Biomedical subjects

M Bader

Publications and source records attributed to M Bader.

At least 109 records · Page 6Linked to original sources

PubMed1996

Permanent inhibition of angiotensinogen synthesis by antisense RNA expression.

The renin-angiotensin system plays a pivotal role in blood pressure regulation. Recent molecular biological findings led to the new concept that in addition to the classic endocrine system, local tissue systems may also play an important role in cardiovascular diseases such as hypertension. In particular, the brain renin-angiotensin system was shown to influence the central control of blood pressure and is thought to contribute to the hypertensive phenotype of genetically hypertensive rat models. To identify the physiological role of these local systems, we established an antisense strategy to downregulate the expression of the precursor hormone angiotensinogen (AOGEN) in cell culture, which can also be used to establish transgenic rat lines. Plasmids encoding an RNA sequence complementary to the rat AOGEN mRNA under control of different viral and tissue-specific promoters were constructed and transfected into an AOGEN-expressing cell line. A competitive reverse transcription-polymerase chain reaction method was established for the quantification of AOGEN mRNA. Depending on the level of antisense RNA, the expression of the AOGEN gene was reduced down to 22% of control levels. Furthermore, the secretion of AOGEN protein was totally abolished. These results clearly demonstrate that the antisense constructs used are functional in reducing the AOGEN gene expression in vivo and can be used for the production of transgenic rats.

Angiotensinogen↗

PubMed1996

Transgenic rats: tools to study the function of the renin-angiotensin system.

1. The development of the transgenic technology for the rat allowed the evaluation of gene functions in the cardiovascular system in vivo. New insights have been gained particularly in the functions of the renin-angiotensin system (RAS), as most transgenic rat models established so far carry genes of this system. 2. TGR(mREN2)27 is a rat harbouring the mouse Ren-2 gene and exhibiting fulminant hypertension. The plasma RAS in this animal is down-regulated; however, the tissue-specific production of angiotensin II is activated (e.g. in the adrenal gland, the brain and the vessel wall). The physiological consequences of this activation, which finally leads to hypertension, can be studied in TGR(mREN2)27, rendering it a valuable tool in the functional analysis of tissue RAS. 3. TGR(hREN) and TGR(hAOGEN) carry the human genes for renin and angiotensinogen, respectively. In these animals the species-specific interaction of the two proteins and the expression pattern of the genes can be studied. Furthermore, these animals can be used to test renin-inhibitory drugs for use in antihypertensive therapy. 4. Further refinement of transgenic methodology (e.g. by the development of gene targeting in rats), should enhance our understanding of the functions of the RAS in cardiovascular regulation.

Angiotensin Receptor Antagonists↗

PubMed1996

Angiotensinogen messenger RNA stabilization by angiotensin II.

OBJECTIVE: To further characterize the molecular mechanism whereby angiotensin II stabilizes the angiotensinogen messenger (m)RNA through binding studies of the previously isolated polysomal stabilizing protein to partial and mutagenized sequences of the 3' untranslated region of the gene and to explore its importance to rodent genetic hypertension. DESIGN: Analysis of angiotensinogen mRNA mutants for half-life and binding to a polysomal protein with a molecular weight of 12000. METHODS: Protein/RNA interactions were determined in band shift assays employing radiolabelled 3' untranslated region of angiotensinogen mRNA. Measurement of the mRNA half-life used a cell-free incubation system and 3' untranslated region DNA sequences were polymerase chain reaction (PCR) cloned and sequenced. Sequences of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rat (SHR) strains were compared. Point mutations were introduced by site directed mutagenesis. RESULTS: The angiotensinogen 3' untranslated region exhibited specific binding to the polysomal 12000 molecular weight protein which, in an in vitro incubation system, increased 10-fold the half-life of full-length angiotensinogen mRNA; no effect was observed with 3' deleted angiotensinogen mRNA indicating a regulatory function of protein at the 3' untranslated region. Sequence analysis of PCR amplified DNA fragments identified a (G-->C) point mutation in the La Jolla colony SHR. Following introduction of this point mutation into wild-type 3' untranslated regions, protein binding significantly increased (wild-type binding constant, 19 mumol/l; mutant binding constant 3.5 mumol/l), indicating that this point mutation affects 3' untranslated region secondary structure, binding of the RNA stabilizing protein and, consequently, the half-life of angiotensinogen mRNA. Deletion of a U-rich region (position 1609-1613, UCCUU) expressed twice in the 3' untranslated region almost completely abolished protein binding suggesting this sequence as one part of the putative binding motif in the 3' untranslated region. CONCLUSIONS: Angiotensin II regulates hepatic angiotensinogen synthesis and secretion by inhibiting degradation of angiotensinogen mRNA by the action of a polysomal protein. Mutations in the 3' untranslated region mRNA coding sequence alter binding and half-life and may significantly affect the half-life of angiotensinogen mRNA thereby altering the secretion rate of angiotensinogen.

