Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM, Orai and TRPC channels

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca2+-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca2+-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.

Estudo quantitativo da matriz extracelular e células do músculo liso de bexigas urinária após traumatismo raquimedular / Quantitative study of the extracellular matrix and smooth muscle cells of the urinary bladder after spinal cord injury

Lesões na inervação do trato urinário inferior ocasionado por traumatismo raquimedular afetam geralmente o músculo detrusor e o esfíncteres uretrais. Estas alterações acarretam problemas basicamente de incontinência urinária e aumento da pressão intravesical, decorrente deste traumatismo, trazendo consequências para o funcionamento do sistema urinário superior. Quantificar os elementos fibrosos da matriz extracelular e fibras musculares das bexigas neurogênicas hiper-reflexas comparando-as com bexigas normais. Foram utilizadas 6 amostras de bexigas neurogênicas de indivíduos que foram submetidos a cirurgia de reparação por cistoenteroplastia realizados pelo serviço de urologia do Hospital Municipal Souza Aguiar, estas amostras foram fixadas imediatamente em solução tamponada de formalina a 10%. O controle com amostras iguais as do estudo extraída de cadáveres cuja causa morte não relacionava-se ao sistema urogenital macroscópicamente. O material foi submetido as seguintes técnicas histoquímicas: H&E, van Gieson e Resorcina Fucsina resorcina de Weigert com prévia oxidação pela oxona. Imunohistoquímica: anti-elastina. A observação dos cortes corados pelo van Gieson demonstrou uma diminuição significativa do músculo liso de 13% e aumento do colágeno em 72% e as fibras do sistema elástico um aumento de 101%. Conclusão. Nas bexigas neurogênicas hiper-reflexas o músculo detrusor e os elementos fibrosos da matriz foram profundamente modificados. As fibras do sistema elástico foram as mais afetadas.

Lesions on lower urinary tract innervations caused by spinal cord injuries usually affect the detrusor muscle and urethral sphincter. Beside the smooth muscle fibers the collagen fibers and elastic system fibers, fibrous components of the extracellular matrix of the bladder wall, are strongly related to vesicle bladder compliance. For this reason the aim of this work is to quantify the fibrous elements of the extracellular matrix and muscle fibers of the neurogenic bladder hyperreflexia. Samples of neurogenic bladder were obtained from six men who had previously undergone surgical repair. The control group samples (n=6) were similarly obtained from patients whose deaths were not related to the urogenital system. The samples were stained using the following histochemical techniques: H&E, Van Gieson, Weigert and Sirius Red. Sections stained with Sirius Red were observed under polarization light microscopy to characterize possible different kinds of collagen. Immunohistochemical technique was used to characterize and quantify the elastic system fibers. Quantification analysis was performed by stereological methods. An increase of 72% of the collagen was observed. Nevertheless, the most significant difference observed was the raising of 101% of the elastic system fibers. Contrary the smooth muscle fibers showed a decrease of 13%. In the neurogenic bladder with detrusor hyperreflexia the fibrous elements of the extracellular matrix and smooth muscle fibers were greatly modified. The elastic system fibers seem to be the most affected in this disease.

Use of a latex biomembrane for bladder augmentation in a rabbit model: biocompatibility, clinical and histological outcomes

PURPOSE: To investigate histological features and biocompatibility of a latex biomembrane for bladder augmentation using a rabbit model. MATERIAL AND METHODS: After a partial cystectomy, a patch of a non-vulcanized latex biomembrane (2x4 cm) was sewn to the bladder with 5/0 monofilament polydioxanone sulfate in a watertight manner. Groups of 5 animals were sacrificed at 15, 45 and 90 days after surgery and the bladder was removed. The 5-µm preparations obtained from grafted area and normal bladder were stained with hematoxylin-eosin. Immunohistochemical staining was performed with a primary antibody against alpha-actin to assess muscle regeneration. RESULTS: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. Macroscopically, after 90 days, the latex biomembrane was not identifiable and the patch was indistinguishable from normal bladder. A bladder stone was found in one animal (6.6 percent). On the 90th day, histology revealed continuity of transitional epithelium of host bladder tissue on the patch area. At this time, the muscle layers were well organized in a similar fashion to native bladder muscle layers. The inflammatory process was higher on grafted areas when compared to controls: 15 days - p < 0.0001, 45 days - p < 0.001, and 90 days - p < 0.01. The anti alpha-actin immunoexpression peaked at 45 days, when the graft was observed covered by muscle cells. CONCLUSION: The latex biomembrane is biocompatible and can be used in models for bladder augmentation in rabbits. It promotes epithelium and muscle regeneration without urinary leakage.

