Real-Time Monitoring of Somatostatin Receptor-cAMP Signaling in Live Pituitary

Abstract

Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by Gs-coupled receptors in living cells. In contrast, real-time imaging of Gi-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual Gi protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

Somatostatin depresses the excitability of subicular bursting cells: Roles of inward rectifier K+ channels, KCNQ channels and Epac

“…We therefore tested the roles of Epac2 and found that application of a selective Epac2 inhibitor, partially blocked SST-mediated outward HCs, suggesting that Epac2 is at least partially responsible for SST-mediated hyperpolarization. Consistent with our results, activation of SST receptors inhibits Epac (Jacobs et al, 2010) and Epac inhibits GIRK channels in medial prefrontal cortex pyramidal neurons (Witkowski, Rola, & Szulczyk, 2012). Third, cAMP by itself can interacts with ion channels to alter RMPs.…”

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Furthermore, by crossing the CAG-EPAC1-camps mice with SSTR2 Ϫ/Ϫ mice, the authors successfully established a major role of SSTR2 in mediating the effects of octreotide, a clinically used somatostatin analog. This study demonstrates that transgenic EPAC sensor mice can be crossbred with other genetic murine models for dissecting functions of various GPCRs under physiological conditions (460).…”

“…Following these studies and using the same transgenic mice, cAMP imaging in living pituitary slices and primary pituitary cells was conducted to evaluate the contribution of G␣ i -coupled somatostatin receptors (SSTRs) in the regulation of cAMP levels under physiological conditions (460). In somatotropic cells, somatostatin and dopamine receptors are the predominant G␣ i -coupled receptors, but whose actions on cAMP signaling have been challenging for realtime live cell imaging studies.…”

Genetically-encoded tools for cAMP probing and modulation in living systems

“…All modern FRET sensors for cAMP are genetically-encoded proteins and hence can be used not only in cell cultures, but in laboratory animals as well, providing insights into cAMP life-cycle in non-perturbed microenvironment ( Nikolaev et al, 2006 ; Calebiro et al, 2009 ; Jacobs et al, 2010 ; Sprenger et al, 2015 ). The ratiometric nature of FRET brings additional benefits, allowing to correct for cell-to-cell variation in sensor expression levels and to minimize the effects of auto-fluorescence and light scattering.…”