RESUMO
The G protein-coupled receptor (GPCR) signaling pathways mediated by trimeric G proteins have been extensively elucidated, but their associated regulatory mechanisms remain unclear. Parathyroid hormone (PTH)/PTH-related protein receptor (PTHR) is a GPCR coupled with Gs and Gq Gs activates adenylyl cyclases (ACs), which produces cAMP to regulate various cell fates. We previously showed that cell surface expression of PTHR was increased by its direct interaction with a subcortical cytoskeletal protein, 4.1G, whereas PTHR-mediated Gs/AC/cAMP signaling was suppressed by 4.1G through an unknown mechanism in human embryonic kidney (HEK)293 cells. In the present study, we found that AC type 6 (AC6), one of the major ACs activated downstream of PTHR, interacts with 4.1G in HEK293 cells, and the N-terminus of AC6 (AC6-N) directly and selectively binds to the 4.1/ezrin/radixin/moesin (FERM) domain of 4.1G (4.1G-FERM) in vitro. AC6-N was distributed at the plasma membrane, which was disturbed by knockdown of 4.1G. An AC6-N mutant, AC6-N-3A, in which three consecutive arginine residues are mutated to alanine residues, altered both binding to 4.1G-FERM and its plasma membrane distribution in vivo. Further, we overexpressed AC6-N to competitively inhibit the interaction of endogenous AC6 and 4.1G in cells. cAMP production induced by forskolin, an adenylyl cyclase activator, and PTH-(1-34) was enhanced by AC6-N expression and 4.1G-knockdown. In contrast, AC6-N-3A had no impact on forskolin- and PTH-(1-34)-induced cAMP productions. These data provide a novel regulatory mechanism that AC6 activity is suppressed by the direct binding of 4.1G to AC6-N, resulting in attenuation of PTHR-mediated Gs/AC6/cAMP signaling.
Assuntos
Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Adenilil Ciclases/genética , Sítios de Ligação , Membrana Celular/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Mutação , Ligação Proteica , Transdução de SinaisRESUMO
The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.
Assuntos
Actinas/fisiologia , Cílios/fisiologia , Dineínas/metabolismo , Linhagem Celular , Clatrina/fisiologia , Dinaminas , Dineínas/genética , Endocitose , Células Epiteliais , Humanos , Fosforilação , Multimerização Proteica , Retina/citologiaRESUMO
Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.
Assuntos
Ácidos Indolacéticos/farmacologia , Túbulos Renais/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fenilbutiratos/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Genetic analyses have revealed an important association between A-kinase anchoring proteins (AKAPs) and the intracellular calcium modulating system. AKAP5, also known as AKAP79/150, is an anchoring protein between PKA and voltage-dependent calcium channels, ryanodine receptor-2, phospholamban and other molecules. The aim of the present study was to elucidate the physiological importance of AKAP5 in the creation of cardiac rhythm using AKAP5-null mice. ECG analysis showed a normal sinus rhythm and a decreased responsiveness to isoproterenol in AKAP5-null mice compared with wild-type mice. Analysis of heart rate variability revealed that the R-R interval was unstable in AKAP5-null mutants and that the low-frequency components had decreased, indicating that the tonus of the sympathetic nervous system was affected. Furthermore, the atrium of the AKAP5-null mice showed a decreased positive inotropic response to isoproterenol, indicating the involvement of AKAP5 in a PKA-dependent pathway. Thus, our present study revealed that AKAP5 plays a significant role in the regulation of sympathetic nerve activities.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Encéfalo/fisiologia , Frequência Cardíaca/fisiologia , Coração/inervação , Coração/fisiologia , Sistema Nervoso Simpático/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Animais , Camundongos , Camundongos KnockoutRESUMO
The histamine system is involved in the regulation of the autonomic nervous system. We used gene-targeted mice to investigate the role of histamine receptors in the regulation of the sympathetic nervous system. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed histamine H1, H2, and H3 receptor expression in the superior cervical ganglion, which contains sympathetic nerve cell bodies. We measured the heart rate variability (HRV), the changes in the beat-to-beat heart rate, which is widely used to assess autonomic activity in the heart. H1 blockade attenuated the baroreflex-mediated changes in heart rate in wild-type (WT) mice, whereas the heart rate response to H2- and H3-specific blockers was unaffected. l-Histidine decarboxylase (HDC) expression in the superior cervical ganglion of H1R-null mice was higher than that in WT controls, whereas the enzyme levels in H2R- and H3R-null mice were not significantly different from those in the WT. All mutant mice (H1R-, H2R-, and H3R-null mice) showed normal electrocardiogram (ECG) patterns with little modification in ECG parameters and the expected response to the ß-adrenergic blocker propranolol. Similar to our findings in WT mice, H1 blockade attenuated the baroreflex-mediated heart rate change in H1R-null mice, whereas the heart rate response was unaffected in H2R- and H3R-null mice. The HRV analysis revealed relatively unstable RR intervals, an increased standard deviation of the interbeat interval (SDNN), and low-frequency (LF) component in H1R-null mice compared with the other groups, suggesting that sympathetic nerve activity was altered in H1R-null mice. Taken together, our findings indicate that H1 receptors play a major role in the regulation of sympathetic nerve activity.
