RESUMEN
The authors describe the case of an 89-year-old patient with a one-month history of pain in the right knee and a pathological fracture of the distal femur. Excisional biopsy showed a classical osteogenic sarcoma. The patient died after six months of palliative surgical and oncological therapy; her primary disease generalised and progressed with numerous metastases. The authors discuss the possibilities of histological diagnostics of osteogenic sarcoma and its differential diagnosis. They draw attention to a possible occurrence of this type of sarcoma also in elderly patients, and not only in adolescents.
Asunto(s)
Neoplasias Femorales/diagnóstico , Osteosarcoma/diagnóstico , Anciano de 80 o más Años , Diagnóstico Diferencial , Femenino , Neoplasias Femorales/cirugía , Humanos , Osteosarcoma/cirugía , Cuidados PaliativosRESUMEN
Both inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the cardiac protective peptide adrenomedullin (AM) are increased in cardiac tissues and plasma in patients with myocardial infarction (MI) and chronic heart failure. Recently they have been increasingly recognized as important factors in the pathophysiology of MI and resultant congestive heart failure. Compared with sham-operated spontaneously hypertensive rats (SHR), we investigated myocardial immunoreactivity of TNF-alpha and AM and also their mutual relations in vivo in SHR+MI. Residual myocardial depression after MI was studied also in isolated perfused hearts. In chronic experiments, 24 and 48 h after permanent ligation of the descending anterior branch of the left coronary artery, we examined hemodynamics, plasma and myocardial peptide levels. Left ventricular function was assessed in isolated perfused hearts subjected to "global ischemia and reperfusion" and after induction of "calcium paradox". Circulating and myocardial TNF-alpha concentrations increased early after MI in SHR. Studies with global ischemia and calcium paradox in isolated heart showed early myocardial depression and calcium-dependent gradual increase of left-ventricular end-diastolic pressure. In the SHR+MI myocardial AM concentrations were increased 9- and 49-fold after respective 24 h and culminated 48 h following MI. Circulating and myocardial AM was increased in SHR+MI in association with TNFalpha-induced myocardial depression. The both studied cardiac parameters displayed the beneficial effect of the enhanced myocardial AM concentration.
Asunto(s)
Adrenomedulina/metabolismo , Infarto del Miocardio/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adrenomedulina/sangre , Animales , Calcio/metabolismo , Hipertensión/sangre , Hipertensión/complicaciones , Hipertensión/metabolismo , Masculino , Infarto del Miocardio/sangre , Infarto del Miocardio/etiología , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Ratas , Ratas Endogámicas SHR , Factores de Tiempo , Factor de Necrosis Tumoral alfa/sangre , Función Ventricular IzquierdaRESUMEN
The cytostatic cytokine tumor necrosis factor-alpha (TNF-alpha) and proliferative hormone adrenomedullin (AM) are abundantly expressed in human tumors. However, little is known about mechanism (s) through which TNF-alpha and AM exert their regulatory effects, especially in the regulation of proliferative activity in malignant cells. Also the role played by TNF-alpha in pathogenesis and treatment of cancer (targeted cancer therapy) remains less understood. The purpose of this study was therefore to characterize the significance of TNF-alpha induced apoptosis with down-regulation of plasma-membrane TNF-alpha receptors and up-regulation of AM receptors with increased production of human AM mRNA, i.e. mechanisms that subsequently control aberrant cellular proliferation in malignant cells. Cytotoxicity, and the whole cell ligand binding assays for TNF-alpha and AM receptors, and RIA-assays of AM production were accomplished in control experiments using pharmacologically pretreated HeLa cells. AM increased proliferation of HeLa cells and AM antagonist (Ala6,21)AM(22-52) significantly antagonized this increase. TNF-alpha inhibitor of cell growth actinomycin-D significantly increased cytotoxicity of TNF-alpha in HeLa cells. Hypoxia increased TNF-alpha production and increased surface-membrane [125I]AM binding. Tumor promotor PMA and histamine down-regulated specific binding of [125I]TNF- alpha on HeLa cells. Mitogenic peptide endothelin-1 increased and specific ET-1 antagonist BQ123 and significantly reduced AM binding. Production of AM in HeLa cells markedly increased after exposure to hypoxia >ET-1 >PMA. BAY11-7082 at concentrations that inhibited IkappaB phosphorylation and thus nuclear translocation and surface membrane TNF-alpha expression increased AM specific binding. Pretreatment of cells inhibitor of HMGCoA reductase inhibitor VULM1457 significantly increased the total number of specific [125I]AM binding sites on HeLa cells. These results suggest relative and contradictory TNF-alpha and AM surface-membrane receptor signaling in HeLa cells and findings reveal a novel proliferative mechanisms that control AM production and thus oncogenic signaling in cells. This implies that several putative inhibitors of TNF-alpha and AM signaling may be considered in oncology for treatment of tumors otherwise nonresponding to cytostatic therapy.
