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 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.
Asunto(s)
Membrana Celular/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Tráquea/metabolismo , Animales , Atropina/farmacología , Western Blotting , Calmodulina/metabolismo , Bovinos , Membrana Celular/efectos de los fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Soluciones Hipotónicas , Concentración 50 Inhibidora , Cinética , Músculo Liso/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Alcaloides de la Vinca/farmacologíaRESUMEN
Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.