Receptor Messenger RNA Expression Research Articles

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Trans-resveratrol attenuates high fatty acid-induced P2X7 receptor expression and IL-6 release in PC12 cells: possible role of P38 MAPK pathway.

Diabetic neuropathy (DNP) is the most common chronic complication of diabetes. Elevated free fatty acids (FFAs) have been recently recognized as major causes of inflammation and are relevant to the functional changes of nerve system in diabetes. Trans-resveratrol (RESV), a polyphenolic natural compound, has long been acknowledged to have anti-inflammation properties and may exert a neuroprotective effect on neuronal damage in diabetes, while the mechanisms underlying are largely unknown. Our previous study on differential PC12 cells cultured with high FFAs has shown chronic FFAs overload increased PC12 interleukin (IL)-6 release mediated by P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine triphosphate (ATP); a high FFA-induced activation of P38 mitogen-activated protein kinase (MAPK) pathway was pointed to be a potential underlying mechanism. Data from this study indicated that RESV, in a dose-dependent manner, reduced high FFA-induced IL-6 release by impeding the activation of P2X7 receptor, as shown by the results that both high FFA-elevated P2X7 receptor messenger RNA (mRNA) and protein expression as well as high FFA-evoked [Ca(2+)]i in response to 3'-O-(4-benzoyl) benzoyl-ATP (a selective P2X7 receptor agonist) were significantly attenuated. Meanwhile, high FFA-induced activation of P38 MAPK, an essential prerequisite for high FFA-activated P2X7 receptor and subsequent IL-6 release, was also dose-dependently abrogated by RESV. Furthermore, RESV may hamper the activation of P38a MAPK (one paramount P38 isoform) via forming hydrogen bonding with Thr175 residue, surrounding the two residues (Thy180 and Tyr182) essential for canonical activation of P38a MAPK. Taken together, RESV could inhibit high FFA-induced inflammatory IL-6 release mediated by P2X7 receptor through deactivation of P38 MAPK signaling pathway. All these results outline the potential mechanisms involved in the neuroprotective roles of RESV and highlight the clinical application of RESV in treatment of inflammation in relation to DNP.

In Preeclampsia, Maternal Third Trimester Subcutaneous Adipocyte Lipolysis Is More Resistant to Suppression by Insulin Than in Healthy Pregnancy

Obesity increases preeclampsia risk, and maternal dyslipidemia may result from exaggerated adipocyte lipolysis. We compared adipocyte function in preeclampsia with healthy pregnancy to establish whether there is increased lipolysis. Subcutaneous and visceral adipose tissue biopsies were collected at caesarean section from healthy (n=31) and preeclampsia (n=13) mothers. Lipolysis in response to isoproterenol (200 nmol/L) and insulin (10 nmol/L) was assessed. In healthy pregnancy, subcutaneous adipocytes had higher diameter than visceral adipocytes (P<0.001). Subcutaneous and visceral adipocyte mean diameter in preeclampsia was similar to that in healthy pregnant controls, but cell distribution was shifted toward smaller cell diameter in preeclampsia. Total lipolysis rates under all conditions were lower in healthy visceral than subcutaneous adipocytes but did not differ after normalization for cell diameter. Visceral adipocyte insulin sensitivity was lower than subcutaneous in healthy pregnancy and inversely correlated with plasma triglyceride (r=-0.50; P=0.004). Visceral adipose tissue had lower ADRB3, LPL, and leptin and higher insulin receptor messenger RNA expression than subcutaneous adipose tissue. There was no difference in subcutaneous adipocyte lipolysis rates between preeclampsia and healthy controls, but subcutaneous adipocytes had lower sensitivity to insulin in preeclampsia, independent of cell diameter (P<0.05). In preeclampsia, visceral adipose tissue had higher LPL messenger RNA expression than subcutaneous. In conclusion, in healthy pregnancy, the larger total mass of subcutaneous adipose tissue may release more fatty acids into the circulation than visceral adipose tissue. Reduced insulin suppression of subcutaneous adipocyte lipolysis may increase the burden of plasma fatty acids that the mother has to process in preeclampsia.

