Naloxone (NX) potentiated epinephrine (EPI) induced submaximal vasoconstriction in canine renal and skeletal muscle arterial segments, yet had no vasoconstrictor action alone. Developed tension generated in-vitro by 4 x 1mm. O.D. rings from 1st degree branches of canine femoral arteries was expressed as % of KCI induced maximum response. NX (10^-5 M) potentiated EPI induced submaximal contractions (34.2%) significantly more than contractions induced by norepinephrine, phenylephrine, lofexidine, ADH, KCI and serotonin (13.8,13.4,4.7,13.5,14.4 and 11.4% respectively). The NX response was unaffected by beta-adrenergic blockade and NX did not reverse an isoproterenol mediated vasodilation. Alphaadrenergic blockade with phentolamine completely eliminated EPI plus NX induced vasoconstriction. After washout, vessels exposed to EPI plus NX relaxed by 50% significantly faster than vessels exposed to EPI alone (18.5 and 27.9 min respectively). EPI induced vasoconstrictions were potentiated by 10^-5 M corticosterone (49.0%) which inhibits extraneuronal catecholamine uptake, but not by 10^-7 M desipramine (1.1%) which inhibits neuronal uptake. EPI induced vasoconstrictions were also potentiated by 10^-4 M pyrogallol (33.0%) which inhibits catechol-o-methyl transferase activity, but not by 10^-5 M pargyline (-1.1%) which inhibits monoamine oxidase activity. The NX effect was endothelium independent. The dose-response of various opioid receptor agonists and antagonists were compared to the NX response. A specific opioid receptor subclass could not be identified as the mediator of the NX effect. The ED_50s for NX (3.7x^-6 M) and (+)NX (8.1x^-7M) indicated a significant stereoselectivity for the (+)enantiomer. A variety of sigma receptor ligands, steroids and steroid metabolites were tested for the ability to augment EPI vasoconstrictions. Several of the opioid, sigma and steroid ligands, all with polycyclic structures, induced responses similarto those of NX. NX exerted its effect independent of traditional opiate receptors and may have influenced the cellular uptake or degradation of EPI. Endogenous compounds with sigma or steroid activity may modulate these processes in-vivo.

Introdução: Este estudo prospectivo, randomizado, duplo-cego investigou, em valvopatas, alterações hemodinâmicas durante o tratamento odontológico com o uso do anestésico local contendo adrenalina e sem a mesma. Métodos: O estudo foi conduzido na Unidade de Odontologia do Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (Brasil 2004-2005). Os pacientes foram alocados em dois grupos através de tabela de números aleatórios: LSA (lidocaína a 2% sem adrenalina, n= 31, 42.2 ± 10.3 anos) e LCA (lidocaína a 2% com adrenalina 1:100.000, n= 28, 40.3 ± 10.9 anos). O volume anestésico foi registrado. Um monitor multiparamétrico DIXTAL (São Paulo- Brasil) registrou os valores da pressão arterial sistêmica, freqüência cardíaca, saturação de oxigênio e traçado eletrocardiográfico. Registrou-se também o volume anestésico aplicado em cada procedimento realizado. Resultados: 22 homens e 37 mulheres foram incluídos. Valores da pressão arterial sistêmica, freqüência cardíaca e saturação de oxigênio, antes, durante e após administração da anestesia local não mostraram diferenças estatísticas entre os dois grupos (P > 0.05). Arritmias observadas em alguns pacientes antes do início do tratamento odontológico não sofreram alterações de morfologia ou gravidade após injeção anestésica. Relatos de dor durante a realização do procedimento odontológico foram mais freqüentes no grupo LSA com conseqüente aumento no volume de anestésico local administrado. Conclusão: lidocaína 2% com adrenalina 1:100.000 mostrou maior eficácia anestésica em comparação com a lidocaína 2% sem adrenalina sem causar alterações hemodinâmicas em pacientes portadores de valvopatias. / Introduction: This prospective, randomized double-blinded study investigated hemodynamic changes in valvular cardiac patients during dental treatment with the use of a local anesthesia containing epinephrine. Methods: The study was conducted in the Dental Department of the Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (Brazil 2004- 2005). Patients were allocated into two groups through an aleatory numbered table: PL (plain 2% lidocaine, n= 31) and LE (2% lidocaine with 1:100.000 epinephrine, n= 28). The anesthetic amount was registered. DIXTAL monitor (São Paulo- Brazil) captured blood pressure, heart rate, oxygenation and electrocardiogram records. Results: 22 men and 37 women were included (LE age 40.3 ± 10.9 and PL age 42.2 ± 10.3). Blood pressure, heart rate and pulse oximetry values before, during and after local anesthesia injection did not show any difference between the two groups (P > 0.05). Any arrhythmias observed in some patients prior to dental anesthesia did not suffer alterations of shape or gravity after it. Complains of pain during dental procedure were more often in the PL group with a higher amount of local anesthesia needed. Conclusion: 2% lidocaine with epinephrine 1:100.000 showed a superior anesthetic efficiency without leading to hemodynamics changes in patients with cardiac valvular compromise.

