A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates

doi:10.1016/0003-2697(92)90045-9

Analytical Biochemistry

Volume 203, Issue 1, 15 May 1992, Pages 76-82

https://doi.org/10.1016/0003-2697(92)90045-9 Get rights and content

Abstract

A method for the separation of cyclic AMP from adenosine and polyvalent adenine nucleotides is described. The method consists of the sequential elution of adenosine and cyclic AMP from a single column of acidic aluminum oxide (alumina) with dilute hydrochloric acid and ammonium acetate. Adenosine, adenine, xanthine, and hypoxanthine are rapidly eluted with the application of 0.005 n hydrochloric acid while cyclic AMP remains adsorbed to the alumina. A subsequent application of 0.1 m ammonium acetate elutes more than 90% of the cyclic AMP. Under these conditions, polyvalent nucleotides (AMP, ADP, and ATP) remain adsorbed to the alumina. The method permits the measurement of adenylylcyclase activity using [³H]ATP as the labeled substrate. The same technique can be used to measure the accumulation of cyclic AMP in intact cells after labeling the ATP pool with [³H]adenine. With slight modification, the technique can be used to measure the activity of cyclic-AMP phosphodiesterase using [³H]cyclic AMP as the substrate. The proposed technique provides rapid, highly reproducible assays using inexpensive, disposable columns.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.
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However, several properties may bias the results, and assay conditions need to be carefully determined. The cell-free adenylyl cyclase assay described here follows established procedures, and separation of products is done according to Alvarez and Daniels (1992) with some modification. Accumulated cAMP is separated from ATP by batch chromatography using ion exchange or alumina columns.
The mammalian sperm flagellum is an example of organelles where sensory and signaling functions are integrated with motility. Structurally related to other ciliary appendages, it has unique cytoskeletal structures that serve to assemble signaling complexes as well as components of metabolic pathways. Flagellar motility is regulated by signaling events that control sperm ion milieu, energy production, and classical second messenger-dependent phosphorylation cascades. Here, we will concentrate on the cAMP signaling pathway associated with flagellar motility. We will describe methods to analyze the properties of a unique adenylyl cyclase termed sAC (gene name Sacy, Adcy10), which plays an essential function in cAMP accumulation in spermatozoa. This soluble adenylyl cyclase (sAC) is a sensor for bicarbonate, pH, and Ca^2 + and is likely involved in coupling energy homeostasis with signaling events. We will also describe methods to investigate the macromolecular complexes that bring together sAC and other signaling molecules.
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The effects of the antidepressant drugs clomipramine (CLOM), desipramine (DMI), tianeptine (TIAN) and of norfluoxetine (NORF, the active metabolite of fluoxetine), were investigated in CHO cells expressing human β₂ adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene to determine their actions on cyclic AMP-driven gene transcription.
After 18 h of exposure, CLOM, DMI and NORF, but not TIAN, had biphasic effects on 1 μM isoprenaline-stimulated SPAP fsproduction with concentrations between 10 nM and 1 μM enhancing the maximal (E_max) SPAP response, without changing EC₅₀ values, but higher concentrations produced marked inhibitory effects.
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Citation Excerpt :
Thus, any increase in intracellular cyclic AMP should provoke the appearance of SPAP in the extracellular medium. Cyclic AMP assays were conducted according to the single column separation method reported by Alvarez and Daniels [16] using cells grown to confluency on 24-well cluster dishes. Where required for 18 h pre-incubation, 20 μM DMI was applied to the cells at the same time as [3H]-adenine (1 μCi per well) for overnight labelling.
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The results suggest a molecular target for DMI that lies downstream of CREB phosphorylation. Whether the inhibitory action of DMI is common to naturally expressed CRE-driven genes involved in adaptive responses to antidepressants in vivo remains to be determined.

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A separation method for the assay of adenylylcyclase, intracellular cyclic AMP, and cyclic-AMP phosphodiesterase using tritium-labeled substrates

Abstract

Anal. Biochem

Anal. Biochem

Anal. Biochem

Anal. Biochem

Anal. Biochem