 ) homozygosity reduces active origin density in primary embryonic fibroblasts (MEFs) in a C57BL/6J (B6) background. We found that this is associated with an intrinsically lower level of active origins in this background compared to others. B6 Mcm4 ( chaos3/chaos3 ) cells proliferate slowly due to p53-dependent upregulation of p21. In fact, the development of B6 Mcm4 ( chaos3/chaos3 ) mice is impaired and a significant fraction of them die at birth. While inactivation of p53 restores proliferation in B6 Mcm4 ( chaos3/chaos3 ) MEFs, it paradoxically does not rescue animal lethality. These findings indicate that a reduction of licensed origins may cause a more profound effect on cell types with lower densities of active origins. Moreover, p53 is required for the development of mice that suffer from intrinsic replication stress.

Walking Attainment in Very Low Birth Weight Infants in Japan.

OBJECTIVE: To clarify the corrected age of walking attainment in very low birth weight infants by birth weight and gestational age, and determine perinatal factors affecting the delay in walking attainment. METHOD: This was a longitudinal study. We investigated walking attainment and perinatal factors in 145 very low birth weight infants without neurological abnormalities (mean birth weight 1019.3 ± 299.7 g, gestational age 29.0 ± 2.9 weeks). The study infants were stratified by birth weight (group A: <1,000 g, group B: 1,000 g≤, <1,500 g) and gestational age (group I: <28 weeks, group II: 28 weeks≤, <37 weeks) and were compared using unpaired t-tests. Furthermore, we examined the perinatal factors that affect the delay in walking attainment using multiple regression analysis. RESULTS: Of the walking attainment, infants in Group A were older than those in Group B (50th percentile, 15.8 vs. 14.7 months). Infants in Group I were older than those in Group II (50th percentile, 16.0 vs. 14.8 months). Using multiple regression analysis with walking attainment age as the dependent variable, the duration of mechanical ventilation was found to be significantly related. CONCLUSION: Very low birth weight infants with light weight and short gestational age have delayed walking attainment, and longer duration of mechanical ventilation increases the risk of delay.

Effects of continued positioning pillow use until a corrected age of six months on cranial deformation and neurodevelopment in preterm infants: A prospective case-control study.

BACKGROUND: Preterm infants have a high risk of cranial deformity resulting from external pressures. Such deformity is associated with delayed neurodevelopment. AIMS: We aimed to clarify the effects of continuous use of positioning pillows on cranial deformity and neurodevelopment in preterm infants. METHODS: This prospective case-control study was conducted between November 2018 and August 2019. The continuous use of a pillow was initiated after discharge from the neonatal intensive care unit, up to a corrected age of six months. Preterm infants weighing less than 1800 g without neurological abnormalities were included in the study. Patients were divided into two groups: non-pillow group (NP-group) and pillow group (P-group). The primary outcome was the Bayley Scales of Infant Development III (BSID-III) score. We compared asymmetrical cranial deformity and the BSID-III scores in the two groups at a corrected age of six months using the Fisher's exact test and unpaired t-test, respectively. RESULTS: There were 19 preterm infants (mean gestational age 32.5 ± 1.9 weeks, birth weight 1461.3 ± 244.7) eligible during the study period. The P-group (n = 11) showed asymmetrical cranial deformity at six months less frequently than the NP-group (n = 8) (p = 0.001, Fisher's exact test). Infants in the P-group had significantly higher scores on the BSID-III cognitive scales (95.0 ± 8.4 vs. 86.9 ± 2.6; p = 0.02, unpaired t-test) and fine motor scores on the motor scales (8.6 ± 2.2 vs. 6.6 ± 0.7, p = 0.02, unpaired t-test). CONCLUSIONS: Continuous pillow use in preterm infants is effective in reducing cranial deformity and improved cognitive and fine motor skills. TRIAL REGISTRATION: UMIN Clinical Trials Registry, trial no. UMIN000034400 (http://www.umin.ac.jp/ctr/index.htm).

