RESUMO
We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Norbornanos/farmacologia , Receptores de GABA-A/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cobaias , Camundongos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Dados de Sequência Molecular , Estrutura Molecular , Ratos , Ratos Wistar , Receptores de GABA-A/química , Receptores de GABA-A/genética , Rodenticidas/farmacologia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Decreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhotic cardiomyopathy, but their molecular mechanisms remain unclear. To study beta-adrenergic-stimulated contractility and beta-adrenergic gene expression patterns, 20 Wistar Kyoto rats were treated with carbon tetrachloride to induce cirrhosis and 20 rats were used as controls. Left ventricular contractility was recorded in electrically driven isolated hearts perfused at constant flow with isoproterenol (10(-10) to 10(-6) M). A cardiac gene expression profile was obtained using a microarray for the myocyte adrenergic pathway. The cardiac contractility maximal response to isoproterenol was significantly reduced in cirrhotic rats in comparison to control rats, whereas the half-maximal effective concentration was not different. In cirrhotic rats, cardiac gene expression analysis showed a significant overexpression of G protein alpha-inhibiting subunit 2 (Galpha(i2)), cyclic nucleotide phosphodiesterase (PDE2a), regulator of G-protein signaling 2 (RGS2), and down-expression of adenylate cyclase (Adcy3). These results indicate that overexpression of Galpha(i2), PDE2a, and RGS2 down-regulates the beta-adrenergic signaling pathway, thus contributing to the pathogenesis of cirrhotic cardiomyopathy.
Assuntos
Cardiomiopatias/genética , Regulação da Expressão Gênica/fisiologia , Cirrose Hepática/complicações , Receptores Adrenérgicos beta/genética , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Tetracloreto de Carbono , Cardiomiopatias/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Isoproterenol/farmacologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Proteínas RGS/genética , Proteínas RGS/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptores Adrenérgicos beta/metabolismo , Transdução de SinaisRESUMO
It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity. Our results show that: (i) norbormide isomers affect PTP in a rat-selective fashion; however, no relevant differences between lethal and non-lethal forms are observed suggesting that drug regulation of PTP-activity and lethality in rats are unrelated phenomena; (ii) a (phenylvinyl)pyridine moiety represents the key element conferring the PTP-activating effect; (iii) cationic derivatives of rat-active compounds accumulate in the matrix via the membrane potential and activate the PTP also in mouse and guinea pig mitochondria. These findings suggest that the norbormide-sensitive PTP-target is present in all species examined, and is presumably located on the matrix side. The species-selectivity may depend on the unique properties of a transport system allowing drug internalisation in rat mitochondria.
Assuntos
Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Norbornanos/toxicidade , Animais , Cobaias , Isomerismo , Camundongos , Norbornanos/química , Permeabilidade/efeitos dos fármacos , RatosRESUMO
OBJECTIVE: Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. METHODS: Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. RESULTS: AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. CONCLUSION: Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.
Assuntos
Antimetabólitos/farmacologia , Ácido Ascórbico/farmacologia , Hipertensão/prevenção & controle , NADPH Oxidases/metabolismo , Vitaminas/farmacologia , Zidovudina/farmacologia , Animais , Antioxidantes/metabolismo , Western Blotting/métodos , Ativação Enzimática/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , NADPH Oxidase 2 , NADPH Oxidases/análise , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Proteína Quinase C/metabolismo , Ratos , Ratos Endogâmicos WKY , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Ouabain, an inhibitor of the Na+/K+-ATPase, has been reported to affect the secretory activity of the adrenal cortex, and especially of zona glomerulosa (ZG). However, conflicting results were obtained, depending on the experimental condition used since ouabain appears to interact with angiotensin-II (Ang-II) and its action to be influenced by the electrolyte balance. Hence, we investigated the effects of prolonged (4-month) infusion with ouabain on the rat adrenal cortex. Ouabain raised the plasma concentrations of aldosterone, corticosterone and endothelin-1 (ET-1), without affecting either systolic blood pressure (SBP) or plasma renin activity (PRA). The treatment caused a marked hypertrophy of ZG and ZG cells, which mainly ensued from increases in the volume of the mitochondrial and smooth-endoplasmic-reticulum compartments, where the enzymes of steroid synthesis are located. Conversely, the volume of the lipid-droplet compartment, which stores cholesterol utilized in steroid-hormone production, underwent a striking decrease. Zona fasciculata and its parenchymal cells were not affected. Basal and maximally agonist (ACTH, Ang-II and ET-1)-stimulated in vitro mineralocorticoid secretion from adrenal slices was also notably enhanced by ouabain administration. Collectively, these findings indicate that prolonged treatment with ouabain selectively stimulates the growth and steroidogenic capacity of the rat adrenal ZG. The possibility that the activation of the renin-angiotensin system may be involved in this effect of ouabain is ruled out by the lack of significant changes in SBP and PRA. Instead, our results suggest the possible involvement of ET-1, the plasma level of which is elevated in ouabain-infused rats.
