Age-Related Changes in Ang II Receptor Localization and Expression in the Developing Auditory Pathway.

We studied Ang II receptor localization in different nuclei of the auditory system, by means of binding autoradiography, during brain development. The inferior colliculus (IC), a large midbrain structure which serves as an obligatory synaptic station in both the ascending and descending auditory pathways, exhibited high Ang II AT2 binding at all ages (P0, P8, P15, P30), being maximal at P15. These observations were confirmed by in situ hybridization and immunofluorescence at P15, demonstrating that AT2 receptor mRNA localized at the same area recognized by AT2 antibodies and anti ß III-tubulin suggesting the neuronal nature of the reactive cells. Ang II AT1 receptors were absent at early developmental ages (P0) in all nuclei of the auditory system and a low level was observed in the IC at the age P8. AT2 receptors were present at ventral cochlear nucleus and superior olivary complex, being higher at P15 and P8, respectively. We also explored the effect of prenatal administration of Ang II or PD123319 (AT2 antagonist) on binding of Ang II receptors at P0, P8, P15. Both treatments increased significantly the level of AT2 receptors at P0 and P8 in the IC. Although total binding in the whole IC from P15 animals showed no difference between treatments, the central nucleus of the IC exhibited higher binding. Our results supports a correlation between the timing of the higher expression of Ang II AT2 receptors in different nuclei, the onset of audition and the establishment of neuronal circuits of the auditory pathway.

Inhibition of DNA topoisomerase I and growth inhibition of human cancer cell lines by an oleanane from Junellia aspera (Verbenaceae).

DNA topoisomerases and DNA polymerases are enzymes that play a crucial role in DNA metabolism events such as replication, transcription, recombination, and chromosome segregation during mitosis. Thus, DNA topoisomerases and DNA polymerases inhibitors could be expected to have antitumor effects. Naturally occurring triterpenoids isolated from Junellia aspera (Gillies & Hook; Moldenke) (Verbenaceae) were assayed for human DNA topoisomerase I and Taq DNA polymerase inhibitory activities. Maslinic acid (2) and its diacetyl derivative (7) showed human DNA topoisomerase I inhibitory activity with IC50 values in the range of 76-80 microM and growth inhibition against various human solid tumour cell lines with GI50 values in the range of 5-18 microM. The triterpene frames could be used for screening new inhibitors of the enzyme, and computer-simulated drug design using the frame and pocket structure of enzyme may in theory be a possible approach to develop new inhibitors.

Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in San Luis

Essential hypertension is considered a multifactorial trait resulting from a combination of environmental and genetic factors. The angiotensin II type 1 receptor mediates the vasoconstrictor and growth-promoting effects of Ang II. The A1166C polymorphism of the AT1 receptor gene may be associated with cardiovascular phenotypes, such as high arterial blood pressure, aortic stiffness, and increased cardiovascular risk. We investigated the association between this A1166C polymorphism and hypertension in hypertense and normotense subjects from San Luis (Argentina) by mismatch PCR-RFLP analysis. Hypertense patients exhibited significant increases in lipid related values and body mass index. The frequency of occurrence of the C1166 allele was higher among patients with hypertension (0.19) than in the control group (0.06). No significant association was found between this polymorphism and essential hypertension in the study population, although the AC genotype prevalence was higher in patients with hypertension and positive family history of hypertension (32%) than in control subjects (12%). Patients with the A1166C polymorphism exhibited higher levels of serum total cholesterol, LDL-cholesterol and BMI than in control subjects. Taken together the genotype and biochemical parameters and considering the restrictive selection criteria used, the present results suggest a correlation between AT1 A1166C gene polymorphism and risk of cardiovascular disease.

Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in San Luis

Essential hypertension is considered a multifactorial trait resulting from a combination of environmental and genetic factors. The angiotensin II type 1 receptor mediates the vasoconstrictor and growth-promoting effects of Ang II. The A1166C polymorphism of the AT1 receptor gene may be associated with cardiovascular phenotypes, such as high arterial blood pressure, aortic stiffness, and increased cardiovascular risk. We investigated the association between this A1166C polymorphism and hypertension in hypertense and normotense subjects from San Luis (Argentina) by mismatch PCR-RFLP analysis. Hypertense patients exhibited significant increases in lipid related values and body mass index. The frequency of occurrence of the C1166 allele was higher among patients with hypertension (0.19) than in the control group (0.06). No significant association was found between this polymorphism and essential hypertension in the study population, although the AC genotype prevalence was higher in patients with hypertension and positive family history of hypertension (32%) than in control subjects (12%). Patients with the A1166C polymorphism exhibited higher levels of serum total cholesterol, LDL-cholesterol and BMI than in control subjects. Taken together the genotype and biochemical parameters and considering the restrictive selection criteria used, the present results suggest a correlation between AT1 A1166C gene polymorphism and risk of cardiovascular disease.(AU)

Inhibition of Taq DNA polymerase by catalpol.

