Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1β and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus. .

O microambiente tumoral como alvo terapêutico: avaliação do efeito de antagonistas do receptor de bradicinina tipo 1 em melanoma murino / Tumor microenvironment as therapeutic target: evaluation of the effect of bradkinin receptor 1 antagonists in murine melanoma

O microambiente tumoral tem sido considerado um importante alvo para terapias anticâncer. Recentemente, o receptor de bradicinina tipo 1 (BKR1), o qual é expresso em leucócitos e células endoteliais, foi implicado na progressão de diversos cânceres, incluindo o melanoma... Dois diferentes métodos foram utilizados na seleção de genes diferencialmente expressos (differentially expressed genes -DEGs): o test “t de Student” e o teste Limma. Um total de 87 DEGs foram selecionadas com fold change >1,5 e valor de p<0,05. Alguns desses genes, envolvidos em importantes processos como angiogênese, invasão, metástase e quimioresistência, têm sido validados por PCR em tempo real, como: transglutaminase-2, HSP-1b, neuropilina-2, serpine 1, fator de crescimento do tecido conjuntivo (Ctgf) e serglicina. Em um segundo conjunto de experimentos foi avaliado o efeito de antagonistas de BKR1 em alterações de permeabilidade vascular e distribuição de droga fluorescente (doxorrubicina) no microambiente do tumor. Os antagonistas de BKR1, R-954 e R-715, causaram redução da penetração de doxorrubicina no microambiente tumoral após tratamento por 2 horas, precedido ou não de tratamento crônico (diário), entretanto após 6 horas de tratamento foi observado um aumento da quantidade de doxorrubicina, comparado ao controle, seguido de redução após 24 horas de tratamento. Já em tumores EMT6 (sarcoma mamário murino) após 2 horas de tratamento com esses antagonistas foi possível observar um aumento da quantidade de doxorrubicina no microambiente tumoral. Esses resultados sugerem mecanismos pelos quais as complexas interações entre as células tumorais e as células do hospedeiro podem resultar na resistência tumoral aos quimioterápicos e a consequente falha dos tratamentos convencionais.

Tumor microenvironment has been considered an important target for anticancer therapies. Recently, the bradykinin receptor type 1 (BKR1), which is expressed on leukocytes and endothelial cells, has been implicated in the progression of several cancers, including melanoma. In this work we have shown that there is a delay in BKR1 knockout mice melanoma engraftment and we also observed higher survival rates in these animals compared to wild type mice. We have investigated the effects of selectives BKR1 antagonists evaluating its combination with the chemotherapeutic agent dacarbazine (DTIC). B16-F10 melanoma cells were injected subcutaneously in C57bl/6 mice, which were treated with DTIC (2mg/kg) every 3 days, R-954 (1mg/kg) daily, DTIC plus R-954 or PBS, as control. Mice were sacrificed at day 12 of treatment; tumors were excised and processed for RNA extraction. Treatments with R-954, DTIC or R-954 plus DTIC did not reduced tumor mass. However, the combined treatment increased the global survival of mice bearing tumor. Microarray experiments were performed using GeneChip Mouse 430 (Affymetrix). Data was normalized using the RMA method. Two different methods were employed for selection of differentially expressed genes (DEGs):the Student's T-test and the Limma package. A total of 87 DEGs were identified with fold change>1,5 and p value<0,05. Some of these genes, involved in important process as angiogenesis, invasion, metastasis and chemoresistance, had been validated by real time PCR as; transglutaminase-2, HSP-1b, neuropilin-2, serpine 1, connective tissue growth factor and serglycin. In a second set of experiments we evaluated the effect of antagonists BKR1 in vascular permeability alterations and distribution of fluorescent drug (doxorubicin) in tumor microenvironment. BKR1 antagonists, R-954 and R-715, caused a reduction of the penetration of doxorubicin in the tumor microenvironment after treatment for 2 hours, followed or not by chronic treatment (daily), but after 6 hours of treatment was observed an increase in the amount of doxorubicin, compared to the control, followed by reduction after 24 hours of treatment. However, in EMT6 tumors (murine mammary sarcoma) after 2 hours of treatment with these antagonists we observed an increased amount of doxorubicin in tumor microenvironment. These results suggest mechanisms by which the complex interactions between tumor and BKR1 positive host cells may result in tumor resistance to chemotherapeutic agents and the consequent failure of regular treatments.