Human bitter perception correlates with bitter receptor messenger RNA expression in taste cells

Alleles of the receptor gene TAS2R38 are responsible in part for the variation in bitter taste perception of 6-n-propylthiouracil (PROP) and structurally similar compounds (eg, glucosinolates in cruciferous vegetables). At low concentrations, people with the PAV ("taster" amino acid sequence) form of TAS2R38 perceive these bitter compounds, whereas most with the AVI ("nontaster" amino acid sequence) form do not; heterozygotes (PAV/AVI) show the widest range of bitter perception. The objectives were to examine individual differences in expression of PAV-TAS2R38 messenger RNA (mRNA) among heterozygotes, to test the hypotheses that the abundance of allele-specific gene expression accounts for the variation in human bitter taste perception, and to relate to dietary intake of bitter-tasting beverages and foods. Heterozygous individuals (n = 22) provided psychophysical evaluation of the bitterness of PROP, glucosinolate-containing broccoli juice, non-glucosinolate-containing carrot juice, and several bitter non-TAS2R38 ligands as well as dietary recalls. Fungiform taste papillae were examined for allele-specific TAS2R38 expression by using quantitative polymerase chain reaction. PAV-TAS2R38 mRNA expression was measured in 18 of 22 heterozygous subjects. Relative expression varied widely and positively correlated with ratings of bitterness intensity of PROP (P = 0.007) and broccoli juice (P = 0.004) but not of the control solutions carrot juice (P = 0.26), NaCl (P = 0.68), caffeine (P = 0.24), or urea (P = 0.47). Expression amounts were related to self-reported recent and habitual caffeine intake (P = 0.060, P = 0.005); vegetable intake was too low to analyze. We provide evidence that PAV-TAS2R38 expression amount correlates with individual differences in bitter sensory perception and diet. The nature of this correlation calls for additional research on the molecular mechanisms associated with some individual differences in taste perception and food intake. The trial was registered at clinicaltrials.gov as NCT01399944.

How systemic inflammation modulates adenosine metabolism and adenosine receptor expression in humans in vivo

Adenosine modulates inflammation and prevents associated organ injury by activation of its receptors. During sepsis, the extracellular adenosine concentration increases rapidly, but the underlying mechanism in humans is unknown. We aimed to determine the changes in adenosine metabolism and signaling both in vivo during experimental human endotoxemia and in vitro. We studied subjects participating in three different randomized double-blind placebo-controlled trials. In order to prevent confounding by the different pharmacological interventions in these trials, analyses were performed on data of placebo-treated subjects only. Intensive care research unit at the Radboud University Nijmegen Medical Center. In total, we used material of 24 healthy male subjects. Subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide) intravenously. Following experimental endotoxemia, endogenous adenosine concentrations increased. Expression of 5'ectonucleotidase messenger RNA was upregulated (p = .01), whereas adenosine deaminase messenger RNA was downregulated (p = .02). Furthermore, both adenosine deaminase and adenosine kinase activity was significantly diminished (both p ≤ .0001). A2a and A2b receptor messenger RNA expression was elevated (p = .02 and p = .04, respectively), whereas messenger RNA expression of A1 and A3 receptors was reduced (both, p = .03). In vitro, lipopolysaccharide dose-dependently attenuated the activity of both adenosine deaminase and adenosine kinase (both p ≤ .0001). Adenosine metabolism and signaling undergo adaptive changes during human experimental endotoxemia promoting higher levels of adenosine thereby facilitating its inflammatory signaling.

Chronic Activation of Corticotropin-Releasing Factor Type 2 Receptors Reveals a Key Role for 5-HT1A Receptor Responsiveness in Mediating Behavioral and Serotonergic Responses to Stressful Challenge

BackgroundThe corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation.MethodsTransgenic mice overexpressing the highly specific CRFR2 ligand urocortin 3 (UCN3OE) were analyzed for stress-related behaviors and hypothalamic-pituitary-adrenal axis responses. Responses to 5-HT receptor agonist challenge were assessed by local cerebral glucose utilization, while 5-HT and 5-hydroxyindoleacetic acid content were quantified in limbic brain regions.ResultsMice overexpressing urocortin 3 exhibited increased stress-related behaviors under basal conditions and impaired retention of spatial memory compared with control mice. Following acute stress, unlike control mice, they exhibited no further increase in these stress-related behaviors and showed an attenuated adrenocorticotropic hormone response. 5-HT and 5-hydroxyindoleacetic acid content of limbic nuclei were differentially regulated by stress in UCN3OE mice as compared with control mice. Responses to 5-HT type 1A receptor challenge were significantly and specifically reduced in UCN3OE mice. The distribution pattern of local cerebral glucose utilization and 5-HT type 1A receptor messenger RNA expression levels suggested this effect was mediated in the raphé nuclei.ConclusionsChronic activation of CRFR2 promotes an anxiety-like state, yet with attenuated behavioral and hypothalamic-pituitary-adrenal axis responses to stress. This is reminiscent of stress-related atypical psychiatric syndromes such as posttraumatic stress disorder, chronic fatigue, and chronic pain states. This new understanding indicates CRFR2 antagonism as a potential novel therapeutic target for such disorders.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells

To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for invitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An invitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines.

