RESUMEN
Angiotensin (Ang) I converting enzyme (ACE) inhibitors represent a major advance in the treatment of congestive heart failure, and tissue, rather than circulating ACE, may be their major site of action. However, assessments of tissue ACE inhibition in treated patients has not always supported this contention. In these studies, ACE activity was measured in homogenates of sampled tissue by biochemical methods. In the present study, using a model system, we have examined the validity of these tissue-sampling methods. Functional ACE activity was determined by comparing positive inotropic responses to [Pro10]Ang I in either vehicle-pretreated or ACE inhibitor-pretreated papillary muscles. [Pro10]Ang I elicits a response, which is entirely dependent on ACE-mediated conversion to Ang II. The ACE inhibitors studied were captopril, enalaprilat, lisinopril, and quinaprilat. In a parallel study, papillary muscle ACE activity was also measured in homogenates using [125I]MK-351A (a radiolabeled ACE inhibitor) binding. The studies indicate that the tissue-sampling method significantly underestimated functional ACE inhibition in hamster papillary muscles (p < 0.001). Kinetic studies indicated that the half-time for the dissociation of [3H]enalaprilat and [3H]lisinopril from hamster ventricular ACE was 4.5 and 6.2 minutes, respectively. The dissociation of [3H]quinaprilat was biphasic (half-time, 47 and 90 minutes), indicating that the two active sites of somatic ACE differ in their ability to bind to this inhibitor. The rapid rate of ACE inhibitor dissociation suggests that, during the time taken to assay ACE activity biochemically, the enzyme becomes "disinhibited," leading to an underestimation of functional ACE inhibition. ACE inhibitor dissociation rates were partially predictive of the duration of functional ACE inhibition in papillary muscles; other factors that appeared to contribute were "tissue trapping" of the inhibitor and de novo synthesis of ACE in papillary muscles. Quantification of tissue ACE inhibition and its relation to drug efficacy must, therefore, involve a careful consideration of these factors to avoid artifacts in clinical decision making and in assessments of pathogenic mechanisms involved in congestive heart failure.
Asunto(s)
Angiotensina I/análisis , Captopril/farmacología , Dipéptidos/farmacología , Enalaprilato/farmacología , Isoquinolinas/farmacología , Músculos Papilares/enzimología , Tetrahidroisoquinolinas , Angiotensina I/análogos & derivados , Angiotensina I/efectos de los fármacos , Angiotensina I/metabolismo , Angiotensina II/análisis , Angiotensina II/efectos de los fármacos , Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Animales , Captopril/metabolismo , Cricetinae , Dipéptidos/metabolismo , Enalaprilato/metabolismo , Humanos , Isoquinolinas/metabolismo , Lisinopril , Masculino , Mesocricetus , Músculos Papilares/químicaRESUMEN
The human heart is a target organ for the octapeptide hormone, angiotensin II (Ang II). Recent studies suggest that the human heart contains a dual pathway of Ang II formation in which the major Ang II-forming enzymes are angiotensin I-converting enzyme (ACE) and chymase. Human heart chymase has recently been purified and its cDNA and gene cloned. This cardiac serine proteinase is the most efficient and specific Ang II-forming enzyme described. To obtain insights into the cardiac sites of chymase-dependent Ang II formation, we examined the cellular localization and regional distribution of chymase in the human heart. Electron microscope immunocytochemistry using an anti-human chymase antibody showed the presence of chymase-like immunoreactivity in the cardiac interstitium and in cytosolic granules of mast cells, endothelial cells, and some mesenchymal interstitial cells. In the cardiac interstitium, chymase-like immunoreactivity is associated with the extracellular matrix. In situ hybridization studies further indicated that chymase mRNA is expressed in endothelial cells and in interstitial cells, including mast cells. Tissue chymase levels were determined by activity assays and by Western blot analyses. Chymase levels were approximately twofold higher in ventricles than in atria. There were no significant differences in chymase levels in ventricular tissues obtained from non-failing donor hearts, failing ischemic hearts, or hearts from patients with ischemic cardiomyopathy. These findings suggest that a major site of chymase-dependent Ang II formation in the heart is the interstitium and that cardiac mast cells, mesenchymal interstitial cells, and endothelial cells are the cellular sites of synthesis and storage of chymase. In the human heart, because ACE levels are highest in the atria and chymase levels are highest in ventricles, it is likely that the relative contribution of ACE and chymase to cardiac Ang II formation varies with the cardiac chamber. Such differences may lead to differential suppression of cardiac Ang II levels during chronic ACE inhibitor therapy in patients with congestive heart failure.
