Constitutive activity of somatostatin receptor subtypes

The five somatostatin receptors (SSTR1-5) are G-protein-coupled receptors, coupling to G(αi/0) subunits to regulate pathways including inhibiting adenylate cyclase activity and reduce intracellular cAMP levels and decrease intracellular calcium levels. In the pituitary gland, somatostatin actions, mediated through SSTR1, 2, 3, and 5, are inhibition of growth hormone, thyrotropin hormone, and adrenocorticotropin hormone release and to a lesser extent, inhibition of cell growth. Establishment of constitutive SSTRs action suggests that abundant pituitary SSTR expression contributes to pituitary function in maintaining homeostasis, aside from the SSTR response to episodic hypothalamic somatostatin release. In this chapter, we describe an experimental approach to directly and indirectly demonstrate constitutive SSTR activity by altering receptor density in AtT20 mouse pituitary corticotroph tumor cells, utilizing small interference RNA to knock receptor expression down or stable SSTRs transfection to overexpress selective receptor levels. We describe methodical validation for each of the approaches and the use of a sensitive cAMP assay to analyze consequences of changing membrane receptor number in the absence of an added ligand.