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ABSTRACT The β-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (−)-|3H|-dihydroalprenolol. Pretreatment of these membranes with either the alkylating agent... more
ABSTRACT The β-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (−)-|3H|-dihydroalprenolol. Pretreatment of these membranes with either the alkylating agent N-ethylmaleimide or with β-adrenergic agonists does not affect (−)-|3H|-dihydroalprenolol binding to the receptor sites. However, the simultaneous presence of both types of products causes a 50% decline in the number of binding sites. A less pronounced decline occurs when the membranes are pretreated with N-ethylmaleimide in presence of the partial agonist (−)-phenylephrine, and no decline in the presence of the antagonist (−)-|3H|-dihydroalprenolol. β-adrenergic agonists thus appear to induce a conformational change of their receptor, with results in an increased susceptibility to inactivation by N-ethylmaleimide.
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Objectives: In diabetes, cardiovascular complications are associated with endothelial dysfunction and oxidative stress. Empagliflozin (Empa), as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i) in clinical development,... more
Objectives: In diabetes, cardiovascular complications are associated with endothelial dysfunction and oxidative stress. Empagliflozin (Empa), as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i) in clinical development, offers a promising novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with Empa could improve endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated oxidative stress. Research Design and Methods: Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection Empa was administered via drinking water for 7 weeks. Results: Treatment with Empa (10 and 30 mg/kg/d), showed reduction of blood glucose and a normalization of endothelial dysfunction (aortic rings) in diabetic rats and a reduced oxidative stress in aortic vessels (dihydroethidine staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence). Additionally, the pro-inflammatory phenotype and glucotoxicity in diabetic animals was normalized by SGLT2i therapy. Conclusion: In this study we could demonstrate that Empa improves hyperglycemia and prevents the development of endothelial dysfunction and oxidative stress in type 1 diabetic rats. Future studies will investigate the underlying mechanisms of these antioxidant and anti-inflammatory effects with special emphasis on low-grade inflammation, glucotoxicity and oxidative stress, all of which contributes to cardiovascular complications.
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S cells are pluripotential cells that have the ability to differentiate into cell lineages that are determined by trophic factors and other biological cues. There is growing evidence in neonatal medicine which suggests understanding stem... more
S cells are pluripotential cells that have the ability to differentiate into cell lineages that are determined by trophic factors and other biological cues. There is growing evidence in neonatal medicine which suggests understanding stem cell physiology may offer a unique pathway leading to decreased pathology. Researchers have proposed bone marrow-derived stem cell use in the treatment of chronic lung disease (Alphonse et al., 2012). In a similar fashion, mesenchymal stem cells also protected against lung disease (Tian et al., 2008). In these studies stem cells show a reparative mechanism that is currently not well described. Mesenchymal stem cells have also been shown to decrease cellular insults in an animal model of necrotizing enterocolitis (Tayman et al 20011). Stem cells have been administered intranasally and shown to have a protective effect on brain injury (van Velthaven 2010). Our laboratory has uncovered a potentially beneficial pathway that involves the biosynthesis of glutathione in stem cells. Most neonatal diseases involve some form of oxidative stress. Glutathione, a tripeptide, is the endogenous tool used to combat oxidative stress. This pathway involves the regulation of the cystine glutamate exchanger (System Xc) which controls the most critical step in glutathione biosynthesis. This pathway is one of the mechanisms that stem cells use to exert its’ protective effect.
