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1.
Nat Commun ; 11(1): 4328, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859919

RESUMO

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's anti-depressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.


Assuntos
Ketamina/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Proliferação de Células , Cistite/induzido quimicamente , Modelos Animais de Doenças , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Camundongos , Camundongos Knockout , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Músculo Liso/patologia , Oócitos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Bexiga Urinária/patologia , Xenopus
2.
Am J Pathol ; 190(10): 2067-2079, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32679229

RESUMO

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1-/-, alias Thbs1-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1-/- compared with WT mice.


Assuntos
Síndromes do Olho Seco/patologia , Células Epiteliais/metabolismo , Inflamação/metabolismo , Células Musculares/patologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Modelos Animais de Doenças , Síndromes do Olho Seco/metabolismo , Células Epiteliais/patologia , Camundongos Endogâmicos C57BL , Células Musculares/metabolismo , Músculo Liso/metabolismo
3.
Am J Physiol Renal Physiol ; 319(2): F257-F283, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628539

RESUMO

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and ultimately determines the two main attributes of the organ: urine storage and voiding. The two functions are facilitated by UBSM relaxation and contraction, respectively, which depend on UBSM excitability shaped by multiple ion channels. In this review, we summarize the current understanding of key ion channels establishing and regulating UBSM excitability and contractility. They include excitation-enhancing voltage-gated Ca2+ (Cav) and transient receptor potential channels, excitation-reducing K+ channels, and still poorly understood Cl- channels. Dynamic interplay among UBSM ion channels determines the overall level of Cav channel activity. The net Ca2+ influx via Cav channels increases global intracellular Ca2+ concentration, which subsequently triggers UBSM contractility. Here, for each ion channel type, we describe UBSM tissue/cell expression (mRNA and protein) profiles and their role in regulating excitability and contractility of UBSM in various animal species, including the mouse, rat, and guinea pig, and, most importantly, humans. The currently available data reveal certain interspecies differences, which complicate the translational value of published animal research results to humans. This review highlights recent developments, findings on genetic knockout models, pharmacological data, reports on UBSM ion channel dysfunction in animal bladder disease models, and the very limited human studies currently available. Among all gaps in present-day knowledge, the unknowns on expression and functional roles for ion channels determined directly in human UBSM tissues and cells under both normal and disease conditions remain key hurdles in the field.


Assuntos
Canais Iônicos/metabolismo , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Bexiga Urinária/metabolismo , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Contração Muscular/fisiologia
4.
Am J Physiol Heart Circ Physiol ; 319(2): H507-H518, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32706268

RESUMO

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.


Assuntos
Linfa/fisiologia , Vasos Linfáticos/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Canais de Cátion TRPV/metabolismo , Temperatura , Animais , Diafragma , Feminino , Técnicas In Vitro , Vasos Linfáticos/efeitos dos fármacos , Masculino , Morfolinas/farmacologia , Músculo Liso/efeitos dos fármacos , Periodicidade , Pirróis/farmacologia , Ratos , Ratos Wistar , Rutênio Vermelho/farmacologia , Transdução de Sinais , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Fatores de Tempo
5.
J Cancer Res Ther ; 16(3): 647-652, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32719283

RESUMO

The leiomyoma is a benign smooth-muscle neoplasm commonly found in the female genital tract, gastrointestinal tract, or skin. Leiomyomas of the oral cavity are unusual. Oral leiomyomas are uncommon due to the paucity of the smooth muscle in the mouth (except in blood vessels) and thus the involvement of jaw bones is extremely rare. Leiomyomas have been classified as solid angiomyoma, angioleiomyoma (vascular leiomyoma), and epithelioid variants. Angioleiomyomas are benign mesenchymal tumors derived from smooth muscle, which rarely occur in the oral cavity. Malignant transformation probably does not occur but careful histopathologic examination is still necessary to differentiate these benign lesions from their malignant counterparts due to different prognosis. Although uncommon in the maxilla and mandible, they should be included in the differential diagnosis of radiolucent lesions of jaw bones. An extensive search of literature was carried out on the Medline-PubMed and Google Scholar database using the keywords such as leiomyoma, angioleiomyoma, jaw bones, maxilla, mandible, intra-osseous to thoroughly search and collect all the reported cases of intraosseous leiomyoma (but our search was not limited to these terms only). To the best of our knowledge, only 23 cases of intraosseous leiomyomas have been reported so far in the jaw bones, among which only 8 belonged to angioleiomyomas. Herein, we report the 9th case of intraosseous angioleiomyoma, one of the variants of leiomyoma and overall 24th intraosseous leiomyoma in a 6-year-old female child, together with conventional histopathologic and immunohistochemical findings.


