Regulation of vascular angiotensin II receptors by EGF

After vascular endothelial injury, angiotensin II (ANG II) playsa role in the resulting hypertrophic response, and expression ofepidermal growth factor (EGF) is enhanced. Therefore, we tested thepossibility that EGF regulates vascular ANG II action and receptorexpression. Incubation of cultured aortic vascular smooth muscle cells(VSMC) with EGF (or basic fibroblast growth factor but notplatelet-derived growth factor isoforms) resulted inconcentration-dependent (1-50 ng/ml EGF), time-dependent (>8 h),and reversible decreases in ANG II surface receptor density. Forexample, a 50% reduction was observed after exposure to 50 ng/ml EGFfor 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 hresulted in a 77% reduction in ANG II-stimulated inositol phosphateformation. EGF not only prevented but also reversed ANG II receptorupregulation by 100 nM corticosterone. The specifictyrosine kinase inhibitor tyrphostin A48 (50 µM) reducedEGF-stimulated thymidine incorporation and EGF-stimulatedphosphorylation of mitogen-activated protein kinase but did not preventEGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristateacetate (100 nM for 24 h) prevented EGF from reducing ANG II receptordensity. In summary, EGF is a potent negative regulator of vascular ANGII surface receptor density and ANG II action by mechanisms that do notappear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reducedsurface ANG II density cannot be eliminated by the present studies.

     

Characterization of angiotensin II receptors in the rat fetus

The presence of AII receptors during early and late embryonic development was studied by binding of 125I[Sar1, Ile8] AII to whole mouse blastocysts and membrane-rich fractions from rat conceptuses, 7 to 21 days in gestation. In early mouse embryos there was no detectable binding under a variety of experimental conditions. However, in late gestation rat fetuses, specific and high affinity binding was observed, with a concentration of sites similar in membranes from whole and eviscerated fetuses. Using less than 100 micrograms of membrane protein, binding was time and temperature dependent, maintaining equilibrium from 30 to 120 min at 23 degrees C and it was enhanced by addition of Mg+2 up to 5 mM, EGTA 2 mM and dithiothreitol up to 2.5 mM. Scatchard analysis of the binding data indicated Kd values ranging between 0.7 and 0.9 nM. Binding was first detectable at day 10 (14.3 +/- 2.3 fmol/mg), increasing to 104 +/- 16, 2,625 +/- 168, 5,993 +/- 152 and 5,902 +/- 92 by days 12, 15, 18, and 21 of gestational age, respectively. Since the functional significance of these binding sites depends on the availability of the agonist ligand, acid extracts from eviscerated 10-day-old fetuses were analyzed for the presence of AII. Measurement of AII by radioimmunoassay revealed immunoreactive AII-like material (845 pg/g of tissue), with an elution pattern identical to that of AII standard in a Sephadex G-50 column. This material was bioactive, as demonstrated by its ability to displace 125I[Sar1, Ile8]AII from adrenal glomerulosa membranes, an effect which was abolished by pretreatment of the extract with AII antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.     

Autoradiographic localization of angiotensin II receptors in the rat brainstem

The microscopic localization of angiotensin II receptors in the rat brainstem has been accomplished utilizing in vitro receptor autoradiographic techniques. These receptors are highly localized to discrete nuclear regions of the brainstem. Significant densities of autoradiographic grains, indicating the presence of specifically bound [125I]-angiotensin II, were observed in regions of the film corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, nucleus intercalatus, nucleus commissuralis, substantia gelatinosa of the trigeminal nerve and the inferior olivary nucleus. Previous immunohistochemical studies have indicated the presence of immunoreactive angiotensin II in several of these regions, especially those concerned with central cardiovascular regulatory mechanisms. These results provide additional evidence in support of the hypothesized role of angiotensin II as a central neuromodulator in the regulation of systemic blood pressure.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Developmental changes in brain angiotensin II receptors in the rat.

AII binding and distribution were measured in rat brain during development by autoradiographic techniques using radioiodinated [Sar1,Ile8]AII. At all ages, from 2 days to 7 weeks, binding was present in the circumventricular organs, and areas related to pituitary hormone secretion and modulation of sympathetic activity. At early stages of development, AII binding was transiently expressed in a number of motor- and sensory-related areas. These findings support a role for AII in the control of water intake and autonomic activity at all stages of development, and suggest that the peptide may be involved in the maturation of neuronal function during development.     

Effect of angiotensin II on vascular resistance in whole-body perfused dogfish

• 1.1. The effect of angiotensin II (AII), norepinephrine (NE), epinephrine (E) and isoproterenol (ISO) was observed on the branchial and systemic circulations in a whole-body-pump perfused dogfish preparation.
• 2.2. NE and E increased systemic blood flow resistance, but decreased branchial resistance.
• 3.3. ISO decreased both systematic and branchial blood flow resistance.
• 4.4. AII had no significant effect on either branchial or systemic resistance.

     

Reduced number of adrenal angiotensin II receptors in the spontaneously hypertensive rat

[¹²⁵I]angiotensin II binding to adrenal subcellular particles was compared between spontaneously hypertensive and Wistar-Kyoto normotensive control rats. The number of angiotensin II receptors was reduced in adrenals of spontaneously hypertensive rats (P < 0.05) without a change in the affinity of angiotensin II for the binding sites at animal ages of 5, 10 and 15 weeks. This decline of receptor content antedated the abrupt rise in blood pressure noted between 5 and 10 weeks. The data suggest the presence of an alteration of the receptor number in the renin-angiotensin- aldosterone system in the spontaneously hypertensive rat.     

