Mouse Anterior Pituitary Cell Line Research Articles

Vasopressin messenger ribonucleic acid regulation via the protein kinase A pathway.

Activation of vasopressin (VP) gene expression in vivo by osmotic stimuli results in an increase in both messenger RNA (mRNA) content and polyadenylate [poly(A)] tail length. VP gene transcription in vitro is stimulated by protein kinase A (PKA) activation. To examine the role of PKA in the regulation of VP mRNA poly(A) metabolism, constructs of the rat VP gene were permanently transfected into the mouse anterior pituitary cell line, AtT-20. Treatment with forskolin of cells expressing the intact VP gene resulted in increased VP gene transcription, an increase in the content of VP mRNA, and a shift toward VP mRNA species with longer poly(A) tails accompanied by the loss of VP mRNA species with shorter poly(A) tails. We uncoupled the PKA-stimulated appearance of long-tailed species from the disappearance of short-tailed species, suggesting that the size shift was caused by a coincident, but uncoupled net increase in VP mRNA species with elongated poly(A) tails and net loss of mRNA species with short poly(A) tails. These data indicate that activation of the PKA second-messenger pathway both enhances transcription of the VP gene and causes an increase in the average length of VP mRNA poly(A) tails. This latter effect, by shifting upwards the average poly(A) tail size, could result in increased translational efficiency or stability of VP mRNA, thereby providing an additional mechanism by which PKA may enhance gene expression.

Differential regulation of the cloned kappa and mu opioid receptors

To directly compare the regulation of the cloned κ and μ opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K + current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the κ and the μ receptor to this somatostatin receptor. We show that the κ receptor readily uncoupled from the K + current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the κ receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K + current was not altered by κ receptor desensitization. The μ receptor uncoupled from the K + current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K + current after μ receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the μ receptor but not the κ receptor from the K + current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the κ receptor displays homologous regulation, whereas the μ receptor undergoes only a heterologous form of desensitization. μ Receptor desensitization may lead to the alterations of diverse downstream events, whereas κ receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the μ receptor could be relevant to the addictive properties of μ agonists. Comparison of κ and μ receptor regulation may help define the properties of the μ receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the κ and μ receptors to two different effectors in the same mammalian expression system.

Effect of S(−)- and R(+)-salsolinol on the POMC gene expression and ACTH release of an anterior pituitary cell line

Tetrahydroisoquinolines (TIQs) are thought to play an important role in the process of development of alcohol dependence. Being a condensation product between the alcohol metabolite acetaldehyde and dopamine they might be involved in the balance of the opioid system as well as the reward system. Therefore, the influence of the TIQ salsolinol (SAL) on the pro-opiomelanocortin (POMC) gene expression was investigated using the AtT-20 mouse anterior pituitary tumor cell line. Our results show a significant decrease in the POMC gene expression by the S(−)-enantiomer of SAL. The basal secretion of adrenocorticotropin (ACTH) as well as the corticotropin-releasing factor (CRF)-stimulated ACTH release remained unchanged after R(+)- and S(−)-SAL treatment. Interestingly, it was clearly shown that a reduction of intracellular cAMP level occurred after the treatment of the cells with S(−)-SAL whereas R(+)-SAL did not affect the cAMP production. The obtained results suggest that S(−)-SAL is possibly involved in the establishment of the opioid deficiency in alcoholics.

Corticotropin release-inhibiting factor is preprothyrotropin-releasing hormone-(178-199).

ACTH is the major regulator of the body's adaptive response to stress and the physiological stimulus for glucocorticoid secretion. In addition to the known negative feedback regulation of ACTH by glucocorticoids, a hypothalamic corticotropin release-inhibiting factor (CRIF) that inhibits ACTH synthesis and secretion has been postulated, but not identified. We previously reported that transfection of prepro-TRH complementary DNA into the mouse anterior pituitary tumor cell line AtT-20 results in inhibition of basal and corticotropin-releasing hormone (CRH)-stimulated ACTH synthesis and secretion, suggesting that one or more of the cryptic peptides encoded within the prepro-TRH precursor has CRIF activity. To narrow the choice of peptides responsible for CRIF activity, we first deleted specific sequences within the prepro-TRH complementary DNA and transfected these constructs into AtT-20 cells. Deletion of sequences encoding amino acids 119-229 resulted in the loss of CRIF activity. Of the peptides encoded within this region, prepro-TRH-(178-199), a 22-amino acid peptide, inhibited basal and CRH-stimulated ACTH synthesis and secretion in cultured primary anterior pituitary cells. As this peptide is processed from prepro-TRH in vivo, is found in the external zone of the median eminence, and is secreted from hypothalamic slices in vitro, prepro-TRH-(178-199) fulfills the criteria for a physiological CRIF. The significance of TRH and CRIF sharing a common precursor opens new areas of research in the integrated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release-inhibiting factor is preprothyrotropin-releasing hormone-(178-199)

