RESUMO
OBJECTIVES: To compare uterine artery pulsatility index (PI) obtained at 11 + 0 to 13 + 6 weeks of gestation in singleton and twin pregnancies and to evaluate changes in PI values of twin pregnancies developing pre-eclampsia (PE) or small-for-gestational age (SGA) of either one or both fetuses. METHODS: Uterine artery PI was measured in 421 twin pregnancies (384 dichorionic and 37 monochorionic) and in 500 singleton pregnancies. The measured mean and lowest uterine artery PI values were converted to multiples of the expected normal median (MoM) after correction for maternal body mass index, ethnicity and gestational age. The median PI-MoM values of twins were compared with those of singleton pregnancies. In twin pregnancies, PI-MoM values were analyzed according to chorionicity, development of early-onset (< 34 weeks) or late-onset (≥ 34 weeks) PE and SGA of one or both twins. RESULTS: Uterine artery PI-MoM was significantly lower in twin compared with singleton pregnancies (mean K = 174.31, P < 0.0001, lowest K = 139.27, P < 0.0001). However, there were no significant differences in the uterine artery PI-MoM values between monochorionic and dichorionic twins. The uterine artery PI in twin pregnancies that developed early-onset PE (P < 0.001) and SGA of both twins (P < 0.05) was higher than the uterine artery PI in uncomplicated twin pregnancies, whereas no differences were found for late PE or SGA of one twin. CONCLUSIONS: First-trimester placental impedance to flow, as assessed by uterine artery Doppler examination, is reduced in twin pregnancies, with no differences related to chorionicity. The relative increase of uterine artery PI found in twin pregnancies that developed early PE and SGA of both twins suggests that first-trimester uterine artery assessment may be useful in identifying such complications.
Assuntos
Recém-Nascido Pequeno para a Idade Gestacional/fisiologia , Pré-Eclâmpsia/fisiopatologia , Gravidez de Gêmeos/fisiologia , Artéria Uterina/fisiologia , Adolescente , Adulto , Velocidade do Fluxo Sanguíneo/fisiologia , Estudos de Casos e Controles , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Retardo do Crescimento Fetal/fisiopatologia , Idade Gestacional , Humanos , Idade Materna , Pré-Eclâmpsia/diagnóstico por imagem , Gravidez , Resultado da Gravidez , Primeiro Trimestre da Gravidez , Fluxo Pulsátil , Gêmeos Dizigóticos , Gêmeos Monozigóticos , Ultrassonografia Doppler , Ultrassonografia Pré-Natal , Adulto JovemRESUMO
Psoriasis is a chronic inflammatory skin disease affecting approximately 2-3 percent of the world population; it is characterised by hyperproliferation and hyperplasia of the superficial layers of the epidermis. Inappropriate signals released by the immune system determine an altered keratinocyte differentiation, resulting in the formation of desquamating, thickened, inflamed and erythematous plaques. The aim of this investigation was to study the pharmacological activity and safety of three low dose cytokines, Guna-Interleukin 4, Guna-Interleukin 10 and Guna-Interleukin 11 at the concentration of 10 fg/ml in patients affected by moderate to slight psoriasis vulgaris. The multicenter, double-blind, randomized, placebo-controlled clinical trial involved 48 patients who were enrolled and followed up according to a 8-month experimental project. All patients received, according to a cross-over model, either the experimental treatment or placebo, alternatively. Globally, in the 41 evaluated patients it was observed a PASI significant reduction (Friedman test: p=0.00960). The DLQI too decreased significantly in all subjects compared to baseline (Friedman test: p=0.00007). The safety of the treatment with three low dose cytokines administered simultaneously was proved; no adverse event was reported during the whole trial.
Assuntos
Interleucina-10/uso terapêutico , Interleucina-11/uso terapêutico , Interleucina-4/uso terapêutico , Psoríase/tratamento farmacológico , Adulto , Idoso , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Interleucina-10/efeitos adversos , Interleucina-11/efeitos adversos , Interleucina-4/efeitos adversos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To describe a novel algorithm, based on the new display technology 'OmniView', developed to visualize diagnostic sagittal and coronal planes of the fetal brain from volumes obtained by three-dimensional (3D) ultrasonography. METHODS: We developed an algorithm to image standard neurosonographic planes by drawing dissecting lines through the axial transventricular view of 3D volume datasets acquired transabdominally. The algorithm was tested on 106 normal fetuses at 18-24 weeks of gestation and the visualization rates of brain diagnostic planes were evaluated by two independent reviewers. The algorithm was also applied to nine cases with proven brain defects. RESULTS: The two reviewers, using the algorithm on normal fetuses, found satisfactory images with visualization rates ranging between 71.7% and 96.2% for sagittal planes and between 76.4% and 90.6% for coronal planes. The agreement rate between the two reviewers, as expressed by Cohen's kappa coefficient, was > 0.93 for sagittal planes and > 0.89 for coronal planes. All nine abnormal volumes were identified by a single observer from among a series including normal brains, and eight of these nine cases were diagnosed correctly. CONCLUSIONS: This novel algorithm can be used to visualize standard sagittal and coronal planes in the fetal brain. This approach may simplify the examination of the fetal brain and reduce dependency of success on operator skill.
