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MOLECULAR PHARMACOLOGY 52:491-498 (1997).

Persistent Activation by and Receptor Reserve for an Irreversible A₁-Adenosine Receptor Agonist in DDT₁ MF-2 Cells and in Guinea Pig Heart

Jiahui Zhang, Luiz Belardinelli, Kenneth A. Jacobson, Deborah H. Otero, and Stephen P. Baker

Departments of Pharmacology (J.Z., L.B., D.H.O., S.P.B) and Medicine (L.B.), University of Florida, Gainesville, Florida 32610, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (K.A.J.).

The p- and m-isothiocyanate adenosine derivatives N⁶-[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 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 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 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 [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.