Methods For Measurement Of cAMP

Many drugs and hormones interact with plasma membrane receptors to induce changes in the production of intracellular second messengers. The first such messenger to be identified was cAMP discovered by Sutherland and Rall in the late 1950s [17]. As for other signaling molecules, the levels of intracellular cAMP are tightly regulated. Production of cAMP is controlled through the adenylyl cyclase family of enzymes, which convert adenosine triphosphate (ATP) to cAMP and inorganic pyrophosphate. These enzymes are activated or inhibited via direct interaction with Ga subunits and, for some isoforms, with calcium and calmodulin. Following Gs-coupled receptor activation, GTP-bound Gas exerts a positive effect on adenylyl cyclase catalysis. Cyclic AMP is produced and is able to bind to and activate cAMP- dependent protein kinase A (PKA) within the cell, initiating phosphorylation events that regulate target enzymes and transcription factors (Fig. 9.2). Following G; -coupled receptor activation, GTP-bound Gai exerts a negative effect on enzyme catalytic activity. Degradation of cAMP is controlled by cAMP phosphodiesterase (PDE) enzymes, which catalyze the hydrolysis of the 3 ester bond of the cAMP to form 5. adenosine monophosphate (AMP). There are several PDE isoforms located in different subcellular compartments and various tissue types; the isoforms are activated by a number of mechanisms. The most widespread mechanism is phosphorylation by cAMP- dependent protein kinases. Thus, cAMP-dependent PDE enzymes act as an important negative feedback system on the receptor-mediated signaling cascade, regulating the extent of changes in intracellular cAMP concentrations [18].

Measurement of adenylyl cyclase activity is carried out by quantitative determination of cAMP produced from ATP as a substrate. However, in the plethora of available methods to study variations in the cAMP levels occurring in cells, tissues, or organic fluids, a first distinction has to be made. Indeed, some assays start from labeled ATP as a substrate while others employ nonlabeled ATP. In the following paragraphs, this criterion will be adopted.

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