ACTH is the major regulator of the body's adaptive response to stress and the physiological stimulus for glucocorticoid secretion. In addition to the known negative feedback regulation of ACTH by glucocorticoids, a hypothalamic corticotropin release-inhibiting factor (CRIF) that inhibits ACTH synthesis and secretion has been postulated, but not identified. We previously reported that transfection of prepro-TRH complementary DNA into the mouse anterior pituitary tumor cell line AtT-20 results in inhibition of basal and corticotropin-releasing hormone (CRH)-stimulated ACTH synthesis and secretion, suggesting that one or more of the cryptic peptides encoded within the prepro-TRH precursor has CRIF activity. To narrow the choice of peptides responsible for CRIF activity, we first deleted specific sequences within the prepro-TRH complementary DNA and transfected these constructs into AtT-20 cells. Deletion of sequences encoding amino acids 119-229 resulted in the loss of CRIF activity. Of the peptides encoded within this region, prepro-TRH-(178-199), a 22-amino acid peptide, inhibited basal and CRH-stimulated ACTH synthesis and secretion in cultured primary anterior pituitary cells. As this peptide is processed from prepro-TRH in vivo, is found in the external zone of the median eminence, and is secreted from hypothalamic slices in vitro, prepro-TRH-(178-199) fulfills the criteria for a physiological CRIF. The significance of TRH and CRIF sharing a common precursor opens new areas of research in the integrated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release inhibiting factor is encoded within prepro-TRH

Corticotropin synthesis and secretion is under negative feedback regulation by glucocorticoids. In addition a hypothalamic factor inhibiting ACTH synthesis and secretion, named corticotropin release inhibiting factor (CRIF), has been postulated but not identified. Here we report that transient transfection of a rat prepro-TRH cDNA into the mouse anterior pituitary tumor cell line AtT-20 inhibits the synthesis and secretion of both basal and CRH-stimulated ACTH. Thus, CRIF appears to be encoded by the same precursor as TRH, suggesting a coordinated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release inhibiting factor is encoded within prepro-TRH.

Corticotropin synthesis and secretion is under negative feedback regulation by glucocorticoids. In addition a hypothalamic factor inhibiting ACTH synthesis and secretion, named corticotropin release inhibiting factor (CRIF), has been postulated but not identified. Here we report that transient transfection of a rat prepro-TRH cDNA into the mouse anterior pituitary tumor cell line AtT-20 inhibits the synthesis and secretion of both basal and CRH-stimulated ACTH. Thus, CRIF appears to be encoded by the same precursor as TRH, suggesting a coordinated regulation of pituitary-adrenal and pituitary-thyroid functions.

Secretion in AtT-20 cells stably transfected with soluble synaptotagmins.

Synaptotagmin (p65) is an integral membrane secretory vesicle-specific protein with two cytoplasmic repeats homologous to the C2 regulatory domain of protein kinase C. Synaptotagmin has been implicated in the regulation of neurotransmitter release from nerve growth factor-differentiated PC12 cells and from synapses in Drosophila, Caenorhabditis elegans, and squid. To address the function of synaptotagmin in endocrine cells, fragments of rat synaptotagmin I were stably expressed in the mouse anterior pituitary cell line AtT-20. The logic of these experiments is that the fragments may interfere with the endogenous synaptotagmin machinery, thus producing a dominant-negative phenotype. Transfected cells expressed the expected fragments that were comprised of either the first C2 repeat, the second C2 repeat, or the entire cytoplasmic domain. The fragments were localized to both soluble and membrane-associated cellular fractions, despite the absence of the transmembrane domain. The second C2 repeat was shown to coimmunoprecipitate with endogenous synaptotagmin, suggesting that protein-protein interactions are mediating the membrane association of the fragments. These fragments had no effect on the targeting of regulated secretory vesicles or on regulated secretion as assayed by the release of ACTH and [3H]choline. Constitutive secretion assayed by the release of glycosaminoglycan side chains was also unaffected, as was the endocytic pathway monitored by the uptake and clearance of transferrin. These data suggest either the existence of a redundant pathway in secretion or that regulated membrane traffic in endocrine cells does not require synaptotagmin.