Assuntos
Receptores Histamínicos H1/metabolismo , Sistema Nervoso Simpático/fisiologia , Animais , Eletrocardiografia , Deleção de Genes , Frequência Cardíaca , Histamina/metabolismo , Histidina Descarboxilase/genética , Camundongos , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/genética , Regulação para CimaRESUMO
Genetic analyses have revealed an important association between P/Q-type calcium channel activities and hereditary neurological disorders. The P/Q-type channels are composed principally of heterologous multimeric subunits including CaV2.1 and CaVß4. Of these, the ß4 subunit is thought to play a significant role in channel physiology, because a mouse line mutant in that subunit (the lethargic mouse: lh) exhibits a severe ataxic phenotype. The aim of the present study was to elucidate the physiological importance of the ß4 subunit. ECG analysis showed that the T wave was high in 8-week-old lh mutants; this may be associated with hyperkalemia. Upon pharmacological ECG analysis, 2-3-week-old lh mutants exhibited reduced responses to a ß-blocker and a muscarinic receptor antagonist. Analysis of heart rate variability revealed that the R-R interval was unstable in lh mutants and that both the low- and high-frequency components had increased in extent, indicating that the tonus of both the sympathetic and parasympathetic nervous systems was modified. Thus, our present study revealed that the ß4 subunit played a significant role in regulation of sympathetic and parasympathetic nerve activities.
Assuntos
Canais de Cálcio Tipo N/genética , Coração/inervação , Coração/fisiologia , Mutação , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Sequência de Bases , Canais de Cálcio Tipo N/metabolismo , Genótipo , Frequência Cardíaca , Camundongos , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Orexin, also known as hypocretin, is a secreted neuropeptide implicated in the regulation of sleep and food intake. In the present study, we examined the importance of orexin in regulation of the sympathetic nervous system using an orexin/ataxin-3 transgenic (OXTg) rat, which has a minimal number of orexin neurons. RT-PCR analysis identified expression of prepro-orexin and orexin receptor-1 (OX1R) in the superior cervical ganglion (SCG), and expression of another receptor (OX2R) was marginal in the wild-type rat. The orexin/ataxin-3 transgenic rat showed increased expression of OX1R and OX2R, whereas expression of prepro-orexin was undetectable, suggesting a compensatory increase in both receptors. In the ECG recording (R-R interval), orexin/ataxin-3 transgenic rats showed decreased responsiveness to the ß-adrenergic blocker propranolol. Furthermore, OXTg rats had deteriorated R-R interval regulation, indicating involvement of the orexin system in sympathetic nerve regulation. This was accompanied by decreased baroreflex and responsiveness to ß-adrenergic blocker in blood pressure recording, also suggesting involvement of the orexin system in sympathetic nerve regulation. Histological examination revealed hypotrophic changes in the transgenic heart, suggesting involvement of the orexin system in cardiac development. Taken together, our present results indicate involvement of the orexin system in sympathetic nerve control.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neuropeptídeos/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Pressão Sanguínea/fisiologia , Eletrocardiografia , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neuropeptídeos/genética , Orexinas , Ratos , Ratos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.
