RESUMO
INTRODUCTION: Trace amine-associated receptors (TAARs) are a family of G protein-coupled receptors and are widely distributed in the body. Activation of TAAR1 by specific agonists can produce a variety of physiological effects centrally and peripherally. The objective of this study was to investigate the vasodilator effect of two selective TAAR1 agonists 3-iodothyronamine (T1AM) and RO5263397 in the isolated perfused rat kidney preparation. METHODS: Kidneys were isolated and perfused with Krebs' solution, gassed with 95% oxygen and 5% carbon dioxide, through the renal artery. RESULTS: In preparations pre-constricted with methoxamine (5 × 10-6
Assuntos
Aminas , Vasodilatadores , Ratos , Animais , Vasodilatadores/farmacologia , Triptaminas , Receptores Acoplados a Proteínas G/agonistas , RimRESUMO
We investigated whether chronic administration of nano-sized polyamidoamine (PAMAM) dendrimers can have beneficial effects on diabetes-induced vascular dysfunction by inhibiting the epidermal growth factor receptor (EGFR)-ERK1/2-Rho kinase (ROCK)-a pathway known to be critical in the development of diabetic vascular complications. Daily administration of naked PAMAMs for up to 4â¯weeks to streptozotocin-induced diabetic male Wistar rats inhibited EGFR-ERK1/2-ROCK signaling and improved diabetes-induced vascular remodeling and dysfunction in a dose, generation (G6â¯>â¯G5) and surface chemistry-dependent manner (cationic > anionic > neutral). PAMAMs, AG1478 (a selective EGFR inhibitor), or anti-EGFR siRNA also inhibited vascular EGFR-ERK1/2-ROCK signaling in vitro. These data showed that naked PAMAM dendrimers have the propensity to modulate key (e.g. EGFR) cell signaling cascades with associated pharmacological consequences in vivo that are dependent on their physicochemical properties. Thus, PAMAMs, alone or in combination with vasculoprotective agents, may have a beneficial role in the potential treatment of diabetes-induced vascular complications.
Assuntos
Dendrímeros/administração & dosagem , Diabetes Mellitus Experimental/fisiopatologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Nanopartículas/administração & dosagem , Transdução de Sinais , Remodelação Vascular , Quinases Associadas a rho/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Dendrímeros/química , Diabetes Mellitus Experimental/sangue , Glucose/toxicidade , Masculino , Artérias Mesentéricas/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nanopartículas/química , Norepinefrina/farmacologia , Tamanho da Partícula , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacosRESUMO
The effects of naked polyamidoamine (PAMAM) dendrimers on renin-angiotensin system (RAS) signaling via Angiotensin (Ang) II-mediated transactivation of the epidermal growth factor receptor (EGFR) and the closely related family member ErbB2 (HER2) were investigated. In primary aortic vascular smooth muscle cells, a cationic fifth-generation (G5) PAMAM dendrimer dose- and time-dependently inhibited Ang II/AT1 receptor-mediated transactivation of EGFR and ErbB2 as well as their downstream signaling via extracellular-regulated kinase 1/2 (ERK1/2). Inhibition even occurred at noncytotoxic concentrations at short (1 h) exposure times and was dependent on dendrimer generation (G7 > G6 > G5 > G4) and surface group chemistry (amino > carboxyl > hydroxyl). Mechanistically, the cationic G5 PAMAM dendrimer inhibited Ang II-mediated transactivation of EGFR and ErbB2 via inhibition of the nonreceptor tyrosine kinase Src. This novel, early onset, intrinsic biological action of PAMAM dendrimers as inhibitors of the Ang II/AT1/Src/EGFR-ErbB2/ERK1/2 signaling pathway could have important toxicological and pharmacological implications.