Asunto(s)
Neoplasias/metabolismo , Péptidos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adrenomedulina , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , RadioinmunoensayoRESUMEN
The proliferative peptide adrenomedullin (AM) has a wide distribution in a variety of tissues and cells. The mechanism how the AM gene is regulated in cells is not yet known. The renal cortex, renal vascular smooth muscles, glomeruli and tubular epithelial cells are very sensitive to hypoxia. Renal hypoxia produces acute renal tubular necrosis and markedly induces AM expression in damaged cells. However, little information is available regarding the possible pathophysiological production and release of renal tubular AM. Regulation of membrane-bound AM receptors in renal cells has not yet been systematically studied. To elucidate the potential pathological role of human AM we examined the production and release of AM, as well as the characteristics of surface membrane AM receptors in cultured monkey renal tubular epithelial cells (RC) exposed to hypoxia, induced with endothelin-1, and subjected to glucose deprivation. Exposure of RC to hypoxia (1 % O(2), 5 % CO(2) in N(2)), and to phorbol 12-myristate 13-acetate (PMA) increased production and secretion of AM and increased specific [(125)I]AM binding on RC. Metabolic stress (1 % glucose in the cultivation medium) and preincubation of RC with rival peptide endothelin-1 significantly reduced immunoreactive-AM in a conditioned medium and whole cell surface membrane AM binding on RC. Altogether, our data suggest that the AM is involved in the adaptation of renal tubular cells to hypoxia. Increased expression of AM mRNA and regulation of AM receptors in metabolic stress may function as an important autocrine/paracrine regulator(s) of renal tubular epithelial cells.
Asunto(s)
Adrenomedulina/metabolismo , Glucosa/deficiencia , Receptores de Péptidos/metabolismo , Adrenomedulina/genética , Animales , Hipoxia de la Célula/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Dactinomicina/farmacología , Endotelina-1/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Expresión Génica/fisiología , Humanos , Túbulos Renales/citología , Túbulos Renales/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Ésteres del Forbol/farmacología , Ensayo de Unión Radioligante , Receptores de Adrenomedulina , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Acyl-CoA:cholesterol acyltransferase (ACAT) is an important enzyme in the pathways of cholesterol esterification. It has been shown that new ACAT inhibitor 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM1457) significantly reduced atherogenic activity in animal experimental atherosclerosis. Proliferative hormone adrenomedullin (AM) has been shown to be released in response to hypoxia, however, its role in cellular protection has remained elusive. The effect of increased local production of AM in cells and resultant down-regulation of AM receptors has not been investigated yet. We hypothesized that increased expression of AM in hypoxic cells was the result of excessive AM production with resultant AM receptor down-regulation, surface-membrane protein degradation and that the new specific ACAT inhibitor would reduce AM induction in hypoxia and thus proliferation of cells. In order to investigate specific cellular AM signaling and protection induced by VULM1457, we characterized specific surface-membrane [125I]AM receptors expressed on cells, evaluated AM secretion (RIA assays), AM mRNA expression in cultured cells (RT-PCR analysis) and proliferation (incorporation of [3H]thymidine) in control, hypoxic and metabolically stressed human hepatoblastoma cell lines exposed to gradually increasing concentrations of VULM1457. The new ACAT inhibitor VULM1457 in concentration 0.03 and 0.1 micromol/l significantly down-regulated specific AM receptors on HepG2 cells, reduced AM secretion of HepG2 cells exposed to hypoxia. These results suggest that VULM1457, as new member of ACAT family of inhibitors could negatively regulate cell proliferation induced by AM, which may correlate with down-regulation of membrane-bound AM receptors on HepG2 cells, and moreover, with the induction and expression of AM in hypoxia.