Nomenclature for atrial peptides.
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Atrial Natriuretic Factor↗
Biomedical subjects
J D Baxter
Publications and source records attributed to J D Baxter.
The primary structure of coho salmon growth hormone and its cDNA.
Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3'-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.
Amino Acid Sequence↗
Identification of atrial natriuretic factor gene transcripts in the central nervous system of the rat.
Atrial natriuretic factor (ANF) gene transcripts have been identified in the hypothalamus, brainstem, and cerebral cortex of the rat. The hypothalamic transcripts are similar in overall size (approximately 1100 nucleotides), 5' terminus, 3' terminus, and nucleotide sequence to their counterparts synthesized in the cardiac atria. The 5' terminus of the hypothalamic transcripts, determined by both S1 nuclease and primer-extension analysis, was located approximately 20 nucleotides downstream from the TATAAAA sequence that is thought to dictate the start of transcription. Similar 5' termini were identified in RNA from the brainstem and cerebral cortex. The 3' termini of the hypothalamic transcripts mapped approximately 10 nucleotides downstream from each of two successive AATAAA sequences that are believed to provide the cleavage signal required for subsequent polyadenylylation. The same 3' termini were present in atrial, ventricular, lung, and pituitary RNA. To confirm the identity of the hypothalamic gene transcript, a partial-length cDNA clone encoding ANF was isolated from a hypothalamic cDNA library and sequenced. This hypothalamic clone was identical to an analogous atrial cDNA clone at each of its 612 nucleotide positions. These findings suggest that ANF peptides, previously identified in specific loci of the hypothalamus and pontine tegmentum, are synthesized locally in these tissues and support a role for this peptide in the central regulation of cardiovascular physiology.
Amino Acid Sequence↗
Differential ability of various plasmid DNAs to sequester inhibitors of RNA polymerase III transcription.
Deletion mutants of the Drosophila tRNA(Arg) gene that lack A-box promoter sequences are not transcribed in several cell-free systems; however, they are actively expressed in vivo in Xenopus oocytes (Sharp et al., 1983a). We show that two A-box deletion mutants of the tRNA(Arg) gene can be transcribed by a HeLa cell-free transcription system if it is preincubated with various DNAs, indicating that an inhibitor is responsible for the lack of mutant tRNA gene transcription. Optimal mutant transcription rescue, and presumably optimal binding of inhibitor, is facilitated by the presence of an active RNA polymerase II promoter in the preincubating DNA. Plasmid DNAs containing RNA polymerase III or weak RNA polymerase II promoters are of intermediate rescue efficiency, and pBR322 DNA is least efficient. Competition studies indicate that the stability of the inhibitor-DNA complex formed initially is apparently increased if the preincubating DNA contains an active RNA polymerase II promoter. Thus, HeLa whole-cell lysates contain a specific inhibitor(s) of RNA polymerase III transcription that primarily affects weakened RNA polymerase III promoters (e.g., A-box deletion mutants) and binds preferentially to DNAs containing an active RNA polymerase II promoter. Yet this apparent sequestration of inhibitor by Class II templates does not appear to inhibit their subsequent transcription by RNA polymerase II. These data raise the possibility that there may be interactions between the RNA polymerase II and III transcription machinery.
Animals↗
Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.
Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.
Animals↗
Increased synthesis and release of atrial peptide during DOCA escape in conscious dogs.
The escape from the sodium-retaining effects of prolonged mineralocorticoid treatment in animals and humans was first noted over 40 yr ago, but despite intense study the mechanisms responsible for the escape phenomenon have not been identified. Putative "natriuretic hormones" have been proposed to account for the escape phenomenon. To determine whether atrial natriuretic peptides (ANP) could participate in the escape phenomenon, the mineralocorticoid deoxycorticosterone acetate (DOCA) was administered to conscious dogs for 14 days. Escape was accompanied by a doubling of plasma ANP concentration and four- to sevenfold increases in cardiac ANP messenger RNA. There were also significant increases in mean arterial blood pressure during the last 8 days of DOCA treatment. Thus increases in the synthesis and secretion of ANP and increases in atrial pressure may represent mechanisms that contribute to the escape from mineralocorticoid-induced sodium retention.
Animals↗
The gene for the atrial natriuretic factor is expressed in the aortic arch.
The gene for atrial natriuretic factor is expressed within the adventitial cells of the rat aortic arch. Atrial natriuretic factor transcripts, similar in overall size (1100-1200 nucleotides) and 5'-termini to those found in the atria, were identified in the arch. Much lower levels (approximately 10-20%) of these transcripts were present in distal thoracic aorta. Atrial natriuretic factor peptide was localized by immunocytochemistry to the adventitia of the arch in regions thought to harbor the aortic baroreceptors. These data suggest a previously unsuspected role for the peptide in regulating systemic blood pressure through the baroreceptor reflex.
Animals↗
Insulin regulation of rat growth hormone gene expression.