Assuntos
Angiomioma/patologia , Neoplasias Mandibulares/patologia , Doenças Raras/patologia , Actinas/metabolismo , Angiomioma/metabolismo , Angiomioma/cirurgia , Antígenos CD34/metabolismo , Biomarcadores Tumorais/metabolismo , Criança , Feminino , Humanos , Neoplasias Mandibulares/metabolismo , Neoplasias Mandibulares/cirurgia , Músculo Liso/metabolismo , Músculo Liso/patologia , Doenças Raras/metabolismo , Doenças Raras/cirurgia
6.
PLoS Comput Biol ; 16(7): e1008069, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716940

RESUMO

Nitric oxide (NO) is a gaseous signaling molecule that plays an important role in neurovascular coupling. NO produced by neurons diffuses into the smooth muscle surrounding cerebral arterioles, driving vasodilation. However, the rate of NO degradation in hemoglobin is orders of magnitude higher than in brain tissue, though how this might impact NO signaling dynamics is not completely understood. We used simulations to investigate how the spatial and temporal patterns of NO generation and degradation impacted dilation of a penetrating arteriole in cortex. We found that the spatial location of NO production and the size of the vessel both played an important role in determining its responsiveness to NO. The much higher rate of NO degradation and scavenging of NO in the blood relative to the tissue drove emergent vascular dynamics. Large vasodilation events could be followed by post-stimulus constrictions driven by the increased degradation of NO by the blood, and vasomotion-like 0.1-0.3 Hz oscillations could also be generated. We found that these dynamics could be enhanced by elevation of free hemoglobin in the plasma, which occurs in diseases such as malaria and sickle cell anemia, or following blood transfusions. Finally, we show that changes in blood flow during hypoxia or hyperoxia could be explained by altered NO degradation in the parenchyma. Our simulations suggest that many common vascular dynamics may be emergent phenomena generated by NO degradation by the blood or parenchyma.


Assuntos
Encéfalo/fisiologia , Circulação Cerebrovascular , Óxido Nítrico/metabolismo , Anemia Falciforme/fisiopatologia , Arteríolas , Transfusão de Sangue , Sistema Livre de Células , Simulação por Computador , Difusão , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Hemodinâmica , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Malária/fisiopatologia , Mitocôndrias/metabolismo , Músculo Liso/metabolismo , Oscilometria , Distribuição de Poisson , Transdução de Sinais , Vasodilatação
7.
PLoS One ; 15(6): e0234330, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516331

RESUMO

Presbyopia and myopia research shows a growing interest in ciliary muscle biometry using optical coherence tomography (OCT). Until now, segmentation of the ciliary muscle is often performed manually using either custom-developed programs or image processing software. Here we present a novel software for semi-automatic segmentation of the ciliary muscle. It provides direct import of OCT images in DICOM format, a standardized procedure for segmentation, image distortion correction, the export of anatomical ciliary muscle landmarks, like ciliary muscle apex and scleral spur, as well as a continuous thickness profile of the ciliary muscle as a novel way of analysis. All processing steps are stored as XML files, fostering documentation and reproducibility of research through the possibility of replicating the analysis. Additionally, CilOCT supports batch processing for the automated analysis of large numbers of images and the respective data export to tabulated text files based on the stored XML files. CilOCT was successfully applied in several studies and their results will be summarized in this paper.


Assuntos
Corpo Ciliar/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Tomografia de Coerência Óptica/métodos , Algoritmos , Biometria/métodos , Humanos , Músculo Liso/diagnóstico por imagem , Músculo Liso/metabolismo , Reprodutibilidade dos Testes , Retina/diagnóstico por imagem , Esclera/diagnóstico por imagem , Software
8.
Am J Pathol ; 190(9): 1843-1858, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479820