Expression of cone transducin, Gz alpha, and other G-protein alpha-subunit messenger ribonucleic acids in pancreatic islets.

The G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins that play important roles in signal transduction and whose expression is regulated in a tissue-specific manner. Here we have surveyed the expression of G-protein alpha-subunits in mouse pancreatic islets. Degenerate oligonucleotide primers corresponding to conserved primary sequences in known G alpha-subunits were used in a reverse transcriptase-polymerase chain reaction, and the amplified complementary DNA (cDNA) fragments were subcloned and sequenced. Over 100 clones were analyzed, from which we determined that islet cells express at least seven G alpha-subunits: G8 alpha, Gi1 alpha or Gi3 alpha, Gi2 alpha, G11 alpha, G14 alpha, Gz alpha, and Gt2 alpha (cone transducin). In particular, the identification of Gz alpha and Gt2 alpha was of interest in that previous studies had indicated that the expression of Gz alpha was restricted mainly to the brain, retina, and adrenal gland, whereas Gt2 alpha was expressed predominantly in retinal cone photoreceptors. By Western blot analysis, we estimated that the amount of Gz alpha protein present in mouse islets was about 40% of that in retina. To further investigate the expression of Gt2 alpha, mouse Gt2 alpha cDNA was cloned from a retinal library and sequenced. The cDNA was used as a probe for Northern blot analysis, and the results confirmed that mouse islets contained a substantial level of Gt2 alpha messenger RNA (mRNA), albeit less than that found in retina (approximately 5-fold lower). Gt2 alpha mRNA was also shown to be present in a clonal mouse pancreatic alpha-cell line (alpha TC1-6) as well as in adrenal gland, pituitary, and a clonal mouse anterior pituitary cell line (AtT20). In situ hybridization revealed that Gt2 alpha mRNA was expressed essentially throughout the islet, suggesting that it is normally expressed in the abundant islet beta-cells and possibly others. In situ analysis also showed that Gt2 alpha mRNA expressed in the pituitary was limited to the intermediate and anterior lobes. We conclude that islet cells express multiple G-proteins, including several that are normally expressed at high levels in certain neuronal cells.

Expression of cone transducin, Gz alpha, and other G-protein alpha- subunit messenger ribonucleic acids in pancreatic islets

The G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins that play important roles in signal transduction and whose expression is regulated in a tissue-specific manner. Here we have surveyed the expression of G-protein alpha-subunits in mouse pancreatic islets. Degenerate oligonucleotide primers corresponding to conserved primary sequences in known G alpha-subunits were used in a reverse transcriptase-polymerase chain reaction, and the amplified complementary DNA (cDNA) fragments were subcloned and sequenced. Over 100 clones were analyzed, from which we determined that islet cells express at least seven G alpha-subunits: G8 alpha, Gi1 alpha or Gi3 alpha, Gi2 alpha, G11 alpha, G14 alpha, Gz alpha, and Gt2 alpha (cone transducin). In particular, the identification of Gz alpha and Gt2 alpha was of interest in that previous studies had indicated that the expression of Gz alpha was restricted mainly to the brain, retina, and adrenal gland, whereas Gt2 alpha was expressed predominantly in retinal cone photoreceptors. By Western blot analysis, we estimated that the amount of Gz alpha protein present in mouse islets was about 40% of that in retina. To further investigate the expression of Gt2 alpha, mouse Gt2 alpha cDNA was cloned from a retinal library and sequenced. The cDNA was used as a probe for Northern blot analysis, and the results confirmed that mouse islets contained a substantial level of Gt2 alpha messenger RNA (mRNA), albeit less than that found in retina (approximately 5-fold lower). Gt2 alpha mRNA was also shown to be present in a clonal mouse pancreatic alpha-cell line (alpha TC1-6) as well as in adrenal gland, pituitary, and a clonal mouse anterior pituitary cell line (AtT20). In situ hybridization revealed that Gt2 alpha mRNA was expressed essentially throughout the islet, suggesting that it is normally expressed in the abundant islet beta-cells and possibly others. In situ analysis also showed that Gt2 alpha mRNA expressed in the pituitary was limited to the intermediate and anterior lobes. We conclude that islet cells express multiple G-proteins, including several that are normally expressed at high levels in certain neuronal cells.

