RESUMEN
OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.
Asunto(s)
Glucosa/metabolismo , Isoenzimas/metabolismo , Proteínas Musculares , Miocardio/metabolismo , Obesidad/metabolismo , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Transporte Biológico , Fenómenos Biomecánicos , Peso Corporal , Radioisótopos de Carbono , Membrana Celular/enzimología , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Femenino , Transportador de Glucosa de Tipo 4 , Glucólisis , Corazón/fisiología , Cinética , Proteínas de Transporte de Monosacáridos/metabolismo , Ratas , Ratas Zucker , TritioRESUMEN
BACKGROUND: In the obesity model of the Zucker rat, myocardial protein kinase C (PKC) activation by phorbol ester is impaired. The influence of obesity on myocardial cell signaling was investigated by studying the activation of PKC isozymes and MAP kinases (MAPK) p38 and p42/44 as well as the induction of ANP mRNA. METHODS: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) at different time periods. Immunodetectable PKC isozymes, phosphorylated-MAPK, and ANP mRNA were determined by Western and Northern blots, respectively. RESULTS: PMA promoted a marked transient translocation of ventricular PKCalpha from the cytosol to the membranes within 10 minutes in lean rats, whereas it had a much weaker effect in obese rats. Moreover, PMA induced a significant activation of PKCdelta in lean but not in obese rat hearts. After PKC activation, increases in phosphorylation levels of myocardial p38 and p42 MAPK were approximately 3-fold higher in lean rats than in obese animals. Concerning the induction of ANP, PMA transiently tripled ANP mRNA within 60 minutes in lean but not in obese rats. CONCLUSIONS: In the genetically obese Zucker rat, the myocardial signal transduction cascade PKC-MAPK-ANP mRNA seems to be markedly impaired. It can be speculated that this abnormal cardiac cell signaling in obese rats reflects an early phase in the cardiac pathogenesis accompanying obesity.
Asunto(s)
Factor Natriurético Atrial/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , Obesidad/genética , Obesidad/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Femenino , Técnicas In Vitro , Proteína Quinasa C/metabolismo , Ratas , Ratas Zucker , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The heart-kidney-adrenal theme at the 42(nd) Journées Internationales d'Endocrinologie Clinique - Henri-Pierre Klotz held in Paris in April 1999 was chosen for at least two reasons. First, cardiovascular signs and symptoms are part of the most important and frequent endocrinopathies whose course is often characterized by cardiovascular complications. Secondly, the treatment of hypertension, heart failure and vascular nephropathies, as well as the prevention of atherosclerosis, call for homonal treatment or therapeutic intervention with agents acting upon endocrine systems that regulate cardiac, renal and vascular functions. The endocrinology specialist should therefore have full knowledge of the latest progress in the fields of the physiological and pathophysiological function of these systems, of the cellular mode of action of the hormones targeting the vardiovascular system, their genesis in classical endocrine organs, and also their local formation and action in tissues. Finally, it is important to be aware of the new therapeutic approaches opened by these recent developments.
Asunto(s)
Glándulas Suprarrenales/fisiopatología , Enfermedades del Sistema Endocrino/fisiopatología , Corazón/fisiopatología , Riñón/fisiopatología , Animales , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/fisiopatología , Enfermedades Cardiovasculares/terapia , Enfermedades del Sistema Endocrino/complicaciones , Enfermedades del Sistema Endocrino/terapia , Humanos , Transducción de SeñalRESUMEN
Epidermal growth factor (EGF) was tested for its ability to promote hypertrophic responses in neonatal rat ventricular cardiomyocytes. Exposure of these cells to 100 n m EGF for 2-18 h resulted in a time-dependent increase in protein synthesis reaching 174+/-18% of control values at 18 h. After 30 min stimulation, the mRNA levels of c-jun and c-fos were also increased 20- and 36-fold, respectively. We also investigated EGF-induced activation of Stat (signal transducers and activators of transcription) proteins as well as the possible interactions of this signaling pathway with the p38 and p42/44 MAP kinases cascades. EGF did not activate Stat1 and Stat3, but did induce a rapid and transient activation of Stat5, which corresponded mainly to Stat5b DNA-binding. The EGF-promoted Stat5 DNA-binding was decreased in a concentration-dependent manner by the p38 MAPK inhibitor SB 203580 (IC(50)=1.2 microm), whereas it was tripled by 50 micro m PD 98059, an inhibitor of the p42/44 MAPK cascade. This is the first demonstration that EGF increases protein synthesis and early response gene expression in cardiomyocytes, responses considered as markers of hypertrophy in these cells. The results further show that EGF activates Stat5, that this response requires p38 MAPK stimulation, and it is negatively modulated by p42/44 MAPK.