Asunto(s)
Angiotensina II/biosíntesis , Miocardio/enzimología , Serina Endopeptidasas/análisis , Adolescente , Adulto , Secuencia de Bases , Southern Blotting , Quimasas , Endotelio/citología , Femenino , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/etiología , Ventrículos Cardíacos/inmunología , Humanos , Hibridación in Situ , Masculino , Mastocitos/enzimología , Microscopía Electrónica , Persona de Mediana Edad , Datos de Secuencia Molecular , Miocardio/química , Miocardio/ultraestructura , ARN/análisis , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Fracciones Subcelulares/enzimologíaRESUMEN
1. The effect of bullfrog angiotensin I [Asp1, Val5, Asn9] angiotensin I, (AT I) on short-circuit current (SCC) on isolated toad skin and aorta contractility was examined. 2. AT I increased SCC in toad skin, the effect was partially inhibited by angiotensin-converting enzyme inhibitor (ACEI) teprotide. 3. AT I induced contractile responses in isolated rings of toad aorta. This effect was partially inhibited by captopril and completely blocked by the peptide antagonist [Sar1, Ile8] angiotensin II. 4. Present results indicate that this homologue AT I would act in amphibian tissues by conversion to AT II.
Asunto(s)
Angiotensina I/análogos & derivados , Angiotensina I/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Transporte Biológico Activo/efectos de los fármacos , Bufonidae , Contracción Isométrica/efectos de los fármacos , Riñón/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Rana catesbeiana , Absorción Cutánea/efectos de los fármacosRESUMEN
The renin-angiotensin system originally was thought to be responsible for only renovascular hypertension, but the development and use of various inhibitors of this system have produced much evidence for its participation in many forms of hypertensive disease. Tissue renin-angiotensin system also may play a major role in blood pressure control. Chronic clinical as well as animal use of converting enzyme inhibitors results in levels of angiotensin II that are equivalent to those found in the normotensive state and higher than those found in the very acute phase of treatment. The source of this conversion possibly may be due to enzymes unrelated to angiotensin converting enzyme. One such enzyme is a very highly specific serine protease isolated from human cardiac tissue. This enzyme exists in human ventricular tissue at levels four to five times that of angiotensin converting enzyme. During chronic treatment of patients with heart failure, angiotensin I levels become high, and heart tissue levels of angiotensin II may become elevated because of the conversion to angiotensin II by this serine protease. This conversion in turn may possibly increase inotropy of the heart, whereas the peripheral resistance remains low because of the reduction of angiotensin II in the circulation.
Asunto(s)
Angiotensina II/biosíntesis , Miocardio/metabolismo , Angiotensina I/metabolismo , Angiotensina II/antagonistas & inhibidores , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , HumanosRESUMEN
Human heart chymase, a chymotrypsin-like serine proteinase that hydrolyzes the Phe8-His9 bond in angiotensin I (Ang I) to yield the octapeptide hormone angiotensin II (Ang II) and His-Leu, is the most specific, efficient Ang II-forming enzyme described. Other mammalian chymases display a much broader substrate specificity. To better define its substrate specificity, we have mapped the extended substrate-binding site of human heart chymase using Ang I analogs. The enzyme has a preference for aromatic amino acids phenylalanine, tyrosine, and tryptophan at the P1 site. At the S2 subsite there is a significant preference for proline over hydrophobic or hydrophilic amino acids. There is no clear preference for hydrophobic or hydrophilic amino acids at the S'1 and S'2 subsites, but an Ang I analog containing a P'1 proline is not hydrolyzed and one with a P'2 proline is hydrolyzed poorly. An increasing reduction in reactivity occurs when the P position amino acids in Ang I are deleted sequentially from the N terminus. An increase or decrease in the length of the His-Leu leaving group also produces a marked decrease in reactivity. No single determinant in Ang I is preeminently required for efficient catalysis, but several factors acting synergistically appear to be important. Thus, we propose that ideal substrates for human heart chymase should contain the structure nXaa-Pro-[Phe, Tyr, or Trp]-Yaa-Yaa, where n greater than or equal to 6; Xaa = any amino acid; Yaa = any amino acid except proline. This structure exists in Ang I and neurotensin, both of which are good substrates for human heart chymase. These findings indicate that the selection of the scissile bond by the extended substrate-binding site of human heart chymase is more restricted than that in other chymases.