Temperature effect on the action of corticotropin-releasing hormone (CRH) on secretion of immunoreactive beta-endorphin (IRβEP) in TM3 and AtT-20 cell lines

Testicular function is sensitive to chemical and thermal stresses. To investigate the effects of small temperature changes on CRH-stimulated βEP release, we employed TM3 cells, a mouse prepubertal Leydig cell line that secretes irβEP. To monitor βEP secretion from these cells we used the reverse hemolytic plaque assay. After 3.5 hr incubation of cells with hormone, the EC 50 of the CRH dose-response curve at 34°C and 37°C were 0.1 nM and 1 nM, respectively. For comparison, we also investigated the effect of temperature on CRH-stimulated βEP release from a non-testicular cell line, AtT-20, a mouse anterior pituitary cell line. Using radioimmunoassay to measure irβEP levels in the media of AtT-20 cells, the EC 50 S for the CRH dose-response curve at 34°C and 37°C were 0.2 nM and 2 nM, respectively, at 1 h. After 3.5 h this temperature dependent difference in EC 50 was still observed. These results suggest that CRH receptors or post-receptor actions in Leydig cells and anterior pituitary corticotropes are sensitive to small temperature changes.

Phorbolester stimulates the activity of human corticotropin-releasing hormone gene promoter via 3',5'-cyclic adenosine monophosphate response element in transiently transfected chicken macrophages

We previously characterized the transcriptional activity of the human CRH (hCRH) gene promoter in the mouse anterior pituitary cell line AtT20. Here, we show that phorbolester stimulated through the cAMP response element (CRE), located between -227 and -220 relative to the putative cap site, about 2.6-fold the expression of a chimeric hCRH-chloramphenicol-acetyl-transferase gene which was transiently transfected into chicken macrophages (HD11 cell line). The induction was dependent on the cell types, and was not observed when AtT20 were used as target cells. Elevated mouse c-fos protein expressed from a cotransfecting plasmid pRSVfos or human c-jun protein expressed from RSVjun led to 2.1-fold and 3.1-fold stimulation of the activity of the hCRH promoter. Tetradecanoyl-phorbol-13-acetate stimulated the c-fos and c-jun transactivation by a factor of 10 and 2.6, indicating a modification of c-fos and c-jun is required for the transactivation induced by protein kinase C activation. Mutation within the cAMP response element abolished this transcriptional activation. This result suggests an involvement of the protein kinase C pathway in the regulation of the CRH gene expression.

Phorbolester stimulates the activity of human corticotropin-releasing hormone gene promoter via 3',5'-cyclic adenosine monophosphate response element in transiently transfected chicken macrophages.

We previously characterized the transcriptional activity of the human CRH (hCRH) gene promoter in the mouse anterior pituitary cell line AtT20. Here, we show that phorbolester stimulated through the cAMP response element (CRE), located between -227 and -220 relative to the putative cap site, about 2.6-fold the expression of a chimeric hCRH-chloramphenicol-acetyl-transferase gene which was transiently transfected into chicken macrophages (HD11 cell line). The induction was dependent on the cell types, and was not observed when AtT20 were used as target cells. Elevated mouse c-fos protein expressed from a cotransfecting plasmid pRSVfos or human c-jun protein expressed from RSVjun led to 2.1-fold and 3.1-fold stimulation of the activity of the hCRH promoter. Tetradecanoyl-phorbol-13-acetate stimulated the c-fos and c-jun transactivation by a factor of 10 and 2.6, indicating a modification of c-fos and c-jun is required for the transactivation induced by protein kinase C activation. Mutation within the cAMP response element abolished this transcriptional activation. This result suggests an involvement of the protein kinase C pathway in the regulation of the CRH gene expression.

Immunomodulatory ionophore copolymers, T150R1 and T130R2, induce corticotropin from anterior pituitary cells.

