RESUMEN
The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.
Asunto(s)
Nicotiana , Proteínas de Plantas , Secuencia de Aminoácidos , Moduladores del Transporte de Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inhibidores de Proteasas , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
The mechanosensitivity of cells depends on the lipid-protein interactions of the plasma membrane. Affectations in the lipid region of the plasma membrane affect the transduction of mechanical forces, and any molecule that modifies the biophysical integrity of the lipid bilayer can alter the mechanical activity of the proteins inside the membrane. To understand whether inhibitors of mechanically activated ion channels affect the mechanical properties of the plasma membrane, we evaluated the rigidity of the membrane of sensory neurons of the DRG of mice using a variant of the scanning ion conductance microscopy method, which allows us to calculate the Young's modulus of individual cells before and after the perfusion of different doses of Gd3+, ruthenium red and GsMTx-4. Our results suggest that these molecules compromise the membrane by increasing the Young's modulus value, which indicates that the membrane becomes more rigid; these compounds act through different mechanisms and by a non-specific manner, each one shows a certain preference for specific cell subpopulations, depending on their cell size and their reactivity to isolectin B4. Our results support the idea that the biophysical properties that result from the interactions that arise in the membranes are part of the mechanotransduction process.
Asunto(s)
Membrana Celular/metabolismo , Moduladores del Transporte de Membrana/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructura , Animales , Cadmio/metabolismo , Línea Celular , Células Cultivadas , Módulo de Elasticidad , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Mecanotransducción Celular , Ratones , Rojo de Rutenio/metabolismo , Transducción de Señal , Venenos de Araña/metabolismoRESUMEN
Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.
Asunto(s)
Venenos de Anfibios/toxicidad , Bufanólidos/toxicidad , Bufonidae/metabolismo , Riñón/efectos de los fármacos , Moduladores del Transporte de Membrana/toxicidad , Glándula Parótida/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Venenos de Anfibios/química , Venenos de Anfibios/aislamiento & purificación , Venenos de Anfibios/metabolismo , Animales , Bufanólidos/química , Bufanólidos/aislamiento & purificación , Bufanólidos/metabolismo , Bufonidae/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Cuba , Humanos , Hidroxilación , Riñón/enzimología , Cinética , Espectroscopía de Resonancia Magnética , Masculino , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/aislamiento & purificación , Moduladores del Transporte de Membrana/metabolismo , Estructura Molecular , Ríos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa de Ion Secundario , Espectrometría de Masas en TándemRESUMEN
Fipronil is an insecticide used to control pests in animals and plants that can causes hepatotoxicity in animals and humans, and it is hepatically metabolized to fipronil sulfone by cytochrome P-450. The present study aimed to characterize the effects of fipronil (10-50µM) on energy metabolism in isolated perfused rat livers. In fed animals, there was increased glucose and lactate release from glycogen catabolism, indicating the stimulation of glycogenolysis and glycolysis. In the livers of fasted animals, fipronil inhibited glucose and urea production from exogenous l-alanine, whereas ammonia and lactate production were increased. In addition, fipronil at 50µM concentration inhibited the oxygen uptake and increased the cytosolic NADH/NAD⺠ratio under glycolytic conditions. The metabolic alterations were found both in livers from normal or proadifen-pretreated rats revealing that fipronil and its reactive metabolites contributed for the observed activity. The effects on oxygen uptake indicated that the possible mechanism of toxicity of fipronil involves impairment on mitochondrial respiratory activity, and therefore, interference with energy metabolism. The inhibitory effects on oxygen uptake observed at the highest concentration of 50µM was abolished by pretreatment of the rats with proadifen indicating that the metabolites of fipronil, including fipronil sulfone, acted predominantly as inhibitors of respiratory chain. The hepatoxicity of both the parent compound and its reactive metabolites was corroborated by the increase in the activity of lactate dehydrogenase in the effluent perfusate in livers from normal or proadifen-pretreated rats.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Metabolismo Energético/efectos de los fármacos , Insecticidas/toxicidad , Hígado/efectos de los fármacos , Moduladores del Transporte de Membrana/toxicidad , Pirazoles/toxicidad , Animales , Biotransformación/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Transporte de Electrón/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Glucogenólisis/efectos de los fármacos , Glucólisis/efectos de los fármacos , Técnicas In Vitro , Insecticidas/metabolismo , Hígado/metabolismo , Masculino , Moduladores del Transporte de Membrana/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Consumo de Oxígeno/efectos de los fármacos , Perfusión , Proadifeno/farmacología , Pirazoles/metabolismo , Ratas Wistar , Urea/metabolismoRESUMEN
Diffusion is not the main process by which drugs are disposed throughout the body. Translational movements of solutes given by different energy-consuming mechanisms are required in order to dispose them efficiently. Membrane transportation and cardiac output distribution are two effective processes to move the molecules among different body sites. Gastrointestinal-blood cycling constitutes a supplementary way to regulate the distribution of molecules between the non-hepatic organs and the liver. Any change in the relative supply of drug molecules among eliminating organs could modify their clearance from the body. Either the nonlinear phenytoin (PHT) pharmacokinetic response or the influence that carbamazepine (CBZ) exerts on PHT exposure could be explained throughout their efflux transporter inducer abilities. Cardiac output distribution difference between the individuals might also explain the dual CBZ-over-PHT interaction response. Finally, valproic acid (VPA) pharmacokinetics can be understood by adding to these mechanisms of transportation its ability to cross the mitochondrial membrane of the hepatocyte.