Asunto(s)
Angiotensina II/metabolismo , Miocardio/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Quimasas , Humanos , Hidrólisis , Cinética , Datos de Secuencia Molecular , Neurotensina/metabolismo , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.
Asunto(s)
Miocardio/enzimología , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cromatografía Líquida de Alta Presión , Quimasas , Clonación Molecular , ADN , Humanos , Intrones , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Serina Endopeptidasas/metabolismoRESUMEN
Angiotensin II (Ang II) receptors, estimated by the specific binding of the peptide Ang II receptor antagonist [125I] [Sar1,Ile8]Ang II, are localized on multiple ovarian structures, including follicular granulosa cells. Using the Ang II receptor subtype-selective nonpeptide antagonists, DuP 753 [selective for the type 1 Ang II (AT1) receptor] and PD 123319 [selective for the type 2 Ang II (AT2) receptor], we show that follicular granulosa cells, in vivo and in vitro, exclusively express the AT2 receptor. To understand the function of Ang II in ovarian follicles, we compared the biochemical properties and transmembrane signaling pathways of the granulosa cell AT2 receptor with those properties generally associated with Ang II receptors found in the adrenal zona glomerulosa, where the AT1 receptor predominates. The mol wt of the granulosa cell AT2 receptor (approximately 79,000), estimated by affinity cross-linking studies, is similar to that of the adrenal zona glomerulosa Ang II receptor. Like the adrenal zona glomerulosa Ang II receptor, binding inhibition studies show that the granulosa cell AT2 receptor binds Ang II and Ang III with high affinity (IC50, approximately 0.5 nM for both peptides), but not Ang-(1-7) (IC50, approximately 0.5 microM) or Ang-(1-5) (IC50, greater than 10 microM). However, unlike the adrenal zona glomerulosa Ang II receptor, the granulosa cell AT2 receptor does not undergo agonist-induced endocytosis. Further, Ang II does not affect basal or stimulated inositol phosphate production, intracellular Ca2+ mobilization, or adenylyl cyclase or guanylyl cyclase activity in granulosa cells. The granulosa cell AT2 receptor does not appear to directly interact with guanine nucleotide binding regulatory proteins, since agonist dissociation from the AT2 receptor is unaffected by the GTP analog guanosine 5'-O-(3-thiotriphosphate); in contrast, the AT1 receptor appears to directly interact with guanine nucleotide binding regulatory protein, because agonist dissociation from the AT1 receptor is stimulated by guanosine 5'-O-(3-thiotriphosphate). These studies clearly demonstrate that the granulosa cell AT2 receptor is functionally distinct from the well characterized adrenal zona glomerulosa Ang II receptor. The exclusive presence of the AT2 receptor on the granulosa cell makes it an ideal cell type for studying the potential, but as yet unknown, function of this receptor.
Asunto(s)
Células de la Granulosa/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/biosíntesis , Estrógenos/biosíntesis , Femenino , Hormona Folículo Estimulante/farmacología , Proteínas de Unión al GTP/fisiología , Células de la Granulosa/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Fosfatos de Inositol/biosíntesis , Peso Molecular , Progesterona/biosíntesis , Inhibidores de Proteasas/farmacología , Ratas , Ratas Endogámicas , Receptores de Angiotensina/química , Zona Glomerular/metabolismoRESUMEN
Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.
