Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle

The changes in protein phosphorylation associated with bovine tracheal smooth muscle contraction were studied by labeling intact muscle strips with [32P]PO4(3-) and analyzing the phosphoproteins by two-dimensional gel electrophoresis. Among 20 to 30 phosphoproteins resolvable with the two-dimensional electrophoresis system, the phosphorylation of 12 proteins was reproducibly affected by treatment with carbachol, in a time-dependent manner. Five of these proteins have been identified as 20-kDa myosin light chain, caldesmon, synemin, and two isoelectric variants of desmin. The other 7 are low molecular weight (Mr less than 40,000) cytosolic proteins. One cytosolic protein and myosin light chain are quickly but transiently phosphorylated by carbachol, the peak of myosin light chain phosphorylation being at about 1 min after agonist addition. In contrast, both variants of desmin, synemin, caldesmon, and 5 cytosolic proteins are phosphorylated at varying rates and remain phosphorylated for the duration of carbachol action. These "late" phosphorylation changes occur simultaneously with the dephosphorylation of one cytosolic protein. These carbachol-induced phosphorylation changes, like the contractile response, appear to be calcium-dependent. The addition of 12-deoxyphorbol 13-isobutyrate, a protein kinase C activator, causes a dose-dependent, sustained contraction of tracheal smooth muscle which develops more slowly than that induced by carbachol. This contractile response is associated with the same protein phosphorylation changes as those observed after prolonged carbachol treatment. In contrast, forskolin, an adenylate cyclase activator and a potent smooth muscle relaxant, induces the phosphorylation protein 3 and one variant of desmin. These observations strongly suggest that different phosphoproteins may be mediators of tension development and tension maintenance in agonist-induced contraction of tracheal smooth muscle.     

Actomyosin isolated from bovine tracheal smooth muscle.

A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of "classical" procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP, and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.     

Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins.

1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.     

Phosphoprotein phosphatase in bovine tracheal smooth muscle. Multiple fractions and multiple substrates.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) from bovine tracheal smooth muscle extracts was isolated and its activity determined using two [32P]phosphorylated proteins as substrates, i.e. phosphorylated histone (H-P) and a phosphorylated muscle specific substrate protein (MS-P) for the tracheal smooth muscle protein kinase. The enzyme was purified by the use of DEAE-cellulose followed by a two stage chromatography on a histone-Sepharose affinity column. Elution from the affinity column resolved the phosphoprotein phosphatase into four activity fractions. While fractions expressed phosphatase activity against both tested substrates the relative amounts of either activity varied. The ratio of activity towards H-P to activity towards MS-P changed from 11.5 to 0.12. The characterization of four phosphoprotein phosphatase fractions was based on the differences found in the following parameters: substrate specificity; sensitivity to NaF; influences of nucleotides (ATP, 5'-AMP, cyclic AMP, cyclic GMP) and the requirement of Mn2+ for maximal activity. Mg2+, Ba2+ or Ca2+ could not substitute for Mn2+.     

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.     

Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle

We describe thebiochemical properties of an eicosanoid-modulated Clchannel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its openprobability (P_o). After a purification protocolinvolving wheat-germ agglutinin affinity and anion-exchangechromatography, the proteins were sequentially inserted into liposomes,which were then fused into PLBs. Functional and biochemicalcharacterization tests confirm that the Cl channel is a55-kDa glycosylated monomer with voltage- and Ca²⁺concentration-independent activity. 5,6- and 8,9-EET decreased theconductance of the native channel (control conductance: 70 ± 5 pSin asymmetrical 50 mM trans/250 mM cis CsCl) in aconcentration-dependent manner, with respective 50% inhibitoryconcentration values of 0.31 and 0.42 µM. These regioisomerssimilarly decreased the conductance of the purified channel (controlconductance value: 75 ± 5 pS in asymmetrical 50 mMtrans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P_o of the nativechannel with respective 50% inhibitory concentration values of 0.27 and 0.30 µM but failed to alter the P_o of thepurified protein. Thus we suggest that the effects of these EETs onchannel conductance likely result from direct interactions ofEET anions with the channel pore, whereas the alterationof P_o requires a lipid environment of specificcomposition that is lost on solubilization and purification of the protein.

     

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.     