O anestésico local é o medicamento mais utilizado na Odontologia e sua associação com vasoconstrictores aumenta a duração da anestesia, diminuindo seus efeitos sistêmicos. A hipertensão e o tabagismo são freqüentes na população, sendo responsáveis por complicações sistêmicas. A felipressina, por não interferir com receptores simpáticos, poderia ser um vasoconstrictor indicado para pacientes hipertensos. O objetivo deste trabalho foi estudar a reatividade cardiovascular de animais simultaneamente hipertensos e fumantes passivos aos agentes vasoconstritores associados aos anestésicos locais, verificando também o efeito do tratamento com atenolol. Foram utilizados ratos Wistar machos, divididos em 5 grupos: 1) normotensos não fumantes; 2) normotensos fumantes passivos; 3) hipertensos não fumantes; 4) hipertensos fumantes passivos; 5) hipertensos fumantes passivos tratados com atenolol. A hipertensão renal foi induzida pela remoção do rim direito e instalação de clip de prata (abertura 0,25mm) na artéria renal esquerda, após anestesia com quetamina e xilazina. Os ratos fumantes passivos foram colocados diariamente por 10 minutos, durante 28 dias, em caixa de madeira de 30cmX25cmX15cm dividida em dois compartimentos. Em um deles, eram acesos 10 cigarros e no outro ficavam os animais. A tampa da caixa era fechada e um sistema de ventilação lançava fumaça dos cigarros para o compartimento dos ratos, num fluxo de 10l/min. Após medida indireta da pressão arterial, 14 dias após a cirurgia, o grupo tratado com atenolol foi medicado durante 14 dias seguintes (90 mg/Kg) por gavage. No 28o dia, todos receberam catéter de polietileno na artéria carótida esquerda (para medida de pressão) e outro na veia jugular direita (para injeção de drogas). Para os 5 grupos foram utilizadas: adrenalina (80, 160, 320, 640 e 1280ng) e felipressina (0,125, 0,25, 0,5, 1, 2 e 3 x 10-3UI). O catéter arterial era conectado a transdutor de pressão e o registro realizado por software específico. Foram analisadas: menor resposta hipotensora, maior resposta hipertensora e duração de resposta para cada dose. Os dados foram analisados por análise de variância de medidas repetidas, seguida do teste de Tuckey ou Holm-Sidack, com nível de significância de 5%. Os resultados mostraram que o fumo passivo reduziu significativamente a resposta vasodilatadora produzida pela adrenalina, em animais normotensos e hipertensos, potencializou suas respostas hipertensoras e aumentou a duração das respostas à adrenalina, ampliadas ainda mais pelo tratamento com atenolol. O tratamento com atenolol promoveu aumento adicional das respostas hipertensoras à adrenalina nos hipertensos-fumantes. A felipressina não apresentou ações vasodilatadoras e suas ações hipertensoras foram potencializadas pelo fumo passivo, em amplitude e duração. O atenolol não promoveu aumento adicional da amplitude das respostas à felipressina. Nos animais hipertensos, o tratamento com atenolol associado ao fumo passivo teve efeito expressivo, aumentando significativamente a duração total das respostas à felipressina. A felipressina, quando comparada à adrenalina, não apresentou efeitos hipotensores diretos, a resposta hipertensora máxima foi nitidamente inferior e a duração das respostas à felipressina foi o dobro da adrenalina. Dessa forma, a felipressina se torna uma droga interessante na hipertensão, devido a sua capacidade de promover vasoconstrição prolongada, sem potencializar a atividade simpática sistêmica. / The local anesthetic is the most common drug in dentistry and the associated vasoconstrictors increase the duration of anesthesia, decreasing its systemic effects. Hypertension and smoking are problems commonly found in the general population, being responsible for systemic complications. Felypressin, a vasoconstrictor that does not interact with sympathetic receptors, could be indicated to hypertensive patients. This study investigated the cardiovascular reactivity of hypertensive and passive smoker animals under atenolol treatment to epinephrine and felypressin. Male wistar rats were divided into five groups: 1) normotensive and non-smokers, 2) normotensive and passive smokers, 3) hypertensive and non-smokers, 4) hypertensive and passive smokers; 5) hypertensive, passive smokers and treated with atenolol. Renal hypertension was induced by removal of the right kidney and installation of a silver clip (with 0.25-mm opening) in the left renal artery, after anesthesia with ketamine and xylazine. The passive smoker rats were placed, 10 minutes per day, during 28 days in a 