Assuntos
Algoritmos , Encéfalo/patologia , Doenças do Sistema Nervoso Central/diagnóstico por imagem , Doenças do Sistema Nervoso Central/patologia , Imageamento Tridimensional/métodos , Ultrassonografia Pré-Natal/métodos , Adolescente , Adulto , Encéfalo/anatomia & histologia , Encéfalo/embriologia , Feminino , Humanos , Aumento da Imagem , Pessoa de Meia-Idade , Gravidez , Segundo Trimestre da Gravidez , Adulto JovemRESUMO
OBJECTIVE: To investigate umbilical vein blood flow (UVBF) during the first trimester in pregnancies with low serum pregnancy-associated plasma protein-A (PAPP-A) levels and to relate umbilical vein (UV) diameter, time-averaged maximum velocity (TAMXV) and UVBF values to the subsequent development of fetal intrauterine growth restriction (IUGR). METHODS: UVBF assessment was performed at 11 + 0 to 13 + 6 weeks' gestation in 102 singleton pregnancies with PAPP-A concentrations of < 0.3 multiples of the median. UV diameter, UV-TAMXV and UVBF were calculated and analyzed in relation to pregnancy outcome. RESULTS: Pregnancy outcomes were: 51 pregnancies with birth weight ≥ 10(th) centile (Group A), 30 pregnancies with birth weight < 10(th) centile with normal Doppler in the umbilical artery throughout gestation (Group B) and 21 pregnancies with birth weight < 10(th) centile and abnormal umbilical artery Doppler later in gestation (Group C). No differences were found in PAPP-A levels between groups. Group C fetuses exhibited significantly lower values of UV-TAMXV (z-score - 1.99 SDs, t = 8.527, P ≤ 0.0001) and UVBF (z-score - 0.97 SDs, t = 7.420, P ≤ 0.0001) in comparison with normal reference ranges, while no differences were found in Groups A or B. CONCLUSIONS: Decreased UV-TAMXV and UVBF at 11 + 0 to 13 + 6 weeks' gestation identify fetuses at risk of developing IUGR among pregnancies with low levels of PAPP-A.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Retardo do Crescimento Fetal/fisiopatologia , Proteína Plasmática A Associada à Gravidez/metabolismo , Veias Umbilicais/fisiopatologia , Adolescente , Adulto , Biomarcadores/sangue , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/diagnóstico por imagem , Humanos , Recém-Nascido , Gravidez , Resultado da Gravidez , Primeiro Trimestre da Gravidez , Proteína Plasmática A Associada à Gravidez/análise , Ultrassonografia Doppler , Ultrassonografia Pré-Natal , Veias Umbilicais/irrigação sanguínea , Veias Umbilicais/diagnóstico por imagem , Adulto JovemRESUMO
OBJECTIVE: A low combined cardiac output (CCO) to the placenta (placenta/CCO fraction) has been reported in growth-restricted (IUGR) fetuses, but the temporal sequence of these modifications in relation to other changes in the fetal circulation is unknown. The aim of this study was to evaluate the placenta/CCO fraction in relation to other hemodynamic changes in fetuses at risk of developing IUGR. METHODS: We studied 340 singleton nulliparous pregnancies characterized at 20-24 weeks by abnormal uterine artery pulsatility index (PI) values (> 95(th) centile). At this gestational age we measured fetal biometry and Doppler waveforms from the umbilical artery (UA), middle cerebral artery (MCA), ductus venosus (DV), umbilical vein (UV) and outflow tracts of both ventricles. The diameters of the semilunar valves and UV were measured and CCO (left cardiac + right cardiac outputs) and UV blood flow were calculated. The placenta/CCO fraction was calculated as UV flow as a percentage of CCO. RESULTS: There were 283 pregnancies with birth weight >or= 10(th) centile and normal UA-PI throughout gestation (Group A), 34 with birth weight < 10(th) centile and normal UA-PI throughout gestation (Group B) and 23 with birth weight < 10(th) centile and abnormal UA-PI developing later in gestation (Group C). At 20-24 weeks there were no differences among the three groups in fetal biometric parameters, PI values from the UA, MCA and DV, and CCO. UV flow and placenta/CCO fraction were significantly lower in Group C compared with Group A (UV flow delta value = - 1.439, P < 0.0001; placenta/CCO fraction delta value = - 1.74, P < 0.0001) but not in Group B. CONCLUSIONS: Our data suggest that, in fetuses developing IUGR secondary to placental compromise, UV flow and placental/CCO fraction are already reduced by 20-24 weeks, and that this reduction occurs earlier than do modifications in fetal size and arterial and venous PI values.