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Clofibrato/análogos & derivados , Péptidos/metabolismo , Receptores de Péptidos/metabolismo , Esterol O-Aciltransferasa/antagonistas & inhibidores , Adrenomedulina , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Clofibrato/administración & dosificación , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Humanos , Receptores de AdrenomedulinaRESUMEN
Previous studies on anti-cancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human uterus HeLa nad murine leukemia L1210 cell lines. Cytotoxicity was measured using in vitro techniques and cell morphology changes were examined by light microscopy in both cytostatic and cytocidal concentration ranges. The IC50 was found to be less than 4 microg/ml, a limit put forward by NCI for classification of the compound as a potential anti-cancer drug. The microscopy examination indicated that at cytocidal concentrations the HeLa and L120 cells died apoptotically. The comparative analysis revealed that berberine belongs to the camptothecin family of drugs characterized by the ability to induce DNA topoisomerase poisoning and hence apoptotic cell death. Although the cytotoxic potency of berberine was found to be several orders of magnitude lower compared to camptothecin, its significance may increase in future in view of the lack of unwanted side effects characteristic for camptothecin compounds currently in clinical use for treatment of cancer.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Berberina/toxicidad , Animales , Recuento de Células , División Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Leucemia L1210/tratamiento farmacológico , Proteínas de Neoplasias/biosíntesisRESUMEN
The contrasting pattern of cardiac inotropy induced by human peptide endothelin-1 (ET-1) has not been satisfactorily explained. It is not clear whether ET-1 is primarily responsible for increased myocardial ET-1 expression and release with resultant inotropic effects, or for the induction of myocardial hypertrophy and heart failure. There are at least two subtypes of endothelin receptors (ET(A) and ET(B)) and the inotropic effects of ET-1 differ depending on the receptor involved. Along with some other groups, we reported significant subtype-ET(B) endothelin receptor down-regulation in human cardiac cells preincubated with endothelin agonists (Drímal et al. 1999, 2000). The present study was therefore designed to clarify the subtype-selective mechanisms underlying the inotropic response to ET-1 and to its ET(B)-selective fragment (8-21)ET-1 in the isolated rat heart. The hearts were subjected to (1-21)ET-1 and to (8-21)ET-1, or to 30 min of stop-flow ischemia followed by 40 min of reperfusion, both before and after selective blockade of endothelin receptors. The present study revealed that both peptides, ET-1 and its (8-21)ET-1 fragment, significantly reduced coronary blood flow in nmolar and higher concentrations. The concomitant negative inotropy and chronotropy were marked after ET-1, while the infusion of the ET-1(8-21) fragment produced a slight but significant positive inotropic effect. Among the four endothelin antagonists tested in continuous infusion only the non-selective PD145065 and ET(B1/B2) selective BQ788 (in molar concentrations) slightly reduced the early contractile dysfunction of the heart induced by ischemia, whereas ET(A)-selective PD155080 partially protected the rat heart on reperfusion.