RESUMO

The progression of Crohn disease to intestinal stricture formation is poorly controlled, and the pathogenesis is unclear, although increased smooth muscle mass is present. A previously described rat model of trinitrobenzenesulfonic acid-induced colitis is re-examined here. Although inflammation of the mid-descending colon typically resolved, a subset showed characteristic stricturing by day 16, with an inflammatory infiltrate in the neuromuscular layers including eosinophils, CD3-positive T cells, and CD68-positive macrophages. Closer study identified CD163-positive, CD206-positive, and arginase-positive cells, indicating a M2 macrophage phenotype. Stricturing involved ongoing proliferation of intestinal smooth muscle cells (ISMC) with expression of platelet-derived growth factor receptor beta and progressive loss of phenotypic markers, and stable expression of hypoxia inducible factor 1 subunit alpha. In parallel, collagen I and III showed a selective and progressive increase over time. A culture model of the stricture phenotype of ISMC showed stable hypoxia inducible factor 1 subunit alpha expression that promoted growth and improved both survival and growth in models of experimental ischemia. This phenotype was hyperproliferative to serum and platelet-derived growth factor BB, and unresponsive to transforming growth factor beta, a prominent cytokine of M2 macrophages, compared with control ISMC. We identified a hyperplastic phenotype of ISMC, uniquely adapted to an ischemic environment to drive smooth muscle layer expansion, which may reveal new targets for treating intestinal fibrosis.


Assuntos
Doença de Crohn/patologia , Intestinos/patologia , Macrófagos/metabolismo , Músculo Liso/patologia , Animais , Constrição Patológica/induzido quimicamente , Constrição Patológica/patologia , Hiperplasia/induzido quimicamente , Hiperplasia/metabolismo , Hiperplasia/patologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Ratos , Ratos Sprague-Dawley , Ácido Trinitrobenzenossulfônico/toxicidade
9.
Prostate ; 80(11): 831-849, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32449814

RESUMO

INTRODUCTION: Prostate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin-linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real-time polymerase chain reaction (RT-PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide), the integrin α2ß1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide), or the integrin α4ß1/α9ß1 inhibitor BOP (N-(benzenesulfonyl)- l-prolyl- l-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt). RESULTS: Western blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, ß1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT-PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate-specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and ß1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency-dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT-3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT-3033 at 32 Hz). The thromboxane A2 analog U46619-induced concentration-dependent contractions, which were inhibited by Cpd22 and BTT-3033 (around 67% by Cpd22 and 39% by BTT-3033 at 30 µM U46619). Endothelin-1 induced concentration-dependent contractions, which were not affected by Cpd22 or BTT-3033. Noradrenaline and the α1 -adrenergic agonists methoxamine and phenylephrine-induced concentration-dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT-3033. BOP did not change EFS- or agonist-induced contraction. CONCLUSIONS: Integrin α2ß1 and ILK inhibitors inhibit neurogenic and thromboxane A2 -induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.


Assuntos
Integrina alfa2beta1/antagonistas & inibidores , Músculo Liso/efeitos dos fármacos , Próstata/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Dipeptídeos/farmacologia , Humanos , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Piperazinas/farmacologia , Próstata/metabolismo , Próstata/fisiologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Tromboxano A2/metabolismo , Vasoconstritores/farmacologia
10.
Arch Biochem Biophys ; 688: 108403, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32418893

RESUMO

Myopia is a main cause of preventable or treatable visual impairment, it has become a major public health issue due to its increasingly high prevalence worldwide. Currently, it is confirmed that the development of myopia is associated with the disorders of accommodation. As a dominant factor for accommodation, ciliary muscle contraction/relaxation can regulate the physiological state of the lens and play a crucial role in the development of myopia. To investigate the relationship between myopia and ciliary muscle, the guinea pigs were randomly divided into a normal control (NC) group and a negative lens-induced myopia (LIM) group, and the animals in each group were further randomly assigned into 2-week (n = 18) and 4-week (n = 21) subgroups in accordance with the duration of myopic induction of 2 and 4 weeks, respectively. In the present study, right eyes of the animals in LIM group were covered with -6.0 D lenses to induce myopia. Next, we performed the haematoxylin and eosin (H&E) staining to observe the pathological change of ciliary muscle, determined the contents of adenosine triphosphate (ATP) and lactate acid (LA), and measured the Na+/K+-ATPase expression and activity in ciliary muscles in both NC and LIM groups. Moreover, we also analyzed the potassium ion (K+) flux in ciliary muscles from 4-week NC and LIM guinea pigs. As a result, we found that the arrangements of ciliary muscles in LIM guinea pigs were broken, dissolved or disorganized; the content of ATP decreased, whereas the content of LA increased in ciliary muscles from LIM guinea pigs. Monitoring of K+ flux in ciliary muscles from LIM guinea pigs demonstrated myopia-triggered K+ influx. Moreover, we also noted a decreased expression of Na+/K+-ATPase (Atp1a1) at both mRNA and protein levels and reduced activity in ciliary muscles from LIM guinea pigs. Overall, our results will facilitate the understanding of the mechanism associated with inhibitory Na+/K+-ATPase in lens-induced myopia and which consequently lead to the disorder of microenvironment within ciliary muscles from LIM guinea pigs, paving the way for a promising adjuvant approach in treating myopia in clinical practice.