Regulation of angiotensin II receptors in cultured rat ovarian granulosa cells by follicle-stimulating hormone and angiotensin II

The regulation of ovarian granulosa cell angiotensin II (Ang-II) receptor formation and progesterone secretion by follicle-stimulating hormone (FSH) and Ang-II was studied in cultured cells prepared from hypophysectomized, diethylstilbestrol-treated immature rats. Ang-II receptors (estimated by the specific cell binding of the Ang-II receptor antagonist 125I-[Sar1,Ile8]Ang-II) were present on freshly prepared granulosa cells and increased by over 2-fold (to 2150 binding sites/cell; KD = 0.5 nM) when cultured in serum-free medium for 48 h. FSH prevented the normal increase in Ang-II receptor expression. Maximal FSH-dependent decrease in Ang-II receptors and increase in progesterone secretion occurred at 100 ng/ml FSH. The inhibitory effect of FSH on granulosa cell Ang-II receptor content was partially mimicked by the cAMP analogue 8-bromo-cAMP, since 8-bromo-cAMP suppressed (by 96%) Ang-II receptor content to a greater extent than FSH (by 60%). Granulosa cell Ang-II receptor content was not modified by progesterone or 17 beta-estradiol, but was decreased by testosterone (by 35%). Ang-II also produced a decrease in granulosa cell Ang-II receptor content, but did not modify progesterone secretion or aromatase activity. The effect of Ang-II on granulosa cell Ang-II receptor content was mimicked by the Ca2+ ionophore A23187, but not by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, suggesting that an elevation of cytosolic Ca2+ may be important for the homologous down-regulation of the Ang-II receptor. These data show homologous and heterologous down-regulation of granulosa cell Ang-II receptors. If these regulatory mechanisms exist in the FSH-sensitive healthy follicle, our findings suggest that in the process of maturation, healthy and dominant follicles may become decoupled from angiotensinergic influences.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.     

Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells.

The binding sites and biochemical effects of angiotensin (A) II were investigated in rat pheochromocytoma (PC12W) cells. Sarcosine1, [125I]-tyrosine4, isoleucine8-AII ([125I]-SI-AII) bound to a saturable population of sites on membranes with an equilibrium dissociation constant (Kd) of 0.4 nM and a binding site maximum of 254 fmol/mg protein. Competitive displacement of [125I]-SI-AII by agonists and antagonists elucidated a rank order of potency of AIII greater than or equal to AII greater than PD 123177 greater than AI greater than [des-Phe]AII [AII(1-7)] much greater than DuP 753. The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate did not alter the binding affinity or slope of the inhibition curves for AI, AII, AIII, or AII(1-7). Treatment of PC12W cells with AII or AIII did not affect the free intracellular calcium concentration, phosphoinositide metabolism, arachidonate release, cyclic GMP, or cyclic AMP concentrations. [125I]-AII binding sites remained on the cell surface and were not internalized after 2 h at 37 degrees C. Angiotensin II did not stimulate tyrosine, serine, or threonine phosphorylation. Northern analysis of PC12W mRNA with an AT1 receptor gene probe failed to produce an RNA:DNA hybrid at low stringency. These data indicate that PC12W cells express a homogeneous population of AT2 binding sites which differ significantly from AT1 receptors in signal transduction and molecular structure. AT2 sites may act via potentially novel, biochemical pathways or, alternatively, be vestigial receptors.     

Heterogeneity of angiotensin II receptors in membranes of developing rat metanephros

Specific and high affinity binding sites for angiotensin II were demonstrated in the membranes of the developing rat metanephros during the second half of pregnancy and in the newborn by binding studies with 125I angiotensin II. Only one type of angiotensin receptor was found during intrauterine life while after birth two classes of angiotensin receptors were present in the membranes of the cortical renal tissue.     

Regulation of angiotensin II receptors levels during rat induced pulpitis

A change in the microcirculatory hemodynamic is one of the most important events in inflammation. In the dental pulp, which is a connective tissue surrounded by a mineralized dentine substrate, disturbance in the blood flow as well as plasma extravasation may increase the pulp pressure and cause local ischemia. The octapeptide angiotensin II (AngII) regulates vascular tone and stimulates the release of pro-inflammatory cytokines by acting through the AT1 and AT2 receptors. The AT1 receptor is responsible for the classical effects of AngII. The AT2 receptor is involved in other effects, such as vasodilation. Therefore, we aimed to evaluate the role of AT1 and AT2 receptors on the pulpal inflammation. The pulp tissue was mechanically exposed and after different periods the teeth were extracted and submitted to histopathological and RT-PCR analyses. The histological sections showed a number of congested and dilated blood vessels associated with a notable presence of inflammatory cells. RT-PCR data revealed that the AT1 receptor was down-regulated at 24 h after the pulp exposure. The AT2 receptor expression was up-regulated by a 9-hour period, and then decreased between 12- and 24-hour periods. It was demonstrated that the renin-angiotensin system plays an important role in the pulpal inflammation, with regulation of AngII receptor levels.     

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.