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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regulation of human corticotropin-releasing hormone (hCRH) gene promoter activity by inducers of cAMP was investigated by transient transfection with a construct containing the hCRH gene promoter fused to the chloramphenicol acetyltransferase gene. Expression of hCRH-chloramphenicol acetyltransferase was strongly enhanced by forskolin in the neuroblastoma SK-N-MC and choriocarcinoma JAR cell lines. Overexpression of the catalytic subunit of protein kinase A dispensed the need for forskolin, and cotransfection of cAMP-responsive element-binding protein cDNAs enhanced forskolin-dependent expression of the hCRH promoter. Progressive 5'-end deletions of the hCRH promoter delineated a cAMP- responsive region between -226 and -164 base pairs. This fragment contained the sequence TGACGTCA at -221 base pairs, consistent with the consensus motif for a CRE. A homologous oligonucleotide responded to cAMP when cloned in either orientation in front of the thymidine kinase promoter. However, the level of constitutive and inductive cAMP expression was dependent on the cell line and on intrinsic properties of the promoter. Mutation of the wild type CRH-CRE sequence into an AP-1 site (TGAGTCA) completely abolished stimulation by cAMP. In contrast, coexpression of the catalytic subunit of protein kinase A dispensed the need for stimulation with forskolin, which showed that the CRH-CRE oligonucleotide served as a functional equivalent of the native CRE element.
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PMID:Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter. 148 Jan 79

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.
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PMID:Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells. 156 25

The electrophysiological actions of corticotropin-releasing hormone (CRH) on myenteric neurons from the guinea-pig ileum were studied by intracellular microelectrode recording. CRH, when applied by micropressure ejection or in the medium (0.2-20 nM) evoked prolonged depolarization in 21 of 42 S/type 1 neurons and in 28 of 40 AH/type 2 neurons. These responses were associated with increased input resistance and augmented excitability. The post-spike hyperpolarization in AH/type 2 cells was suppressed during the CRH-evoked responses. The reversal potential of the response to CRH was about -90 mV, consistent with the closure of potassium channels by the peptide. The CRH-induced depolarization was prevented by incubation in 10 microM 5'-N-ethylcarboxamidoadenosine (NECA, an adenosine analog) suggesting that the response was mediated by stimulation of adenylate cyclase and elevation of cAMP. CRH reduced the amplitude of fast nicotinic excitatory postsynaptic potentials. This appeared to be a postsynaptic action because the peptide also reduced the responses to exogenously applied acetylcholine. These results suggest that CRH can directly influence intestinal function by acting on myenteric neurons.
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PMID:Corticotropin-releasing hormone excites myenteric neurons in the guinea-pig small intestine. 161 64

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.
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PMID:Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells. 164 51

An ideal in vitro model for the study of endocrine functions would be one in which cells could propagate in culture and express their specialized functions. Most endocrine studies to date have relied on primary cell culture or on the use of tumor cell lines. This report describes the characterization of three endocrine cell lines immortalized by transfecting endocrine cells with a temperature-sensitive mutant SV40 virus. Rabbit endometrium (HRE-H9), human placenta (SPA209-10) and rat pituitary (RP) cells were immortalized by SV40 virus, a temperature-sensitive (ts) mutant in the A gene, which encodes the large tumor antigen that is required for the maintenance of transformation. The transformed phenotype of the SV40 tsA mutant-immortalized cell line can be reversed simply by a shift in temperature. At the permissive temperature (34 degrees C), all three types of cells exhibited a transformed phenotype, which is characterized by high cell density growth and by the overgrowth of nontransformed cell layers. However, at the non-permissive temperature (40 degrees C) these cells reverted to a non-transformed phenotype as demonstrated by a marked decrease in the overgrowth of nontransformed layers and by the expression of differentiated functions. At the non-permissive temperature (40 degrees C), the endometrial cell line was capable of synthesizing beta-endorphin, and it exhibited hormonally regulated expression of the transfected hybrid uteroferrin gene construct. The human placenta cell line was capable of secreting GnRH upon stimulation by cAMP, forskolin, theophyllin, PGE, catecholamine and Ca++ channel stimulators. Moreover, the rat pituitary cell line was capable of synthesizing and secreting growth hormone (GH) which was stimulated by GHRH and cAMP. The advantage of the temperature-sensitive cell lines is that a single cell line is the source of both the normal and transformed states; thus, studies are internally controlled. These results demonstrate that tsA mutants of SV40 virus are the best available agents for immortalizing mammalian endocrine cells that retain differentiated functions.
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PMID:Characterization of endocrine cell lines immortalized by a temperature-sensitive mutant SV40. 165 33