Assuntos
Olho/metabolismo , Homeostase/fisiologia , Músculo Liso/metabolismo , Miopia/metabolismo , Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Olho/patologia , Cobaias , Ácido Láctico/metabolismo , Masculino , Músculo Liso/patologia , Miopia/patologia , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo
11.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L185-L195, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432920

RESUMO

S100A4 is a low-molecular-mass (12 kDa) EF-hand Ca2+-binding S100 protein that is expressed in a broad range of normal tissue and cell types. S100A4 can be secreted from some cells to act in an autocrine or paracrine fashion on target cells and tissues. S100A4 has been reported in the extracellular fluids of subjects with several inflammatory diseases, including asthma. Airway smooth muscle plays a critical role in airway inflammation by synthesizing and secreting inflammatory cytokines. We hypothesized that S100A4 may play an immunomodulatory role in airway smooth muscle. Trachealis smooth muscle tissues were stimulated with recombinant His-S100A4, and the effects on inflammatory responses were evaluated. S100A4 induced the activation of Akt and NF-κB and stimulated eotaxin secretion. It also increased the expression of RAGE and endogenous S100A4 in airway tissues. Stimulation of airway smooth muscle tissues with IL-13 or TNF-α induced the secretion of S100A4 from the tissues and promoted the expression of endogenous receptors for advanced glycation end products (RAGE) and S100A4. The role of RAGE in mediating the responses to S100A4A was evaluated by expressing a mutant nonfunctional RAGE (RAGEΔcyto) in tracheal muscle tissues and by treating tissues with a RAGE inhibitor. S100A4 did not activate NF-κB or Akt in tissues that were expressing RAGEΔcyto or treated with a RAGE inhibitor, indicating that S100A4 mediates its effects by acting on RAGE. Our results demonstrate that inflammatory mediators stimulate the synthesis and secretion of S100A4 in airway smooth muscle tissues and that extracellular S100A4 acts via RAGE to mediate airway smooth muscle inflammation.


Assuntos
Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Músculo Liso/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Transdução de Sinais , Animais , Quimiocinas/metabolismo , Cães , Interleucina-13/metabolismo , Modelos Biológicos , Fator de Necrose Tumoral alfa/metabolismo
12.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L943-L952, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32233794

RESUMO

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.


Assuntos
Dinoprostona/metabolismo , Músculo Liso/metabolismo , Canal de Cátion TRPA1/genética , Traqueia/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Estimulação Elétrica , Regulação da Expressão Gênica , Cobaias , Histamina/farmacologia , Humanos , Indometacina/farmacologia , Isotiocianatos/farmacologia , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Cloreto de Potássio/farmacologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Respiração Artificial , Transdução de Sinais , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/metabolismo , Tetrodotoxina/farmacologia , Traqueia/efeitos dos fármacos , Nervo Vago/fisiologia
13.
Pharm Biol ; 58(1): 257-264, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32208946

RESUMO

Context: Curcumin, the active component of Curcuma longa L. (Zingiberaceae), exhibits a wide variety of biological activities including vasodilation and anti-inflammation.Objective: The relaxant effect of curcumin in tracheal smooth muscle (TSM) was not examined so far, thus, this study was designed to assess the relaxant effect of curcumin on rat TSM and examine the underlying mechanism(s) responsible for this effect.Materials and methods: TSM was contracted by KCl (60 mM) or methacholine (10 µM), and cumulative concentrations of curcumin (12.5, 25, 50, and 100 mg/mL) or theophylline (0.2, 0.4, 0.6, and 0.8 mM, as positive control) were added to organ bath. The relaxant effect of curcumin was examined in non-incubated or incubated tissues with atropine (1 µM), chlorpheniramine (1 µM), indomethacin (1 µM), and papaverine (100 µM).Results: In non-incubated TSM, curcumin showed significant relaxant effects on KCl-induced contraction in a concentration-dependent manner (p < 0.001 for all concentrations). The relaxant effects of curcumin 12.5, 25, and 50 mg/mL were significantly lower in atropine-incubated tissue compared to non-incubated TSM (p < 0.05 to p < 0.001). A significant difference was observed in EC50 between atropine-incubated (48.10 ± 2.55) and non-incubated (41.65 ± 1.81) tissues (p < 0.05). Theophylline showed a significant relaxant effect on both KCl and methacholine-induced contraction in a concentration-dependent manner (p < 0.001 for all cases).Conclusions: The results indicated a relatively potent relaxant effect of curcumin on TSM, which was less marked than the effect of theophylline. Calcium channel blocking and/or potassium channel opening properties of curcumin may be responsible for TSM relaxation.


