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1.
J Am Coll Cardiol ; 79(24): 2367-2378, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35710187

RESUMO

BACKGROUND: Heterogeneity in diagnostic criteria and provocation protocols has posed challenges in understanding the safety of coronary provocation testing with intracoronary acetylcholine (ACh) for the contemporary diagnosis of epicardial and microvascular spasm. OBJECTIVES: We examined the safety of testing and subgroup differences in procedural risks based on ethnicity, diagnostic criteria, and provocation protocols. METHODS: PubMed and Embase were searched in November 2021 to identify original articles reporting procedural complications associated with intracoronary ACh administration. The primary outcome was the pooled estimate of the incidence of major complications including death, myocardial infarction, ventricular tachycardia/fibrillation, and shock. RESULTS: A total of 16 studies with 12,585 patients were included in the meta-analysis. The overall pooled estimate of the incidence of major complications was 0.5% (95% CI: 0.0%-1.3%) without any reports of death. Exploratory subgroup analyses revealed that the pooled incidence of major complications was significantly higher in the studies that followed the contemporary diagnosis criteria for epicardial spasm defined as ≥90% diameter reduction (1.0%; 95% CI: 0.3%-2.0%) but significantly lower in Western populations (0.0%; 95% CI: 0.0%-0.45%). The rate of positive epicardial spasm and the incidence of major complications were similar between provocation protocols using the maximum ACh doses of 100 µg and 200 µg. CONCLUSIONS: Intracoronary ACh administration for the contemporary diagnosis of epicardial and microvascular spasm is a safe procedure. Moreover, excellent safety records are observed in Western populations primarily presenting with myocardial ischemia and/or infarction with nonobstructive coronary arteries. This study will help standardize ACh testing to improve clinical diagnosis and ensure procedural safety.


Assuntos
Acetilcolina , Vasoespasmo Coronário , Acetilcolina/efeitos adversos , Angiografia Coronária/métodos , Vasoespasmo Coronário/induzido quimicamente , Vasoespasmo Coronário/diagnóstico , Vasos Coronários/diagnóstico por imagem , Humanos , Metanálise como Assunto , Espasmo , Fibrilação Ventricular
2.
Front Immunol ; 13: 893844, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35711456

RESUMO

Acetylcholine (ACh) from neuronal and non-neuronal sources plays an important role in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection and therefore sought to determine whether this also plays a role in allergic inflammation. Rora Cre+ Chat LoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues were analyzed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 h after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in a more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for the release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing the expression of IL-5, IL-13, and subsequent eosinophilia. Depletion of ACh reduced macrophages with an alternatively activated M2 phenotype and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia but suppressing neutrophilia through reduced chemokine expression.


Assuntos
Eosinofilia , Pneumonia , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Imunidade Inata , Inflamação/metabolismo , Interleucina-33/metabolismo , Pulmão , Linfócitos , Camundongos
3.
Cells ; 11(11)2022 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-35681486

RESUMO

Irritable bowel syndrome (IBS) is a chronic functional bowel disorder characterized by intestinal dysmotility. Changes in intestinal microbiota (dysbiosis) can lead to alterations in neuro-muscular functions in the gut. Toll-like receptors (TLRs) 2 and 4 recognize intestinal bacteria and are involved in the motor response induced by gastrointestinal (GI) neurotransmitters. Acetylcholine (ACh) is a well-known neurotransmitter involved in the regulation of GI motility. This study aimed to evaluate the role of TLR2 and TLR4 in the intestinal motor-response induced by ACh in the mouse ileum, as well as the expression and function of the muscarinic and nicotinic ACh receptors. Muscle contractility studies showed that the contractions induced by ACh were significantly lower in TLR2-/- and TLR4-/- with respect to WT mice. In WT mice, the contractions induced by ACh were reduced in the presence of AF-DX AF-DX 116 (a muscarinic ACh receptor (mAChR) M2 antagonist), 4-DAMP (a mAChR M3 antagonist), mecamylamine (a nicotinic AChR receptor (nAChR) α3ß4 antagonist) and α-bungarotoxin (a nAChR α7 antagonist). In TLR2-/- mice, the contractions induced by ACh were increased by AF-DX 116 and mecamylamine. In TLR4-/- mice, the contractions induced by ACh were reduced by α-bungarotoxin and 4-DAMP. The mRNA and protein expressions of M3 and α3 receptors were diminished in the ileum from TLR2-/- and TLR4-/- with respect to WT mice. However, the levels of mRNA and protein of ß4 were diminished only in TLR4-/- but not in TLR2-/- mice. In conclusion, our results show that TLR2 and TLR4 modulates the motor responses to ACh in the mouse ileum. TLR2 acts on muscarinic M2 and M3 and nicotinic α3ß4 ACh receptors, while TLR4 acts on muscarinic M3 and nicotinic α3ß4 and α7 ACh receptors.