Interstitial cells of Cajal and intestinal function in Trinidadian children / Células intersticiales de Cajal y función intestinal en niños trinitenses

OBJECTIVE: This study was carried out to compare the density of the interstitial cells of Cajal (ICCs) in the bowel wall of children with Hirschsprung's disease (HD), anorectal malformations (ARM) and normal controls in Trinidad and Tobago. SUBJECTS AND METHOD: Segments of bowel wall excised from eight children with HD, three controls and two children with ARM were immunostained with c-Kit primary antibody. Cells with features of ICCs were counted. RESULTS: All three controls and the two children with ARM had dense distribution of ICCs. Most children (6/8;75%) with HD had markedly reduced counts in aganglionic bowel. Two (25%) also had a decrease in ganglionic bowel. Possible influences were patient age and gender and the level of bowel sectioned. CONCLUSION: Analysis of this sample suggests that immunostaining for c-Kit positive cells might be a useful screening test in the assessment of bowel motility disorders. The possible effects of age, gender and the level of bowel sampled await determination.

OBJETIVO: Este estudio se llevó a cabo con el propósito de comparar la densidad de las células intersticiales de Cajal (CIC) en las paredes intestinales de niños con la enfermedad de Hirschprung (EH), y malformaciones anorectales (MAR), frente a controles normales en Trinidad Tobago. SUJETOS Y MÉTODOS: Segmentos de las paredes intestinales les fueron extirpados a ocho niños con EH; tres controles y dos niños con MAR fueron inmunoteñidos con anticuerpo primario c-kit. Se contaron las células con características de CIC. RESULTADOS: Los tres controles y los dos niños con MAR presentaban una distribución densa de CICs. La mayor parte de los niños (6/8; 75%) con EH tuvieron conteos marcadamente reducidos de intestino agangliónico. Dos niños (25%) también tuvieron una disminución de intestino gangliónico. Entre las influencias posibles se cuentan la edad y el género del paciente así como el nivel de intestino seccionado. CONCLUSIÓN: El análisis de esta muestra sugiere que la inmunotinción para células c-kit positivas podría ser un útil test de pesquisaje a la hora de evaluar desórdenes en la motilidad intestinal. Los efectos posibles de la edad, el género y el nivel de intestino muestreado, están pendientes de determinación.

Avaliação quantitativa das fibras elásticas na doença pulmonar obstrutiva crônica / Quantitative assessment of elastic fibers in chronic obstructive pulmonary disease

OBJETIVO: Quantificar fibras elásticas (FE), músculo liso (ML) e linfócitos T CD4+ e CD8+ na doença pulmonar obstrutiva crônica (DPOC) estável. MÉTODOS: Biópsias cirúrgicas foram obtidas de 15 pacientes com DPOC, 18 tabagistas sem limitação do fluxo aéreo e 14 não tabagistas. FE, ML e células T CD4+ e CD8+ foram quantificados através de métodos histológicos e imuno-histoquímicos. RESULTADOS: Não foi observada diferença estatisticamente significativa das FE nos três grupos (p > 0,05). Tanto a quantidade de FE por unidade de área pulmonar (mm²), quanto o percentual destas fibras por tecido pulmonar foram semelhantes nos três grupos. Foi encontrado aumento da quantidade de ML em pacientes com DPOC quando comparados a tabagistas (p = 0,003) e não tabagistas (p = 0,009). Houve tendência de aumento das células T CD8+ nos pacientes com DPOC. O total de células T CD4+ estava diminuído nos pacientes com DPOC quando comparados aos tabagistas (p = 0,015) e não tabagistas (p = 0,003). Observou-se fraca correlação entre estas células e a relação entre o volume expiratório forçado no primeiro segundo e a capacidade vital forçada (r² = 0,003). CONCLUSÕES: A quantidade de FE foi semelhante nos três grupos estudados. A hipertrofia/hiperplasia muscular da parede das vias aéreas foi encontrada tanto em pacientes com DPOC quanto em tabagistas, indicando que o remodelamento ocorra também nos tabagistas sem limitação do fluxo aéreo. Houve diminuição da relação CD4/CD8 em pacientes com DPOC.