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (KD congruent to 2.4 +/- 0.3 10(-9) M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65,000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (KD congruent to 2.6 +/- 0.4 10(-10) M) and low capacity (2030 +/- 390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time- and dose-dependent. The maximal effects were observed at 10(-10) to 10(-9) M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.
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PMID:Characterization and regulation of angiotensin and corticotropin receptors on cultured bovine adrenal cells. 165 29

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A)+ RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. These were detected at 48 h and reached a maximum 72 h after microinjection of 20-40 ng of adrenal mRNA. In response to 1 microM ACTH, total cAMP production increased within 2.5 min and reached half-maximal and maximal levels (5-fold greater than basal) at 10 and 75 min, respectively, and then remained elevated for up to 5 h. Extracellular cAMP levels were much lower but showed prominent linear increases from almost undetectable levels, with 70- and 150-fold increases evident at 1 and 2 h, respectively. The half-maximal concentration (ED50) for stimulation of cAMP formation was 5 x 10(-8) M ACTH-(1-24); the ED50 for ACTH-(1-17) was 5 x 10(-7) M, and no response was observed with ACTH-(1-10). Size fractionation of rat adrenal poly(A)+ RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1- to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.
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PMID:Adrenocorticotropin receptors: functional expression from rat adrenal mRNA in Xenopus laevis oocytes. 165 48

We have reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.
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PMID:Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E. 165 49

The long-term effects of angiotensin-II (A-II) and corticotropin (ACTH) on bovine adrenal fasciculata cells (BAC) were studied. Cells were pretreated for 3 days with either A-II or ACTH followed by an examination of the acute steroidogenic response to both hormones as well as the ability to convert several steroid precursors to cortisol and corticosterone. ACTH pretreatment caused a marked increase in cortisol output associated with a decrease in corticosterone secretion in response to both hormones leading to a 50-fold decrease in the corticosterone/cortisol ratio compared to control cells. After incubation with saturating concentrations (5 X 10(-5) M) of 22 R-hydroxycholesterol, pregnenolone or progesterone, ACTH-pretreated cells produced more cortisol than corticosterone whereas the contrary was observed in control cells. However, the conversion of 17 alpha-hydroxyprogesterone and 11-deoxycortisol to cortisol by ACTH-pretreated cells was lower than by control cells. Thus, the main effects of ACTH were a marked increase of 17 alpha-hydroxylase and a small but significant decrease of 21-hydroxylase and 11 beta-hydroxylase activities. A-II pretreatment produced, in a concentration-dependent manner, a down-regulation of its own receptors and homologous and heterologous steroidogenic desensitization. At maximal concentrations (10(-6) M) A-II reduced by 70% its own receptors while the steroidogenic response to A-II and ACTH was reduced by 95% and 75%, respectively. However, the coupling of A-II receptors to phosphoinositide pathway and to Ca2+ influx, as well as its potentiation effect on ACTH-induced cAMP production were similar in control and A-II pretreated cells. Moreover, the conversion of several steroid precursors to corticosterone was similar in control cells and A-II-pretreated cells, whereas the conversion to cortisol was reduced by approximately 30% due mainly to a decrease of 17 alpha-hydroxylase activity. Thus, the marked steroidogenic desensitization induced by A-II is most likely related to some alteration located beyond the activation of the two branches of the phosphoinositide pathway and before the first steps of steroidogenesis.
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PMID:Opposite effects of angiotensin-II and corticotropin on bovine adrenocortical cell steroidogenic responsiveness. 166 31

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.
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PMID:Growth factor-mediated regulation of aromatase activity in human skin fibroblasts. 167 98


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