RESUMEN
Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The ß-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Células Musculares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Cola (estructura animal)/metabolismo , Animales , Canales de Calcio Tipo L/química , Relación Dosis-Respuesta a Droga , Masculino , Células Musculares/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Proteínas Quinasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Cola (estructura animal)/citología , Cola (estructura animal)/efectos de los fármacosRESUMEN
Substantial experimental evidences support the hypothesis that dietary flavonoid intake has a favourable impact on cardiovascular diseases such as systemic, arterial hypertension and coronary artery diseases, which represent the leading cause of morbidity and mortality worldwide. The biological effects of flavonoids involve complex biochemical interactions with numerous, specific, cellular and molecular targets. K+ channels, fine modulators of both cardiac action potential and vascular cell membrane potential, represent one of these targets. Overexpression, downregulation or dysfunction of these channel proteins are the cause of many cardiovascular diseases. Therefore, it appears of particular interest a detailed analysis of the flavonoid potential, direct/indirect modulation of cardiovascular K+ channels as these natural compounds ingested with the diet, despite extensive gut metabolism, may accumulate at cellular level in the form of the parent aglycones. The present review will portray their effects on cardiovascular K+ channels. Molecular docking was used to strengthen experimental evidences and describe flavonoid-channel interactions at molecular level.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Flavonoides/farmacología , Canales de Potasio/fisiología , Animales , Humanos , Simulación de Dinámica MolecularRESUMEN
Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca(2+) antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 µM (FRA77, GDE6, GDE19) and >10 µM (verapamil) in EA.hy926 cells, or >10 µM (FRA77, GDE6, GDE19) and at 100 µM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K(+) (K60)-induced contraction at concentrations ≥1 and ≥3 µM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 µM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.
Asunto(s)
Aminas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ésteres/farmacología , Simulación del Acoplamiento Molecular , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Aminas/química , Aminas/metabolismo , Animales , Sitios de Unión , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Ésteres/química , Ésteres/metabolismo , Humanos , Masculino , Potenciales de la Membrana , Estructura Molecular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Unión Proteica , Ratas , Ratas Wistar , Relación Estructura-Actividad , Técnicas de Cultivo de Tejidos , Vasodilatadores/química , Vasodilatadores/metabolismo , Verapamilo/metabolismo , Verapamilo/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca(2+) currents carried by the Cav 1.2 channels [ICa1.2 ] of rat cardiomyocytes. As only few drugs stimulate ICa1.2 , this study was designed to analyse the effects of MuA on vascular Cav 1.2 channels. EXPERIMENTAL APPROACH: Vascular activity was assessed on rat aorta rings mounted in organ baths. Cav 1.2 Ba(2+) current [IBa1.2 ] was recorded in single rat aorta and tail artery myocytes by the patch-clamp technique. Docking at a 3D model of the rat, α1c central pore subunit of the Cav 1.2 channel was simulated in silico. KEY RESULTS: In rat aorta rings MuA, at concentrations ≤14.2 µM, increased 30 mM K(+) -induced tone and shifted the concentration-response curve to K(+) to the left. Conversely, at concentrations >14.2 µM, it relaxed high K(+) depolarized rings and antagonized Bay K 8644-induced contraction. In single myocytes, MuA stimulated IBa1.2 in a concentration-dependent, bell-shaped manner; stimulation was stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activation curve. MuA docked at the α1C subunit central pore differently from nifedipine and Bay K 8644, although apparently interacting with the same amino acids of the pocket. Neither Bay K 8644-induced stimulation nor nifedipine-induced block of IBa1.2 was modified by MuA. CONCLUSIONS AND IMPLICATIONS: Murrayafoline A is a naturally occurring vasoactive agent able to modulate Cav 1.2 channels and dock at the α1C subunit central pore in a manner that differed from that of dihydropyridines.
Asunto(s)
Alcaloides/metabolismo , Canales de Calcio Tipo L/fisiología , Carbazoles/metabolismo , Endotelio Vascular/metabolismo , Simulación del Acoplamiento Molecular/métodos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Alcaloides/química , Alcaloides/farmacología , Animales , Canales de Calcio Tipo L/química , Carbazoles/química , Carbazoles/farmacología , Relación Dosis-Respuesta a Droga , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Endotelio Vascular/efectos de los fármacos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Técnicas de Cultivo de Órganos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas WistarRESUMEN
Proteins, with the large variety of chemical groups they present at their molecular surface, are a class of molecules which can be very informative on most of the possible solute-solvent interactions. Hen egg white lysozyme has been used as a probe to investigate the complex solvent dynamics occurring at the protein surface, by analysing the results obtained from Nuclear Magnetic Resonance, X-ray diffractometry and Molecular Dynamics simulations. A consistent overall picture for the dynamics of water molecules close to the protein is obtained, suggesting that a rapid exchange occurs, in a picosecond timescale, among all the possible hydration surface sites both in solution and the solid state, excluding the possibility that solvent molecules can form liquid-crystal-like supramolecular adducts, which have been proposed as a molecular basis of 'memory of water'.
Asunto(s)
Proteínas del Huevo/metabolismo , Muramidasa/metabolismo , Conformación Proteica , Agua/metabolismo , Animales , Pollos , Simulación por Computador , Cristalografía por Rayos X , Modelos Químicos , Modelos Moleculares , SolventesRESUMEN
Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity.
Asunto(s)
Aprotinina/química , Péptidos/química , Espectroscopía de Resonancia MagnéticaRESUMEN
The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.