Asunto(s)
Endotelina-1/análogos & derivados , Endotelina-1/farmacología , Insuficiencia Cardíaca/fisiopatología , Corazón/fisiología , Contracción Miocárdica/fisiología , Secuencia de Aminoácidos , Animales , Circulación Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Dioxoles/farmacología , Endotelina-1/antagonistas & inhibidores , Endotelina-1/química , Endotelinas/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Insuficiencia Cardíaca/etiología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Humanos , Técnicas In Vitro , Masculino , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Isquemia Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/prevención & control , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Perfusión , Piperidinas/farmacología , Ratas , Ratas Wistar , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Izquierda/fisiología , Presión Ventricular/efectos de los fármacos , Presión Ventricular/fisiologíaRESUMEN
UNLABELLED: Clearance of human peptide endothelin-1 (ET-1) has been proposed to follow a receptor pathway involving a cascade of ET-1 receptor endocytosis and lysosomal degradation by a family of proteinases expressed constitutively by most cells. Genetically distinct endopeptidases produce ET-1 and degrade mature peptide. The ET-1 degradation products were considered to be inactive, however, recent evidence suggests that ET-1 fragments sustain most of the homeostatic response produced by parent peptides. The purpose of this study was to establish whether the overall structure of human ET-1 or the structure of its C-terminus is responsible for the subtype-selectivity, down-regulation and clearance of endothelin, and whether D-aminoacid substitution in the moiety of synthetic peptide is involved in effective ET-1 antagonism in coronary vascular smooth muscle. To characterize specific mechanism(s) leading to subtype-selective ET-receptor down-regulation and/or to ET-1 antagonism, ligand binding studies were accomplished with radioactive human (1-21)ET-1 and with C-terminal ET-1 fragments, both peptide agonists and antagonists, in adult male porcine coronary artery vascular smooth muscle (CVSM). The subcellular membranes of CVSM were isolated by isopycnic gradient centrifugation. Exposure of porcine coronary artery to exogenous ET-1 induced endothelin-ETB selective down-regulation. ETA-mediated subtype-ETB down-regulation was observed with distribution of ligand-ETB receptor complexes in light, endosomal, membranes. The ETA selective PD151242 significantly attenuated [3H]-thymidine incorporation, and the ETB selective antagonist BQ788 blocked down-regulation observed in porcine vascular fibroblasts (PF). Preincubation of coronary arteries with ETB selective BQ3020 was accompanied with a more intense down-regulation. CONCLUSION: our data are indicative of short-term ETB selective down-regulation of endothelin receptors in coronary vascular smooth muscle after exposure to ET-1. The presence in the carboxy-terminus of (Ala11,15) substitution in peptide fragments IRL1620 and BQ3020 determined the differential specificity of ETB-receptor coupling and was important for subtype-ETB-receptor down-regulation. The activation of the dominating ETA-receptor by ET-1 facilitated mitogenic responses to ET-1 in porcine vascular fibroblasts.
Asunto(s)
Regulación hacia Abajo/fisiología , Endotelina-1/análogos & derivados , Endotelina-1/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Fraccionamiento Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Endotelina-1/administración & dosificación , Endotelina-1/química , Humanos , Técnicas In Vitro , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Masculino , Músculo Liso Vascular/efectos de los fármacos , Sensibilidad y Especificidad , Estereoisomerismo , PorcinosRESUMEN
The aim of the present study was to investigate the effect of dietary supplementation with the pyridoindole antioxidant stobadine on kidney status and function in streptozotocin-induced diabetic rats. Diabetic male Wistar rats were fed a standard diet for 32 weeks or a diet supplemented with stobadine (0.05% w/w). The diabetic state was characterized by significantly elevated plasma levels of glucose, HbA1c and urea, severe reduction of total body weight and relatively enlarged kidneys. Elevated levels of conjugated dienes were recorded in the diabetic kidney confirming the presence of oxidative stress in diabetic animals. All diabetic rats showed marked proteinuria and albuminuria along with elevated excretion of the enzyme N-acetyl-beta-D-glucosaminidase. Long-term treatment of diabetic animals with stobadine significantly reduced total proteinuria, albuminuria and enzymuria, yet left the overall physical and glycemic status unaffected. It reduced oxidative damage of kidney tissue as shown by decreased conjugated diene level, and decreased matrix collagen cross-linking, as indicated by decreased breaking time values of rat tail tendons. These beneficial effects of stobadine, supported also by histological findings, may be brought about by virtue of the combination of its antioxidant potential with other effects, e.g., the postulated cholesterol-lowering ability or its ability to alter vascular reactivity and reduce the vascular tone.