Staged Bilateral Total Knee Arthroplasty in a Patient with Charcot Knees: A Case Report.

INTRODUCTION: Charcot arthropathy causes severe progressive and destructive joint disease. With the development of prostheses and surgical techniques, orthopedic surgeons have a greater opportunity to use total knee arthroplasty (TKA) to treat Charcot knee. However, consensus is lacking regarding prosthesis choice. Here, we present a case of staged bilateral TKA in a patient with bilateral Charcot knees in a different way. CASE REPORT: We report a case of a 64-year-old woman with bilateral Charcot knees. Her knee joints had become increasingly unstable with severe deformity over 1 year, and she has been unable to walk for1 month before hospitalization. We performed the first TKA of the right knee using rotating hinge prosthesis with a combination of autologous bone graft and metal tibial block augmentation; lateral release and patellar replacement were also performed. Three months after the first surgery, we performed the second TKA for her left knee using a constrained condylar prosthesis with a combination of lateral retinaculum release and patella replacement. At the 6-month follow-up, both knees were stable and in good alignment. There were no signs of loosening or fracture. The patient experienced no pain and was able to ambulate in her household using a walker. CONCLUSION: TKA using constrained condylar or rotating hinge prosthesis effectively treats Charcot knee. Surgeons must acquire both appropriate alignment and ligament stability rather than the range of motion to ensure increased longevity of the prosthesis by performing various surgical techniques. Careful follow-up is needed, but Charcot knee patients can have good outcomes with TKA.

Functional screen of human MCM2-7 variant alleles for disease-causing potential.

Origin licensing builds a fundamental basis for genome stability in DNA replication. Recent studies reported that deregulation of origin licensing is associated with replication stress in precancerous lesions. The heterohexameric complex of minichromosome maintenance proteins (MCM2-7 complex) plays an essential role in origin licensing. Previously, we reported the recovery of the first viable Mcm mutant allele (named Mcm4(Chaos3)) in mice. The Mcm4(Chaos3) allele destabilizes the MCM2-7 complex, leading to chromosome instability and the formation of spontaneous tumors in Mcm4(Chaos3) homozygous mice. Supporting our finding, a recent study reported that mice with reduced expression of MCM2 die with lymphomas within the first few months after birth. These data strongly suggest that mutant Mcm2-7 genes are cancer-causing genes with nearly complete penetrance in mice. This could be the case for humans as well. Nevertheless, related investigations have not been undertaken due to the essential nature of the MCM2-7 genes. To circumvent this problem, we focused on the variant alleles of human MCM2-7 genes derived from single nucleotide polymorphisms. We created a total of 14 variant alleles in the corresponding genes in Saccharomyces cerevisiae. The phenotypic consequence was assayed for minichromosome loss, a surrogate phenotype for genome instability and cancer susceptibility. This screen identified a MCM5 variant allele with pathogenic potential. This allele deserves further investigations on its effect on cancer development in human populations.

Mitotic DNA Synthesis Is Differentially Regulated between Cancer and Noncancerous Cells.

Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.

Brain nuclear factor kappa B is involved in the corticotropin-releasing factor-induced central activation of sympatho-adrenomedullary outflow in rats.

Using urethane-anesthetized rats, we examined whether an activation of nuclear factor kappa B is involved in the corticotropin-releasing factor-induced increase in plasma levels of catecholamines. An intracerebroventricularly administered corticotropin-releasing factor (1.5 nmol/animal)-induced increase of plasma catecholamines was dose-dependently reduced by pyrrolidine dithiocarbamate (a nuclear factor kappa B antagonist) (1 and 9 nmol/animal, intracerebroventricularly) and SN50 (a peptide inhibiting nuclear factor kappa B translocation) (9 and 18 nmol/animal, intracerebroventricularly), while SN50M (an inactive control peptide for SN50, 19 nmol/animal, intracerebroventricularly) had no effect on the corticotropin-releasing factor-induced elevation of both catecholamines. Furthermore, the corticotropin-releasing factor-induced responses were also attenuated by rosiglitazone (a peroxisome proliferator-activated receptor-gamma agonist)(50 nmol/animal, intracerebroventricularly). These results suggest the involvement of brain nuclear factor kappa B in the corticotropin-releasing factor-induced central activation of the sympatho-adrenomedullary outflow in rats.