OBJECTIVE: To quantify elastic fibers (EFs) and smooth muscle (SM) cells, as well as CD4+ and CD8+ T lymphocytes, in stable chronic obstructive pulmonary disease (COPD). METHODS: Surgical specimens were obtained from 15 COPD patients, 18 smokers without airflow limitation, and 14 nonsmokers. Histological and immunohistochemical methods were employed in order to quantify EFs, SM cells, CD4+ T cells, and CD8+ T cells. RESULTS: There was no significant difference in EF numbers among the three groups (p > 0.05). The number of EFs per unit area of lung tissue (mm²) and the percentage of EFs in the lung tissue were similar among the three groups. The numbers of SM cells were found to be higher in the COPD patients than in the smokers (p = 0.003) or in the nonsmokers (p = 0.009). There was a tendency toward an increase in CD8+ T-cell counts in the COPD patients. In specimens collected from the COPD patients, CD4+ T-cell counts were lower than in those collected from the smokers (p = 0.015) or from the nonsmokers (p = 0.003). There was a weak correlation between CD4+ T-cell count and the ratio of forced expiratory volume in one second to forced vital capacity (r² = 0.003). CONCLUSIONS: The EF counts were similar among the three groups. Hypertrophy/hyperplasia of airway wall SM cells was found in the COPD patients and in the smokers, indicating that airway remodeling occurs in smokers. The CD4/CD8 ratio was lower in the COPD patients.

Expression of heat stress protein 70 mRNA in patients with chronic obstructive pulmonary disease and its significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70 (Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0.24 +/- 0.11 and 0. 42 +/- 0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P < 0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9 +/- 9.9 vs 44.8 +/- 15.3, P < 0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0.85, P < 0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

Smooth Muscle Cells Transplantation is better than Heart Cells Transplantation for Improvement of Heart Function in Dilated Cardiomyopathy

Muscle cell transplantation may delay or prevent cardiac dilation in dilated cardiomyopathy. The present study was designed to compare the effects of the heart function of smooth muscle cell (SMCs) auto-transplantation and heart cell (CMs) allo-transplantation in dilated cardiomyopathic hamsters, and to determine which cells are better for cell transplantation. CMs and SMCs were isolated from BIO 53.58 hamsters, and cultured for transplantation. CMs, SMCs (4 X 10(6) cells each) or culture medium were transplanted into 17 weeks old BIO 53.58 hamsters to achieve CM transplantation (CMTx), SMC transplantation (SMCTx), and controls (Con) (N=10 each). Cyclosporine (5 mg/Kg) was administered subcutaneously to CMTx. Healthy hamsters (sham, N=6) were used to compare heart functions. Four weeks after transplantation, heart function was evaluated in all groups using a Langendorff perfusion apparatus. Histology demonstrated severe focal myocardial necrosis in the dilated cardiomyopathic hearts. CMTx and SMCTx formed huge muscle tissue in the dilated myocardium. Sham, SMCTx, and CMTx had a better heart function than Con (p < 0.01), and SMCTx had a better peak systolic pressure (p < 0.05) and developed pressure (p < 0.05) than CMTx at any balloon volume. However, sham and SMCTx were not statistically different. SMCTx and CMTx formed muscle tissue and produced better heart function in the cardiomyopathic hearts, and SMCTx showed better systolic and developed pressures than CMTx, even though they were similar in other functions. Significantly, SMCTx had heart functions, which were similar to those of healthy hamster's hearts.

Relaxant effect of 6-deoxyclitoriacetal on smooth muscle preparations.

The pharmacological effect of 6-deoxyclitoriacetal (6-DA), a rotenoid compound isolated from the roots of Clitoria macrophylla Wall. (Papilionaceae), was examined on different smooth muscle preparations. 6-Deoxyclitoriacetal 0.2 mg/ml produced a significant decrease in the spontaneous contraction of isolated rat uterus. It also suppressed the contraction induced by acetylcholine 5x10(-6) M and oxytocin 5x10(-3) IU/ml. The cumulative contractile responses of rat aortic strips caused by serotonin 10(-8)-10(-4) M and norepinephrine 10(-11)-10(-7) M were reduced by 6-DA 0.4 mg/ml. In calcium free Kreb's solution, 6-DA inhibited the aortic contraction produced by a cumulative dose of calcium chloride (0.1-30 mM). In guinea-pig ileum, 6-DA 0.15 mg/ml exerted the spasmolytic activity by inhibition of the contractile response evoked by various contractile agents e.g. acetylcholine 10(-9)-10(-5) M, serotonin 10(-9)-10(-5) M and histamine 10(-9)-10(-5) M. All of the results indicated that 6-DA could induce a smooth muscle relaxant effect by interference with intracellular calcium metabolism.

