Vascular function in arteries of intertidal fish Girella laevifrons (Kyphosidae) / Função vascular nas artérias do peixe marinho Girella laevifrons (Kyphosidae)

Preliminary studies showed that dorsal artery contraction mediated by acetylcholine (ACh) is blocked with indomethacin in intertidal fish (Girella laevifrons). Our objective was to characterise the cholinergic pathway in several artery vessels of the G. laevifrons. Afferent and efferent branchial, dorsal and mesenteric arteries were dissected of 6 juvenile specimens, isometric tension studies were done using dose response curves (DRC) for Ach (10–13 to 10–3 M), and cholinergic pathways were obtained by blocking with atropine or indomethacin. CRC to ACh showed a pattern of high and low sensitivity. Furthermore, these contractions were blocked in the presence of atropine and indomethacin in all vessels. Our results suggest that contraction observed with acetylcholine is mediated by receptors that activate a cyclooxygenase contraction pathway.

Estudos preliminares mostraram que a contração da artéria dorsal mediada por acetilcolina (ACh) é bloqueada com indometacina em peixes marinhos Girella laevifrons. Nosso objetivo foi caracterizar a via colinérgica em várias artérias de G. laevifrons. Artérias aferentes e eferentes branquiais, dorsais e mesentéricas foram dissecadas de 6 espécimes juvenis. Os estudos de tensão isométrica foram feitos utilizando-se a curva dose - resposta (CDR) para Ach (10–13 a 10–3M), e identificaram-se as vias colinérgicas, bloqueando com atropina e indometacina. CRC para ACh mostrou um padrão de alta e baixa sensibilidade. Essas contrações foram bloqueadas na presença de atropina e indometacina em todas as artérias avaliadas. Nossos resultados sugerem que a contração observada com acetilcolina é mediada por receptores muscarínicos que ativam uma ciclo-oxigenase.

Resistance Exercise Restores Endothelial Function and Reduces Blood Pressure in Type 1 Diabetic Rats / Exercício Resistido Restaura a Função Endotelial e Reduz a Pressão Arterial de Ratos Diabéticos Tipo 1

Background: Resistance exercise effects on cardiovascular parameters are not consistent. Objectives: The effects of resistance exercise on changes in blood glucose, blood pressure and vascular reactivity were evaluated in diabetic rats. Methods: Wistar rats were divided into three groups: control group (n = 8); sedentary diabetic (n = 8); and trained diabetic (n = 8). Resistance exercise was carried out in a squat device for rats and consisted of three sets of ten repetitions with an intensity of 50%, three times per week, for eight weeks. Changes in vascular reactivity were evaluated in superior mesenteric artery rings. Results: A significant reduction in the maximum response of acetylcholine-induced relaxation was observed in the sedentary diabetic group (78.1 ± 2%) and an increase in the trained diabetic group (95 ± 3%) without changing potency. In the presence of NG-nitro-L-arginine methyl ester, the acetylcholine-induced relaxation was significantly reduced in the control and trained diabetic groups, but not in the sedentary diabetic group. Furthermore, a significant increase (p < 0.05) in mean arterial blood pressure was observed in the sedentary diabetic group (104.9 ± 5 to 126.7 ± 5 mmHg) as compared to that in the control group. However, the trained diabetic group showed a significant decrease (p < 0.05) in the mean arterial blood pressure levels (126.7 ± 5 to 105.1 ± 4 mmHg) as compared to the sedentary diabetic group. Conclusions: Resistance exercise could restore endothelial function and prevent an increase in arterial blood pressure in type 1 diabetic rats. .