Asunto(s)
Albuminuria/tratamiento farmacológico , Antioxidantes/farmacología , Carbolinas/farmacología , Colágeno/química , Diabetes Mellitus Experimental/tratamiento farmacológico , Nefropatías Diabéticas/tratamiento farmacológico , Riñón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Acetilglucosaminidasa/orina , Animales , Glucemia/análisis , Carbolinas/uso terapéutico , Riñón/metabolismo , Riñón/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
BACKGROUND: There is still considerable uncertainty regarding sensitivity of arterial blood pressure to endogenous peptides in renal hypertension. Many pathological processes including hypertension have been shown to be associated with release of endothelin-1 (ET-1). However the role of ET-1 in regulation of arterial blood pressure in hypertension is still controversial. OBJECTIVES: The role of endothelin-1 (ET-1) and angiotensin-II (AT-II) in malignant phase of renovascular hypertension has been assessed on the basis of arterial blood pressure increase and ETA receptor density measurements in Glodblatt-hypertensive rats (RVH). RESULTS: The arterial blood pressure response to sympatomimetic amines, vasopressors, the plasma ET-1 and AT-II levels as well as renal subtype-ETA receptor density were significantly increased in RVH rats with malignant hypertension. The dominance of vasopressor ETA receptors in RVH rats suggest the contribution of endothelin peptides to malignant renovascular hypertension. (Tab. 1, Fig. 7, Ref. 25.)
Asunto(s)
Acetilcolina/farmacología , Angiotensina II/sangre , Presión Sanguínea/efectos de los fármacos , Endotelina-1/sangre , Hipertensión Renovascular/fisiopatología , Norepinefrina/farmacología , Simpatomiméticos/farmacología , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Animales , Hipertensión Renovascular/sangre , Masculino , Ratas , Ratas WistarRESUMEN
The characteristics of specific binding of human [125I]Tyr(13)-endothelin-(1-21), [125I]-Tyr(13)-Suc-[Glu(9),Ala(11, 15)]-endothelin-(8-21), ([125I]IRL-1620) and endothelin ET(A) receptor antagonist [125I]Tyr(3)-(N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-Leu]-1Me )-D-Trp ([125I]PD151242) (number of sites and their affinity) and proliferation responses to exogenous endothelin receptor agonists (endothelin-1 and the endothelin ET(B) receptor-selective, truncated N-acetyl-[Ala(11,15)]-endothelin-(6-21) analogue BQ3020) were determined in cultured human fibroblasts and in tumorigenic HeLa cells. The cells were pre-incubated with equimolar concentrations of human endothelin-1 or its truncated analogue BQ3020. After pre-incubation (2 h), both peptides induced down-regulation of surface-membrane endothelin-1 receptors. This process was specific for endothelin ET(B) receptors and was much more intensive in tumorigenic cells. BQ3020, acting mostly through its C-terminus, induced nearly maximal endothelin ET(B) receptor down-regulation in HeLa cells. Staurosporine, a wide spectrum protein kinase inhibitor, significantly reduced, and N-[N-[N-[2, 6-dimethyl-1piperidinyl)carbonyl]-4-Me-L-Leu]-1-(methoxycarbonyl)- D-t ryptophanyl]-D-norleucine (BQ788), an endothelin ET(B) receptor antagonist, attenuated the down-regulation of endothelin receptors induced by endothelin receptor agonists. The down-regulation of endothelin ET(B) receptors was prevented by pre-incubation of the cells with the lysosomal enzyme blocker chloroquine. The endothelin-1-induced cell proliferation was attenuated by pre-incubation of the cells with the non-selective endothelin receptor antagonist Ac-D-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-glycine-3,3-D-diphenyl-Ala-Leu-Asp-Ile-Ile-Trp (PD142893) and it was only partially reduced by the endothelin ET(A) receptor-selective endothelin antagonist PD151242.
Asunto(s)
Regulación hacia Abajo/genética , Neoplasias/metabolismo , Receptores de Endotelina/biosíntesis , Receptores de Endotelina/genética , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Endotelina-1/metabolismo , Endotelinas/farmacología , Fibroblastos/metabolismo , Células HeLa , Humanos , Cinética , Ligandos , Membranas/metabolismo , Neoplasias/genética , Fragmentos de Péptidos/farmacología , Receptor de Endotelina B , Receptores de Endotelina/efectos de los fármacosRESUMEN
The authors present a group of 60 acromioclavicular dislocations of Tossy III type in 59 patients whom they operated on in the period of 1992-1996. Within the use of a variety of methods of the surgery of acromioclavicular dislocation they achieved the best results by a timely suture of the coracoclavicular ligament and stabilization of the clavicula by a Bosworth screw. They have been using this method most frequently since 1990 as when performed in time and technically correctly it is very efficient despite its certain drawbacks. The authors emphasize mainly technical aspects of a timely acromioclavicular stabilization by a cancellous screw. In the conclusion they formulate principles of the use of the method of stabilization by a screw after Bosworth for the achievement of a good functional result. Key words: acromioclavicular dislocation, acromioclavicular stabilization by a screw after Bosworth.