Asunto(s)
Angiotensina II/biosíntesis , Miocardio/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Angiotensina I/metabolismo , Animales , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Quimasas , Endopeptidasas/genética , Ventrículos Cardíacos/enzimología , Humanos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Neuropéptidos/metabolismo , Péptidos/síntesis química , Inhibidores de Proteasas/farmacología , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
Ovarian angiotensin I (Ang I)-converting enzyme (ACE), estimated by the specific binding of the ACE inhibitor [125I]iodo-MK-351A, is localized on multiple ovarian structures, including follicular granulosa cells, corpora lutea, terminal epithelium, and ovarian blood vessels, but total ovarian ACE does not display a cyclic pattern of variation during the rat estrous cycle. We have previously shown that ACE is localized on the granulosa cell layer of a subpopulation of rat ovarian follicles. Our present study shows that ovarian granulosa cells contain high affinity [binding site affinity (Kd), approximately 90 pM] and low capacity [binding site density (Bmax), approximately 12 fmol/2.5 X 10(5) cells] [125I]iodo-MK-351A-binding sites and convert [125I]iodo-Ang I to [125I]iodo-Ang II (greater than 85% of this conversion was inhibited by the ACE inhibitor captopril). Throughout the rat estrous cycle, 94-100% of developing follicles and 89-96% of atretic follicles contained high levels of ACE; however, ACE was either not observed or its levels were very low in preovulatory follicles. These findings indicate the presence of high levels of biologically active ACE on the surface of granulosa cells and suggest a potential role for follicular ACE in early stages of follicular maturation and atresia. Although ACE is known to process a variety of peptides found within the ovary, and these peptides may have opposing effects on follicular maturation, we attempted to define the cumulative effect of ACE inhibition on follicular maturation. Short and long term (6- and 14-day) infusions of captopril (6-day, 30.5 +/- 3.5 ova; 14-day, 28.5 +/- 7.5 ova) in immature rats, in which ovulation was induced by sequential treatments with PMSG and hCG, did not significantly affect ovulation compared with that in vehicle-infused control rats (6-day, 22.4 +/- 2.4 ova; 14-day, 20.8 +/- 3.1 ova), suggesting that ACE inhibition does not modify the follicular selection process in a way that affects ovulation. This may explain the lack of any reports of adverse effects of clinically used ACE inhibitors on ovulation.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Estro/fisiología , Folículo Ovárico/enzimología , Ovario/enzimología , Ovulación/efectos de los fármacos , Peptidil-Dipeptidasa A/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animales , Sitios de Unión , Captopril/farmacología , Membrana Celular/enzimología , Gonadotropina Coriónica/metabolismo , Dipéptidos/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Células de la Granulosa/enzimología , Radioisótopos de Yodo , Ratas , Ratas EndogámicasRESUMEN
Reduced preload and afterload to the heart are important effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of congestive heart failure. However, since angiotensin II (Ang II) directly increases the strength of myocardial contraction, suppression of Ang II formation by ACE inhibitors could potentially reduce the beneficial effects of Ang II on the failing heart. To study how ACE inhibition suppresses cardiac Ang II formation in man, we characterized ACE-dependent and ACE-independent Ang II-forming pathways in eight normal and 24 failing human hearts obtained at cardiac transplantation. Ang II-forming activity in left ventricular (LV) membrane preparations was assessed by measuring the conversion of [125I]angiotensin I (Ang I) to [125I]Ang II. LV [125I]Ang II-forming activity in normal hearts (35.5 +/- 2.7 fmol/min/mg, n = 8) was not different from that in hearts from patients with ischemic cardiomyopathy (25.5 +/- 2.9 fmol/min/mg, n = 9) and was 48% lower (p less than 0.001) in hearts from patients with idiopathic cardiomyopathy (18.5 +/- 1.9 fmol/min/mg, n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Angiotensina II/fisiología , Gasto Cardíaco Bajo/fisiopatología , Corazón/fisiopatología , Adolescente , Adulto , Angiotensina I/sangre , Angiotensina I/metabolismo , Angiotensina II/sangre , Captopril/farmacología , Femenino , Corazón/fisiología , Ventrículos Cardíacos , Humanos , Masculino , Persona de Mediana Edad , Miocardio/enzimología , Peptidil-Dipeptidasa A/metabolismo , Valores de Referencia , Glycine max/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
