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1.
Life Sci ; 256: 117986, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32585245

RESUMO

AIMS: HSP70, a molecular chaperone, helps to maintain proteostasis. In muscle biology, however, evidence suggests HSP70 to have a more versatile range of functions, as genetic deletion of its inducible genes impairs Ca2+ handling, and consequently, cardiac and skeletal muscle contractility. Still, it is unknown whether HSP70 is involved in vascular reactivity, an intrinsic physiological mechanism of blood vessels. Therefore, we designed this study to test the hypothesis that proper vascular reactivity requires the assistance of HSP70. MAIN METHODS: We performed functional studies in a wire-myograph using thoracic aorta isolated from male Sprague Dawley rats. Experiments were conducted with and without an HSP70 inhibitor as well as in heat-stressed vessels. The expression levels of HSP70 were evaluated with Western blotting. NO and ROS levels were assessed with fluorescence microscopy. KEY FINDINGS: We report that blockade of HSP70 weakens contraction in response to phenylephrine (dose-response) in the aorta. Additionally, we demonstrated that inhibition of HSP70 affects the amplitude of the fast and of the slow components of the time-force curve. Corroborating these findings, we found that inhibition of HSP70, in vessels over-expressing this protein, partly rescues the contractile phenotype of aortic rings. Furthermore, we show that blockade of HSP70 facilitates relaxation in response to acetylcholine and clonidine without affecting the basal levels of NO and ROS. SIGNIFICANCE: Our work introduces an additional physiological role for HSP70, the assistance of vascular reactivity, which highlights this protein as a new player in vascular physiology, and therefore, uncovers a promising research avenue for vascular diseases.


Assuntos
Aorta Torácica/fisiologia , Endotélio Vascular/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Músculo Liso Vascular/fisiologia , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/agonistas , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fenilefrina/farmacologia , Nucleosídeos de Purina/farmacologia , Ratos , Ratos Sprague-Dawley , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia
2.
Nature ; 581(7807): 204-208, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405000

RESUMO

It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by the α9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Imunidade Humoral/imunologia , Baço/imunologia , Baço/inervação , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Neurônios Adrenérgicos/metabolismo , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Hemocianinas/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária , Masculino , Camundongos , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Estresse Psicológico/imunologia , Estresse Psicológico/metabolismo , Linfócitos T/imunologia
3.
Am J Physiol Heart Circ Physiol ; 318(6): H1371-H1378, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32330091

RESUMO

Microvascular dysfunction often precedes other age-related macrovascular conditions and predicts future cardiovascular risk. Sirtuin 1 (Sirt1) has recently emerged as a protein that protects the vasculature and reduces the risk of cardiovascular diseases. We tested the hypothesis that lower Sirt1 during childhood is associated with a reduced microvascular function during adulthood. Thirty-four adults (34 ± 3 yr) from the Augusta Heart Study returned to participate in the present clinical observational study. Sirt1 was assessed in samples collected during both adulthood and participants' childhood (16 ± 3 yr), and data were divided based on childhood Sirt1 concentrations: <3 ng/dL (LowCS; n = 16) and ≥3 ng/dL (HighCS; n = 18). MVF was evaluated in all of the adults using laser-Doppler flowmetry coupled with three vascular reactivity tests: 1) local thermal hyperemia (LTH), 2) post-occlusive reactive hyperemia (PORH), and 3) iontophoresis of acetylcholine (ACh). The hyperemic response to LTH was significantly (P ≤ 0.044) lower in the LowCS than in the HighCS group. Similarly, the LowCS also exhibited an ameliorated (P ≤ 0.045) response to the PORH test and lower (P ≤ 0.008) vasodilation in response to iontophoresis of ACh when compared with the HighCS. Positive relationships were identified between childhood Sirt1 and all MVF reactivity tests (r≥0.367, P ≤ 0.004). Novel observations suggest that lower Sirt1 during childhood is associated with premature microvascular dysfunction in adulthood. These findings provide evidence that Sirt1 may play a critical role in microvascular function and have therapeutic potential for the prevention of age-associated vascular dysfunction in humans.NEW & NOTEWORTHY With a longitudinal cohort, novel observations from the present study demonstrate that individuals who had lower Sirt1 early in life exhibit premature microvascular dysfunction during adulthood and may be at higher risk to develop CVD. These results provide experimental evidence that Sirt1 may play an important role in microvascular function with age and represent a potential therapeutic target to prevent premature vascular dysfunction.