RESUMEN
Binding of endothelin (ET) peptides to their respective receptors with resulting proliferation of vascular smooth muscle has been implicated in the pathogenesis of arterial hypertension and atherosclerosis. Recently it was hypothesized that endothelin- (ET-1) bound to its two membrane receptors (ET(A) and ET(B)) continues to activate signal transducing proteins in cells. It was also shown that pyridoindole stobadine stabilized lysosomal membranes in myocardium in early ischemia. Therefore we decided to study the effects of stobadine on specific, subtype-selective binding and subsequent degradation of human, synthetic [125I]-ET-1 in human fibroblasts (HF). Our results indicate that stobadine significantly potentiated ET-1 binding by reductive ET(B) selective degradation of ET-1 in HF. Hence, it is very plausible that stobadine may modulate endogenous endothelin and its intracellular mitogenic and chemotactic factors, principally by affecting two presumably related processes, participating in the proliferative and mitogenic response, (1) potentiation of signal trasduction from ET(A) receptors, and (2) subtype-ET(B) selective intracellular processing.
Asunto(s)
Antioxidantes/farmacología , Carbolinas/farmacología , Endotelina-1/metabolismo , Azepinas/farmacología , Células Cultivadas , Cloroquina/farmacología , Antagonistas de los Receptores de Endotelina , Endotelinas/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Radioisótopos de Yodo , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Mitógenos/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/agonistas , Receptores de Endotelina/metabolismoRESUMEN
Many physiological and pathological processes in the cardiac tissue have been shown to be associated with a release of endothelin (ET) peptides and with induction of specific ET-receptors and G-protein-coupled ion channels. However, the exact mechanism regulating ET-receptors in the myocardium is controversial. The response to ET-1, the most important member of the ET family, is rapidly attenuated by down-regulation of ET-receptors. The internalization of ET-1 bound to two subclasses of specific receptors (ET(A) and ET(B)) that are abundant in the myocardium has been hypothesized to activate and/or inhibit a variety of intracellular signal transducing systems. The [125I]ET-1, BQ-3020 and selective ET-antagonists were used to study the subtype-selective component of regulation of ET-1 receptors in myocardial membranes. We determined the characteristics of [125I]ET-1 binding and [3H]thymidine incorporation in whole cell saturation studies and measured Ca2+ channel induction and the total number of inactive Ca2+ channels in photoaffinity studies with [3H]azidopine. Here we demonstrate four important components of the complex ET-1 response in human, porcine and rat myocardium, leading to aberrant responses of cells. After ET-1 induction, adaptive subtype-ETB selective down-regulation predominated in human embryonic fibroblasts, in porcine membrane vesicles and in microsomal membranes of renal hypertensive rats, with preferential high affinity ET-1 binding to ETA receptors and with the resultant ETA mediated proliferative and mitogenic activation of human fibroblasts. The ET-1 induction was also accompanied by profound inactivation of Ca2+ channels in myocardial membranes.
Asunto(s)
Regulación hacia Abajo , Endotelina-1/metabolismo , Miocardio/metabolismo , Receptores de Endotelina/fisiología , Transducción de Señal , Animales , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , ADN/biosíntesis , Fibroblastos/metabolismo , Humanos , Hipertensión/metabolismo , Radioisótopos de Yodo , Masculino , Microsomas/metabolismo , Miocardio/ultraestructura , Ratas , Ratas Wistar , Receptor de Endotelina A , Receptor de Endotelina B , PorcinosRESUMEN
The affinity of two compounds, closely related to 1,4-dihydropyridines, was determined in competition binding experiments using canine cerebral cortex membranes and [3H]nitrendipine as a radioligand. Some aspects of structure-affinity relationships are discussed in relation to existing models of the dihydropyridine binding site.