Assuntos
Trifosfato de Adenosina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Fibroblastos/efeitos dos fármacos , Ácidos Indolacéticos/química , Ácidos Indolacéticos/farmacologia , Doenças Mitocondriais/tratamento farmacológico , Fenilbutiratos/farmacologia , Análise de Variância , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Fibroblastos/fisiologia , Humanos , Fosforilação Oxidativa , Fenilbutiratos/química , Bibliotecas de Moléculas PequenasRESUMO
Release of growth hormone (GH) from the somatotroph is regulated by binding GH-releasing hormone (GHRH) to its cognate receptor (GHRHR), one of the members of the G protein-coupled receptor (GPCR) superfamily. Proteins bound to the carboxy (C)-terminus of GPCR have been reported to regulate intracellular trafficking and function of the receptor; however, no functionally significant protein associated with GHRHR has been reported. We have identified a protein interacting with C-kinase 1 (PICK1) as a binding partner of GHRHR. In vitro binding assay revealed the PDZ-domain of PICK1 and the last four amino acid residues of GHRHR were prerequisite for the interaction. Further, in vivo association of these proteins was confirmed. Immunostaining data of a stable cell line expressing GHRHR with or without PICK1 suggested the C-terminus of GHRHR promoted cell surface expression of GHRHR and PICK1 affected the kinetics of the cell surface expression of GHRHR. Furthermore, cAMP production assay showed the C-terminus of GHRHR is involved in the regulation of receptor activation, and the interaction of GHRHR with PICK1 may influence intensities of the signal response after ligand stimulation. Thus, the interaction of the C-terminus of GHRHR with PICK1 has a profound role in regulating the trafficking and the signaling of GHRHR. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12287FP].
Assuntos
Proteínas de Transporte/fisiologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/fisiologia , Proteínas Nucleares/fisiologia , Domínios PDZ/fisiologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/fisiologia , Transdução de Sinais/fisiologia , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto , Humanos , Masculino , Proteínas Nucleares/metabolismo , Ligação Proteica , Transporte Proteico , Ratos Sprague-DawleyRESUMO
Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death.
Assuntos
Doxorrubicina , Miócitos Cardíacos , Ratos , Animais , Miócitos Cardíacos/metabolismo , Doxorrubicina/efeitos adversos , Autofagia , Mitocôndrias/metabolismo , Antibióticos Antineoplásicos/farmacologia , Estresse Oxidativo , ApoptoseRESUMO
Myocardial infarction (MI) and subsequent adverse remodeling cause heart failure. Previously we demonstrated a role for Kit ligand (KL) in improving cardiac function post-MI. KL has two major isoforms; KL-1 is secreted whereas KL-2 is predominantly membrane bound. We demonstrate here first that KL-2-deficient mice have worse survival and an increased heart/bodyweight ratio post-MI compared to mice with reduced c-Kit receptor expression. Next we synthesized recombinant lentiviral vectors (LVs) that engineered functional expression of murine KL-1 and KL-2. For in vivo analyses, we directly injected these LVs into the left ventricle of membrane-bound KL-deficient Sl/Sl(d) or wild-type (WT) mice undergoing MI. Control LV/enGFP injection led to measurable reporter gene expression in hearts. Injection of LV/KL-2 attenuated adverse left ventricular remodeling and dramatically improved survival post-MI in both Sl/Sl(d) and WT mice (from 12 to 71% and 35 to 73%, respectively, versus controls). With regard toward beginning to understand the possible salutary mechanisms involved in this effect, differential staining patterns of Sca-1 and Ly49 on peripheral blood (PB) cells from therapeutically treated animals was found. Our data show that LV/KL-2 gene therapy is a promising treatment for MI.
