RESUMO
G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.
Assuntos
Autofagia/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Miostatina/farmacologia , Animais , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Camundongos , Mitocôndrias/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miostatina/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Dexmedetomidine (DEX), a selective agonist of α2-adrenergic receptors, has anti-inflammation properties and potential beneficial effects against trauma, shock, or infection. Therefore, this study aimed to investigate whether DEX might protect against multiple-organ dysfunction in a two-hit model of hemorrhage/resuscitation (HS) and subsequent endotoxemia. Eighty Wistar rats were randomized into four groups: NS (normal saline), HS/L (HS plus lipopolysaccharide), HS/L+D (HS/L plus dexmedetomidine), and HS/L+D+Y (HS/L+D plus yohimbine). Six hours after resuscitation, blood gas (PaO2) and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urine nitrogen (BUN), creatinine (Cr), TNF-α, IL-ß, IL-6, IL-8, IL-10, and nitric oxide (NO) were measured. The histopathology was assayed by staining. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels and heme oxygenase-1 (HO-1) were assayed. The PaO2 levels in HS/L rats were lower whereas the ALT, AST, BUN, Cr, TNF-α, IL-ß, IL-6, IL-8, IL-10, and NO levels were higher compared to the control group. The HS/L+D increased PaO2 and further increased IL-10 and decreased ALT, AST, BUN, Cr, TNF-α, IL-ß, IL-6, IL-8, and NO levels of the HS/L groups. In addition, the MDA in the HS/L groups increased whereas SOD activity decreased compared to the control group. Moreover, the HO-1 expression levels were increased by DEX administration in lung, liver, and kidney tissues. Lungs, livers, and kidneys of the HS/L group displayed significant damage, but such damage was attenuated in the HS/L+D group. All of the above-mentioned effects of DEX were partly reversed by yohimbine. DEX reduced multiple organ injury caused by HS/L in rats, which may be mediated, at least in part, by α2-adrenergic receptors.
Assuntos
Dexmedetomidina/uso terapêutico , Endotoxemia/tratamento farmacológico , Hemorragia/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Ressuscitação , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Endotoxemia/patologia , Hemorragia/patologia , Masculino , Insuficiência de Múltiplos Órgãos/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Fatores de TempoRESUMO
Dexmedetomidine (DEX), a selective agonist of α2-adrenergic receptors, has anti-inflammation properties and potential beneficial effects against trauma, shock, or infection. Therefore, this study aimed to investigate whether DEX might protect against multiple-organ dysfunction in a two-hit model of hemorrhage/resuscitation (HS) and subsequent endotoxemia. Eighty Wistar rats were randomized into four groups: NS (normal saline), HS/L (HS plus lipopolysaccharide), HS/L+D (HS/L plus dexmedetomidine), and HS/L+D+Y (HS/L+D plus yohimbine). Six hours after resuscitation, blood gas (PaO2) and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urine nitrogen (BUN), creatinine (Cr), TNF-α, IL-β, IL-6, IL-8, IL-10, and nitric oxide (NO) were measured. The histopathology was assayed by staining. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels and heme oxygenase-1 (HO-1) were assayed. The PaO2 levels in HS/L rats were lower whereas the ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, IL-10, and NO levels were higher compared to the control group. The HS/L+D increased PaO2 and further increased IL-10 and decreased ALT, AST, BUN, Cr, TNF-α, IL-β, IL-6, IL-8, and NO levels of the HS/L groups. In addition, the MDA in the HS/L groups increased whereas SOD activity decreased compared to the control group. Moreover, the HO-1 expression levels were increased by DEX administration in lung, liver, and kidney tissues. Lungs, livers, and kidneys of the HS/L group displayed significant damage, but such damage was attenuated in the HS/L+D group. All of the above-mentioned effects of DEX were partly reversed by yohimbine. DEX reduced multiple organ injury caused by HS/L in rats, which may be mediated, at least in part, by α2-adrenergic receptors.
