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PubMed1998

Biosynthesis and degradation of bioactive fatty acid amides in human breast cancer and rat pheochromocytoma cells--implications for cell proliferation and differentiation.

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [14C]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 microM for oleamide and 2.1 microM for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB1 receptor selective antagonist SR 141716A (0.5 microM) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [14C]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first time that: (a) induction by nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on arachidonoylethanolamide/oleamide metabolism, (b) arachidonoylethanolamide and oleamide are autacoid suppressors of human breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that oleamide may act as an enhancer of arachidonoylethanolamide actions through competitive inhibition of its degradation.

Amidohydrolases↗

PubMed1999

Evaluation of alpha1-adrenoceptors in the rabbit iris: pharmacological characterization and expression of mRNA.

Subtypes of alpha1-adrenoceptor in rabbit iris have been examined in functional, binding and molecular biological experiments. In functional studies, exogenous and endogenous noradrenaline produced contractions of the iris dilator muscle. The contractile responses to noradrenaline were competitively antagonized by a range of alpha1-adrenoceptor antagonists (pA2 values): prazosin (8.1), WB4101 (8.2), BMY7378 (5.9), YM617 (9.5), JTH-601 (8.8), HV723 (7.8) and KMD-3213 (9.8). The same order of inhibitory potency was seen in the adrenergic responses to electrical stimulation. This affinity profile corresponds well to that of the putative alpha1L-adrenoceptor, which has been proposed in lower urinary tract tissues. In binding studies on rabbit iris membrane however, prazosin, KMD-3213 and WB4101 displayed high affinity (pKd or pKi: 9.6, 10.3, 9.6, respectively), and BMY7378 displayed low affinity (pKi: 6.9). These results show that the binding sites typically correspond to alpha1A-adrenoceptor subtype in character, and we could not detect the significant amount of alpha1L-adrenoceptor subtype. The expression of the three distinct mRNAs that encode proteins of alpha1a-, alpha1b- and alpha1d-adrenoceptors was studied using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the strongest expression of the alpha1a-adrenoceptor, weak expression of the alpha1b-adrenoceptor and undetectable expression of the alpha1d-adrenoceptor. The present study suggests that alpha1A-adrenoceptor is a major subtype detectable in binding and RT-PCR studies in rabbit iris, but that the adrenergic contractions of iris dilator muscle are mediated via activation of alpha1-adrenoceptor subtype having low affinity for prazosin and WB4101.

Adrenergic alpha-Agonists↗

PubMed2001

Ghrelin and growth hormone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adenomas.

OBJECTIVE: The level of growth hormone (GH), growth hormone secretogogue (GHS) and GHS receptor (GHSR) messenger ribonucleic acid (mRNA) expression has been reported as being higher in GH-producing pituitary adenomas than in other types of pituitary adenomas. Recently, ghrelin, an endogenous ligand specific for GHSR, was isolated. Therefore, we attempted to clarify whether ghrelin mRNA is expressed in various types of human pituitary adenoma by competitive reverse transcription-polymerase chain reaction (RT-PCR). We also examined the relationship between the levels of ghrelin or GHSR mRNA and hormonal and tumour characteristics in patients with pituitary adenomas. PATIENTS: Pituitary adenoma tissue was obtained at surgery from 13 patients with acromegaly, 4 with prolactinomas, 5 with gonadotrophin (Gn)-producing adenomas, 4 with non-functioning adenomas, 2 with ACTH-producing adenomas and 2 with TSH-producing adenomas. METHODS: The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. RESULTS: Ghrelin mRNA was detected in all pituitary adenoma tissues examined, with the highest mean level detected in non-functioning adenomas, a moderate level in GH-producing adenomas and Gn-producing adenomas, and the lowest level in prolactinomas. The level of ghrelin mRNA expression in GH-producing adenomas correlated negatively with the size of the adenoma (n = 13) (r = - 0.756, P = 0.0028). Furthermore, the mean level of ghrelin mRNA expression in high-grade (III and IV of Hardy classification) GH-producing adenomas was significantly lower than that in low-grade (I and II) GH-producing adenomas (P = 0.0016). GHSR mRNA was also detected in all pituitary adenomas with the highest mean level in GH-producing adenomas, a moderate level in nonfunctioning adenoma, and the lowest level in prolactinoma and Gn-producing adenomas. CONCLUSIONS: Ghrelin mRNA, in addition to GHSR mRNA, is expressed in various types of pituitary adenoma with different levels of expression in each type. Our findings suggest that ghrelin produced in pituitary adenoma may play some role in the mechanism underlying the development of adenoma cells through autocrine and/or paracrine pathways.