Assuntos
Hiperemia/fisiopatologia , Microcirculação/fisiologia , Microvasos/fisiologia , Sirtuína 1/sangue , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Adolescente , Adulto , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Hiperemia/sangue , Fluxometria por Laser-Doppler , Masculino , Microcirculação/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Adulto Jovem
4.
PLoS Pathog ; 16(4): e1008396, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32243475

RESUMO

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.


Assuntos
Antinematódeos/farmacologia , Ascaris suum/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Faringe/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Ascaris suum/efeitos dos fármacos , Ascaris suum/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Helminto/genética , Faringe/efeitos dos fármacos , Receptores Nicotínicos/genética
5.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L1036-L1055, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32130030

RESUMO

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.


Assuntos
Actinina/genética , Mecanotransdução Celular , Músculo Liso/metabolismo , Paxilina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Traqueia/metabolismo , Acetilcolina/farmacologia , Actinina/metabolismo , Animais , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Cães , Feminino , Regulação da Expressão Gênica , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Paxilina/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Miosinas de Músculo Liso/genética , Miosinas de Músculo Liso/metabolismo , Traqueia/efeitos dos fármacos
6.
Nat Commun ; 11(1): 1073, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103010

RESUMO

Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.


Assuntos
Acetilcolina/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Músculo Esquelético/inervação , Atrofia Muscular/patologia , Receptores Nicotínicos/metabolismo , Acetilcolina/análogos & derivados , Acetilcolina/farmacologia , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Conexina 43/metabolismo , Conexinas/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo
7.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L900-L907, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101015

RESUMO

The hyperconstriction of airway smooth muscle (ASM) is the main driving mechanism during an asthmatic attack. The airway lumen is reduced, resistance to airflow increases, and normal breathing becomes more difficult. The tissue contraction can be temporarily relieved by using bronchodilator drugs, which induce relaxation of the constricted airways. In vitro studies indicate that relaxation of isolated, precontracted ASM is induced by mechanical oscillations in healthy subjects but not in asthmatic subjects. Further, short-term acute asthmatic subjects respond to superimposed pressure oscillations (SIPO) generated in the range of 5-15 Hz with ~50% relaxation of preconstricted sensitized airways. Mechanical oscillations, and specifically SIPO, are not widely characterized in asthmatic models. The objective of this in vivo study is to determine the effects of a range of oscillation patterns similar to our previous acute study differing from normal breathing. Both healthy and sensitized mice were observed, with their responses to SIPO treatments measured during induced bronchoconstriction resulting from acetylcholine (Ach) challenge. SIPO-generated results were compared with data from treatments using the bronchorelaxant isoproterenol (ISO). The study shows that SIPO in the range of 5-20 Hz induces relaxation in chronic sensitized airways, with significant improvements in respiratory parameters at SIPO values near 1.7 cmH2O irrespective of the frequency of generation.


Assuntos
Asma/terapia , Pulmão/imunologia , Músculo Liso/imunologia , Acetilcolina/farmacologia , Alérgenos/administração & dosagem , Animais , Antígenos de Plantas/administração & dosagem , Aspergillus/química , Aspergillus/imunologia , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Fenômenos Biomecânicos/imunologia , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/farmacologia , Modelos Animais de Doenças , Feminino , Fungos/química , Fungos/imunologia , Isoproterenol/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/patologia , Extratos Vegetais/administração & dosagem , Pressão , Pyroglyphidae/química , Pyroglyphidae/imunologia , Testes de Função Respiratória
8.
Braz J Med Biol Res ; 53(2): e9304, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049102

