Bruno Cerrato - Academia.edu (original) (raw)

Papers by Bruno Cerrato

Research paper thumbnail of MAS1 Receptor Trafficking Involves ERK1/2 Activation Through a β-Arrestin2-Dependent Pathway

Hypertension, 2017

The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R traff... more The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell.

Research paper thumbnail of Heteromerization Between the Bradykinin B2 Receptor and the Angiotensin-(1-7) Mas Receptor: Functional Consequences

Hypertension, 2016

The renin-angiotensin system and the kallikrein-kinin system contribute to fluid homeostasis and ... more The renin-angiotensin system and the kallikrein-kinin system contribute to fluid homeostasis and blood pressure regulation. The renin-angiotensin system is composed of 2 arms with opposing functions. The pressor arm, represented by the angiotensin type 1 receptor (AT 1 R), angiotensin-converting enzyme (ACE), and angiotensin II, is responsible for the vasoconstrictive, proliferative, fibrotic, and hypertensive effects of the renin-angiotensin system. In contrast, the second arm exerts depressor and cardiovascular protective effects through angiotensin-(1-7) (Ang-(1-7)), the Ang-(1-7)-specific receptor Mas that transduces the main physiological actions of Ang-(1-7), and ACE2 that catalyzes the generation of Ang-(1-7) from angiotensin II. 1-3 Bradykinin, generated by the kallikrein-kinin system, exerts cardioprotective, vasodilatory, and depressor properties through B2 receptor (B2 R) stimulation. 4,5 Several studies showed the existence of a cross talk between the renin-angiotensin system and kallikrein-kinin system. 6,7 For instance, kallikrein not only catalyzes the generation of kinin from kininogen, but also acts as a prorenin-activating enzyme leading to an increase in angiotensin II. 6 In addition , ACE not only generates the vasopressor angiotensin II but also responsible for the proteolytic degradation of bradykinin. 6,7 Not only was a cross talk between both systems reported at the enzymatic level, but also between the components of these systems. For example, Ang-(1-7) exerts kinin-like effects and Abstract-Bradykinin B 2 receptor (B 2 R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2 R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B 2 R and MasR formed a constitutive heteromer, which was not modified by their agonists. B 2 R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B 2 R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B 2 R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2 R antagonist. Concerning serine/ threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2 R-MasR but not in B2 R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2 R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2 R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2 R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties. (Hypertension. 2016;68:00-00.

Research paper thumbnail of Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

Journal of Neurochemistry, 2012

Research paper thumbnail of Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

Journal of Neurochemistry, 2012

Research paper thumbnail of Neuromodulatory role of angiotensin-(1–7) in the central nervous system

Clinical Science, 2013

Ang-(1-7) [angiotensin-(1-7)] constitutes an important functional end-product of the RAS (renin-a... more Ang-(1-7) [angiotensin-(1-7)] constitutes an important functional end-product of the RAS (renin-angiotensin system) endogenously formed from AngI (angiotensin I) or AngII (angiotensin II) through the catalytic activity of ACE2 (angiotensin-converting enzyme 2), prolyl carboxypeptidase, neutral endopeptidase or other endopeptidases. Ang-(1-7) lacks the pressor, dipsogenic or stimulatory effect on aldosterone release characteristic of AngII. In contrast, it produces vasodilation, natriuresis and diuresis, and inhibits angiogenesis and cell growth. At the central level, Ang-(1-7) acts at sites involved in the control of cardiovascular function, thus contributing to blood pressure regulation. This action may result from its inhibitory neuromodulatory action on NE [noradrenaline (norepinephrine)] levels at the synaptic cleft, i.e. Ang-(1-7) reduces NE release and synthesis, whereas it causes an increase in NE transporter expression, contributing in this way to central NE neuromodulation. Thus, by selective neurotransmitter release, Ang-(1-7) may contribute to the overall central cardiovascular effects. In the present review, we summarize the central effects of Ang-(1-7) and the mechanism by which the peptide modulates NE levels in the synaptic cleft. We also provide new evidences of its cerebroprotective role.