Assuntos
Motilidade Gastrointestinal , Íleo , Receptores Muscarínicos , Receptores Nicotínicos , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Acetilcolina/farmacologia , Animais , Bungarotoxinas , Colinérgicos , Íleo/fisiologia , Mecamilamina , Camundongos , Antagonistas Muscarínicos/farmacologia , RNA Mensageiro/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
4.
Nutrients ; 14(12)2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35745206

RESUMO

One of the symptoms of Alzheimer's disease (AD) is low acetylcholine level due to high acetylcholinesterase (AChE) activity. For this reason, AChE inhibitors are used in the treatment of AD, the prolonged use of which may cause a cholinergic crisis. There is a need to search for safe natural AChE inhibitors. The study analyzed 16 hydroxybenzoic acids using calorimetry and docking simulation as AChE inhibitors. All tested compounds were shown to inhibit the hydrolysis of ACh. The best properties were shown by methyl syringinate, which acted as competitive inhibitor at a catalytic site. The tested compounds also interacted with the anionic or peripheral binding site known to block ß-amyloid plaques formation. The activity of the tested hydroxybenzoic acids IC50 ranged from 5.50 to 34.19 µmol/µmol of AChE, and the binding constant Ka from 20.53 to 253.16 L/mol, which proves their reversible, non-toxic effect, and activity at physiological concentrations.


Assuntos
Doença de Alzheimer , Inibidores da Colinesterase , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Calorimetria , Inibidores da Colinesterase/química , Humanos , Hidroxibenzoatos , Simulação de Acoplamento Molecular
5.
Cell Rep ; 39(9): 110874, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35649378

RESUMO

Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) have been implicated in the extinction of drug associations, as well as related plasticity in medium spiny neurons (MSNs). However, since most previous work relied on artificial manipulations, whether endogenous acetylcholine signaling relates to drug associations is unclear. Moreover, despite great interest in the opposing effects of dopamine on MSN subtypes, whether ChIN-mediated effects vary by MSN subtype is also unclear. Here, we find that high endogenous acetylcholine event frequency correlates with greater extinction of cocaine-context associations across male mice. Additionally, extinction is associated with a weakening of glutamatergic synapses across MSN subtypes. Manipulating ChIN activity bidirectionally controls both the rate of extinction and the associated plasticity at MSNs. Our findings indicate that NAc ChINs mediate drug-context extinction by reducing glutamatergic synaptic strength across MSN subtypes, and that natural variation in acetylcholine signaling may contribute to individual differences in extinction.


Assuntos
Cocaína , Acetilcolina , Animais , Colinérgicos/farmacologia , Cocaína/farmacologia , Interneurônios , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia
6.
J Nucl Med ; 63(Suppl 1): 33S-44S, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35649648

