RESUMO
BACKGROUND: Some cannabinoids, a family of compounds derived from Cannabis sativa (marijuana), have previously shown vasodilator effects in several studies, a feature that makes them suitable for the generation of a potential treatment for hypertension. The mechanism underlying this vasodilator effect in arteries is still controversial. In this report, we explored how the synthetic cannabinoids ACPA (CB1-selective agonist) and JWH-133 (CB2-selective agonist) regulate the vascular tone of rat superior mesenteric arteries. METHODS: To screen the expression of CB1 (Cannabinoid receptor 1) and CB2 (Cannabinoid receptor 2) receptors in arterial rings or isolated smooth muscle cells obtained from the artery, immunocytochemistry, immunohistochemistry, and confocal microscopy were performed. In addition, the effects on vascular tone induced by the two cannabinoids were tested in isometric tension experiments in rings obtained from superior mesenteric arteries. The participation of voltage and calcium-activated potassium channel of big conductance (BKCa) and the role of nitric oxide (NO) release on the vascular effects induced by ACPA and JWH-133 were tested. RESULTS: CB1 and CB2 receptors were highly expressed in the rat superior mesenteric artery, in both smooth muscle and endothelium. The vasodilation effect shown by ACPA was endothelium-dependent through a mechanism involving CB1 receptors, BKCa channel activation, and NO release; meanwhile, the vasodilator effect of JWH-133 was induced by the activation of CB2 receptors located in smooth muscle and by a CB2 receptor-independent mechanism inducing NO release. CONCLUSIONS: CB1 and CB2 receptor activation in superior mesenteric artery causes vasorelaxation by mechanisms involving BKCa channels and NO release.
Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 â¼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.
Assuntos
Miócitos Cardíacos/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Células HEK293 , Humanos , Quinacrina/farmacologiaRESUMO
Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.
Assuntos
Cloroquina/metabolismo , Cloroquina/farmacologia , Bloqueadores dos Canais de Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Sítios de Ligação/genética , Linhagem Celular , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/síntese química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Propriedades de Superfície , TransfecçãoRESUMO
Se estudiaron las propiedades antagonistas del calcio de 2 nuevas 1,4- dihidropiridinas sintéticas (I y II). Ambos compuestos disminuyeron en gran medida las contracciones de anillos de aorta de conejo y con menor potencia inhibieron las contracciones de papilares de ventrículo derecho de rata, acortaron la duración del potencial de acción cardíaco a 0 mV y redujeron la corriente de calcio tipo L en cardiomiocitos ventriculares aislados de rata. La inhibición de la corriente de calcio tipo L fue dependiente del potencial, con un incremento de la potencia de acción en más de un orden de magnitud de concentración en células parcialmente despolarizadas (-50 mV). Estos resultados permiten concluir que las 1,4- dihidropiridinas I y II poseen acciones antagonistas del calcio vasoselectivas marcadas y este mecanismo está dado por el bloqueo de la corriente de calcio tipo L de manera dependiente del potencial
Assuntos
Coelhos , Ratos , Bloqueadores dos Canais de Cálcio , Di-HidropiridinasRESUMO
Many drugs block delayed rectifier K+ channels and prolong the cardiac action potential duration. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) K+ channels expressed in cells HEK-293 and Xenopus oocytes by maprotiline. The IC50 determined at 0 mV on HERG expressed HEK-293 cell and oocytes was 5.2 and 23.7 microM, respectively. Block of HERG expressed in oocytes by maprotiline was enhanced by progressive membrane depolarization and accompanied by a negative shift in the voltage dependence of channel activation. The potency of maprotiline was reduced 7-fold by point mutation of a key aromatic residue (F656T) and 3-fold for Y652A, both located in the S6 domain. The mutation Y652A inverted the voltage dependence of HERG channel block by maprotiline. Together, these results suggest that voltage-dependent block of HERG results from gating dependent changes in the accessibility of Y652, a critical component of the drug binding site.
Assuntos
Antidepressivos de Segunda Geração/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Maprotilina/farmacologia , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Estimulação Elétrica , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/fisiologia , Feminino , Expressão Gênica , Humanos , Potenciais da Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Mutação Puntual , XenopusRESUMO
Berberine prolongs the duration of cardiac action potentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, I(Kr) and I(Ks). Here we have studied the effects of berberine on hERG (I(Kr)) and KCNQ1/KCNE1 (I(Ks)) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC50 for berberine was 3.1 +/- 0.5 microM on hERG compared with 11 +/- 4% decreases on KCNQ1/KCNE1 channels by 100 microM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC50 = 80 +/- 5 microM) compared with KCNQ1/KCNE1 channels (approximately 20% block at 300 microM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by berberine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists.
Assuntos
Berberina/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Sítios de Ligação , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Canal de Potássio KCNQ1/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , XenopusRESUMO
La estimulación de los receptores -adrenérgicos juega un papel esencial en el control del estado inotrópico del corazón, además de ser un factor arritmogénico durante la isquemia cardíaca. Sin embargo, su papel en el control de los movimientos de Ca2+ en la célula cardíaca es poco conocido. Nuestro objetivo fue, por tanto, estudiar los efectos de la estimulación a1-adrenérgica sobre las corrientes de Ca2+ tipos T y L (LCaT e ICaL respectivamente) en células cardíacas aisladas de corazón de rana. La fenilefrina (10 mmol/L) en presencia de propranolol (1 mmol/L), incrementó ICaL e ICaT. Este incremento fue completamente bloqueado por el prazosín (0,1 mmol/L). El bloqueo de la actividad de las proteínas G impidió el incremento de ICaT e ICaL por la fenilefrina. El bloqueo de la fosfolipasa C impidió las acciones a1-adrenérgicas sobre ICaL pero no totalmente sobre ICaT. Se concluye que la estimulación a1-adrenérgica incrementa ICaL por una vía que involucra fosfolipasa C, mientras que sobre ICaT el control pudiera ser más directo a través de las proteínas G