Assuntos
Baixo Débito Cardíaco/diagnóstico por imagem , Retardo do Crescimento Fetal/etiologia , Placenta/irrigação sanguínea , Ultrassonografia Doppler de Pulso/métodos , Ultrassonografia Pré-Natal/métodos , Útero/irrigação sanguínea , Adulto , Artérias/diagnóstico por imagem , Artérias/fisiopatologia , Feminino , Idade Gestacional , Frequência Cardíaca Fetal/fisiologia , Hemodinâmica , Humanos , Recém-Nascido , Gravidez , Fluxo Pulsátil/fisiologia , Estudos RetrospectivosRESUMO
The zona glomerulosa cell of the adrenal cortex produces mineralocorticoids in response to physiological stimuli (angiotensin II and extracellular K(+)) activating the Ca(2+) messenger system. The mechanisms underlying the generation of the Ca(2+) signal have been analyzed extensively and recent developments have contributed to bridging the gap between intracellular signals and activation of the biological function. This article summarizes the current knowledge on the intracellular targets of the Ca(2+) messenger, obtained mainly in bovine glomerulosa cells. Ca(2+) appears to exert a dual effect, both at the intramitochondrial level and at the nuclear level, where it activates steroidogenic acute regulatory protein (StAR) gene transcription.
RESUMO
Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (approximately 8 s) mobilization of an ionomycin-sensitive intracellular Ca2+ pool. A second component of Ca2+ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca2+ concentration ([Ca2+]i). Thereafter, influx of Ca2+ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca2+]i during the second phase of GnRH action. Addition of inositol 1,4,5-trisphosphate (IP3) to permeabilized pituitary cells resulted in a Ca2+ transient, released from a nonmitochondrial pool, which maintained ambient free Ca2+ concentration around 170 nM in an ATP-dependent mechanism. Successive stimulations of the cells with IP3 produced an attenuated response. Elevation of the gonadotroph [Ca2+]i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca2+ channels by the dihydropyridine Ca2+-agonist [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nM) response being reached by 10(-8) M of the drug, both [Ca2+]i elevation and GnRH-induced LH release were inhibited similarly (40-50%) by the dihydropyridine Ca2+-antagonist nifedipine. The results indicate that peak [Ca2+]i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP3 formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Citosol/metabolismo , Éteres/farmacologia , Exocitose , Feminino , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/farmacologia , Ionomicina , Cinética , Nifedipino/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
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.
Assuntos
Fator Natriurético Atrial/farmacologia , Cálcio/farmacologia , Fosfoproteínas/genética , Transcrição Gênica/efeitos dos fármacos , Zona Glomerulosa/metabolismo , Aldosterona/biossíntese , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Colesterol/metabolismo , Feminino , Mitocôndrias/metabolismo , Fosfoproteínas/biossíntese , Pregnenolona/biossíntese , RNA Mensageiro/metabolismoRESUMO
In the human premonocytic line U937, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) induces a functional NADPH oxidase, that is responsive to both phorbol esters and opsonized zymosan. The chemotactic peptide f-Met-Leu-Phe (fMLP) did not, however, induce superoxide generation by these cells. This was not due to the absence of receptors for fMLP. Although there was no significant binding of [3H]-fMLP to undifferentiated U937 cells, preincubation with 1,25-(OH)2D3 induced expression of specific and saturable binding sites. Moreover, fMLP induced a rapid and reversible rise in cytosolic free Ca2+ concentration ([Ca2+]i) in 1,25-(OH)2D3-treated U937 cells, but not in control or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3)-treated cells. This [Ca2+]i response was dependent on concentrations of both fMLP and 1,25-(OH)2D3 and was observed at physiologic concentrations of the hormone (approximately 25 pM). The rise in [Ca2+]i induced by fMLP in 1,25-(OH)2D3-treated U937 cells was blocked by pertussis toxin and presumably mediated by inositol (1,4,5)-trisphosphate generation. These results indicate that in U937 cells differentiated with 1,25-(OH)2D3, inositol phosphate-mediated [Ca2+]i responses to fMLP are uncoupled from NADPH oxidase activation.