Insulin has been shown previously to inhibit basal and glucocorticoid- or T3-stimulated rat GH (rGH) synthesis, secretion, and mRNA levels in cultured rat pituitary tumor cells (GH3 cells) or pituitaries. The effects of insulin on rGH gene expression in GH3 cells were examined in greater detail in the current studies. Cells were deinduced for 5 days in medium devoid of steroids, T3, and insulin. Cells were then treated for 48 h with insulin (5 X 10(-9) M), dexamethasone (Dex; 10(-6) M), T3 (10(-8) M), insulin plus Dex, or insulin plus T3. When media and hormones were not replaced daily the results were similar to those obtained previously. Insulin decreased both basal and glucocorticoid-stimulated rGH mRNA levels to approximately 70% of control levels, as measured by cytoplasmic dot hybridization. By contrast, when media and hormones were replaced daily, rGH mRNA levels increased by 1.5 to 7-fold in response to insulin in the absence or presence of Dex or T3, measured by both cytoplasmic dot hybridization and RNA (Northern) blotting. Dex increased rGH mRNA levels under both sets of conditions, verifying the specific nature of the insulin influence. Maximum rGH gene expression was achieved after a 48-h exposure to insulin. The observed insulin effects were probably mediated through insulin rather than insulin-like growth factor I or II receptors, since the concentration of insulin employed was near the Kd of the hormone for its receptor measured in the same cells. These results suggest that insulin is capable of regulating rGH gene expression. The action of insulin can be either positive or negative and is influenced by the metabolic state of the cell.
Animals↗
A pituitary-specific factor interacts with an upstream promotor element in the rat growth hormone gene.
The expression of the rat GH (rGH) gene is limited to the anterior pituitary. Using a DNase I footprinting assay, we have sought to identify specific protein-DNA interactions in the rGH 5'-flanking DNA that may be important in conferring tissue-specific use of the rGH promoter. We have identified a nuclear factor from rGH-secreting GC (rat pituitary) cells which interacts in the rGH 5'-flanking DNA between positions -94 to -62 relative to the start site of rGH gene transcription. This factor, which we have named GC1, is undetectable in nuclear extracts of non-rGH-producing rat fibroblast (Rat-2), or HeLa cells. Mutation of the GC1 binding region in the rGH 5'-flanking DNA results in only a small (30%) decrease in the ability of the rGH promoter to drive the expression of a linked marker gene when transfected into GC cells. GC1 represents, therefore, a novel type of promoter binding factor which is gene-specific and whose distribution is tissue-specific, but which is nonessential for the basal expression of its linked gene. Such a factor may, however, play a role in the modulation of rGH gene expression either through its interaction with other regulatory proteins or ligands or after enzyme modification.
Animals↗
Glucocorticoid control of rat growth hormone gene expression: effect on cytoplasmic messenger ribonucleic acid production and degradation.
The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of [3H]uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: 1) pulse-chase studies and 2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production.
Animals↗
Hormonal regulation of expression of the endogenous and transfected human growth hormone gene.
Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenoma↗
Control elements in the human renin gene.
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Amnion↗
The human growth hormone gene is negatively regulated by triiodothyronine when transfected into rat pituitary tumor cells.
Human growth hormone (hGH) gene expression and regulation by thyroid and glucocorticoid hormones were examined after its stable introduction into rat anterior pituitary tumor cells. Transcripts from the hGH gene were correctly initiated and could be positively regulated by glucocorticoids. In contrast to the endogenous rat GH gene and a transfected, modified rat GH gene, the hGH gene was negatively regulated by triiodothyronine. Analysis of hGH gene 5'-flanking deletions indicated that a distal regulatory element is required for efficient transcription. Thus promoter, glucocorticoid, and thyroid hormone control elements are present on the transfected hGH gene, whose expression may be negatively controlled by thyroid hormone.
Animals↗
Atrial natriuretic peptide mRNA is regulated by glucocorticoids in vivo.
In these studies glucocorticoids were found to increase the plasma levels of atrial natriuretic peptide (ANP) as well as the expression of the ANP gene in the Sprague-Dawley rat. Plasma ANP rose two-fold after 48 hrs. of exposure to dexamethasone (1 mg/day) in both intact and adrenalectomized animals. This was accompanied by a 1.5-2.0 fold increase in the levels of atrial and ventricular ANP transcripts. Deoxycorticosterone acetate (5 mg/day), administered on the same schedule, failed to increase either plasma ANP levels or cardiac ANP mRNA accumulation. These effects suggest that ANP may have a potential role as a mediator of glucocorticoid activity in the cardiovascular system and support the hypothesis that ANP is a glucocorticoid-regulated gene.
Animals↗
Glucocorticoids regulate the expression of a rat growth hormone gene lacking 5' flanking sequences.