Assuntos
Angiotensina II/farmacologia , Dendrímeros/farmacologia , Receptores ErbB/metabolismo , Poliaminas/farmacologia , Polímeros/farmacologia , Receptor ErbB-2/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Linhagem Celular , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
Hydrogen sulfide (H2S) has been reported to have beneficial effects in different pathological conditions. OBJECTIVES: the effects of chronic treatment of diabetic rats with GYY4137 (slow releasing H2S donor) or NaHS (fast releasing H2S donor) on the reactivity of the mesenteric bed to vasoactive agonists and the changes in its downstream effectors, ERK1/2 and p38 MAP Kinase have been investigated. In addition, the levels of nitric oxide (NO) and H2S in all groups were measured. METHODS: diabetes was induced by a single intraperitoneal (ip) injection of streptozotocin (STZ; 55 mg/kg). Sprague Dawley (SD; n = 10-12/group) rats were randomly divided into six groups: control, STZ-induced diabetic rats, GYY4137-treated control, NaHS-treated control, GYY4137-treated diabetic, and NaHS-treated diabetic. After 28 days of treatment, rats were sacrificed and mesenteric beds were isolated for functional or biochemical studies. The vascular reactivity of the perfused mesenteric bed to norepinephrine, carbachol and sodium nitroprusside were determined by measurement of changes in perfusion pressure. Western blotting was performed to measure the protein expression of ERK1/2, p38, eNOS, and H2S biosynthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase. NO and H2S levels were measured in all groups in isolated mesenteric tissues or plasma. RESULTS: diabetes resulted in a significant increase in vasoconstrictor responses to norepinephrine (e.g., 129.6 ± 6.77 mmHg in diabetic vs 89.3 ± 8.48 mmHg in control at 10-7 dose), and carbachol-induced vasodilation was significantly reduced in diabetic mesenteric bed (e.g., 68.9 ± 4.8 mmHg in diabetic vs 90.6 ± 2.2 mmHg in control at 10-7 dose). Chronic treatment of the diabetic rats with GYY4137 resulted in a significant improvement in the response to norepinephrine (e.g., 86.66 ± 8.04 mmHg in GYY4137-treated diabetic vs 129.6 ± 6.77 mmHg in untreated diabetic at 10-7 dose) or carbachol (e.g., 84.90 ± 2.48 mmHg in GYY4137-treated diabetic vs 68.9 ± 4.8 mmHg in untreated diabetic at 10-7 dose). The biochemical studies showed a marked reduction of the protein expression of ERK and p38 and a significant upregulation of the expression of eNOS and H2S synthesizing enzymes after chronic treatment with GYY4137. Plasma levels of NO and H2S were significantly elevated after treatment with GYY4137. However, H2S production in the mesenteric bed showed a marginal elevation in diabetic tissues compared to controls. CONCLUSION: the results indicate that GYY4137 may be a novel therapeutic tool to prevent diabetes-associated vascular dysfunction.
Assuntos
Diabetes Mellitus Experimental , Sulfeto de Hidrogênio , Animais , Carbacol , Cistationina/uso terapêutico , Cistationina gama-Liase , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Sulfeto de Hidrogênio/metabolismo , Morfolinas , Óxido Nítrico/metabolismo , Nitroprussiato , Norepinefrina , Compostos Organotiofosforados , Ratos , Ratos Sprague-Dawley , Estreptozocina , Sulfetos , Vasoconstritores/uso terapêutico , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The in vivo impact of two generation 6 cationic polyamidoamine (PAMAM) dendrimers on cellular signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK), as well as their relationship to epidermal growth factor receptor (EGFR), were studied in the normal and/or diabetic rat kidney. A single 10mg/kg/i.p administration of Polyfect (PF; with an intact branching architecture) or Superfect (SF; with a fragmented branching architecture) modulated renal ERK1/2 and p38 MAPK phosphorylation in a dendrimer-specific and animal model-dependent manner. AG1478 treatment (a selective EGFR inhibitor) confirmed that renal ERK1/2 and p38 MAPK signaling was downstream of EGFR. Surprisingly, both PAMAMs induced hyperphosphorylation of ERK1/2 and p38 MAPK (at 1 or 5mg/kg) despite inhibiting EGFR phosphorylation in the diabetic kidney. PAMAMs did not alter renal morphology but their effects on p38 MAPK and EGFR phosphorylation were reversed by ex vivo treatment of kidneys with the anti-oxidant, Tempol. Thus, PAMAMs can intrinsically modulate signaling of mitogen-activated protein kinases (MAPKs) depending on the type of dendrimer (fragmented vs intact branching architecture) and animal model (normal vs diabetic) used and likely occurs via an EGFR-independent and oxidative-stress dependent mechanism. These findings might have important toxicological implications for PAMAM-based delivery systems.
Assuntos
Dendrímeros/farmacologia , Diabetes Mellitus Experimental/metabolismo , Rim/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Glicemia , Óxidos N-Cíclicos/farmacologia , Diabetes Mellitus Experimental/patologia , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Rim/patologia , Masculino , Fosforilação/efeitos dos fármacos , Quinazolinas/farmacologia , Ratos , Marcadores de Spin , Tirfostinas/farmacologiaRESUMO
Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1-7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1-7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1-7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1-7). Ang-(1-7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1-7) that could be prevented by D-Pro7-Ang-(1-7) in VSMC. These data suggest that Ang-(1-7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.
