Pharmacological distinction between soluble and transmembrane adenylyl cyclases

Abstract

The second messenger cAMP is involved in a number of cellular signaling pathways. In mammals, cAMP is produced by either the hormonally responsive, G protein-regulated transmembrane adenylyl cyclases (tmACs) or by the bicarbonate- and calcium-regulated soluble adenylyl cyclase (sAC). To develop tools to differentiate tmAC and sAC signaling, we determined the specificity and potency of commercially available adenylyl cyclase inhibitors. In cellular systems, two inhibitors, KH7 and catechol estrogens, proved specific for sAC, and 2',5'-dideoxyadenosine proved specific for tmACs. These tools provide a means to define the specific contributions of the different families of adenylyl cyclases in cells and tissues, which will further our understanding of cell signaling.

Fig. 1.

Structures of compounds used in this study. Dideoxyadenosine 3′-triphosphate (ddATP), 2′,5′-Dideoxyadenosine (ddAdo), 9-(tetrahydrofuryl)-adenine (SQ 22,536), 9-β-d-arabinosyladenine (vidarabine), 9-cyclopentyladenine (9-CP), cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride (MDL-12,330a), 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazoline (NKY80), 2-(1H-benzo[d]imidazol-2-ylthio)-N′-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), and catechol derivatives of estrogen, such as 2-hydroxyestradiol (2-CE) or 4-hydroxyestradiol (4-CE).

Fig. 2.

Inhibition of purified rat sAC_t activity. (A) Rat sAC_t in vitro adenylyl cyclase activity in the presence of 2.5 mM ATP and 10 mM MnCl₂ and the indicated concentrations of KH7 (red; IC₅₀ = 3 μM) or ddATP (blue; IC₅₀ = 400 µM); (B) ddAdo (blue) or MDL-12,330a (purple; IC₅₀ = 240 µM) (shown with KH7 for comparison); (C) SQ 22,536; (D) 9-CP; (E) NKY80; or (F) vidarabine. Graphs are representative assays repeated at least twice; values represent activity normalized to sAC_t activity in the absence of any inhibitor. Curves are nonlinear fits generated by Prism.

Fig. 3.

Inhibition of sAC and tmACs in cellular lysates. (A, inset) Western blot of sAC in whole-cell lysates (10 µg) from parental HEK293 cells and 4-4 cells using the anti-sAC monoclonal antibody R21 (Zippin et al., 2003). (A–H) Adenylyl cyclase activities in whole-cell lysates from 4-4 cells (red) assayed in the presence of 2.5 mM ATP, 10 mM MnCl₂, and 500 µM IBMX or from 293 cells (blue) assayed in the presence of 2.5 mM ATP, 10 mM MnCl₂, 500 µM IBMX, and 50 µM forskolin in the presence of the indicated concentrations of (A) KH7, (B) ddATP, (C) ddAdo, (D) MDL-12,330a, (E) SQ 22,536, (F) 9-CP, (G) NKY80, or (H) vidarabine. Graphs are representative assays repeated at least twice; values represent activity normalized to activity in each whole-cell lysate in the absence of any inhibitor. Curves are nonlinear fits generated by Prism.

Fig. 4.

Inhibition of sAC and tmACs in intact cells. Cellular cAMP accumulation in 4-4 cells (red) or in forskolin-stimulated (50 µM) sAC KO MEFs (blue) in the presence of 500 µM IBMX and 50 µM dipyridamole and the indicated concentrations of (A) KH7, (B) ddAdo, (C) 2-CE, or (D) 4-CE. Graphs are representative assays repeated at least twice; values represent activity normalized to cAMP accumulation in each cell line in the absence of any inhibitor. Curves are nonlinear fits generated by Prism.