New tool highlights activity of key cellular signal

November 15, 2004

Scientists at Johns Hopkins and the University of Texas Medical Branch have created a new tool that easily reveals when and where a key cellular signal is active. The development, described in the early edition of the Proceedings of the National Academy of Sciences, should speed identification of the signal's triggers and effects in normal processes and in conditions such as asthma, allergy, inflammation, lung disease and heart disease.

The tool -- a special fluorescent protein -- probes the activity of cyclic AMP in living cells and represents biology's growing application of a fluorescent phenomenon to study the molecular changes that reveal cells' inner workings.

Much like a child might pass along a visitor's request to a grown-up, cyclic AMP carries messages from hormones or other molecules "knocking" at the cell's door to proteins inside the cell. But because cyclic AMP uses just a handful of proteins to pass on many messages, scientists have had a hard time figuring out how it can trigger the right cellular response to each one.

"Scientists suspected that timing and location of cyclic AMP activity was important, but there was no easy way to study cyclic AMP inside cells in real time and in real space," says Jin Zhang, Ph.D., senior author of the study and an assistant professor of pharmacology and molecular sciences and of neuroscience in Johns Hopkins' Institute for Basic Biomedical Sciences. "This new fluorescent protein can be directed to the nucleus or to other parts of the cell, so we can now follow cyclic AMP activity in real time and space."

The new fluorescent protein takes advantage of the fact that fluorescent molecules can "talk" to one another when they are close together, affecting the color of light emitted -- a phenomenon called fluorescent resonance energy transfer (FRET). If the distance between the fluorescent molecules changes, the color of light emitted may change as well.

In the early 1990s, biologists began harnessing this phenomenon to study molecular changes in cells. A team led by Roger Tsien, Ph.D., at the University of California San Diego created the first FRET probe of cyclic AMP activity by attaching small fluorescent molecules to the ends of a protein called PKA. Once activated by cyclic AMP, PKA breaks in two, separating its two fluorescent "caps." The first all-protein fluorescent version of PKA was developed a few years later.

But the PKA-based probes, which Zhang used as a postdoctoral fellow at the University of California San Diego, were difficult to use because PKA is made of four parts. For a simpler and easier to use probe, Lisa DiPilato, now a second-year graduate student in pharmacology, added fluorescent caps -- one cyan, the other yellow -- to a one-piece protein called Epac that is also activated by cyclic AMP.

By using a special microscope to measure how the fluorescence of the probe changed in response to cyclic AMP, DiPilato proved the probe's ability. Addition of a genetic "address label" then allowed her to direct the fluorescent probe to go to particular places in cells -- keeping it at the cell membrane, sending it to the nucleus, or directing it to the cell's power plant, the mitochondria.

"The probe has already provided new information," says Zhang, who arrived at Hopkins in September 2003. "The probe itself shows that Epac changes shape when it's activated, which had not been directly observed. And while some scientists had suggested cyclic AMP could get into the mitochondria, our studies are the first to show that it's there."

Stimulating cyclic AMP production in a human embryonic kidney cell line containing the targeted fluorescent Epac proteins caused an immediate build-up of cyclic AMP and activation of Epac at the cell membrane, where cyclic AMP is produced, the researchers report. Within seconds, build-up of cyclic AMP also began in the cytoplasm of the cell, the nucleus and the mitochondria.

Because cyclic AMP does so many things, understanding how it works has broad implications, says Zhang. For example, the main enzyme that degrades cyclic AMP is being targeted by drug companies for potential application in inflammation, allergy, asthma or chronic lung disease, conditions that may be helped by increasing cyclic AMP. More recently, altered regulation of cyclic AMP has been tied to the risk of stroke occurrence and to heart conditions including dilated cardiomyopathy.

Zhang says her lab will use the new and other fluorescent probes to study the role of cyclic AMP in smooth muscle cells, such as those in blood vessels, in inflammation, and in fat cells, where it may be involved in diabetes-related processes.
-end-
The Hopkins researchers were supported by start-up funds from the Department of Pharmacology and Molecular Sciences and the W.M. Keck Center. Authors on the paper are DiPilato and Zhang of Johns Hopkins, and Xiaodong Cheng of the University of Texas Medical Branch.

On the Web: http://www.pnas.org

Johns Hopkins Medicine

Related Inflammation Articles from Brightsurf:

3D printed stents that treat inflammation
POSTECH Professor Dong-Woo Cho's research team develops bioink-loaded esophageal stents for treating radiation esophagitis.

New cause of inflammation in people with HIV identified
A new study led by researchers at Boston Medical Center examined what factors could be contributing to this inflammation, and they identified the inability to control HIV RNA production from existing HIV DNA as a potential key driver of inflammation.

Maltreatment tied to higher inflammation in girls
New research by a University of Georgia scientist reveals that girls who are maltreated show higher levels of inflammation at an early age than boys who are maltreated or children who have not experienced abuse.

A protein that controls inflammation
A study by the research team of Prof. Geert van Loo (VIB-UGent Center for Inflammation Research) has unraveled a critical molecular mechanism behind autoimmune and inflammatory diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis.

Inflammation in the brain linked to several forms of dementia
Inflammation in the brain may be more widely implicated in dementias than was previously thought, suggests new research from the University of Cambridge.

Social isolation could cause physical inflammation
Social isolation could be associated with increased inflammation in the body new research from the University of Surrey and Brunel University London has found.

Hydrogels control inflammation to help healing
Researchers test a sampling of synthetic, biocompatible hydrogels to see how tuning them influences the body's inflammatory response.

Why beta-blockers cause skin inflammation
Beta-blockers are often used to treat high blood pressure and other cardiovascular diseases.

The 'inflammation' of opioid use
New research correlates inflammation in the brain and gut to negative emotional state during opioid withdrawal.

Using a common anticonvulsant to counteract inflammation
The interaction between a chromosomal protein called HMGB1 and a cellular receptor called RAGE is known to trigger inflammation.

Read More: Inflammation News and Inflammation Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.