Smoke signals and flag formations have served the messaging needs of mankind for centuries. These ingenious modes of communication are akin to the intricate networks of intercellular signaling; both have not only withstood the test of time, they will also stand up to any power outage or the most heinous computer virus. Deciphering the codes and cascades of intercellular crosstalk unveils how the clock of life ticks—and what happens when it stops.
Cell signaling, also referred to as signal transduction, usually starts with hormones, neurotransmitters, or cytokines. These so-called first messengers bind to membrane-associated protein receptors, such as G protein-coupled receptors, tyrosine kinase receptors, or ion channel receptors. These receptors touch off a non-protein molecule, called a second messenger, which transduces the signal to the cell’s messaging network of proteins. The subsequent cascade of protein activations and alterations transmits the message to its final destination in the cytoplasm or nucleus. By mapping out the components of these communication networks, researchers can find potential drug targets to interfere with errant messages that could cause disease.
Second messengers have garnered particular attention since they were discovered by Earl Sutherland. Studying the hormone epinephrine, Sutherland found that it caused the activation of the enzyme phosphorylase, which led to the polymerization of glucose into glycogen. But the effect was not direct. On the hunt for the intermediate component of the pathway, Sutherland became the first to come across the second messenger cyclic AMP. In 1971, he earned the Nobel Prize for this discovery and other work in cell signaling.
Cyclic AMP (cAMP), adenosine 3',5' cyclic mononucleotide, is one of the most studied second messengers. Involved in activating or inhibiting enzymatic activity and gene expression, cAMP is synthesized from ATP by membrane-bound adenylate cyclase, which is either stimulated or inhibited by its associate G-protein couple receptor. cAMP is one of the hydrophilic second messengers, which are located in the cytosol. Others include cyclic GMP and calcium ions. Hydrophobic second messengers diffuse from the plasma membrane; these include diacylglycerol and inositol triphosphate (IP3). Second messengers also come in the form of gasses, such as nitric oxide and carbon monoxide.
Researchers can use the changing amounts of second messengers to study drug candidates and their effect on cell signaling. A relatively straightforward method is to assess the presence and amount of second messengers using ELISA-type assays, usually relying on beads as the solid substrate. For example, anti-cAMP antibody can be tethered to beads, along with beads conjugated to streptavidin. The competition between exogenously added biotinylated cAMP and cAMP produced by stimulated cells causes a change in detectable signal.
Assay kits, such as those below, have been designed with your busy schedule in mind. Many are compatible with various automated instruments and formats, such as the popular 96-well plates. You’ll have results before you know it; you need only choose how to communicate them—and in the event of a technical malfunction, you might consider a more traditional option. (Yodeling, anyone?)
BioVision’s cAMP and cGMP Assay Kits are the competitive immunoassays for the quantitative determination of cAMP and cGMP levels, respectively. cAMP is one of the most important “second messengers” involved as a modulator of physiological processes, such as regulating neuronal, glandular, cardiovascular, immune mechanism, nervous system, cell growth and differentiation. A number of hormones are known to activate cAMP through the action of the enzyme adenylate cyclase which converts ATP to cAMP. cGMP has been shown to be present at levels typically 10-100 fold lower than cAMP in most tissues and is formed by the action of the enzyme guanylate cyclase on GTP.