Assuntos
Canais de Cálcio/metabolismo , Curcumina/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Canais de Potássio/metabolismo , Traqueia/efeitos dos fármacos , Animais , Curcuma/química , Curcumina/isolamento & purificação , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Músculo Liso/metabolismo , Músculo Liso/fisiopatologia , Ratos Wistar , Traqueia/metabolismo , Traqueia/fisiopatologia
14.
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
15.
Pharmacol Rep ; 72(1): 179-187, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32016843

RESUMO

BACKGROUND: Benign prostatic hyperplasia (BPH) is associated with obesity and prostatic inflammation. The present study investigated the participation of toll-like receptor 9 (TLR9) in obesity-induced BPH, focusing on metabolic impairments, damage-associated molecular patterns (DAMP) levels and prostatic oxidative stress generation. METHODS: C57BL/6 (WT) and TLR9 mutant male mice were fed with regular or high-fat diet for 12 weeks. Metabolic profile, functional protocols, reactive-oxygen species (ROS) generation, prostatic histological analysis and DAMP levels were analyzed. Western blotting for prostatic TLR9 signaling pathway was also performed. RESULTS: BPH in WT obese animals was characterized by increased prostate weight, smooth muscle hypercontractility and prostatic epithelial hyperplasia. Higher epididymal fat weight and prostatic ROS generation along with increased fasting glucose, triglyceride and circulating DAMP levels were also observed in WT obese group. Conversely, TLR9 mutant obese animals exhibited lower epididymal fat weight, fasting glucose and triglyceride levels associated with reduced prostate hypercontractility, prostatic ROS and circulating DAMP levels. However, TLR9 mutant obese mice were not protected from obesity-associated prostatic overgrowth and epithelial hyperplasia. Interestingly, TLR9 mutant lean mice exhibited augmented fasting glucose and prostatic ROS levels compared with WT lean mice. Despite increased prostatic expression of TLR9 in WT obese mice, no differences were seen in MyD88 expression between groups. CONCLUSION: Improved obesity-induced BPH-related prostatic smooth muscle hypercontractility in TLR9 obese mice may be associated with amelioration in the metabolic profile, ROS and DAMP generation. Therefore, TLR9 could be a valuable target to improve obesity-associated metabolic disorders and prostate smooth muscle hypercontractility in BPH.


Assuntos
Obesidade/complicações , Estresse Oxidativo/fisiologia , Hiperplasia Prostática/fisiopatologia , Receptor Toll-Like 9/genética , Alarminas/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Hiperplasia Prostática/etiologia , Hiperplasia Prostática/genética , Espécies Reativas de Oxigênio/metabolismo
17.
J Vis Exp ; (155)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32065126

RESUMO

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 °C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.


Assuntos
Cátions/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Fenantrenos/metabolismo , Feminino , Humanos , Masculino , Músculo Liso/citologia
18.
Drug Res (Stuttg) ; 70(2-03): 107-111, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31896158

RESUMO

The effect of methanol extract of P. americana seeds on isolated ileal smooth muscle was studied for isometric response using 10 adult rabbits of both sexes. Reactivity and agonist-antagonist responses of rabbit ileum to the extract were determined in this study. The affinity, effective concentration to give 50% response (EC50) and maximum response were calculated from the concentration response curves (CRC) obtained. The result for the reactivity study showed the seed extract of P. americana caused concentration dependent relaxation of isolated rabbit ileum with threshold responses at concentration of 1×10-9 mg/ml and 120 mg/ml respectively. The extract-antagonist study showed an upward and right shift in CRC in the presence of phenoxybenzamine, a non-selective adrenergic antagonist, with the EC50 increased from 5.01 mg/ml to 12.59 mg/ml and affinity decreased from 0.20 to 0.08. Extract-antagonist study also showed a right and upward shift in the CRC with a greater magnitude in the presence of prazosin, an α1-adrenergic antagonist, with EC50 increased from 0.32 mg/ml to 25.12 mg/ml and a consequential decrease in the affinity from 3.13 to 0.04. In the presence of propranolol, a ß-adrenergic antagonist, a downward and left shift in the CRC was observed with the EC50 and PA2 remaining constant at 0.1 mg/ml and 10 respectively. P. americana concentration-dependently reduced or inhibited gastric motility, increasing transit time which is important for food absorption, thus a pro-nutritive and antispasmodic effect. The interaction with α1-adrenoceptors is beneficially in heart failure management. The plant can be developed as a drug candidate for management of hypertension.