30cmX25cmX15cm wood box divided into two compartments. Ten cigarettes were lit in one compartment, and the rats were placed in the other. The box lid was closed and a ventilation system threw the cigarette smoke to the rat compartment. After indirect measurement of blood pressure, 14 days after the surgery, the group of rats treated with atenolol was medicated during the following fourteen days (90 mg/kg) by gavage. On the 28th day, a polyethylene catheter was inserted into the left carotid artery (for direct blood pressure measurements) and into the right jugular vein (for drug injection). The groups received epinephrine (80, 160, 320, 640 and 1280ng) or felypressin (0.125, 0.25, 0.5, 1, 2 and 3 x 10-3UI). The arterial catheter was connected to a pressure transducer and recording was made by a specific computer software. The following parameters were analyzed for all groups: lower hypotensive response, higher hypertensive response and duration of response for each dose. Data were statistically analyzed by repeated measures analysis of variance, followed by Tukey test or Holm-Sidack test, at a significance level of 5%. The results showed that passive smoking significantly decreased the vasodilator response produced by epinephrine in normotensive and hypertensive animals, increasing their hypertensive responses and increased the duration of response to epinephrine, that was further increased by atenolol treatment. Atenolol treatment increased the hypertensive responses in hypertensive-smokers rats. The felypressin did not show vasodilator responses and its hypertensive responses were increased by passive smoking. The atenolol did not cause additional increase in felypressin responses. In hypertensive animals, the atenolol treatment associated with passive smoking had expressive effects, significantly increasing the total duration of response to felypressin. Felypressin, when compared with epinephrine, did not show direct hypotensive effects, the higher hypertensive responses were smaller and the duration of response to felypressin was twice the epinephrine time. Then, felypressin becomes an interesting drug to hypertensive patients, due to its capacity to promote prolonged vasoconstrictor effect without increasing the sympathetic nerve activity.

No description available.

O anestésico local é o medicamento mais utilizado na Odontologia e sua associação com vasoconstrictores aumenta a duração da anestesia, diminuindo seus efeitos sistêmicos. A hipertensão e o tabagismo são freqüentes na população, sendo responsáveis por complicações sistêmicas. A felipressina, por não interferir com receptores simpáticos, poderia ser um vasoconstrictor indicado para pacientes hipertensos. O objetivo deste trabalho foi estudar a reatividade cardiovascular de animais simultaneamente hipertensos e fumantes passivos aos agentes vasoconstritores associados aos anestésicos locais, verificando também o efeito do tratamento com atenolol. Foram utilizados ratos Wistar machos, divididos em 5 grupos: 1) normotensos não fumantes; 2) normotensos fumantes passivos; 3) hipertensos não fumantes; 4) hipertensos fumantes passivos; 5) hipertensos fumantes passivos tratados com atenolol. A hipertensão renal foi induzida pela remoção do rim direito e instalação de clip de prata (abertura 0,25mm) na artéria renal esquerda, após anestesia com quetamina e xilazina. Os ratos fumantes passivos foram colocados diariamente por 10 minutos, durante 28 dias, em caixa de madeira de 30cmX25cmX15cm dividida em dois compartimentos. Em um deles, eram acesos 10 cigarros e no outro ficavam os animais. A tampa da caixa era fechada e um sistema de ventilação lançava fumaça dos cigarros para o compartimento dos ratos, num fluxo de 10l/min. Após medida indireta da pressão arterial, 14 dias após a cirurgia, o grupo tratado com atenolol foi medicado durante 14 dias seguintes (90 mg/Kg) por gavage. No 28o dia, todos receberam catéter de polietileno na artéria carótida esquerda (para medida de pressão) e outro na veia jugular direita (para injeção de drogas). Para os 5 grupos foram utilizadas: adrenalina (80, 160, 320, 640 e 1280ng) e felipressina (0,125, 0,25, 0,5, 1, 2 e 3 x 10-3UI). O catéter arterial era conectado a transdutor de pressão e o registro realizado por software específico. Foram analisadas: menor resposta hipotensora, maior resposta hipertensora e duração de resposta para cada dose. Os dados foram analisados por análise de variância de medidas repetidas, seguida do teste de Tuckey ou Holm-Sidack, com nível de significância de 5%. Os resultados mostraram que o fumo passivo reduziu significativamente a resposta vasodilatadora produzida pela adrenalina, em animais normotensos e hipertensos, potencializou suas respostas hipertensoras e aumentou a duração das respostas à adrenalina, ampliadas ainda mais pelo tratamento com atenolol. O tratamento com atenolol promoveu aumento adicional das respostas hipertensoras à adrenalina nos hipertensos-fumantes. A felipressina não apresentou ações vasodilatadoras e suas ações hipertensoras foram potencializadas pelo fumo passivo, em amplitude e duração. O atenolol não promoveu aumento adicional da amplitude das respostas à felipressina. Nos animais hipertensos, o tratamento com atenolol associado ao fumo passivo teve efeito expressivo, aumentando significativamente a duração total das respostas à felipressina. A felipressina, quando comparada à adrenalina, não apresentou efeitos hipotensores diretos, a resposta hipertensora máxima foi nitidamente inferior e a duração das respostas à felipressina foi o dobro da adrenalina. Dessa forma, a felipressina se torna uma droga interessante na hipertensão, devido a sua capacidade de promover vasoconstrição prolongada, sem potencializar a atividade simpática sistêmica. / The local anesthetic is the most common drug in dentistry and the associated vasoconstrictors increase the duration of anesthesia, decreasing its systemic effects. Hypertension and smoking are problems commonly found in the general population, being responsible for systemic complications. Felypressin, a vasoconstrictor that does not interact with sympathetic receptors, could be indicated to hypertensive patients. This study investigated the cardiovascular reactivity of hypertensive and passive smoker animals under atenolol treatment to epinephrine and felypressin. Male wistar rats were divided into five groups: 1) normotensive and non-smokers, 2) normotensive and passive smokers, 3) hypertensive and non-smokers, 4) hypertensive and passive smokers; 5) hypertensive, passive smokers and treated with atenolol. Renal hypertension was induced by removal of the right kidney and installation of a silver clip (with 0.25-mm opening) in the left renal artery, after anesthesia with ketamine and xylazine. The passive smoker rats were placed, 10 minutes per day, during 28 days in a 30cmX25cmX15cm wood box divided into two compartments. Ten cigarettes were lit in one compartment, and the rats were placed in the other. The box lid was closed and a ventilation system threw the cigarette smoke to the rat compartment. After indirect measurement of blood pressure, 14 days after the surgery, the group of rats treated with atenolol was medicated during the following fourteen days (90 mg/kg) by gavage. On the 28th day, a polyethylene catheter was inserted into the left carotid artery (for direct blood pressure measurements) and into the right jugular vein (for drug injection). The groups received epinephrine (80, 160, 320, 640 and 1280ng) or felypressin (0.125, 0.25, 0.5, 1, 2 and 3 x 10-3UI). The arterial catheter was connected to a pressure transducer and recording was made by a specific computer software. The following parameters were analyzed for all groups: lower hypotensive response, higher hypertensive response and duration of response for each dose. Data were statistically analyzed by repeated measures analysis of variance, followed by Tukey test or Holm-Sidack test, at a significance level of 5%. The results showed that passive smoking significantly decreased the vasodilator response produced by epinephrine in normotensive and hypertensive animals, increasing their hypertensive responses and increased the duration of response to epinephrine, that was further increased by atenolol treatment. Atenolol treatment increased the hypertensive responses in hypertensive-smokers rats. The felypressin did not show vasodilator responses and its hypertensive responses were increased by passive smoking. The atenolol did not cause additional increase in felypressin responses. In hypertensive animals, the atenolol treatment associated with passive smoking had expressive effects, significantly increasing the total duration of response to felypressin. Felypressin, when compared with epinephrine, did not show direct hypotensive effects, the higher hypertensive responses were smaller and the duration of response to felypressin was twice the epinephrine time. Then, felypressin becomes an interesting drug to hypertensive patients, due to its capacity to promote prolonged vasoconstrictor effect without increasing the sympathetic nerve activity.

Introdução: Este estudo prospectivo, randomizado, duplo-cego investigou, em valvopatas, alterações hemodinâmicas durante o tratamento odontológico com o uso do anestésico local contendo adrenalina e sem a mesma. Métodos: O estudo foi conduzido na Unidade de Odontologia do Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (Brasil 2004-2005). Os pacientes foram alocados em dois grupos através de tabela de números aleatórios: LSA (lidocaína a 2% sem adrenalina, n= 31, 42.2 ± 10.3 anos) e LCA (lidocaína a 2% com adrenalina 1:100.000, n= 28, 40.3 ± 10.9 anos). O volume anestésico foi registrado. Um monitor multiparamétrico DIXTAL (São Paulo- Brasil) registrou os valores da pressão arterial sistêmica, freqüência cardíaca, saturação de oxigênio e traçado eletrocardiográfico. Registrou-se também o volume anestésico aplicado em cada procedimento realizado. Resultados: 22 homens e 37 mulheres foram incluídos. Valores da pressão arterial sistêmica, freqüência cardíaca e saturação de oxigênio, antes, durante e após administração da anestesia local não mostraram diferenças estatísticas entre os dois grupos (P > 0.05). Arritmias observadas em alguns pacientes antes do início do tratamento odontológico não sofreram alterações de morfologia ou gravidade após injeção anestésica. Relatos de dor durante a realização do procedimento odontológico foram mais freqüentes no grupo LSA com conseqüente aumento no volume de anestésico local administrado. Conclusão: lidocaína 2% com adrenalina 1:100.000 mostrou maior eficácia anestésica em comparação com a lidocaína 2% sem adrenalina sem causar alterações hemodinâmicas em pacientes portadores de valvopatias. / Introduction: This prospective, randomized double-blinded study investigated hemodynamic changes in valvular cardiac patients during dental treatment with the use of a local anesthesia containing epinephrine. Methods: The study was conducted in the Dental Department of the Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (Brazil 2004- 2005). Patients were allocated into two groups through an aleatory numbered table: PL (plain 2% lidocaine, n= 31) and LE (2% lidocaine with 1:100.000 epinephrine, n= 28). The anesthetic amount was registered. DIXTAL monitor (São Paulo- Brazil) captured blood pressure, heart rate, oxygenation and electrocardiogram records. Results: 22 men and 37 women were included (LE age 40.3 ± 10.9 and PL age 42.2 ± 10.3). Blood pressure, heart rate and pulse oximetry values before, during and after local anesthesia injection did not show any difference between the two groups (P > 0.05). Any arrhythmias observed in some patients prior to dental anesthesia did not suffer alterations of shape or gravity after it. Complains of pain during dental procedure were more often in the PL group with a higher amount of local anesthesia needed. Conclusion: 2% lidocaine with epinephrine 1:100.000 showed a superior anesthetic efficiency without leading to hemodynamics changes in patients with cardiac valvular compromise.

