PubMed Health⌕ Search

Biomedical subjects

P A Doris

Publications and source records attributed to P A Doris.

At least 19 recordsLinked to original sources
PubMed2001

High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise.

Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 microl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5' exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping.

Alleles↗
PubMed2001

G-protein beta3 subunit and alpha-adducin polymorphisms and risk of subclinical and clinical stroke.

BACKGROUND AND PURPOSE: Essential hypertension is a significant risk factor for stroke. Genes contributing to interindividual variation in blood pressure levels and essential hypertension status may play a role in the etiology of stroke either through their effects on blood pressure levels or through separate pathways. For this reason, we sought to examine the association between the alpha-adducin (ADD1) G/W460 and G-protein beta3 subunit (GNbeta3) 825C/T polymorphisms and subclinical and clinical stroke in the Atherosclerosis Risk in Communities (ARIC) Study. METHODS: Subclinical stroke was determined by cerebral MRI. Subclinical cerebral infarct cases (n=202) were compared with a stratified random sample (MRI-CRS) identified from individuals participating in the MRI examination (n=211). Incidence of clinical ischemic stroke was determined by following the ARIC cohort for an average of 7.2 years for potential cerebrovascular events; 231 validated clinical ischemic strokes were identified. A stratified random sample of the ARIC cohort (CRS) (n=984) was used as the comparison group for the clinical cases. RESULTS: The frequency of the ADD1 W460 allele was determined for the subclinical cases (0.12), MRI-CRS (0.16), clinical cases (0.14), and CRS (0.17). The frequency of the GNbeta3 825T allele was determined in whites and blacks, respectively, for the subclinical cases (0.26, 0.73), MRI-CRS (0.31, 0.75), clinical cases (0.36, 0.72), and CRS (0.30, 0.72). The ADD1 W460 and GNbeta3 825T alleles were not significantly associated with subclinical stroke. The ADD1 W460 allele was also not a significant predictor of clinical stroke. The GNbeta3 825T allele was significantly associated with clinical stroke in whites after adjustment for age and sex (hazard rate ratio, 1.45; 95% CI, 1.05 to 2.00) and after further adjustment for multiple stroke risk factors (hazard rate ratio, 1.68; 95% CI, 1.18 to 2.41). The GNbeta3 825T allele was not significantly associated with clinical stroke in blacks for either adjustment model. CONCLUSIONS: The GNbeta3 gene 825C/T polymorphism is significantly associated with incident clinical ischemic stroke in a white middle-aged American population, but not in blacks. This association does not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status.

Age Distribution↗
PubMed2001

Abnormalities of sodium pump function in hypertension and the role of endogenous cardiotonic steroids.

Elevated arterial blood pressure is a common heritable susceptibility in the human population. The high penetrance of this trait in industrialized societies may be influenced by the interactions of environmental factors and common genetic variants. This review examines the role of the renal sodium pump (sodium, potassium-ATPase, NKA) in hypertension and its integration into mechanisms of body sodium balance. In particular, renal NKA provides an appealing target by which inherited factors caninfluence renal sodium reabsorption. Recent work has indicated how some such genetic mechanisms may function. In this paper, the capacity of renal NKA to integrate environmental and heritable factors to increase blood pressure are examined.

Absorption↗
PubMed2000

Use of single nucleotide polymorphisms for gene discovery in hypertension.

Genetic susceptibility to common diseases is likely influenced by common DNA variants with small-to-moderate effects, rather than by rare mutations with large effects. The majority of such common variation occurs in the form of single nucleotide polymorphisms (SNPs). DNA sequence variation, including SNPs, is created and maintained by the action of evolutionary forces such as mutation, recombination, and selection. Over time and space, these forces have shaped the relationships between the contemporary organization of the human genome and disease susceptibility in the population at large. Single nucleotide polymorphisms have emerged as valuable new tools to uncover these relationships. In particular, their use in both genome-wide and gene-based association studies holds great promise for the identification of genes and gene variations involved in predisposition to essential hypertension. New advances in biotechnology are making possible large-scale discovery of SNP variation and rapid application of this information to disease gene discovery.