Asunto(s)
Cardiomegalia/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas de la Leche , Transactivadores/metabolismo , Animales , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Ventrículos Cardíacos/citología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Ratas Wistar , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Intraarticular therapy with corticosteroids can cause systemic effects such as decreased concentration of plasma cortisol, but whether this might place a patient at risk from stress induced acute adrenal failure is not known. We describe a patient who presented with lethargy, hyponatremia, and then with acute abdomen. The diagnosis of acute adrenal crisis was related to suppression of the hypothalamic-pituitary-adrenal axis by intraarticular use of corticosteroid. This was confirmed by a low basal cortisol concentration and by a short Synacthen test that elicited an increase in plasma cortisol concentration from 36 to 481 nmol/l. Within 24 h of receiving 37.5 mg of hydrocortisone, the patient rapidly improved.
Asunto(s)
Corticoesteroides/efectos adversos , Enfermedades de las Glándulas Suprarrenales/inducido químicamente , Condrocalcinosis/tratamiento farmacológico , Corticoesteroides/administración & dosificación , Enfermedades de las Glándulas Suprarrenales/fisiopatología , Anciano , Anciano de 80 o más Años , Condrocalcinosis/patología , Femenino , Humanos , Rodilla/patologíaRESUMEN
Incubation of spontaneously beating ventricular cardiomyocytes from neonatal rats with prostaglandin E(2) (0.1 microM) or forskolin (0.1 microM) simultaneously increased the rate of cellular contraction and atrial natriuretic peptide (ANP) secretion. Both responses were maximal within 10-20 min of application and were accompanied by three- to fourfold increases in cAMP formation. By contrast, a higher regimen of forskolin (10 microM) promoted a 20- to 30-fold increase in basal cAMP production, which was accompanied by the abolition of contractile activity and ANP release. Low regimens of forskolin (0.1 microM) doubled the occurrence of cytosolic Ca(2+) transients associated with monolayer contraction, whereas higher regimens of forskolin (10 microM) completely suppressed Ca(2+) transients. Moreover, in quiescent cultures that were pretreated with ryanodine, tetrodotoxin, nifedipine, or butanedione monoxime, prostaglandin E(2) (0.1 microM) and forskolin (0.1 microM) failed to elicit significant ANP secretion, suggesting that cAMP-elevating agents promote ANP secretion to a great extent via an increase in cellular contraction frequency in ventricular cardiomyocytes.