Centrally administered N-methyl-d-aspartate evokes the adrenal secretion of noradrenaline and adrenaline by brain thromboxane A2-mediated mechanisms in rats.

Plasma adrenaline mainly originated from adrenaline-containing cells in the adrenal medulla, while plasma noradrenaline reflects the release from sympathetic nerves in addition to the secretion from noradrenaline-containing cells in the adrenal medulla. The present study was undertaken to characterize the source of plasma catecholamines induced by centrally administered N-methyl-d-aspartate with regard to the brain prostanoid, using urethane-anesthetized rats. Intracerebroventricularly (i.c.v.) administered N-methyl-d-aspartate (1.0, 5.0, 10.0 nmol/animal) dose-dependently elevated plasma levels of noradrenaline and adrenaline. The N-methyl-d-aspartate (5.0 nmol/animal, i.c.v.)-induced elevation of both catecholamines was reduced by dizocilpine maleate (5 nmol/animal, i.c.v.), a non-competitive N-methyl-d-aspartate receptor antagonist. Indomethacin (0.6 and 1.2 micromol/animal, i.c.v.), an inhibitor of cyclooxygenase, dose-dependently reduced the N-methyl-d-aspartate (5.0 nmol/animal, i.c.v.)-induced elevation of both catecholamines. The N-methyl-d-aspartate-induced response was dose-dependently attenuated by furegrelate (0.9 and 1.8 micromol/animal, i.c.v.), an inhibitor of thromboxane A2 synthase. Furthermore, the acute bilateral adrenalectomy abolished the N-methyl-d-aspartate-induced responses, indicating that the source of increase in plasma noradrenaline evoked by N-methyl-d-aspartate is due to secretion from the adrenal gland and not due to release from sympathetic nerve terminals. These results suggest that centrally administered N-methyl-d-aspartate induces the secretion of noradrenaline and adrenaline from adrenal medulla by the brain thromboxane A2-mediated mechanisms in rats.

Role of brain nicotinic acetylcholine receptor in centrally administered corticotropin-releasing factor-induced elevation of plasma corticosterone in rats.

The present study was undertaken to clarify the central mechanisms involved in the intracerebroventricularly administered corticotropin-releasing factor-induced elevation of plasma corticosterone in urethane- and alpha-chloralose-anesthetized rats using microdialysis and immunohistochemical techniques. When corticotropin-releasing factor was given at 0.5, 1.5, and 3.0 nmol/animal intracerebroventricularly, it dose-dependently increased noradrenaline release but not adrenaline release in the hypothalamic paraventricular nucleus. The 1.5 nmol/animal dose of corticotropin-releasing factor-induced noradrenaline release was attenuated by CP-154,526 (butyl-ethyl-{2,5-dimethyl-7-(2,4,6 trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amine), a selective corticotropin-releasing factor receptor 1 antagonist, at 1.3 micromol/animal, intracerebroventricularly, and was also abolished by phentolamine at 0.66 micromol/animal, intracerebroventricularly. In addition, the corticotropin-releasing factor-induced elevation of noradrenaline release in the hypothalamic paraventricular nucleus and plasma corticosterone were abolished by hexamethonium, a non-selective nicotinic acetylcholine receptor antagonist, at 1.8 micromol/animal, intracerebroventricularly, and alpha-conotoxin MII, a potent alpha(3)beta(2) nicotinic acetylcholine receptor antagonist, at 30 nmol/animal, i.c.v. Corticotropin-releasing factor at 1.5 nmol/animal, i.c.v. evoked a significant expression of Fos, an immediate-early transcription factor in neurons, on the dopamine-beta-hydroxylase-containing neurons and alpha(3) nicotinic acetylcholine receptor subunit-expressing neurons in the locus coeruleus, but not in the medullary A(1) and A(2) regions containing noradrenergic neurons. These results suggest that centrally administered corticotrophin-releasing factor elevates plasma corticosterone by the corticotropin-releasing factor 1 receptor and alpha(3) subunit-containing nicotinic acetylcholine receptor (probably alpha(3)beta(2) nicotinic acetylcholine receptor) mediated activation of the locus coeruleus noradrenergic neurons projecting to the paraventricular nucleus in rats.

