9-(tetrahydro-2-furyl)-adenine and 2--5--dideoxyadenosine

We evaluated the efficacy, potency, and selectivity of the three most commonly used adenylate cyclase (AC) inhibitors in a battery of cell lines constructed to study signaling via three discrete cAMP sensors identified in neuroendocrine cells. SQ22,536 [9-(tetrahydrofuryl)-adenine] and 2',5'-dideoxyadenosine (ddAd) are effective and potent AC inhibitors in HEK293 cells expressing a cAMP response element (CRE) reporter gene, and MDL-12,330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride] is not. Neuroscreen-1 (NS-1) cells were used to assess the specificity of the most potent AC inhibitor, SQ22,536, to block downstream cAMP signaling to phosphorylate CREB (via PKA); to activate Rap1 (via Epac); and to activate ERK signaling leading to neuritogenesis (via the newly described neuritogenic cAMP sensor NCS). SQ22,536 failed to inhibit the effects of cAMP analogs 8-Br-cAMP and 8-CPT-2'-O-Me-cAMP on PKA-mediated CREB activation/phosphorylation and Epac-mediated Rap1 activation, indicating that it does not inhibit these cAMP pathways beyond the level of AC. On the other hand, SQ22,536, but not ddAd, inhibited the effects of cAMP analogs 8-Br-cAMP and 8-CPT-cAMP on ERK phosphorylation and neuritogenesis, indicating that it acts not only as an AC blocker, but also as an inhibitor of the NCS. The observed off-target actions of SQ22,536 are specific to cAMP signaling: SQ22,536 does not block the actions of compounds not related to cAMP signaling, including ERK induction by PMA, and ERK activation and neuritogenesis induced by NGF. These data led us to indicate a second target for SQ22,536 that should be considered when interpreting its effects in whole cell and in vivo experiments.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dideoxyadenosine; ets-Domain Protein Elk-1; Extracellular Signal-Regulated MAP Kinases; Guanine Nucleotide Exchange Factors; HEK293 Cells; High-Throughput Screening Assays; Humans; Imines; Neurites; Neuroendocrine Cells; Phosphorylation; Receptors, G-Protein-Coupled; Signal Transduction

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 microM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific Calpha2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

Topics: Adenine; Adenosine; Adenylyl Cyclases; Animals; Calcium; Catecholamines; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Dideoxyadenosine; Enzyme Inhibitors; Imines; Isoquinolines; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Kinase Inhibitors; Signal Transduction; Sperm Motility; Sperm Tail; Sulfonamides

It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl(-) conductance (G(Cl)) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G(Cl) is independent of protein kinase A. We activated G proteins and monitored CFTR G(Cl) in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 microM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G(Cl) required Mg(2+) and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G(Cl) was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 microM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 microM); and 3) independent of Ca(2+), suggesting that Ca(2+)-dependent protein kinase C and Ca(2+)/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10(-6) M forskolin plus 10(-6) M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G(Cl) endogenously via a cAMP-independent pathway in this native absorptive epithelium.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adult; Calcium; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Dideoxyadenosine; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; In Vitro Techniques; Male; Phosphorylation; Sweat Glands

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Apoptosis; Caspase 3; Caspases; Cyclic AMP; Cysteine Endopeptidases; Dideoxyadenosine; DNA Fragmentation; Dolichol Phosphates; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Humans; Kinetics; Leukemia, Monocytic, Acute; Tumor Cells, Cultured

It is uncertain whether adenosine 3',5'-cyclic monophosphate (cAMP) or the inositol-calcium pathway mediates the stimulation of bone resorption by parathyroid hormone (PTH). Incubation of bone organ cultures with cAMP analogues and forskolin has not resolved this question because of the cellular inhomogeneity of bone and the consequent presence of adenylate cyclase-linked receptors for both PTH and calcitonin, hormones with opposite effects on bone resorption. We have used two new inhibitors of adenylate cyclase, 9-(tetrahydro-2-furyl)adenine (SQ 22536) and 2',5'-dideoxyadenosine (DDA), to directly reassess the role of cAMP in PTH-stimulated osteolysis. SQ 22536 (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blocked PTH stimulation of cAMP production measured in the absence of a phosphodiesterase blocker. In the presence of 1 mM 3-isobutyl-1-methylxanthine, half-maximal inhibition of PTH-induced cAMP production occurred with 0.2 mM SQ and 0.1 mM DDA, respectively. These concentrations of SQ and DDA had no effect on PTH-stimulated 45Ca release from calvaria, although both agents inhibited bone resorption when present at concentrations of 1-2 mM. At these levels, SQ and DDA caused equivalent inhibition of 45Ca release stimulated by 1,25-dihydroxyvitamin D3 but did not affect basal 45Ca release or [3H]-phenylalanine incorporation. It is concluded that substantial blockade of PTH-induced cAMP production does not affect this hormone's stimulation of bone resorption, which is therefore likely to be mediated by another intracellular messenger system, possibly calcium. In millimolar concentrations, SQ and DDA appear to be nonspecific blockers of osteoclastic bone resorption.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclase Inhibitors; Animals; Bone and Bones; Bone Resorption; Calcium; Cyclic AMP; Dideoxyadenosine; DNA Replication; In Vitro Techniques; Indomethacin; Isomerism; Kinetics; Mice; Parathyroid Hormone; Phenylalanine; Protein Biosynthesis; Theophylline