Abstract

The p- and m-isothiocyanate adenosine derivativesN ⁶-[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl]amino]ethyl]amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]adenosine (p- and m-DITC-ADAC) were examined for irreversible agonist effects at the A₁-adenosine receptor (A₁-AdoR) in DDT₁ MF-2 (DDT) cells and a functional A₁-AdoR response in the guinea pig isolated heart. The p- and m-DITC-ADAC inhibited (−)-isoproterenol stimulated cAMP accumulation in DDT cells in the low nanomolar range, and the maximal responses elicited by both compounds were similar to that forN ⁶-cyclopentyladenosine. Once established, thep-DITC-ADAC-mediated inhibition of cAMP accumulation in DDT cells was not affected by the addition of the AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX). Pretreatment of DDT cells with p-DITC-ADAC (1 μm), followed by washing, reduced [³H]CPX binding to the A₁-AdoR by 44% without altering the K_d value for the radioligand to the remaining receptors. The relationship between irreversible A₁-AdoR occupancy byp-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 [³H]CPX to the guinea pig ventricular membranes afterm-DITC-ADAC treatment and washing was reduced by 35%. The A₁-AdoR occupancy response relationship form-DITC-ADAC to prolong the SH interval indicated a small (10–20%) receptor reserve. Both p -andm-DITC-ADAC seem to be irreversible full agonists at the A₁-AdoR and may prove to be useful probes to further investigate A₁-AdoR structure-function relationships.