RESUMEN

Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Ca2+o)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Ca2+o or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Ca2+o-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gq and G(i). High Ca2+o increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2 (cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Ca2+o)-induced ERK1/2 activation and reduced cPLA2 phosphorylation in both cell types, documenting MAPK's role in cPLA2 activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through G(i)- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2 activation.

Asunto(s)

Calcio/metabolismo , Riñón/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Glándulas Paratiroides/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Calcio/farmacología , Bovinos , Inhibidores Enzimáticos/farmacología , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Glándulas Paratiroides/citología , Glándulas Paratiroides/efectos de los fármacos , Toxina del Pertussis , Fosfolipasas A/efectos de los fármacos , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Fosforilación , Receptores Sensibles al Calcio , Receptores de Superficie Celular/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología

RESUMEN

To determine the role of aldosterone in mediating cardiovascular damage, we performed ablation/replacement experiments with aldosterone in a rat model of cardiac injury. Administration of angiotensin II and Nomega-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthesis inhibitor) to male rats drinking 1% saline caused hypertension, severe biventricular myocardial necrosis, proteinuria, and fibrinoid necrosis of renal and cardiac vessels. Removal of aldosterone by adrenalectomy or through administration of the selective aldosterone antagonist eplerenone markedly reduced the cardiac and renal damage without significantly altering blood pressure. Aldosterone infusion in adrenalectomized, glucocorticoid-replaced L-NAME/angiotensin II-treated animals restored damage. Thus, we identified aldosterone as a critical mediator of L-NAME/angiotensin II induced vascular damage through mechanisms apparently independent of its effects on systolic blood pressure.

Asunto(s)

Aldosterona/fisiología , Miocardio/patología , Arteria Renal , Enfermedades Vasculares/fisiopatología , Adrenalectomía , Aldosterona/sangre , Angiotensina II/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Inhibidores Enzimáticos/farmacología , Eplerenona , Antagonistas de Hormonas/farmacología , Riñón/patología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Necrosis , Ratas , Ratas Wistar , Renina/sangre , Cloruro de Sodio , Espironolactona/análogos & derivados , Espironolactona/farmacología

RESUMEN

Although endothelial cells produce angiotensin II (ANG II) and endothelin-1 (ET-1), it is not clear whether a single cell produces both peptides, with cosecretion in response to stimulation, or whether different subpopulations of endothelial cells secrete one or the other peptide, with secretion in response to different stimuli. Exposure of cultured coronary microvascular endothelial cells to cycloheximide for 60 min had no effect on ANG II or ET-1 secretion. This result suggested the existence of a preformed intracellular pool of ANG II and ET-1, which is a precondition for regulated secretion. Exposure of endothelial cells to isoproterenol, high extracellular potassium, or cadmium, all of which stimulate peptide secretion via different signaling pathways, significantly (P > 0.001) increased the secretion of both ANG II and ET-1 in a cell size-dependent manner. Sodium nitroprusside and S-nitroso-N-acetyl penicillamine significantly (P > 0.001) decreased ANG II and ET-1 secretion, whereas N(omega)-nitro-L-arginine-methyl ester enhanced it. The similar regulation of ANG II and ET-1 secretion and the presence of both peptides around individual endothelial cells indicate that the autocrine/paracrine regulation of cardiovascular function by endothelial cells is accomplished via cosecretion of ANG II and ET-1.