Base Sequence↗
PubMed2000

Arterial responses in vitro and plasma digoxin immunoreactivity after losartan and enalapril treatments in experimental hypertension.

Treatment with the angiotensin-converting enzyme inhibitor, quinapril, has been shown to normalize increased dihydropyridine sensitivity and impaired potassium relaxation, characteristic features of arterial smooth muscle in spontaneously hypertensive rats, and also reduce the concentration of plasma digoxin-like immunoreactivity in these animals. However, whether angiotensin II receptor blocker therapy can beneficially influence these variables is not known. Therefore, we compared the effects of 10-week losartan and enalapril treatments (15 and 4 mg/kg/day, respectively) on functional responses of mesenteric arterial rings in spontaneously hypertensive rats and Wistar-Kyoto rats. Both losartan and enalapril normalized blood pressure, cardiac mass, and media to lumen ratio without significantly changing the media cross-sectional area in the mesenteric artery of spontaneously hypertensive rats (i.e. induced outward remodelling). The inhibitory effect of the calcium entry blocker nifedipine on calcium-evoked contractions was similar and less marked in arterial preparations from Wistar-Kyoto rats and losartan- and enalapril-treated spontaneously hypertensive rats than in those from untreated spontaneously hypertensive rats. Furthermore, the relaxations of arterial rings induced by the return of potassium to the organ bath (upon precontractions elicited by potassium-free solution) were used to evaluate the function of vascular Na+,K+-ATPase. The rate of potassium relaxation was faster in losartan- and enalapril-treated spontaneously hypertensive rats and all Wistar-Kyoto groups than in untreated spontaneously hypertensive rats, and the response was effectively inhibited by the sodium pump inhibitor ouabain. Both treatments especially augmented the ouabain-sensitive part of the potassium-relaxation in spontaneously hypertensive rats, indicating the involvement of the sodium pump in this response. However, no significant changes in plasma digoxin-like immunoreactivity were observed. In conclusion, the outward remodelling following long-term AT1-receptor blockade and angiotensin-converting enzyme inhibition in spontaneously hypertensive rats was associated with normalization of the increased dihydropyridine sensitivity of arteries. Both losartan and enalapril treatments also augmented arterial potassium relaxation in spontaneously hypertensive rats, suggesting enhanced function of Na+,K+-ATPase, but this effect could not be attributed to changes in circulating sodium pump inhibitor concentration.

Angiotensin Receptor Antagonists↗
PubMed2000

Renal proximal tubule sodium transport and genetic mechanisms of essential hypertension.

Renal sodium re-absorption is a closely regulated process serving to maintain both extracellular fluid volume and arterial blood pressure. Proteins participating in sodium re-absorption and its regulation are therefore important candidate proteins whose genes may contain sequence variation contributing to the inherited tendency for increased arterial blood pressure (essential hypertension). Important insight has come from rare forms of single-gene hypertension in human subjects and from polygenic animal models of genetic hypertension. Both indicate the primacy of altered renal function in the genesis of hypertension, and suggest that genes contributing to the disease are members of the subset of genes expressed in the kidney. This review examines evidence for abnormalities in renal sodium re-absorption in hypertension and focuses on the proximal tubule as a site of relevant dysfunction. Identification of the proteins participating in renal sodium re-absorption and its regulation, particularly those involved in the renal pressure-natriuresis mechanism, will allow gene cloning and sequencing which in turn may lead to the identification of novel gene sequence variation participating in hypertension.

Animals↗
PubMed2000

Cyclophilin B expression in renal proximal tubules of hypertensive rats.