RESUMO

Metabolic syndrome is a multifaceted condition associated with a greater risk of various disorders (e.g., diabetes and heart disease). In a rat model of metabolic syndrome, an acute in vitro application of rosuvastatin causes relaxation of aortic rings. Since the outcome of a subchronic rosuvastatin treatment is unknown, the present study explored its effect on acetylcholine-induced vasorelaxation of aortic rings from rats with metabolic syndrome. Animals were submitted to a 16-week treatment, including a standard diet, a cafeteria-style diet (CAF-diet), or a CAF-diet with daily rosuvastatin treatment (10 mg/kg). After confirming the development of metabolic syndrome in rats, aortic segments were extracted from these animals (those treated with rosuvastatin and untreated) and the acetylcholine-induced relaxant effect on the corresponding rings was evaluated. Concentration-response curves were constructed for this effect in the presence/absence of L-NAME, ODQ, KT 5823, 4-aminopyridine (4-AP), tetraethylammonium (TEA), apamin plus charybdotoxin, glibenclamide, indomethacin, clotrimazole, and cycloheximide pretreatment. Compared to rings from control rats, acetylcholine-induced vasorelaxation decreased in rings from animals with metabolic syndrome, and was maintained at a normal level in animals with metabolic syndrome plus rosuvastatin treatment. The effect of rosuvastatin was inhibited by L-NAME, ODQ, KT 5823, TEA, apamin plus charybdotoxin, but unaffected by 4-AP, glibenclamide, indomethacin, clotrimazole, or cycloheximide. In conclusion, the subchronic administration of rosuvastatin to rats with metabolic syndrome improved the acetylcholine-induced relaxant response, involving stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel pathway.


Assuntos
Acetilcolina/farmacologia , Aorta/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Síndrome Metabólica/fisiopatologia , Rosuvastatina Cálcica/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Masculino , Ratos , Ratos Wistar , Vasodilatadores
9.
Am J Respir Cell Mol Biol ; 62(5): 645-656, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31913659

RESUMO

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle on contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is underinvestigated. SLK (Ste20-like kinase) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated Plk1 (polo-like kinase 1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the nonphosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Pulmão/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Polimerização , Proteínas Serina-Treonina Quinases/metabolismo , Acetilcolina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Adulto , Biocatálise/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Feminino , Histamina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serotonina/farmacologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
10.
Am J Physiol Lung Cell Mol Physiol ; 318(2): L287-L295, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747299

RESUMO

TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.


Assuntos
Anoctamina-1/agonistas , Anoctamina-1/metabolismo , Pulmão/metabolismo , Tono Muscular , Músculo Liso/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Acetilcolina/farmacologia , Animais , Anoctamina-1/genética , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Cloreto de Metacolina/farmacologia , Contração Muscular/efeitos dos fármacos , Tono Muscular/efeitos dos fármacos , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Toxicol Lett ; 321: 21-31, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31830555

RESUMO

Nerve agents inhibit acetylcholinesterase (AChE), leading to a build-up of acetylcholine (ACh) and overstimulation at cholinergic synapses. Current post-exposure nerve agent treatment includes atropine to treat overstimulation at muscarinic synapses, a benzodiazepine anti-convulsant, and an oxime to restore the function of AChE. Aside from the oxime, the components do not act directly to reduce the overstimulation at nicotinic synapses. The false transmitters acetylmonoethylcholine (AMECh) and acetyldiethylcholine (ADECh) are analogs of ACh, synthesised similarly at synapses. AMECh and ADECh are partial agonists, with reduced activity compared to ACh, so it was hypothesised the false transmitters could reduce overstimulation. Synthetic routes to AMECh and ADECh, and their precursors, monoethylcholine (MECh) and diethylcholine (DECh), were devised, allowing them to be produced easily on a laboratory-scale. The mechanism of action of the false transmitters was investigated in vitro. AMECh acted as a partial agonist at human muscarinic (M1 and M3) and muscle-type nicotinic receptors, and ADECh was a partial agonist only at certain muscarinic subtypes. Their precursors acted as antagonists at muscle-type nicotinic, but not muscarinic receptors. Administration of MECh and DECh improved neuromuscular function in the soman-exposed guinea-pig hemi-diaphragm preparation. False transmitters may therefore help reduce nerve agent induced overstimulation at cholinergic synapses.