T150R1 is a synthetic copolymer with Na+ ionophore activity. We demonstrated previously that T150R1, when injected into mice, produces rapid thymic involution with depletion of cortical thymocytes. Elevated serum ACTH and corticosterone levels, as well as abrogation of the effects of T150R1 on the thymus by adrenalectomy and hypophysectomy, suggested a pituitary-mediated mechanism. In this work, we investigated the ability of T150R1, and of the related ionophore copolymer T130R2, to stimulate ACTH in vitro from the mouse anterior pituitary cell line AtT-20. Copolymer-induced ACTH release was dose-, time-, and temperature-dependent. Hormone induction peaked at 30 degrees C for T150R1 and 37 degrees C for T130R2. The temperature dependence of ACTH release paralleled that of ionophore activity measured in red blood cells, providing evidence that the ability to induce ACTH is related to the ionophore property of the copolymers. Peak ionophore activity and hormonal release occurred at the temperatures when the copolymers form partially soluble complexes which interact optimally with cell membranes. Cotreatment with exogenous phospholipase C inhibited the effects of T150R1, which suggests that the enzyme either blocks the insertion of T150R1 into the cell membrane or that the phospholipase C-induced increase in intracellular calcium inhibits the ionophore activity of T150R1. These data support an ionophore mechanism for copolymer-induced ACTH release in which changes in the physicochemical structure of the copolymers may affect their interaction with cell membranes. The data also suggest that direct stimulation of pituitary ACTH accounts for at least some of the in vivo immunomodulatory effects of T150R1.

Effects of glucocorticoid on corticotropin-releasing hormone gene regulation by second messenger pathways in NPLC and AtT-20 cells.

We have examined the regulation of the hypothalamic secretagogue CRH by glucocorticoid and the protein kinase-A and -C second messenger pathways in cultured cells. We show that the human primary liver carcinoma NPLC expresses the endogenous CRH gene. Dexamethasone reduced CRH mRNA levels by more than 90%, with half-maximal suppression at 5 nM. Phorbol ester treatment to activate the protein kinase-C pathway increased CRH mRNA levels up to 30-fold, whereas forskolin treatment to activate the protein kinase-A pathway had no effect. In coincubation experiments, dexamethasone completely suppressed phorbol ester-induced CRH mRNA levels in NPLC cells, maintaining them at the levels seen in untreated cells. We contrasted this regulation with the effects of glucocorticoid on CRH mRNA induction by forskolin in R1, a mouse anterior pituitary cell line (AtT-20) stably transfected with the human CRH gene. Dexamethasone suppressed forskolin-induced CRH mRNA levels by 70% in R1 cells, but only to levels that were still 10-fold greater than those in untreated cells. These results suggest that CRH induction in vivo by ligands that act via protein kinase-A may be less effectively suppressed by glucocorticoid feedback than CRH induction by ligands that act via protein kinase-C. This differential effect of glucocorticoid on CRH mRNA regulation could help explain the abnormal CRH production observed in clinical disorders such as anorexia nervosa and major depression.

Characterization of a neuronal interleukin-1 receptor and the corresponding mRNA in the mouse anterior pituitary cell line AtT-20.

Interleukin-1 (IL-1) mediates numerous responses on the mouse anterior pituitary cell line AtT-20. We have studied the ligand binding properties of the IL-1 receptors (IL-1R) of the AtT-20 cells and found that they possess 1.5 x 10(3) receptors/cell with a Kd of 0.15 nM for [125I]IL-1 alpha. Using oligonucleotide primers, which define a 236 bp region of the cDNA coding for the cytosolic part of the mouse T-cell IL-1R, and cDNA prepared from AtT-20 cells, we obtained by polymerase chain reaction (PCR) a DNA fragment which was shown to possess an identical sequence to that of the corresponding region of the mouse T-cell IL-1R. Thus the AtT-20 pituitary cell line IL-1 receptor appears to be similar to those found on mouse T-cells and fibroblasts.