Role of brain adrenoceptors in the corticortopin-releasing factor-induced central activation of sympatho-adrenomedullary outflow in rats.

We investigated the role played by catecholamine-dependent pathways in modulating the ability of centrally administered corticotropin releasing factor (CRF) to activate sympatho-adrenomedullay outflow, using urethane-anesthetized rats. The CRF (1.5 nmol/animal, i.c.v.)-induced elevations of both plasma noradrenaline and adrenaline were attenuated by phentolamine (a non-selective alpha adrenoceptor antagonist) [125 and 250 microg (0.33 and 0.66 micromol)/animal], Heat (a selective alpha(1) adrenoceptor antagonist) [10 and 30 microg (30 and 90 nmol)/animal, i.c.v.] and clonidine (a selective alpha(2) adrenoceptor agonist) [100 microg (0.375 micromol)/animal, i.c.v.]. On the other hand, the CRF (1.5 nmol/animal, i.c.v.)-induced elevation of both catecholamines was not influenced by RS 79948 (a selective alpha(2) adrenoceptor antagonist) [10 and 30 microg (7.2 and 72 nmol)/animal, i.c.v.]. Furthermore, the CRF (1.5 nmol/animal, i.c.v.)-induced elevation of noradrenaline was attenuated by sotalol (a non-selective beta adrenoceptor antagonist) [125 and 250 microg (0.4 and 0.8 micromol)/animal, i.c.v.], while that of adrenaline was not influenced by sotalol. These results suggest that centrally administered CRF-induced elevation of plasma noradrenaline is mediated by an activation of alpha(1) and beta adrenoceptors in the brain, and that of plasma adrenaline is mediated by an activation of alpha(1) adrenoceptors in the brain. Furthermore, central alpha(2) adrenoceptors are involved in modulating the CRF-induced elevation of both plasma catecholamines.

Role of brain prostanoids in glucagon-like peptide-1-induced central activation of sympatho-adrenomedullary outflow in rats.

1. The aim of the present study was to characterize the source of plasma catecholamines induced by centrally administered glucagon-like peptide-1 (GLP-1), with regard to brain prostanoids, in urethane-anaesthetized rats. 2. Glucagon-like peptide-1 and other compounds were administered intracerebroventricularly (i.c.v.) and blood samples were collected via a cannula inserted into the femoral artery. Catecholamines were extracted from plasma with activated alumina and were assayed electrochemically using high-performance liquid chromatography. 3. At 0.3, 1.0 and 3.0 nmol/animal, GLP-1 dose-dependently elevated plasma levels of noradrenaline and adrenaline and the 1.0 nmol GLP-1-induced response was dose-dependently reduced by 5 and 10 nmol/animal exendin (5-39), a selective GLP-1 receptor antagonist. The GLP-1-induced elevation of concentrations of both catecholamines was abolished by 1.2 micromol/animal indomethacin, an inhibitor of cyclo-oxygenase, whereas 1.2 micromol/animal baicalein, a lipoxygenase inhibitor, had no effect. 4. Both furegrelate (1.8 micromol/animal; an inhibitor of thromboxane A(2) synthase) and (+)S-145 (625 nmol/animal; a thromboxane A(2) receptor antagonist) attenuated the GLP-1-induced increases in plasma adrenaline concentrations, but had no effect on the increases in plasma noradrenaline. The GLP-1-induced increase in plasma adrenaline concentrations was abolished by acute bilateral adrenalectomy, but the procedure had no effect on increases in plasma noradrenaline. 5. These results suggest that, in rats, centrally administered GLP-1 induces the secretion of adrenaline from the adrenal medulla by brain thromboxane A(2)-mediated mechanisms, whereas the peptide evokes the release of noradrenaline from sympathetic nerves by brain prostanoids via mechanisms other than those mediated by thromboxane A(2).