Adenoma↗

PubMed1997

Erythropoietin gene regulation depends on heme-dependent oxygen sensing and assembly of interacting transcription factors.

Studies on erythropoietin (Epo) gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia. Both in vivo and in Hep3B cells. Epo production is induced not only by hypoxia but also by certain transition metal (cobalt and nickel) and by iron chelation. When Hep3B cells were incubated in an iron deficient medium, Epo mRNA expression was enhanced fourfold compared to Hep3B cells in iron enriched medium. Epo induction by cobalt was inversely related to iron concentration in the medium, indicating competition between the two metals. Under hyperbaric oxygen, cobalt induction of erythropoietin mRNA was modestly suppressed while nickel induction was markedly enhanced. These recent observations support the proposal that the oxygen sensor is a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. The up-regulation of Epo gene transcription by hypoxia depends on at least two known DNA binding transcription factors, HIF-1 and HNF-4, which bind to cognate response elements in a critical approximately 50 bp 3' enhancer. Hypoxia induces HIF-1 binding. HNF-4, an orphan nuclear receptor constitutively expressed in kidney and liver, binds downstream of HIF-1 and cooperates with HIF-1, contributing importantly to high level and perhaps tissue specific expression. The C-terminal activation domain of HNF-4 binds to the beta subunit of HIF-1. The C-terminal portion of the alpha subunit of HIF-1 binds specifically to p300, a general transcriptional activator. Hypoxic induction of the endogenous Epo gene in Hep3B cells as well as an Epo-reporter gene was fully inhibited by E1A, an adenovirus protein that binds to and inactivates p300, but only slightly by a mutant E1A that fails to bind to p300. Moreover, overexpression of p300 enhanced hypoxic induction. Thus, it is likely that in hypoxic cells, p300 or a related family member plays a critical role in forming a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3' enhancer to the apparatus on the promoter responsible for the initiation of transcription.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2004

Cytokine and inducible nitric oxide synthase mRNA expression during experimental murine cryptococcal meningoencephalitis.

The immune events that take place in the central nervous system (CNS) during cryptococcal infection are incompletely understood. We used competitive reverse transcription-PCR to delineate the time course of the local expression of mRNAs encoding a variety of cytokines and inducible nitric oxide synthase (iNOS) during progressive murine cryptococcal meningoencephalitis and assessed the CNS inflammatory response using immunohistochemistry. Interleukin 18 (IL-18), transforming growth factor beta1, and IL-12p(40) mRNAs were constitutively expressed in the brains of infected and uninfected mice; IL-2 mRNA was not detected at any time. Increased levels of transcripts corresponding to IL-1 alpha, tumor necrosis factor alpha (TNF-alpha), and iNOS were detected as early as day 1 postinfection, with TNF-alpha rising by approximately 30-fold and iNOS increasing by approximately 5-fold by day 7. Each remained at these levels thereafter. IL-4, IL-6, and gamma interferon transcripts were detected on day 5, and IL-1 beta and IL-10 transcripts were detected beginning on day 7. Once detected, each remained at a relatively constant level through 28 days of infection. This cytokine profile does not suggest a polarized Th1 or Th2 response. Immunohistochemistry did not reveal inflammatory infiltrates before day 7, despite the presence of cryptococci. Intraparenchymal abscesses with inflammatory cells in their peripheries were found beginning on day 10. The infiltrates were comprised primarily of cells expressing CD4, CD8, or CD11b; low numbers of cells expressing CD45R/B220 were also present. The persistence of Cryptococcus observed in the CNS may result from an ineffective immune response, perhaps owing to an insufficient anticryptococcal effector function of endogenous glial cells resulting from competing pro- and anti-inflammatory cytokines. These data detail the immune response in the brain and could be important for the future design of specific immunomodulatory therapies for this important opportunistic infection.