Asunto(s)

Angiotensina II/metabolismo , Vasos Coronarios/metabolismo , Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Especificidad de Anticuerpos , Cadmio/farmacología , Tamaño de la Célula , Células Cultivadas , Vasos Coronarios/citología , Cicloheximida/farmacología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Immunoblotting , Inmunohistoquímica , Masculino , Microcirculación/citología , Microcirculación/metabolismo , Donantes de Óxido Nítrico/farmacología , Potasio/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Vasodilatadores/farmacología

RESUMEN

Parathyroid cells have an intracellular machinery for parathyroid hormone (PTH) secretion that is inversely regulated by the extracellular calcium concentration (Ca2+o). The recently characterized Ca2+o-sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor mediating the inhibitory effects of high Ca2+o on PTH secretion. The CaR's precise cell surface localization and the signal transduction pathway(s) mediating its inhibitory effects on PTH secretion have not been characterized fully. Here, we demonstrate that the CaR resides within caveolin-rich membrane domains in bovine parathyroid cells. Chief cells within bovine parathyroid glands exhibit a similar pattern of staining for caveolin-1 and for alkaline phosphatase, a glucosylphosphatidylinositol-anchored protein often enriched in caveolae. Purified caveolin-enriched membrane fractions (CEMF) from bovine parathyroid cells are highly enriched in the CaR and alkaline phosphatase. Other signaling proteins, including Gq/11, eNOS, and several protein kinase C isoforms (i.e. alpha, delta, and zeta), are also present in CEMF. Activation of the CaR by high Ca2+o increases tyrosine phosphorylation of caveolin-1 in CEMF, suggesting that CaR-mediated signal transduction potentially involved in Ca2+o-regulated processes in parathyroid cells occur in caveolae-like domains.

Asunto(s)

Calcio/metabolismo , Caveolinas , Proteínas de la Membrana/metabolismo , Glándulas Paratiroides/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Caveolina 1 , Membrana Celular/metabolismo , Células Cultivadas , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Hormona Paratiroidea/metabolismo , Proteína Quinasa C/metabolismo , Propiedades de Superficie , Tirosina/metabolismo

RESUMEN

In extracavernosal vascular tissue, smooth muscle tone is modulated by a balance between angiotensin II (Ang II) and nitric oxide (NO). We hypothesized that these substances also play an important role in regulating cavernosal smooth muscle contractility. We therefore studied the in vitro effects of an Ang II receptor antagonist and a nitric oxide synthase (NOS) inhibitor on the contractile effects of Ang II, phenylephrine, and electrical field stimulation in canine corpus cavernosum. Ang II caused a dose-dependent contraction of cavernosal smooth muscle which was inhibited by the Ang II receptor antagonist and augmented by NOS inhibition. Contractions induced by phenylephrine or electrical stimulation were diminished by the Ang II receptor antagonist, and augmented by NOS inhibition. We conclude that Ang II plays an important role in modulating cavernosal smooth muscle tone, and that the contractile effect of Ang II on cavernosal tissue is modulated by the local NO environment.

Asunto(s)

Angiotensina II/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Óxido Nítrico/metabolismo , Pene , Antagonistas de Receptores de Angiotensina , Animales , Perros , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Losartán/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fenilefrina/farmacología

