RESUMO
To study whether diabetes mellitus (DM) would cause electrophysiological alterations in nodose ganglion (NG) neurons, we used patch clamp and intracellular recording for voltage and current clamp configuration, respectively, on cell bodies of NG from rats with DM. Intracellular microelectrodes recording, according to the waveform of the first derivative of the action potential, revealed three neuronal groups (A0 , Ainf , and Cinf ), which were differently affected. Diabetes only depolarized the resting potential of A0 (from -55 to -44 mV) and Cinf (from -49 to -45 mV) somas. In Ainf neurons, diabetes increased action potential and the after-hyperpolarization durations (from 1.9 and 18 to 2.3 and 32 ms, respectively) and reduced dV/dtdesc (from -63 to -52 V s-1 ). Diabetes reduced the action potential amplitude while increasing the after-hyperpolarization amplitude of Cinf neurons (from 83 and -14 mV to 75 and -16 mV, respectively). Using whole cell patch clamp recording, we observed that diabetes produced an increase in peak amplitude of sodium current density (from -68 to -176 pA pF-1 ) and displacement of steady-state inactivation to more negative values of transmembrane potential only in a group of neurons from diabetic animals (DB2). In the other group (DB1), diabetes did not change this parameter (-58 pA pF-1 ). This change in sodium current did not cause an increase in membrane excitability, probably explainable by the alterations in sodium current kinetics, which are also induced by diabetes. Our data demonstrate that diabetes differently affects membrane properties of different nodose neuron subpopulations, which likely have pathophysiological implications for diabetes mellitus.
Assuntos
Diabetes Mellitus , Neurônios Aferentes , Ratos , Animais , Neurônios Aferentes/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Potenciais de Ação/fisiologia , SódioRESUMO
Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-ß-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-ß-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-ß-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters.
Assuntos
Evolução Molecular , beta-Lactamases/genética , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Biocatálise/efeitos dos fármacos , Cefalexina/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Cinética , Testes de Sensibilidade Microbiana , Mutação/genética , Periplasma/metabolismo , Temperatura , Zinco/metabolismoRESUMO
OBJECTIVES: Croton nepetaefolius is a medicinal plant useful against intestinal disorders. In this study, we elucidate the effects of its essential oil (EOCN) on sympathetic neurons, with emphasis on the interaction of EOCN- and histamine-induced effects. METHODS: The effects of EOCN and histamine were studied in guinea-pig celiac ganglion in vitro. KEY FINDINGS: Histamine significantly altered the resting potential (E(m)) and the input resistance (R(i)) of phasic neurons (from -56.6 +/- 1.78 mV and 88.6 +/- 11.43 MOmega, to -52.9 +/- 1.96 mV and 108.6 +/- 11.00 MOmega, respectively). E(m), R(i) and the histamine-induced alterations of these parameters were not affected by 200 microg/ml EOCN. The number of action potentials produced by a 1-s (two-times threshold) depolarising current and the current threshold (I(th)) for eliciting action potentials (rheobase) were evaluated. Number of action potentials and I(th) were altered by histamine (from 2.6 +/- 0.43 action potentials and 105.4 +/- 11.15 pA to 6.2 +/- 1.16 action potentials and 67.3 +/- 8.21 pA, respectively). EOCN alone did not affect number of action potentials and I(th) but it fully blocked the histamine-induced modifications of number of action potentials and I(th). All the effects produced by histamine were abolished by pyrilamine. CONCLUSIONS: EOCN selectively blocked histamine-induced modulation of active membrane properties.
Assuntos
Óleo de Cróton/farmacologia , Gânglios Simpáticos/efeitos dos fármacos , Bloqueadores Ganglionares/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Histamina/farmacologia , Óleos Voláteis/farmacologia , Potenciais de Ação , Animais , Cobaias , Antagonistas dos Receptores Histamínicos H1/farmacologia , Técnicas In Vitro , Cinética , Masculino , Pirilamina/farmacologiaRESUMO
Caffeine-induced Ca2+ transients (CICTs) in rabbit nodose ganglion neurons (NGNs) are produced by two distinct mechanisms: release from intracellular stores via ryanodine receptors and Ca2+ influx across the plasma membrane, due to activation of an unknown receptor. In isolated rat NGNs, we used single-cell microfluorimetry to measure changes in intracellular Ca2+ and to test whether TRPV1 receptors underlie the Ca2+ influx pathway. Caffeine (10 mM) evoked CICTs in all NGNs tested (n = 47) averaging 365 +/- 32 nM. CICTs were partially dependent upon a Ca2+ influx pathway that ranged between 33% and 98% of the total Ca2+ transient. Application of two selective TRPV1 antagonists significantly attenuated CICTs. The peak average amplitudes of CICTs in Ca2+-free Locke solution and Ca2+-free Locke solution with IRTX or with BCTC were not significantly different from one another (n = 5 and 7, respectively). These observations suggest that caffeine can induce Ca2+ influx by activating TRPV1 channels.