DNA polymerases have recently emerged as important cellular targets for chemical intervention in the development of anti-cancer agents. This report describes a PCR assay as a method to investigate the action mechanism of the inhibition of Taq DNA polymerase by catalpol. This inhibition was not primer or template specific, nor was it due to chelation of Mg2+ ions. In assays of hyperchromicity of double-stranded DNA, catalpol did not affect melting profile. The inhibitory effect of catalpol does not appear to depend on DNA concentration. In contrast, increasing dNTP concentration rescue the Taq DNA polymerase activity, suggestingthat catalpol acts in a competitive way with dNTPs at the binding site of the enzyme. Theoretical calculations reinforce the experimental data and the proposed mode of action of catalpol.

Structural and electronic properties of tyrosine kinases inhibitors.

Protein tyrosine kinases (TKs) regulate cell proliferation, cell differentiation, and play a fundamental role in signal transduction pathway. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases was related to diseases such as cancer, atherosclerosis and psoriasis. For the present study, we selected a number of structurally related ATP-binding site inhibitors of EGF-receptors of diverse classes. Molecular properties of competitive inhibitors are key features for the action mechanism of these compounds. We performed a theoretical study at the RHF/6-311G* level of theory, in order to correlate the molecular parameters with the biological inhibitory activities. Species stability as evaluated by ionization potentials as well as the E(HOMO)-E(LUMO) energy gap, is in very good correlation with higher inhibitory potency (IP). The most active species, 1, 5, 6,10,11 and 12 exhibited strongly negative charged atoms over the C6 and C7 positions, the higher IP, higher mu and higher energy gap. In summary, a good correlation was observed between the molecular parameters, such as ionization potential, dipolar moment and E(HOMO)-E(LUMO) energy gap and inhibitory potency, suggesting that these properties play an important role for the interaction at the ATP-binding site of EGF-receptors.

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem.

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.

Angiotensin II receptors induce tyrosine dephosphorylation in rat fetal membranes.

Recently, it has become clear that many of the intracellular signals mediated by Ang II receptors are similar to the signaling pathways activated by receptor tyrosine kinases. In the present paper, we are reporting a full characterization of Ang II receptors in rat fetal membranes. We assayed binding of the Ang II antagonist [125I]Sar1Ile8Ang II and the AT2 specific competitor [(125)1]CGP42112. Both ligands exhibited a rapid equilibrium and a high specificity for Ang II receptors. Competition studies confirmed the presence of both receptor subtypes, with a predominance of AT2 receptors and the following order of potency: CGP42112>Ang II>Losartan>PD123177. Immunoblotting studies of tyrosine phosphoproteins showed that Ang II (10(-6)M) mediates a rapid reduction in tyrosine phosphorylation of several proteins with apparent molecular masses in the range of 30-45 kDa. Increasing concentrations of Ang II (10(-9) - 10(-6)M) showed a dose-response behavior, suggesting a clear physiological role of the observed effect. The response, blocked by Losartan and PD123177, seems to be mediated by both receptor subtypes. These results clearly indicate that both Ang II receptors mediate tyrosine dephosphorylation in early stages of development and support a role of these receptors in growth and development.

Angiotensin II receptor expression in the conduction system and arterial duct of neonatal and adult rat hearts.