Assuntos
Acetilcolina/análogos & derivados , Antídotos/farmacologia , Colina/análogos & derivados , Inibidores da Colinesterase/envenenamento , Diafragma/inervação , Agentes Neurotóxicos/envenenamento , Neurotransmissores/farmacologia , Intoxicação por Organofosfatos/tratamento farmacológico , Soman/envenenamento , Sinapses/efeitos dos fármacos , Acetilcolina/síntese química , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Antídotos/síntese química , Células CHO , Linhagem Celular Tumoral , Colina/síntese química , Colina/farmacologia , Cricetulus , Agonismo Parcial de Drogas , Cobaias , Humanos , Masculino , Neurotransmissores/síntese química , Intoxicação por Organofosfatos/enzimologia , Intoxicação por Organofosfatos/fisiopatologia , Receptores Colinérgicos/efeitos dos fármacos , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapses/enzimologia
12.
Plant Biol (Stuttg) ; 22(3): 357-365, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31811780

RESUMO

Acetylcholine (ACh) is believed to improve plant growth. However, regulation at biochemical and molecular levels is largely unknown. The present study investigated the impact of exogenously applied ACh (10 µm) on growth and chlorophyll metabolism in hydroponically grown Nicotiana benthamiana under salt stress (150 mm NaCl). Salinity reduced root hydraulic conductivity while ACh-treated seedlings exhibited a significant increase, resulting in increased relative water content. Salinity induced a reduction in chlorophyll biosynthetic intermediates, such as protoporphyrin-IX, Mg-photoporphyrin-IX and protochlorophyllide, which were significantly ameliorated in the presence of ACh. This influence of ACh on chlorophyll synthesis was confirmed by up-regulation of HEMA1, CHLH, CAO and POR genes. Gas exchange parameters, i.e. stomatal conductance, internal CO2 concentration and transpiration rate, increased with ACh, thereby alleviating the salinity effects on photosynthesis. In addition, the salinity-induced enhancement of lipid peroxidation declined after ACh treatment through modulation of the activity of the assayed antioxidant enzymes (superoxide dismutase and peroxidase). Importantly, ACh significantly reduced the uptake of Na and increased uptake of K, resulting in a decline in the Na/K ratio. Results of the present study indicate that ACh can be effective in ameliorating NaCl-induced osmotic stress, altering chlorophyll metabolism and thus photosynthesis by maintaining ion homeostasis, hydraulic conductivity and water balance.


Assuntos
Acetilcolina , Fotossíntese , Estresse Salino , Plântula , Tabaco , Acetilcolina/farmacologia , Clorofila/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Estresse Salino/efeitos dos fármacos , Plântula/efeitos dos fármacos , Tabaco/efeitos dos fármacos
13.
Int J Radiat Biol ; 96(2): 236-244, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31633438

RESUMO

Purpose: Pharmacological medications can reduce the radiation damage in the organism when applied in the stage before or after exposure to radiation. Cholinergic drugs are a category of pharmaceutical agents acting on the neurotransmitter acetylcholine, the primary neurotransmitter in the parasympathetic nervous system. In this investigation, some gamma radiation interaction parameters namely mass attenuation coefficients (µρ), effective atomic number (Zeff) and electron densities (Nel) of 12 cholinergic system drugs have been calculated in the energy range 1 KeV-100 GeV. In addition, gamma-ray energy absorption (EABF) and exposure (EBF) of buildup factors have been computed using the five-parameter geometric progression (G-P) fitting formula for investigated drugs in the energy range 0.015-15 MeV, and for penetration depths up to 40 mean free path (mfp).Materials and methods: In order to perform these calculations, data obtained from WinXCom computer program were used. The computed µρ values were then used to calculate the effective atomic numbers and electron density of the investigated drugs. To compute the buildup factors, the G-P fitting parameters were determined by the method of interpolation from the equivalent atomic number, 'Zeq'Results and Conclusions: It has been concluded that effective atomic number and electron density of malathion is bigger than the other drugs and the variations in values of Zeff and Nel for all drugs depend on chemical compositions and photon energy where the K-absorption edge of elements may affect the energy dependence of Zeff and Nel. It should also be noted that the buildup of photons is less in malathion and carbachol and is more in tabun and parathion compared with other drugs. Photon interaction parameters evaluated in the present study may be beneficial in radiation dosimetry and therapy.