We examined the hypothesis that the positive inotropic effect of angiotensin I (Ang I) may be retained in the presence of angiotensin converting enzyme inhibitors so that it may have a direct beneficial effect on the heart. Accordingly, isolated perfused hearts (Langendorff preparation) of 300-day-old cardiomyopathic hamsters (a model of spontaneous cardiomyopathy) and age-matched normal hamsters (controls) were infused with Ang I in the presence of captopril; propranolol was added to the perfusing medium to block catecholamine-mediated effects of angiotensins on the heart. Left ventricular developed pressure and the rate of increase in left ventricular developed pressure increased significantly (p less than 0.001) in both the cardiomyopathic and the normal hamster heart despite concomitant reduction in myocardial flow rate favoring a direct inotropic effect of Ang I in both normal and myopathic hearts; these changes were significantly higher by almost threefold in the cardiomyopathic than in the normal hamsters (p less than 0.01) and were blocked by the angiotensin II (Ang II) antagonist [Sar1,Thr8]Ang II. Comparing dose-left ventricular contractility response curves for Ang I and Ang II, ED50 for responses was identical in both normal and myopathic hearts, whereas peak responses to Ang II were double those to Ang I in normal hearts but were almost identical in the myopathic hearts. Binding of [125I]Ang II in six cardiomyopathic and four normal hamster hearts was of high affinity, but there was no evidence for Ang I-saturable high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Angiotensina I/farmacología , Captopril/farmacología , Gasto Cardíaco Bajo/fisiopatología , Cardiomiopatías/fisiopatología , Corazón/fisiopatología , Contracción Miocárdica/efectos de los fármacos , Angiotensina II/antagonistas & inhibidores , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Angiotensinas/fisiología , Animales , Cricetinae , Sinergismo Farmacológico , Corazón/efectos de los fármacos , Masculino , Mesocricetus , Miocardio/metabolismo , Receptores de Angiotensina/metabolismoRESUMEN
To demonstrate the existence and help clarify the function of angiotensin II (Ang II) receptors in the human heart, we characterized the cardiac Ang II receptor and examined the levels and distribution of ventricular Ang II receptors in normal (n = 6) and failing (n = 14) hearts. Ang II receptors were characterized using the Ang II receptor agonist [125I]Ang II. Cardiac [125I]Ang II-binding sites were of high affinity (Kd, approximately 1 nmol/L) and low capacity (Bmax, approximately 3 fmol/mg membrane protein) and were pharmacologically specific [IC50 values for Ang II, [Sar1,Ile8]Ang II, and Ang III were 1.2, 3.0, and 400 nmol/L, respectively; the inactive Ang II metabolite Ang-(1-5), at a concentration of 1 mumol/L, inhibited [125I]Ang II binding by less than 10%]. These characteristics of cardiac [125I]Ang II-binding sites are similar to those of previously characterized mammalian heart Ang II receptors. In normal adult donor hearts (n = 5), Ang II receptor density in the left ventricle [LV, 2.90 +/- 1.40 (+/- SE) fmol/mg] was similar to that in the right ventricle (RV, 3.82 +/- 1.10 fmol/mg). The ventricular Ang II receptor density in adult patients with idiopathic (LV, 1.77 +/- 0.35 fmol/mg; RV, 1.58 +/- 0.29 fmol/mg; n = 8) or dilated cardiomyopathy (LV, 2.00 +/- 0.58 fmol/mg; RV, 2.56 +/- 0.52 fmol/mg n = 5) was similar to that in the normal heart. Ventricular Ang II receptors, localized by autoradiography using the Ang II receptor antagonist [125I]-[Sar1,Ile8]Ang II, were consistently found in the myocardium, cardiac adrenergic nerves, and coronary vessels of normal and failing ventricles. In human ventricles Ang II receptor levels were not correlated with age. Because ventricular Ang II receptor density in a normal neonatal human heart and that in a heart from an adolescent patient with idiopathic cardiomyopathy were more than 10-fold and more than 5-fold higher, respectively, than in normal adult ventricles, we investigated whether postnatal changes occur in ventricular Ang II receptors in rats. In male and female rats ventricular Ang II receptor density was about 2-fold higher in 1-day-old rats compared to that in 10-day-old or peripubertal rats. These data suggest developmental regulation of ventricular Ang II receptors. Our findings suggest that direct and neural angiotensinergic inputs to the myocardium play a role in the regulation of cardiac function in man and that these inputs are preserved in the failing heart.