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We are overwhelmed to announce “3rd International Conference on Diabetes, Hypertension & Metabolic Syndrome” which is going to be held during February 2425, 2020 at Tokyo, Japan. The Conference will be organized around the theme... more
We are overwhelmed to announce “3rd International Conference on Diabetes, Hypertension & Metabolic Syndrome” which is going to be held during February 2425, 2020 at Tokyo, Japan. The Conference will be organized around the theme “Collection of advanced therapies: Prevention of Diabetes & Metabolic syndrome”. This conference highlights the latest and exciting innovations in Diabetes & its Treatment. Diabetes 2020 Conference invites all renowned scientists, endocrinologists, surgeons, dietitians, radiation therapists, general physicians, primary health care specialists, talented young scientists, pharmaceutical industrial delegates and student communities across the globe to attend International Diabetes congress
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Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and... more
Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and tissues. We show here that, in intact PC12W cells which express only AT2 receptors, Ang II significantly decreases basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentration. This effect is mimicked by the AT2 selective agonist CGP 42112, and is not prevented by the AT1 selective antagonist losartan, indicating that this is an AT2 receptor mediated response. The lack of effect of the phosphodiesterase (PDE) inhibitor IBMX shows that this mechanism does not involve PDE stimulation. This is confirmed by the finding that neither Ang II or CGP 42112 affect the Ca++/calmodulin dependent cGMP PDE activity. Furthermore Ang II and CGP 42112 have no effect on nitroprusside-stimulated cGMP levels in these cells, thus ruling out interactions between the AT2 receptor and soluble guanylate cyclase. These data indicate that the AT2 receptor mediated decrease of cGMP is due to the selective inhibition of particulate guanylate cyclase (pGC) activity. In an accompanying paper we report that interaction of Ang II with the AT2 receptor in the same cells results in the stimulation of phosphotyrosine phosphatase (PTPase) activity. Interestingly, the PTPase inhibitors sodium orthovanadate and phenylarsine oxyde, but not the Ser/Thr phosphatase inhibitor okadiac acid, inhibitthe Ang II and CGP 42112 induced decreases in cellular cGMP concentration. These findings suggest that stimulation of PTPase activity may be involved in the regulation of pGC activity via AT2 receptors.
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The β1-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (-)-[3H]dihydroalprenolol. Binding of β-adrenergic agonists to these receptor sites sensitizes them to... more
The β1-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (-)-[3H]dihydroalprenolol. Binding of β-adrenergic agonists to these receptor sites sensitizes them to inactivation by the alkylating agent N -ethylmaleimide. A dose- and time-dependent decrease of 45 to 60% of the sites is commonly observed. Binding of (-)-3H-dihydroalprenolol and β-adrenergic agonists to the remaining sites occurs with the same characteristics as for the untreated receptor population. Kinetic experiments reveal that the rate of inactivation is proportional to the concentration of N -ethylmaleimide (between 5.5 and 450 µM). In contrast, the rate of inactivation reaches a plateau value when increasing the concentration of the agonist. The rate of inactivation is half-maximal in presence of 1.3 µM (-)-epinephrine or 20 µM (+)-epinephrine. This marked stereospecificity, along with the close identity of the above concentrations with the equilibrium dissociation constant ( KD ) of the epinephrine isomers for binding to the β-receptor (i.e., 2.0 µM for (-)-epinephrine and 21 µM for (+)-epinephrine) indicate that N -ethylmaleimide inactivates the agonist-bound form of the receptor. The second-order rate constant ( k 2) of the inactivation process, in the presence of 15 β-adrenergic ligands, was found to correlate with their capability to stimulate the adenylate cyclase activity, i.e., "intrinsic activity." Since all tested ligands were able to cause a complete and dose-dependent displacement of bound (-)-[3H]dihydroalprenolol, it is likely that both the intrinsic activity and k 2 of each adrenergic ligand reflect an inherent property of the ligand-bound receptor. The proportionality between k 2 and the intrinsic activity further suggests that β-adrenergic agonists "induce" or "favor" a conformational change of the receptor, resulting in adenylate cyclase activation and the uncovering of an alkylable group which becomes exposed to N -ethylmaleimide in the active conformation.
