Persistent activation by and receptor reserve for an irreversible A1-adenosine receptor agonist in DDT1 MF-2 cells and in guinea pig heart.

The p- and m-isothiocyanate adenosine derivatives N6-[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl ]am ino]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 for N6-cyclopentyladenosine. Once established, the p-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 microM), followed by washing, reduced [3H]CPX binding to the A1-AdoR by 44% without altering the Kd value for the radioligand to the remaining receptors. The relationship between irreversible A1-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 microM) prolonged the stimulus to His bundle (SH) interval by 2.1-fold; this response could be prevented by the antagonist 8-cyclopentyltheophylline (5 microM). 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 after m-DITC-ADAC treatment and washing was reduced by 35%. The A1-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 A1-AdoR and may prove to be useful probes to further investigate A1-AdoR structure-function relationships.