Cortical Glutamic Acid Decarboxylase 67 Deficiency Results in Lower Cannabinoid 1 Receptor Messenger RNA Expression: Implications for Schizophrenia

Levels of cannabinoid 1 receptor (CB1R) messenger RNA (mRNA) and protein, which are expressed most heavily in the cholecystokinin class of γ-aminobutyric acid (GABA) neurons, are lower in the dorsolateral prefrontal cortex in schizophrenia, and the magnitude of these differences is strongly correlated with that for glutamic acid decarboxylase 67 (GAD(67)) mRNA, a synthesizing enzyme for GABA. However, whether this correlation reflects a cause-effect relationship is unknown. Using quantitative in situ hybridization, we measured CB1R, GAD(67), and diacylglycerol lipase alpha (the synthesizing enzyme for the endocannabinoid 2-arachidonoylglycerol) mRNA levels in the medial prefrontal cortex of genetically engineered GAD(67) heterozygous (GAD(67)(+/-)), CB1R heterozygous (CB1R(+/-)), CB1R knockout (CB1R(-/-)), and matched wild-type mice. In GAD(67)(+/-) mice, GAD(67) and CB1R mRNA levels were significantly reduced by 37% and 16%, respectively, relative to wild-type mice and were significantly correlated across animals (r = .61; p = .01). In contrast, GAD(67) mRNA levels were unaltered in CB1R(+/-) andCB1R(-/-) mice. Expression of diacylglycerol lipase alpha mRNA, which is not altered in schizophrenia, was also not altered in any of the genetically engineered mice. The findings that reduced GAD(67) mRNA expression can induce lower CB1R mRNA expression support the hypothesis that lower cortical levels of CB1Rs in schizophrenia may partially compensate for deficient GAD(67)-mediated GABA synthesis by reducing endogenous cannabinoid suppression of GABA release.

Postnatal Serotonin Type 2 Receptor Blockade Prevents the Emergence of Anxiety Behavior, Dysregulated Stress-Induced Immediate Early Gene Responses, and Specific Transcriptional Changes that Arise Following Early Life Stress

BackgroundEarly life adverse experience contributes to an enhanced vulnerability for adult psychopathology. Recent evidence indicates that serotonin type 2 (5-HT2) receptor function, implicated in the pathophysiology of mood and anxiety disorders, is significantly enhanced in the maternal separation model of early life stress. We examined whether postnatal 5-HT2 receptor blockade would prevent the consequences of maternal separation on anxiety behavior and dysregulated gene expression.MethodsControl and maternally separated litters received treatment with the 5-HT2 receptor antagonist, ketanserin, or vehicle during postnatal life and were examined for effects on adult anxiety behavior, adult stress-induced immediate early gene expression responses, and transcriptional changes within the prefrontal cortex during postnatal life and in adulthood.ResultsTreatment with ketanserin during postnatal life blocked the long-lasting effects of maternal separation on anxiety behavior in the open field test and the elevated plus maze. Further, the dysregulated adult stress-induced expression pattern of the immediate early gene, Arc, observed in maternally separated animals was also prevented by postnatal ketanserin treatment. Ketanserin treatment normalized the alterations in the expression of specific genes in the prefrontal cortex of maternally separated animals, including changes in serotonin type 2A receptor messenger RNA expression during postnatal life and in genes associated with G-protein signaling in adulthood.ConclusionsPostnatal treatment with the 5-HT2 receptor antagonist, ketanserin, blocked specific consequences of maternal separation, including anxiety behavior and dysregulated gene expression in the prefrontal cortex. Our results suggest that enhanced 5-HT2 receptor function may contribute to the emergence of anxiety behavior and perturbed stress responses following early life stress.

Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells

To determine that anti-Müllerian hormone (AMH) has been shown to inhibits E(2) production in rodents and in luteinized granulosa cells (GC). We determined whether this occurs in human cells most highly expressing AMH (i.e., from small antral follicles) and whether this is an effect on aromatase promoter activity. We also investigated the effects of AMH on other factors determining FSH sensitivity. Granulosa cells were exposed to AMH with and without gonadotropins for 48 hours. University laboratory. Not applicable. None. Aromatase and FSH receptor messenger RNA expression measured using real time quantitative polymerase chain reaction (PCR). Aromatase promoter II activity measured using a luciferase assay. Estradiol, inhibin A and B, and vascular endothelial growth factor production were measured in the conditioned medium. The AMH decreased gonadotropin-stimulated aromatase expression and decreased forskolin-stimulated aromatase in KGN cells and this effect was through a dose-dependent inhibition of promoter II. Surprisingly, AMH also reduced FSH receptor mRNA expression. High AMH doses had no effect on inhibin B, whereas a low dose stimulated production. There was no effect on inhibin A or vascular endothelial growth factor. The AMH inhibits factors affecting FSH sensitivity. As AMH levels decrease with follicle growth, this inhibition would be removed. The AMH overproduction in anovulatory polycystic ovaries (PCO) may therefore restrict folliculogenesis by an inhibitory effect on FSH sensitivity, thereby contributing to anovulation.

Protective Effects of Erythropoietin on Myocardial Infarction in Rats: The Role of AMP-Activated Protein Kinase Signaling Pathway

Erythropoietin (EPO) has protective effects on myocardial infarction (MI). This study was to test whether EPO administrated after MI is as effective as EPO administrated before MI and to determine the role of AMP-activated protein kinase (AMPK) in the cardioprotective effects of EPO. Recombinant human EPO (5000IU/kg) was intraperitoneally injected 12hours before MI (pretreatment) or 30minutes after MI (posttreatment). The levels of serum enzymes were assayed at 24 and 72hours after MI. The infarct size was determined by nitro blue tetrazolium staining. The microarchitecture damages were evaluated by electron microscopy. EPO receptor messenger RNA and protein levels were determined by RT-PCR and immunohistochemistry, respectively. Activation of AMPK and nuclear factor kappa B was determined by immunoblotting. An AMPK-specific inhibitor, compound C, was used to inhibit AMPK activation in vivo. The authors found that both pretreatment and posttreatment of EPO successfully attenuated the serum enzyme levels, reduced the infarct size and ameliorated the microarchitecture damage. Moreover, both pretreatment and posttreatment of EPO decreased the EPO receptor messenger RNA and protein expressions, which were up-regulated in MI. More importantly, under MI conditions, EPO further increased the phosphorylation of AMPK and suppressed the activation of nuclear factor kappa B. Moreover, when AMPK was blocked by compound C, the cardioprotective effect of EPO was significantly attenuated (P < 0.01). Thus, this study demonstrates that both pretreatment and posttreatment of EPO are cardioprotective in an AMPK-dependent manners, providing the first evidence that the AMPK signaling pathway is involved in the cardioprotective effects of EPO.

CHANGES IN ODORANT RECEPTOR MESSENGER RNA EXPRESSION ASSOCIATED WITH MATURATION, REPRODUCTIVE HORMONES, AND HOMING IN SOCKEYE SALMON (ONCORHYNCHUS NERKA)

