In rabbit arteries, acetylcholine (ACh) stimulates an endothelial hyperpolarization that is conducted into the media via myoendothelial gap junctions to reduce contractile tone. E ndogenous production of cAMP facilitates electrotonic signalling via gap junctions, so that P-site inhibitors of adenylyl cyclase such as dideoxyadenosine (ddA) attenuate EDHF-type smooth muscle hyperpolarizations and relaxation (Griffith et al., 2002). Since the addition of phosphate groups to ddA enhances its ability to inhibit adenylyl cyclase, we have investigated whether a lipophilic ‘masked phosphate’ phenyl methoxyalaninyl phosphoramidate derivative of 2',3'-ddA (2',3'-ddA-PMAPh) is a more potent inhibitor of the EDHF phenomenon than the parent nucleoside . The first step in the intracellular metabolism of 2',3'- ddA-PMAPh is a carboxylesterase mediated hydrolysis, followed by spontaneous elimination of phenol. Subsequent enzymatic cleavage of the phosphorus-nitrogen bond delivers the nucleotide monophosphate 2',3'- dd-5'- AMP directly, thereby bypassing conversion of 2',3'- ddA to 2',3'- dd-5'- AMP by adenosine kinase or via dideoxyinosine (Siddiqui et al., 1999 ) .

Arteries were obtained from male NZW rabbits (2-2.5kg) killed with sodium pentobarbitone (120mg/kg; i.v.) and iliac artery strips held adventitia down in an organ chamber superfused (2 ml/min at 37 °C) with oxygenated Holmans solution. All experiments were performed in the presence of 300 µM L-NAME and 10 µM indomethacin. The resting membrane potential of subintimal smooth muscle cells, measured by a sharp electrode intracellular technique, was -45.2 ±2.4 mV (n=20). Subintimal smooth muscle hyperpolarizations evoked by 3 µM ACh were 18.0 ±2.6 mV and were maximally attenuated by ~50% in the presence of 200-500 µM 2',3'-ddA, whereas 200 µM 2',3'-ddA-PMAPh effectively abolished smooth muscle hyperpolarization (n=4-8, P<0.05). The IC₅₀ value for 2',3'- ddA-PMAPh inhibition was correspondingly decreased by some 7-fold compared to 2',3'- ddA, being estimated as 12.6 mM (95% confidence limits 8.2-18.6) and 86.2 µM (95% confidence limits 72.1-102.6), respectively. Inhibition of ACh-evoked hyperpolarizations by 2',3'-ddA or 2',3'-ddA-PMAP (at 200 µM) was overcome in each case by the cAMP phosphodiesterase inhibitor IBMX (30 µM, n=4). Control experiments with the corresponding phosphoramidate derivative of adenosine (300 µM) failed to reveal inhibitory effects against ACh-evoked hyperpolarizations. In whole cell patch clamp studies the resting membrane potential of endothelial cells was -45.0 ±3.2 mV (n=6) and did not differ significantly from the corresponding subintimal smooth muscle membrane potential. Neither 200 µM 2',3'-ddA nor 200 µM 2',3'-ddA-PMAPh affected resting endothelial membrane potential or hyperpolarizations stimulated by 3 µM ACh.

In conclusion, we have shown that a phosphoramidate pronucleotide derivative of dideoxyadenosine, 2',3'- ddA-PMAPh, is a potent inhibitor of the EDHF phenomenon and is capable of abolishing smooth muscle hyperpolarizations without affecting the mechanisms that underpin endothelial hyperpolarization. Inhibition of hyperpolarization was linked to blockade of adenylyl cyclase as it was prevented by inhibition of cAMP hydrolysis with the cAMP phosphodiesterase inhibitor IBMX. Modifications in the lipophilicity of dideoxyadenosine and its direct intracellular delivery as a mononucleotide may thus enhance the ability to inhibit adenylyl cyclase and depress electrotonic signalling via myoendothelial gap junctions.

Griffith et al. (2002). Proc. Natl. Acad. Sci. U.S.A.,99, 6392-6397
Siddiqui et al. (1999). Bioorg. Med. Chem. Lett., 9, 2555-2560