RESUMO

As a neuromodulator, the neurotransmitter acetylcholine plays an important role in cognitive, mood, locomotor, sleep/wake, and olfactory functions. In the pathophysiology of most neurodegenerative diseases, such as Alzheimer disease (AD) or Lewy body disorder (LBD), cholinergic receptors, transporters, or enzymes are involved and relevant as imaging targets. The aim of this review is to summarize current knowledge on PET imaging of cholinergic neurotransmission in neurodegenerative diseases. For PET imaging of presynaptic vesicular acetylcholine transporters (VAChT), (-)-18F-fluoroethoxybenzovesamicol (18F-FEOBV) was the first PET ligand that could be successfully translated to clinical application. Since then, the number of 18F-FEOBV PET investigations on patients with AD or LBD has grown rapidly and provided novel, important findings concerning the pathophysiology of AD and LBD. Regarding the α4ß2 nicotinic acetylcholine receptors (nAChRs), various second-generation PET ligands, such as 18F-nifene, 18F-AZAN, 18F-XTRA, (-)-18F-flubatine, and (+)-18F-flubatine, were developed and successfully translated to human application. In neurodegenerative diseases such as AD and LBD, PET imaging of α4ß2 nAChRs is of special value for monitoring disease progression and drugs directed to α4ß2 nAChRs. For PET of α7 nAChR, 18F-ASEM and 11C-MeQAA were successfully applied in mild cognitive impairment and AD, respectively. The highest potential for α7 nAChR PET is seen in staging, in evaluating disease progression, and in therapy monitoring. PET of selective muscarinic acetylcholine receptors (mAChRs) is still in an early stage, as the development of subtype-selective radioligands is complicated. Promising radioligands to image mAChR subtypes M1 (11C-LSN3172176), M2 (18F-FP-TZTP), and M4 (11C-MK-6884) were developed and successfully translated to humans. PET imaging of mAChRs is relevant for the assessment and monitoring of therapies in AD and LBD. PET of acetylcholine esterase activity has been investigated since the 1990s. Many PET studies with 11C-PMP and 11C-MP4A demonstrated cortical cholinergic dysfunction in dementia associated with AD and LBD. Recent studies indicated a solid relationship between subcortical and cortical cholinergic dysfunction and noncognitive dysfunctions such as balance and gait in LBD. Taken together, PET of distinct components of cholinergic neurotransmission is of great interest for diagnosis, disease monitoring, and therapy monitoring and to gain insight into the pathophysiology of different neurodegenerative disorders.


Assuntos
Doença de Alzheimer , Doença por Corpos de Lewy , Acetilcolina , Colinérgicos , Progressão da Doença , Humanos , Tomografia por Emissão de Pósitrons/métodos , Transmissão Sináptica
7.
Biol Pharm Bull ; 45(6): 675-683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650095

RESUMO

Immune cells such as T and B cells, monocytes and macrophages all express most of the cholinergic components of the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase (AChE). Because of its efficient cleavage by AChE, ACh synthesized and released from immune cells acts only locally in an autocrine and/or paracrine fashion at mAChRs and nAChRs on themselves and other immune cells located in close proximity, leading to modification of immune function. Immune cells generally express all five mAChR subtypes (M1-M5) and neuron type nAChR subunits α2-α7, α9, α10, ß2-ß4. The expression pattern and levels of mAChR subtypes and nAChR subunits vary depending on the tissue involved and its immunological status. Immunological activation of T cells via T-cell receptor-mediated pathways and cell adhesion molecules upregulates ChAT expression, which facilitates the synthesis and release of ACh. At present, α7 nAChRs expressed in macrophages are receiving much attention because they play a central role in anti-inflammatory cholinergic pathways. However, it now appears that through modification of cytokine synthesis, Gq/11-coupled mAChRs play a prominent role in regulation of T cell proliferation and differentiation and B cell immunoglobulin class switching. It is anticipated that greater understanding of Gq/11-coupled mAChRs on immune cells will provide an opportunity to develop new and effective treatments for immunological disorders.


Assuntos
Acetilcolinesterase , Receptores Muscarínicos , Acetilcolina , Colina O-Acetiltransferase/metabolismo , Colinérgicos , Imunidade
8.
PLoS One ; 17(6): e0268644, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35657915

RESUMO

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances.


Assuntos
Células Acinares , Cálcio , Acetilcolina/farmacologia , Animais , Cálcio na Dieta , Ceruletídeo , Camundongos , Microscopia Confocal , Pâncreas
9.
PLoS One ; 17(6): e0269618, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35657974

RESUMO

Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.