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The uterine contractile response to adrenergic agonists or sympathetic stimulation is influenced dramatically by the hormonal milieu. Rabbit uterine contraction is mediated by alpha 1-adrenoceptors, whereas relaxation in response to the... more
The uterine contractile response to adrenergic agonists or sympathetic stimulation is influenced dramatically by the hormonal milieu. Rabbit uterine contraction is mediated by alpha 1-adrenoceptors, whereas relaxation in response to the same stimulus is mediated by beta 2-adrenoceptors. Whether uterine contractility is increased or decreased by adrenergic stimulation is determined by the gonadal steroids estrogen and progesterone: uterine contraction prevails in the estrogen-dominant or the ovariectomized animal, but in the progesterone-dominant rabbit, uterine relaxation is observed. In previous studies, we have demonstrated that changes in the concentration or agonist affinity of these adrenoceptors cannot account for the changes in contractile response. In the present studies, we tested whether sex steroids might alter beta-adrenergic response by acting on events distal to receptor occupancy, and whether this could explain the conversion of contractile response. We found that myometrial cAMP generation is potently stimulated by beta-agonists in progesterone-treated and also in ovariectomized animals, but this stimulation is absent after estrogen treatment. Similar, but smaller, changes were observed for cAMP generation in response to prostaglandin E2 and forskolin. Stimulation of adenylate cyclase in uterine particulates by agents which act on the guanyl nucleotide-sensitive stimulatory transducer, Gs, is unchanged after estrogen treatment. However, specific labeling of Gs catalyzed by cholera toxin is reduced in membrane particulates from estrogen-treated animals. Recombination of extracts of uterine membranes from the differently treated animals also suggested qualitative differences in Gs. We conclude that at least one component of the adenylate cyclase cascade beyond the beta-adrenoceptor, i.e., Gs, is a target for ovarian steroids; estrogen reduces Gs labeling and beta-adrenoceptor-mediated cAMP production. However, uterine Gs labeling and cAMP production are similar in ovariectomized and in progesterone-treated rabbits. Since these uteri exhibit different contractile responses, the observed changes are not sufficient to explain sex steroid-mediated conversion of myometrial contractile response.
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The radiolabeled β-adrenergic antagonists (-)-[3H]-dihydroalprenolol binds to a single class of noncooperative sites on turkey erythrocyte membranes. These sites have previously been identified as the functional β-adrenergic receptors.... more
The radiolabeled β-adrenergic antagonists (-)-[3H]-dihydroalprenolol binds to a single class of noncooperative sites on turkey erythrocyte membranes. These sites have previously been identified as the functional β-adrenergic receptors. Treatment of the membranes with the reducing agent dithiothreitol causes a decrease in the number of binding sites, without affecting the affinity of (-)-[3H]dihydroalprenolol for the remaining sites. The binding activity is partially restored by extensive washing of the dithiothreitol-treated membranes. No restoration occurs when the wash buffer contains 2 mM N-ethylmaleimide or 10 mM reduced glutathione. The effect of dithiohreitol is mimicked by a hundredfold higher concentration of the monosulfydryl derivatives: reduced glutathione, cysteine, and mercaptoethanol. In contrast, treatment of the membranes with the metal chelators ethylenediamine tetraacetate and ethylene glycol bis(β-amino-ethyl ether)N,N'-tetraacetic acid (10 mM) does not affect (-)-[3H]dihydroalprenolol binding. Kinetic data indicate that dithiothreitol inactivates the β-receptors according to a bimolecular reaction mechanism, with a second order rate constant (k2) of approximately 1.27 M-1 x S-1 at 30°C. The data suggest that dithiothreitol inactivates the β-receptors by reducing one or more disulfide bonds. Both β-adrenergic agonists and antagonists cause an effective protection of the (-)-[3H]dihydroalprenolol binding sites against inactivation by dithiothreitol. The protection is dose-dependent, and linearly related to the fraction of receptor sites occupied by the tracer. The protection is stereospecific for both agonists (epinephrine) and antagonists (propranolol) and reflects, for the same concentration of agonists, the order of affinities for the receptor. The α-adrenergic agents clonidine (agonist) and phentolamine (antagonist), and the nonbioactive compound pyrocatechol do not confer an appreciable protection at concentrations as high as 100 μM. Receptor protection by β-adrenergic agonists and antagonists proceed by causing a conformational change of the receptors as to bury the disulfide bonds or by shielding bonds located near or at the binding site of the receptor.