Assuntos
Calcitriol/farmacologia , Monócitos/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/biossíntese , Monócitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Superóxidos/biossínteseRESUMO
The adipocyte-derived hormone leptin is a central modulator of food intake, metabolism and neuroendocrine functions. It is also involved in a physiological loop linking the activity of the hypothalamo-pituitary-adrenal axis and adipose tissue. At the adrenal level, leptin has been shown to antagonize the effects of ACTH on glucocorticoid biosynthesis by decreasing the expression of various enzymes of the steroid biosynthetic pathway. The steroidogenic acute regulatory protein regulates cholesterol delivery to the P450(scc) enzyme, a process that is rate limiting in steroid hormone biosynthesis. We have demonstrated here that leptin significantly inhibits the expression of steroidogenic acute regulatory protein in primary cultures of rat adrenocortical cells. This inhibition was observed at both the protein and mRNA levels. In contrast, leptin was not found to interfere with the expression of the cytosolic enzyme cholesterol ester hydrolase or with that of the mitochondrial enzyme P450(scc). In addition, we observed the anticipated stimulation of cAMP production by ACTH in the presence of leptin, suggesting that it does not interfere with intracellular ACTH signaling. In summary, our data provide evidence that the interplay existing between leptin and ACTH in vivo is mediated at least partially via a direct and opposite modulation of steroidogenic acute regulatory protein, a key factor in the adrenal steroid biosynthetic pathway. This effect of leptin could also be relevant to other steroidogenic tissues.
Assuntos
Glucocorticoides/antagonistas & inibidores , Leptina/farmacologia , Fosfoproteínas/antagonistas & inibidores , Hormônio Adrenocorticotrópico/farmacologia , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , AMP Cíclico/metabolismo , Feminino , Fosfoproteínas/genética , Pregnenolona/antagonistas & inibidores , Pregnenolona/biossíntese , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Angiotensin-II (Ang-II), K+, and ACTH are important stimulators of aldosterone secretion that require Ca2+ influx to be active. However, Ang-II and K+ are linked to the Ca2+ messenger system, while ACTH is coupled to the cAMP pathway. Peripheral-type binding sites for benzodiazepines are particularly abundant in steroidogenic tissues and have been proposed to be involved in the steroidogenic action of ACTH in Y-1 adrenocortical cells. We report here that in adrenal glomerulosa cells, peripheral-type [4'-chlor-diazepam (CDZ), 1-(2-chlorophenyl)N-methyl-N-(1-methylpropyl)3-isoquinolinecarboxamid e (RP 52028), and flunitrazepam], but not a central-type (flumazenil) benzodiazepine reversibly abolished the stimulation of aldosterone output induced by Ang-II or K+, while they had no significant effect on basal aldosterone secretion. This inhibitory effect depended upon drug concentration (IC50 30 microM for CDZ) and affected the potencies of both stimulators, without altering their respective EC50 values. Similar results were obtained when aldosterone production was stimulated with ACTH, forskolin, or (Bu)2cAMP. Aldosterone production from exogenous 25-hydroxycholesterol or progesterone was partially inhibited by CDZ. In glomerulosa cells loaded with a fluorescent Ca2+ probe, benzodiazepines blocked Ca2+ influx triggered by K+ or Ang-II without affecting the release of Ca2+ from intracellular stores induced by Ang-II. T- and L-type Ca2+ channel activities, monitored with the patch-clamp technique, were both inhibited within the same range of concentrations as aldosterone synthesis and Ca2+ influx. These results indicate that in adrenal zona glomerulosa cells, peripheral-type benzodiazepines block Ca2+ influx through voltage-activated channels. The combined action of peripheral-type benzodiazepines on calcium influx and precursor conversion may be responsible for the observed inhibition of Ang-II-, K(+)-, or ACTH-induced aldosterone secretion.
