Abstract
Cyclic AMP (cAMP) and Ca2+ are two ubiquitous second messengers in transduction pathways downstream of receptors for hormones, neurotransmitters and local signals. The availability of fluorescent Ca2+ reporter dyes that are easily introduced into cells and tissues has facilitated analysis of the dynamics and spatial patterns for Ca2+ signaling pathways. A similar dissection of the role of cAMP has lagged because indicator dyes do not exist. Genetically encoded reporters for cAMP are available but they must be introduced by transient transfection in cell culture, which limits their utility. We report here that we have produced a strain of transgenic mice in which an enhanced cAMP reporter is integrated in the genome and can be expressed in any targeted tissue and with tetracycline induction. We have expressed the cAMP reporter in β-cells of pancreatic islets and conducted an analysis of intracellular cAMP levels in relation to glucose stimulation, Ca2+ levels, and membrane depolarization. Pancreatic function in transgenic mice was normal. In induced transgenic islets, glucose evoked an increase in cAMP in β-cells in a dose-dependent manner. The cAMP response is independent of (in fact, precedes) the Ca2+ influx that results from glucose stimulation of islets. Glucose-evoked cAMP responses are synchronous in cells throughout the islet and occur in 2 phases suggestive of the time course of insulin secretion. Insofar as cAMP in islets is known to potentiate insulin secretion, the novel transgenic mouse model will for the first time permit detailed analyses of cAMP signals in β-cells within islets, i.e. in their native physiological context. Reporter expression in other tissues (such as the heart) where cAMP plays a critical regulatory role, will permit novel biomedical approaches.
Highlights
Cyclic adenosine 39-59-monophosphate has been recognized as an intracellular second messenger for decades, a detailed understanding of its regulation, compartmentalization, range of subcellular targets, and interactions with other second messenger systems has been difficult to reach, especially in complex tissues
An early microinjectable reporter for cellular cyclic adenosine 39-59-monophosphate (cAMP) was constructed by labeling Protein Kinase A subunits with fluors for Fluorescence Resonance Energy Transfer (FRET) [1]
The original genetically encoded cAMP reporter [3] consists of two chimeric proteins, Yellow Fluorescent Protein fused to the Protein Kinase A (PKA) Catalytic subunit (C-YFP) and Cyan
Summary
Cyclic adenosine 39-59-monophosphate (cAMP) has been recognized as an intracellular second messenger for decades, a detailed understanding of its regulation, compartmentalization, range of subcellular targets, and interactions with other second messenger systems has been difficult to reach, especially in complex tissues. This contrasts with analyses on another ubiquitous second messenger, Ca2+, in large part due to the availability of indicator dyes that are readily introduced into cells and tissues. Encoded reporters have been produced using the cAMP-binding domains of Protein Kinase A [2,3] or Epac [4], fused to spectrum-shifted variants of Green Fluorescent Protein (GFP). Tissues from transgenic animals endogenously expressing functional reporters offer the advantage of examining tissues with interacting cell types ex vivo or in vivo
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