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Research Interests: Genetics, Chemistry, Medicine, Multidisciplinary, Magnesium, and 15 moreCerebral Cortex, Animals, Male, Rat Brain, Rats, Adenylate Kinase, Receptor, Reserpine, Erythrocyte Membrane, Structure activity Relationship, Gtp Binding Proteins, Nucleotides, Isoproterenol, Sodium Butyrate, and Antidepressive agents
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Angiotensin-II (AII), which stimulates steroidogenesis in bovine adrenocortical (BAC) cells through the phosphoinositides pathway, activates p42-p44 mitogen-activated protein kinases (MAPKs) after 5 min of treatment (EC50 = 0.1 nM). This... more
Angiotensin-II (AII), which stimulates steroidogenesis in bovine adrenocortical (BAC) cells through the phosphoinositides pathway, activates p42-p44 mitogen-activated protein kinases (MAPKs) after 5 min of treatment (EC50 = 0.1 nM). This activation is 1) completely inhibited by the AII receptor AT1 subtype antagonist Dup 753 (10 microM), but unaffected by the AT2 antagonist PD 123177; 2) not reproduced by the AT2 agonist CGP 42112A; 3) insensitive to pretreatment with pertussis toxin; and 4) abolished by a 48-h preexposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM), which down-regulates protein kinase-C activity. Fibroblast growth factor-2, a potent mitogen for BAC cells, which acts through its tyrosine kinase receptor, also activates MAPK (EC50 = 0.3 in a TPA-insensitive manner, while exhibiting no detectable effect on BAC cell steroidogenesis. In contrast, ACTH, which stimulates steroidogenesis via cAMP and inhibits BAC cell proliferation, does not stimulate MAPK. Indeed, ACTH completely blocks (IC50 = 0.01 nM) the stimulation of MAPK by AII, fibroblast growth factor-2, or TPA. Therefore, bovine adrenocortical cells provide an example of positive and negative hormonal regulation of MAPK activity through a cross-talk between the inositide-, cAMP-, and growth factor-activated tyrosine kinase pathways.
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This article deals especially with radioligand studies performed in conjunction with group-specific reagents as probes for determining receptor conformational status, this being an aspect of agonist-mediated excitation and of ‘population’... more
This article deals especially with radioligand studies performed in conjunction with group-specific reagents as probes for determining receptor conformational status, this being an aspect of agonist-mediated excitation and of ‘population’ diversity. One such probe is the alkylating agent N-ethylmaleimide (NEM). It causes, with agonists present, inactivation of β-adrenergic receptors and affinity changes in muscarinic receptors. The approaches and the results obtainable are exemplified first for β-adrenergic receptors, e.g. with turkey erythrocytes and human adipose membranes, and then for muscarinic receptor sites (‘RH’ and ‘RL’ in particular) in rat forebrain. The approaches are potentially applicable to other hormone or neurotransmitter types of receptor.
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Protein tyrosine kinases (PTKs) are a family of enzymes sharing a highly conserved catalytic domain which phosphorylates substrate proteins on tyrosine residues. PTKs play a major role in the transduction of the mitogenic signal and are... more
Protein tyrosine kinases (PTKs) are a family of enzymes sharing a highly conserved catalytic domain which phosphorylates substrate proteins on tyrosine residues. PTKs play a major role in the transduction of the mitogenic signal and are involved in the control of cell proliferation, differentiation, and transformation processes. PTKs can be subdivided into two major types: membrane associated PTKs consisting essentially of growth factor receptors (receptor tyrosine kinases or RTKs) and cytosolic PTKs involved in the intracellular transduction of mitogenic and differentiation signals. From January 1988 to January 1992, PTK activity was assayed in cytosolic fractions prepared from 350 T1-T2, N0-N1 M0, breast carcinomas. Enzymatic activity was measured using phosphate transfer from [32P]-ATP to poly-Glu-Tyr as an artificial substrate. According to our previously reported pilot study, we chose a cut-off value of 12 pmol 32P incorporated min-1 mg-1 protein, corresponding to the median value. We found positive PTK levels (> or = 12 pmol/min/mg) to be correlated with a loss of differentiation according to Scarff-Bloom grade (p < 0.001), negative PR (p = 0.03) and ER status (p = 0.04). With a median follow-up of 30 months (0-82), patients with a positive PTK level presented a smaller 3-year disease free survival than in the PTK negative group of patients (p = 0.07). In Cox multivariate analysis including pT, pN, Scarff-Bloom grade, PR and ER, PTK activity does not emerge as a significant prognostic factor.