Paralleling the classic circulating system, recent evidence has demonstrated the presence of a cardiac renin-angiotensin system, as well as the synthesis of angiotensin II in the heart. Two receptors for angiotensin II have been identified and classified as AT1 and AT2. The proportions of these receptor subtypes vary with the tissues, species and stage of development. From the results of other studies, it might be generalized that the expression of angiotensin II receptors and the proportion of AT2 receptor subtype are much higher in fetal and neonatal tissues than in the same tissues from an adult. The aim of this study was to specifically evaluate the AT1/AT2 ratio in the neonatal and adult conduction systems of rat hearts by means of quantitative autoradiogrphy. In the neonatal hearts, angiotensin II binding sites were highly concentrated in the vasculature, arterial duct, and conduction system, whereas their concentrations were barely detectable in the myocardium. Incubation with selective angiotensin II receptor ligands (losartan and CGP 42112) revealed that AT2 was the major subtype in vasculature (86 +/- 3%) and conduction system (73 +/- 4%). In the adult conduction system, the total expression of angiotensin II receptors was greatly reduced meanwhile the AT1 receptors represented the major proportion of the binding sites (80 +/- 3%). Our results demonstrated that the pattern of angiotensin II receptor expression in the conduction system of the rat heart is developmentally regulated. We suggest, as others have already, that the renin-angiotensin system plays a role during the early stage of cardiac development.

Sympathetic innervation modulates the expression of angiotensin II receptors in embryonic rat heart grafted in oculo.

Angiotensin II acts as a cardiac growth factor, and causes both inotropic and chronotropic changes within the heart. In the present study, we used an in oculo model system to examine the effects of sympathetic innervation on the density of cardiac angiotensin II receptors. Quantitative autoradiography was used to determine the density of angiotensin II receptors in embryonic rat hearts grafted into either sympathetically innervated or sympathetically denervated eye chambers of adult host rats. The density of specific binding to angiotensin II receptors was nearly three-fold higher in sympathetically non-innervated compared to sympathetically innervated heart grafts (30.8 +/- 4.2 v 11.5 +/- 3.2 fmol/mg protein). Specific binding to angiotensin II receptors in heart grafts was displaced by addition of the AT1 receptor antagonist losartan, but not by addition of the AT2 receptor competitor PD 123177. Thus, only AT1 receptors were present in sympathetically innervated and sympathetically non-innervated embryonic rat hearts grafted in oculo. We conclude that changes in sympathetic innervation caused changes in the density of cardiac angiotensin II receptors in the present study. Our results may have implications for growth and function not only during cardiac development, but also during cardiac disease.

Selective peptide and nonpeptide ligands differentially bind to angiotensin II AT2 receptor and a non-angiotensin II CGP42112 binding site.

[125I]CGP42112 [Nic-Tyr-(epsilon-CBZ (benzyloxycarbonyl)-Arg)Lys-His-Pro-Ile] does not only recognize angiotensin II AT2 receptors, but has also the capacity to label a high-affinity, non-angiotensin II binding site, selectively associated with macrophages and activated microglia. We have searched for the structural requirements of the novel CGP42112 binding site, and compared these with the requirements for binding to the angiotensin II AT2 site. We designed a series of CGP42112 analogs and evaluated the new compounds by using binding assays on rat spleen (CGP42112 site) and rat fetal (angiotensin II AT2 site) membranes. The non-peptidic analog Z-Arg(Pmc)OH (N alpha CBZ-NG-2,2,5,7,8-pentamethylchroman-6-sulphonyl-L-Arg), the side chain of CGP42112 substituted on the guanidinium group, was selective in recognizing the CGP42112 site, and did not displace binding from the angiotensin II AT2 site. This is a potential lead compound for development of CGP42112 site-selective analogs. Conversely, the CGP42112 analog lacking the CBZ-group (Nic-Tyr-(Ac-Arg)Lys-His-ProOH, III) and the peptide Nic-Tyr-Lys-His-Ala-HisOH (VI), were selective for the angiotensin II AT2 site, and recognized the CGP42112 site poorly. Our results demonstrate that the structural requirements for the nonangiotensin II CGP42112 and the angiotensin II AT2 binding sites are different. We propose that the CBZ group and the free carboxyl terminal group, together with their spatial orientation, are key components of the molecule for the interaction in the non-angiotensin CGP42112 binding pocket.

Quantification and localisation of angiotensin II receptors and angiotensin converting enzyme in the developing rat heart.