Assuntos
Injeções/métodos , Lentivirus/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Fator de Células-Tronco/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Miocárdio/patologia , Fator de Células-Tronco/fisiologiaRESUMO
A highly sensitive determination method was established for catecholamines (norepinephrine (NE), epinephrine, and dopamine) with high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection. In this study, the method was applied to mouse plasma, and it was determined that only 10 microl of mouse plasma was necessary for the selective and reproducible determination of catecholamines. Studies were then conducted in acute cardiovascular effects of sodium nitroprusside, nicardipine, captopril (angiotensin-converting enzyme (ACE) inhibitor), candesartan, and olmesartan (type 1 angiotensin receptor antagonists (AT(1) antagonists)) by this method. Sodium nitroprusside and nicardipine elevated plasma NE concentrations significantly, whereas the ACE inhibitor and the AT(1) antagonists did not change plasma NE concentrations in anesthetized mice. These results suggested that angiotensin II-induced augmentation may be mainly carried through the central baroreflex pathway.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Catecolaminas/sangue , Frequência Cardíaca/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Compostos de Bifenilo , Captopril/farmacologia , Cromatografia Líquida de Alta Pressão , Imidazóis/farmacologia , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicardipino/farmacologia , Nitroprussiato/farmacologia , Oxalatos/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tetrazóis/farmacologia , Fatores de TempoRESUMO
Histamine H2 receptor (H2R) is a member of G protein-coupled receptor family. Agonist stimulation of H2R results in several cellular events including activation of adenylate cyclase and phospholipase C, desensitization of the receptor, activation of extracellular signal-regulated kinases ERK1/2, and receptor endocytosis. In this study, we identified a GTPase dynamin as a binding partner of H2R. Dynamin could associate with H2R both in vitro and in vivo. Functional analyses using dominant-negative form of dynamin (K44E-dynamin) revealed that cAMP production and the following H2R desensitization are independent of dynamin. However, the agonist-induced H2R internalization was inhibited by co-expression of K44E-dynamin. Furthermore, activation of extracellular-signal regulated kinases ERK1/2 in response to dimaprit, an H2R agonist, was attenuated by K44E-dynamin. Although H2R with truncation of 51 amino acids at its carboxy-terminus did not internalize after agonist stimulation, it still activated ERK1/2, but the degree of this activation was less than that of the wild-type receptor. Finally, K44E dynamin did not affect ERK1/2 activation induced by internalization-deficient H2R. These results suggest that the agonist-induced H2R internalization and ERK1/2 activation are partially dynamin-dependent. Furthermore, ERK1/2 activation via H2R is likely dependent of the endocytotic process rather than dynamin itself.
Assuntos
Dinaminas/metabolismo , Endocitose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos H2/efeitos dos fármacos , Receptores Histamínicos H2/metabolismo , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/biossíntese , Dimaprit/farmacologia , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptores Histamínicos H2/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Histamine H3 receptor (H3R), one of G protein-coupled receptors (GPCRs), has been known to regulate neurotransmitter release negatively in central and peripheral nervous systems. Recently, a variety of intracellular proteins have been identified to interact with carboxy (C)-termini of GPCRs, and control their intracellular trafficking and signal transduction efficiencies. Screening for such proteins that interact with the C-terminus of H3R resulted in identification of one of the chloride intracellular channel (CLIC) proteins, CLIC4. The association of CLIC4 with H3R was confirmed in in vitro pull-down assays, coimmunoprecipitation from rat brain lysate, and immunofluorescence microscopy of rat cerebellar neurons. The data from flowcytometric analysis, radioligand receptor binding assay, and cell-based ELISA indicated that CLIC4 enhanced cell surface expression of wild-type H3R, but not a mutant form of the receptor that failed to interact with CLIC4. These results indicate that, by binding to the C-terminus of H3R, CLIC4 plays a critical role in regulation of the receptor cell surface expression.
Assuntos
Membrana Celular/metabolismo , Cerebelo/metabolismo , Canais de Cloreto/metabolismo , Neurônios/metabolismo , Receptores Histamínicos H3/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células PC12 , Ratos , Ratos WistarRESUMO
The dramatic increase of oxytocin (OT) receptor (OTR) in the myometrium as well as circulating progesterone withdrawal has been thought to be the most important factor in the induction and accomplishment of parturition since delivery fails in prostaglandin F2alpha receptor (FP) knockout (FP KO) mice. The expression levels of OTR mRNA/protein were not dramatically increased in the near-term uteri of FP KO mice. However, OT-induced myometrial contractions and the concentration-response curves in FP KO in vitro were almost similar to those in wild-type (WT) mice. OT-infusion (0.3 U/day) enabled FP KO mice to experience successful delivery, and furthermore the duration until the onset was hastened by a higher dose of OT (3 U/day). The plasma progesterone levels of FP KO females were maintained at high levels, but decreased during labor by OT-infusion (3 U/day). These results suggest that OT has potentials to induce strong myometrial contractions in uterus with low expression levels of OTR and luteolysis in ovary, which enabled FP KO females to undergo successful delivery.