Identificação de um novo sítio de produção da enzima conversora de angiotensina I(ECA) nas células IMCD e estudo da interação do receptor de brasicinina e da ECA utilizando inibidores da ECA / Identification of a new site of angiotensin-I converting enzyme (ACE) production in IMCD cells and study of interation between the bradykinin receptors and ACE using ACE inhibitors

Enzima conversora de angiotensina I (ECA) ou kininase II é uma dipeptidil- carboxipepitidase (EC 3.4.15.1) que tem um importante papel na regulação de pressão sangüínea, cliva o dipeptídeo C-terminal da angiotensina I(AI)i produzindo o potente peptídeo vasopressor angiotensina II (AII) e inativa bradicinina (BK), um potente vasodilatador. Baseados no estudo de Casarini e cols. (1997) no qual um novo sítio produção de ECA foi localizado no ducto coletor, o objetivo deste trabalho foi purificar e caracterizar na cultura de células mIMCD-3 a presença e/ou síntese da ECA, enfatizando desta forma um papel fisiológico importante para esta enzima nestas células, uma vez que todo sistema calicreína-cininas encontra-se presente nesta região. Outro objetivo foi estudar a interação da ECA com os receptores B2 da BK utilizando inibidores da ECA. Inicialmente, isolamos a atividade da ECA presente nas células mIMCD no meio de cultura, por gel filtração em coluna empacotada com resina AcA-34 previamente calibrada. Cada isoforma foi recromatografada em coluna afinidade Lisinopril-sepharose. As enzimas isoladas do meio foram denomina M1 e M2, e as ECAs extraídas das células, C1 e C2. As massas moleculares determinadas por eletroforese em gel de poliacrilamida foram: M1, 60 kDa e M2 117 kDa (isoformas secretadas) e C1, 63 kDa e C2, 130 kDa (isoformas intracelulares). A expressão protéica foi verificada por Western Blotting utilizando anticorpo monoclonal 9B9, nas isoformas purificadas e nas frações do citoplasma da membrana e do núcleo…(au)

Expression of Bradykinin Receptors and Effects of Bradykinin in the Interstitial Cells of Cajal from Mouse Small Intestine / 체질인류학회지

Interstitial Cells of Cajal (ICC) are pacemaker cells that generates slow waves and drive spontaneous mechanical contractions of gastrointestinal smooth muscle. Slow waves are generated the periodic activation of spontaneous inward currents (pacemaker currents). We studied the modulation of pacemaker activities by bradykinin (10-8 M) in cultured ICC with the whole cell patch-clamp technique, and the localization of bradykinin-2 receptor-immunoreactivity using double labelling immunohistochemistry in the murine small intestine. Externally applied bradykinin produced membrane depolarization in current-clamping mode. At a -70 mV of holding potential bradykinin increased tonic inward pacemaker currents. Double labelling with bradykinin-2 receptor and and c-kit was shown that ICC expressed the bradykinin-2 receptor-immunoreactivity. These results suggest that bradykinin modulates electrical activities of ICC via bradykinin-2 receptor, which may regulate gastrointestinal motility.

Mecanismo de transporte iônico, em íleo de coêlho, induzido por mecrocistina_LR de microcystis aeruginosa: participação de macrófagos, IL 1 ß, TNF a e mediadores pro-inflamatórios [Manuscrito] / Ion transport mechanism in rabbit ileum induced by Microcystis aeruginosa mecrocistina_LR of: participation of macrophages, IL 1 beta, TNF a and pro-inflammatory mediators [manuscript]

Este trabalho teve-se como principal objetivo, avaliar os efeitos eletrogênicos em preparações de íleo de coelho fixadas em câmaras de Üssing, em presença de sobrenadante de macrófagos (S.MφS) estimulados com microcistina-LR MCLR de Microcystis aeruginosa. S.MφS estimulados com MCLR (3,2. 10-7M; 9,6.10-7M e 3,2.10-6M), produz, de foram dose-dependente; sendo que a variação temporal (t) da corrente de curto-circuito (Isc), pode ser descrita por uma equação do tipo Isc = a. ekt. para um coeficiente de correlação r = 0,9988 e Iscmaximo = 128,16 =- 14,54 μacm-2. Posteriormente, observou-se que os processos metabólicos associados à gênese do fator de " secreção intestinal" (FSI), a partir de macrófagos estimulados, requer a participação de uma proteína G sensível à toxina pertusis ativa. Verificou-se também que inibidores de síntese proteica, proteases, fosfolipase A2, cicloxigenases, lipoxigenases; síntese de TNF-α e antagonista do PAF, reduziram a síntese de FSI. Com o emprego de anticorpos monoclonais, verificou-se que IL-1β era o principal FSI; como também, que macrófagos estimulados com MCLR, nas concentrações acima, reduziu IL-1β e TNF-α, de forma dose-dependente...