RESUMEN

PURPOSE: Although Angiotensin II (Ang II) is a major modulator of regional blood flow in the extracavernosal segments of the vascular bed, its role in erectile function is unknown. The corpus cavernosum penis is a modified vascular tissue that contains endothelial and smooth muscle cells. In other segments of the vascular bed, these cell types produce Ang II. Therefore, we explored the presence and function of an Ang II producing paracrine system in the corpus cavernosum. METHODS: The angiotensin content of the human corpus cavernosum was measured by radioimmunoassay. The distribution pattern of Ang II containing cells within the corpus cavernosum was assessed by an immunohistochemical technique, and the rate of its secretion was determined by superfusion. The effects of Ang II and its antagonist, losartan, on intracavernosal pressure were determined under in vivo conditions, in anesthetized dogs. RESULTS: Human corpus cavernosum contained 1178 +/- 223 (SEM) fmol Ang II, 528 +/- 171 fmol Ang I, 475 +/- 67 fmol des-asp-Ang I, and 1897 +/- 371 fmol des-asp-Ang II/gm. tissue (n = 4). Ang II was found mainly in endothelial cells lining blood vessels and smooth muscle bundles within the corpus cavernosum. Superfused cavernosal tissue secreted immuno-reactive Ang II (Ang II(ir)) at a rate of 57 +/- 36.5 fmol Ang II(ir)/gm. tissue/minute (n = 10). The amount of Ang II released per gram of tissue in an hour was 3-fold greater than the Ang II content/gm. tissue, suggesting a local production of Ang II. Papaverine and prostaglandin E1 suppressed Ang II secretion significantly (p <0.001, p = 0.013). The responsiveness to inhibition was a function of the initial rate of Ang II secretion. Tissue samples with a high rate of secretion were less responsive to the inhibitors than tissue that secreted small amounts of Ang II (n = 6). In anesthetized dogs, intra-cavernosal injection of Ang II terminated spontaneous erections, while losartan increased the intracavernosal pressure in a dose dependent manner up to the mean arterial pressure (n = 4). CONCLUSIONS: The corpus cavernosum produces and secretes physiologically relevant amounts of Ang II. The rate of Ang II secretion can be modulated by pharmacologic agents that regulate cytosolic calcium levels and are used clinically to treat erectile dysfunction. Intracavernosal injection of Ang II causes contraction of cavernosal smooth muscle and terminates spontaneous erection in anesthetized dog, while administration of an Ang II receptor antagonist results in smooth muscle relaxation and thus erection.

Asunto(s)

Angiotensina II/fisiología , Erección Peniana/fisiología , Pene/fisiología , Alprostadil/farmacología , Anestesia , Angiotensina II/administración & dosificación , Angiotensina II/análisis , Angiotensina II/antagonistas & inhibidores , Animales , Perros , Humanos , Inyecciones , Losartán/farmacología , Masculino , Papaverina/farmacología , Pene/química , Pene/citología , Pene/efectos de los fármacos

RESUMEN

Basic fibroblast growth factor (bFGF; fibroblast growth factor-2) and angiotensin II (ANG II), among other peptide signaling autacoids (cytokines), are known to regulate the phenotypic adaptation of cardiac muscle to physiological stress. The cell type(s) in cardiac muscle responsible for ANG II synthesis and secretion and the role of endogenous cytokines in the regulation of bFGF induction remain unclear. With the use of confluent, serum-starved, low-passage cultures of cardiac microvascular endothelial cells (CMEC), ANG II could be detected in cellular lysates and in medium conditioned by these cells with the use of high-performance liquid chromatography followed by radioimmunoassay. The secretion of angiotensins by individual CMEC could be detected with a cell-blot assay technique. ANG II secretion was decreased by brefeldin A, an agent that interrupts constitutive and regulated secretory pathways for peptide autacoid/ hormone synthesis, suggesting de novo synthesis, activation, and secretion of angiotensins by CMEC. In primary isolates of adult rat ventricular myocytes (ARVM) and CMEC, ANG II, acting at ANG II type 1 receptors in both cell types, was found to increase bFGF mRNA levels measured by ribonuclease protection assay. Endothelin-1 (ET-1), which is known to be synthesized by CMEC, and bFGF itself, which has been detected in both ARVM and CMEC, increased bFGF transcript levels in both cell types. Interleukin-1beta (IL-1beta), which like ANG II and ET-1 is known to activate mitogen-activated protein kinases in both ARVM and CMEC, increased bFGF mRNA levels only in cardiac myocytes. Thus cytokines such as ANG II, ET-1, bFGF, and IL-1beta locally generated by cellular constituents of cardiac muscle, including CMEC, regulate bFGF mRNA levels in a cell type-specific manner.