Animals↗

PubMed2003

Orexin-A immunoreactivity and prepro-orexin mRNA expression in hyperphagic rats induced by hypothalamic lesions and lactation.

Orexins are endogenous neuropeptides that potently facilitate appetite and food consumption. In the present study, we examined orexin immunoreactivity and prepro-orexin mRNA expression in the lateral hypothalamus by immunohistochemistry and competitive reverse transcription-polymerase chain reaction (RT-PCR) methods in different models of hyperphagia in rats. Hyperphagia was induced by lesions of either the ventromedial hypothalamus (VMHL) or the paraventricular nucleus (PVNL), and we also compared lactating rats to nonlactating controls. Both VMHL and PVNL increased food intake and body weight compared to shams. On day 7 post lesion, serum leptin and insulin concentrations exhibited 3.2- and 2.8-fold increases in VMHL rats, and nonsignificant 1.8- and 1.8-fold increases in PVNL rats; there were significant decreases (48% and 33%) in lactating rats on day 12 postpartum compared to controls, respectively. Serum glucose concentrations were not significantly changed compared to controls in these rats. Quantification by image analysis suggests that VMHL significantly decreased the number and mean staining intensity of orexin-A immunoreactive neurones compared to those in the sham-lesioned group; while PVNL did not change orexin-A immunoreactivity. Competitive RT-PCR analysis showed that VMHL significantly decreased the prepro-orexin mRNA expression compared to those in the sham-lesioned group, and PVNL did not change it. Lactating rats on days 11-12 of lactation had significantly greater number and mean staining intensity of orexin-A immunoreactive neurones, prepro-orexin mRNA expression food intake and body weight than nonlactating postpartum rats. Thus, changes in orexin-A immunoreactivity and prepro-orexin mRNA expression were not consistent between the hyperphagia models. These results suggest that the hyperphagia from VMHL or PVNL and lactating rats differ in their involvement of orexin-A, and the change in circulating leptin and insulin concentrations may be involved in the change of orexin-A immunoreactivity in these rats.

Animals↗

PubMed2001

Expression of the human beta(3)-adrenergic receptor gene in SK-N-MC cells is under the control of a distal enhancer.

Mechanisms of transcriptional regulation of the human beta(3)-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses beta(3)- and beta(1)-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5'-flanking region of the human beta(3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act together to achieve full transcriptional activity. Mutational analysis, antibody supershift, and electrophoretic mobility shift assay competition experiments indicated that element A binds the transcription factor Sp1, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-MC and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positive cis-regulatory elements located 6.5 kb upstream from the translation start site may play an important role in transcriptional regulation of the human beta(3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-specific expression of the beta(3)-adrenergic receptor gene.

Adipose Tissue, Brown↗

PubMed1995

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice.

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.

Animals↗

PubMed2001

Abrogation of tumor necrosis factor-alpha converting enzyme inhibits embryonic lung morphogenesis in culture.

TNF-alpha converting enzyme (TACE)-mediated cell surface protein ectodomain cleavage constitutes an important cellular regulatory mechanism during mammalian lung development. Herein, we have found that TAPI, a synthetic inhibitor of TACE, inhibits embryonic mouse lung branching morphogenesis in culture. To further investigate the biological significance of TACE as a shedding enzyme during early lung organogenesis, we have devised an antisense oligonucleotide to specifically block endogenous TACE gene expression at both transcriptional and translational levels in embryonic mouse lung explant culture. Addition of TACE antisense oligonucleotide resulted in a concentration-dependent reduction in lung branching morphogenesis in culture, whereas both scrambled and sense control oligonucleotides showed no adverse effects on lung growth. Furthermore, both aquaporin-5 (Aqp5) and surfactant protein-C (SP-C) mRNA expression and protein immunoreactivity were significantly inhibited in cultured mouse lungs treated with TACE antisense oligonucleotide, indicating defective epithelial cell differentiation in embryonic lungs with decreased TACE expression. TACE is known to be involved in the proteolytic release of TGF-alpha, an EGF family stimuli critical for lung growth and maturation. We therefore tested the possibility that a lack of diffusable TGF-alpha, due to TACE deficiency, contributes to the inhibitory lung morphogenesis in the presence of TACE antisense oligonucleotide in lung culture. Soluble TGF-alpha, when included in the lung culture, rescued the TACE antisense oligonucleotide-treated lungs from inhibition of both lung branching morphogenesis and lung epithelial cell differentiation, suggesting an impaired release of circulating regulators necessary for lung development in the absence of TACE gene expression. Our findings provide evidence that TACE-mediated membrane protein shedding is indispensable for normal lung branching morphogenesis and cytodifferentiation, probably through regulating the availability of positive cytokines/growth factors essential for lung organogenesis such as TGF-alpha.