Assuntos
Endotelina-1/sangue , Inibidores Enzimáticos/farmacologia , Ouabaína/farmacologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/fisiologia , Aldosterona/sangue , Animais , Pressão Sanguínea , Corticosterona/sangue , Inibidores Enzimáticos/administração & dosagem , Humanos , Masculino , Ouabaína/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Endogâmicos WKY , Renina/metabolismo , Zona Glomerulosa/anatomia & histologiaRESUMO
OBJECTIVE: We investigated the effects of zidovudine (AZT) on cardiac and vascular smooth muscle function and morphology in rats. METHODS: Four adult male Wistar-Kyoto rats received AZT in drinking water for 240 days; four rats served as controls. Echocardiographic examination and systolic blood pressure (SBP) measurement were performed. At the end of treatment the rats were sacrificed, their hearts were weighed and vascular smooth muscle contractile and relaxing properties were evaluated in vitro on endothelium-intact aortic rings. Morphological studies were performed on cardiac and aortic myocytes by light and electron microscopy. RESULTS: AZT-treated rats (AZT-Rs) showed higher SBP, greater heart weight and, as revealed by echocardiography, greater interventricular septum thickness. Electron microscopy revealed mitochondrial swelling in myocardiocytes in AZT-Rs. Reduced response to contractile stimuli and enhanced relaxation in response to charbacol were observed in the aortic rings of AZT-Rs. The aortic myocytes of AZT-Rs contained apparently unaffected ultrastructural features, but light microscopy suggested their marked enlargement. CONCLUSIONS: AZT treatment for 240 days in rats induces a modest increase in SBP, hypertrophy of the interventricular septum and modified vascular smooth muscle responsiveness. The role of mitochondria in these AZT-induced cardiovascular alterations remains to be established.
Assuntos
Fármacos Anti-HIV/farmacologia , Coração/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Aorta/efeitos dos fármacos , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Ecocardiografia , Endotélio Vascular/efeitos dos fármacos , Ventrículos do Coração/ultraestrutura , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos WKY , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
1 Aim of the present study was to investigate the effects of norbormide, a selective vasoconstrictor agent of the rat peripheral vessels, on the whole-cell voltage-dependent L-type Ca(2+) current (I(Ca(L))) of freshly isolated smooth muscle cells from the rat caudal artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2 Norbormide decreased L-type Ca(2+) current in a concentration- and voltage-dependent manner, without modifying the threshold and the maximum of the current-voltage relationship. Norbormide-induced I(Ca(L)) inhibition was reversible upon wash-out. 3 Norbormide both shifted the voltage dependence of the steady-state inactivation curve to more negative potentials by about 16 mV, without affecting the activation curve, and decreased the slope of inactivation. Norbormide, however, did not modify both the activation and the inactivation kinetics of the I(Ca(L)). 4 Norbormide decreased I(Ca(L)) progressively during repetitive step depolarizations, with inhibition depending on the stimulation frequency (use-dependent block) as well as on the holding potential. 5 Addition of 50 micro M norbormide caused the contraction of all freshly isolated cells and also of those impaled with the perforated method, but not of those impaled with the conventional method (i.e. dialysed). 6 In conclusion, these results prove norbormide to be a vascular L-type Ca(2+) channel inhibitor, which preferentially acts on the inactivated and/or open state of the channel. In rat caudal artery smooth muscle, however, this mechanism does not result in a vasodilating effect since it is overwhelmed by the mechanism underlying norbormide-induced vasoconstriction.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Cálcio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Norbornanos/farmacologia , Vasoconstritores/farmacologia , Animais , Aorta , Canais de Cálcio Tipo L/fisiologia , Separação Celular , Masculino , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , Técnicas de Patch-Clamp , RatosRESUMO
The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.
Assuntos
Ácido Araquidônico/metabolismo , Endotelina-1/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Fosfolipases A/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Artérias , Proteínas de Transporte/farmacologia , Cicloexanonas/farmacologia , Interações Medicamentosas , Antagonistas dos Receptores de Endotelina , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Norepinefrina/farmacologia , Fosfolipases A2 , Fosforilação , Ratos , Ratos Wistar , Cauda , TrítioRESUMO
Callipeltin A is a cyclic depsidecapeptide isolated from the marine sponges Callipelta sp. and Latrunculia sp. that has been previously shown to increase the force of contraction of guinea-pig atria through the inhibition of Na+/Ca2+ exchanger (NCX). We investigated the effect of callipeltin A on guinea-pig aortic rings contracted by procedures that activate NCX in "calcium entry mode". Callipeltin A did not inhibit these contractions. Resting aorta responded to callipeltin A with a remarkable contraction that was concentration-dependent (EC50 0.44microM). This contraction was not inhibited by the calcium channel blocker verapamil and was not mediated by the activation of alpha-adrenergic or endothelin-1 receptors. Pre-incubation of aortic rings with 0.5mM amiloride, an inhibitor of NCX, completely prevented callipeltin A-induced contraction. Furthermore, callipeltin A (EC50 0.51microM) increased Na+ efflux of Na-loaded erythrocytes. 1H and 13C NMR resonances of callipeltin A revealed small but significant changes in the titration with K+ and Na+ salts. It is suggested that the effect of callipeltin A on cardiac and vascular preparations is linked to a Na-ionophore action.
