RESUMO
The corneal endothelium is a fluid-transporting epithelium. As other similar tissues, it displays an electrical potential of ~1 mV (aqueous side negative) across the entire layer [transendothelial potential difference (TEPD)]. It appears that this electrical potential is mainly the result of the transport of anions across the cell layer (from stroma to aqueous). There is substantial evidence that the TEPD is related linearly to fluid transport; hence, under proper conditions, its measure could serve as a measure of fluid transport. Furthermore, the TEPD is not steady; instead, it displays a spectrum of frequency components (0-15 Hz) recognized recently using Fourier transforms. Such frequency components appear due to charge-separating (electrogenic) processes mediated by epithelial plasma membrane proteins (both ionic channels and ionic cotransporters). In particular, the endothelial TEPD oscillations of the highest amplitude (1-2 Hz) were linked to the operation of so-called sodium bicarbonate cotransporters. However, no time localization of that activity could be obtained with the Fourier methodology utilized. For that reason we now characterize the TEPD using wavelet analysis with the aim to localize in time the variations in TEPD. We find that the mentioned high-amplitude oscillatory components of the TEPD appear cyclically during the several hours that an endothelial preparation survives in vitro. They have a period of 4.6 ± 0.4 s on average (n=4). The wavelet power value at the peak of such oscillations is 1.5 ± 0.1 mV(2) Hz on average (n = 4), and is remarkably narrow in its distribution.
Assuntos
Líquidos Corporais/metabolismo , Endotélio Corneano/fisiologia , Modelos Biológicos , Análise de Ondaletas , Animais , Simulação por Computador , Campos Eletromagnéticos , HumanosRESUMO
Pregnancy is often associated with oxidative stress (OS) and lower antioxidant defences, which are both implicated in the pathophysiology of preeclampsia, free radical-induced birth defects, and abortions, as well as gestational diabetes mellitus (GDM), where products of lipid peroxidation are increased. The molecular target(s) of increased oxygen free radicals and consequent lipid peroxidation in the human placenta remains ill defined. The human syncytiotrophoblast (hST) expresses abundant polycystin-2 (PC2, TRPP2), a TRP-type Ca(2+)-permeable non-selective cation channel. Here, we explored the effect of reactive oxygen species (ROS) on PC2 channel activity of term hST. Apical membranes of the hST were reconstituted in a lipid bilayer chamber. Addition of either hydrogen-peroxide (H(2)O(2)) or tert-butyl hydroperoxide (tBHP) to the cis chamber (intracellular side) rapidly and completely inhibited PC2-mediated cation channel activity in reconstituted hST vesicles. A dose-response titration with increasing concentrations of H(2)O(2) gave an IC(50)=131 nM. The effect of H(2)O(2) on the isolated protein from in vitro transcribed/translated material was significantly different. H(2)O(2) inhibited PC2 cation channel activity, with a much lower affinity (IC(50)=193 microM). To correlate these findings with H(2)O(2)-induced lipid peroxidation, TBARS where measured in hST apical membranes incubated with H(2)O(2). Increased TBARS by exposure of hST apical membranes to H(2)O(2) (625 microM) returned to control value in the presence of catalase (167 microg/ml). Taken together these data indicate that ROS affect PC2 channel function by targetting both membrane lipids and the channel protein. Thus, OS in human pregnancy may be linked to dysregulation of channels such as PC2, which allow the transport of Ca(2+) into the placenta. Oxidative complications in pregnancy may implicate dysfunctional cation transfer between mother and fetus.