Research Interests: Breast Cancer, Biology, Medicine, Multivariate Analysis, Humans, and 15 moreFemale, Protein Tyrosine Kinase, Enzyme, Aged, Middle Aged, Pilot study, Adult, Breast carcinoma, Prognosis, Cell Proliferation, Enzymatic Activity, Growth factor receptor, Breast Neoplasms, Disease Free Survival, and Protein tyrosine kinases
Bovine fasciculata cells in culture (BAC) express both AT1 and AT2 angiotensin receptors. The role and signaling pathways of this latter receptor are still the subject of debate. We found that in BAC stimulation of cortisol (F) production... more
Bovine fasciculata cells in culture (BAC) express both AT1 and AT2 angiotensin receptors. The role and signaling pathways of this latter receptor are still the subject of debate. We found that in BAC stimulation of cortisol (F) production by angiotensin II (A II) is accounted for by both receptor subtypes. We have investigated the potential AT2 signalling pathways involved in this response. As previously described in other cells, we found this receptor to mediate inhibition of ANP stimulated cGMP production through a phosphodiesterase independent pathway. This phenomenon does however not appear to be involved in cortisol production as this response was not affected by the addition of 8-Br-cGMP or ANP. It was however abolished after down-regulation of PKC by phorbol esters, but not by Gi inhibition with pertussis toxin. Moreover and as opposed to the AT1 mediated response, AT2 receptor stimulation potentiated K+ induced F production. In conclusion, these observations suggest that the AT2 pathway which mediates F production requires intact PKC and might involve a Gi independent stimulation of Ca++ or K+ channels.
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Research Interests: Physiology, Biology, Medicine, Signal Transduction, HSP, and 15 moreAnimals, Male, Phosphorylation, Medical Physiology, Rats, Heat stress, Isoenzymes, Heat Shock Proteins, Protein Kinase, Heat Shock Protein, Protein Transport, Protein Kinase C, Pharmacology and pharmaceutical sciences, In Vitro Techniques, and signalling pathway
... A. Donny Strosberg 1 , Georges Vauquelin 1 , Odile Durieu-Trautmann 2 , Colette Delavier-Klutchko 2 ... 10, 4, G. Fischer, Stuttgart 7 Hfhling, HJ, Steffens, H., Stamm, G. and Mays, U. (1976 ... in conformation or an... more
... A. Donny Strosberg 1 , Georges Vauquelin 1 , Odile Durieu-Trautmann 2 , Colette Delavier-Klutchko 2 ... 10, 4, G. Fischer, Stuttgart 7 Hfhling, HJ, Steffens, H., Stamm, G. and Mays, U. (1976 ... in conformation or an 'anchoring effect' caused by the agonist binding may constitute ...
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Reactive nitrogen species (RNS) have been shown to play a major role in the pathophysiology of hypoxic-ischemic cerebral injury. Using a novel sensitive ELISA allowing the quantification of nitrated albumin (nitroalbumin) in plasma, we... more
Reactive nitrogen species (RNS) have been shown to play a major role in the pathophysiology of hypoxic-ischemic cerebral injury. Using a novel sensitive ELISA allowing the quantification of nitrated albumin (nitroalbumin) in plasma, we tested the hypothesis that perinatal asphyxia increases nitrating RNS generation by verifying whether the concentration of one of its target proteins is correlated with the clinical outcome. We assayed nitroalbumin in 114 plasma samples collected during the first hour, at day 1, and at day 4 of life from 48 term newborns suffering from perinatal asphyxia and correlated this marker with neurological and systemic neonatal outcomes. Nitroalbumin levels at day 1, but not at days 0 and 4, were significantly increased in patients who developed moderate or severe encephalopathy compared to those who had a normal neurological evolution or developed mild encephalopathy (median: 14.4 ng/ml versus 7.3 ng/ml, respectively). In contrast, nitroalbumin concentration at day 1 was not associated with systemic complications. First-hour and fourth-day nitroalbumin concentrations did not differ with respect to the neonatal neurological course. At day 0, nitroalbumin levels also correlated with circulating leukocytes. We conclude that plasma nitroalbumin seems to be a specific marker of neurological injury after perinatal asphyxia and may serve as a secondary end-point in neuroprotective clinical trials.