Assuntos
Neoplasias do Colo , MicroRNAs , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Derivados da Atropina , Colinérgicos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Cinética , MicroRNAs/genética , Antagonistas Muscarínicos/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
PLoS One ; 17(6): e0269310, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35653352

RESUMO

BACKGROUND: Children and adult with sickle cell disease (SCD) display priapism associated with low nitric oxide (NO) bioavailability and oxidative stress in penis. AIM: This study aimed to evaluate the effects of hybrid compound RVT-FxMe, derived from resveratrol bearing a NO-donor subunit, on two murine model that display priapism phenotype, SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice. METHODS: Wild-type, SCD, and eNOS-/- mice were treated with RVT-FxMe (25 mg/kg/d, 2 weeks). OUTCOMES: Hematological parameters, concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as to electrical field stimulation (EFS), were obtained in mice corpus cavernosum strips. RESULTS: Corpus cavernosum relaxations to SNP and EFS were increased in eNOS-/- group, which were normalized by RVT-FxMe treatment. SCD mice exhibited an excessive CC relaxant response induced by ACh, EFS and SNP RVT-FxMe treatment did not change the increased relaxant responses to ACh, EFS and SNP in corpus cavernosum from SCD group. CLINICAL TRANSLATION: Excess of plasma hemoglobin in SCD may interfere in pharmacological activity of NO donors compounds. STRENGTH/LIMITATIONS: While mechanistic data with promising potential is showed, the current study is not without limitations. RVT-FxMe effects in the mid- and long-term warrant complementary studies. CONCLUSION: Treatment with RVT-FxMe reversed the enhanced NO-cGMP-mediated CC relaxations in eNOS-/- mice, but not in SCD mice; it is likely that excess of plasma hemoglobin in SCD mice act to inactivate NO before it reaches soluble guanylyl cyclase, avoiding restoration of NO bioavailability in penis.


Assuntos
Anemia Falciforme , Priapismo , Acetilcolina/farmacologia , Anemia Falciforme/complicações , Anemia Falciforme/tratamento farmacológico , Animais , Hemoglobinas , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Óxido Nítrico , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Priapismo/tratamento farmacológico , Priapismo/etiologia , Resveratrol/farmacologia
11.
Int J Mol Sci ; 23(11)2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35682752

RESUMO

The native function of amyloid-ß (Aß) peptides is still unexplored. However, several recent reports suggest a prominent role of Aß peptides in acetylcholine homeostasis. To clarify this role of Aß, we have reported that Aß peptides at physiological concentrations can directly enhance the catalytic efficiency of the key cholinergic enzyme, choline acetyltransferase (ChAT), via an allosteric interaction. In the current study, we further aimed to elucidate the underlying ChAT-Aß interaction mechanism using in silico molecular docking and dynamics analysis. Docking analysis suggested two most probable binding clusters on ChAT for Aß40 and three for Aß42. Most importantly, the docking results were challenged with molecular dynamic studies of 100 ns long simulation in triplicates (100 ns × 3 = 300 ns) and were analyzed for RMSD, RMSF, RoG, H-bond number and distance, SASA, and secondary structure assessment performed together with principal component analysis and the free-energy landscape diagram, which indicated that the ChAT-Aß complex system was stable throughout the simulation time period with no abrupt motion during the evolution of the simulation across the triplicates, which also validated the robustness of the simulation study. Finally, the free-energy landscape analysis confirmed the docking results and demonstrated that the ChAT-Aß complexes were energetically stable despite the unstructured nature of C- and N-terminals in Aß peptides. Overall, this study supports the reported in vitro findings that Aß peptides, particularly Aß42, act as endogenous ChAT-Potentiating-Ligand (CPL), and thereby supports the hypothesis that one of the native biological functions of Aß peptides is the regulation of acetylcholine homeostasis.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Acetilcolina/metabolismo , Sítio Alostérico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Colina O-Acetiltransferase/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular
12.
J Immunother Cancer ; 10(6)2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35705312