Assuntos
Relaxamento Muscular/efeitos dos fármacos , Persea/química , Extratos Vegetais/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Íleo/efeitos dos fármacos , Íleo/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Extratos Vegetais/administração & dosagem , Coelhos , Receptores Adrenérgicos alfa 1/metabolismo , Sementes
19.
Am J Physiol Renal Physiol ; 318(2): F496-F505, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904286

RESUMO

Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca2+ influx via voltage-dependent Ca2+ channels in USM cells, modulated by the activation of Ca2+-activated Cl- channels encoded by Ano1. However, recent findings in the mouse have suggested that USM tone is voltage independent, relying on Ca2+ influx through Orai channels via store-operated Ca2+ entry (SOCE). We explored if this pathway also occurred in the pig using isometric tension recordings of USM tone. Pig USM strips generated myogenic tone, which was nearly abolished by the Cav1.2 channel antagonist nifedipine and the ATP-dependent K+ channel agonist pinacidil. Pig USM tone was reduced by the Orai channel blocker GSK-7975A. Electrical field stimulation (EFS) led to phentolamine-sensitive contractions of USM strips. Contractions of pig USM were also induced by phenylephrine. Phenylephrine-evoked and EFS-evoked contractions of pig USM were reduced by ~50-75% by nifedipine and ~30% by GSK-7975A. Inhibition of Ano1 channels had no effect on tone or EFS-evoked contractions of pig USM. In conclusion, unlike the mouse, pig USM exhibited voltage-dependent tone and agonist/EFS-evoked contractions. Whereas SOCE plays a role in generating tone and agonist/neural-evoked contractions in both species, this dominates in the mouse. Tone and agonist/EFS-evoked contractions of pig USM are the result of Ca2+ influx primarily through Cav1.2 channels, and no evidence was found supporting a role of Ano1 channels in modulating these mechanisms.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Contração Isométrica , Músculo Liso/metabolismo , Uretra/metabolismo , Animais , Benzamidas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Sinalização do Cálcio/efeitos dos fármacos , Estimulação Elétrica , Feminino , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Fenilefrina/farmacologia , Pirazóis/farmacologia , Sus scrofa , Uretra/efeitos dos fármacos
20.
PLoS One ; 15(1): e0227672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945134

RESUMO

A large number of pre-clinical and developmental investigations involve experimental vertebrate animals, of which mice have emerged as a favored organism. Recognition of the differences between humans and mice is essential for assessment of the relevance of animal studies to humans. The primary purpose of this study was to gauge the conservation between human and mouse vascular smooth muscle cell (VSMC) proteins mined from an analysis of the Human Protein Atlas. Two comparison were made: a) immunohistochemistry for 16 proteins in brain, heart, esophagus, bladder, stomach, lung, kidney, and aorta enabled comparison between human and mouse of protein localization in VSMC and non-vascular SMC; and b) multi-species primary protein sequence analysis of an expanded set vascular molecules enabled comparison between VSMC sequences among vertebrate species. In total, three dimensions of diversity were uncovered. First, a significant number of factors show human/mouse differences in cellular expression; these differences occurred in both VSMC and non-vascular SMC in an organ and cell-type dependent fashion. Many markers demonstrated notable cell-to-cell and regional heterogeneity in VSMC of the aorta and non-vascular SMC of the esophagus, bladder, and stomach. Second, species specificity can arise by genetic deletions as exemplified by the human protein adipogenesis regulatory factor (ADIRF), which is not present due to a large sequence gap in mice. Third, we describe significant cross-species protein sequence divergence in selected VSMC proteins which may result in altered orthologue function. In a sample of 346 vascular molecules, 15% demonstrate incomplete vertebrate species gene conservation. Divergence of predicted human/mouse VSMC protein sequences is higher than for endothelial proteins in all species examined. In the future, each of these three cross-species differences could be neutralized using gene manipulation, resulting in improved translational potential of murine experimental models.


Assuntos
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Animais , Biomarcadores/metabolismo , Sequência Conservada , Bases de Dados de Proteínas , Variação Genética , Humanos , Imuno-Histoquímica , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Especificidade da Espécie , Distribuição Tecidual
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