OBJECTIVE: The aim was to determine the quantification and localisation of angiotensin II receptors and angiotensin converting enzyme (ACE) in the developing rat heart. METHODS: Quantitative autoradiography was used to determine the density of [125I]Sar1-angiotensin II binding to angiotensin II receptors and the density of [125I]351A binding to ACE. RESULTS: Angiotensin II receptors were first detected in the myocardium on embryonic day 14, and reached a maximum density within the first postnatal week. During the perinatal period, the density of angiotensin II receptors was twofold higher in atrial than in ventricular myocardium [27(SEM 7) v 11(1) fmol.mg-1 protein]. By adulthood, however, the density of angiotensin II receptors did not differ between atria and ventricles. A high density of angiotensin II receptors was first detected in the cardiac vasculature on embryonic day 19, with binding density decreasing during development. During the embryonic and early postnatal periods, most of the binding in the cardiac vasculature was to AT2 receptors. ACE was first detected in the myocardium on the day of birth, with the density of binding to ACE increasing during development. On postnatal day 56, the density of binding to ACE was fourfold higher in atrial than in ventricular myocardium [99(22) v 22(4) fmol.mg-1 protein]. A moderate density of binding to ACE was first detected in the cardiac vasculature and heart valves on embryonic day 19, with binding density increasing during development. By adulthood, the density of binding to ACE was 10- to 25-fold higher in the cardiac vasculature and heart valves than in the myocardium. CONCLUSIONS: The density of angiotensin II receptors and the density of binding to ACE are developmentally regulated in the heart. Both AT1 and AT2 receptors were present in the rat heart by late gestation and could, therefore, mediate the effects of angiotensin II on early cardiac growth and development.

Kidney angiotensin II receptors and converting enzyme in neonatal and adult Wistar-Kyoto and spontaneously hypertensive rats.

The aim of the present study was to correlate the development of the renin angiotensin system (RAS) in the kidney of the rat with the development of genetic hypertension. Immature (1-week-old) and adult (12-week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive kidney rats (SHR) were used for quantification of angiotensin II (ANG II) receptors and angiotensin converting enzyme (ACE) binding sites using quantitative autoradiography. In both neonatal and adult animals of either strain, ANG II receptors were of AT1 subtype. In all kidney areas of 1-week-old rats. ANG II receptor density was higher in SHR than WKY. Binding density increased with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer medulla of 12-week-old WKY, binding was significantly higher than that present in age-matched SHR. [125I]351A binding to ACE was highest in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to ACE was higher in WKY than in SHR strain. No differences in ACE binding were found between adult SHR and WKY rats, with the exception of the inner stripe of the outer medulla, where no binding was detected in SHR. Our results support the hypothesis that the RAS in kidney is developmentally regulated and is involved in the development and maintenance of genetic hypertension in SHR.

Purification and characterization of angiotensin II AT2 receptors from neonatal rat kidney.

Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a nearly homogeneous state after solubilization from neonatal rat kidney membranes with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid. Comparable IC50 values for the soluble extract (0.32 nM) and membranes (0.31 nM) were obtained by competition curves with 125I-labeled CGP42112, a selective AT2 ligand. Binding to AT2 receptors in the soluble extract was not sensitive to dithiothreitol. AT2 receptors were further purified by gel filtration and a CGP42112 Sepharose affinity column. Ang II AT2 receptors were selectively eluted with 5 microM CGP42112 at 4 degrees C, and a single band with an apparent molecular mass of 71 kDa was obtained after SDS/PAGE. Two-dimensional electrophoresis confirmed the purity of the protein and an isoelectric point of 5.3-5.5 was obtained. A highly selective elution of the AT2 receptors from the affinity column was performed with 5 nM 125I-labeled CGP42112 at room temperature after the column was treated with 1 microM losartan in the presence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociation of the radiolabeled protein was insensitive to losartan but was enhanced by CGP42112, PD123177, Ang II, and [Sar1]Ang II. In summary, Ang II AT2 receptors were purified by CGP42112 affinity chromatography and selective elution and retain the pharmacological specificity of particulate receptors.

Glomerular angiotensin II receptor subtypes during development of rat kidney.