Fundamento: Os efeitos do exercício resistido sobre os parâmetros cardiovasculares não são consistentes. Objetivos: Foram avaliados os efeitos do exercício resistido sobre as alterações na glicemia, reatividade vascular e pressão arterial de ratos diabéticos. Métodos: Ratos Wistar foram divididos em três grupos: grupo controle (n = 8), diabético sedentário (n = 8) e diabético treinado (n = 8). O exercício resistido foi realizado no aparelho de agachamento para ratos e consistiu em três séries de dez repetições com uma intensidade de 50%, três vezes por semana, durante 8 semanas. As alterações na reatividade vascular foram avaliadas em anéis de artéria mesentérica superior. Resultados: Foi observada uma redução significativa da resposta máxima dos relaxamentos induzidos por acetilcolina no grupo diabético sedentário (78,1% ± 2) e um aumento do grupo diabético treinado (95 ± 3%), sem alterar a potência. Na presença de NG-nitro-L-arginina metil éster, os relaxamentos induzidos por acetilcolina foram significativamente reduzidos nos grupos controle e diabético treinado, mas não no grupo diabético sedentário. Além disso, foi observado um aumento significativo (p < 0,05) da pressão arterial média no grupo diabético sedentário de 104,9 ± 5 para 126,7 ± 5 mmHg, quando comparado ao grupo controle. Por outro lado, o grupo diabético treinado apresentou redução significativa (p < 0,05) nos níveis da pressão arterial média de 126,7 ± 5 mmHg para 105,1 ± 4 mmHg, quando comparado ao diabético sedentário. Conclusões: O exercício resistido foi capaz de restaurar a funcionalidade endotelial e impedir o aumento da pressão arterial em ratos com ...

Histological, biochemical and pharmacologycal characterization of the gastric muscular layer in Chagas disease / Caracterização histológica, bioquímica e farmacológica da musculatura gástrica na doença de Chagas

PURPOSE: To assess in vitro the correlation between the number of neurons and the sensitivity to cholinergic drugs and acetylcholinesterase activity in chagasic patients. METHODS: A 3x1 cm strip of the muscle layer of the anterior part of the stomach, always close to the angular incisure, was removed from 10 chronic chagasic patients (6 men) submitted to megaesophagus or megacolon surgery and from 10 non-chagasic patients (4 men) submitted to other types of surgery (control group), aged on average 52.3 and 50.1 years, respectively, for histological and pharmacological studies. The action of cholinergic drugs was investigated in isolated preparations according to the superfusion method of Ferreira and Costa, and acetylcholinesterase activity was determined by the method of Ellman. For neuron count, the strips were cut into 8 µm sections according to the method standardized by Alcântara. RESULTS: There was a difference in number of neurons between the chagasic (5,6) and control (7,3) groups. Acetylcholinesterase activity, in moles of hydrolyzed substrate per minute per gram tissue, was reduced in chagasic patients (4,32) compared to the controls (7,30). No hypersensitivity of the gastric musculature to cholinergic drugs was detected, with a reduced maximum response to carbachol and betanechol in the chagasic group. CONCLUSIONS: The reduction of neurons in the myenteric plexus of the stomach of chronic chagasic patients can be demonstrated even in the absence of clinical chagasic gastropathy. The hypersensitivity of the gastric musculature to cholinergic drugs probably depends on intense denervation. The reduced acetylcholinesterase activity demonstrates the involvement of the cholinergic innervation in the stomach of chronic chagasic patients. There was no correlation between number of neurons, sensitivity to cholinergic drugs and acetylcholinesterase activity in the gastric musculature of chagasic and non-chagasic patients.

