RESUMO
Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na(+), K(+)-ATPase inhibition. Present results show that CNP did not affect either (3)H-dopamine uptake in renal tissue or Na(+), K(+)-ATPase activity; meanwhile, Ang-(1-7) was able to increase (3)H-dopamine uptake and decreased Na(+), K(+)-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na(+), K(+)-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on (3)H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on (3)H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on (3)H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na(+), K(+)-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.
RESUMO
The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Etanol/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sinapses/efeitos dos fármacos , Sinapses/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Forma do Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/fisiologia , Ratos Wistar , Sinapses/fisiologiaRESUMO
1. Since we previously reported that angiotensin-(1-7) [Ang-(1-7)] increases or inhibits norepinephrine (NE) release in rat atria or hypothalamus, respectively, the present work was undertaken to investigate the effect of the heptapeptide on NE neuronal uptake and metabolism in atria and hypothalamus isolated from rats. 2. Ang II (1-10 microM) caused a decrease in neuronal NE uptake in both atria and hypothalami isolated from rats. On the contrary, tissues incubated with [3H]NE in the presence of 0.1-10 microM Ang-(1-7) showed no modification in [3H]NE content with respect to the control group, suggesting that the heptapeptide did not modify [3H]NE neuronal uptake. 3. To study the effect of the heptapeptide on NE catabolism, monoamine-oxidase (MAO) and catechol-O-methyltransferase (COMT) activities were determined. Pretreatment of the tissue with Ang-(1-7) (0.1-1.0 microM) showed a tendency to diminish MAO activity in rat atria, while no significant changes were observed in hypothalamic MAO activity. Moreover, the heptapeptide (0.1-1.0 microM) did not affect central COMT activity with respect to the control group. 4. Present results allow us to conclude that Ang-(1-7) interacts with noradrenergic neurotransmission by increasing or inhibiting NE release at the peripheral and central levels, respectively, without affecting either the neurotransmitter neuronal uptake or catabolism.
Assuntos
Angiotensina I/farmacologia , Hipotálamo/metabolismo , Miocárdio/metabolismo , Neurônios/metabolismo , Norepinefrina/metabolismo , Fragmentos de Peptídeos/farmacologia , Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Transporte Biológico/efeitos dos fármacos , Catecol O-Metiltransferase/metabolismo , Feminino , Átrios do Coração , Monoaminoxidase/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Angiotensin (Ang)-(1-7) elicits a facilitatory presynaptic effect on peripheral noradrenergic neurotransmission, and because biological responses to the heptapeptide on occasion are tissue specific, the present investigation was undertaken to study its action on noradrenergic neurotransmission at the central level. In rat hypothalamus labeled with [(3)H]-norepinephrine, 100 to 600 nmol/L Ang-(1-7) diminished norepinephrine released by 25 mmol/L KCl. This effect was blocked by the selective angiotensin type 2 receptor antagonist PD 123319 (1 micromol/L) and by the specific Ang-(1-7) receptor antagonist ([D-Ala(7)]Ang-(1-7) (1 micromol/L) but not by losartan (10 nmol/L to 1 micromol/L), a selective angiotensin type 1 receptor antagonist. The inhibitory effect on noradrenergic neurotransmission caused by Ang-(1-7) was prevented by 10 micromol/L N(omega)-nitro-L-arginine methylester, an inhibitor of nitric oxide synthase activity, and was restored by 100 micromol/L L-arginine, precursor of nitric oxide synthesis. Methylene blue (10 micromol/L), an inhibitor of guanylate cyclase considered as the target of nitric oxide action, as well as Hoe 140 (10 micromol/L), a bradykinin B(2)-receptor antagonist, prevented the inhibitory effect of the heptapeptide on neuronal norepinephrine release, whereas no modification was observed in the presence of 0.1 to 10 micromol/L indomethacin, a cyclooxygenase inhibitor. Our results indicate that Ang-(1-7) has a tissue-specific neuromodulatory effect on noradrenergic neurotransmission, being inhibitory at the central nervous system by a nitric oxide-dependent mechanism that involves angiotensin type 2 receptors and local bradykinin production.