ADAM Proteins↗

PubMed2000

Diabetes attenuates the response of the lumbospinal noradrenergic system to idazoxan.

Allodynia is a common feature of painful diabetic neuropathy. This phenomenon appears to be under endogenous noradrenergic control and can be ameliorated effectively by alpha(2)-adrenoceptor agonists. Accordingly, diabetic lumbospinal noradrenergic dynamics was evaluated using high performance liquid chromatography with electrochemical detector (HPLC-ECD), in vitro ligand binding and RT-PCR-based techniques. Streptozotocin (STZ)-treated and Goto-Kakizaki (GK) diabetic rats were included, respectively, as models for type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus. The data from these studies revealed that lumbospinal norepinephrine (NE) release, as indicated by the 3-methoxy-4-hydroxyphenyl glycol (MHPG)/NE ratio, was decreased as a function of diabetes. Similarly, the binding density of [3H] p-aminoclonidine and the level of expression of mRNA transcripts encoding for the alpha(2A)-adrenoceptor subtype and noradrenergic transporter were also reduced in this disease state. Analogous findings were obtained in non-diabetic Wistar rats rendered hypercortisolemic by the subcutaneous implantation of slow releasing pellets containing a supraphysiological dose of glucocorticoid (GC). Tactile allodynia was consistently observed in STZ- and GC-treated animals. The responsiveness of alpha(2)-adrenoceptors to idazoxan (alpha(2)-adrenoceptor antagonist) indicated a dose-dependent enhancement of noradrenergic transmission in lumbar segments of normal spinal cord. In stark contrast, this neurochemical action of idazoxan was attenuated in diabetic and hypercortisolemic animals. The institution of insulin therapy ameliorated diabetes-related abnormalities in lumbospinal noradrenergic dynamics. Overall, the current finding suggests that diabetic and hypercortisolemic allodynic symptoms may stem from, at least in part, down-regulation of alpha(2)-adrenoceptors in these disease states.

Adrenergic alpha-Antagonists↗

PubMed2004

Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes.

OBJECTIVE: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility, induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to stress and exogenous apoptotic agents. METHODS: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye vital staining, respectively. RESULTS: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is expressed in human cardiomyocytes; and competitive binding studies using (125)I-labelled ghrelin showed efficient constitutive expression of GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to AraC was preceded by GH treatment. CONCLUSIONS: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes, probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.

Animals↗

PubMed1995

Accelerated binding of secretory leukoprotease inhibitor to human leukocyte elastase mediated by single-stranded sites in DNA from tracheobronchial mucus.

We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.

Binding Sites↗

PubMed2000

The glucocorticoid receptor is essential for induction of cytochrome P-4502B by steroids but not for drug or steroid induction of CYP3A or P-450 reductase in mouse liver.