Assuntos
Angiotensina I/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Receptores ErbB/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Receptores ErbB/antagonistas & inibidores , Glucose/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fosforilação , Proto-Oncogene Mas , Ratos , Ratos Wistar , Transdução de Sinais , Vasodilatadores/farmacologiaRESUMO
Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling-an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine.
Assuntos
Dendrímeros/farmacologia , Receptores ErbB/efeitos dos fármacos , Poliaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
The epidermal growth factor receptors, EGFR and EGFR2 (ErbB2), appear important mediators of diabetes-induced vascular dysfunction. We investigated whether targeted dual inhibition of EGFR and ErbB2 with Lapatinib would be effective in treating diabetes-induced vascular dysfunction in a rat model of type 1 diabetes. In streptozotocin-induced diabetes, chronic 4-week oral or acute, ex vivo, administration of Lapatinib prevented the development of vascular dysfunction as indicated by the attenuation of the hyper-reactivity of the diabetic mesenteric vascular bed (MVB) to norephinephrine without correcting hyperglycemia. Chronic in vivo or acute ex vivo Lapatinib treatment also significantly attenuated diabetes-induced increases in phosphorylation of EGFR, ErbB2, ERK1/2, AKT, ROCK2 and IkB-alpha as well as normalized the reduced levels of phosphorylated FOXO3A, and eNOS (Ser1177) in the diabetic MVB. Similar results were observed in vascular smooth muscle cells (VSMCs) cultured in high glucose (25 mM) treated with Lapatinib or small interfering RNA (siRNA) targeting the ErbB2 receptor. Lapatinib also prevented high glucose-induced apoptosis in VSMC. Thus, Lapatinib corrects hyperglycemia-induced apoptosis and vascular dysfunction with concomitant reversal of diabetes or high glucose-induced signaling changes in EGFR/ErbB2 and downstream signaling pathways implying that targeted dual inhibition of EGFR/ErbB2 might be an effective vasculoprotective treatment strategy in diabetic patients.
Assuntos
Apoptose/efeitos dos fármacos , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/fisiopatologia , Receptores ErbB/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Quinazolinas/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/induzido quimicamente , Angiopatias Diabéticas/metabolismo , Receptores ErbB/metabolismo , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Lapatinib , Masculino , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptor ErbB-2/metabolismoRESUMO
The effect of monocrotaline-induced pulmonary hypertension on alpha(1)-adrenoceptor-mediated contractions of pulmonary artery segments was studied. In control and monocrotaline-treated rats, noradrenaline evoked concentration-dependent contractions of the pulmonary artery. There was no change in the potency and affinity of noradrenaline but the maximum response and receptor reserve were significantly reduced. Noradrenaline-induced contractions were competitively antagonized by prazosin, 2-(2-6dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) and 8-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9 dione dihydrochloride (BMY 7378) with pA(2) values of 9.64+/-0.16, 9.45+/-0.10 and 8.30+/-0.14, respectively. These antagonists also competitively antagonized noradrenaline-induced contractions of pulmonary artery segments isolated from rats with monocrotaline-induced pulmonary hypertension. The pA(2) values were 9.66+/-0.11 (prazosin), 9.62+/-0.09 (WB 4101) and 8.47+/-0.15 (BMY 7378). Chloroethylclonidine (CEC) shifted noradrenaline concentration-response curve to the right and depressed the maximum response. There was no difference between the effects of CEC in both groups. It was therefore concluded that pulmonary hypertension significantly reduced noradrenaline-induced contractions of the rat pulmonary artery without affecting the sensitivity. Studies with receptor-selective antagonists confirmed that alpha(1)D-adrenoceptor subtype is the predominant receptor subtype in the pulmonary artery and this was maintained in this disease state.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1 , Hipertensão Pulmonar/fisiopatologia , Norepinefrina/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos alfa 1 , Animais , Relação Dose-Resposta a Droga , Hipertensão Pulmonar/induzido quimicamente , Masculino , Monocrotalina/toxicidade , Prazosina/farmacologia , Artéria Pulmonar/fisiologia , Ratos , Receptores Adrenérgicos alfa 1/classificação , Receptores Adrenérgicos alfa 1/fisiologia , Vasoconstrição/fisiologiaRESUMO
Our main objective was to study the interaction of BKCa channel modulators with adrenergic agonists UK 14304 and noradrenaline (NA), acting on alpha1-adrenoceptors, in the rat aorta and how this is affected by receptor reserve. NA and UK 14304 evoked concentration-dependent contractions of the rat aorta. UK 14304 was a partial agonist relative to NA in this preparation. The BK(Ca) channel blocker tetraethylammonium (TEA, 1 mM) and opener NS 1619 (3 x 10(-5) M) modulated NA- and UK 14304-induced contractions, and were more effective on UK 14304-induced contractions. TEA (1 mM) increased the maximum response to NA and UK 14304 by about 13% and 300%, respectively, while NS 1619 (3 x 10(-5) M) reduced the maximum response to UK 14304 by about 81% compared to 31% for noradrenaline. The effect of TEA on the noradrenaline concentration-response curve was increased after treatment of the aorta with phenoxybenzamine (PBZ), an irreversible alpha1-adrenoceptor antagonist, to reduce receptor reserve. We concluded that the interaction of BKCa channel modulators with alpha1-adrenergic agonists in the rat aorta was influenced by receptor reserve.
