Vol. 300, Issue 3, 910-917, March 2002

Functional Characterization of Adenosine Receptors and Coupling to ATP-Sensitive K⁺ Channels in Guinea Pig Urinary Bladder Smooth Muscle

Sujatha M. Gopalakrishnan, Steven A. Buckner, Ivan Milicic, Duncan R. Groebe, Kristi L. Whiteaker, David J. Burns, Usha Warrior and Murali Gopalakrishnan

Advanced Technology (S.M.G., D.G., D.J.B., U.W.) and Neuroscience Research (S.A.B., I.M., K.L.W., M.G.), Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois

Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (K_ATP) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the K_ATP channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5'-N-ethyl-carboxamido adenosine (NECA; log EC₅₀ = 7.97) ~ 2-p-(2-carboxethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) ~ 2-chloro-N⁶-cyclopentyladenosine (CCPA; 5.51) ~ N⁶-cyclopentyladenosine ~ N⁶-(R)-phenylisopropyladenosine > 2-chloro- N⁶-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A_2A-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K⁺-stimulated guinea pig bladder strips, NECA (log EC₅₀ = 6.41) ~ CGS-21680 (6.38) > 2-chloro adenosine (5.90) CCPA ~ 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A_2A receptor-mediated activation of K_ATP channels via adenylate cyclase and elevation of cAMP.