Asunto(s)
Factor Natriurético Atrial/metabolismo , AMP Cíclico/fisiología , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/biosíntesis , Citosol/metabolismo , Dinoprostona/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Concentración Osmolar , Ratas , Ratas WistarRESUMEN
Microdomains of high cytosolic free Ca(2+) concentration in the proximity of mitochondria might have an important role in the stimulation of steroidogenesis in bovine adrenal glomerulosa cells. In the present study we have investigated local changes of free Ca(2+) concentration near the outer mitochondrial membrane ([Ca(2+)](om)) under stimulation with angiotensin II (Ang II) and K(+). Glomerulosa cells in primary culture were transfected with a recombinant cDNA encoding the N-terminal region of the human translocase protein 20 of the outer mitochondrial membrane, in frame with the Ca(2+)-sensitive photoprotein aequorin. This chimaeric aequorin (TomAeq) was associated with mitochondria-enriched subcellular fractions of transfected COS-7 cells and was susceptible to proteinase K, showing that it was targeted to the outer mitochondrial membrane, facing the cytosolic space. In bovine adrenal glomerulosa cells transfected with TomAeq cDNA, Ang II induced a transient [Ca(2+)](om) peak reaching 1.42+/-0.28 microM, which decreased immediately to the basal resting value. The peak response to Ang II was strikingly lower than the peak response of mitochondrial free Ca(2+) concentration, which increased to 5.4+/-1.2 microM. The smaller response of [Ca(2+)](om) to Ang II compared with the elevated matrix response did not result from buffering effects of the organelle, from altered mechanisms of intramitochondrial Ca(2+) transport or from differences in the affinity of the chimaeric aequorins for Ca(2+). This approach has allowed us to follow perimitochondrial Ca(2+) homeostasis in bovine glomerulosa cells under stimulation with Ca(2+)-mobilizing agonists and to reveal a strong gradient of Ca(2+) concentration between the mitochondrial matrix and the immediate environment of the organelle.
Asunto(s)
Aequorina/metabolismo , Calcio/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Zona Glomerular/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Angiotensina II/farmacología , Animales , Células COS , Bovinos , Humanos , Membranas Intracelulares/efectos de los fármacos , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Potasio/farmacología , Proteínas Recombinantes/metabolismo , Zona Glomerular/citología , Zona Glomerular/efectos de los fármacosAsunto(s)
Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/tratamiento farmacológico , Diagnóstico Diferencial , Humanos , Hiperaldosteronismo/epidemiología , Hiperaldosteronismo/metabolismo , Tamizaje Masivo/métodos , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , PrevalenciaRESUMEN
In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.
Asunto(s)
Angiotensina II/metabolismo , Canales de Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología , Aldosterona/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L , Bovinos , Células Cultivadas , Losartán/farmacología , Técnicas de Placa-Clamp , Potasio/farmacología , Zona GlomerularRESUMEN
In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Canales de Calcio/metabolismo , Receptores de Angiotensina/metabolismo , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Angiotensina II/farmacología , Animales , Calcio/metabolismo , Canales de Calcio/clasificación , Canales de Calcio/efectos de los fármacos , Bovinos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Genisteína/farmacología , Técnicas In Vitro , Modelos Biológicos , Potasio/farmacología , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.
Asunto(s)
Aldosterona/biosíntesis , Bencimidazoles/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Tetrahidronaftalenos/farmacología , Zona Glomerular/efectos de los fármacos , Animales , Calcio/antagonistas & inhibidores , Bovinos , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Mibefradil , Nicardipino/farmacología , Técnicas de Placa-Clamp , Pimozida/farmacología , Cloruro de Potasio/farmacología , Transducción de Señal/efectos de los fármacos , Zona Glomerular/metabolismoRESUMEN
In salt-losing congenital adrenal hyperplasia (CAH), continuous therapy with glucocorticoids and 9 alpha-fluorohydrocortisone (9 alpha-F) remains the golden rule. Previous reports showed a growth promoting effect of 9 alpha-F therapy. In addition, 9 alpha-F seemed to have a negligible glucocorticoid action. To confirm these facts, we analyzed the clinical data and the biological markers of control of therapy in two groups of patients with salt-losing CAH aged from 2 to 12 years: group I: before (time 0) and 6 months after the increase in 9 alpha-F dosage (time +6); group II: at time 0 and time +6 but without change in 9 alpha-F dosage. Groups were similar in terms of mean age, bone age and hydrocortisone dose. The mean dose of 9 alpha-F was 68.2 +/- 5.0 micrograms/m2/d at time 0 and was increased to 98.6 +/- 7.7 micrograms/m2/d at time +6 in group I; it remained similar in group II. In group I, height velocity decreased significantly from 8.1 +/- 0.6 at time 0 to 6.3 +/- 0.3 cm/yr at time +6 (p < 0.01) while in group II there was no significant change. In group I, plasma renin activity decreased from 10.4 +/- 1.6 at time 0 to 3.9 +/- 1.1 ng/ml/h at time +6 (p < 0.005) and showed no change in group II. These preliminary results suggest that careful monitoring of 9 alpha-F is essential to control a proper growth rate.