Asunto(s)
Canales de Calcio/metabolismo , Dihidropiridinas , Proteínas del Tejido Nervioso/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Corteza Cerebral/metabolismo , Dihidropiridinas/síntesis química , Perros , Técnicas In Vitro , Masculino , Modelos Biológicos , Proteínas del Tejido Nervioso/efectos de los fármacos , Nitrendipino/farmacocinética , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
The effects of endothelin-1 (ET-1) on surface membrane Ca2+ channels were studied on cultured human embryonal vascular smooth muscle cells (VSMC) and on isolated rat aorta using photoaffinity labelling with DHP Ca2+ channel antagonist (-)-[3H]-azidopine (AZI). The AZI-labelled saturable population of sites on VSMC with Bmax = 1.59 +/- 0.10 pmol/mg of protein and KD = 5.40 +/- 1.70 nmol/l; and 1.32 +/- 0.11 pmol/mg w.w. and KD = 1.09 +/- 0.20 nmol/l in isolated rings of the rat aorta. Preincubation with ET-1 (0.1, 1.0 and 10 nmol/l) increased (in a concentration-dependent manner) the total number of sites specifically photolabelled on VSMC. The number of sites labelled with AZI on ET-1 preincubated VSMC increased markedly when divalent cations (Ca2+ or Mg2+ in other experiments) were present in the incubation medium. Specific photolabelling also significantly increased in VSMC pretreated with intrinsically photoreactive nifedipine. A protein kinase C inhibitor staurosporine, added to the incubation medium, significantly reduced the enhanced specific photolabelling after ET-1. The increase in specific photolabelling after ET-1 preincubation (+197 +/- 46%; P < 0.05) was also observed in rings of the rat aorta and it was significantly reduced after preincubation with S-(+)-niguldipine.
Asunto(s)
Azidas , Canales de Calcio/efectos de los fármacos , Dihidropiridinas , Endotelina-1/farmacología , Músculo Liso Vascular/efectos de los fármacos , Marcadores de Afinidad , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Sitios de Unión/efectos de los fármacos , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Cinética , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Estaurosporina/farmacologíaRESUMEN
With the use of the photoactivable dihydropyridine (DHP)-type calcium (Ca2+ channel antagonist (-)-[3H]-azidopine, a specific photoaffinity probe for Ca2+ channels, we tested the hypothesis of the existence of a separate subsite in the DHP receptor region on native polarized, stimulated depolarized and UV irradiated green monkey renal (GMR) cells preincubated with selected DHPs. Our results demonstrate that specific binding of (-)-[3H]-azidopine on GMR cells is of high affinity, stereoselective and dependent mainly on the inactivation of the membrane bound Ca2+ channel. Preincubation of the GMR cells with the DHP Ca2+ channel agonist BAY-K-8644 significantly reduced specific photolabeling. The site-directed free radicals generated after UV irradiation in DHP-preincubated renal cells inactivated Ca2+ channels and did not significantly affect the specific photoincorporation of (-)-[3H]-azidopine. (+)-Niguldipine, a DHP with the voluminous substituent on the DHP ring, significantly reduced the photolabeling. Low affinity labeling was partially prevented in (+)-nimodipine and (+)-niguldipine preincubated photoirradiated cells. The results strongly support the existence of central and peripheral subsites of the DHP region on GMR cells, with the former incorporating on photoactivation the intrinsically photoactive DHPs and with the latter labeled with a side chain bearing nitrene-generating photoreactive group, the photoaffinity probe, (-)-[3H]-azidopine.
Asunto(s)
Marcadores de Afinidad/metabolismo , Azidas/metabolismo , Dihidropiridinas/metabolismo , Riñón/metabolismo , Proteínas Musculares/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Marcadores de Afinidad/química , Animales , Azidas/química , Unión Competitiva/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L , Línea Celular , Chlorocebus aethiops , Dihidropiridinas/química , Dihidropiridinas/farmacología , Radicales Libres , Riñón/citología , Riñón/efectos de los fármacos , Riñón/efectos de la radiación , Cinética , Ligandos , Potenciales de la Membrana , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/efectos de la radiación , Nimodipina/farmacología , Rayos UltravioletaRESUMEN
The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.