Event Abstract Back to Event CHANGES IN ODORANT RECEPTOR MESSENGER RNA EXPRESSION ASSOCIATED WITH MATURATION, REPRODUCTIVE HORMONES, AND HOMING IN SOCKEYE SALMON (ONCORHYNCHUS NERKA) Andrew Dittman1*, Darran May2, Larissa Felli2 and Penny Swanson1 1 Northwest Fisheries Science Center, NOAA Fisheries, United States 2 University of Washington, United States Seasonal migrations for reproduction are common in many mid-high latitude animals and for many species there are strong links between hormones, reproduction and migration. In addition, hormones associated with sexual maturation may modulate the sensory systems involved in migratory and reproductive behaviors. The homing migration of Pacific salmon from oceanic feeding grounds back to their river of origin to spawn provides an ideal model for studying the endocrine control of neuronal plasticity and sensory physiology involved in migration. The final stages of salmon homing migrations are governed by olfactory discrimination of homestream odors that juvenile salmon learn (imprint to) prior to their seaward migrations. One aspect of the imprinting and homing process involves long-term sensitization of the peripheral olfactory system to specific odorants associated with their natal stream. Concurrent with these homing migrations, salmon experience dramatic changes in hormones associated with gametogenesis and final maturation. In this study, we examined changes in odorant receptor (OR) mRNA expression in the olfactory epithelium of sockeye salmon exposed to specific odorants during sensitive periods for imprinting. We then examined the patterns of OR mRNA expression during final maturation, the period when salmon would be homing in the wild. Developmental increases in OR mRNA expression were correlated with surges in plasma luteinizing hormone (LH) and reproductive steroids that occurred just prior to final maturation. Differences in OR mRNA expression levels between imprinted and unimprinted fish were apparent prior to the surge in LH and final maturation and were consistent with increased sensitivity to imprinted odors during the period salmon would be homing to their natal streams. Acknowledgements Supported by the Bonneville Power Administration and the NWFSC) Keywords: imprinting, maturation, Olfaction, Salmon Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Invited Symposium Topic: Ectohormones Citation: Dittman A, May D, Felli L and Swanson P (2011). CHANGES IN ODORANT RECEPTOR MESSENGER RNA EXPRESSION ASSOCIATED WITH MATURATION, REPRODUCTIVE HORMONES, AND HOMING IN SOCKEYE SALMON (ONCORHYNCHUS NERKA). Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00129 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Andrew Dittman, Northwest Fisheries Science Center, NOAA Fisheries, Seattle, United States, andy.dittman@noaa.gov Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrew Dittman Darran May Larissa Felli Penny Swanson Google Andrew Dittman Darran May Larissa Felli Penny Swanson Google Scholar Andrew Dittman Darran May Larissa Felli Penny Swanson PubMed Andrew Dittman Darran May Larissa Felli Penny Swanson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Intensive Statin Therapy: A Favorable Adjunct to the Improvement of Small-Diameter Vascular Grafts

To assess the effect of intensive statins therapy on the outcome of small-diameter vascular prosthesis, we investigated whether atorvastatin treatment (30 mg/d) could accelerate the re-endothelialization process and improve the patency rate in a canine infrarenal abdominal aorta-expanded polytetrafluoroethylene (ePTFE) bypass model. Furthermore, we also evaluated the effect of atorvastatin on the migratory and adherent capacity of circulating endothelial progenitor cells (EPCs) in vitro. Improved patency was confirmed by Doppler sonography and arteriography. Histological and scanning electron microscopy illustrated enhanced re-endothelialization process. Treatment with atorvastatin enhanced the circulating pool of EPCs with fortified migratory and adherent capacity. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that atorvastatin treatment increased endothelial nitric oxide synthase (eNOS) and kinase insert domain receptor (KDR) messenger RNA (mRNA) expression in cultured EPCs and neointima. In conclusion, intensive statin therapy could be considered a favorable option to improve small-diameter vascular graft patency.

Galanin Mediates the Pathogenesis of Cerulein-Induced Acute Pancreatitis in the Mouse

Acute pancreatitis (AP) is characterized by pancreatic microcirculatory and secretory disturbances. As galanin can modulate pancreatic vascular perfusion, we sought to determine if galanin plays a role in AP. Acute pancreatitis was induced in wild-type and galanin gene knockout mice by intraperitoneal injections of cerulein. The severity of AP was evaluated (plasma amylase and lipase, myeloperoxidase activity, and acinar cell necrosis) with and without treatment with galanin or the antagonist galantide. Galanin receptor messenger RNA expression in mouse pancreas was measured by reverse transcription-polymerase chain reaction and Western blot analysis. Galantide ameliorated AP, reducing all indices by 25% to 40%, whereas galanin was without effect. In galanin knockout mice, all indices of AP were reduced 25% to 50% compared with wild-type littermates. Galanin administration to the knockout mice exacerbated AP such that it was comparable with the AP induced in the wild-type mice. Conversely, administration of galantide to the galanin knockout mice did not affect the AP, whereas AP was ameliorated in the wild-type mice. The 3 galanin receptor subtypes are expressed in mouse pancreas, with receptor subtype 3 expression predominating. These data implicate a role for galanin in AP and suggest a potential clinical application for galanin antagonists in treatment.