Assuntos
Aldosterona/biossíntese , Benzodiazepinas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Zona Glomerulosa/metabolismo , Animais , Bário/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Concentração Osmolar , Zona Glomerulosa/citologiaRESUMO
Ovine adrenal fasciculata cells (OAC) responded to ACTH but were resistant to the steroidogenic action of angiotensin-II (A-II), while bovine adrenal fasciculata cells (BAC) responded to this hormone as well as to ACTH. However both cell types contained specific A-II binding sites (120,000 +/- 14,000 and 85,000 +/- 10,000 sites per cell for OAC and BAC, respectively) of similar high affinity [dissociation constant (KD) congruent to 2 x 10(-9) M]. Moreover, in both cell types, A-II receptors were coupled to intracellular effectors since A-II: 1) stimulated the accumulation of inositol phosphates, although the effects in BAC were higher than in OAC; 2) enhanced the influx and the efflux of 45Ca2+; 3) increased cytosolic free Ca2+ concentration ([Ca2+]i); 4) potentiated ACTH-induced cAMP production; and 5) induced A-II receptor loss. Both cell types appear to have an active protein kinase C since the phorbol ester 4 beta-phorbol 12-myristate-13-acetate potentiates ACTH-induced cAMP production and caused A-II receptor loss. In addition, 4 beta-phorbol 12-myristate-13-acetate and Ca2+ ionophore enhanced the steroid production by BAC but had no effect on OAC. These results indicated that the steroidogenic refractoriness of OAC to A-II might involve some step(s) beyond the initial activation of the two branches of the phosphoinositide pathway, activation of protein kinase C and increase of [Ca2+]i, and before conversion of cholesterol to pregnenolone.
Assuntos
Glândulas Suprarrenais/metabolismo , Angiotensina II/farmacologia , Receptores de Angiotensina/metabolismo , Esteroides/biossíntese , Glândulas Suprarrenais/citologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Ativação Enzimática , Fosfatos de Inositol/metabolismo , Membranas Intracelulares/metabolismo , Concentração Osmolar , Proteína Quinase C/metabolismo , Ovinos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Angiotensin-II (AngII)-induced Ca2+ influx in adrenal glomerulosa cells, a signal necessary for the stimulation of steroidogenesis by the hormone, is believed to involve two distinct mechanisms: 1) opening of voltage-operated Ca2+ channels, and 2) activation of a capacitative Ca2+ entry pathway that is dependent on calcium release from intracellular stores. Nicardipine, a dihydropyridine calcium antagonist, has been used to investigate the role of these Ca2+ entry mechanisms in the steroidogenic response to AngII. As demonstrated with the patch-clamp technique, micromolar concentrations of nicardipine completely blocked voltage-operated Ca2+ channel activity of both T- and L-types. This agent similarly inhibited the rise of cytosolic free calcium concentration induced by potassium, but did not significantly affect the response to thapsigargin, an activator of the capacitative pathway. Nicardipine reduced by only 22% the calcium influx stimulated by AngII, and the nicardipine-insensitive part of this response was abolished after exhausting the intracellular Ca2+ stores with thapsigargin. Similarly, aldosterone secretion induced by AngII was only partially inhibited (40%) by nicardipine at concentrations that completely abolished the steroidogenic response to potassium. Thapsigargin by itself was able to stimulate aldosterone production, an action highly potentiated by physiological concentrations of extracellular potassium. These data strongly suggest that the major part of the calcium influx response to AngII, leading to aldosterone formation, involves a capacitative calcium entry pathway activated by the release of calcium from intracellular stores. This mechanism of calcium influx could be responsible for some features of aldosterone response to the hormone, such as its poor sensitivity to dihydropyridines or its potentiation by potassium.
