Persistent Activation by and Receptor Reserve for an Irreversible A₁-Adenosine Receptor Agonist in DDT₁MF-2 Cells and in Guinea Pig Heart

Abstract

SUMMARYThe p-and m-isothiocyanate adenosine derivatives N 6 - [4-[[[4-[[[[2-[[[(p-(m value for the radioligand to the remaining receptors. The relationship between irreversible A 1 -AdoR occupancy by p-DITC-ADAC and inhibition of cAMP accumulation revealed a relatively large receptor reserve (64%) for the maximal response. In guinea pig isolated hearts, m-DITC-ADAC (5 μM) prolonged the stimulus to His bundle (SH) interval by 2.1-fold; this response could be prevented by the antagonist 8-cyclopentyltheophylline (5 μM). However, after the SH interval prolongation was established, extensive washout or the addition of 8-cyclopentyltheophylline had little reversal effect on the m-DITC-ADAC response. Binding of [ 3 H]CPX to the guinea pig ventricular membranes after m-DITC-ADAC treatment and washing was reduced by 35%. The A 1 -AdoR occupancy response relationship for m-DITC-ADAC to prolong the SH interval indicated a small (10-20%) receptor reserve. Both p -and m-DITC-ADAC seem to be irreversible full agonists at the A 1 -AdoR and may prove to be useful probes to further investigate A 1 -AdoR structure-function relationships.Adenosine is a ubiquitous modulator of physiological processes that acts to both increase tissue oxygen supply and reduce metabolic demand. The actions of adenosine are mediated The AdoR regulates the activity of several cellular components, including inhibition of adenylyl cyclase activity, stimulation of phospholipases A 2 and C, and guanylyl cyclase, and regulates the ion permeability of calcium and potassium channels (2-4). The A 2 -AdoRs, on the other hand, mediate stimulation of adenylyl cyclase activity (1, 2). Although less is known about the A 3 -AdoR, it has been reported that this AdoR subtype mediates an inhibition of adenylyl cyclase and a stimulation of phospholipase C, resulting in an elevation of inositol phosphates (5, 6). The characterization, function, and regulation of the AdoRs have been greatly facilitated by the development of potent and selective agonists and antagonists (1,7,8). HHS Public AccessChemoreactive ligands are useful pharmacological probes in structure and function studies of receptors (9). These ligands are usually composed of two components: a pharmacophore and a reactive moiety. The pharmacophore is usually derived from known agonists or antagonists, which provide affinity and selectivity for the receptor. The reactive moiety allows covalent incorporation of the ligand into the receptor, which prevents ligand dissociation (9). These ligands have been used in a variety of studies, including determination of receptor reserve (10, 11), receptor subtype discrimination (12, 13), basal receptor turnover (14, 15), and ligand binding site mapping (16,17).Jacobson et al. (18) have synthesized a series of potential irreversible ligands for the adenosine receptor by modifying the high affinity agonist pharmacophore ADAC with substituents containing reactive electrophilic groups. Coupling of ADAC with 1,3-or 1,4-phenylene DITC produced p-and m-DITC-ADAC, which were sho...

Chemical Probes for the Adenosine Receptors

“…Concerning the A 1 AR subtype, examples of covalent agents are reported and summarized in Figure 4 [40][41][42].…”

“…In particular the N 6 substituted derivative of adenosine, named m-DITC-ADAC (4-isothiocyanatophenylaminothiocarbonyl, DITC; adenosine amine congener or (11), proved to be a good irreversible agonist for A 1 AR. It was able to mimic ischemic preconditioning in rabbits [40] and also prolong the stimulus of the His bundle (SA) interval by 2.1 fold in guinea pig isolated hearts [41].…”

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

“…Previous studies have shown that A 1 AR‐mediated inhibition of cAMP accumulation in DDT cells is dominant over β 2 AR stimulation 3. 22 As these bivalent compounds have higher affinity and functional potencies for the A 1 AR over the β 2 AR, the lack of β 2 AR stimulation in the absence of DPCPX or with G protein‐uncoupled A 1 AR is likely due to their preferential binding to and activation of the inhibitory A 1 AR. Interestingly, all of the active bivalent compounds were full agonists at the A 1 AR, but partial agonists at the β 2 AR.…”

“…Receptor binding assays : The preparation of DDT 1 MF‐2 cell membranes and the ability of the test compounds to displace [ 3 H]‐8‐cyclopentyl‐1,3‐dipropylxanthine from the A 1 AR were determined as previously described 22. The ability of the test compounds to displace [ 125 I]‐(−)‐iodopindolol from the β 2 AR in the DDT cell membranes was determined as previously described 3.…”

Electrochemistry and Voltammetric Determination of Adenosine with N‐Hexylpyridinium Hexafluorophosphate Modified Electrode

“…It modulates physiological functions in heart and brain, regulates oxygen supply during cell stress, and is useful in the regulation renal function [2,3]. The amounts of adenosine in the urine and plasma samples can also be the marker of some diseases such as carcinoma or liver diseases [4].…”