Assuntos
Animais , Masculino , Ratos , Ressuscitação , Endotoxemia/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Dexmedetomidina/uso terapêutico , Hemorragia/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Fatores de Tempo , Biomarcadores/sangue , Ratos Wistar , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Endotoxemia/patologia , Modelos Animais de Doenças , Hemorragia/patologia , Insuficiência de Múltiplos Órgãos/patologiaRESUMO
We have previously demonstrated that low dose of inhaled dopamine (0.5-2 microg kg(-1) min(-1)) induces broncodilatacion in patients with acute asthma attack, suggesting that this dopamine effect is mediated by dopaminergic rather than by adrenergic receptors. To understand better these dopamine effect, rat tracheal smooth muscle was used as a model to evaluate the responses of beta2-, alpha1-, alpha2-adrenergic and DA1 and DA2 dopaminergic antagonists. Tracheal rings from male Sprague-Dawley rats (n = 90) were excised and placed in an organ bath containing modified Krebs-Ringer bicarbonate buffer at 37 degrees C, and gassed with O2 (95%) and CO2 (5%). Contractile responses were recorded with an isometric transducer in a polygraph (Letica, Spain). Contraction was induced by accumulative doses of acetylcholine (0.1, 0.3, 1, 3, 10 mM) or by electric field stimulation (10 Hz at 2 milliseconds), and accumulative doses of dopamine were added to the bath. Low concentration (0.1-0.3 mM) elicited a small initial contraction, followed by a marked relaxation. Cholinergic contraction was completely reversed at 6 mM of dopamine. This biphasic dopaminergic response was not blocked by incubation with beta2-adrenergic antagonist propranolol (0.1 microM), alpha1-antagonist, terazosin (0.1 mM), alpha2-antagonist, yohimbine (0.1 mM), or by DA2 antagonist metoclopramide (1-8 mM); DA1 antagonist SCH23390 (0.1 microM) produced a sustained increase of basal tone but did not block initial dopaminergic contraction and partially inhibited bronchodilator effect of dopamine. Dopaminergic relaxation in rat trachea is mediated by DA1 rather than by DA2 receptors; and adrenergic receptors are not involved in such dopamine-induced response. Finally, DA1 antagonist SCH23390 exerts intrinsic contractile activity on airway smooth muscle that deserves further research.
Assuntos
Dopamina/farmacologia , Contração Muscular/efeitos dos fármacos , Receptores de Dopamina D1/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Acetilcolina/administração & dosagem , Acetilcolina/farmacologia , Animais , Dopamina/administração & dosagem , Relação Dose-Resposta a Droga , Estimulação Elétrica , Técnicas In Vitro , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Traqueia/metabolismo , Vasodilatadores/administração & dosagem , Vasodilatadores/farmacologiaRESUMO
We recently observed the enhanced serine and matrix metalloproteinase (MMP) activity in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) rat and the cleavage of membrane receptors in the SHR by MMPs. We demonstrate in vivo that MMP-7 and MMP-9 injection leads to a vasoconstrictor response in microvessels of rats that is blocked by a specific MMP inhibitor (GM-6001, 1 microM). Multiple pathways may be responsible. Since the beta(2)-adrenergic receptor (beta(2)-AR) is susceptible to the action of endogenous MMPs, we hypothesize that MMPs in the plasma of SHRs are able to cleave the extracellular domain of the beta(2)-AR. SHR arterioles respond in an attenuated fashion to beta(2)-AR agonists and antagonists. Aorta and heart muscle of control Wistar rats were exposed for 24 h (37 degrees C) to fresh plasma of male Wistar and WKY rats and SHRs with and without doxycycline (30 microM) and EDTA (10 mM) to reduce MMP activity. The density of extracellular and intracellular domains of beta(2)-AR was determined by immunohistochemistry. The density of the extracellular domain of beta(2)-AR is reduced in aortic endothelial cells and cardiac microvessels of SHRs compared with that of WKY or Wistar rats. Treatment of the aorta and the heart of control Wistar rats with plasma from SHRs, but not from WKY rats, reduced the number of extracellular domains, but not intracellular domains, of beta(2)-AR in aortic endothelial cells and cardiac microvessels. MMP inhibitors (EDTA and doxycycline) prevented the cleavage of the extracellular domain. Thus MMPs may contribute to the reduced density of the extracellular domain of beta(2)-AR in blood vessels and to the increased arteriolar tone of SHRs compared with normotensive rats.