Angiotensin II↗

PubMed1996

Phase I trial of 9-cis retinoic acid in adults with solid tumors.

Retinoids have been shown to be potent inhibitors of epithelial carcinogenesis. Recent evidence has demonstrated that retinoid actions are mediated through nuclear receptors, which are proteins encoded by the retinoic acid receptor and retinoid X receptor gene families. These receptors are activated by binding to specific retinoids; of the known naturally occurring retinoids, 9-cis retinoic acid is unique in its ability to bind to both receptor families. Because of its unique receptor-binding characteristics, 9-cis retinoic acid may have biological activity not possible with other retinoids. For this reason, we conducted a Phase I trial of 9-cis retinoic acid in adult patients with solid tumors. Twenty-two patients were treated twice daily with p.o. 9-cis retinoic acid at doses ranging from 20 mg/m2/day to 150 mg/m2/day. The patients had non-small cell lung cancer (n = 8), breast cancer (n = 5), colorectal cancer (n = 3), head and neck cancer (n = 2), nonmelanoma skin cancer (n = 2), or ovarian cancer (n = 2). The dose-limiting (WHO grade III) toxic effects, which occurred at the 150-mg/m2/day dose level, were headaches and diarrhea. Less severe (grades I and II) toxic effects included cheilitis, dry skin, conjunctivitis, fatigue, hypertriglyceridemia, alkaline phosphatase elevation, myalgia/arthralgia, and hypercalcemia. Of the 15 patients evaluable for tumor response, no objective responses were observed. Pharmacokinetic analysis revealed a reduction in peak 9-cis retinoic acid plasma levels with chronic administration. Based on this study, the recommended Phase II dose of 9-cis retinoic acid in adult patients with solid tumors is 100 mg/m2/day administered in a divided dose twice daily.

Adult↗

PubMed1995

Expression of the mouse and rat mas proto-oncogene in the brain and peripheral tissues.

We isolated the mas proto-oncogene from a mouse genomic library. Sequence analysis showed that it contains an open reading frame without intervening sequences. The amino acid sequence deduced confirms the seven-transmembrane-domain structure and exhibits 97% and 91% amino acid homology with the rat and the human Mas, respectively. In mice and rats, mas mRNA was detected in the testis, kidney, heart, and in the brain regions: hippocampus, forebrain, piriform cortex, and olfactory bulb. Testicular mas mRNA from rats increases markedly during development, while cerebellar mRNA is high postnatally but completely disappears at later stages. We conclude that the product of the mouse mas gene may be involved in the development of the brain and testis.

Amino Acid Sequence↗

PubMed1995

Transcriptional silencer in intron I of the rat renin gene.

In the present study we investigated the influence of intron I of the rat renin gene on the transcriptional activity of its promoter in cell culture. The presence of intron I abolished the transcription of reporter genes (luciferase and lacZ) in the non-renin-expressing human embryonic kidney cell line 293, while it did not significantly affect the activity of the rat renin promoter in rat sceletal myoblast line L8 expressing renin. We conclude from these results that intron I of the rat renin gene contains a tissue-specific silencer element probably also responsible for the transcriptional repression of the endogeneous renin gene in 293 cells.

Animals↗

PubMed1995

Analysis of N-alkylated amino acids in human hemoglobin: evidence for elevated N-methylvaline levels in smokers.

