RESUMO
Muscarinic activation of bovine tracheal smooth muscle (BTSM) is involved in cyclic guanosine monophosphate (cGMP) production mediated through soluble (sGC) and membrane-bound (mGC) guanylyl cyclases. A muscarinic- and NaCl-sensitive mGC exists in BTSM regulated by muscarinic receptors coupled to G proteins. To identify the mGCs expressed in BTSM, reverse transcriptase/polymerase chain reaction (RT-PCR) from total RNA was performed using degenerate oligonucleotides for amplification of a region conserved among GC catalytic domains. Cloning of amplification products revealed that 76% of all BTSM GC transcripts corresponded to the sGC beta1 subunit and 24% to the B-type (C-type NP 1-22 [CNP]-sensitive) GC receptor. cGMP production by BTSM membrane and soluble fractions confirmed that sGC activity is 3-fold with respect to mGC activity. RT-PCR using specific oligonucleotides revealed that A (atrial NP-sensitive) and C (guanylin-sensitive) mGC subtypes are also expressed in BTSM. Stimulation of basal plasma membrane GC activity by CNP was higher than that by ANP, whereas guanylin showed no effect, indicating that CNP-sensitive guanylyl cyclase (GC-B) is the predominant functional BTSM mGC subtype. Strong adenosine triphosphate inhibition of CNP-stimulated mGC activity supports the finding that the tracheal mGC isoform belongs to the natriuretic peptide-sensitive mGCs. Additionally, CNP was able to reverse the chloride inhibition of BTSM mGC activity, suggesting that this is a novel G protein-coupled GC-B receptor.
Assuntos
Hormônios Gastrointestinais , Guanilato Ciclase/metabolismo , Músculo Liso/efeitos dos fármacos , Peptídeo Natriurético Tipo C/farmacologia , Traqueia/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , GMP Cíclico/fisiologia , Primers do DNA , Ativação Enzimática , Guanilato Ciclase/química , Guanilato Ciclase/genética , Dados de Sequência Molecular , Músculo Liso/enzimologia , Peptídeos Natriuréticos , Peptídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Frações Subcelulares/enzimologia , Traqueia/enzimologiaRESUMO
Membrane-bound guanylyl cyclase (GC) is regulated by muscarinic receptors (mAChRs). Carbamylcholine (CC) induces a "dual" biological response on GC activity. Thus, an activation is observed at 0.1 nM and a maximal response at 1 nM CC. However, at higher agonist concentration (> 100 nM), there is an agonist-dependent inhibition of GC. This CC dual response is affected by 4-DAMP and HDD (M3 antagonists), which produce a right-shift of the CC curve; the maximal CC dose response with 4-DAMP is more potent than that with HDD. Moreover, AFDX-DS (an M2 antagonist) increases basal activity and decreases the agonist-dependent inhibition. Neither the CC response nor the CC maximal dose responses are affected by pirenzepine (PZ, M1 antagonist). The agonist-dependent stimulation of GC activity is inhibited by 4-DAMP showing a -log IC50 = 8.4 +/- 0.4, while AFDX116 DS poorly inhibits such activity with a -log IC50 = 5.0 +/- 0.2. The agonist-independent (basal) GC activity also was inhibited by 4-DAMP, in a dose-dependent manner, with an IC50 = 8.5 +/- 0.2. Nonetheless, other muscarinic antagonists (PZ and HDD) were not able to inhibit this basal GC. Pertussis toxin treatment produces a complete blockade of the agonist-dependent inhibition of GC with a full expression of the agonist-dependent activation of membrane-bound GC. These results indicate that membrane-bound GC is regulated by muscarinic agents through two opposite signaling pathways; one involves the activation of GC via an M3 mAchR coupled to a PTX-insensitive G protein, while the GC inhibition is mediated through a PTX-sensitive Gi/o protein possibly coupled to an M2 mAChR.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanilato Ciclase/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Carbacol/farmacologia , Bovinos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Músculo Liso/citologia , Músculo Liso/metabolismo , Toxina Pertussis , Piperidinas/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais , Traqueia/citologia , Traqueia/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
In this work, we show evidence to support the existence of a Ca2+ calmodulin (CAM) dependent protein kinase and a substrate, a 17 kilodaltons (KDA) polypeptide being both associated to sarco(endo)plasmic reticulum vesicles from tracheal smooth muscle. Anti-CAM drugs such as compound 48/80 inhibited this protein kinase activity and this inhibition was reversed in the presence of Ca2+CAM. Moreover, as a result of this phosphorylation, there is a significant increase in the ATP dependent Ca2+ transport in these sarco(endo)plasmic vesicles.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Retículo Endoplasmático/enzimologia , Transporte de Íons , Músculo Liso/enzimologia , Fosforilação , Retículo Sarcoplasmático/enzimologia , Frações Subcelulares/enzimologia , Especificidade por SubstratoRESUMO
Protons generated inside the cells during metabolic activity have to be extruded through active mechanisms from the intracellular to the extracellular space. One of the systems involved in proton transport across membranes are the V-ATPases, which are oligomeric complexes that have been found in several subcellular organelles energizing such organelle through a proton gradient and a membrane potential. In this paper, a V-ATPase activity has been described at the plasma membranes fractions isolated from airway smooth muscle. This activity was measured as a Cl- stimulated Mg2+ ATPase. This Cl- activating effect was also shared by others halogens as I- and Br- but not F-. This Cl- stimulated ATPase is a nucleotide triphosphatase being unable to hydrolyze mono and dinucleotides. The divalent cations showed the following sequence of activation (Mg2+ > Mn2+ > Ca2+) of the Cl- activated Mg2+ ATPase. This Cl- stimulated Mg2+ ATPase was insensitive to ouabain, vanadate, sodium azide and rutamicina. NEM (N-ethylmaleimide) partially inhibited this activity but a complete inhibition was observed with p-CMB (p-chloromercurbenzoate ). Several specific proton transport inhibitors were employed to show the presence of a H+ pump activity. Thus, the strong inhibition induced by DCCD suggest the existence of hydrophobic subunits related to a proton channel. In addition, protonophores as 1799 and FCCP stimulated the Cl- stimulated ATPase indicating the presence of a H+ pump in these plasma membranes vesicles. The chloride requirement could be explained by the existence of a chloride conductor coupled to the proton pump (H+ ATPase-type V) due to the inhibitory effect of duramycin.(ABSTRACT TRUNCATED AT 250 WORDS)