We used quantitative autoradiography to investigate distribution of angiotensin II (ANG II) receptor subtypes during development of the kidney in the rat. In fetal, newborn, and 3-day-old rats, immature glomeruli in the form of comma and S-shaped bodies, located in the nephrogenic zone of the renal cortex, expressed only the angiotensin AT2 receptor subtype. Conversely, the juxtamedullary glomeruli, in more advanced developmental stages, expressed only the AT1 subtype. Similarly, maturing and fully developed glomeruli, present in 1-, 2-, and 8-wk-old rats, expressed only AT1 receptors. In the kidney medulla, there was a similar change in ANG II receptor subtype expression, with the AT2 subtype expressed earlier and the AT1 subtype later during development. Our results demonstrate a selective expression of ANG II receptor subtypes during kidney development. We have found glomerular and medullary AT1 receptors only at developmental stages when kidney function has matured. Conversely, AT2 receptors are expressed only in immature structures, suggesting that they may have a role during kidney organogenesis.

Enhanced expression of angiotensin II receptor subtypes and angiotensin converting enzyme in medroxyprogesterone-induced mouse mammary adenocarcinomas.

Angiotensin II receptors of the AT1 subtype were very highly expressed in medroxyprogesterone-induced ductal adenocarcinomas of the mammary gland in BALB/c mice. AT1 receptors are associated only to neoplastic epithelial cells. Lobular adenocarcinomas expressed very few AT1 receptors and expressed AT2 receptors only in areas corresponding to peritumoral connective tissue. Binding to angiotensin converting enzyme was present in all adenocarcinomas studied and was higher in ductal than in lobular adenocarcinomas. Normal mammary gland did not express either angiotensin II receptors or angiotensin converting enzyme. The present results are the first demonstration of angiotensin receptor subtypes and converting enzyme in mammary adenocarcinomas differentially expressed in tumors of ductal and lobular origin. Localization of receptor subtypes could be useful to study the differentiation of mammary cells during experimental mammary carcinogenesis in mice.

Structure-activity relationship on excitatory amino acid receptors.

It is generally accepted that the carboxylic amino acids L-Glu and L-Asp play a role as excitatory neurotransmitters at the CNS. In the search of a structure-activity relationship we have made a theoretical conformational analysis with a mechanics molecular model on active compounds on QUIS and NMDA-receptors. We consider the COO-...COO- distance in these compounds as the more important one in order to discuss the different mode of action of this compound at the excitatory amino acid receptors. On QUIS-receptors: We had found that Glu, Asp and QUIS are very flexible molecules that can easily pass from one conformation to another and to get the distance of 3A degrees between the two COO- groups. All these compounds show an important population in conformations with this distance. The antagonist GDEE had 90% of its whole population in folded conformations with a distance of about 3 A degrees between COO-...COO- groups. On NMDA-receptors: IBO is a rigid analogue and the distance COO-...COO- is about 4A degrees at the different conformations found. Asp, Glu, beta-Amglu and NMDA have a considerable flexibility and they can easily adopt the distance required. They leave a considerable population in conformations that present a COO-...COO- distance about 4A degrees. We propose that folded conformations are important for activity on QUIS-receptors, whereas extended conformations are the important on NMDA-receptors.

Structure-activity relationship on excitatory amino acid receptors

Actualmente se acepta que los aminoácidos carboxílicos L-Glu y L-Asp tienen un rol como neurotransmisores excitatorios en el sistema nervioso contral (CNS). En el presente trabajo hemos realizado un análisis teórico conformacional con un modelo de mecánica molecular sobre compuestos activos en QUIS y NMDA-receptores, en la búsqueda de una correlación estructrura-actividad. En estos compuestos, consideramos la distancia COO-..COO- como la más importante para el análisis de la actividad de los mismos sobre los distintos receptores de aminoácidos excitatorios. Sobre los QUIS-receptores: Hemos encontrado que Glu, Asp son móleculas muy flexibles que pueden pasar con facilidad de una conformación a otra y alcanzar la distancia de 3A- entre los dos grupos COO-. Todos estos compuestos muestran una importante población en conformaciones con esta distancia. El antagonista GDEE tiene un 90% de la población total en conformaciones plegadas con una distancia de alrededor de 3A- entre los grupos COO-. Sobre los NMDA-receptores: IBO es un análogo rígido y la distancia COO-..CO- es alrededor de 4A- en las diferentes conformaciones encontradas. Asp. Glu, ß-Amglu y NMDA tienen una considerable flexibilidad y pueden adoptar fácilmente las distancia requeridas. Ellos poseen una considerable población en conformaciones que presentan una distancia COO-..COO- de alrededor de 4A-. Se propone que conformaciones plegadas son importantes para la actividad en QUIS-receptors, mientras conformaciones extendidas serían importantes en NMDA-receptors