Células de músculo liso: organo blanco en cardiología

La proliferación rápida de células de músculo liso vascular (CMLV) es un fenómeno que ha sido reconocido como evento central de varias formas de enfermedad vascular: Aterosclerosis, hipertensión arterial, restenosis coronaria luego de angioplastia percutánea. Avances recientes en las últimas dos décadas, han generado hipótesis claras en cuanto a la génesis de estas tres patologías, donde la CMLV son común denominador. La presencia de múltiples factores que interactúan en el organismo (mecánicos, hemodinámicos, celulares, plasmáticos, nerviosos, hormonales, humorales, etc) sobre las CMLVs para su recambio constante de fenotipo, reflejan la complejidad en la fisiopatología vascular. La importancia en el conocimiento de la CMLV y su comportamiento ante el trauma es cada vez mayor y se refleja en el entendimiento de la enfermedad y en el gran adelanto de las diferentes formas terapéuticas

Subcellular distribution and pharmacological characterization of muscarinic receptors in tracheal smooth muscle

: Subcellular fractions isolated from tracheal smooth muscle have been identified using biochemical markers and measuring the [3H]QNB muscarinic receptor binding activity in these fractions. This muscarinic receptor (mAchR) activity was slightly enriched 1.6 times in the crude mitochondrial fraction (M), 2.6 times in the crude microsomal fraction (P), and greatly enriched in the highly purified plasma membranes fractions, being 5.3 times in a heavy plasma membrane fraction designed as P2 and 9.1 times in a light plasma membrane fraction named P1 fraction. The muscarinic receptor subtypes present in the subcellular fractions were identified using competition experiments. The binding of five selective antagonists, pirenzepine, AF-DX 116, hexahydrodifenidol, methoctramine and 4-DAMP were examined. In this sense, the M1 antagonist pirenzepine showed pKi's values between 6.44-7.45 and the M2 antagonist AF-DX 116 showed pKi's values ranging from 6.75 to 7.45 being the lowest pKi's values here described. The antagonist hexahydrodifenidol showed higher affinities than pirenzepine-derivated compounds with pKi's values from 7.25 to 7.65. The antagonist 4-DAMP exhibited pKi's values from 8.18-8.41. Finally, methoctramine showed similar affinities as 4-DAMP, with pKi's ranging from 8.09 to 8.22 suggesting the existence of M2 receptors in these fractions. These data suggest that M2 mAchR are present in all articulate fractions here studied. It is important to emphasize that the M2 muscarinic receptor presents in the light plasma membrane fraction (P1) shows poor selectivity towards the muscarinic antagonists being different from the M2 mAchRs associated with other subcellular fractions isolated from bovine tracheal smooth muscle

Leiomiócitos parasitados pelo Trypanosoma Cruzi nas formas congênita e crônica adquirida da doença de Chagas / Smooth muscle cells infected by Trypanosoma Cruzi in congenital and chronic acquired forms of Chagas disease

Os leiomiócitos parassitados pelo T. cruzi, de vasos umbilicais e placentários de casos congênitos da doença de Chagas, foram comparados com células semelhantes da veia suprarrenálica de chagásicos crônicos. Observou-se que algumas destas células, em ambos os grupos, apresentavam envoltório visto à microscopia de fase, nas preparaçöes näo coradas, e à microscopia de luz comum, quando coradas pelo PAS e pela HE. A prata-metenamina cora apenas o envoltório das células parasitadas dos casos de doença de Chagas congênita. A técnica do picro-sirius, associada à microscopia de luz polarizada, mostra que ambos os grupos de leiomiócitos parasitados podem apresentar delicada membrana com birrefrigência discreta "verde limäo", enquanto que un envoltório espesso com forte birrefrigência amarelada ou brancacente é visto apenas nos leiomiócitos fetais. A técnica da peroxidase anti-peroxidase cpara T. cruzi mostra acúmulo de material PAP-positivo formando uma faixa interrompida que se coloca mais internamente à faixa PAS-positivo, em ambas as formas da doença. As modificaçöes nucleares e citoplasmáticas das células fetais parasitadas (Chagas congênito) e dos adultos (chagásicos crônicos) säo muito semelhantes, destacando-se o gigantismo nuclear, o aparecimento de grânulos algumas vezes PAP-positivo, e a desproporçäo entre o aumento do volume celular e o número de parasitas. É possível que o estado de imunodeficiência, localizado na glândula suprarrenal do adulto e o tecido fetal tenham algum papel na gênese dessa peculiaridade das células parasitadas, comuns na doença de Chagas congênita e na forma crônica adquirida