Asunto(s)

Angiotensina II/metabolismo , Circulación Coronaria , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Miocardio/metabolismo , Angiotensina II/farmacología , Animales , Células Cultivadas , Endotelina-1/farmacología , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ventrículos Cardíacos , Microcirculación , Miocardio/citología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Most parathyroid adenomas and some pathological parathyroid glands from patients with primary parathyroid hyperplasia or severe uremic secondary/tertiary hyperparathyroidism show an elevated set-point [the extracellular Ca2+ concentration (Ca2+o) half-maximally inhibiting PTH secretion]. In the present study, we investigated whether expression of the Ca2+o-sensing receptor protein recently cloned from bovine parathyroid, a key component in Ca2+o-regulated PTH release, is altered in primary and uremic hyperparathyroidism. Using immunohistochemistry with specific antireceptor antibodies, we compared immunoreactivity of the receptor protein in 14 adenomas, biopsies of 24 normal glands from this same group of patients, and 8 hyperplastic parathyroid glands from 2 individuals with uremic hyperparathyroidism. The results show a substantial reduction in the intensity of immunostaining for the receptor protein that averaged nearly 60% for both adenomas and hyperplastic glands, as quantitated by image analysis. Although normal glands from normocalcemic controls were not available, the intensity of receptor staining in normal glands from patients with adenomas was comparable to that in normal bovine, rat, and mouse parathyroid glands. There was considerable variation in staining intensity among different pathological parathyroid glands, even in those from the same patient with secondary hyperparathyroidism. In addition, both adenomas and hyperplastic glands had, in some cases, isolated chief cells and groups of cells, sometimes around the periphery of an abnormal gland, with receptor staining equivalent to that of normal parathyroid cells, whereas the bulk of the cells in the same gland showed a marked decrease in staining. Thus, there is a variable, but substantial, reduction in the immunoreactivity of the Ca2+o-sensing receptor protein in both parathyroid adenomas and uremic hyperparathyroidism, as assessed by immunohistochemistry, that probably results from reduced expression of the receptor protein and may contribute to the increase in the set-point often observed in these patients.

Asunto(s)

Adenoma/patología , Proteínas de Unión al Calcio/metabolismo , Hiperparatiroidismo/patología , Glándulas Paratiroides/metabolismo , Glándulas Paratiroides/patología , Neoplasias de las Paratiroides/patología , Uremia/patología , Adenoma/metabolismo , Animales , Anticuerpos , Biopsia , Proteínas de Unión al Calcio/análisis , Bovinos , Humanos , Hiperparatiroidismo/etiología , Hiperparatiroidismo/metabolismo , Hiperplasia , Inmunohistoquímica , Ratones , Glándulas Paratiroides/citología , Neoplasias de las Paratiroides/metabolismo , Ratas , Valores de Referencia

RESUMEN

To address the question as to how zona glomerulosa (ZG) cell angiotensin II (Ang II) secretion is regulated, we developed an immuno-cell blot assay to measure its secretion from single cells. We compared these results with those obtained from population studies using a superfusion system. Modulation of Ang II secretion was investigated acutely (by administrating potassium [K+] or captopril) and chronically (by feeding the animals low or high sodium diets). The area of secretory cells, halo areas, and halo intensities varied widely but were highly significantly correlated (P < 0.001) with each other. A disproportionate amount of Ang II was secreted by a small number of large cells. When K+ concentration was increased from 3.6 to 0 mM, superfused ZG cells increased their Ang II secretion 2.32 +/- 0.59-fold. Administration of captopril reduced the K(+)-stimulated Ang II secretion 1.24 +/- 0.07 fold. These findings were reflected in the cell blot assay as a change in the frequency distribution of halo area by K+ and captopril in the same direction as in the population study. In both conditions, the percentage of secretory cells did not change significantly from control. Superfused ZG cells from rats on a low sodium diet secreted 1.85 +/- 0.58-fold more Ang II than cells from sodium-loaded rats (p < 0.05, n = 6). The cell blot assay confirmed these findings with sodium restriction significantly increasing (P < 0.001) both the halo area and its frequency distribution to a larger portion of high secreting cells. However, in contrast to acute treatment with K+ or captopril, the number of secretory cells also doubled. Thus, the individual ZG cell uses two mechanisms to modify Ang II production. In response to acute stimulation and suppression, the amount of Ang II secreted per cell is modified without changing the number of secretary cells. With chronic stimulation, both the amount of Ang II secreted per cell and the number of secretary cells increase.