Assuntos
Angiotensina I/farmacologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Óxido Nítrico/fisiologia , Norepinefrina/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Hipotálamo/citologia , Indometacina/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cloreto de Potássio/farmacologia , Prostaglandinas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de AngiotensinaRESUMO
Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).
Assuntos
Angiotensina II/metabolismo , Angiotensina I/metabolismo , Córtex Renal/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Angiotensina/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Sítios de Ligação , Imidazóis/farmacologia , Radioisótopos do Iodo , Losartan/farmacologia , Masculino , Piridinas/farmacologia , Ensaio Radioligante , Ratos , Ratos WistarRESUMO
The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.
Assuntos
Angiotensina II/farmacologia , ATPase de Ca(2+) e Mg(2+)/metabolismo , Fragmentos de Peptídeos/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Angiotensina I , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Miocárdio/enzimologia , Ratos , Ratos Wistar , Ovinos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/enzimologiaRESUMO
In rat atria isolated with their cardioaccelerans nerves and labeled with [3H]norepinephrine, exposure to 1 x 10(-7) mol/L angiotensin II (Ang II) and 1 x 10(-7) mol/L Ang-(1-7) increased the release of radioactivity elicited by nerve stimulation (0.5-millisecond-long square-wave pulses at 2 Hz during 2 minutes) by 90% and 60%, respectively. The facilitatory effect on noradrenergic neurotransmission caused by both peptides was stereospecifically prevented by N omega-nitro-L-arginine methyl ester (1 x 10(-4) mol/L), an inhibitor of nitric oxide synthase that catalyzes the conversion of L-arginine to nitric oxide, as well as by 1 x 10(-5) mol/L methylene blue, a substance that inhibits the guanylate cyclase considered as the final target of nitric oxide action. On the other hand, the precursor of nitric oxide synthesis. L-arginine (1 x 10(-3) mol/L), reversed the prevention produced by N omega-nitro-L-arginine methyl ester on the increased release of norepinephrine caused by Ang II and Ang-(1-7). The present results suggest that nitric oxide could be involved in the neuromodulatory function elicited by both Ang II and Ang-(1-7) in rat atria. The physiological role of this observation is still under study.
Assuntos
Angiotensina II/fisiologia , Óxido Nítrico/fisiologia , Norepinefrina/metabolismo , Fragmentos de Peptídeos/fisiologia , Simpatomiméticos/metabolismo , Angiotensina I , Animais , Interações Medicamentosas , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Ratos , Ratos WistarRESUMO
We examined the effects of angiotensin II (Ang II) and Ang-(1-7) on the release of [3H]norepinephrine elicited by nerve stimulation (2 Hz, 0.5 millisecond, for 2 minutes) in rat atria isolated with their cardioaccelerans nerves. The stimulation-induced release of [3H]norepinephrine was increased 50% by 3 x 10(-8) mol/L of either peptide. No further increase in [3H]norepinephrine release was observed with peptide concentrations up to 3 x 10(-7) mol/L. This effect was completely blocked by the nonselective angiotensin receptor antagonist saralasin (1 x 10(-7) mol/L). The type 1 angiotensin receptor antagonist DuP 753 (1 x 10(-6) mol/L) entirely prevented the increases in [3H]norepinephrine caused by Ang II and Ang-(1-7). On the other hand, the type 2 angiotensin receptor antagonist PD 123319 (1 x 10(-6) mol/L) prevented the increase in [3H]norepinephrine release elicited by Ang-(1-7) but not by Ang II. These results suggest that Ang-(1-7), like Ang II, could have a neuromodulatory function in rat atria via activation of specific angiotensin receptor subtypes, which could be the subtype 1 angiotensin receptor for Ang II and subtypes 1 and 2 for Ang-(1-7).