RESUMO

BACKGROUND: Recent studies have demonstrated that T cells can induce vasodilation in a choline-acetyltransferase dependent manner, leading to an increase in T cell migration to infected tissues in response to viral infection, but its role in cancer is unclear. Choline acetyltransferase catalyzes the production of acetylcholine from choline and acetyl-CoA, however, acetylcholine is challenging to quantify due to its extremely short half-life while choline is stable. This study aims to correlate serum choline levels in patients with advanced solid tumors receiving pembrolizumab with treatment outcomes. METHODS: Blood samples were collected at baseline and at week 7 (pre-cycle 3) in patients treated with pembrolizumab in the INvestigator-initiated Phase 2 Study of Pembrolizumab Immunological Response Evaluation phase II trial (NCT02644369). Samples were analyzed for choline and circulating tumor DNA (ctDNA). Multivariable Cox models were used to assess the association between choline and overall survival (OS) and progression-free survival (PFS) when including ΔctDNAC3 (the change in ctDNA from baseline to cycle 3), cohort, PD-L1 expression and tumor mutation burden (TMB). An independent validation cohort from the LIBERATE study (NCT03702309) included patients on early phase trials treated with a PD-1 inhibitor. RESULTS: A total of 106 pts were included in the analysis. With a median follow-up of 12.6 months, median PFS and OS were 1.9 and 13.7 months, respectively. An increase in serum choline level at week 7 compared with baseline (ΔcholineC3) in 81 pts was significantly associated with a better PFS (aHR 0.48, 95% CI 0.28 to 0.83, p=0.009), and a trend toward a better OS (aHR 0.64, 95% CI 0.37 to 1.12, p=0.119). A combination of ΔctDNAC3 and ΔcholineC3 was prognostic for both OS and PFS. Multivariable analyses show ΔcholineC3 was a prognostic factor for PFS independent of ΔctDNAC3, cohort, PD-L1 and TMB. In the independent validation cohort (n=51), an increase in serum choline at cycle 2 was associated with a trend to improved PFS. CONCLUSIONS: This is the first exploratory report of serum choline levels in pan-cancer patients receiving pembrolizumab. The association between improved PFS and ΔcholineC3 suggests a possible role for the cholinergic system in the regulation of antitumor immunity. Further pre-clinical and clinical studies are required to validate this finding. TRIAL REGISTRATION NUMBER: NCT03702309.


Assuntos
Antineoplásicos Imunológicos , DNA Tumoral Circulante , Neoplasias , Acetilcolina/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos Imunológicos/efeitos adversos , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/genética , Colina/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Intervalo Livre de Progressão
13.
Clin Transl Med ; 12(6): e890, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35758323

RESUMO

BACKGROUND: Acetylcholine (ACh) and norepinephrine (NE) are representative neurotransmitters of parasympathetic and sympathetic nerves, respectively, that antagonize each other to coregulate internal body functions. This also includes the control of different kinds of hormone secretion from pancreatic islets. However, the molecular mechanisms have not been fully elucidated, and whether innervation in islets is abnormal in diabetes mellitus also remains unclear. METHODS AND RESULTS: Immunofluorescence colocalization and islet perfusion were performed and the results demonstrated that ACh/NE and their receptors were highly expressed in islet and rapidly regulated different hormones secretion. Phosphorylation is considered an important posttranslational modification in islet innervation and it was identified by quantitative proteomic and phosphoproteomic analyses in this study. The phosphorylated islet proteins were found involved in many biological and pathological processes, such as synaptic signalling transduction, calcium channel opening and insulin signalling pathway. Then, the kinases were predicted by motif analysis and further screened and verified by kinase-specific siRNAs in different islet cell lines (αTC1-6, Min6 and TGP52). After functional verification, Ksr2 and Pkacb were considered the key kinases of ACh and NE in insulin secretion, and Cadps, Mlxipl and Pdcd4 were the substrates of these kinases measured by immunofluorescence co-staining. Then, the decreased expression of receptors, kinases and substrates of ACh and NE were found in diabetic mice and the aberrant rhythm in insulin secretion could be improved by combined interventions on key receptors (M3 (pilocarpine) or α2a (guanfacine)) and kinases (Ksr2 or Pkacb). CONCLUSIONS: Abnormal innervation was closely associated with the degree of islet dysfunction in diabetic mice and the aberrant rhythm in insulin secretion could be ameliorated significantly after intervention with key receptors and kinases in the early stage of diabetes mellitus, which may provide a promising therapeutic strategy for diabetes mellitus in the future.


