A Novel Pde1a Coupled To M(2)Achr At Plasma Membranes From Bovine Tracheal Smooth Muscle

Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M(2)AChR pharmacological profile. Therefore, a novel Ca2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58 kDa) being the 58 kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [H-3]cGMP and [H-3] cAMP exhibiting a higher affinity as K-m (mu M) for cGMP than cAMP but being close values with V-max cAMP/cGMP ratio of 1.5. The co-factor Mg2+ showed similar K-(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC50 of 4.9 and 4.6 mu M) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K-(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M(2)AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed.