Persistent Activation by and Receptor Reserve for an Irreversible A1-Adenosine Receptor Agonist in DDT1 MF-2 Cells and in Guinea Pig Heart
- Departments of 1Pharmacology (J.Z., L.B., D.H.O., S.P.B) and2Medicine (L.B.), University of Florida, Gainesville, Florida 32610, and3Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (K.A.J.).
Abstract
The p- and m-isothiocyanate adenosine derivativesN6-[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 forN6-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 Kd 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.
Footnotes
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Send reprint requests to: Stephen P. Baker, Ph.D., University of Florida, College of Medicine, Department of Pharmacology and Therapeutics, P.O. Box 100267, Gainesville, FL 32610. E-mail:sbaker{at}college.med.ufl.edu
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This work was supported by a Grant-in-Aid from the American Heart Association, Florida Affiliate, and National Institutes for Health Grant HL35272.
- Abbreviations:
- AdoR
- adenosine receptor
- AV
- atrioventricular
- CPA
- N6-cyclopentyladenosine
- CPT
- 8-cyclopentyltheophylline
- CPX
- 8-cyclopentyl-1,3-dipropylxanthine
- DITC
- diisothiocyanate
- HBSS
- Hanks’ balanced salt solution
- NECA
- 5′-N-ethylcarboamidoadenosine
- SH
- stimulus-to-His bundle
- ADAC
- N6-[4-[[[4-[[[(2-aminoethyl)-amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]adenosine
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- Received February 7, 1997.
- Accepted May 28, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