Assuntos
Depsipeptídeos , Ionóforos/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Sódio/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Aorta , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Cobaias , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Músculo Liso Vascular/fisiologiaRESUMO
We have investigated the effects of the rat-specific vasoconstrictor agent norbormide on the mechanical and electrophysiological properties of rat non-vascular smooth muscles. Norbormide (50 microM) did not affect the resting tone of urinary bladder, tracheal, and duodenal rings. In all tissues, KCl-induced concentration-response curves were shifted downward by norbormide (5 and 50 microM). In urinary bladder and tracheal rings, norbormide inhibited contractile responses to carbachol only at the higher concentration (50 microM). In single gastric fundus myocytes, 50 microM norbormide inhibited L-type Ca(2+) current (I(Ca(L))) by about 60%, neither affecting both activation and inactivation rates of the current nor the current-voltage curve along the voltage axis. Our results indicate that rat non-vascular smooth muscles are relaxed by norbormide with a mechanism likely involving a reduction of Ca(2+) entry through L-type Ca(2+) channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Norbornanos/farmacologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Relaxamento Muscular/fisiologia , Músculo Liso/fisiologia , Ratos , Ratos Endogâmicos WKY , Vasoconstritores/farmacologiaRESUMO
UNLABELLED: Norbormide is a vasoconstrictor of rat peripheral arteries and a relaxant in rat aorta. To characterise norbormide actions within the rat vascular tree we have investigated its effects on the contractile function of rings from several arteries and veins. A maximal norbormide concentration (50 microM) failed to contract thoracic aorta and carotid artery, whereas in pulmonary artery, abdominal aorta, iliac, caudal, and femoral arteries it induced a contractile effect that was respectively 4.8 +/- 0.6, 18.4 +/- 1.5, 39 +/- 5, 144 +/- 7, and 260 +/- 22% of that induced by 90 mM KCl. In pulmonary, carotid, and iliac arteries, and in thoracic and abdominal aorta, 50 microM norbormide inhibited KCl-induced responses. Norbormide (50 microM) contracted all veins investigated. The effect, expressed as % of KCl-induced contraction, was 121 +/- 25, 154 +/- 14.5, 154 +/- 18.2, 203 +/- 19, and 267 +/- 33 for pulmonary vein, thoracic and abdominal vena cava, iliac and jugular veins, respectively. In jugular vein, as previously shown in rat caudal artery, norbormide contraction was abolished in Ca2+-free medium, was unaffected by the Ca2+ channel blocker nifedipine, and was relaxed by SK&F 96365, a blocker of store-operated Ca2+ channels. IN CONCLUSION: i) rat veins represent the main target for contractile norbormide action; ii) in both artery and veins norbormide contractions are generally inversely related to the calibre of the vessel; iii) norbormide-induced contraction is mediated by the same mechanism/s in arteries and veins; iiii) in norbormide-contracted arteries the drug activates both contractile and relaxing mechanisms.
Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Norbornanos/farmacologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Animais , Artérias/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Imidazóis/farmacologia , Contração Isométrica , Masculino , Nifedipino/farmacologia , Ratos , Ratos Wistar , Veias/efeitos dos fármacosRESUMO
We investigated whether chronic zidovudine (AZT) administration in rats could impair cardiac function by affecting intercellular junctions and whether vitamin C could prevent these possible effects. Rats were treated for 8 months with AZT, vitamin C, and AZT plus vitamin C. Cardiac fractional shortening (FS) was assessed by echocardiographic examination, intercellular junctions morphology was detected by electron microscopy (EM) and immunocytochemistry (ICC). AZT-treated rats showed a reduced FS that was not prevented by vitamin C. EM revealed that AZT treatment did not affect coronary endothelial intercellular junctions whereas it caused an enlargement of fascia adherens of the intercalated discs that was prevented by vitamin C. AZT treatment did not induce either alterations of gap junctions morphology or distribution of connexin-43, the major protein expressed in the gap junctions. We conclude that AZT treatment may be potentially deleterious to the heart by inducing a ROS-mediated damage of cardiac intercalated discs.
RESUMO
Endogenous ouabain may play a role in the control of cardiovascular system function. In this study, we investigated the effects of a long-term ouabain treatment on basal and endothelin-1 (ET-1)-induced phosphorylation of cardiac and vascular extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), which are involved in several cardiac and vascular physiologic and pathologic conditions. Our results show that the hearts from ouabain-treated rats have a higher basal level of ERK-1 and ERK-2 phosphorylation compared with untreated rats. Perfusion of the hearts with ET-1 increased ERK-1 and ERK-2 phosphorylation both in ouabain-treated and in control rats, with a larger stimulatory effect in ouabain-treated animals. On the contrary, exposure of endothelium-free tail artery to ET-1 increased ERKs phosphorylation both in treated and untreated rats, but this effect was blunted in ouabain-treated rats. These findings demonstrate that ouabain treatment has opposite effects on basal and ET-1-induced ERKs phosphorylation in the heart and in the tail artery of the rat.