Research paper thumbnail of Angiotensin-(1–7) upregulates central nitric oxide synthase in spontaneously hypertensive rats

Brain Research, 2012

Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous... more Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous system (SNS) activity. Since Ang (1-7) exerts an inhibitory neuromodulatory effect on the SNS through a NO-mediated mechanism, we tested the hypothesis that Ang (1-7) alters centrally nitric oxide synthase (NOS) activity and expression in spontaneously hypertensive rats (SHR). Since NOS activity is altered in relation to the development of hypertension in rats, we evaluated the effect of Ang-(1-7) on hypothalamic NOS activity in two different ages in SHR, corresponding to a prehypertensive phase (3-4 weeks) and a established hypertension (13-14 weeks) and compared with age-matched Wistar-Kyoto (WKY) rats. NOS activity was measured by the conversion of [ 3 H]L-arginine to citrulline .

Research paper thumbnail of Protective axis of the renin–angiotensin system in the brain

Clinical Science, 2014

The RAS (renin-angiotensin system) is composed of two arms: the pressor arm containing AngII (ang... more The RAS (renin-angiotensin system) is composed of two arms: the pressor arm containing AngII (angiotensin II)/ACE (angiotensin-converting enzyme)/AT1Rs (AngII type 1 receptors), and the depressor arm represented by Ang-(1-7) [angiotensin-(1-7)]/ACE2/Mas receptors. All of the components of the RAS are present in the brain. Within the brain, Ang-(1-7) contributes to the regulation of BP (blood pressure) by acting at regions that control cardiovascular function such that, when Ang-(1-7) is injected into the nucleus of the solitary tract, caudal ventrolateral medulla, paraventricular nucleus or anterior hypothalamic area, a reduction in BP occurs; however, when injected into the rostral ventrolateral medulla, Ang-(1-7) stimulates an increase in BP. In contrast with AngII, Ang-(1-7) improves baroreflex sensitivity and has an inhibitory neuromodulatory role in hypothalamic noradrenergic neurotransmission. Ang-(1-7) not only exerts effects related to BP regulation, but also acts as a cerebroprotective component of the RAS by reducing cerebral infarct size and neuronal apoptosis. In the present review, we provide an overview of effects elicited by Ang-(1-7) in the brain, which suggest a potential role for Ang-(1-7) in controlling the central development of hypertension.

Research paper thumbnail of MAS1 Receptor Trafficking Involves ERK1/2 Activation Through a β-Arrestin2-Dependent Pathway

Hypertension, 2017

The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R traff... more The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell.

Research paper thumbnail of Heteromerization Between the Bradykinin B2 Receptor and the Angiotensin-(1-7) Mas Receptor: Functional Consequences

Hypertension, 2016

The renin-angiotensin system and the kallikrein-kinin system contribute to fluid homeostasis and ... more The renin-angiotensin system and the kallikrein-kinin system contribute to fluid homeostasis and blood pressure regulation. The renin-angiotensin system is composed of 2 arms with opposing functions. The pressor arm, represented by the angiotensin type 1 receptor (AT 1 R), angiotensin-converting enzyme (ACE), and angiotensin II, is responsible for the vasoconstrictive, proliferative, fibrotic, and hypertensive effects of the renin-angiotensin system. In contrast, the second arm exerts depressor and cardiovascular protective effects through angiotensin-(1-7) (Ang-(1-7)), the Ang-(1-7)-specific receptor Mas that transduces the main physiological actions of Ang-(1-7), and ACE2 that catalyzes the generation of Ang-(1-7) from angiotensin II. 1-3 Bradykinin, generated by the kallikrein-kinin system, exerts cardioprotective, vasodilatory, and depressor properties through B2 receptor (B2 R) stimulation. 4,5 Several studies showed the existence of a cross talk between the renin-angiotensin system and kallikrein-kinin system. 6,7 For instance, kallikrein not only catalyzes the generation of kinin from kininogen, but also acts as a prorenin-activating enzyme leading to an increase in angiotensin II. 6 In addition , ACE not only generates the vasopressor angiotensin II but also responsible for the proteolytic degradation of bradykinin. 6,7 Not only was a cross talk between both systems reported at the enzymatic level, but also between the components of these systems. For example, Ang-(1-7) exerts kinin-like effects and Abstract-Bradykinin B 2 receptor (B 2 R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2 R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B 2 R and MasR formed a constitutive heteromer, which was not modified by their agonists. B 2 R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B 2 R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B 2 R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2 R antagonist. Concerning serine/ threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2 R-MasR but not in B2 R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2 R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2 R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2 R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties. (Hypertension. 2016;68:00-00.