Assuntos
Aldosterona/biossíntese , Angiotensina II/farmacologia , Cálcio/metabolismo , Zona Glomerulosa/metabolismo , Aldosterona/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Bovinos , Células Cultivadas , Nicardipino/farmacologia , Potássio/farmacologia , Terpenos/farmacologia , Tapsigargina , Zona Glomerulosa/efeitos dos fármacosRESUMO
The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.6]GnRH. GnRH elevated [Ca2+]i only in gonadotroph-enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca2+]i in mammotroph- and somatotroph-enriched cells fractions, respectively. A rapid increase (first phase) in [Ca2+]i induced by GnRH was observed in Ca2+-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca2+ to the medium induced a second slower rise in [Ca2+]i (plateau phase). Addition of K+ caused a rapid rise in [Ca2+]i, which was dependent on extracellular Ca2+, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca2+]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca2+]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca2+]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca2+ pools and partly from influx of Ca2+ across the cell membrane.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Adeno-Hipófise/metabolismo , Aminoquinolinas , Animais , Separação Celular/métodos , Centrifugação/métodos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Feminino , Corantes Fluorescentes , Técnicas In Vitro , Cinética , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Angiotensina II/metabolismo , Antígenos CD36/metabolismo , Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Zona Glomerulosa/metabolismo , Angiotensina II/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Células Cultivadas , Humanos , Receptores Depuradores , Receptores Depuradores Classe B , Transdução de Sinais/efeitos dos fármacosRESUMO
Calcium influx into adrenal glomerulosa cells is a key event during the stimulation of aldosterone secretion by physiological increases in extracellular potassium concentrations. Two types of voltage-operated calcium channels, T- and L-types, are present on bovine glomerulosa cells, but their respective functions are not yet clearly defined. Using the patch-clamp method in the perforated patch configuration combined with microfluorimetry of cytosolic calcium, we demonstrate that L-type channels are exclusively responsible for the sustained elevation of cytosolic calcium observed upon stimulation with extracellular potassium, even at low, physiological concentrations of this agonist. In contrast, aldosterone secretion appears closely related to T-type channel activity. Moreover, when the activity of each channel type is selectively modulated by pharmacological agents, such as dihydropyridines or zonisamide, the cytosolic calcium response can be clearly dissociated from the steroidogenic response. Similarly, modulation of T channel activation by protein kinase C results in a parallel inhibition of aldosterone secretion, without any effect on the levels of cytosolic free calcium. This direct functional link between T-type calcium channel activity and steroidogenesis suggests a model in which calcium entering the cell through these channels bypasses the cytosol to activate intramitochondrial steps of aldosterone biosynthesis.
Assuntos
Canais de Cálcio/fisiologia , Zona Glomerulosa/fisiologia , Aldosterona/biossíntese , Compostos de Anilina , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio Tipo L , Bovinos , Células Cultivadas , Citosol/metabolismo , Venenos Elapídicos/farmacologia , Corantes Fluorescentes , Ionomicina/farmacologia , Cinética , Análise dos Mínimos Quadrados , Potenciais da Membrana/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Cloreto de Potássio/farmacologia , Análise de Regressão , Acetato de Tetradecanoilforbol/farmacologia , XantenosRESUMO
The properties of angiotensin II receptors were studied in isolated rat anterior pituitary cells prepared by trypsin digestion. Angiotensin II bound in a time- and temperature-dependent manner to pituitary cells, with Kd of 4.1 x 10(-9) M. The heptapeptide, des-Asp1-angiotensin II, had only one-tenth of the affinity of the octapeptide (Ki = 5.5 x 10(-8) M). These two peptides displayed a similar potency ratio in their ability to stimulate ACTH release from pituitary cells. These results indicate that angiotensin II may play a regulatory role in controlling ACTH secretion from the pituitary gland.
Assuntos
Angiotensina II/metabolismo , Adeno-Hipófise/metabolismo , Receptores de Angiotensina/metabolismo , Receptores de Superfície Celular/metabolismo , Angiotensina II/farmacologia , Animais , Ligação Competitiva , Feminino , Técnicas In Vitro , Cinética , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Tetrandrine, an alkaloid extracted from a Chinese medicinal herb traditionally used in hypertension treatment, inhibited aldosterone production induced in bovine adrenal glomerulosa cells by either potassium ion, angiotensin II, or ACTH in a concentration-dependent manner (IC50 = 10 microM). The inhibition of the response to potassium by tetrandrine had a pattern very similar to that of nickel, a blocker of T-type calcium channels. In addition, tetrandrine prevented calcium influx induced by potassium or angiotensin II without affecting the calcium release phase stimulated by the hormone. The effect of tetrandrine on voltage-activated barium currents was investigated using the whole cell configuration of the patch clamp technique. T-type currents were isolated by recording the slowly deactivating currents elicited during repolarization of the cell to -65 mV after various depolarizing pulses. These currents were blocked by micromolar concentrations of the drug. The voltage sensitivity of channel activation was not affected by tetrandrine; nevertheless, the drug significantly slowed the deactivation of the current. The action of tetrandrine did not require the activation of the channel. Tetrandrine also affected L-type currents, as assessed after inactivating T channels for 100 msec, but at higher concentrations of the drug. Thus, tetrandrine affects with a similar potency aldosterone production, calcium influx, and T-type calcium channel activity. This finding strongly suggests a role for these channels in calcium signaling and control of steroidogenesis in adrenal glomerulosa cells.