Calcium-independent contraction and sensitization of airway smooth muscle by p21-activated protein kinase

2003 ◽  
Vol 284 (5) ◽  
pp. L863-L870 ◽  
Author(s):  
P. K. McFawn ◽  
L. Shen ◽  
S. G. Vincent ◽  
A. Mak ◽  
J. E. Van Eyk ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Force Production ◽  
Force Generation ◽  
Glutathione S Transferase ◽  
Tonic Smooth Muscle ◽  
P21 Activated Kinase ◽  
Incremental Increase ◽  
Constitutively Active

In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca2+-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433–23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca2+-independent contractions and/or Ca2+ sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca2+ (pCa > 9), constitutively active glutathione- S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to ∼40% of the maximal force generated by Ca2+ at pCa 4.4. In addition, GST-mPAK3 enhanced Ca2+ sensitivity of contraction by increasing force generation by 80% at intermediate Ca2+ concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3K297R had no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser657) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca2+-independent contractions and produces Ca2+ sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.

Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis

2000 ◽  
Vol 88 (4) ◽  
pp. 1338-1345 ◽  
Author(s):  
Pasquale Chitano ◽  
Jizhong Wang ◽  
Carrie M. Cox ◽  
Newman L. Stephens ◽  
Thomas M. Murphy
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Force Production ◽  
Internal Resistance ◽  
Force Generation ◽  
Stress Generation ◽  
Shortening Velocity ◽  
Smooth Muscle Function ◽  
Old Adult ◽  
Force Velocity

Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length ( l o) to obtain maximum active stress (Po) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load ( V o) and the value α ⋅ γ/β, an index of internal resistance to shortening (Rsi), were calculated (α, β, and γ are the constants of the equation). Poincreased little with maturation, whereas the rate of stress generation increased significantly (0.40 ± 0.03, 0.45 ± 0.03, 0.51 ± 0.03 P o/s for 1 wk, 3 wk, and adult animals). V o slightly increased early with maturation to decrease significantly later (1.79 ± 0.67, 2.45 ± 0.92, and 0.55 ± 0.09 l o/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 ± 5.19, 8.99 ± 3.01, and 32.07 ± 5.54 mN ⋅ mm−2 ⋅ l o −1 ⋅ s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V o with age. An elevated V o may contribute to the incidence of airway hyperresponsiveness in healthy juveniles.

scholarly journals β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent

2014 ◽  
Vol 289 (33) ◽  
pp. 23065-23074 ◽  
Author(s):  
Sarah J. Morgan ◽  
Deepak A. Deshpande ◽  
Brian C. Tiegs ◽  
Anna M. Misior ◽  
Huandong Yan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Airway Smooth Muscle ◽  
Kinase A

Qualitative immunoblot analysis of PKC isoforms expressed in airway smooth muscle

1997 ◽  
Vol 272 (4) ◽  
pp. L603-L607 ◽  
Author(s):  
H. Togashi ◽  
C. A. Hirshman ◽  
C. W. Emala
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Intracellular Signaling ◽  
Immunoblot Analysis ◽  
Calcium Dependent ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Dependent Activity ◽  
Trachealis Muscle

Protein kinase C (PKC) was originally identified as a single serine/ threonine protein kinase with calcium- and phospholipid-dependent activity, but more recently PKC has been found to consist of a family of multiple isoenzymes with different biochemical characteristics, substrates, and cofactor requirements. PKC is particularly important in regulating airway smooth muscle (ASM) tone. Although a previous investigation has demonstrated PKC-beta, -delta, -epsilon, -theta and -zeta in canine trachealis muscle, additional PKC isoforms have not been characterized in ASM. Therefore, immunoblot analysis using nine isotype-specific antibodies was used to further characterize the expression of PKC isoforms in porcine ASM. In addition to the previously described beta-, delta-, epsilon-, and zeta-isoforms in ASM, the calcium-dependent alpha-isoform, and the calcium- and diacylglycerol-independent isoforms iota/lambda and mu were identified. This study demonstrates multiple PKC isoforms in porcine ASM that can participate in intracellular signaling pathways in this tissue.

Protein kinase C is involved in enhanced airway smooth muscle cell growth in hyperresponsive rats

2000 ◽  
Vol 278 (1) ◽  
pp. L59-L67 ◽  
Author(s):  
M. E. Zacour ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Growth ◽  
Airway Smooth Muscle ◽  
Western Blotting ◽  
Growth Response ◽  
Airway Smooth Muscle Cell ◽  
Kinase C ◽  
Potential Risk Factors

Fischer rat airway smooth muscle (ASM) models two potential risk factors for asthma: hyperresponsiveness to contractile agonists and to growth stimuli. The aim of this study was to identify the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not be accounted for by phospholipase C, mitogen-activated protein kinases, or tyrosine kinase activities as assessed by pharmacological inhibition and Western blotting. In contrast, depletion of phorbol ester-sensitive isoforms of the serine/threonine kinase protein kinase C (PKC) removed the difference in growth response between the rat strains. Additionally, FBS selectively induced serine/threonine phosphorylation of a 115-kDa protein in Fischer ASM cells. Enhanced activation of PKC-βI and decreased activation of PKC-δ in Fischer compared with Lewis cells following FBS stimulation were suggested by Western blotting of membrane and cytosolic fractions. The data are consistent with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.

Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells

1997 ◽  
Vol 272 (5) ◽  
pp. L865-L871 ◽  
Author(s):  
B. Tolloczko ◽  
Y. L. Jia ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Airway Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
High Concentrations

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.

Selected Contribution: Mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells

2000 ◽  
Vol 89 (5) ◽  
pp. 2092-2098 ◽  
Author(s):  
Paul G. Smith ◽  
Chaity Roy ◽  
Steven Fisher ◽  
Qi-Quan Huang ◽  
Frank Brozovich
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Force Production ◽  
Muscle Cells ◽  
Calcium Sensitivity ◽  
Airway Smooth Muscle Cells ◽  
Maximal Force

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6 ± 0.4 in the strain cells compared with 5.9 ± 0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6 ± 2.9 vs. 5.7 ± 3.1 μN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.

Sign in / Sign up

Export Citation Format

Share Document