Asunto(s)

Angiotensina II/biosíntesis , Sistema Renina-Angiotensina , Renina/biosíntesis , Zona Glomerular/metabolismo , Angiotensina II/análisis , Angiotensina II/metabolismo , Animales , Captopril/farmacología , Dieta Hiposódica , Femenino , Técnicas In Vitro , Potasio/farmacología , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Renina/análisis , Renina/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Sodio en la Dieta/farmacología , Factores de Tiempo , Zona Glomerular/efectos de los fármacos

RESUMEN

PTH secretion from dispersed bovine parathyroid cells maintained in culture becomes progressively less responsive to changes in the extracellular Ca2+ concentration (Ca2+o) over several days. We have now investigated whether this change in secretory control is associated with alterations in the expression of the Ca2+o-sensing receptor (BoPCaR) recently cloned from bovine parathyroid, which plays a central role in Ca2+o-regulated PTH secretion. BoPCaR messenger RNA levels dropped rapidly in cultured bovine parathyroid cells, as assessed by Northern analysis, decreasing by 78% within 18 h and remaining low for at least 4 days. The level of receptor protein decreased to a comparable extent (approximately 72-82%) after 3-4 days in culture, as determined by immunocytochemistry with specific antibodies directed at the extracellular domain of the receptor. The half-time for the reduction in receptor protein (approximately 2 days) was considerably longer, however, than that for BoPCaR messenger RNA, but was comparable to that for the loss of sensitivity of PTH secretion to Ca2+o. Indeed, there was a close linear correlation between maximal suppressibility of PTH secretion and the intensity of staining for the receptor protein (r = 0.88; P = 0.004). We conclude that alterations in the expression of BoPCaR could explain much of the reduced responsiveness of cultured bovine parathyroid cells to Ca2+o.

Asunto(s)

Calcio/farmacología , Expresión Génica , Glándulas Paratiroides/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Animales , Bovinos , Células Cultivadas , Técnicas para Inmunoenzimas , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio , Receptores de Superficie Celular/metabolismo

RESUMEN

Multiple observations suggest local control of renal function via an intrarenal renin-angiotensin system, including evidence for local angiotensin (Ang) II production. Our first goal was to examine renal tissue Ang I:Ang II relations to ascertain whether Ang II formation differs in the circulation and in renal tissue. We have recently shown an authentic Ang II/Ang I ratio of 1.5:1 in renal lymph, the opposite of the Ang II:Ang I relation in plasma. Our second goal was to examine the influence of maximal angiotensin converting enzyme inhibition on these relations in plasma and in renal tissue. We used two converting enzyme inhibitors with differing lipid solubility, on the premise that tissue penetration and action might differ on that basis. We measured Ang I and Ang II in plasma and renal tissue of rats given an intravenous dose of either vehicle, enalapril, or ramipril, over a wide dose range, from 0.1 to 10.0 mg/kg i.v. Renal and plasma angiotensin concentrations were measured by high-performance liquid chromatography and radioimmunoassay. Whereas the Ang I concentration in normal rat plasma (273 +/- 84 fmol/mL) was over threefold the plasma Ang II concentration (83 +/- 12 fmol/mL), the ratio was reversed in the kidney (Ang II, 178 +/- 12 versus Ang I, 91 +/- 3 fmol/g; P < .001). Although ramipril and enalapril induced an indistinguishable dose-related acute fall in blood pressure and plasma Ang II concentration, lower enalapril doses were less effective in reducing renal tissue Ang I:Ang II conversion and Ang II concentration (P < .025).(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Angiotensina II/metabolismo , Angiotensina I/metabolismo , Enalapril/farmacología , Riñón/efectos de los fármacos , Ramipril/farmacología , Angiotensina I/sangre , Angiotensina II/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Riñón/metabolismo , Masculino , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley

