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http://hdl.handle.net/10872/13989
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Título : | A NOVEL PDE1A COUPLED TO M2AChR AT PLASMA MEMBRANES FROM BOVINE TRACHEAL SMOOTH MUSCLE |
Autor : | Mastromatteo, Patrizzia Placeres, Fabiola Alfonzo-González, Marcelo Alfonzo, Ramona Lippo de Becemberg, Itala Alfonzo R., Marcelo J. |
Palabras clave : | Muscarinic antagonists asthma airway smooth muscle m2AChR |
Fecha de publicación : | Jan-2009 |
Editorial : | Journal of Receptors and Signal Transduction |
Resumen : | 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 M2AChR
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 [3H]cGMP and [3H]cAMP exhibiting a higher affinity as
Km (μM) for cGMP than cAMP but being close values with Vmax 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 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 M2AChR. A
model of this novel signal transducing cascade for the regulation of cyclic nucleotides
levels at BTSM is proposed. |
URI : | http://hdl.handle.net/10872/13989 |
Aparece en las colecciones: | Artículos Publicados
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