The mechanism of contraction by 2-chloroadenosine in cat detrusor muscle cells

Purpose: Four adenosine receptors (ARs), designated A(1)AR (A, adenosine receptor), A(2a)AR (A(2a) adenosine receptor), A(2b)AR (A(2b) adenosine receptor), and A(3)AR (A(3) adenosine receptor), have been cloned from various species, but the contraction mechanism via A(1)ARs in cat; detrusor muscle cell is not well known. Materials and Methods: We examined the cellular mechanism using an A(1)AR agonist 2-chloroadenosine (2-CA) in cat detrusor cell isolated by enzymatic digestion. To examine which phospholipase mediates the contraction, we used phospholipase inhibitors. Results: The adenosine analog potency order is R-N(6)-phenylisopropyladenosine (R-PLA) > 5'-N-ethylcarbosamine adenosine (NE CA) > 2-chloroadenosine (2-CA) > S-N(6)-phenylisopropyladenosine (S-PIA). The ratio of equi-effective concentrations of R-PIA/S-PIA was 58.2. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM) shifted to the right the concentration-response curves of 2-CA. These results indicate A(1)ARs mediate 2-CA induced contraction in cat detrusor muscle. G proteins (G(i1), G(i2), G(i3), G(o), G(s), and G(q)) in cat detrusor muscle were detected by immunoblot analysis. Pertussis toxin (PTX) inhibited 2-CA induced contraction. In permeabilized cells, antibodies against G(alpha i3) antagonized 2-CA induced contraction, suggesting that the contraction is mediated by G(i3) protein. A phosphatidylinositol-specific phospholipase C (PLC) inhibitor, neomycin, reduced 2-CA induced contraction, but a phospholipase D (PLD) inhibitor, rho-chloromercuribenzoic acid, and a phospholipase A, (PLA(2)) inhibitor, dimethyl-eicosa-dienoic acid (DEDA), had no effect. we found the presence of the main PLC isozymes, PLC-beta 1, PLC-beta 3, and PLC-gamma 1. 2-CA induced contraction in permeabilized cells was inhibited by PLC-beta 3 but not by PLC-beta 1 or PLC-gamma 1 antibody. These results imply that A(1)ARs are coupled to PLC-beta 3 via PTX-sensitive G(i3) protein. Sr(2+) medium and thapsigargin, which replaces intracellular Ca and deplete intracellular calcium stores respectively, inhibited 2-CA induced contraction. Conclusions: These results suggest that A(1)ARs mediating 2-CA induced contraction exist in cat detrusor muscle and the contraction depends on a PTX-sensitive G(i3) protein, PLC-beta 3 and the release of intracellular Ca.(2+).