Rat growth hormone (rGH) gene expression is regulated by glucocorticoids in vivo and in cultured pituitary cells. After the co-transfer of a plasmid containing the rGH gene into mouse L-cells (with or without the simian virus 40 enhancer), little or no normal rGH mRNA is produced. Instead, the predominant rGH gene transcripts are about 0.75 kilobase pairs (kb); these lack the first two exons of the rGH but possess a 3' end that terminates accurately. Nevertheless, the levels of these transcripts are increased by glucocorticoids. When all of the rat sequences 5' to an Xho1 site located 7 nucleotides downstream from the rGH gene physiological cap site are deleted and the mutant gene introduced into L-cells, the transfectant cell lines still produce the 0.75-kb transcripts; however, in addition, these cells produce a more abundant 1.1-kb mRNA that has a 3' terminus similar to that of rGH mRNA, but whose 5' termini begin in the region of, but not at, the initiation site used in pituitary cells. Both of these transcripts are increased 3- to 5-fold by 1 microM concentration of the glucocorticoid dexamethasone. These data indicate that 1) deletion of all of the rGH gene 5' flanking sequences allows formation of approximately full length transcripts and 2) sequences containing information for regulation of rGH gene expression in L-cells by glucocorticoids are contained in the structural portion of the gene and/or the 3' flanking sequence.
Animals↗
Mechanisms of glucocorticoid hormone action.
This report summarizes our studies, in context with the results of other laboratories, of the molecular mechanisms of glucocorticoid hormone action. The receptors for these steroids are comprised of single polypeptide chains of about 90,000 molecular weight. Binding of agonist steroids to the receptor induces a conformational change to an active receptor form that is followed by a second change in the glucocorticoid-receptor complex, termed activation, that alters the charge of the complex and results in its binding to specific sites on the DNA termed glucocorticoid regulatory elements (GREs). The GRE on the human metallothionein-IIA gene is located in the 5'-flanking DNA. It can function independently of the gene's promoter, and when ligated upstream from the herpes simplex virus (HSV) thymidine kinase (TK) gene promoter, can activate it. The binding of the glucocorticoid-receptor complex to the GRE probably alters chromatin structure over a limited span to facilitate RNA polymerase action. The regulation by glucocorticoids of growth hormone gene expression is more complex. The steroid appears to elicit both transcriptional and posttranscriptional influences that are also affected by thyroid hormone. Also the glucocorticoid influences appear to be exerted in part through DNA structures located downstream from the transcriptional initiation site. A GRE has been defined in intron A of the hGH gene through gene transfer and DNA binding experiments. Finally, gene transfer experiments suggest that pituitary-specific factors influence the ability of glucocorticoids to affect GH gene expression.
Adenosine Triphosphate↗
Stable accumulation of a rat truncated repeat transcript in Xenopus oocytes.
To define potential mechanisms of expression of middle-repetitive DNA, Xenopus oocytes were employed to examine the rat type 2 and truncated repeat (TR) elements contained in an intron and in the 3'-flanking region of the rat growth hormone gene. These repeats contain significant sequence and structural homology to tRNA genes and, thus, may represent tRNA pseudogenes. Transcripts from the type 2 elements do not accumulate in the cytosol and are found predominantly in the nucleus, whereas those from TR DNA are expressed in the cytosol of neural and pituitary tissues. In HeLa cell extracts, the rat growth hormone type 2 sequences initiate RNA polymerase III transcription resulting in multiple transcripts of 175-970 nucleotides; some of these also contain TR sequences that are present only as downstream structures since the rat growth hormone-TR DNA lacks promoter activity. In Xenopus oocytes the same template also results in multiple transcripts, but with time a single, homogeneous 73-base RNA preferentially accumulates. This RNA probably arises from larger repetitive DNA transcripts as assessed by the kinetics of its formation, its 5' terminus, and the injection of transcripts generated in HeLa cell-free extracts into the oocytes. Sequence analysis of the 73-base RNA suggests that it is a TR transcripts derived from the TR region with tRNA homology. Stable type 2 transcripts were not detected. Thus, type 2 elements are transcribed in the oocytes, but RNAs from them are degraded whereas discrete TR DNA transcripts can be derived from larger RNA molecules and can accumulate in the cytosol due to their preferential stability. These findings indicate that posttranscriptional control mechanisms can operate to direct differential expression of closely related repetitive DNAs and suggest that structures similar to tRNA contained within the TR sequences may allow them to accumulate preferentially in the cytoplasm.
Animals↗
Characterization of human prorenin expressed in mammalian cells from cloned cDNA.
Human preprorenin was synthesized in Chinese hamster ovary (CHO) cells transfected with an expression vector containing renin cDNA sequences. These cells secrete an inactive form of renin (EC 3.4.23.15) that can be activated by trypsin. This inactive renin is precipitable by antibody generated against purified human renal renin and also by antisera generated to a synthetic peptide derived from the amino acid sequence of the pro segment of preprorenin (anti-propeptide), indicating that the secreted inactive enzyme is a form of prorenin. Analysis of [35S]methionine-labeled proteins immunoprecipitated from CHO cell conditioned culture medium indicates that prorenin is expressed in CHO cells as two distinct forms that differ in their degree of glycosylation. In vitro trypsin activation of prorenin cleaves approximately 4.5 kDa from the protein, rendering it unreactive with the antipropeptide antiserum but still recognizable by anti-renal renin antibody. These results show directly that the prorenin expressed by CHO cells is an inactive enzyme that is activated by trypsin cleavage of the pro segment. The ability to express human renin in this form will allow for the purification of both active and inactive forms of the enzyme in quantities sufficient for detailed physiological and structural studies.
Animals↗