RESUMO
The aim of our work was to investigate the presence of non-selective cation channels (NSCC) in freshly isolated smooth muscle cells from the human umbilical artery (HUA), one of the vessels involved in fetal-placental circulation. We studied the electrophysiological properties of NSCC using the patch-clamp technique in whole-cell configuration, and their possible role in the contractile state of intact vessels' rings. Recording with a high intracellular Cs(+) solution and a near physiological extracellular saline solution, we found a Gd(3+)-sensitive current (IC(50) = 1.05 microM) with a linear current-voltage relationship showing a reversal potential (E(rev)) of -2.1 +/- 1.2 mV (n =15 cells). La(3+) (100 microM) and Mg(2+) (5 mM) also blocked this current. In such conditions, inward currents were carried by Na(+) and Ca(2+); hence, a Na(+)-free solution inhibited only inward current (-67.3 +/- 11.4%, at -40 mV, n = 7, p < 0.05) and a Ca(2+)-Na(+)-free solution decreased the current even further with respect to values obtained in Na(+)-free solution (-69.8 +/- 8.8% at -40 mV, n = 9, p < 0.05). The permeability ratios (P(X)/P(Cs(+))) for monovalent and divalent cations were 1, 0.9, 0.7, 0.7, 0.7, and 0.5 where X = Cs(+), Na(+), Li(+), Ca(2+), Ba(2+) and Tris(+), respectively. In intact tissue, a 0 Ca(2+) extracellular solution, Gd(3+) (100-250 microM), La(3+) (200 microM) and Mg(2+) (5 mM) induced vasorelaxation in non-stimulated HUA rings.
Assuntos
Cátions/metabolismo , Canais Iônicos/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artérias Umbilicais/metabolismo , Cordão Umbilical/irrigação sanguínea , Relação Dose-Resposta a Droga , Eletrofisiologia , Gadolínio/metabolismo , Gadolínio/farmacologia , Humanos , Lantânio/metabolismo , Lantânio/farmacologia , Magnésio/metabolismo , Magnésio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Artérias Umbilicais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
An extraction methodology based on cloud point phase separation of non-ionic surfactants has been developed for the preconcentration of ppb amounts of gadolinium in urine as a prior step to its determination by an absorptiometric procedure. A method based on the formation of complexes with 2-(3,5-dichloro-2-pyridylazo)-5-dimethylaminophenol was used for the extraction of Gd(III) in the surfactant-rich phase of non-ionic surfactant polyethyleneglycolmono-p-nonylphenylether (PONPE 7.5). The variables affecting the combined preconcentration-absorptiometric method have been evaluated and optimised. The extraction efficiency, linearity, and the limit of detection (LOD) of the method were determined. The optimised procedure was applied to determine total and free Gd(III) contents in real urine samples of patients after the NMR imaging diagnostic examination with contrast agent.
Assuntos
Meios de Contraste/metabolismo , Gadolínio/metabolismo , Gadolínio/urina , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
Ca2+ and Gd3+ stimulated the GTPase activity of chicken brain tubulin 13- and 26-fold, respectively. Mg2+, Tb3+, and Na+ had no effect. This GTPase activity showed a saturation behavior with Ca2+ and Gd3+ with a maximal activity of 0.26 +/- 0.026 and 1.15 +/- 0.78 nmol min-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 +/- 0.18 and 0.011 +/- 0.007 mM, respectively. In the presence of Ca2+, the GTPase activity was proportional to tubulin concentration in the range 0.9-31.8 microM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca2+ were 0.71 +/- 0.1 and 0.049 +/- 0.043 mM, respectively. The similarity of the Ca2+ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not directly involved in the mechanism of inhibition of Ca2+ -dependent tubulin assembly. This inhibition could be better explained by the formation of a nonfunctional conformational state of tubulin induced by Ca2+ that is responsible for the GTPase activity. Quenching of the intrinsic fluorescence of tryptophan induced by Ca2+ showed an apparent dissociation constant of 0.14 +/- 0.005 mM, in the range of values determined through tubulin polymerization inhibition or through the induction of GTPase activity by Ca2+. Acrylamide-induced quenching of the intrinsic fluorescence showed values of the Stern-Volmer constants of 5.4 +/- 0.12 and 5.0 +/- 0.15 M-1 in the absence and presence of Ca2+, respectively. These results support the hypothesis that the inhibition of tubulin polymerization and the induction of the GTPase activity by Ca2+ is mediated by a conformational change. Ca2+ failed to induce depolymerization of GDP-AIF4-microtubules; this could be explained by a model in which Ca-tubulin is unable to assemble into microtubules and the rate of dissociation of GDP-Pi-tubulin from the microtubule ends is extremely slow compared with the rate of GDP-subunit dissociation, supporting the concept that the GTP- and GDP-Pi-tubulin cap at the ends of microtubules regulates their dynamic instability.