Asunto(s)
Cardiopatías/metabolismo , Miocardio/metabolismo , Receptores de Angiotensina/análisis , Adolescente , Adulto , Factores de Edad , Animales , Autorradiografía , Sitios de Unión , Niño , Preescolar , Vasos Coronarios/metabolismo , Femenino , Atrios Cardíacos/crecimiento & desarrollo , Cardiopatías/patología , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/crecimiento & desarrollo , Histocitoquímica , Humanos , Lactante , Recién Nacido , Masculino , Miocardio/patología , Ratas , Ratas Endogámicas , Receptores de Angiotensina/fisiologíaRESUMEN
To demonstrate the existence and help clarify the function of renin in the rat ovary, we have characterized rat ovarian renin and examined ovarian renin levels during different stages of the rat estrous cycle. We show that high concentrations of active renin are present in the rat ovary (2.9 ng angiotensin I/h/mg). Ovarian renin activity has a pH optimum of about 7.0 and is due to a glycosylated aspartyl protease with an apparent mol wt of 39,000. These properties of rat ovarian renin are identical to previously characterized rat kidney renin. In PMSG-treated immature and adult 5-day cycling rats, ovarian renin was increased about 2-fold at estrus. At all stages of the estrous cycle in the 5-day cycling rat, the ratio of active to inactive ovarian renin was about 3:1, whereas about 90% of the renin in plasma was inactive. In the hypophysectomized diethylstilbestrol-treated rat ovary, over 90% of active renin remained in the residual ovary after the granulosa cells had been expressed, suggesting a theca-interstitial localization for renin. These studies indicate that active renin exists in the rat ovary, that its levels are cyclically increased at estrus, and that this increase may be due to enhanced local production and activation of the renin precursor. These findings greatly strengthen the concept of a functional renin-angiotensin system in the rat ovary.
Asunto(s)
Estro/metabolismo , Ovario/metabolismo , Renina/metabolismo , Sulfato de Amonio , Animales , Precipitación Química , Cromatografía , Dietilestilbestrol/farmacología , Ácido Edético/farmacología , Estro/efectos de los fármacos , Etilmaleimida/farmacología , Femenino , Gonadotropinas Equinas/farmacología , Concentración de Iones de Hidrógeno , Hipofisectomía , Ovario/efectos de los fármacos , Fluoruro de Fenilmetilsulfonilo/farmacología , Inhibidores de Proteasas , Ratas , Ratas EndogámicasRESUMEN
Based on the observation that high levels of renin and angiotensin II (Ang II) are found in the adrenal zona glomerulosa (ZG), it has been postulated that Ang II is formed intracellularly by the renin-converting enzyme cascade in this tissue. To test this hypothesis, we examined renin-angiotensin system components in subcellular fractions of the rat adrenal ZG. Renin activity and immunoreactive-Ang II (IR-Ang II) were observed in vesicular fractions but were not colocalized. In addition, angiotensinogen, angiotensin I, and converting enzyme were not observed in the renin or IR-Ang II-containing vesicular fractions. These data do not support the hypothesis that Ang II is formed intracellularly within the renin-containing vesicles of the ZG. Rather, since modulatable renin release from adrenal ZG slices was observed and renin activity was found in dense vesicular fractions (33-39% sucrose), it is likely that Ang II formation in the ZG is extracellular and initiated by the release of vesicular renin. Receptor-mediated endocytosis and subsequent degradation of Ang II in ZG lysosomes have been shown by others. The presence of IR-Ang II in light vesicular fractions (15% sucrose) and the finding of a high correlation between ZG IR-Ang II and Ang II receptor levels suggest that the primary occurrence of this peptide in the ZG is by receptor-mediated endocytosis. In ZG lysosomal fractions 125I-labeled Ang II was degraded to 125I-labeled des-[Phe8]Ang II. Since Ang II antibodies do not recognize des-[Phe8]Ang II, these findings explain why IR-Ang II in the ZG is due predominantly to Ang II and not to its C-terminal immunoreactive fragments.