Assuntos
Agonistas Adrenérgicos/metabolismo , Aorta Torácica/metabolismo , Canais de Potássio/metabolismo , Receptores Adrenérgicos/metabolismo , Agonistas Adrenérgicos/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas In Vitro , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Masculino , Norepinefrina/metabolismo , Norepinefrina/farmacologia , Ratos , Ratos Endogâmicos WKY , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologiaRESUMO
Polyamidoamine (PAMAM) dendrimers are cationic branch-like macromolecules that may serve as drug delivery systems for gene-based therapies such as RNA interference. For their safe use in the clinic, they should ideally only enhance drug delivery to target tissues and exhibit no adverse effects. However, little is known about their toxicological profiles in terms of their interactions with cellular signal transduction pathways such as the epidermal growth factor receptor (EGFR). The EGFR is an important signaling cascade that regulates cell growth, differentiation, migration, survival and apoptosis. Here, we investigated the impact of naked, unmodified Superfect (SF), a commercially available generation 6 PAMAM dendrimer, on the epidermal growth factor receptor (EGFR) tyrosine kinase-extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway in human embryonic kidney (HEK 293) cells. At concentrations routinely used for transfection, SF exhibited time and dose-dependent stimulation of EGFR and ERK1/2 phosphorylation whereas AG1478, a selective EGFR tyrosine kinase antagonist, inhibited EGFR-ERK1/2 signaling. SF-induced phosphorylation of EGFR for 1h was partly reversible upon removal of the dendrimer and examination of cells 24 later. Co-treatment of SF with epidermal growth factor (EGF) ligand resulted in greater EGFR stimulation than either agent alone implying that the stimulatory effects of SF and the ligand are synergistic. Dendrimer-induced stimulation of EGFR-ERK1/2 signaling could be attenuated by the antioxidants apocynin, catalase and tempol implying that an oxidative stress dependent mechanism was involved. These results show for the first time that PAMAM dendrimers, aside from their ability to improve drug delivery, can modulate the important EGFR-ERK1/2 cellular signal transduction pathway - a novel finding that may have a bearing on their safe application as drug delivery systems.
Assuntos
Dendrímeros/toxicidade , Receptores ErbB/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu), a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor) family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem) or acute, ex-vivo (10(-6), 10(-5) M) administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB) to the vasoconstrictor, norephinephrine (NE) and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y) residues (Y1221/1222, Y1248 and Y877) in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs) and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC) that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications.
Assuntos
Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/patologia , Sistema de Sinalização das MAP Quinases , Receptor ErbB-2/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Células Cultivadas , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Ratos , Ratos Wistar , Ativação Transcricional , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.