Rat cyclophilin-like protein (Cy-LP) is a candidate hypertension gene initially identified by differential hybridization and implicated in renal mechanisms of salt retention and high blood pressure. We report the molecular characterization of rat cyclophilin B (CypB) and demonstrate, through sequence analysis and an allele-specific polymerase chain reaction primer assay, that CypB but not Cy-LP is expressed in rat kidney. CypB is an endoplasmic reticulum-localized prolyl-isomerase that interacts with elongation initiation factor 2-beta, an important regulator of protein translation and a central component of the endoplasmic reticulum stress response to hypoxia or ATP depletion. Active renal transport of sodium is increased in the spontaneously hypertensive rat (SHR), and there is evidence that this coincides with hypoxia and ATP depletion in the renal cortex. In the present studies we have examined expression of CypB in rat proximal tubules, which contributes to the increased renal sodium reabsorption in this model of hypertension. We report that CypB transcript abundance is significantly elevated in proximal convoluted tubules from SHR compared with the control Wistar-Kyoto strain. This upregulation occurs in weanling animals and precedes the development of hypertension, indicating that it is not a simple response to hypertension in SHR. Further, CypB expression is also higher in a proximal tubule cell line derived from SHR compared with a similar line derived from Wistar-Kyoto rats, indicating that this difference is genetically determined. No sequence differences were observed in the CypB cDNA from these 2 strains. These observations suggest that a genetically determined alteration in proximal tubules from SHR occurs that leads to increased expression of CypB. In view of evidence linking CypB to the regulation of elongation initiation factor-2, the upregulation of CypB may result from metabolic stress.

Animals↗
PubMed2000

Mammalian bufadienolide is synthesized from cholesterol in the adrenal cortex by a pathway that Is independent of cholesterol side-chain cleavage.

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.

Adrenal Cortex↗
PubMed1999

Altered sodium pump alpha and gamma subunit gene expression in nephron segments from hypertensive rats.

OBJECTIVE: To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. DESIGN: A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. METHODS: We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. RESULTS: Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. CONCLUSION: The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.

Animals↗
PubMed1999

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia.

OBJECTIVE: To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma. DESIGN AND METHODS: Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries. RESULTS: In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery. CONCLUSIONS: Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Adult↗
PubMed1998

Modeling and analysis of competitive RT-PCR.

The present studies demonstrate a theoretical and practical framework for the accurate quantitation of gene expression in RNA extracted from microscopic tissue samples. The approaches are developed around competitive RT-PCR techniques. Assay performance has been examined and validated at both the RT and PCR steps. Our analysis of RT transcription efficiency for a number of native and competitor combinations shows that this property can differ, even for very similar templates. However, this difference is consistent and, once identified and measured, can be removed as an obstacle to accuracy. Using mathematical modeling, we have examined the simulated co-amplification of native and competitor templates in PCR. Useful insights have emerged from such modeling which indicate that differences in initial amplification efficiency and the rate of decay of amplification efficiency during the reaction can rapidly lead to inaccuracy, even while the slope and linearity of log plots of the competitor input and reaction product ratios are close to ideal. Finally, we show here that competitive RT-PCR reactions do not have to remain in the log-linear phase of PCR in order to accomplish accurate and precise quantification. Using appropriate competitors sharing primer binding sites and high internal sequence similarity, identical amplification efficiencies are preserved throughout the reaction. Reaction products, including heteroduplexes formed between native and competitor templates as reactions progress to plateau, can be identified and quantified accurately using the new technique of denaturing HPLC (dHPLC). This analytical technique allows the accuracy of competitive RT-PCR to be preserved beyond the linear phase. The technique has high sensitivity and precision and target abundances as low as 100 copies could be reliably estimated.

Animals↗
PubMed1998

Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography.

We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against native RNA resulted in the expected linear relationships which had slopes of unity. Quantitation could be performed with similar precision in single tube comparisons in which the initial abundance of the native transcript was calculated by knowledge of the final reaction product ratio and the initial competitor input level. The assay system is highly accurate, i.e. the measured level of gene expression reflected the actual copy number of the gene present in the sample. This was demonstrated by performing reactions in which known amounts of native transcript were quantified and the amount estimated by the assay was shown to be the same as the known amount added to the reaction. A similar approach has been devised for examining the relative levels of alternatively spliced isoforms. In this system, primers were selected to produce reaction products which served as their own internal competitors (by spanning the alternative splice site). Hormonal dependence of the ratio of abundance of two isoforms of the rabbit RUSH-1 gene was demonstrated.

Alternative Splicing↗
PubMed1998

Endogenous sodium pump inhibitors and blood pressure regulation: an update on recent progress.