Assuntos
Acetilcolina/farmacologia , Acetilcolina/efeitos da radiação , Colinérgicos/farmacologia , Colinérgicos/efeitos da radiação , Raios gama , Algoritmos , Carbacol/farmacologia , Carbacol/efeitos da radiação , Cloro/química , Elétrons , Malation/farmacologia , Malation/efeitos da radiação , Modelos Estatísticos , Organofosfatos/farmacologia , Organofosfatos/efeitos da radiação , Paration/farmacologia , Paration/efeitos da radiação , Fósforo/química , Fótons , Probabilidade , Doses de Radiação , Radiometria , Espalhamento de Radiação , Software
14.
Am J Physiol Regul Integr Comp Physiol ; 318(1): R148-R155, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31577152

RESUMO

Despite remission of clinical symptoms postpartum, women who have had preeclampsia demonstrate microvascular endothelial dysfunction, mediated in part by increased sensitivity to angiotensin II (ANG II). Angiotensin-(1-7) [Ang-(1-7)] is an endogenous inhibitor of the actions of ANG II and plausible druggable target in women who had preeclampsia. We therefore examined the therapeutic potential of Ang-(1-7) in the microvasculature of women with a history of preeclampsia (PrEC; n = 13) and parity-matched healthy control women (HC; n = 13) hypothesizing that administration of Ang-(1-7) would increase endothelium-dependent dilation and nitric oxide (NO)-dependent dilation and decrease ANG II-mediated constriction in PrEC. Using the cutaneous microcirculation, we assessed endothelium-dependent vasodilator function in response to graded infusion of acetylcholine (ACh; 10-7 to 102 mmol/L) in control sites and sites treated with 15 mmol/L NG-nitro-l-arginine methyl ester (l-NAME; NO-synthase inhibitor), 100 µmol/L Ang-(1-7), or 15 mmol/L l-NAME + 100 µmol/L Ang-(1-7). Vasoconstrictor function was measured in response to ANG II (10-20-10-4 mol/L) in control sites and sites treated with 100 µmol/L Ang-(1-7). PrEC had reduced endothelium-dependent dilation (P < 0.001) and NO-dependent dilation (P = 0.04 vs. HC). Ang-(1-7) coinfusion augmented endothelium-dependent dilation (P < 0.01) and NO-dependent dilation (P = 0.03) in PrEC but had no effect in HC. PrEC demonstrated augmented vasoconstrictor responses to ANG II (P < 0.01 vs. HC), which was attenuated by coinfusion of Ang-(1-7) (P < 0.001). Ang-(1-7) increased endothelium-dependent vasodilation via NO synthase-mediated pathways and attenuated ANG II-mediated constriction in women who have had preeclampsia, suggesting that Ang-(1-7) may be a viable therapeutic target for improved microvascular function in women who have had a preeclamptic pregnancy.


Assuntos
Angiotensina I/farmacologia , Endotélio Vascular/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Pré-Eclâmpsia , Acetilcolina/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Gravidez
15.
Hypertension ; 75(1): 218-228, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31760883