Assuntos
Hipertensão/enzimologia , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Aorta/enzimologia , Arteríolas/enzimologia , Arteríolas/fisiopatologia , Pressão Sanguínea , Western Blotting , Vasos Coronários/enzimologia , Modelos Animais de Doenças , Hipertensão/fisiopatologia , Imuno-Histoquímica , Infusões Intra-Arteriais , Masculino , Metaloproteinase 7 da Matriz/administração & dosagem , Metaloproteinase 9 da Matriz/administração & dosagem , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Fatores de Tempo , VasoconstriçãoRESUMO
The sympathetic nervous system plays an important role in wound healing, but its mechanism of action is poorly understood. The aim of this study was to investigate the effects of beta- and alpha-adrenoceptor blockade on cutaneous wound healing. Male rats were treated with propranolol (beta1- and beta2-antagonist), atenolol (beta1-antagonist), or phentolamine (alpha1- and alpha2-antagonist) dissolved in drinking water. A full-thickness excisional lesion was created and the wound area was measured. Fourteen days after wounding, lesions and adjacent skin were removed, formalin-fixed, and paraffin-embedded. Sections were stained with hematoxylin-eosin and toluidine blue, and immunostained for alpha-smooth muscle actin and proliferating cell nuclear antigen. Wound contraction was delayed in propranolol- and atenolol-treated animals but not in phentolamine-treated animals. Reepithelialization was decreased only in propranolol-treated animals. beta1- and beta2-adrenoceptor blockade delayed leukocyte migration, epidermal and connective tissue cell proliferation, myofibroblastic differentiation, and mast cell migration. The volume density of blood vessels was increased in the propranolol- and atenolol-treated animals compared with controls. The levels of matrix metalloproteases (MMP-2 and MMP-9) decreased in the propranolol- and atenolol-treated animals. alpha1- and alpha2-adrenoceptor blockade only affected leukocyte migration, epithelial and connective tissue cell proliferation, and pro-MMP-9 levels. In conclusion, beta-1 and beta-2, but not alpha-1 and alpha-2, adrenoceptor blockade delays cutaneous wound healing.
Assuntos
Modelos Animais de Doenças , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Receptores Adrenérgicos beta 1/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Cicatrização/fisiologia , Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1 , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/farmacologia , Animais , Atenolol/farmacologia , Tecido de Granulação/fisiologia , Imuno-Histoquímica , Masculino , Fentolamina/farmacologia , Propranolol/farmacologia , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Pele/lesões , Estatísticas não ParamétricasRESUMO
This study was designed to study the effects of angiotensin converting enzyme inhibitors (ACEI) following treatment with trandolapril (0.3 mg kg(-1) day(-1)) on carotid arterial responsiveness in normotensive Wistar rats. Carotid arteries were obtained from control or trandolapril-treated animals and mounted in an isolated organ bath. Reactivity to angiotensin II (Ang II), phenylephrine (Phe) and KCl was studied. Agonist concentration-response curves were constructed in either the absence or presence of the endothelium or after incubation with L-NAME (10(-6) M), HOE140 (10(-7) M) or indomethacin (10(-5) M). Trandolapril treatment decreased the Ang II and Phe potencies in carotid arteries, but did not affect the maximal response. The KCl responses (potency and Emax) were similar in both control and trandolapril-treated arteries. The absence of endothelium increased the response to both agonists in control and trandolapril-treated arteries; however, the inhibitory component from the endothelial layer of the Phe response was greater in trandolapril-treated animals than in control animals. The presence of L-NAME or HOE140 abolished the changes in the potency values of trandolapril-treated animals. The presence of indomethacin did not change the effect of trandolapril on the potency values of both agonists. We conclude that trandolapril treatment decreased the carotid arterial reactivity in normotensive rats and that this effect is endothelium-dependent. Furthermore, the involvement of B(2)-receptors and NO production, but not of prostaglandins, is suggested in this mechanism.