To investigate the contribution of cigarette smoking to the levels of N-methylvaline and N-hydroxyethylvaline in hemoglobin we analyzed samples from 32 smokers and 37 nonsmokers. The average background levels of the nonsmoking individuals were determined to be 1175 +/- 176 pmol N-methylvaline/g globin, ranging from 722 to 1516 pmol/g globin, and 46 +/- 12 pmol N-hydroxyethylvaline/g globin, ranging from 19 to 64 pmol/g globin. A significant correlation (P < 0.001) was found between both amino acids and the amount of cigarettes smoked per day, with an increase of 42 pmol N-methylvaline/g globin/cigarette per day and 11 pmol N-hydroxyethylvaline/g globin/cigarette per day. No influence of age, sex, and occupational exposure was observed. Furthermore, the levels of N-hydroxyethylvaline and N-methylvaline correlated for smokers but not for nonsmokers, indicating cigarette smoking as a common source for both adducts. To our knowledge, this is the first time N-methylvaline levels are reported to differ significantly between smokers and nonsmokers in the general population. Especially the analysis of N-methylvaline following low-level exposures to methylating agents should therefore take into consideration the influence of individual smoking habits. Additionally, the results of our study confirm the reliability of N-hydroxyethylvaline as an indicator of individual cigarette consumption. We successfully applied a new calibration technique to the analysis of N-hydroxyethylvaline, introducing a commercially available and well-defined dipeptide standard. The observed levels of N-hydroxyethylvaline in the samples are in line with those reported in the literature and verify the applicability of our calibration method.

Adult↗

PubMed1995

Chronic dexamethasone treatment suppresses hypertension development in the transgenic rat TGR(mREN2)27.

INTRODUCTION: The transgenic rat TGR(mREN2)27 is a monogenetic rat model in hypertension research. Integration of mouse Ren-2 gene into the rat genome led to fulminant hypertension despite suppressed plasma and kidney renin concentrations. Renin is highly expressed in extrarenal tissues, especially throughout the adrenal cortex. AIMS AND METHODS: Because plasma and urinary corticosteroid concentrations are elevated during the development of hypertension in these rats, we investigated the effect of dexamethasone on blood pressure, adrenal renin and steroid metabolism. RESULTS: A daily injection of 100 micrograms/kg dexamethasone for 8 weeks was capable of suppressing the development of hypertension in the transgenic rats. The same regimen did not alter blood pressure in Sprague-Dawley control rats. Plasma concentrations of adrenocorticotrophic hormone (ACTH)-dependent steroids (corticosterone and 18-hydroxydeoxycorticosterone) decreased markedly in both strains treated with dexamethasone, but more pronouncedly in transgenic rats. Surprisingly, plasma aldosterone concentrations increased exclusively in the transgenic rats, and not in control rats, treated with dexamethasone. The decrease in corticosterone and 18-hydroxydeoxycorticosterone production was accompanied by a decrease in the abundance of the messenger RNA (mRNA) encoding the rate-limiting enzyme in steroidogenesis (P450scc cholesterol side-chain cleavage) and a decrease in the mRNA encoding P450c11 beta (11 beta-hydroxylase). The increase in aldosterone was accompanied by a massive increase in the abundance of the mRNA encoding zona glomerulosa-specific P450c11AS (aldosterone synthase), which was not increased in control rats. CONCLUSION: We conclude that ACTH-dependent steroids other than the mineralocorticoid aldosterone are responsible for the development of hypertension in the transgenic rat.

Adrenal Glands↗

PubMed1995

Angiotensin II receptor blockade in TGR(mREN2)27: effects of renin-angiotensin-system gene expression and cardiovascular functions.