Asunto(s)
Dihidropiridinas/metabolismo , Riñón/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Marcadores de Afinidad , Animales , Azidas , Sitios de Unión , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Células Cultivadas , Chlorocebus aethiops , Dihidropiridinas/farmacología , Riñón/efectos de los fármacos , Cinética , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/metabolismo , Nisoldipino/farmacología , Nitrendipino/farmacología , Propanolaminas/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMEN
The effect of the new beta-adrenolytic drug Bl 343 Ac (propyl-3-acetyl-4-[(2-hydroxy-3-isopropylamino) propoxy] carbanylate hydrochloride) was tested in preparations of the guinea-pig atrium and compared with the effects of propranolol and metipranolol. BL 343 Ac has an affinity to beta-adrenergic receptors, expressed as the pA2 value, which is comparable to those of propranolol and metipranolol. To achieve the slope of the Schild plot not different from unity, it was necessary to prolong incubation of the drug with the tissue to 75 min. At the concentrations of 10(-5) and 10(-4) mol/l and higher, BL 343 Ac was found to have negative chronotropic and inotropic effects, respectively. IN atria isolated from guinea-pigs and rats premedicated with reserpine, application of 10(-8) and 10(-7) mol/l of Bl 343 Ac exhibited a slightly stimulatory effect on the chronotropy. A positive inotropic effect of Bl 343 Ac was observed on the left guinea-pig atria after prolongation of the incubation time of the drug with the tissue up to 75 min. This inotropic effect of Bl 343 Ac may be associated with an increased concentration of cyclic adenosine monophosphate (cAMP), observed after 1-h incubation with the drug (from 0.78 +/- 0.05 to 1.13 +/- 0.09 pmol/mg w.w. cAMP). Bl 343 Ac, similarly to propranolol and metipranolol, prolonged the refractory period in the atrial preparations starting with the concentration of 10(-6) mol/l. In the ventricular preparations, Bl 343 Ac shortened the refractory period up to the concentration of 10(-5) mol/l, similarly to propranolol.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Carbamatos/farmacología , Corazón/efectos de los fármacos , Animales , Gatos , AMP Cíclico/metabolismo , Femenino , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Isoproterenol/farmacología , Masculino , Metipranolol/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Músculos Papilares/efectos de los fármacos , Propranolol/farmacología , Ratas , Ratas Wistar , Periodo Refractario Electrofisiológico/efectos de los fármacos , Reserpina/farmacología , Estimulación QuímicaRESUMEN
Two phenylalkylamine Ca2+ channel ligands, (+/-)-[3H]verapamil ((+/-)-[3H]V) (-)-[3H]desmethoxyverapamil ((-)-[3H]DV), were employed in whole cell binding assays to characterize the specific high affinity binding sites on Ca2+ channels, their cooperativity and modulations induced on cultured human embryonal vascular smooth muscle preparation (VSM) by: 1) Beta-adrenergic stimulation of the cell, 2) exposure to high K+ concentration, 3) exposure to high concentration of Mg2+ ions, 4) the presence of a benzothiazepine Ca2+ channel antagonist and modulator d-cis-diltiazem, and 5) guanylylimidodiphosphate. The total amounts of specific (+/-)-[3H]V and (-)-[3H]DV binding sites present on VSM cells increased significantly after beta-adrenergic receptor activation, following cell membrane depolarization induced by high concentrations of K+, in the presence of Ca2+ chelator Na3EDTA, and after incubation of VSM cells with a benzothiazine-type Ca2+ channel blocker d-cis-diltiazem. A marked reduction of (-)-[3H]DV binding was observed after permanent G-protein activation by a nonhydrolyzable analog of guanylylimidodiphosphate, after incubation of the cells with norepinephrine, and after incubation of VSM cells with millimolar concentration of Mg2+. The results suggest the existence of multiple modulations of specific (-)-[3H]DV binding sites on Ca2+ channel corresponding to the way of activation of the cell and also to the immediate "state" of the membrane bound Ca2+ channels present on VSM cells, the positive heterotropic interaction after beta-adrenergic stimulation, the homotropic positive allosteric interaction induced by d-cis-diltiazem and pure noncompetitive inhibition induced by guanylylimidodiphosphate. The presence of high concentrations of Mg2+ inhibited whereas the presence of Ca2+ chelator, of ethylenediamine-tetraacetic acid sodium salt, significantly increased the total number of specific high affinity (-)-[3H]DV binding sites on VSM cells.