Interleukin-1 potentiation of β-endorphin secretion and the dynamics of interleukin-1 internalization in pituitary cells

1. This study demonstrates that human recombinant Interleukin-1 (IL-1) stimulates β-endorphin release and potentiates the secretion of β-endorphin in both a mouse anterior pituitary cell line AtT-20 and rat pituitary cell cultures. 2. In pituitary cell cultures, prolonged treatment with phorbol ester had no effect on IL-1-induced β-endorphin release, but abolished the potentiating effects of IL-1 on vasopressin-induced β-endorphin secretion. 3. The enhancement of CRF-stimulated β-endorphin release by IL-1 was also reduced in normal pituitary cell cultures following depletion of protein kinase C. 4. The late IL-1-induced secretion of β-endorphin does not require the continuous presence of the cytpkine. 5. Incubation of monolayers with 125I-IL-1α (10 −9M) at 8°C and then at 37°C for various times revealed that IL-1α was internalized. There was a progressive increase in the ratio of cytoplasmic to cell-surface-associated 125I-IL-1α. 6. These results indicate that the IL-1-induced β-endorphin release and its potentiation of β-endorphin secretion involves internalization of this cytokine, perhaps via cell surface IL-1 receptors.

Interleukin 1 induces beta-endorphin secretion via Fos and Jun in AtT-20 pituitary cells.

Previous work had shown that interleukin 1 (IL-1), after a long period of treatment, stimulates beta-endorphin release and potentiates the effects of secretagogues in AtT-20 cells, a mouse anterior pituitary cell line. Treatment of AtT-20 cells with IL-1 induced a transient and early stimulation of mRNA expression by both immediate-early protooncogenes Fos and Jun (mouse c-fos and c-jun). The effect appeared within 30 min, and returned to basal levels after 2 hr. Desensitization of protein kinase C by phorbol ester pretreatment had no effect on the ability of IL-1 to induce Fos and Jun mRNA expression. Somatostatin, an inhibitor of cAMP and beta-endorphin secretion, did not reduce the IL-1 effect on Fos and Jun mRNA expression. Addition to AtT-20 cells of antisense oligonucleotides to Fos and Jun abolished the secretion induced by IL-1. These results indicate that immediate-early signals Fos and Jun are involved in IL-1-induced beta-endorphin secretion in AtT-20 cells.

Glucocorticoid Repression of 3',5'-Cyclic-Adenosine Monophosphate-Dependent Human Corticotropin-Releasing-Hormone Gene Promoter Activity in a Transfected Mouse Anterior Pituitary Cell Line

The regulation of the expression of the human corticotropin-releasing-hormone gene (hCRH) was studied in a mouse anterior pituitary cell line (AtT20) after transiently transfection with a chimeric gene containing the hCRH gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Expression of the chimeric hCRH-CAT gene in AtT20 cells was enhanced by the cAMP analog (8-bromo-cAMP) about 5-fold but not by phorbol 12-myristate-13-acetate. The cAMP phosphodiesterase inhibitor isobutylmethylxanthine also strongly stimulated 15-fold the expression of the chimeric hCRH-CAT gene. Coincubation of cAMP analog and isobutylmethylxanthine resulted in a moderate 2-fold synergistic enhancement of CAT activity. Sequence comparison of the hCRH gene revealed a core sequence for a cAMP responsive element 5'-TGACGTCA-3' at -221 relative to the cap site. This regulatory element also confers cAMP inducibility on a heterologous promoter when placed upstream of the thymidine kinase promoter from herpes simplex virus. Finally, treatment with 0.5 microM dexamethasone reduced CAT activity about 2.0-fold in cAMP-stimulated cells. This result suggests that cAMP and glucocorticoids coordinately control hCRH gene expression.

Low temperature blocks exit of pro‐opiomelanocortin from the endoplasmic reticulum but not subsequent delivery to the site of prohormone processing

The effect of reduced temperature on the delivery of the prohormone pro-opiomelanocortin (POMC) to the site of prohormone processing was investigated in the mouse anterior pituitary cell line AtT20. At 20 degrees C processing was substantially inhibited and was almost completely arrested at 18 degrees C. Earlier studies with membrane glycoproteins indicated that at these temperatures protein movement was blocked at the level of exit from the Golgi apparatus. In contrast it was found here that the inhibition of processing at reduced temperature was due to the retention of POMC in the endoplasmic reticulum. When POMC was allowed to progress to the Golgi before temperature was reduced, subsequent processing was only slightly retarded by incubation at 18 degrees C. This indicates either that Golgi exit is not inhibited at this temperature, or that the processing apparatus exists in the Golgi. A surprising incidental result was that when held in the endoplasmic reticulum at low temperature POMC is apparently subject to post-translational N-linked glycosylation.