Assuntos
Complicações do Diabetes/metabolismo , Receptores ErbB/metabolismo , Miocárdio/metabolismo , Proteínas Quinases/metabolismo , Receptor ErbB-2/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Glicemia/análise , Dimerização , Imunoprecipitação , Masculino , Fosforilação , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: The transactivation of the epidermal growth factor (EGF) receptor appears to be an important central transduction mechanism in mediating diabetes-induced vascular dysfunction. Angiotensin-(1-7) [Ang-(1-7)] via its Mas receptor can prevent the development of hyperglycaemia-induced cardiovascular complications. Here, we investigated whether Ang-(1-7) can inhibit hyperglycaemia-induced EGF receptor transactivation and its classical signalling via ERK1/2 and p38 MAPK in vivo and in vitro. EXPERIMENTAL APPROACH: Streptozotocin-induced diabetic rats were chronically treated with Ang-(1-7) or AG1478, a selective EGF receptor inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in primary cultures of vascular smooth muscle cells (VSMCs). KEY RESULTS: Diabetes significantly enhanced phosphorylation of EGF receptor at tyrosine residues Y992, Y1068, Y1086, Y1148, as well as ERK1/2 and p38 MAPK in the mesenteric vasculature bed whereas these changes were significantly attenuated upon Ang-(1-7) or AG1478 treatment. In VSMCs grown in conditions of high glucose (25 mM), an Src-dependent elevation in EGF receptor phosphorylation was observed. Ang-(1-7) inhibited both Ang II- and glucose-induced transactivation of EGF receptor. The inhibition of high glucose-mediated Src-dependant transactivation of EGF receptor by Ang-(1-7) could be prevented by a selective Mas receptor antagonist, D-Pro7-Ang-(1-7). CONCLUSIONS AND IMPLICATIONS: These results show for the first time that Ang-(1-7) inhibits EGF receptor transactivation via a Mas receptor/Src-dependent pathway and might represent a novel general mechanism by which Ang-(1-7) exerts its beneficial effects in many disease states including diabetes-induced vascular dysfunction.
Assuntos
Angiotensina I/farmacologia , Receptores ErbB/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glucose/genética , Glucose/metabolismo , Hiperglicemia/complicações , Hiperglicemia/genética , Hiperglicemia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Beta1- and beta2-adrenoceptors mediate relaxation in the oviductal smooth muscle. This study examines the existence and function of beta3-adrenoceptors in the human oviduct. Ring segments of the oviduct were set up for isometric tension recording. The effect of isoprenaline and BRL 37344 on smooth muscle tone was examined. The expression of beta3-adrenoceptors in the oviduct was also examined. Isoprenaline and BRL 37344 concentration-dependently relaxed circular muscles of the oviduct. BRL 37344 was less potent than isoprenaline and was a partial agonist. Propranolol shifted isoprenaline but not BRL 37344 concentration-response curve to the right without reducing the maximum response. Cyanopindolol (1 micromol/l), a beta3-adrenoceptor antagonist, shifted the isoprenaline concentration response curve to the right. The -log K(B) value of 7.8 indicates activation of beta3-adrenoceptors by isoprenaline. mRNA for beta3-adrenoceptors was expressed in the oviduct. These results suggest that beta3-adrenoceptors, mediating relaxation, are expressed in the human oviduct.
Assuntos
Tubas Uterinas/fisiologia , Receptores Adrenérgicos beta 3/biossíntese , Agonistas de Receptores Adrenérgicos beta 3 , Antagonistas de Receptores Adrenérgicos beta 3 , Adulto , Relação Dose-Resposta a Droga , Etanolaminas/farmacologia , Tubas Uterinas/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Isoproterenol/farmacologia , Pessoa de Meia-Idade , Relaxamento Muscular/efeitos dos fármacos , Pindolol/análogos & derivados , Pindolol/farmacologia , Propranolol/farmacologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Noradrenaline-induced pulmonary artery contraction was reduced in monocrotaline-treated rats. The possibility that this could be due to alterations in the rho kinase pathway was examined in this study. A combination of nifedipine (10(-6) M) and thapsigargin (10(-6) M) attenuated noradrenaline-induced contraction significantly more in artery segments from monocrotaline-treated rats than in artery segments from control rats indicating a reduced role for calcium sensitization in artery segments from monocrotaline-treated rats. In artery segments permeabilized with ionomycin, CaCl(2) (1.25 mmol/l) produced significantly greater contraction in monocrotaline treated rats compared with control rats. Addition of noradrenaline (10(-5) M) to the bath produced further contractions in both groups. However, noradrenaline-induced contraction was less in monocrotaline-treated rats compared with controls. Y-27632 concentration dependently relaxed ring segments of pulmonary artery pre-contracted with noradrenaline (10(-5)M). The pIC(50) values were 6.46+/- 0.09 (n=5) 5.81+/- 0.06 (n=5) in control and pulmonary hypertensive rings, respectively. The maximum relaxation to Y-27632 was significantly higher in monocrotaline-treated rats. ROCK II was the predominant isoform of rho kinase expressed in the pulmonary artery. The level of expression was increased in rats treated with monocrotaline. These results would suggest that while basal rho kinase activity was elevated in monocrotaline-induced pulmonary hypertension, noradrenaline-induced contraction was attenuated, suggesting poor coupling of the receptor activation to rho kinase activation.
