Endogenous ouabain, sodium balance and blood pressure.
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Adrenal Cortex↗
Biomedical subjects
L K Lewis
Publications and source records attributed to L K Lewis.
Chronic ouabain infusion does not cause hypertension in sheep.
Ouabain is claimed to be a hormone of adrenal origin, capable of raising arterial pressure in rats. We infused ouabain in conscious sheep under carefully controlled circumstances to determine its effects on blood pressure, urine electrolytes, and vasoactive hormones. Eight healthy ewes were studied while taking a constant intake of dietary sodium and potassium. Ouabain infusion at 0.25 mg daily over 22 days reduced heart rate and arterial pressure and had no effect on pressor responsiveness to incremental intravenous infusions of angiotensin II. Ouabain induced minor, but statistically significant, decrements in urine volume, urinary sodium excretion, plasma renin and angiotensin II concentrations, and a rise in plasma aldosterone and cortisol. Plasma ouabain levels averaged 1.37 +/- 0.28 nmol/l during ouabain infusion. In conclusion, high-dose chronic ouabain infusion in sheep did not elevate arterial pressure or alter pressor responsiveness to angiotensin II, was antidiuretic and antinatriuretic, and induced minor perturbations in circulating renin, angiotensin II, aldosterone, and cortisol.
Adrenocorticotropic Hormone↗
Ouabain: a new steroid hormone?
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Adrenal Cortex Hormones↗
Long-term ouabain administration does not alter blood pressure in conscious Sprague-Dawley rats.
1. We tested the ability of ouabain to cause chronic hypertension by continuously infusing ouabain for 28 days (miniosmotic pump implantation; i.p.). The blood pressure and metabolic effects of sham (150 mmol/L NaCl; n = 12) or ouabain infusion (10 micrograms/kg per day; n = 14; 100 micrograms/kg per day; n = 14) were examined in conscious Sprague-Dawley rats. 2. Plasma ouabain concentrations measured after 28 days of ouabain infusion were as follows: sham, not detectable (n = 11); ouabain 10 micrograms/kg per day, 0.60 +/- 0.07 nmol/L (n = 14); and ouabain 100 micrograms/kg per day, 7.17 +/- 0.57 nmol/L (n = 14; P < 0.001). 3. Sham or ouabain infusion did not alter food intake, bodyweight, water intake or urine output in conscious rats. 4. Blood pressure was not altered by sham treatment. Ouabain at 10 micrograms/kg per day or 100 micrograms/kg per day did not produce consistent rises in blood pressure. Ouabain at 10 micrograms/kg per day increased blood pressure on treatment day 12 only (+6 mmHg; P < 0.05), while at 100 micrograms/kg per day blood pressure increased on treatment days 16 (+9 mmHg; P < 0.05) and day 18 (+8 mmHg; P < 0.05) only. There was no significant difference in blood pressure between sham and ouabain groups. 5. Renal blood flow was decreased in rats infused with ouabain at 10 micrograms/kg per day (2.0 +/- 0.3 mL/min per 100 g bodyweight; n = 5; P < 0.01) and 100 micrograms/kg per day (2.2 +/- 0.4 mL/min per 100 g bodyweight; n = 7; P < 0.05) compared with sham treatment (3.5 +/- 0.2 mL/min per 100 g bodyweight; n = 6). Renal vascular resistance was increased in rats treated with ouabain at 10 micrograms/kg per day (65.5 +/- 12.6 mmHg/mL per min per 100 g bodyweight; n = 5; P < 0.01) and 100 micrograms/kg per day (66.0 +/- 15.6 mmHg/mL per min per 100 g bodyweight; n = 7; P < 0.05) compared with sham treatment (32.6 +/- 2.5 mmHg/mL per min per 100 g bodyweight; n = 6). 6. High plasma concentrations of ouabain do not cause consistent increases in blood pressure in conscious Sprague-Dawley rats.
Animals↗
Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli.
A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.
Amino Acid Sequence↗
Regional distribution of estradiol receptors in growing antlers.