Asunto(s)
Hiperplasia Suprarrenal Congénita/tratamiento farmacológico , Hiperplasia Suprarrenal Congénita/metabolismo , Monitoreo de Drogas , Fludrocortisona/administración & dosificación , Mineralocorticoides/administración & dosificación , Cloruro de Sodio/metabolismo , Hiperplasia Suprarrenal Congénita/patología , Estatura/efectos de los fármacos , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Fludrocortisona/uso terapéutico , Hormonas/sangre , Humanos , Masculino , Mineralocorticoides/uso terapéutico , Resultado del TratamientoRESUMEN
World-wide, the incidence of amiodarone-induced thyroid dysfunction is estimated to be between 2% and 24%. More than 50% of patients who receive long-term amiodarone have abnormal results on thyroid function tests. However, most of these patients are euthyroid. In a euthyroid amiodarone-treated patient, an increase in serum thyroxine levels (total and free) and in serum reverse T3 levels and a decrease in serum T3 levels (total and free), are observed. TSH levels are within the normal range with chronic treatment (more than 3 months). The aim of this study was to identify the frequency of amiodarone prescription by cardiologists, to learn about their clinical practice in screening for thyroid dysfunction, and to formulate guidelines for clinical investigation of thyroid function before and during amiodarone treatment. A questionnaire was sent to 27 cardiologists in private practice in the Geneva area and to 12 cardiologists in Geneva University Hospital (19 and 7 questionnaires returned respectively). Cardiologists in private practice prescribe amiodarone quite frequently (once to twice per month and more). Clinical investigation prior to treatment involves history (95%), clinical examination (63%) or thyroid function tests (37% routinely, 16% where there is clinical suspicion of thyroid dysfunction). Thyroid function is investigated by determination of TSH (100%), free T4 (68%), free T3 (50%), total T3 (32%) or total T4 (26%). Antithyroid antibodies are measured only by 10% of the cardiologists in private practice. Cardiologists would restrict amiodarone prescription in cases of clinical hyperthyroidism (90%), clinical hypothyroidism (74%), in the presence of goitre in a hyper- or hypothyroid patient (79 and 63% respectively), and if antithyroid antibodies were positive (32%). During amiodarone treatment 84% of cardiologists in private practice repeat thyroid function tests occasionally and 16% only in the presence of clinical signs. Hypothyroidism is a reason for withdrawing amiodarone treatment for 58% of the cardiologists and 37% would prescribe substitution. Hyperthyroidism is a reason for immediately suspending treatment for 90% of the cardiologists, only 5% would continue and 16% would prescribe antithyroid drugs. In conclusion, there is considerable divergence of opinion among cardiologists concerning investigation and interpretation of thyroid function before and during amiodarone treatment. Simple and practicable guidelines are needed.
Asunto(s)
Amiodarona/uso terapéutico , Antiarrítmicos/uso terapéutico , Hipertiroidismo/inducido químicamente , Infarto del Miocardio/tratamiento farmacológico , Taquicardia Supraventricular/tratamiento farmacológico , Taquicardia Ventricular/tratamiento farmacológico , Glándula Tiroides/efectos de los fármacos , Amiodarona/efectos adversos , Antiarrítmicos/efectos adversos , Antiarrítmicos/farmacocinética , Femenino , Humanos , Hipertiroidismo/prevención & control , Masculino , Pruebas de Función de la Tiroides , Tirotropina/sangre , Tiroxina/sangreRESUMEN
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.