Cytochrome P-4503A, CYP2B, and P-450 reductase are induced by glucocorticoids, antiglucocorticoids such as pregnenolone 16alpha-carbonitrile, and drugs such as rifampin and phenobarbital. Although the pregnane X receptor is reported to mediate steroid and drug activation of CYP3A via a conserved cis-element in CYP3A genes, discrepancies exist between the induction of the endogenous CYP3A genes and the activation of the pregnane X receptor. It is a formal possibility that the glucocorticoid receptor may account for some of these discrepancies. To determine the requirement in vivo of the glucocorticoid receptor in expression of CYP3A and CYP2B, we compared the induction of these proteins in the livers of normal mice and mice with a targeted mutation in the glucocorticoid receptor. Mice lacking the glucocorticoid receptor show no difference in constitutive hepatic expression of CYP3A but show a decrease in the level of CYP2B. Glucocorticoid receptor-deficient mice challenged with either dexamethasone or pregnenolone 16alpha-carbonitrile failed to induce CYP2B proteins, whereas CYP2B was readily induced in (+/+) mice. In contrast, CYP3A and P-450 reductase proteins were induced by either inducer in wild-type and glucocorticoid receptor-null mice. Similarly, rifampin induced CYP3A in either wild-type or glucocorticoid receptor-null mice. Despite reports that rifampin is a nonsteroidal ligand for the human glucocorticoid receptor, rifampin failed to induce tyrosine aminotransferase in mice regardless of glucocorticoid receptor genotype, and rifampin did not compete for ligand binding to either mouse or human glucocorticoid receptor. Phenobarbital induced CYP3A, CYP2B, and P-450 reductase in all mice, but the amplitude of induction was diminished 37% in glucocorticoid receptor-null mice. Thus, there are distinctly different essential requirements of CYP3A, CYP2B, and P-450 reductase genes for the glucocorticoid receptor in their induction by steroids and drugs.

Aldosterone↗

PubMed2002

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats.

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.

Animals↗

PubMed2007

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function.

UNLABELLED: PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor. INTRODUCTION: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand-receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. MATERIALS AND METHODS: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. RESULTS: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (IC(max) and EC(max)) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. CONCLUSIONS: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.

Amino Acid Sequence↗

PubMed1994

Inverse relationship between plasma cholestanol concentrations and bile acid synthesis in sitosterolemia.

We investigated the relationship between plasma cholestanol (5 alpha-dihydrocholesterol) concentrations and the activity and mRNA levels of cholesterol 7 alpha-hydroxylase, the rate-controlling enzyme for bile acid synthesis, in three female sitosterolemic homozygotes. In this lipid storage disease, large amounts of plant sterols and cholestanol accumulate because of hyperabsorption and endogenous synthesis, respectively. Plasma cholestanol concentrations were 14 times greater in the three sitosterolemic homozygotes than the mean for five control subjects. To investigate the cholestanol biosynthetic pathway, tracer doses of two putative precursors, [1,2-3H]4-cholesten-3-one and [4-14C]7 alpha-hydroxycholesterol were injected intravenously into a homozygote, and radioactivity was sought in cholestanol, bile acids, cholesterol, and sitosterol fractions isolated from plasma and bile. Tritium was concentrated only in cholestanol; neither cholesterol, sitosterol nor bile acids were derived from [1,2-3H]4-cholesten-3-one. In contrast, bile acids were labeled exclusively with 14C from [4-14C]7 alpha-hydroxycholesterol; no 14C radioactivity was detected in cholestanol. Mathematical analysis of specific activity versus time curves for [3H]cholestanol revealed very slow decay, large exchangeable pools, and enhanced synthesis in the sitosterolemic homozygote. Measurements of cholesterol 7 alpha-hydroxylase activity were 39% lower in whole liver microsomes from three sitosterolemic homozygotes that contained 19% plant sterols as compared to the mean value for six control microsomal specimens that contained 0.1% plant sterols. Removal of the excess plant sterols from the microsomes, in vitro, normalized microsomal cholesterol 7 alpha-hydroxylase activity in the homozygotes but did not affect enzyme activity in the controls. Equal amounts of cholesterol 7 alpha-hydroxylase mRNA were detected in the livers of both control and sitosterolemic subjects. Bile acid malabsorption after ileal bypass surgery stimulated cholesterol 7 alpha-hydroxylase activity 78% in sitosterolemic whole liver microsomes and reduced plasma cholesterol, sitosterol, and cholestanol levels 61%, 55% and 91%, respectively, producing a pronounced decrease in the cholestanol/cholesterol ratio without changing the sitosterol/cholesterol ratio. These results demonstrate that increased cholestanol is synthesized from 4-cholesten-3-one and not 7 alpha-hydroxycholesterol in sitosterolemia. Enhanced pools and plasma concentrations are related inversely to hepatic cholesterol 7 alpha-hydroxylase activity. Competitive inhibition of cholesterol 7 alpha-hydroxylase by the large microsomal plant sterol pool diverts cholesterol into cholestanol. Alternatively, stimulating cholesterol 7 alpha-hydroxylase activity after ileal bypass surgery markedly diminished plasma cholestanol levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1992

Quinoxaline derivatives: structure-activity relationships and physiological implications of inhibition of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated currents and synaptic potentials.