Research Interests: Free Radicals, Clinical Trial, Oxidative Stress, Medicine, Free Radical, and 15 moreHumans, Albumin, Anesthesia, Biochimie, Biological markers, Encephalopathy, Enzyme Linked Immunosorbent Assay, Brain Damage, Asphyxia neonatorum, Biochemistry and cell biology, Cohort Studies, Neonatal Encephalopathy, Free Radical Biology, Nitrotyrosine, and Asphyxia
Research Interests: Biology, Medicine, Multidisciplinary, Free Radical, Humans, and 15 moreInsulin, Animals, Nitrates, Insulin signaling, Phosphorylation, Mitogen Activated Protein Kinase, PLoS one, Protein kinase B, Active site, Nitric Oxide Synthase, Protein Kinase, Angiotensin II, MAP Kinase, GLUT, and Muscle fibers skeletal
Development of specific angiotensin II receptor ligands has recently provided evidence for the existence of two angiotensin II receptor subtypes, termed AT1 and AT2, which differ in their signal transduction mechanisms and in the effects... more
Development of specific angiotensin II receptor ligands has recently provided evidence for the existence of two angiotensin II receptor subtypes, termed AT1 and AT2, which differ in their signal transduction mechanisms and in the effects they mediate. In brain, both receptor subtypes are present. Most of the known central actions of angiotensin II, for example the regulation of blood pressure and of electrolyte and water balance, seem to be mediated by the AT1 receptor, while the role of the AT2 receptor is still an enigma. This review by Thomas Unger and colleagues summarizes the current knowledge and latest hypotheses in this rapidly developing field.
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Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [ 125 I]Sar 1 Ile 8 angiotensin II ([ 125 I]sarilesin) as a tracer and ANG II, sarilesin and the subtype... more
Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [ 125 I]Sar 1 Ile 8 angiotensin II ([ 125 I]sarilesin) as a tracer and ANG II, sarilesin and the subtype selective ligands DuP 753 (AT 1 ) and CGP 42112A (AT 2 ) ...
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Research Interests: Endocrinology, Chemistry, Kinetics, Calcium, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Internal Medicine, Glioma, Animals, Calcium channels, Neuroblastoma, CHEMICAL SCIENCES, Time Factors, Receptor, Angiotensin II, Gtp Binding Proteins, cyclic GMP, Medical and Health Sciences, and Angiotensin Receptor Antagonists
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Research Interests: Endocrinology, Biology, Medicine, Biological Sciences, Cell line, and 14 moreCell Differentiation, Molecular and cellular biology, Internal Medicine, Low Dose, Epidermal Growth Factor, Neurite, Receptor, Cell Proliferation, Losartan, Nerve Growth Factor, Growth Factor, Angiotensin II, Cell Growth, and Medical and Health Sciences
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SCOPUS: NotDefined.jinfo:eu-repo/semantics/publishe
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Research Interests: Physics and Humanities
Research Interests: Molecular Medicine, Biology, Medicine, Gene expression, Cell Division, and 15 moreSignal Transduction, Molecular, Cell line, Animals, Epidermal Growth Factor, Rats, Retinoic Acid, Transforming Growth Factor Beta, Receptor, Growth Inhibition, Growth Factor, Angiotensin II, Ligands, Down-Regulation, and Transforming Growth Factor
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Using Chinese Hamster Ovary cells expressing human AT(1) receptors cells (CHO-hAT(1)), it was previously shown that insurmountable inhibition of the angiotensin II response by non-peptide antagonists is related to the duration of their... more
Using Chinese Hamster Ovary cells expressing human AT(1) receptors cells (CHO-hAT(1)), it was previously shown that insurmountable inhibition of the angiotensin II response by non-peptide antagonists is related to the duration of their receptor occupancy. In the present study it was shown that these antagonists displayed similar binding characteristics to endogenously expressed AT(1) receptors in human adrenal cortex cells (NCI-h295) and renal vascular smooth muscle cells (HVSMC). Competition binding studies with [(3)H]candesartan for NCI-h295 cells, with [(125)I]Sar(1)-Ile(8) angiotensin II for HVSMC and with both radioligands for CHO-hAT(1) cells displayed the same potency order for unlabelled antagonists: candesartan>EXP3174>irbesartan>losartan. The AT(2) receptor antagonist PD123319 displayed low potency in all instances. The apparent half-lives of the antagonist-AT(1) receptor complexes in NCI-h295 cells and HVSMC were comparable to those obtained under identical conditions with CHO-hAT(1) cells. Angiotensin II increased the inositol phosphate accumulation dose dependently with half-maximal response at 17.4+/-1.6nM for NCI-h295 cells and 4.5+/-0.8nM for HVSMC. Pre-incubation of the cells with losartan only produced concentration-dependent rightward shifts of the angiotensin II concentration-response curve. The maximal response was decreased by 85-92% with candesartan, 70-88% with EXP3174 and 60% with irbesartan. The similar binding and inhibitory properties of these antagonists among the investigated cell types validates the use of CHO-hAT(1) cells for investigating pharmacological properties of human AT(1) receptors.