Assuntos
Diabetes Mellitus Experimental , Ilhotas Pancreáticas , Acetilcolina/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Insulina/metabolismo , Ilhotas Pancreáticas/inervação , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Neurotransmissores/metabolismo , Proteômica
14.
Reprod Toxicol ; 111: 178-183, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35671880

RESUMO

Alcohol has been demonstrated to impair maternal uterine arterial adaptations in Fetal Alcohol Spectrum Disorder (FASD) animal models. However, the exact mechanism remains inconclusive. We hypothesized that phosphatidic acid (PA), a direct target of alcohol metabolism, would alleviate alcohol-induced vascular dysfunction of the maternal uterine artery. Mean fetal weight, and crown-rump length of the alcohol administered rats were ~9 % and 7.6 % lower than the pair-fed control pups, respectively. Acetylcholine (Ach)-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P < 0.05). Supplementation of 10-5 M PA reversed alcohol-induced vasodilatory deficit; no difference was detected after PA treatment between pair-fed control and alcohol groups (P = 0.37). There was a significant interaction between PA concentrations and alcohol exposure (PA X Alcohol effect, P < 0.0001). Pair-wise comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats, with % relaxation significantly improved at PA concentrations > 10-7 M (P < 0.05). Alcohol significantly reduced vasodilatory P-Ser1177 endothelial nitric oxide synthase (eNOS) levels in the uterine artery (↓90.7 %; P = 0.0029). PA treatment significantly reversed P-Ser1177 eNOS level in alcohol uterine arteries (153.7 %↑; P = 0.005); following ex vivo PA, there was no difference in P-Ser1177 eNOS levels between Control and Alcohol. Neither alcohol treatment nor PA affected total eNOS levels. Our data provide the first evidence of the interaction of alcohol and PA in rat maternal uterine artery vascular function and demonstrates PA's relationship with the eNOS system. Overall, the current study demonstrates that PA may be a promising therapeutic molecule of interest in alcohol-related gestational vascular dysfunction.


Assuntos
Ácidos Fosfatídicos , Artéria Uterina , Acetilcolina/farmacologia , Animais , Endotélio Vascular/metabolismo , Etanol/toxicidade , Óxido Nítrico Sintase Tipo III/metabolismo , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Ratos , Vasodilatação
15.
Clin Sci (Lond) ; 136(12): 973-987, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35678315

RESUMO

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.


Assuntos
Nicotina , Receptor Nicotínico de Acetilcolina alfa7 , Acetilcolina/metabolismo , Administração por Inalação , Animais , Aorta Torácica/efeitos dos fármacos , Feminino , Masculino , Camundongos , Nicotina/administração & dosagem , Artéria Pulmonar/efeitos dos fármacos , Regulação para Cima , Vasodilatação/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
16.
Environ Health Prev Med ; 27(0): 27, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35753805

RESUMO

BACKGROUND: Ulcerative colitis (UC) is related to stress, but few studies have evaluated the influence of stress on factors affecting colon contractility in rats with UC. Also, there have been no studies investigating beneficial effects of linalyl acetate (LA), the major component of lavender essential oil, in repeatedly stressed-ulcerative colitis rats. Therefore, we investigated the differences in factors affecting colon contractility of UC rats with or without repeated restraint stress (RRS) and the effects of LA on these parameters in repeatedly stressed-UC rats. METHODS: Rats were assigned to following groups: control, RRS, UC, RRS+UC, and RRS+UC treated with LA or sulfasalazine. To induce UC, rats were administered 2% dextran sodium sulfate (DSS) water on days 1-5, followed by tap water on days 6-15 and DSS water on days 16-20. RRS was induced by immobilizing rats for 2 hr/day on days 1-20. LA or sulfasalazine were daily administered on days 16-20. RESULTS: Disease activity index (DAI) was markedly increased in RRS+UC. Serum interleukin-6 levels and acetylcholine-induced colon contraction were higher in RRS+UC than in control, RRS and UC. Colon nitrite levels also significantly increased in RRS+UC compared to the control and RRS. Blood pressure (BP) was higher in RRS+UC than in the control and UC. Both LA and sulfasalazine was effective in decreasing DAI, colon nitrite levels, acetylcholine-induced colon contraction in RRS+UC. Sulfasalazine significantly reduced serum IL-6 levels in RRS+UC with decreasing tendency in RRS+UC treated by LA. Only LA significantly reduced BP in RRS+UC. CONCLUSIONS: Our findings emphasize the importance of stress management in UC patients. Also, LA may be beneficially used in repeatedly stressed-UC patients with high BP.