RESUMO

Extracellular vesicles (EVs) have been described as novel biomarkers and bioactivators in vascular dysfunction in hypertension. However, the mechanism(s) by which EVs affect vascular function is unknown. To examine the effects of EVs on endothelial-dependent vasodilation (acetylcholine), we isolated circulating EVs from platelet-poor plasma using a low centrifugation speed (17 000g) and mesenteric resistance arteries from 12-week-old normotensive WKYs (Wistar-Kyoto rats) and SHRs (spontaneously hypertensive rats). Arteries were cannulated on a pressure myograph, and EVs were added to the vessel lumen and circulating bath. We found that circulating EVs from normotensive WKY reduced vasodilation of normotensive WKY arteries but had no effect on hypertensive SHR arteries. In contrast, EVs from hypertensive SHR failed to reduce vasodilation of arteries from both WKY and SHR. The restraining effect on vasodilation by EVs from normotensive WKY may be mediated by inhibition of eNOS (endothelial NO synthase), as addition of L-nitro-arginine methyl ester did not provide any additive effect. Moreover, circulating EVs from normotensive 6-week-old SHR-an age where SHRs have not yet developed hypertension-had similar restraining effect on vasodilation. In addition, delipidation of EVs did not alter the restraining effect of EVs from WKY but did restore the restraining effect of EVs from SHR. Finally, EVs from normotensive humans also restrained vasodilation of normotensive mouse arteries-an effect not observed in EVs from hypertensive humans. Taken together, our data support a vasoactive role of EVs that is altered in hypertension.


Assuntos
Endotélio Vascular/fisiopatologia , Vesículas Extracelulares/metabolismo , Hipertensão/fisiopatologia , Vasodilatação/fisiologia , Acetilcolina/farmacologia , Animais , Pressão Sanguínea , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Hipertensão/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
16.
J Trace Elem Med Biol ; 57: 40-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31557574

RESUMO

BACKGROUND: Mercury is an established environmental toxicant reported to cause reproductive disorders in women, however, its direct action on myometrial activity is yet to be understood. Earlier we have reported the underlying mechanism of mercury-induced myometrial contractions following in vitro exposure; however, no such information on the effect of mercury on myometrial activity following in vivo exposure is available, therefore, the present study was undertaken. OBJECTIVE: Present study was designed to evaluate the effect of mercury on myometrial activity following in vivo exposure of rats and unravel the possible underlying mechanism. METHODS: Female Wistar rats were orally exposed to mercury (5, 50 and 500 µg/L in drinking water) for 28 days to investigate the toxicodynamics of mercuric chloride (HgCl2)-induced alterations in myometrial activity. Response of the isolated myometrial strips to different spasmogens was recorded using polyphysiograph. Blood and uterine calcium, mercury, iron and zinc levels were estimated by atomic absorption spectrophotometry. Blood biochemicals and serum hormonal profiles (estradiol, progesterone) were also determined. RESULTS: No systemic toxicity of mercury was observed in any of the treatment groups (5, 50 and 500 µg/L) in terms of alterations in body weight, organ weights, blood biochemical parameters including hormonal profile. Interestingly, mercury at 5 µg/L concentration significantly increased the receptor-dependent (PGF2α-induced) and receptor-independent (CaCl2-induced and high K+-depolarizing solution-induced) myometrial contractions and it was coupled with corresponding increase in the uterine calcium levels. However, mercury at higher dose levels (50 and 500 µg/L) did not significantly alter the myometrial response. CONCLUSION: Our results evidently suggest that mercury at low level (5 µg/L) produced detrimental effect on myometrial activity by altering calcium entry into the smooth muscle and/or the release of calcium from intracellular stores without causing any apparent systemic toxicity in rats.


Assuntos
Mercúrio/sangue , Acetilcolina/farmacologia , Animais , Sinalização do Cálcio/fisiologia , Estradiol/sangue , Feminino , Progesterona/sangue , Ratos , Ratos Wistar , Contração Uterina/efeitos dos fármacos , Útero/metabolismo
17.
J Pharmacol Sci ; 142(2): 60-68, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31843508

RESUMO

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.


Assuntos
Acetaminofen/farmacologia , Antipiréticos/farmacologia , Asma/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Acetilcolina/química , Acetilcolina/farmacologia , Animais , Asma/tratamento farmacológico , Brônquios/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Nifedipino/farmacologia , Potássio/metabolismo
18.
Medicina (Kaunas) ; 55(12)2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817916