Rapid progress has occurred recently in understanding the origin, chemistry, synthesis, control, and actions of endogenous materials that may be ligands for the cardiac glycoside binding site on the mammalian sodium pump (Na,K-ATPase). The present paper reviews this progress and examines in detail the evidence supporting ouabain-like and bufodienolide-like compounds as functioning in endogenous control of sodium pump activity, renal sodium excretion, blood pressure, and cardiovascular contractility. Other novel compounds identified in this search as potentially influencing natriuresis and blood pressure are also discussed.

Animals↗
PubMed1997

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Alternative Splicing↗
PubMed1997

Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR.

MOTIVATION: We have developed software to assist in the computation of gene expression from data obtained in competitive reverse transcription-polymerase chain reaction (RT-PCR). This report describes the mathematical basis of competitive RT-PCR and discusses the criteria which must be met to permit accurate estimations of gene expression to be obtained using this technique. RESULTS: The software that has been developed assists in both the assessment of assay performance (specifically in establishing the equality of amplification efficiency of the native and competitor templates) and in the routine analysis of data obtained in quantitation of gene expression by competitive RT-PCR. The software is a 100 kb module which functions as a Microsoft Excel add-in. It is compatible with both Windows and Mac versions of Excel 5 and Excel 7 on the Windows 95 platform, and employs the spreadsheet, statistical and graphing capabilities incorporated into Excel. AVAILABILITY: The software can be downloaded from http://www.grad.ttuhsc.edu/archive/. A brief summary in both HTML and Microsoft Word 6 format of the installation and use of the software is also located at this website.

Algorithms↗
PubMed1996

Role of endogenous cardiac glycosides in the spontaneously hypertensive rat--antagonism by active immunization.

The effects of simultaneous active immunization against two cardiac glycoside drugs, digoxin and proscillaridin, have been examined in young spontaneously hypertensive and Wistar-Kyoto rats. Control animals were immunized with protein carrier only. Animals were studied from 5 weeks to 13 weeks of age. Effectiveness of immunization to produce antibody responses was assessed at the end of the study by estimating the titer of antibodies in plasma against both of the antigens. Robust antibody responses were obtained. Immunization had no effect on the normal growth of these animals. Further, immunization against cardiac glycosides did not change blood pressure in either strain of animals. Blood pressure in the SHR increased as anticipated as the weanling animals grew to maturity. These studies indicate that active immunization against cardiac glycosides does not alter blood pressure in the SHR in spite of strong evidence for increased levels of endogenous cardiac glycosides in this strain.

Animals↗
PubMed1996

Ouabain production by cultured adrenal cells.

The adrenal cortex releases a sodium pump inhibitor. The present studies tested whether this material was endogenous and identical to ouabain by 1) studying the production of ouabain in long term cultures of adrenocortical cells, 2) seeking evidence that ouabain might be taken up from exogenous sources by adrenocortical cells, 3) examining the release of adrenocortical cells loaded with exogenous ouabain, 4) attempting to stimulate ouabain steroidogenesis in cultured adrenocortical cells, and 5) performing further chemical analysis on ouabain immunoreactivity released by cultured adrenocortical cells. Our results indicate that ouabain immunoreactivity is present in conditioned medium from both murine Y-1 adrenocortical cultures and primary bovine adrenocortical cell (BAC) cultures. We also found that BACs bind and internalize [3H]ouabain. Bound [3H]ouabain is released from BACs by both receptor dissociation and cytoplasmic release of internalized [3H]ouabain. Only one isoform of membrane sodium, potassium-adenosine triphosphatase, alpha 1, was expressed in the adrenal. Authentic ouabain was not metabolized during membrane binding or while present intracellularly. Stimulation of steroidogenesis in Y-1 and BAC with 22R-hydroxycholesterol and 25-hydroxycholesterol was performed and confirmed increased steroidogenesis; however, there was no effect on ouabain immunoreactivity content or release. Comparison of the ouabain binding density in cultured BAC, hepatoma cells, and 3T3 fibroblasts indicated that adrenocortical cells have a high ouabain-binding capacity. HPLC studies of the ouabain immunoreactivity released by bovine adrenocortical cells indicated that essentially no authentic ouabain was secreted. The present studies confirm that both BAC and Y-1 cultures release a ouabain-like material that differs in structure from authentic plant ouabain and is not a product of cholesterol side-chain cleavage.

Adrenal Cortex↗