Asunto(s)
Mutación de Línea Germinal , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasia Endocrina Múltiple/genética , Mutación , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas , Sustitución de Aminoácidos , Exones , Femenino , Tamización de Portadores Genéticos , Humanos , Intrones , Masculino , Neoplasia Endocrina Múltiple/clasificación , Neoplasia Endocrina Múltiple Tipo 1/clasificación , Mutación Missense , Linaje , Mutación Puntual , Eliminación de SecuenciaRESUMEN
Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.
Asunto(s)
Factor Natriurético Atrial/farmacología , Calcio/farmacología , Fosfoproteínas/genética , Transcripción Genética/efectos de los fármacos , Zona Glomerular/metabolismo , Aldosterona/biosíntesis , Angiotensina II/farmacología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Colesterol/metabolismo , Femenino , Mitocondrias/metabolismo , Fosfoproteínas/biosíntesis , Pregnenolona/biosíntesis , ARN Mensajero/metabolismoRESUMEN
We studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in epidermal growth factor (EGF)-induced prostacyclin (PGI2) production in cultured, spontaneously-beating neonatal ventricular rat cardiomyocytes. To this purpose, the effect of EGF on cardiomyocyte MAPK phosphorylation, MAPK activity and PGI2-production were investigated, and compared to those induced by the PKC activator 4 beta phorbol 12-myristate 13-acetate (PMA). Both EGF (0.1 microM) and PMA (0.1 microM) induced the rapid and reversible phosphorylation of 42 KDa-MAPK in ventricular cardiomyocytes, responses that were accompanied by transient increases in MAPK activity (190-230% of control values within 5 min), and two- to three-fold increases in PGI2 formation. The tyrosine kinase inhibitors lavendustin (1 microM) and genistein (10 microM) strongly inhibited EGF-induced MAPK activation and PGI2-formation, but had no effect on PMA-stimulated responses. Experiments with the PKC inhibitor CGP 41251 (1 microM) or with PKC-downregulated cells demonstrated that in contrast to the PMA-stimulated responses, EGF-induced MAPK activation and PGI2-production were PKC-independent processes. Investigating the role of MAPK in EGF- and in PMA-promoted PGI2-formation, we found that the MAPK-inhibitor 6-thioguanine (500 microM), as well as the MAPK-kinase-inhibitor PD98059 (50 microM) abolished both EGF- and PMA-stimulated PGI2-production in cardiomyocytes. Our results indicate that MAPK-activation is at the basis of both growth factor receptor and PKC-dependent eicosanoid-formation in ventricular cardiomyocytes, where EGF-induced prostaglandin-production takes place via a PKC-independent pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Factor de Crecimiento Epidérmico/farmacología , Epoprostenol/biosíntesis , Ventrículos Cardíacos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Análisis de Varianza , Animales , Células Cultivadas , Activación Enzimática , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Fosforilación , Ratas , Ratas WistarRESUMEN
Angiotensin II (AngII) plays a crucial role in the control of aldosterone biosynthesis in adrenal glomerulosa cells through the stimulation of two distinct Ca2+ entry pathways: (1) opening of voltage-operated calcium channels, and (2) activation of a capacitative Ca2+ entry that is dependent on calcium release from intracellular pools. Adrenocorticotrophic hormone (ACTH), on the other hand, a major hormonal regulator of steroidogenesis, induces an increase in intracellular cAMP through the activation of a G-protein-coupled adenylyl cyclase. Recent studies have demonstrated that the rise in cAMP induced by ACTH can be potentiated by AngII in bovine glomerulosa cells. The aim of the present study was to investigate the mechanism of AngII action on ACTH-induced cAMP production. In primary cultures of bovine glomerulosa cells, we found that AngII (100 nM), which had no effect by itself on cAMP production, significantly potentiated maximal