This study of estrogen receptors (ER) was carried out to confirm their presence and to determine their localisation in antler bones. Partially grown antlers were amputated from red deer (Cervus elaphus) stags, the skin removed, and samples taken of periosteum, cartilaginous tissue including perichondrium, and bone. Capacity and binding of free ER in the samples were calculated by Scatchard analysis of data obtained from a radioreceptor assay which utilised [3H]estradiol as tracer. High affinity ER (ka 1.3-3.4 x 10(10)/M) were detected in all tissues sampled with the exception of bone. Receptor capacity ranged from 12-74 fmol/mg protein, ranking the tissues for capacity in the following descending order: periosteum, cartilage, calcified cartilage. These results demonstrate the presence of ER in growing antlers and indicate regional localization of the receptors within these structures. The absence of ER in bone tissue within the antler suggests that the effect of estradiol on stimulation of mineralization in this tissue is indirect and must occur via its binding to the non-calcified tissues of antlers, e.g., periosteum, perichondrium, and cartilage.
Animals↗
Acute effects of intravenous ouabain in healthy volunteers.
1. The influence of single-dose intravenous ouabain on haemodynamics, renal function and vasoactive hormones was investigated in healthy subjects. 2. After ouabain injection there was a significant reduction in minimum and average hourly heart rates. Diastolic blood pressure fell immediately after ouabain, without a significant change in systolic blood pressure. 3. There was no change in renal blood flow, glomerular filtration rate, hourly urine volume or sodium and potassium excretion. 4. Ouabain injection was associated with a fall in plasma angiotensin II levels and a rise in plasma adrenaline levels, but plasma noradrenaline levels were unchanged. Both plasma atrial and brain natriuretic peptide levels were increased after ouabain administration. 5. The present study demonstrates that ouabain has significant although minor, acute effects on plasma levels of vasoactive hormones. It fails, however, to confirm that ouabain is an acute pressor or natriuretic substance. If it has an important influence on electrolyte and haemodynamic homeostasis, its actions are likely to be slowly developed.
Adult↗
Effect of ouabain on pressor responsiveness in normal man.
To assess the effects of ouabain on pressor and vasoactive hormone responsiveness, 10 healthy volunteers were pretreated with ouabain (0.5 mg i.v. 42 and 18 h before study) or placebo before pressor challenge with angiotensin II (ANG II; 2, 4, and 8 ng.kg-1.min-1 for 30 min/dose) and norepinephrine (NE; 5, 15, and 45 ng.kg-1.min-1 for 15 min/dose). There were no differences at baseline between the two study days regarding mean arterial pressure (MAP) or heart rate. Baseline pulse pressure, however, was significantly greater after ouabain (47 +/- 3 vs. 41 +/- 1 mmHg; P < 0.05). The mean maximum increments in MAP during ANG II and NE infusions were 17.5 +/- 1.1 and 10.5 +/- 1.3 (SE) mmHg, respectively, after ouabain and 19.2 +/- 1.3 and 10.4 +/- 1.5 mmHg after placebo (not significant). The mean heart rate was lower during both infusion periods on the ouabain study day compared with control (P < 0.05). Baseline plasma levels of ANG II, aldosterone, plasma renin activity, atrial and brain natriuretic peptide, guanosine 3',5'-cyclic monophosphate, NE, and epinephrine and achieved levels during the two infusions were similar on the two study days. We conclude that short-term ouabain administration does not alter pressor responsiveness or plasma levels of vasoactive hormones in healthy volunteers.
Adult↗
Ouabain is not detectable in human plasma.
An enzyme-linked immunosorbent assay is described for the measurement of ouabain in human plasma. This assay is specific for ouabain, strophanthidin, and ouabagenin, with other steroids, including digoxin and vasopressor hormones, exhibiting negligible cross-reactivity. Assay sensitivity was 0.06 nmol/L if 1 mL plasma was extracted and less than 0.005 nmol/L when 20 mL plasma was analyzed. Extracted plasma samples showed ouabainlike immunoreactivity that diluted in parallel with the ouabain standard curve. Repeated extraction and assay of single plasma samples, however, did not produce consistent results in the assay. Increased specificity was obtained by high-performance liquid chromatography of sample extracts before assay. When high-performance liquid chromatographic profiles of plasma spiked with ouabain standard or following bolus intravenous injections of ouabain into normal human volunteers were compared with profiles of unspiked plasma, there was no support for the immunoreactive material in the latter samples being ouabain. We propose that if ouabain is present in the human circulation, its concentration is less than 0.005 nmol/L.