Influences of Sex, Incubation Temperature, and Environmental Quality on Gonadal Estrogen and Androgen Receptor Messenger RNA Expression in Juvenile American Alligators (Alligator mississippiensis)1

Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5 degrees C expressed greater AR levels than females incubated at 30 degrees C; dimorphic expression was not observed in animals incubated at 32 degrees C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants.

Altered coronary microvascular serotonin receptor expression after coronary artery bypass grafting with cardiopulmonary bypass

We evaluated roles of serotonin 1B and 2A receptors, thromboxane synthase and receptor, and phospholipases A(2) and C in response to cardiopulmonary bypass. Patients' atrial tissues were harvested before and after cardiopulmonary bypass with cardioplegia (n = 13). Coronary microvessels were assessed for vasoactive response to serotonin with and without inhibitors of serotonin 1B and 2A receptors and phospholipases A(2) and C. Expressions of serotonin receptor messenger RNA were determined with reverse transcriptase polymerase chain reaction. Expressions of serotonin receptors and thromboxane A(2) receptor and synthase proteins were determined with immunoblotting and immunohistochemistry. Microvessel exposure to serotonin elicited 7.3% +/- 2% relaxation before bypass, changing to contraction of -19.2% +/- 2% after bypass (P <.001). Additions of specific serotonin 1B receptor antagonist and inhibitor of phospholipase A(2) resulted in significantly decreased contraction, -8.6% +/- 1% (P < .001) and 2.8% +/- 3% (P = .001), respectively. Serotonin 1B receptor messenger RNA expression increased 1.82 +/- 0.34-fold after bypass (p = .044); serotonin 2A receptor messenger RNA expression did not change. Serotonin 1B but not 2A receptor protein expression increased after bypass by 1.35 +/- 0.7-fold (P = .0413). Thromboxane synthase and receptor expressions were unchanged after bypass. Serotonin 1B receptor increased mainly in arterial smooth muscle. There were no appreciable differences in arterial expressions of thromboxane synthase or receptor. Serotonin-induced vascular dysfunction after cardiopulmonary bypass with cardioplegic arrest may be mediated by increased expression of serotonin 1B receptor and subsequent phospholipase A(2) activation in myocardial coronary smooth muscle.

Predisposition to atherosclerosis and aortic aneurysms in mice deficient in kinin B1 receptor and apolipoprotein E

Kinin B1 receptor is involved in chronic inflammation and expressed in human atherosclerotic lesions. However, its significance for lesion development is unknown. Therefore, we investigated the effect of kinin B1 receptor deletion on the development of atherosclerosis and aortic aneurysms in apolipoprotein E-deficient (ApoE(-/-)) mice. Mice deficient both in ApoE and in kinin B1 receptor (ApoE(-/-)-B(1)(-/-)) were generated and analyzed for their susceptibility to atherosclerosis and aneurysm development under cholesterol rich-diet (western diet) and angiotensin II infusion. Kinin B1 receptor messenger RNA (mRNA) expression was significantly increased in ApoE(-/-) mice after Western-type diet. Although no difference in serum cholesterol was found between ApoE(-/-)-B(1)(-/-) and ApoE(-/-) mice under Western-type diet, aortic lesion incidence was significantly higher in ApoE(-/-)-B(1)(-/-) after this treatment. In accordance, we observed increased endothelial dysfunction in these mice. The mRNA expression of cyclic guanosine monophosphate-dependent protein kinase I, CD-11, F4/80, macrophage colony-stimulating factor, and tumor necrosis factor-alpha were increased in the aorta of double-deficient mice following Western-type diet, whereas the levels of peroxisome proliferator-activated receptor gamma protein and the activity of matrix metalloproteinase-9 activity were decreased. In addition to the increased atherosclerotic lesions, the lack of kinin B(1) receptor also increased the incidence of abdominal aortic aneurysms after angiotensin II infusion. In conclusion, our results show that kinin B(1) receptor deficiency aggravates atherosclerosis and aortic aneurysms under cholesterolemic conditions, supporting an antiatherogenic role for the kinin B(1) receptor.