RESUMEN

Potassium (K+) and angiotensin-II (Ang-II) are two distinct secretagogues for aldosterone release. However, a local adrenal renin-angiotensin system is present, and several studies suggest a complex interaction between K+ and locally produced Ang-II. First, superfusing zona glomerulosa (ZG) cells with K+ stimulates the secretion of both Ang-II and aldosterone. Second, K(+)-stimulated aldosterone secretion can be reduced in the presence of angiotensin-converting enzyme inhibitors. Because angiotensin-converting enzyme inhibitors are not specific inhibitors of the adrenal renin-angiotensin system, we further tested the hypothesis that locally produced Ang-II participates in K(+)-stimulated aldosterone release from rat ZG cells by using a specific Ang-II antagonist. Although type 1 (AT1) and type 2 (AT2) Ang-II receptors are present in ZG cells, only AT1 antagonist has been shown to mediate Ang-II-induced aldosterone secretion. Losartan, a specific AT1 antagonist, was used in this study. In the presence of losartan (10 microM for 9 mM K+ and 100 microM for 5 mM K+), the average aldosterone secretion during 2 h of superfusion with 9 mM K+ and 5 mM K+ was 70.1 +/- 5.4% (n = 5) and 58.5 +/- 2.2% (n = 3), respectively, of that in its absence. Losartan did not alter the amount of Ang-II secreted. The inhibitory effect of losartan lasted longer than 60 min after it was terminated. In summary, our results support the hypothesis that locally produced Ang-II contributes to the aldosterone secretory response to K+ stimulation at both physiological and supraphysiological levels.

Asunto(s)

Aldosterona/metabolismo , Compuestos de Bifenilo/farmacología , Imidazoles/farmacología , Potasio/farmacología , Tetrazoles/farmacología , Zona Glomerular/metabolismo , Angiotensina II/metabolismo , Animales , Femenino , Losartán , Periodicidad , Ratas , Ratas Sprague-Dawley , Zona Glomerular/efectos de los fármacos

RESUMEN

We compared the renal vascular responses to angiotensin converting enzyme inhibition and renin inhibition to assess the influence of angiotensin II (Ang II). We examined the renal and endocrine responses to the renin inhibitor enalkiren, to captopril, and to placebo in nine healthy and nine hypertensive men on a 10-mmol sodium diet. Ang II was infused to assess effects of the agents on renal and adrenal responsiveness to Ang II. Plasma Ang II concentration was suppressed similarly with enalkiren and captopril--an identical level of blockade was achieved. Although renal plasma flow was stable during placebo, a substantial rise was seen with both enalkiren (+133 +/- 26 mL/min per 1.73 m2) and captopril (+99.4 +/- 22.6). There was remarkable intrasubject concordance between the renal plasma flow responses to renin inhibition and converting enzyme inhibition (r = .90, P < .004). The vasodilator response to both agents correlated inversely with the fall in renal plasma flow induced by Ang II alone (r = -.66, P < .05). Both agents significantly enhanced the renal vascular response to Ang II (P = .01), and, furthermore, the renal vasodilator response to captopril predicted the potentiation of the renal plasma flow response to Ang II after either agent (enalkiren: r = .91, P < .001; captopril: r = .56, P < .05). Concordance of the maximal renal plasma flow response to the two agents appeared in the hypertensive men as well. Our results indicate that the acute renal response to captopril largely reflects a reduction in Ang II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Angiotensina II/fisiología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Dipéptidos/farmacología , Renina/antagonistas & inhibidores , Adulto , Aldosterona/sangre , Angiotensina II/farmacología , Presión Sanguínea/efectos de los fármacos , Captopril/farmacología , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Circulación Renal/efectos de los fármacos , Renina/sangre

RESUMEN

We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, "receptor-like" mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca(2+)-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca(2+)-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5-10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29-36% decreases in PIP2 (within 5-10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic "Ca(2+)-mobilizing" hormones, the high Ca(2+)-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Ca(i)) in parathyroid cells.

