ABSTRACT
OBJETIVO: Investigar os efeitos de baixas concentrações de LDL oxidada (LDL-ox) sobre a proliferação e a motilidade espontânea de células endoteliais de artérias coronárias humanas (CEACH) em cultura. MÉTODOS: Culturas de CEACH foram tratadas com baixas concentrações de LDL nativa (LDLn), isolada de plasma humano, e com LDL minimamente oxidada por diferentes métodos químicos, e os efeitos, comparados entre si. RESULTADOS: LDLn não apresentou efeitos deletérios sobre o endotélio em proliferação e na motilidade in vitro de CEACH, porém na mais alta concentração e por tempo mais prolongado inibiu a proliferação celular. As LDL-ox, quimicamente, pela espermina nonoato (ENO) e 3-morfolinosidnonimina (SIN-1) expressaram efeitos inibitórios significativos sobre a proliferação e a motilidade in vitro de CEACH proporcionais às maiores concentrações e graus de oxidação das LDL. CONCLUSÃO: LDL-ox apresenta efeito citotóxico, inibindo a proliferação e a motilidade espontânea de células endoteliais de artérias coronárias humanas em cultura, proporcionalmente à concentração e ao grau de oxidação da LDL, enquanto, LDL nativa é relativamente inócua.
Subject(s)
Humans , Endothelial Cells , Endothelium, Vascular/cytology , Lipoproteins, LDL/pharmacology , Cell Movement , Cell Proliferation , Coronary Vessels/cytology , Endothelial Cells/physiology , Coronary Artery Disease/physiopathology , Coronary Artery Disease/metabolism , Lipoproteins, LDL/physiology , Lipoproteins, LDL/metabolism , Cell Movement/physiologyABSTRACT
It has been reported that a change in the cellular redox state may be involved in the regulation of vascular tone, but the underlying mechanism is not fully understood. The present study was designed to investigate the cellular effect of sulfhydryl modifying agents in the coronary artery of rabbit using the tension measurement and whole cell clamping method. The application of diamide, a sulfhydryl oxidizing agent, relaxed the endothelium denuded coronary arteries in a dose dependent manner. The fact that this diamide-induced relaxation was significantly attenuated by a pretreatment of 4-AP, and the coronary arteries precontracted with 100 mM K+ instead of histamine, suggests the involvement of 4-AP sensitive K+ channels in the diamide-induced relaxation of coronary arteries. Whole cell patch clamp studies revealed that the 4-AP sensitive IdK was significantly enhanced by the membrane permeant oxidizing agents, diamide and DTDP, and were reversed by subsequent exposure to the reducing agent, DTT. Neither the membrane impermeant oxidizing or reducing agents, GSSG or GSH, had any effect on the activity of IdK, indicating that intracellular sulfhydryl modification is critical for modulating IdK activity. The Diamide failed to significantly alter the voltage dependence of the activation and inactivation parameters, and did not change the inactivation process, suggesting that diamide increases the number of functional channels without altering their gating properties. Since IdK has been believed to play an important role in regulating membrane potential and arterial tone, our results about the effect of sulfhydryl modifying agents on coronary arterial tone and IdK activity should help understand the pathophysiology of the diseases, where oxidative damage has been implicated.
Subject(s)
Female , Male , Rabbits , Animals , Arteries/physiology , Arteries/drug effects , Arteries/cytology , Coronary Vessels/physiology , Coronary Vessels/drug effects , Coronary Vessels/cytology , Oxidants/pharmacology , Potassium Channels/physiology , Reducing Agents/pharmacology , Sulfhydryl Compounds/metabolismABSTRACT
The large conductance Ca2+ activated K+ channel (BK channel) has been considered to play an important role in the excitability and contractility of vascular smooth muscle cells. Activation of the BK channel causes the hyperpolarization and relaxation of vascular smooth muscle cells. It has been reported that fatty acids can affect the BK channel activity and its concentration is increased significantly during myocardial ischemia. These reports suggest that fatty acids may contribute to the ischemic coronary vasodilation by increasing the BK channel activity. However, the underlying mechanism of fatty acid-induced activation of the BK channel is still uncertain. In the present study, we measured the effect of fatty acids on the BK channel activity in rabbit coronary smooth muscle cells by using patch clamp method and also examined its underlying mechanism. Arachidonic acid (AA) dissolved in DMSO activated the BK channel in a dose-dependent manner (from 0.5 to 10 microM), and DMSO (0.1%) alone had no effect on the activity of the BK channel. Arachidonic acid activated BK channels in both cell-attached and inside-out patches, but the onset and recovery of this effect were slower in the cell-attached patch configuration. The BK channel activity was also increased by other fatty acids, including myristic acid, linoleic acid, palmitoleic acid and palmitic acid. Long chain fatty acids were more effective than short chain fatty acids (myristic acid), and there was no statistical difference between the effect of saturated (palmitic acid) and unsaturated fatty acids (palmitoleic acid) on the BK channel activity. The concentration of Ca2+ and Mg2+ in the bathing solution had no appreciable effects on the AA-induced increase of BK channel activity. From the above results, it may be concluded that fatty acids directly increase the BK channel activity and may contribute to the ischemic coronary vasodilatation in rabbit coronary smooth muscle cells.