Asunto(s)
Corteza Suprarrenal/citología , Angiotensina II/biosíntesis , Hormona Adrenocorticotrópica/farmacología , Animales , Epinefrina/farmacología , Femenino , Lisosomas , Potasio/farmacología , Ratas , Ratas Endogámicas , Renina/metabolismo , Sistema Renina-AngiotensinaRESUMEN
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.
Asunto(s)
Angiotensina II/farmacología , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Angiotensina II/metabolismo , Animales , Aromatasa/metabolismo , Calcimicina/farmacología , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Estradiol/farmacología , Femenino , Células de la Granulosa/efectos de los fármacos , Cinética , Progesterona/metabolismo , Progesterona/farmacología , Ratas , Ratas Endogámicas , Receptores de Angiotensina/efectos de los fármacos , Testosterona/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Ovarian angiotensin II (Ang II) receptors display a cyclical pattern of variation during the rat estrous cycle. Ang II receptors, estimated by the specific binding of the Ang II receptor antagonist [125I]iodo-[Sar1,Ile8] Ang II to ovarian membranes, were lowest at estrus [binding site density (Bmax) = 35 +/- 2 fmol/mg; binding site affinity (KD) = 2.0 +/- 0.2 nM] and highest at diestrus I (Bmax = 59 +/- 3 fmol/mg; KD = 1.6 +/- 0.1 nM). We have previously shown that Ang II receptors in the rat ovary predominantly exist on the granulosa cell layer of a subpopulation of follicles. Our present studies show that the Ang II receptor-containing follicles in the rat ovary are mainly atretic (approximately 80%) or show signs of early atresia (approximately 15%) during all stages of the estrous cycle. A small number of Ang II receptor-containing follicles were healthy (approximately 5%). In contrast to the Ang II receptor-containing follicles, the FSH receptor-containing follicles were predominantly healthy (greater than 90%). Follicles which contained both Ang II receptors and FSH receptors were mainly early atretic. Since Ang II receptor-containing follicles in the rat ovary were mainly atretic these studies suggest that in the rat Ang II may be a major factor in regulating the function of atretic ovarian follicles.
Asunto(s)
Angiotensina II/metabolismo , Estro/metabolismo , Atresia Folicular , Fase Folicular , Ovario/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Animales , Membrana Celular/metabolismo , Epitelio/metabolismo , Femenino , Hormona Folículo Estimulante/metabolismo , Radioisótopos de Yodo , Folículo Ovárico/anatomía & histología , Folículo Ovárico/metabolismo , Ratas , Ratas Endogámicas , Receptores de HFE/metabolismo , Distribución TisularRESUMEN
Mammalian ovarian follicles contain the enzymes and prohormones necessary to elaborate the active octapeptide hormone angiotensin II. In the rat ovary, angiotensin II receptors are located primarily in the theca interna and granulosa cell layers of a discrete subpopulation of follicles. Angiotensin II stimulates both androgen and estrogen secretion from rat ovarian slices. These findings suggest an autocrine/paracrine role for angiotensin II in ovarian follicular development.