The inhibitory potencies at excitatory amino acid (EAA) receptors of 11 quinoxaline derivatives were evaluated in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in Xenopus oocytes were inhibited competitively by all the quinoxaline derivatives, with apparent Ki values ranging from 0.27 to 300 microM against kainate and from 0.25 to 137 microM against AMPA. An excellent correlation was observed between inhibitory potencies of the quinoxaline derivatives against kainate and AMPA currents, in support of the contention that in this preparation these two agonists act at a single site. All 11 quinoxaline derivatives also inhibited current activated by the combination of glycine and N-methyl-D-aspartate (NMDA), apparently acting at the glycine site, and did so over a narrower range of apparent Ki values (0.37-8.1 microM). The correlation between the quinoxalines' kainate/AMPA potencies and their glycine/NMDA potencies was relatively weak. Thus, the quinoxaline derivatives were all good antagonists of glycine/NMDA currents and displayed a greater range of potencies against kainate and AMPA. The inhibitory effects of the six quinoxaline derivatives most potent in the Xenopus oocyte experiments were also tested against the excitatory postsynaptic field potential (EPSFP) recorded in the pyramidal cell dendritic field of the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways. In slices superfused with "normal" medium (containing 1 mM Mg2+), in which the EPSFP is mediated primarily by non-NMDA receptors, IC50 values correlated closely with the Ki values against kainate/AMPA obtained in oocyte experiments but were approximately 8-fold higher. Similarly, in slices superfused with nominally Mg(2+)-free medium, in which the EPSFP is amplified due to a relief of the Mg2+ block of NMDA receptors, IC50 values correlated closely with the Ki values against glycine/NMDA obtained in oocyte experiments but were 60-fold higher. This comparison of results from the two experimental systems lends further support to the argument that hippocampal synaptic transmission is mediated postsynaptically by kainate/AMPA-type and NMDA/glycine-type EAA receptors that are pharmacologically indistinguishable from those expressed in mRNA-injected Xenopus oocytes. Furthermore, it suggests that EAA receptors in situ may be nearly saturated by high local concentrations of the endogenous ligands, a condition that would contribute substantially to the apparent non-NMDA receptor selectivity of certain quinoxaline derivatives.

Animals↗

PubMed1997

Activation of the aryl hydrocarbon receptor/transcription factor and bone marrow stromal cell-dependent preB cell apoptosis.

In the absence of known endogenous ligands, investigators have exploited ubiquitous environmental pollutants, including polycyclic aromatic hydrocarbons, to gain insight into the physiologic functions of the aryl hydrocarbon (dioxin) receptor/transcription factor (AhR). AhR ligands induce cell transformation and steroid-like immunosuppression, suggesting a role for the AhR in regulation of cell growth and/or function. However, mechanisms through which the AhR influences cells in general and lymphocytes in particular remain unresolved. A murine model of B cell development was created to: 1) examine a role for the AhR in immunosuppression; 2) define mechanisms of AhR ligand immunosuppression; 3) characterize AhR expression in preB cells, in bone marrow stromal cells that support preB cells, or in primary bone marrow B cells; and 4) determine if AhR ligands suppress lymphopoiesis by acting directly on preB cells or indirectly via the microenvironment, as represented by bone marrow stromal cells. Results indicate that: 1) low doses (> or = 10(-8) M) of the prototypic AhR ligand, 7,12-dimethylbenz[a]anthracene (DMBA), induce preB cell apoptosis in 12 to 24 h; 2) alpha-naphthoflavone, an AhR and cytochrome P-450 inhibitor, blocks DMBA-induced apoptosis; 3) AhR mRNA and functional AhR protein are expressed at high levels in bone marrow stromal cells (little or no AhR is present in preB cell lines), and 4) preB cells maintained in rIL-7 do not undergo DMBA-induced apoptosis unless cultured with stromal cells. Results underscore the regulatory role played by bone marrow stromal cells in lymphopoiesis and support the hypothesis that the AhR effects immunosuppression by inducing stromal cells to deliver a death signal to lymphocytes.

9,10-Dimethyl-1,2-benzanthracene↗