In vitro antiproliferative characteristics of flavonoids and diazepam on SNU-C4 colorectal adenocarcinoma cells

The need for beneficial use of sedatives in oncologic patients is increasing. Therefore, in this study, antiproliferative characteristics of herbal and synthetic sedatives were examined in vitro in SNU-C4 human colorectal adenocarcinoma cells. Apigenin (50% inhibition concentration, IC(50) = 1.8 +/- 0.5 microM) and diazepam (IC(50) = 7.0 +/- 0.5 microM) showed concentration-dependent inhibition of SNU-C4 cancer cell survival. Efficacy of cancer cell survival inhibition by apigenin and diazepam was much lower than that of 5-fluorouracil (5-FU), a known chemotherapeutic drug. However, 10(-6) M concentration of apigenin and diazepam potentiated 5-FU-induced cytotoxicity. In SNU-C4 cells, 10(-6) M concentrations of diazepam, flumazenil (Ro15-1788), Ro5-4864, or PK11195, all ligands for central- or peripheral-type benzodiazepine (BZD) receptors, inhibited cell survival like the flavonoid apigenin (4',5,7-trihydroxyflavone) and fisetin (3,7,3',4'-tetrahydroxyflavone). Also like the plant flavonoids, treatment with 10(-6) M concentration of diazepam for 3 days hardly affect the peripheral-type BZD receptor (PBR) messenger RNA (mRNA) expression and inhibited glucose utilization of SNU-C4 cells. Treatment with flavonoids or diazepam for 6 days upregulated PBR mRNA expression and cell cytotoxicity of SNU-C4 cells. Furthermore, treatment with 10(-6) M concentration of apigenin, a natural sedative material originating from traditional herbs, positively modulated BZD-induced antiproliferative cytotoxicity in SNU-C4 cells. Overall, the in vitro antiproliferative activity on SNU-C4 cancer cells of herbal sedatives, such as apigenin, plus additive enhancement of synthetic BZD- and 5-FU-induced antiproliferative activities, were shown. In conclusion, this study provides experimental basis for advanced trial in the future.

Reduced Cortical Cannabinoid 1 Receptor Messenger RNA and Protein Expression in Schizophrenia

Cannabis use is associated with both impaired cognitive functions, including working memory, and an increased risk of schizophrenia. Schizophrenia is characterized by impairments in working memory that are associated with reduced gamma-aminobutyric acid (GABA) neurotransmission in the dorsolateral prefrontal cortex. The cannabinoid 1 receptor (CB1R) is highly expressed in the dorsolateral prefrontal cortex, is contained in the axon terminals of a subpopulation of perisomatic-targeting GABA neurons, and, when activated, suppresses the release of GABA. To determine the potential relationship between CB1R signaling and altered GABA neurotransmission in schizophrenia by evaluating CB1R messenger RNA (mRNA) and protein expression in the dorsolateral prefrontal cortex. In situ hybridization and immunocytochemistry techniques were used to examine the cortical levels of CB1R mRNA and protein, respectively. Brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania. Postmortem brain specimens from 23 pairs of subjects with schizophrenia and age-, sex-, and postmortem interval-matched comparison subjects, as well as brain specimens from 18 macaque monkeys with long-term exposure to haloperidol, olanzapine, or placebo. Optical density measures of CB1R mRNA expression and protein levels and correlations with previously reported glutamic acid decarboxylase 67 and cholecystokinin mRNA measures. Levels of CB1R mRNA were significantly lower by 14.8% in the subjects with schizophrenia. Similarly, CB1R protein levels, assessed by radioimmunocytochemistry and standard immunocytochemistry, were significantly decreased by 11.6% and 13.9%, respectively. Group differences in CB1R mRNA levels were significantly correlated with those in glutamic acid decarboxylase 67 and cholecystokinin mRNA levels. Expression of CB1R mRNA was not changed in antipsychotic-exposed monkeys, and neither CB1R mRNA levels nor protein levels were affected by potential confounding factors in the subjects with schizophrenia. This combination of findings suggests the testable hypothesis that reduced CB1R mRNA and protein levels in schizophrenia represent a compensatory mechanism to increase GABA transmission from perisomatic-targeting cholecystokinin interneurons with impaired GABA synthesis.

Dysregulation of Nociceptin/Orphanin FQ Activity in the Amygdala Is Linked to Excessive Alcohol Drinking in the Rat

Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, with an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (Marchigian Sardinian alcohol-preferring [msP]). The msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats. Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. The NOP receptor messenger RNA expression and binding was upregulated across most brain regions in msP compared with Wistar rats. However, in msP rats [(35)S]GTPgammaS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala. These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted on the basis of the anti-corticotropin releasing factor (CRF) actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.