OBJETIVO: Avaliar in vitro a correlação entre o número de neurônios e a sensibilidade a drogas colinérgicas e a atividade da acetilcolinesterase em pacientes chagásicos. MÉTODOS: Em 10 pacientes chagásicos crônicos (6 homens) submetidos à cirurgia de megaesôfago ou de megacólon e em 10 pacientes não chagásicos (4 homens) submetidos a outros tipos de cirurgia (grupo controle), respectivamente com idade média de 52,3 e 50,1 anos, retirou-se uma tira de 3x1 cm da camada muscular da parede anterior do estômago, sempre junto á cisura angular, que serviu para os estudos histológicos e farmacológicos. A ação de drogas colinérgicas foi feita em preparação isolada de acordo com o método de superfusão de Ferreira e Costa, e a determinação da atividade da acetilcolinesterase pelo método de Ellman. Para a contagem de neurônios a tira muscular foi submetida a cortes de 8 micra segundo método padronizado por Alcântara. RESULTADOS: Houve diferença do número de neurônios entre os grupos chagásico (5,6) e controle (7,3). A atividade da acetilcolinesterase mostrou-se diminuída nos chagásicos (4,32) expressa como número de moles do substrato hidrolisado por minuto por grama de tecido, em relação aos controles (7,30). Não se encontrou hipersensibilidade da musculatura gástrica a drogas colinérgicas, encontrando-se inclusive efeito máximo reduzido ao carbacol e betanecol no grupo chagásico. CONCLUSÕES: A redução de neurônios no plexo mioentérico do estômago de pacientes chagásicos crônicos pode ser demonstrada mesmo na ausência de gastropatia chagásica clínica. A hipersensibilidade da musculatura gástrica a drogas colinérgicas provavelmente depende de desnervação intensa. A redução da atividade da acetilcolinesterase demonstra o comprometimento da inervação colinérgica no estômago de pacientes chagásicos crônicos. Não houve correlação entre número de neurônios, sensibilidade a drogas colinérgicas e atividade da acetilcolinesterase na musculatura gástrica de pacientes chagásicos ou não chagásicos.

Avaliação morfológica e dos mecanismos de mobilização de Ca2+ pela glicose e acetilcolina em células pancreáticas humanas / Morphologic evaluation and Ca2+ mobilization by glicose and acetylcholine in human pancreatic cells

OBJETIVOS: Avaliar a morfologia das organelas e do citoesqueleto em células pancreáticas humanas cultivadas e a mobilização de Ca2+ em resposta à glicose e ACh por medidas fluorimétricas. MATERIAL E MÉTODOS: As células foram semeadas em lamínulas, fixadas e marcadas com uma combinação de fluoróforos: o núcleo foi corado com DAPI e as mitocôndrias, com Mytotracker Red. Foram utilizados faloidina e anticorpos secundários conjugados com Alexa Fluor verde e vermelho fluorescentes (488 e 594) para identificar proteína actina F e receptor muscarínico tipo M3, respectivamente. Para estudar a mobilização de Ca2+, as células foram incubadas com fura-2/AM. RESULTADOS: As células pancreáticas humanas apresentaram morfologia preservada com grande quantidade de mitocôndrias. Na região de maior densidade celular, evidenciou-se as pseudo-ilhotas e os receptores muscarínicos M3. Por meio da elevação da [Ca2+]c, devido à ação da glicose e ACh, mostrou-se preservação da capacidade responsiva a esses estímulos e foi dependente de concentração desses agonistas. A glicose promoveu uma resposta sustentada e a ACh induziu uma resposta bifásica. CONCLUSÃO: As células pancreáticas humanas cultivadas conservaram sua morfologia. A mobilização de Ca2+ em resposta à glicose e a ACh confirma a sua funcionalidade. Os receptores muscarínicos M3 estão presentes nessas células.

AIMS: The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method. MATERIAL AND METHODS: The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization. RESULTS: The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cellsÆ capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response. CONCLUSION: The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.

Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets

Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50 percent. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.

Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-kappaB activation in rats in vitro / 华中科技大学学报（医学）（英德文版）

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Electroacupuncture ameliorates experimental colitis induced by acetic acid in rat

The effect of electroacupuncture (EA) on experimental colitis was investigated in Sprague-Dawley rats. Colitis was induced by intracolonic instillation of 4% acetic acid. EA (2 Hz, 0.05 ms, 2 V for 20min) was applied to bilateral Hoku (LI-4) and Zusanli (ST-36) on 12 hrs and 36 hrs after induction of colitis. EA-treatment significantly reduced the macroscopic damage and the myeloperoxidase activity of colonic samples at 3 days post-induction of colitis. Colitic colon showed a decreased in vitro motility. However, colonic motility of EAtreated group was not significantly different from that of normal group. The anti-inflammatory effect of EA was not inhibited by a glucocorticoid receptor antagonist, RU-486, but suppressed by a beta-adrenoceptor antagonist, propranonol. These results suggest that EA-treatment has a beneficial effect on colitis, and its anti-inflammatory effect is mediated by beta-adrenoceptor activation but not by endogenous glucocorticoiddependent mechanism.

Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (ICCh) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited ICCh dose- dependently with IC50 of 1.1 +/- 0.1 nM (n = 6). GTPgS- induced current, however, was not inhibited by 10 nM 4-DAMP. ICCh was not recorded in pertussis- toxin (PTX)-pretreated smooth muscle cells of gastric antrum. ICCh values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 +/- 3 pA (n = 6) in the control and PTX-treated cells, respectively (P<0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that Go protein is important for activation of ACh-activated NSC channels, our results suggest that ICCh might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.

Carbachol potentiates cholera toxin-induced secretion in a colonic epithelial cell line (HT29-19A) and rat ileal mucosa in vitro.

Recent studies show that enteric nerves are involved in the action of cholera toxin, both in vivo and in vitro. The aim of this study was to investigate in vitro the influence of carbachol, a cholinergic agonist, on the action of cholera toxin. Cultured HT29-19A cell lines and rat ileal mucosa were used in an Ussing chamber for the measurement of short-circuit current induced by cholera toxin. Cyclic AMP was measured from HT29-19A cell lines by standard radio-immunoassay. Pre-treatment of the HT29-19A cell lines with carbachol potentiated cholera toxin-induced secretory response, and enhanced accumulation of cAMP. Carbachol also potentiated the cholera toxin-secretory response in the rat ileal mucosa, but only following pretreatment with the prostaglandin synthesis inhibitor, indomethacin. There was synergistic interaction between cholera toxin and cholinergic neurotransmitter carbachol on the intestinal epithelium. Cholinergic agonists may play a role in regulating the secretory response to the toxin. Such interaction is masked in the intact tissues in vitro due to the release of prostaglandins during isolation.

Fluid and amylase secretion by perfused parotid gland: physio-morphological approach

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly around 60 microL/g-min, whereas amylase secretion exhibited an initial peak, followed by a rapid decrease to reach a plateau. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion accompanied by small increase in oxygen consumption. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop.

Effect of cholinergic agonists on muscular tonus of the lizard small intestine and esophagus

The underlying mechanisms of acetycholine-induced intestinal relaxation in the lizard Liolaemus tenuis tenuis are still unknows. By using a classical model of intestinal recording of isometric contraction and relaxation in conjunction with specific pharmacological tools, this article studies the possible influence of EDRF/NO and nicotinic ganglionar receptors on the Ach-induced relaxation in an effort to elucidate the probable mechanisms involved in ACh effect. It was observed that the relaxation of the lizard intestine elicited by ACh (10(-7) - 4 x 10(-4) M) was not affected by hexametonium (5 x 10(4) M) or tetrodotoxin (10(-6) M). Nicotine (10(-7) to 10(-4) M) induced relaxation was significantly antagonized by hexametonium; however, it was not influenced by tetrodotoxin. These results allow us to discard a neuronal pathway in cholinergic-induced relaxation, suggesting a more direct cholinergic effect on the smooth muscle, perhaps mediated by an unknown substance released by some specialized tissue. N-nitro-L-arginine, used to block NO-synthase and NO production, induced no changes in ACh-induced relaxation. Methylene blue, a soluble guanylate cyclase inhibitor, induced no changes in ACh-induced relaxation. These results allow us to dicard a probable role of EDRF/nitric oxide in the ACh-induced relaxation of lizard small intestine, providing evidence that this mechanism could be different from reported on other species.

Doença de Alzheimer: tratamento farmacológico / Alzeheimer's disease: pharmacological treatment

O tratamento farmacológico da doença de Alzheimer é considerado tomando como base os fatores etiológicos/fisiopatológicos subjacentes ao processo neurodegenerativo. Assim, é dado enfoque aos estudos terapêuticos pré-clínicos em relaçäo aos distúrbios básicos (genéticos, apoptose, placas senis, amaranhados neurofibrilares). O tratamento compensatório com utilizaçäo de estratégias colinérgicas (dando-se destaque ao uso de drogas inibidoras da acetilcolinesterase) e também relacionadas com outros neurotransmissores é analisado, assim como a terapêutica em funçäo de outros aspectos fisiopatológicos (neuroprotetores, antioxidantes, antiinflamatórios, metabólicos, tóxicos). Finalmente säo considerados os aspectos gerais do tratamento dos transtornos de comportamento/psicógicos que acompanham a doença

Comparative effects of hyoscine butylbromide and atropine sulphate on sleep architecture in healthy human volunteers.

The changes in sleep architecture, heart rate and respiratory rate to hyoscine butylbromide (HBB), a peripherally acting anticholinergic was studied. These effects were compared with that of atropine sulphate, a drug known to cross the blood brain barrier. The study followed a single blind cross over design with a one week washout period. Atropine sulphate (0.4 mg) and HBB (10 mg) were given intravenously to ten adult healthy male volunteers before sleep onset. Normal saline was used as control. All night sleep polysomnography was done with the standard montage for sleep staging. Respiration and airflow were also monitored. Rapid eye movement (REM) latency was significantly increased with both the drugs whereas the duration of REM sleep was decreased only with atropine. Slow wave sleep (SWS) was also increased significantly by atropine. There was no change in heart rate, or respiratory rate during any of the sleep stages. HBB affects the initiation of REM sleep whereas atropine affects both its initiation and maintenance.

Functional evidence that the central renin-angiotensin system plays a role in the pressor response induced by central injection of cabachol

We investigated the effects of losartan, an AT1-receptor blocker, and ramipril, a converting enzyme inhibitor, on the pressor response induced by angiotensin II (ANG II) and carbachol (a cholinergic receptor agonist). Male Holtzman rats (250-300 g) with a stainless steel cannula implanted into the lateral ventricle (LV) were used. The injection of losartan (50 nmol/l mul) into the LV blocked the pressor response induced by ANG II (12 ng/1 mul) and carbachol (2 nmol/ 1 mul). After injection of ANG II and carbachol into the LV, mean arterial pressure (MAP) increased to 31 + 1 and 28 + 2 mmHg, respectively. Previous injection of losartan abolished the increase in MAP induced by ANG II and carbachol into the LV (2 + 1 and 5 + 2 mmHg, respectively). The injection of ramipril (12 ng/ 1 mul) prior to carbachol blocked the pressor effect of carbachol to 7 + 3 mmHg. These results suggests an interaction between central cholinergic pathways and the angiotensinergic system in the regulation of arterial blood pressure.

Panorama atual do tratamento farmacológico do déficit cognitivo da doença de Alzheimer: um estudo de revisäo / Actual view of the pharmacological treatment of cognitive deficit of Alzheimer disease: a review

Neste artigo o autor faz uma revisäo das estratégias farmacoterápicas mais recentes, usadas no cenário internacional, para o tratamento do declínio cognitivo progressivo que caracteriza a Doença de Alzheimer. Para tanto, baseia-se no conhecimento atual acerca da etiopatogenia e neuropatologia desta demência. Apesar dos progressos obtidos pela investigaçäo científica, a etiologia desta doença é ainda obscura e os resultados dos tratamento ainda duvidosos, priorizando-se no momento medidas terapêuticas que impliquem na lentificaçäo do processo degenerativo.