Animals↗
An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).
A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.
Animals↗
Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor.
A new genetic screening method has been developed to isolate Escherichia coli promoters which are components of the SOS regulon. Plasmids containing the regulatory regions of polB (dinA) and two new loci, dinG and dinH, were characterized. Galactokinase gene fusion experiments indicated that transcription of these genes is inducible by treatment with mitomycin and conforms to a classical model of SOS regulation involving simple LexA repression. Mapping studies using the E. coli DNA library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) revealed that dinG and dinH are located at 17.8 and 19.8 min on the chromosome, respectively. The nucleotide sequence of the dinH regulatory region contains a segment which is very similar to previously characterized binding sites for LexA protein. An asymmetric, noncanonical 20-bp LexA operator in the cloned dinG promoter region was identified. Additional experiments have revealed that the nucleotide sequence of the gene immediately downstream of the DNA damage-inducible polB locus encodes a polypeptide which has extensive sequence homology to several known and putative DNA and RNA helicase proteins. This gene, which is not regulated by the LexA repressor, has been designated hepA. The predicted amino acid sequence of the product of hepA contains several highly conserved sequence motifs that are also found in enzymes such as the RecQ and UvrB proteins of E. coli and the Rad3 protein of Saccharomyces cerevisiae.
Amino Acid Sequence↗
Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene.
The dinG gene was originally isolated during a search for Escherichia coli promoters which are components of the SOS regulon. The regulatory region of this gene contains a potential binding site for LexA repressor which is quite different from other known sites. All previously described chromosomal LexA operators are imperfect palindromes containing the sequence CTG(N10)CAG. The noncanonical dinG sequence breaks the symmetry and takes the form TTG(N10)CAG. In the present study, a search for mutations within dinGop::galK fusion plasmids which render transcription independent of intracellular levels of LexA has yielded mutations only within this 16-bp sequence. Electrophoretic mobility shift assays performed with purified mutant and wild-type operator fragments revealed that the affinity of LexA for each of the mutant sites is greatly reduced compared with that of the wild type. One of the mutants contained an alteration in the putative promoter of dinG which increased the similarity of the -35 region to the consensus sequence (TTGGCT----TTGACT); the apparent promoter activity of this construct was subsequently found to be approximately eight times higher than that of the wild type in vivo. Additional experiments have established the complete nucleotide sequence of the dinG gene. A long open reading frame located immediately downstream of the asymmetric operator segment which could potentially encode a 72.9-kDa DinG protein was identified.
Amino Acid Sequence↗
BIGPROBE: a computer program that predicts the sequence of long oligonucleotide probes with high reliability.
We have written a computer program, BIGPROBE, which facilitates the design of long nucleic acid probes from the partial or complete amino acid sequence of a protein. BIGPROBE relies upon information on codon usage, intercodon dinucleotide frequency, and potential probe self-complementarity. We have examined the accuracy with which the program predicts coding sequences using sample human and rat genes and probe lengths of 30-60 nucleotides. Rat probe sequences selected by BIGPROBE using either codon usage or dinucleotide frequency data alone averaged 86-92% homology with the known exons of the corresponding gene sequences. Predictive accuracy with rat gene probes could be improved to 89-94%, depending upon probe length, by applying codon usage and dinucleotide frequency data in combination. Similar accuracy was achieved for human genes.
Amino Acid Sequence↗
CT and angiographic correlation of severe neurological disease in toxemia of pregnancy.
Two patients with severe cerebral symptoms of toxemia of pregnancy were examined using computed tomography (CT) and angiography. CT disclosed extensive areas of low attenuation within the cerebral hemispheres. Angiography revealed constriction and narrowing of proximal and peripheral vessels suggesting vasculitis with extensive areas of impaired regional cerebral blood flow. A review of the known pathology and the theories regarding the pathophysiology of the cerebral effects of toxemia is presented.
Adult↗