Age-related changes in the expression of ER-beta mRNA in the female rat brain.

Estrogen is important for numerous physiological actions, most of which are mediated via the nuclear estrogen receptors (ERs), ER-alpha and ER-beta, which modulate the transcription of target genes following estrogen binding. Estrogen functions change with age. In the present study, to reveal the effects of normal aging on ER-beta expression in the brain, we examined ER-beta expression at the transcriptional level using young (10 weeks), middle-aged (12 months) and old (24 months) intact female rats. In situ hybridization using a digoxigenin-labeled RNA probe was used to assess the number of ER-beta mRNA-positive cells in each region in whole brain. ER-beta mRNA-positive cells were detected throughout the brain in young female rats and were reduced in number in the olfactory bulb, cerebral cortex, hippocampus, accumbens nucleus, part of the amygdala and raphe nucleus of middle-aged rats but did not decline further in number in aged animals. By contrast, the number of ER-beta mRNA-positive cells in the hippocampus, caudate putamen, claustrum, accumbens nucleus, substantia nigra and cerebellum was not significantly different between young and middle-aged rats but was decreased in old rats. These results indicate that the expression of ER-beta mRNA in the female rat brain is differentially modulated during aging and that the changes are region specific.

Adrenal adrenaline- and noradrenaline-containing cells and celiac sympathetic ganglia are differentially controlled by centrally administered corticotropin-releasing factor and arginine-vasopressin in rats.

The adrenal glands and sympathetic celiac ganglia are innervated mainly by the greater splanchnic nerves, which contain preganglionic sympathetic nerves that originated from the thoracic spinal cord. The adrenal medulla has two separate populations of chromaffin cells, adrenaline-containing cells (A-cells) and noradrenaline-containing cells (NA-cells), which have been shown to be differentially innervated by separate groups of the preganglionic sympathetic neurons. The present study was designed to characterize the centrally activating mechanisms of the adrenal A-cells, NA-cells and celiac sympathetic ganglia with expression of cFos (a marker for neural excitation), in regard to the brain prostanoids, in anesthetized rats. Intracerebroventricularly (i.c.v.) administered corticotropin-releasing factor (CRF) induced cFos expression in the adrenal A-cells, but not NA-cells, and celiac ganglia. On the other hand, i.c.v. administered arginine-vasopressin (AVP) resulted in cFos induction in both A-cells and NA-cells in the adrenal medulla, but not in the celiac ganglia. Intracerebroventricular pretreatment with indomethacin (an inhibitor of cyclooxygenase) abolished the CRF- and AVP-induced cFos expression in all regions described above. On the other hand, intracerebroventricular pretreatment with furegrelate (an inhibitor of thromboxane A2 synthase) abolished the CRF-induced cFos expression in the adrenal A-cells, but not in the celiac ganglia, and also abolished the AVP-induced cFos expression in both A-cells and NA-cells in the adrenal medulla. These results suggest that centrally administered CRF activates adrenal A-cells and celiac sympathetic ganglia by brain thromboxane A2-mediated and other prostanoid than thromboxane A2 (probably prostaglandin E2)-mediated mechanisms, respectively. On the other hand, centrally administered AVP activates adrenal A-cells and NA-cells by brain thromboxane A2-mediated mechanisms in rats.