Research paper thumbnail of Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

Journal of Neurochemistry, 2012

Research paper thumbnail of Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

Journal of Neurochemistry, 2012

Research paper thumbnail of Neuromodulatory role of angiotensin-(1–7) in the central nervous system

Clinical Science, 2013

Ang-(1-7) [angiotensin-(1-7)] constitutes an important functional end-product of the RAS (renin-a... more Ang-(1-7) [angiotensin-(1-7)] constitutes an important functional end-product of the RAS (renin-angiotensin system) endogenously formed from AngI (angiotensin I) or AngII (angiotensin II) through the catalytic activity of ACE2 (angiotensin-converting enzyme 2), prolyl carboxypeptidase, neutral endopeptidase or other endopeptidases. Ang-(1-7) lacks the pressor, dipsogenic or stimulatory effect on aldosterone release characteristic of AngII. In contrast, it produces vasodilation, natriuresis and diuresis, and inhibits angiogenesis and cell growth. At the central level, Ang-(1-7) acts at sites involved in the control of cardiovascular function, thus contributing to blood pressure regulation. This action may result from its inhibitory neuromodulatory action on NE [noradrenaline (norepinephrine)] levels at the synaptic cleft, i.e. Ang-(1-7) reduces NE release and synthesis, whereas it causes an increase in NE transporter expression, contributing in this way to central NE neuromodulation. Thus, by selective neurotransmitter release, Ang-(1-7) may contribute to the overall central cardiovascular effects. In the present review, we summarize the central effects of Ang-(1-7) and the mechanism by which the peptide modulates NE levels in the synaptic cleft. We also provide new evidences of its cerebroprotective role.

Research paper thumbnail of Angiotensin-(1–7) upregulates central nitric oxide synthase in spontaneously hypertensive rats

Brain Research, 2012

Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous... more Increased blood pressure in hypertension is hypothesized to be caused by high sympathetic nervous system (SNS) activity. Since Ang (1-7) exerts an inhibitory neuromodulatory effect on the SNS through a NO-mediated mechanism, we tested the hypothesis that Ang (1-7) alters centrally nitric oxide synthase (NOS) activity and expression in spontaneously hypertensive rats (SHR). Since NOS activity is altered in relation to the development of hypertension in rats, we evaluated the effect of Ang-(1-7) on hypothalamic NOS activity in two different ages in SHR, corresponding to a prehypertensive phase (3-4 weeks) and a established hypertension (13-14 weeks) and compared with age-matched Wistar-Kyoto (WKY) rats. NOS activity was measured by the conversion of [ 3 H]L-arginine to citrulline .

Research paper thumbnail of Protective axis of the renin–angiotensin system in the brain

Clinical Science, 2014

The RAS (renin-angiotensin system) is composed of two arms: the pressor arm containing AngII (ang... more The RAS (renin-angiotensin system) is composed of two arms: the pressor arm containing AngII (angiotensin II)/ACE (angiotensin-converting enzyme)/AT1Rs (AngII type 1 receptors), and the depressor arm represented by Ang-(1-7) [angiotensin-(1-7)]/ACE2/Mas receptors. All of the components of the RAS are present in the brain. Within the brain, Ang-(1-7) contributes to the regulation of BP (blood pressure) by acting at regions that control cardiovascular function such that, when Ang-(1-7) is injected into the nucleus of the solitary tract, caudal ventrolateral medulla, paraventricular nucleus or anterior hypothalamic area, a reduction in BP occurs; however, when injected into the rostral ventrolateral medulla, Ang-(1-7) stimulates an increase in BP. In contrast with AngII, Ang-(1-7) improves baroreflex sensitivity and has an inhibitory neuromodulatory role in hypothalamic noradrenergic neurotransmission. Ang-(1-7) not only exerts effects related to BP regulation, but also acts as a cerebroprotective component of the RAS by reducing cerebral infarct size and neuronal apoptosis. In the present review, we provide an overview of effects elicited by Ang-(1-7) in the brain, which suggest a potential role for Ang-(1-7) in controlling the central development of hypertension.