Assuntos
Parto Obstétrico , Ocitocina/administração & dosagem , Receptores de Prostaglandina/deficiência , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Bombas de Infusão , Trabalho de Parto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Ocitocina/farmacologia , Gravidez , Prenhez , Progesterona/sangue , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Prostaglandina/metabolismo , Contração Uterina/efeitos dos fármacosRESUMO
ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Clonagem Molecular/métodos , Proteína 4 Homóloga a Disks-Large , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Guanilato Quinases , Cobaias , Imunoprecipitação/métodos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Coelhos , Receptores de GABA-A/metabolismo , Sinapses/ultraestrutura , TransfecçãoRESUMO
We examined the effects of beta-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca(2+)-activated K(+) (K(Ca)) channels in relaxation by beta-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 micromol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 micromol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 micromol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 micromol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of beta(2)-adrenoceptors in the cells. These results suggest that Ca(2+)-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K(Ca) channels could be increased by stimulation of beta(2)-adrenoceptors.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Apamina/farmacologia , Linhagem Celular , Eletrofisiologia , Etanolaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoproterenol/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Bexiga Urinária/metabolismoRESUMO
Human thromboxane A(2) receptor (TP) consists of two alternatively spliced isoforms, TP alpha and TP beta, which differ in their cytoplasmic tails. To examine the functional difference between TP alpha and TP beta, we searched proteins bound to C termini of TP isoforms by a yeast two-hybrid system, and found that proteasome subunit alpha 7 and proteasome activator PA28 gamma interacted potently with the C terminus of TP beta. The binding of TP beta with alpha 7 and PA28 gamma was confirmed by co-immunoprecipitation and pull-down assays. MG-132 and lactacystin, proteasome inhibitors, increased cell-surface expression of TP beta, but not TP alpha. Scatchard analysis of [(3)H]SQ29548 binding revealed that the B(max) was higher in transiently TP alpha-expressing cells than TP alpha-expressing cells. In addition, TP-mediated phosphoinositide hydrolysis was clearly observed in TP alpha-, but not TP beta-expressing cells. These results suggest that TP beta binds to alpha 7 and PA28 gamma, and the cell-surface expression of TP beta is lower than that of TP alpha through the negative regulation of its membrane traffic by proteasomes.
Assuntos
Membrana Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Imunoprecipitação , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fosfatidilinositóis/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Highly active anti-retrovirus therapy (HAART) has been used to block the progression and symptoms of human immunodeficiency virus infection. Although it decreases morbidity and mortality, clinical use of HAART has also been linked to various adverse effects such as severe cardiomyopathy resulting from compromised mitochondrial functioning. However, the mechanistic basis for these effects remains unclear. Here, we demonstrate that a key component of HAART, 3ê-azido-3ê-deoxythymidine (AZT), particularly, its active metabolite AZT-triphosphate (AZT-TP), caused mitochondrial dysfunction, leading to induction of cell death in H9c2 cells derived from rat embryonic myoblasts, which serve as a model for cardiomyopathy. Specifically, treatment with 100µM AZT for 48h disrupted the mitochondrial tubular network via accumulation of AZT-TP. The mRNA expression of dynamin-related protein (Drp)1 and the Drp1 receptor mitochondrial fission factor (Mff) was upregulated whereas that of optic atrophy 1 (Opa1) was downregulated following AZT treatment. Increased mitochondrial translocation of Drp1, Mff upregulation, and decreased functional Opa1 expression induced by AZT impaired the balance of mitochondrial fission vs. fusion. These data demonstrate that AZT-TP causes cell death by altering mitochondrial dynamics.
Assuntos
Fármacos Anti-HIV/toxicidade , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , Zidovudina/toxicidade , Animais , Fármacos Anti-HIV/efeitos adversos , Cardiotoxicidade/etiologia , Linhagem Celular , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Ratos , Zidovudina/efeitos adversosRESUMO
Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.