Characterization of a G protein-coupled guanylyl cyclase-B receptor from bovine tracheal smooth muscle

A G protein-coupled natriuretic peptide-guanylyl cyclase receptor-B (NPR-B) located in plasma membranes from bovine tracheal smooth muscle shows complex kinetics and regulation. NPR-B was activated by natriuretic peptides (CNP-53 > ANP-28) at the ligand extracellular domain, stimulated by Gq-protein activators, such as mastoparan, and inhibited by Gi-sensitive chloride, interacting at the juxtamembrane domain. The kinase homology domain was evaluated by the ATP inhibition of Mn2+-activated NPR-B, which was partially reversed by mastoparan. The catalytic domain was studied by kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogues with modifications of the /gamma phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent solubilization but the mastoparan activation and chloride inhibition of NPR-B disappeared. Our results indicate that NPR-B is a highly regulated nano-machinery with domains acting at cross-talk points with other signal transducing cascades initiated by G protein-coupled receptors and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP, and GTP.     

Parasympathetic innervation of canine tracheal smooth muscle

To investigate whether efferent parasympathetic fibers to the trachealsmooth muscle course through the pararecurrent nerve rather than therecurrent or the superior laryngeal nerve, we stimulated all threenerves in anesthetized dogs. We also recorded the pararecurrentnerve activity response to bronchoconstrictor stimuli and compared itwith pressure changes inside a saline-filled cuff of an endotrachealtube. Electrical stimulation (30 s, 100 Hz, 0.1 ms, 10 mA) increasedtracheal cuff pressure by 21.0 ± 3.2 and 1.3 ± 0.7 cmH₂O for the pararecurrent and the recurrent laryngealnerve, respectively. Stimulation of the superior laryngeal nerveincreased tracheal cuff pressure before, but not after, sectioning ofthe ramus anastomoticus, which connects it to the pararecurrent nerve.Intravenous administration of sodium cyanide increased pararecurrentnerve activity by 208 ± 51% and tracheal cuff pressure by14.4 ± 3.5 cmH₂O. Elevation of end-tidalPCO₂ to 50 Torr increased pararecurrent nerveactivity by 49 ± 19% and tracheal cuff pressure by 8.4 ± 3.6 cmH₂O. Further elevation to 60 Torr increasedpararecurrent nerve activity by 101 ± 33% and tracheal cuffpressure by 11.3 ± 2.9 cmH₂O. These results lead usto the conclusion that parasympathetic efferent fibers reach the smoothmuscle of the canine trachea via the pararecurrent nerve.

     

Temperature dependence of tracheal smooth muscle response to isoproterenol.

An inverse relationship between temperature and the negative inotropic response of tracheal smooth muscle segments to isoproterenol was demonstrated at resting tension and following precontraction with histamine. In addition, the β-blocking effect of propranolol was more pronounced at higher temperatures. These findings suggest that in asthmatic patients fever might be one of the factors which contribute to diminished responsiveness of sympathomimetic bronchodilators.     

Electrical behavior of guinea pig tracheal smooth muscle

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 +/- 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca(2+) channels with nifedipine or by depleting intracellular Ca(2+) stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 microM). In quiescent cells, nifedipine (1 microM) reduced EJPs by 30%, whereas ryanodine (10 microM) reduced EJPs by 93%. These results suggest that both the release of Ca(2+) from intracellular stores and the influx of Ca(2+) through voltage-dependent Ca(2+) channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.     

Evidence against phosphorylation of a cyclic AMP-dependent protein kinase from bovine tracheobronchial smooth muscle.

The data presented in this report are evidence against the autophosphorylation of the cyclic AMP-dependent protein kinase isolated from bovine tracheobronchial smooth muscle. This suggests that there may be a fundamental difference in the regulation in vivo of the protein kinases from bovine heart and tracheobronchial smooth muscle.     

Cooling-induced contraction of guinea pig tracheal smooth muscle

Cooling of isolated guinea pig tracheal smooth muscle from 38 to 28 degrees C over 2.25 min produced a transient contraction followed by sustained relaxation. The cooling-induced contraction was blocked either by pretreatment with ouabain at concentrations of 10(-5) M or greater or by substitution of normal physiological salt solution with K-free solution. In contrast, the contractile response to cooling was not inhibited by pretreatment with phentolamine (10(-5) M), atropine (10(-5) M), tetrodotoxin (3 X 10(-7) M), diphenhydramine (10(-5) M), cromolyn sodium (10(-3) M), indomethacin (3 X 10(-7) M), nifedipine (10(-7) M), or verapamil (3 X 10(-6) M). Addition of NaHCO3 to the bath during cooling, preventing a change in pH of the physiological salt solution, did not affect the cooling-induced contraction. It is concluded that cooling of isolated guinea pig trachea produces a transient ouabain-sensitive contraction, and that the data suggest the contraction is mediated by inhibition of Na-K-ATPase in the smooth muscle rather than through neuronal stimulation or chemical mediator release.