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...

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Several carbostyril-based beta-agonists have been shown to bind tightly to and slowly dissociate from the beta2-adrenoceptor (beta2AR). In the present study, the structural features of 8-hydroxy-5-[2-[(1-phenyl-2-methylprop-2-yl)amino]-1-hydroxyethyl] -carbostyril (11a) which contribute to its binding properties at the beta2AR were investigated using a series of synthesized analogs. The k(off), estimated by the rate of cAMP decline in DDT1 MF-2 (DDT) cells with a reduced receptor density, Ki and ligand-induced receptor reductions were determined. All of the derivatives stimulated cAMP accumulation in DDT cells in the sub to mid nanomolar range and elicited the same maximal stimulation as (-)isoproterenol. Derivatives of 11a with side chain N-substitutions comprising 2-methylbutyl, phenylethyl and isopropyl had higher k(off)-values and lower affinities as compared to 11a. Increasing the number of methylenes between the side chain tertiary alpha carbon and phenyl from 1 in 11a to 3 or reducing the number to 0 also resulted in derivatives with higher k(off)- and Ki-values. In addition, replacement of the 8-hydroxycarbostyril nucleus of 11a with catechol reduced the affinity of the compound for the beta2AR by 48-fold and increased its k(off). Only those derivatives with the lowest k(off)-values induced a decrease in the receptor density of DDT cell membranes following a preincubation and extensive washing. The data show that the 8-hydroxycarbostyril nucleus in conjunction with substitutions on the tertiary alpha carbon of the side chain and positioning of the phenyl group are important characteristics determining the high affinity and slow dissociation of 11a from the beta2AR.