Influence of Bradykinin on Catecholamine Release from the Rat Adrenal Medulla

The present study was undertaken to investigate the effect of bradykinin on secretion of catecholamines (CA) evoked by stimulation of cholinergic receptors and membrane depolarization from the isolated perfused model of the rat adrenal glands, and to elucidate its mechanism of action. Bradykinin (3 X 10 (-8) M) alone produced a weak secretory response of the CA. however, the perfusion with bradykinin (3 X 10 (-8) M) into an adrenal vein of the rat adrenal gland for 90 min enhanced markedly the secretory responses of CA evoked by ACh (5.32 X 10 (-3) M), excess K+ (5.6 X 10 (-2) M, a membrane depolarizer), DMPP (10 (-4) M, a selective neuronal nicotinic agonist) and McN-A-343 (10 (-4) M, a selective M1-muscarinic agonist). Moreover, bradykinin (3 X 10 (-8) M) in to an adrenal vein for 90 min also augmented the CA release evoked by BAY-K-8644, an activator of the dihydropyridine L-type Ca2+ channels. However, in the presence of (N-Methyl-D-Phe7) -bradykinin trifluoroacetate salt (3 X 10 (-8) M), an antagonist of BK2-bradykinin receptor, bradykinin no longer enhanced the CA secretion evoked by Ach and high potassium whereas the pretreatment with Lys- (des-Arg9, Leu8) -bradykinin trifluoroacetate salt (3 X 10 (-8) M), an antagonist of BK1-bradykinin receptor did fail to affect them. Furthermore, the perfusion with bradykinin (3 X 10 (-6) M) into an adrenal vein of the rabbit adrenal gland for 90 min enhanced markedly the secretory responses of CA evoked by excess K+ (5.6 X 10 (-2) M). Collectively, these experimental results suggest that bradykinin enhances the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) and membrane depolarization through the activation of B2-bradykinin receptors, not through B1-bradykinin receptors. This facilitatory effect of bradykinin seems to be associated to the increased Ca2+ influx through the activation of the dihydropyridine L-type Ca2+ channels.

Regulation of the kinin receptors after induction of myocardial infarction: a mini-review

It is well known that the responses to vasoactive kinin peptides are mediated through the activation of two receptors termed bradykinin receptor B1 (B1R) and B2 (B2R). The physiologically prominent B2R subtype has certainly been the subject of more intensive efforts in structure-function studies and physiological investigations. However, the B1R activated by a class of kinin metabolites has emerged as an important subject of investigation within the study of the kallikrein-kinin system (KKS). Its inducible character under stress and tissue injury is therefore a field of major interest. Although the KKS has been associated with cardiovascular regulation since its discovery at the beginning of the last century, less is known about the B1R and B2R regulation in cardiovascular diseases like hypertension, myocardial infarction (MI) and their complications. This mini-review will summarize our findings on B1R and B2R regulation after induction of MI using a rat model. We will develop the hypothesis that differences in the expression of these receptors may be associated with a dual pathway of the KKS in the complex mechanisms of myocardial remodeling.

Vasopressin and bradykinin receptors in the kidney: implications for tubular function

Vasopressin and bradykinin are two of the most important peptides in regulating vascular tone, water, and ionic balance in the body, adn thus they play a key role in controlling blood pressure. In addition to being a potent vasoconstrictor, Vasopressin also has an antidiuretic activity in the kidney, whereas kinins regulate renal blood flow in addition to their vasodilatory and natriuretic activity. We review here the primary evidence for the localization of the vasopressin and kinin receptors and their role in ionic and water regulation in the kidney.

Distribution of bradykinin B2 receptors in target cells of kinin action: visualization of the receptor protein in A431 cells, neutrophils and kidney sections

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Apllication of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue

Mechanism of smooth muscle contraction and relaxation mediated by kinin receptor

The increase in sensitivity of guinea pig preparations to bradykinin (BK) due to stretching occurring with time after mounting was studied by determining the time course of changes in the cell membrane potential, measured with intracellular microelectrodes. A sustained hyperpolarizing effect of BK, which was observed in recently mounted preparations, became transient after 120 min, when it was followed by depolarization, which was much more evident after 4 h of stretching. As a consequence, a parallel increase in the contractile response to BK was also observed. The hyperpolarizing effect was due to the opening of Ca²+-dependent K+ channels sensitive to apamin, since BK dose-response curves done within 1 h of mouting were shifted to the left, becoming similar to dose-response curves obtained 4 h after mounting of the guinea pig ileum preparation. These results were specific for BK, since the potentiating effect of apaming was not observed for acetylcholine. Our results show that the activation of B2 receptors by BK in the isolated guinea pig ileum induce a dual effect - hyperpolarization and depolarization - and that the increase in the contractile response consequent to stretching is probably due to the inactivation of apaminsensitive Ca²+-dependent K+ channels