El glaucoma es una de las principales causas de ceguera parcial o total e irreversible en el mundo. Los avances en su estudio revelan la compleja naturaleza multifactorial de la enfermedad que consiste finalmente en un daño del nervio óptico (neuropatía).El incremento de la presión intraocular (PIO) prolongado en el tiempo es uno de los factores de riesgo más significativo y reconocido para el desarrollo de esta enfermedad. Este incremento puede causar lesiones en el nervio óptico por deformación mecánica y/o cambios fisiopatológicos en una zona de la retina dónde se sitúan las células ganglionares del nervio. La PIO es resultado del balance entre la producción y drenaje del humor acuoso. El humor acuoso es un fluído derivado de plasma sanguíneo, producido en los procesos ciliares, que baña la cámara anterior del ojo para alimentar sus estructuras avasculares y mantener la tensión mecánica adecuada del globo ocular, y es drenado, principalmente, a través de la malla trabecular o trabéculo. Se sabe que el incremento de la contracción de este tejido implica una disminución en la facilidad de drenaje del humor acuoso y un aumento de la PIO. La presencia incrementada de endotelina-1 (ET-1), potente vasoconstrictor, en humor acuoso de pacientes glaucomatosos plantea algunas cuestiones que abordamos en este trabajo de tesis doctoral. Estas sustancias (endotelina-1, bradiquinina) son agonistas de receptores acoplados a proteína Gq que activan la vía de la fosfolipasa C (vía PLC- inositol trifosfato y diacilglicerol) y podrían tener un efecto contráctil, también, sobre la malla trabecular dado que activan la depleción de Ca2+ intracelular. El objetivo principal de este trabajo de tesis doctoral ha sido identificar y caracterizar un tipo de corriente de entrada de calcio extracelular (vía principal de entrada de calcio en células no excitables), llamada corriente 'capacitativa' de Ca2+ o dependiente del vaciado de Ca2+ de los depósitos intracelulares (ICCE o ISOC) y su posible implicación en la función de contracción/relajación de las células trabeculares y en consecuencia del tejido. Identificamos distintos tipos de corrientes capacitativas de Ca2+ en cultivos primarios de células trabeculares en modelo bovino y humano, tras el estímulo con sustancias vasoconstrictoras y tapsigargina. Para la caracterización farmacológica usamos bloqueadores típicos de la ICCE (2-APB, lantánidos, SKF96365) y registramos la evolución de la activación de ICCE mediante registros electrofisiológicos, testeando nuevamente bloqueadores conocidos de este tipo de corrientes. Observamos cómo en algunos casos las corrientes capacitativas son selectivas para el Ca2+ y en otros (vía PLC), en cambio, aparecían formas de corrientes catiónicas no selectivas.Detectamos mediante western blot la presencia de proteínas de canal tipo TRPC (TRPC1 y TRPC4) que forman canales catiónicos en membrana y pueden ser activados vía PLC, posibles mediadores de la ICCE en células trabeculares.La técnica de microscopía de tracción permitió estudiar la evolución de la contracción de la célula por estímulo con sustancias vasoconstrictoras. Obtuvimos medidas cuantitativas y la distribución de fuerzas en la célula durante la contracción. Estos resultados revelaron que la contracción inducida por estas sustancias depende primeramente de la depleción de Ca2+ intracelular y curiosamente, el Ca2+ extracelular regula, disminuyendo, la intensidad de esa contracción. Mostramos en este trabajo, la existencia de la ICCE en células trabeculares y sugerimos el papel de esta vía de entrada de Ca2+ como señal autoreguladora del proceso de contracción celular y su posible efecto en la función evacuadora del tejido. / High concentrations of endothelin-1 (ET-1), one of the most potent vasoconstrictor peptide, has been found in the aqueous humor of glaucomatous eyes. It is said that vasoconstrictor substances as ET-1 or bradykinin could induce contraction of trabecular meshwork tissue (TM) and decreases the aqueous humour outflow facility increasing the intraocular pessure (IOP) and the risk of glaucoma. The aim of this study is the calcium signalling, mainly, the characterization of the calcium influx/entry in these cells, the identification of capacitative calcium entry (ICCE) or, also called, store-operated calcium entry (ISOC) in trabecular cells and their role on cell contraction.We found these currents in cell culture of bovine and human trabecular cells and in a human cell line, too, using fluorescence techniques and electrophysiology recordings. They are stimulated by the intracellular Ca2+ depletion from stores via IP3 (bradykinin, endothelin) and blocking the Ca2+-pump of endoplasmic reticulum (thapsigargin). These currents are blocked or inhibited by certain lanthanids ions, 2-APB or SKF96365. Western blots showed the presence of certain members of the Transient Receptor Potential (TRP) channels subfamily C (TRPC1 and TRPC4). The pharmacology of inhibition of the Ca2+ influx suggests the TRPC channels and store-operated calcium channels as mediators. The measurements of cell traction evolution by traction microscopy maps stimulating with vasoconstrictor substances were useful to determine store-operated Ca2+ entry may not be necessary for cell contraction in TM cells but may contribute to modulate cell contractility and so, the aqueous humour outflow facility.