OBJECTIVE: To study the effect of angiotensin II receptor AT1 blockade on blood pressure, gene expression and pathomorphology of transgenic rats harbouring the mouse Ren-2 gene [TGR(mREN2)27], that develop fulminant hypertension while exhibiting suppressed components of the circulating renin-angiotensin system. DESIGN: TGR(mREN2)27 were treated orally with the newly developed AT1-specific angiotensin receptor antagonist Telmisartan, 4'-[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-yl) methyl]-[1,1'-biphenyl]-2-carboxylic acid, in three doses (0.1, 1 and 3 mg/kg body weight) for 9 weeks. METHODS: The concentrations of the renin-angiotensin system components were analysed in plasma and tissues by radioimmunoassay. Messenger RNA levels for the angiotensinogen and renin genes were quantified by RNAase protection assay in several tissues. Heart hypertrophy and kidney morphology and function were monitored at the end of the treatment. RESULTS: In contrast to 0.1 mg/kg, 1 and 3 mg/kg Telmisartan normalized tail blood pressure measured once a week. Plasma renin and angiotensin II concentration increases were dose-dependent. The renin-angiotensin system genes in various cardiovascular organs were differentially regulated by angiotensin II receptor blockade. Treatment with Telmisartan stimulated angiotensinogen gene expression in the liver, kidney and heart, whereas it remained unchanged in the hypothalamus, thymus and adrenal gland. In the kidney, the expression of the endogenous, but not of the mouse Ren-2 gene, was increased in parallel to the renin concentration. Telmisartan reduced the severe glomerulosclerosis and proteinuria as well as cardiac hypertrophy observed in untreated TGR(mREN2)27 even with the lowest dose of 0.1 mg/kg, at which the blood pressure of the rats still exceeded 225 mmHg and the plasma renin-angiotensin system parameters were unchanged. CONCLUSION: From these experiments using a specific antagonist we can conclude that high blood pressure in TGR(mREN2)27 is angiotensin II-dependent. Furthermore, the expression of the renin-angiotensin system genes seems to be regulated not only by blood pressure and the plasma renin-angiotensin system but also by other, tissue-specific mechanisms. Pathomorphological changes in the kidney and in the heart do not seem to be caused by the systemic hypertension exclusively, but are also influenced by angiotensin II directly.

Adrenal Glands↗

PubMed1995

Differential gene expression of renin and angiotensinogen in the TGR(mREN-2)27 transgenic rat.

Transgenic rats carrying the murine Ren-2 gene represent a monogenetic model of hypertension characterized by low plasma renin and high extrarenal expression of the transgene. The hypothesis has been raised that stimulated local reninangiotensin systems may be responsible for the development of hypertension in this model. This study analyzes the effects of the converting enzyme inhibitor lisinopril, which specifically interferes with the renin-angiotensin system, and the direct vasodilator dihydralazine on the renal and extrarenal expression of renin and angiotensinogen. A comparison of gene expression between heterozygous and homozygous transgenic and normal Sprague-Dawley rats was also performed. We demonstrate high sensitivity of blood pressure toward converting enzyme inhibition in transgenic TGR(mREN-2)27 rats. In the kidney, expression of the transgene and the endogenous renin gene increased, suggesting that both are modulated by lisinopril in a similar manner. On the other hand, blood pressure reduction by dihydralazine did not abolish renal renin suppression in transgenic rats, indicating that mechanisms different from direct effects of blood pressure account for renin suppression. Homozygosity for the transgene led to increased Ren-2 expression and higher blood pressure and had opposite effects on angiotensinogen expression compared with heterozygous rats. Cardiac hypertrophy was reduced by lisinopril but not dihydralazine and was positively correlated with cardiac angiotensinogen expression. Increased angiotensin II in the adrenal gland of TGR(mREN-2)27 rats, which overexpresses the transgene, provides evidence that this leads to enhanced generation of tissue angiotensin II. We conclude that expression of the mouse transgene, the endogenous rat renin gene, and the angiotensinogen gene is subject to differential tissue-specific regulation. Reversal of cardiovascular damage with the converting enzyme inhibitor but not dihydralazine suggests that angiotensin II generated locally may be involved in the pathogenesis of hypertension and structural changes in TGR(mREN-2)27 rats.

Angiotensin II↗

PubMed1995

Contribution of a 12 kDa protein to the angiotensin II-induced stabilization of angiotensinogen mRNA: interaction with the 3' untranslated mRNA.