Assuntos
Colite Ulcerativa , Acetilcolina , Animais , Pressão Sanguínea , Colite Ulcerativa/induzido quimicamente , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Interleucina-6 , Monoterpenos , Nitritos , Ratos , Sulfassalazina , Água
17.
Prog Neurobiol ; 214: 102279, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35513164

RESUMO

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked ß2V287L nAChR subunit. ß2V287L mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness. However, they showed a substantial decrease of the excitatory input to layer V fast-spiking (FS) interneurons, despite a concomitant increase in the number of glutamatergic terminals around the cell soma. Hence, prefrontal hyperexcitability may depend on a permanent impairment of surround inhibition. The effect disappeared when ß2V287L was silenced until postnatal day 15th, suggesting that the transgene selectively affects the maturation of glutamatergic synapses on FS neurons. The other main population of interneurons in layer V was constituted by somatostatin-expressing regular spiking cells. When tested with 10 µM nicotine, these displayed larger somatic nicotinic currents in transgenic mice. Thus, during wakefulness, activation of ß2V287L-containing nAChRs by the high cholinergic tone may counteract hyperexcitability by promoting local inhibition by somatostatin-expressing cells and decreasing the effect of glutamatergic deficit in FS neurons. This interpretation was tested in networks disinhibited by 2 µM bicuculline. Slices expressing ß2V287L were more susceptible to develop synchronized activity in the absence of nicotine. Addition of the drug boosted excitability in the controls, but had little effect in ß2V287L. Our findings suggest why NREM sleep favors ADSHE seizures and nicotine can be palliative in patients.


Assuntos
Epilepsia , Receptores Nicotínicos , Acetilcolina/farmacologia , Animais , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/fisiologia , Nicotina/farmacologia , Córtex Pré-Frontal/metabolismo , Receptores Nicotínicos/metabolismo , Convulsões , Sono/fisiologia , Somatostatina
18.
J Neuroimmunol ; 368: 577895, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35617719

RESUMO

BACKGROUND: LRP4 is a post-synaptic membrane protein that promotes acetylcholine (AChR) clustering on the crest of post-synaptic neuromuscular folds. Autoantibodies against LRP4 are suggested to account for myasthenia gravis (MG) patients negative for antibodies to AChR. OBJECTIVES: To report a clinical experience with service-line LRP4-IgG cell-based testing in electrodiagnostically confirmed MG patients and controls. METHODS: We identified all Mayo patients undergoing MG evaluations with send out LRP4-IgG antibody testing by cell-based assay, having clinical-electrodiagnostic (EDX) testing. To be included, muscle acetylcholine receptor binding (AChR-Bi) and muscle-specific tyrosine kinase (MuSK) antibodies had to be absent prior to LRP4-IgG testing. Follow-up AChR-Bi antibody testing was reviewed. Also tested for LRP4-IgGs were 119 healthy subjects. RESULTS: Identified were 25 generalized MG, 24 ocular MG, and 55 patients initially considered to have MG prior to negative EDX testing. No seronegative patients with EDX confirmed MG had LRP4-IgG positivity but five non-MG patients did: Guillain-Barre syndrome with fatigue (N = 1); multiple cranial neuropathies (N = 1); functional neurologic disorders (N = 3). Of healthy subjects, 4% (5/119) were LRP4-IgG positive (N = 5) or had a borderline result (N = 1). Of MG patients with repeat AChR-Bi testing, 40% (10/25) seroconverted (5 with ocular MG and 5 with generalized MG) (median AChR IgG value: 0.34 nmol/L, range 0.2-20.9 nmol/L, median followup 26 months, range 2-72 months). CONCLUSION: Clinical review of LRP4-IgG commercial cell-based testing suggests lack of diagnostic utility in seronegative EDX-confirmed MG. The clinical utility of LRP4-IgG testing is not substantiated in service line testing. In contrast, repeat testing for AChR-Bi antibodies is shown clinically useful.