Asunto(s)

Calcio/farmacología , Fosfatos de Inositol/metabolismo , Glándulas Paratiroides/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Citosol/metabolismo , Hidrólisis , Inositol 1,4,5-Trifosfato/metabolismo , Litio/farmacología , Magnesio/farmacología , Glándulas Paratiroides/citología , Glándulas Paratiroides/efectos de los fármacos

RESUMEN

In rats, plasma renin activity (PRA) increases sharply, reaching a plateau within hours of sodium restriction. Plasma aldosterone increases gradually, not reaching a plateau for 1-2 days. To determine whether this dissociation is secondary to the time needed to modify adrenal sensitivity to angiotensin II (ANG II) and to assess the role of locally produced ANG II in this process, rats were salt restricted for 0-120 h. Plasma hormone levels were assessed, adrenal ANG II was measured, and basal and ANG II (1 x 10(-8) M)-stimulated steroidogenesis were determined in vitro. Although PRA attained an elevated plateau within 8 h, plasma aldosterone did not peak until after 48 h of sodium depletion. The in vitro aldosterone sensitivity to exogenous ANG II was not apparent until rats had been salt restricted for 16 h. A plateau (4-fold increase above the ANG II response on high salt) was achieved between 24 and 48 h. Adrenal ANG II also exhibited a similar delayed response that correlates significantly with changes in aldosterone biosynthesis and late pathway activity. Thus the dissociation between PRA and plasma aldosterone may be secondary to a lag in the zona glomerulosa's (ZG) steroidogenic response to ANG II as well as a parallel lag in tissue ANG II production, suggesting that changes in tissue ANG II may mediate ZG sensitivity to ANG II during sodium deprivation.

Asunto(s)

Aldosterona/metabolismo , Angiotensina II/metabolismo , Dieta Hiposódica , Renina/sangre , Zona Glomerular/metabolismo , Aldosterona/sangre , Angiotensina II/farmacología , Animales , Corticosterona/sangre , Femenino , Ratas , Ratas Endogámicas

RESUMEN

The cells of the adrenal cortex contain angiotensin-II (AII), but whether this peptide is synthesized there (vs. internalized from the systemic circulation), whether it is secreted, and whether it is important in aldosterone production remain uncertain. To address these issues, we studied AI and AII release from superfused rat adrenal capsules and dispersed glomerulosa cells. Superfused adrenal capsules released 7-fold more AII in 270 min than the capsules originally contained (495 +/- 101 fmol AII/rat released vs. 66 +/- 8 fmol AII/rat tissue content). The amount of AI released in the same period only slightly exceeded the tissue content. In response to higher potassium concentrations in the medium (9 vs. 3.6 mM K+), adrenal capsules and dispersed glomerulosa cells both released significantly more AI and AII into the superfusate. This release of AI and AII was oscillatory. The oscillations occurred in each of 15 experiments, with a period of 45-90 min. Decapsulated adrenal glands (the zona faciculata/reticularis plus medulla) also contained and released AII, but did not respond to potassium stimulation. There was a highly significant correlation between AII and aldosterone release. This was especially apparent if aldosterone secretion was examined during oscillations of AII release (r = 0.97; P less than 0.0001). We conclude that AII is synthesized in the zona glomerulosa and can be released in response to stimuli. The close correlation between AII and aldosterone secretion suggests that locally produced AII may play an important role in aldosterone biosynthesis.

Asunto(s)

Glándulas Suprarrenales/metabolismo , Angiotensina II/metabolismo , Angiotensina I/metabolismo , Potasio/farmacología , Zona Glomerular/metabolismo , Glándulas Suprarrenales/efectos de los fármacos , Aldosterona/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Femenino , Periodicidad , Radioinmunoensayo , Ratas , Ratas Endogámicas , Zona Glomerular/efectos de los fármacos

RESUMEN

To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. We conclude that these sodium-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction.

