BioWorld International Correspondent
LONDON - Therapies that aim to stop calcium from flowing into cells and building up after a stroke might have been barking up the wrong tree. A new study shows that a better therapeutic strategy might be to ensure that the calcium can get out.
The new findings showed that, in the aftermath of a stroke, the proteins in the cell membrane that normally pump calcium out of a cell are broken down by proteases called calpains. If drugs could be developed to inhibit those calpains, the researchers suggested, they might be able to prevent much of the cell death that occurs following a stroke.
Pierluigi Nicotera, director of the Medical Research Council's toxicology unit, based at the University of Leicester in the UK, told BioWorld International: "This study has triggered a change in paradigm. In the past, the emphasis has always been on controlling the entry of calcium into the ischemic cells following stroke, but we have now discovered that the routes by which calcium leaves the cells are impaired in these circumstances."
The discovery is "exciting," Nicotera added, because it helps to explain why therapy aimed solely at decreasing calcium entry in brain cells has been unsuccessful. It will, he predicted, make it possible to identify potential targets for treatment of stroke and other neurodegenerative diseases. "The findings may lead to new drugs, which will treat these conditions successfully," he said.
Nicotera, together with collaborators elsewhere in the UK and in Italy, published an account of the study in the Jan. 28, 2005, issue of Cell. The paper is titled "Cleavage of the Plasma Membrane Na+/Ca2+ Exchanger in Excitotoxicity."
A stroke occurs when the blood supply to part of the brain is cut off, either as a result of a ruptured blood vessel, or due to a clot or other blockage in a blood vessel. When the blood supply stops, the nerve cells that are directly deprived of oxygen quickly die. As a result, they release chemicals, such as neurotransmitters, that they normally use to communicate with each other. The neurotransmitter glutamate spreads to surrounding cells and sets off a process called excitotoxicity, which results in further cell death.
The glutamate stimulates N-methyl-D-aspartate (NMDA) receptors on the nerve cells, triggering an overload of calcium and sodium ions in postsynaptic cells. In neurons in cell culture, and in animal models of stroke, researchers have found that the initial influx of calcium ions triggered by glutamate stimulation of NMDA receptors is followed by a delayed but massive increase in calcium ions, leading to cell death. Other channels also can drive calcium into neurons during ischemia.
The initial calcium influx is within the range that cells normally would cope with. However, the delayed calcium influx is huge and stays that way - suggesting to scientists that the cell's normal way of regulating intracellular levels of calcium have been turned off.
Nicotera and colleagues studied the mechanism of calcium overload in neurons after reduction in blood supply to areas of the rat brain. They found that removing extracellular calcium ions did not reduce the secondary calcium overload once it had occurred. That allowed them to conclude that the secondary calcium overload was not solely due to opening of membrane channels that allowed calcium to flow into the cells.
They then turned their attention to molecules called Na+/Ca2+ exchangers (NCXs), on the grounds that those should be able to extrude large amounts of calcium ions from neurons. When the researchers added small interfering RNA targeted at NCXs - in order to specifically down-regulate the gene encoding the NCX so that the cells would not have any of those molecules in its membrane - they found that those cultured neurons were unable to move even low concentrations of calcium ions to the exterior.
That finding supported that of another team of researchers, who found that mice lacking a functional gene for the NCX molecule were able to extrude calcium ions following stimulation with just glutamate, at a much slower rate than normal.
Further experiments carried out by Nicotera and his team showed that an isoform of NCX, called NCX3, is split by calpains. The researchers also found that overexpressing the natural protein inhibitor of calpains - a molecule called calpastatin - inhibited the cleavage of NCX3.