ACTH-induced cAMP formation in the presence of extracellular calcium (1.2 mM). In contrast, in the absence of extracellular calcium, AngII did not affect ACTH-induced cAMP production. These results suggest that calcium entry into the cell plays an important role in the activation of the cyclase by AngII. The inhibition of voltage-operated calcium channels by nicardipine, a dihydropyridine calcium antagonist blocking both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels, did not significantly affect the potentiating effect of AngII. Moreover, the cAMP response to ACTH was insensitive to activation of these Ca2+ channels induced by potassium ions and, even when cytosolic free-calcium concentration ([Ca2+]c) was kept elevated with the Ca2+ ionophore, ionomycin, no stimulation of adenylyl cyclase was observed at concentrations of [Ca2+]c up to 640 nM. In contrast, thapsigargin, an activator of capacitative Ca2+ influx, mimicked the potentiating effect of AngII on ACTH-induced cAMP formation. In agreement with the characteristics of cAMP modulation by Ca2+ in these cells, the presence of type III adenylyl cyclase was observed by immunodetection in bovine glomerulosa cell membranes. In conclusion, these data suggest a tight coupling between the capacitative Ca2+ influx induced upon stimulation by either AngII or thapsigargin and a calcium-sensitive isoform of adenylyl cyclase, probably type III, in bovine glomerulosa cells.
Asunto(s)
Adenilil Ciclasas/metabolismo , Hormona Adrenocorticotrópica/administración & dosificación , Angiotensina II/administración & dosificación , Calcio/metabolismo , AMP Cíclico/biosíntesis , Zona Glomerular/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Bovinos , Células Cultivadas , Sinergismo Farmacológico , Ionomicina/farmacología , Ionóforos/farmacología , Isoenzimas/metabolismo , Potenciales de la Membrana , Tapsigargina/farmacologíaRESUMEN
The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.
Asunto(s)
Factor Natriurético Atrial/biosíntesis , Calcio/metabolismo , AMP Cíclico/metabolismo , Endotelina-1/farmacología , Corazón/efectos de los fármacos , Miocardio/metabolismo , Alcaloides , Animales , Animales Recién Nacidos , Benzofenantridinas , Calcio/farmacología , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Epoprostenol/biosíntesis , Ventrículos Cardíacos , Cinética , Miocardio/citología , Naftalenos/farmacología , Nifedipino/farmacología , Fenantridinas/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/biosíntesis , Receptores de Endotelina/fisiología , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Tionucleótidos/farmacologíaRESUMEN
OBJECTIVE: To look for a possible relation between the occurrence of heterotopic ossification (HO) and the modifications of the 24-hour prostaglandin E2 (PGE2) urinary excretion. DESIGN: A 5-year prospective study to determine the 24-hour urinary excretion of PGE2 by radioimmunoassay with specific antisera not cross-reacting with TXA2, TXB2, 15-keto-PGE2 alpha, PGI2, 6-keto-PGF1 alpha. SETTING: The laboratory of a division of endocrinology and diabetology of a university hospital. PATIENTS: Of 262 acute spinal cord injury patients screened, 44 were eligible for the study. INTERVENTIONS: Serial diagnostic quantitative bone scannings with technetium 99m Tc methylene diphosphate (99mTc-MDP) and therapeutic assessment of radiotherapy and indomethacin. MEAN OUTCOME MEASURE: Hypothetical increase of PGE2 before and during HO formation. RESULTS: Of 44 patients, 8 developed an HO (18.8%) with concomitant marked increase of the PGE2 excretion for as long as the HO had not reached maturity. The results of the radiotherapy were inconclusive. Indomethacin was shown to be efficacious in holding back or slowing down the HO evolution. CONCLUSIONS: Measurement of the 24-hour PGE2 urinary excretion appears to be a valuable indicator in the early diagnosis of HO. Indomethacin should be considered as an alternative to other existing therapies.