Assuntos
Proteínas Relacionadas a Receptor de LDL , Miastenia Gravis , Acetilcolina , Autoanticorpos , Humanos , Imunoglobulina G/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligação Proteica
19.
Biosensors (Basel) ; 12(5)2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35624580

RESUMO

For decades, acetylcholine (Ach) has been considered a critical biomarker for several degenerative brain diseases, including Alzheimer's, Parkinson's disease, Huntington's disease, and schizophrenia. Here, we propose a wafer-scale fabrication of polyaniline (PAni)-grafted graphene-based field-effect transistors (PGFET) and their biosensing applications for highly sensitive and reliable real-time monitoring of Ach in flow configuration. The grafted PAni provides suitable electrostatic binding sites for enzyme immobilization and enhances the pH sensitivity (2.68%/pH), compared to that of bare graphene-FET (1.81%/pH) for a pH range of 3-9 without any pH-hysteresis. We further evaluated the PGFET's sensing performance for Ach detection with a limit of detection at the nanomolar level and significantly improved sensitivity (~103%) in the concentration range of 108 nM to 2 mM. Moreover, the PGFET exhibits excellent selectivity against various interferences, including glucose, ascorbic acid, and neurotransmitters dopamine and serotonin. Finally, we investigated the effects of an inhibitor (rivastigmine) on the AchE activity of the PGFET. From the results, we demonstrated that the PGFET has great potential as a real-time drug-screening platform by monitoring the inhibitory effects on enzymatic activity.


Assuntos
Técnicas Biossensoriais , Grafite , Acetilcolina , Compostos de Anilina , Técnicas Biossensoriais/métodos , Grafite/química
20.
Int J Mol Sci ; 23(10)2022 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-35628371

RESUMO

Menopause is associated with memory deficits attributed to reduced serum estrogen levels. We evaluated whether an increase in brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF) levels, through transplantation of choline acetyltransferase (ChAT)-overexpressing neural stem cells (F3.ChAT), improved learning and memory in ovariectomized rats. PD13 mouse neuronal primary culture cells were treated with estradiol or co-cultured with F3.ChAT cells; choline transporter1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) expression was evaluated using real-time PCR. The relationship between estrogen receptors (ERs) and neurotrophin family members was analyzed using immunohistochemistry. After the transplantation of F3.ChAT cells into OVx rats, we evaluated the memory, ACh level, and the expression of ER, neurotrophin family proteins, and cholinergic system. Estradiol upregulated CHT1, ChAT, and VAChT expression in ER; they were co-localized with BDNF, NGF, and TrkB. Co-culture with F3.ChAT upregulated CHT1, ChAT, and VAChT by activating the neurotrophin signalling pathway. Transplantation of F3.ChAT cells in OVX animals increased the ACh level in the CSF and improved memory deficit. In addition, it increased the expression of ERs, neurotrophin signaling, and the cholinergic system in the brains of OVX animals. Therefore, the estradiol deficiency induced memory loss by the down-regulation of the neurotrophin family and F3.ChAT could ameliorate the cognitive impairment owing to the loss or reduction of estradiol.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Colina O-Acetiltransferase , Cognição , Células-Tronco Neurais , Acetilcolina/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colina/metabolismo , Colina O-Acetiltransferase/biossíntese , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Colinérgicos/metabolismo , Cognição/fisiologia , Estradiol/metabolismo , Humanos , Transtornos da Memória/metabolismo , Camundongos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Ratos , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
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