Assuntos
Hipertensão Pulmonar/induzido quimicamente , Monocrotalina/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Artéria Pulmonar/enzimologia , Vasoconstrição/efeitos dos fármacos , Amidas/farmacologia , Animais , Cálcio/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Hipertensão Pulmonar/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Norepinefrina/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Vasoconstritores/farmacologia , Quinases Associadas a rhoRESUMO
Vascular reactivity to vasoconstrictors has been observed in preeclamptic vessels. In this investigation, the possible role of endothelin-1 and endoperoxide/thromboxane receptor activation in the exaggerated response of the uterine vascular bed from rats with experimentally induced preeclampsia-like syndrome to noradrenaline was studied. The mean blood pressure in non-pregnant rats was 126.0 +/- 8.7 mm Hg (n = 5) while in pregnant rats, the mean blood pressure was 110.0 +/- 4.7 mm Hg (n = 5). Corresponding values in l-NAME-treated non-pregnant and pregnant rats were 167.5 +/- 6.9 mm Hg (n = 6) and 167.5 +/- 6.9 mm Hg (n = 6). These values were not significantly (P > 0.05) different from each other but were significantly (P < 0.05) different from corresponding values in control rats (not treated with l-NAME). Noradrenaline (10-10-10-6 mol) produced potent and reproducible vasoconstriction in isolated perfused rat uterine vascular bed from l-NAME-treated and untreated pregnant and non-pregnant rats. There was no significant difference in the potency of noradrenaline. However, there was an increase in the absolute maximum response to noradrenaline in uterine vascular bed from l-NAME-treated pregnant rats when compared with the other groups. Noradrenaline-induced vasoconstriction was not significantly affected by AT1-receptor antagonist, ZD 7155 or SB 209670, a potent ETA/ETB receptor antagonist. Vasoconstrictor responses to noradrenaline were however significantly reduced by indomethacin and SQ 29548 in l-NAME-treated pregnant rats. These observations would suggest that in pregnant rats treated with l-NAME, cyclooxygenase products play a significant role in noradrenaline-induced vasoconstriction of this preparation.
Assuntos
Inibidores Enzimáticos/farmacologia , Indometacina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Norepinefrina/farmacologia , Útero/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Interações Medicamentosas , Feminino , Gravidez , Ratos , Ratos Wistar , Útero/efeitos dos fármacosRESUMO
OBJECTIVE: This project was designed to study endothelium-dependent vasodilation in the uterine vascular bed during experimentally induced preeclampsia in rats. METHODS: Uterine vascular beds were isolated from non-pregnant and pregnant rats with or without treatment with adriamycin (ADR) and perfused with physiological solution. Thereafter, vasodilator responses to acetylcholine were recorded. RECORDS: Pregnant ADR-treated rats displayed symptoms of preeclampsia including hypertension and proteinuria. Blood pressure was 110.0 +/- 4.7 mm Hg (n = 5) in control pregnant rats and 136.0 +/- 5.3 mm Hg (n = 5) in ADR-treated pregnant rats, and urinary protein concentrations were 0.35 mg/ml (n = 5) and 13.2 +/- 3.6 mg/ml (n = 9), respectively. Both blood pressure and proteinuria values were significantly (p < 0.05) different between controls and ADR-treated rats. However, acetylcholine-induced dose-dependent vasodilator responses in the vascular beds were not significantly different between the pregnant and nonpregnant rats. Although acetylcholine-induced vasodilation was significantly reduced by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) in both groups, the residual response to acetylcholine was not affected by indomethacin, suggesting that prostanoids were not involved in this response. The L-NAME-resistant component, endothelium-derived hyperpolarizing factor (EDHF), was greater in ADR-treated uterine beds than in those of the controls, indicating a significant contribution from EDHF in these vessels. In the presence of an elevated external potassium ion concentration, acetylcholine produced similar vasodilator responses, indicating that the release of nitric oxide was not impaired. CONCLUSION: These results indicate that endothelium-dependent vasodilation was not impaired in this model of preeclampsia.