RESUMO

Background and Objectives: The potent, endothelium-independent, vasorelaxant effect of ethyl rosmarinate, an ester derivative of rosmarinic acid, makes it of interest as an alternative therapeutic agent for use in hypertension. This study was designed to investigate the effect of ethyl rosmarinate on Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. Materials and Methods: L-NAME was given orally to male Wistar rats for 6 weeks to induce hypertension concurrently with treatment of ethyl rosmarinate at 5, 15, or 30 mg/kgor enalapril at 10 mg/kg Systolic blood pressure (SBP), heart rate, and body weight of all experimental groups were recorded weekly, while the vascular sensitivity and histological changes of the aorta were evaluated at the end of the experiment. Results: For all treatment groups, the data indicated that ethyl rosmarinate significantly attenuated the SBP in hypertensive rats induced by L-NAME, with no significant differences in heart rate and body weight. In addition, the response of vascular sensitivity to acetylcholine (ACh) was improved but there was no significant difference in the response to sodium nitroprusside (SNP). Furthermore, the sensitivity of the aorta to phenylephrine (PE) was significantly decreased. The thickness of the aortic wall did not differ between groups but the expression of endothelial nitric oxide synthase (eNOS) was increased in ethyl rosmarinate- and enalapril-treated groups compared with the hypertensive group. Conclusions: Ethyl rosmarinate is an interesting candidate as an alternative treatment for hypertension due to its ability to improve vascular function and to increase the expression of eNOS similar to enalapril which is a drug commonly used in hypertension.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/efeitos adversos , Hipertensão/induzido quimicamente , NG-Nitroarginina Metil Éster/efeitos adversos , Acetilcolina/farmacologia , Administração Oral , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Cinamatos/uso terapêutico , Depsídeos/uso terapêutico , Enalapril/farmacologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster/administração & dosagem , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Vasodilatadores/farmacologia
19.
PLoS One ; 14(10): e0224367, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31671117

RESUMO

G protein coupled receptors (GPCRs) participate in the majority of signal transduction processes in the body. Specifically, the binding of an external agonist promotes coupling of the GPCR to its G protein and this, in turn, induces downstream signaling. Recently, it was shown that agonist binding to the M2 muscarinic receptor (M2R) and to other GPCRs is voltage dependent. Here we examine, whether the coupling of the M2R to its G protein is also voltage-dependent. We first show, in Xenopus oocytes, that the activity of the M2R in the absence of agonist (constitutive activity) can be used to report the coupling. We then show that the coupling is, by itself, voltage dependent. This novel finding is of physiological importance, as it shows that the actual signal transduction, whose first step is the coupling of the GPCR to its cognate G protein, is voltage dependent.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Acetilcolina/farmacologia , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Oócitos , Técnicas de Patch-Clamp , Receptor Muscarínico M2/fisiologia , Receptores Acoplados a Proteínas-G/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Xenopus , Xenopus laevis
20.
Nat Commun ; 10(1): 5280, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754098

RESUMO

Neocortical choline acetyltransferase (ChAT)-expressing interneurons are a subclass of vasoactive intestinal peptide (ChAT-VIP) neurons of which circuit and behavioural function are unknown. Here, we show that ChAT-VIP neurons directly excite neighbouring neurons in several layers through fast synaptic transmission of acetylcholine (ACh) in rodent medial prefrontal cortex (mPFC). Both interneurons in layers (L)1-3 as well as pyramidal neurons in L2/3 and L6 receive direct inputs from ChAT-VIP neurons mediated by fast cholinergic transmission. A fraction (10-20%) of postsynaptic neurons that received cholinergic input from ChAT-VIP interneurons also received GABAergic input from these neurons. In contrast to regular VIP interneurons, ChAT-VIP neurons did not disinhibit pyramidal neurons. Finally, we show that activity of these neurons is relevant for behaviour and they control attention behaviour distinctly from basal forebrain ACh inputs. Thus, ChAT-VIP neurons are a local source of cortical ACh that directly excite neurons throughout cortical layers and contribute to attention.


Assuntos
Atenção/efeitos dos fármacos , Colinérgicos/farmacologia , Interneurônios/fisiologia , Córtex Pré-Frontal/metabolismo , Acetilcolina/farmacologia , Animais , Atenção/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Colina O-Acetiltransferase/metabolismo , Feminino , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Masculino , Camundongos da Linhagem 129 , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Córtex Pré-Frontal/citologia , Ratos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo
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