Assuntos
Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Artérias Carótidas/efeitos dos fármacos , Indóis/farmacologia , Fenilefrina/farmacologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Artérias Carótidas/fisiologia , Endotélio/fisiologia , Técnicas In Vitro , Indometacina/farmacologia , Masculino , Contração Muscular , NG-Nitroarginina Metil Éster/farmacologia , Peptidil Dipeptidase A/metabolismo , Ratos , Ratos WistarRESUMO
In the present study, we investigated the mechanism by which isoproterenol hyperpolarises membrane potential (MP) in Sertoli cells from seminiferous tubules of 15-day-old rat testes. Modification of MP and resistance (R0) was analysed using conventional intracellular glass microelectrodes. Isoproterenol (2 x 10(-6) M) induced an immediate and significant hyperpolarisation in the Sertoli-cell membrane. The beta2-AR antagonist, butoxamine (1 x 10(-6) M), nullified isoproterenol action. The effect of the beta1 antagonist, metoprolol (1 x 10(-6) M), was light and non-significant. Sulphonylurea glibenclamide inhibition of the K+(ATP) channels suppressed isoproterenol action, and testosterone, while depolarising Sertoli-cell MP closing the K+(ATP) channels through the PLC/PIP2 pathway, reduced beta-AR agonist-induced hyperpolarisation. Also, polycations LaCl3 and spermine reversed isoproterenol's hyperpolarisation effect, probably depolarising the membrane potential through ionic interaction neutralising the action of isoproterenol on K+(ATP) channels. Adenylate cyclase agonist forskolin (0.1 microM) rapidly hyperpolarised Sertoli-cell MP, mimicking the isoproterenol effect. These effects indicate that isoproterenol's action on K+(ATP) channel probably involves the known signalling cascade beta-AR/Gs/AC/cAMP/PKA. These results suggest that the isoproterenol-induced hyperpolarisation is mediated by the opening of K+(ATP) channels in Sertoli cells. This beta-adrenergic hyperpolarisation might play a physiological role in the modulation of MP.
Assuntos
Transportadores de Cassetes de Ligação de ATP/agonistas , Agonistas Adrenérgicos beta/farmacologia , Isoproterenol/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Células de Sertoli/metabolismo , Animais , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais KATP , Lantânio/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Células de Sertoli/efeitos dos fármacos , Testosterona/farmacologiaRESUMO
1. The role of beta2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective hbetaagonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10(-5)M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5)M ICI 118.551, a selective fbeta2antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4)M dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by fbeta2adrenoceptors, with the participation of a cAMP-dependent pathway.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Epinefrina/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Receptores Adrenérgicos beta 2/fisiologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Bucladesina/farmacologia , Galinhas , Clembuterol/farmacologia , Cinética , Proteínas Musculares/efeitos dos fármacos , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 2/efeitos dos fármacosRESUMO
This study examines the mechanisms by which the standardised ethanolic extract of propolis induces relaxation of the guinea-pig trachea in-vitro. In guinea-pig trachea with or without epithelium and contracted by histamine, the propolis extract caused reproducible and graded relaxation, with a mean EC50 value of 3.8 or 10.5 microg mL(-1) and Emax of 100%, respectively. The propolis extract-induced relaxation was markedly reduced (26+/-9 and 96+/-3%) when guinea-pig tracheas were exposed to Krebs solution containing elevated K+ in the medium (40 or 80 mM). Pre-incubation of guinea-pig tracheas with tetraethylamonium (100 mM) or with 4-aminopyridine (10mM) reduced the propolis extract-induced relaxation by 31+/-10% and 28+/-2%. Likewise, apamin (0.1 microM), charybdotoxin (0.1 microM) or iberiotoxin (0.1 microM) caused marked inhibition of propolis extract-mediated relaxation in guinea-pig trachea (percentage of inhibition: 65+/-3%, 60+/-5% and 65+/-9%, respectively). Also, glibenclamide (1 microM) inhibited the relaxant response caused by the propolis extract by 57+/-4%. Omega-conotoxin GIVA (0.1 microM) or capsaicin (1 microM) produced small but significant inhibition (30+/-5% or 47+/-7%, respectively) of the propolis extract-induced relaxation. The vasoactive intestinal peptide (VIP) antagonist D-p-Cl-Phe6,Leu17[VIP] porcine (0.1 microM) inhibited relaxation by 55+/-5%, while propranolol (1 microM) induced a parallel rightward displacement (about 20 fold) of the propolis extract concentration-response curve. Finally, the propolis extract-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-N(G)-nitroarginine (L-NOArg, 100 microM) (48+/-6%), and by the soluble guanylatecyclase inhibitormethylene blue (10 microM) (37+/-6%), whilethe moreselectivesoluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3-alquinoxalin-1-one (ODQ, 1 microM) produced only a parallel (about 3 fold) rightward displacement of the propolis extract concentration-response curve. Collectively, these results support the notion that the propolis extract-mediated relaxation in the guinea-pig trachea involves the release of nitric oxide, probably from sensory neurons, besides the activation of soluble guanylate cyclase and activation of Ca2+- and ATP-sensitive K+ channels. Furthermore, the stimulation of beta2-adrenergic and VIP receptors also seems to account for its relaxant action.