A novel PDE1A coupled to M2AChR at plasma membranes from bovine tracheal smooth muscle.
J Recept Signal Transduct Res
; 36(3): 278-87, 2016.
Article
em En
| MEDLINE
| ID: mdl-26513204
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Traqueia
/
Membrana Celular
/
Receptor Muscarínico M2
/
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1
/
Músculo Liso
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Recept Signal Transduct Res
Assunto da revista:
BIOQUIMICA
/
FISIOLOGIA
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Venezuela
País de publicação:
Reino Unido