Contractility of vascular smooth muscles (VSMs) in resistance arteries determines systemic blood pressure and blood supplies to local tissues, in which ATP sensitive K+ (KATP) channels play a role. The KATP channels that couple metabolic state to cellular activity are activated by multiple hormonal vasodilators and inhibited by vasoconstrictors. To understand the molecular mechanisms for the channel regulation by vasodilators, we studied the effects of β-adrenergic receptors on Kir6.1/SUR2B in HEK cells. Stimulation of β-adrenergic receptors activated the channels, which relied on the GS-protein, adenylyl cyclase, cAMP and PKA system. Using mutational analysis, we scanned all the putative PKA sites on Kir6.1 and SUR2B subunits and identified two residues (Ser1351 and Ser1387) in SUR2B critical for channel activation. In vitro phosphorylation experiments confirmed that Ser1387 but not Ser1351 was phosphorylated in isolated SUR2B peptides. Molecular modeling and molecular dynamics simulations reveal that phosphorylation at Ser1387 causes interdomain movements in SUR2B subunit. Blockage of the movements by engineering a disulfide bond across NBD2 and TMD1 eliminated the PKA-dependent channel activation. We also studied the molecular basis for the inhibition of vascular KATP channels by PKC. In the HEK expression system, we found that the Kir6.1/SUR2B channel but not the Kir6.2/SUR2B was drastically inhibited by PKC stimulation. We constructed Kir6.1/Kir6.2 chimeras and identified two critical protein domains for the Kir6.1 channel inhibition by PKC. The distal C-terminus was the direct target of PKC where multiple phosphorylation sites were identified. These phosphorylation sites were located in a short sequence with stereotypical sequence repeats. Mutation of any decreased the effects of PKC. Joint mutation of all of them prevented the channel inhibition by PKC. The proximal N-terminus is also involved in PKC effects without phosphorylation sites, suggesting it may play a role in channel gating. Thus, this thesis provides experimental evidence for the vascular KATP channel modulation by PKA and PKC. Phosphorylation of the Kir6.1 and SUR2B subunits by PKC and PKA produce inhibition and activation of the vascular KATP channel, respectively, which appears to be one of the molecular bases contributing to vascular tone regulation by both vasoconstricting and vasodilating hormones and neurotransmitters.

The KATP channels play an important role in the membrane excitability and vascular tone regulation. Previous studies indicate that the function of KATP channels is disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrate S-glutathionylation to be a modulation mechanism underlying the oxidant-mediated vascular KATP channel inhibition, the molecular basis for the channel inhibition and the alleviation of the channel inhibition by vasoactive intestinal peptide (VIP). We found that an exposure of isolated mesenteric rings to H2O2 impaired the KATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, micromolar H2O2 or diamide caused a strong inhibition of the vascular KATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH), indicating S-glutathionylation. By co-expressions of Kir6.1 or Kir6.2 with SUR2B subunits, we found that the oxidant sensitivity of the KATP channel relied on the Kir6.1 subunit. Systematic mutational analysis revealed three cysteine residues (Cys43, Cys120 and Cys176) to be important. Among them, Cys176 was prominent, contributing to >80% oxidant sensitivity. Biochemical pull-down assay with biotinylated glutathione ethyl ester (BioGEE) showed that mutations of Cys176 impaired the oxidant-induced incorporation of GSH to the Kir6.1 subunit. Simulation modeling of Kir6.1 S-glutathionylation revealed that after incorporation to residue 176, the GSH moiety occupied a space between slide helix and two transmembrane helices. This prevented the necessary conformational change of the inner helix for channel gating, and retained the channel in its closed state. VIP is a potent vasodilator, and is shown to have protective role against oxidative stress. We found that the channel was strongly augmented by VIP and the channel activation relied on PKA phosphorylation. These results therefore indicate that 1) the vascular KATP channel is strongly inhibited in oxidative stress, 2) S-glutathionylation underlies the oxidant-mediated KATP channel inhibition, 3) Cys176 in the Kir6.1 subunit is the major site for S-glutathionylation, and 4) the Kir6.1/SUR2B channel is activated in a PKA-dependent manner by VIP that has been previously shown to alleviate oxidative stress.

Abstract not available