title: A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle.
abstract: 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 Ca(2+)/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 [(3)H]cGMP and [(3)H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with V(max) cAMP/cGMP ratio of 1.5. The co-factor Mg(2+) showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC(50) of 4.9 and 4.6 μM) 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.
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Patrizzia
authors.initials: P
authors.lastname: Mastromatteo-Alberga
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Placeres-Uray
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Alfonzo-González
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Ramona Gonzalez de
authors.initials: RG
authors.lastname: Alfonzo
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Itala Lippo de
authors.initials: IL
authors.lastname: Becemberg
authors.affiliation: a Sección de Biomembranas, Facultad de Medicina, Universidad Central de Venezuela, Instituto de Medicina Experimental , Caracas , Venezuela.
authors.forename: Marcelo J
authors.initials: MJ
authors.lastname: Alfonzo
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007038:Hypotonic Solutions; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014132:Trachea / Q000378:metabolism*; D014748:Vinca Alkaloids / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D007038:Hypotonic Solutions; D043585:Receptor, Muscarinic M2; D014748:Vinca Alkaloids; C013983:vinpocetine; D001285:Atropine; D000242:Cyclic AMP; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D006152:Cyclic GMP
keywords: Airway smooth muscle; asthma; m2AChR; muscarinic antagonists