Several authors have shown that angiotensin II stimulates hepatic angiotensinogen synthesis in vivo, ex vivo and in vitro. In previous studies we have demonstrated that this effect of angiotensin II depends mainly on a transient inhibition of adenylyl cyclase and is the consequence of a stabilization of angiotensinogen mRNA. In the present study we describe the isolation of a polysomal 12 kDa protein which, in band shift and cross link assays, shows a specific affinity to the 3' untranslated region (3' UTR) of angiotensinogen mRNA and prevents enzymatic degradation of angiotensinogen mRNA in a cell-free incubation system. [32P]UTP-labelled or unlabelled 3' fragments of angiotensinogen mRNA were synthesized on a transcription vector (pGEM5zf+) into which the corresponding DNA sequence was cloned after restriction from vector pRAG 16. Binding of the 12 kDa protein to the radioactively labelled 3' UTR of angiotensinogen mRNA could be displaced by unlabelled 3' UTR mRNA fragments but not by a renin mRNA of comparable length derived from the coding region. The RNA-binding protein appears to be derived from a higher molecular mass precursor (45 kDa) which is preferentially present under reducing conditions in vitro; the active low molecular mass form is evident in the absence of reducing agents. In a cross link experiment we established that a band shift signal which was obtained in the presence of the 45 kDa protein preparation exclusively depends on RNA binding of the active 12 kDa protein. In addition, a phosphorylation step may be involved in the activation of the 12 kDa protein, since its molecular mass and isoelectric point correlate with proteins which were phosphorylated in response to transient decreases of cAMP (induced by guanfacine or angiotensin II) or in response to a direct inhibition of protein kinase A by the cAMP antagonist Rp-cAMP. The importance of phosphorylation reactions for the stabilization of angiotensinogen mRNA was further assessed in a cell-free incubation system of rat liver parenchymal cells. These studies demonstrated that in the presence of acid phosphatase (1 U/ml) the half-life of angiotensinogen was significantly decreased. In the same incubation system the 12 kDa protein increased the half-life of endogenous as well as of exogenous angiotensinogen mRNA three- to fourfold, while no stabilizing effect was apparent when exogenous angiotensinogen mRNA from which the 3' tail had been deleted was added.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclase Inhibitors↗

PubMed1994

Molecular structure and expression of rat bradykinin B2 receptor gene. Evidence for alternative splicing.

Bradykinin is a potent vasodilatory peptide hormone involved in a broad range of physiological actions. While it acts through at least two types of receptors which are named B1 and B2, most of its effects are mediated via activation of the B2 receptor. The gene for this receptor was isolated from a rat genomic library and shown to span more than 28 kilobases, including four introns. The relative positions of the exons were mapped and all exons, intron-exon boundaries, and 5'- and 3'-flanking regions were sequenced. While the 5'-untranslated region of the mRNA is distributed on all four exons, the coding and the 3'-untranslated region are located entirely on the fourth exon. Characterization of the region upstream to the transcriptional start site detected by primer extension analysis shows that the bradykinin B2 receptor promoter contains no typical TATA or CCAAT boxes. Nevertheless, the promoter sequence was shown to be functional in NG108-15 cells transfected with a construct bearing 1.1 kilobases of 5'-flanking sequence fused to a luciferase reporter gene. Reverse transcription-polymerase chain reaction analysis detected two different bradykinin B2 receptor mRNAs containing or lacking exon 3 in all rat tissues tested, providing evidence for alternative splicing of the 5'-untranslated sequence.

Alternative Splicing↗

PubMed1994

Varicella vaccine.

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Chickenpox Vaccine↗

PubMed1994

GC/MS determination of N-phenylvaline, a possible biomarker for benzene exposure in human hemoglobin by the "N-alkyl Edman method".

We report the application of a modified Edman degradation procedure to the analysis of benzene oxide adducts at the N-terminal valine of human hemoglobin (Hb). Benzene oxide is thought to be formed in the liver from benzene and adduct formation with macromolecules is therefore likely to occur. We assumed that benzene oxide could covalently bind to hemoglobin after leaving the hepatic tissue. The "N-alkyl Edman method" was adapted for the approach to investigate this hypothesis. Using capillary gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization, we could not detect N-phenylvaline in blood samples from persons occupationally exposed to benzene. We conclude that adducts of benzene oxide to the N-terminal valine of Hb are not formed in detectable amounts in vivo and consequently are not suitable for biomonitoring purposes. This result clearly indicates that other reactive benzene metabolites have to be taken into account not only in the search for a biomarker but also as the ultimate carcinogenic species.

Air Pollutants, Occupational↗

PubMed1994

Characterization of the Han:SPRD rat model for hereditary polycystic kidney disease.

The Han:SPRD rat model for inherited polycystic kidney disease (PKD) was characterized (clinical parameters, morphology, immunohistochemistry and in situ hybridization). Homozygous animals died of uremia after three to four weeks with severe cystic transformation of virtually all nephrons and collecting ducts (serum urea: 616 +/- 195 mg/dl; kidney-to-body weight ratio: > 20%). In heterozygotes, slow progression of the disease led to death between the 12th and 21st month (median: 17 months; serum urea levels above 200 mg/dl). Kidney enlargement was moderate, and cysts were restricted to the cortex and outer medulla. Immunohistochemical markers showed that approximately 75% of the cysts were derived from the proximal tubule. Cystic transformation started in the proximal tubule with a sharp onset of basement membrane alteration and a loss of epithelial differentiation restricted to small focal areas. In these areas, alpha 1(IV) collagen and laminin B1 mRNA were enhanced as revealed by isotopic and non-isotopic in situ hybridization. Fibroblasts underlying the affected tubular portions were involved in matrix overexpression resulting in subepithelial accumulation of immunoreactive collagen IV and laminin. In later stages of cystic transformation distal nephron segments were affected as well. A reversal in epithelial polarity as judged from Na,K-ATPase-immunoreactivity was not observed. Renal immunoreactive renin-status was significantly decreased. Hematocrit was lowered in heterozygotes (40.4 +/- 5.8 vol% compared to 46.7 +/- 1.99 vol% in controls; P < 0.05) and total renal EPO mRNA was reduced to 36 +/- 14% of the mean value of control animals, whereas serum EPO levels were not significantly altered. We conclude that the Han:SPRD rat is a useful model for the study of human ADPKD since both diseases are similar in several aspects. The model is particularly suitable for the study of epithelial-mesenchymal interactions at the beginning of tubular cystic transformation.

Animals↗

PubMed1994

Mechanism by which angiotensin II stabilizes messenger RNA for angiotensinogen.

The most important specific regulatory mechanism for hepatic angiotensinogen synthesis and secretion is its stimulation by angiotensin II, the effector peptide of the renin-angiotensin system. In the circulating system, this octapeptide is thought to stimulate hepatic angiotensinogen synthesis through a positive feedback loop. In the present study, we have identified the intracellular mechanisms leading to an increase in angiotensinogen messenger RNA (mRNA) and secretion. In a [3H]uridine-dependent pulse and chase system as well as in hepatocytes in which de novo synthesis of mRNA has been blocked by actinomycin D or 5,6-dichlorobenzimidazole riboside, angiotensin II significantly increased the half-life of angiotensinogen mRNA. In contrast, no effect of angiotensin II on the transcription of angiotensinogen mRNA could be observed in a nuclear run-on assay with nuclei from pretreated hepatocytes, whereas dexamethasone, as a positive control, increased the transcription fivefold to sevenfold. We have isolated a 12-kD protein from the polysomal fraction of isolated hepatocytes, which has an affinity to the nontranslated 3' tail of angiotensinogen mRNA. For in vitro transcription of this mRNA fragment, the DNA sequence coding for the nontranslated 3' tail was excised from the vector pRAG 16 and cloned into the transcription vector pGEM 5zf+. Molecular weight and isoelectric point of the mRNA-binding protein correspond to the parameters of a cytosolic protein that becomes phosphorylated by decreased cyclic AMP concentrations as analyzed in [32P]orthophosphate-loaded hepatocytes. In a cytosolic incubation system in which the polysomal fraction was integrated, the mRNA-binding protein increased the half-life of angiotensinogen mRNA significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1994

Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents.

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.

Animals↗

PubMed1994

Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex.

Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.

Adrenal Cortex↗