A sort of biochemical scaffold for a compound that enables blood pressure to be low, heart bypass grafts to remain open and nerves to communicate has been identified by Medical College of Georgia researchers.
Researchers say identifying the framework for how these and other very positive health benefits occur should help them find ways to augment the benefits and identify new treatments for cardiovascular disease, which may result when the support structure falls apart.
"It's a whole new ball game," Dr. John D. Catravas, director of the Vascular Biology Center, said of the findings which contradict previous understanding of how the compound, cyclic GMP, which also helps keep blood vessels open and enables penile erection, is ultimately produced.
Dr. Catravas and Dr. Richard C. Venema, biochemist and molecular biologist, recently received funding from the American Heart Association and the National Institutes of Health to study how the scaffold they have found is assembled.
They found the scaffold while studying the endothelial and smooth muscle cells – the two major cell types within blood vessels – in the vessels of healthy animals. They found, not surprisingly, nitric oxide synthase which makes nitric oxide, the short-lived gas that, in turn, activates the enzyme guanylate cyclase or sGC, an enzyme key to the production of cyclic GMP; in fact, heart drugs such as nitroglycerin work by stimulating sGC to produce cyclic GMP.
They also found the heat shock protein 90, or hsp 90, – one of the ubiquitous heat shock proteins – which makes nitric oxide synthase more efficient in producing nitric oxide.
What they didn't expect was to find that the three substances combined to form an efficient biochemical structure that makes sGC readily available to support the positive benefits of cyclic GMP.
Prior to the findings by MCG researchers, it was believed that only hsp 90 and nitric oxide synthase were packaged together, that the resulting nitric oxide would essentially float away in search of sGC. But in fact, that may be the disassembled structure that can precipitate or augment cardiovascular disease, Dr. Catravas said.
"The way we think it works right now is that this hsp 90 is like a scaffold that allows sGC and nitric oxide synthase to attach," Dr. Catravas said. "You have one nitric oxide synthase sitting on the hsp 90 and you have an sGC molecule, which allows for a very, very close environment for the nitric oxide to move to the sGC. This is very important because nitric oxide has a very short life and there are other compounds inside cells that, as soon as the nitric oxide is produced, they grab it and either inactivate it or turn it into a toxic compound."
And there's the rub. When sufficient nitric oxide isn't available to activate sGC, the unhealthy result can be production of one of the most potent self-made toxins in the body and resulting hypertension, heart and kidney disease and erectile dysfunction. "We propose that when the enzyme sGC is not part of the structure, this contributes to cardiovascular disease," Dr. Catravas said.
"We are saying that one of the reasons nitric oxide is more beneficial than detrimental in the body is because it doesn't have the opportunity to be attacked by other compounds and become a toxin because it's very close to its receptor, to the sGC enzyme that it stimulates and then creates all the good things that it does," he said.
They first confirmed the structure by using a process called immunoprecipitation, in which antibodies were used to try to pull each of the three proteins out of the scaffold, Dr. Venema said. "When we pulled down heat shock protein 90, for example, it also pulled down sGC and nitric oxide synthase because they are all attached."
An NIH grant reviewer wanted to know if this union held up in an intact, living cell as well, rather than in a cell that is ground up for the purposes of immunoprecipitation where a chemical reaction could explain the close proximity of sGC.
So they used a process called co-localization to also study the relationship in a living cell, using fluorescent antibodies to the two proteins: the antibody for hsp 90 was labeled green and an antibody for sGC was labeled red. The resulting yellow glow showed that the two proteins were together in the living endothelial and smooth muscle cell.
The question remained: Does the form they found really affect function? So they also used drugs to interfere with the scaffolding and the activity of the sGC enzyme decreased. "That is when we started thinking we had something important," Dr. Catravas said.
Now they are looking to see exactly where hsp 90 and sGC bind and why sometimes they don't; possibly a mutation in heat shock protein 90 makes it difficult for the other two proteins to attach, they said.
"This is the hypothesis: when these two proteins move apart, that is when you worsen or precipitate cardiovascular disease. We still have to prove this," Dr. Catravas said. "If this is true, it could very well change how we treat those diseases."
American Heart Association and National Institutes of Health funding.
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