Asunto(s)

Glándulas Suprarrenales/metabolismo , Angiotensinas/metabolismo , Sodio en la Dieta/farmacología , Animales , Cromatografía Líquida de Alta Presión , Dieta Hiposódica , Femenino , Ratas , Ratas Endogámicas , Distribución Tisular , Zona Glomerular/metabolismo

RESUMEN

The effects of chronic constriction of the thoracic inferior vena cava (TIVCC) on plasma atrial natriuretic factor (pANF) were studied in conscious dogs (n = 5). TIVCC decreased left and right atrial pressure and led to a decrease in pANF concentration from 199 +/- 12 to 104 +/- 14 pg/ml while plasma renin and vasopressin concentrations increased. These hormonal changes were associated with a significant fall in sodium excretion to less than 5 meg/day. pANF remained suppressed during chronic TIVCC as the dogs expanded their extracellular fluid volume and developed ascites. Acute release of TIVCC resulted in abrupt increases in left and right atrial pressure but only a modest rise in pANF from 96 +/- 16 to 185 +/- 45 pg/ml. The magnitude of the rise in pANF (twofold) contrasted sharply with the eightfold increase in sodium excretion that occurred over the first 24 hours. Our data suggest that decrease in atrial pressure below normal results in a decline in pANF, which, acting in concert with the activated renin-angiotensin system and vasopressin, may contribute to sodium retention. On the other hand, during acute release of TIVCC, which markedly increased atrial pressure and sodium excretion, pANF only returned to control levels. These data suggest that ANF release may be attenuated during chronic reduction in atrial pressure and also raise a question concerning the magnitude of the primary role of ANF in this natriuretic response.

Asunto(s)

Factor Natriurético Atrial/sangre , Vasoconstricción , Vena Cava Inferior/fisiología , Animales , Arginina Vasopresina/sangre , Presión Sanguínea , Perros , Masculino , Renina/sangre , Sodio/metabolismo

RESUMEN

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.

Asunto(s)

Factor Natriurético Atrial/sangre , Espacio Extracelular/fisiología , Animales , Factor Natriurético Atrial/inmunología , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Espacio Extracelular/efectos de los fármacos , Furosemida/farmacología , Sueros Inmunes , Masculino , Radioinmunoensayo , Ratas , Valores de Referencia , Privación de Agua

RESUMEN

Cultured bovine aortic endothelial cells (BAEC) contain renin and angiotensinogen. To examine whether angiotensins are synthesized intracellularly and secreted by these cells, we assayed cell extracts as well as serum-free media of intact confluent BAEC. Angiotensins were identified by their retention time on reverse phase high performance liquid chromatography and direct radioimmunoassay. BAEC and their media contain angiotensin II and angiotensin III. The rate of angiotensin accumulation in the media was a nonlinear function of time; the highest rate occurred in the first 15 minutes. The amount of angiotensin II accumulated in 30 minutes exceeded 200% of the intracellular concentration and that of angiotensin III exceeded 500% of the initial intracellular content. Neither renin nor angiotensinogen could be detected in the media. The viability of these cells was supported by low lactic dehydrogenase activity in the media (less than 0.5% of cellular level). These data suggest that BAEC is capable of synthesizing and secreting angiotensins. We postulate that this endothelial-derived angiotensin system may play an important paracrine or autocrine role in influencing local vascular tone.

Asunto(s)

Angiotensinas/biosíntesis , Endotelio/metabolismo , Membranas Intracelulares/metabolismo , Sistema Renina-Angiotensina , Angiotensina I/biosíntesis , Angiotensina II/biosíntesis , Angiotensina III/biosíntesis , Animales , Línea Celular , Endotelio/citología , Renina/metabolismo