Department of Anesthesiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We determined whether activation of G
proteins can affect the force developed for a given intracellular
Ca2+ concentration ([Ca2+]; i.e., the
Ca2+ sensitivity) by mechanisms in addition to changes in
regulatory myosin light chain (rMLC) phosphorylation. Responses in
-toxin-permeabilized canine tracheal smooth muscle were determined
with Ca2+ alone or in the presence of ACh, endothelin-1
(ET-1), or aluminum fluoride (AlF
regulatory myosin light chain phosphorylation; aluminum fluoride; remodeling; airway inflammation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
STIMULATION OF SMOOTH MUSCLE by contractile agonists increases cytosolic Ca2+ concentration ([Ca2+]i). Ca2+ binds to calmodulin and increases myosin light chain kinase activity, regulatory myosin light chain (rMLC) phosphorylation, actomyosin cross-bridge cycling rate, and force (27). The relationship between [Ca2+]i and rMLC phosphorylation further depends on agonist-induced inhibition of smooth muscle protein phosphatase (SMPP) activity, which increases the amount of force produced for a given [Ca2+]i (i.e., the Ca2+ sensitivity; see Refs. 28 and 29). Membrane receptors that inhibit SMPP are coupled to these enzymes via a cascade of heterotrimeric and monomeric GTP-binding regulatory proteins (G proteins; see Ref. 40). Several studies have suggested that mechanisms in addition to rMLC phosphorylation may also regulate force in smooth muscle (18). Proposed mechanisms include those thought to regulate actomyosin cross-bridge interaction, such as the actin-associated proteins such as caldesmon and calponin (10, 44), and others such as remodeling of the smooth muscle cytoarchitecture to optimize the transduction of ATPase activity to force (15, 23, 31, 33, 41).
If these additional regulatory mechanisms are functionally important and can be activated by receptor agonists, then the relationship between rMLC phosphorylation and force at various [Ca2+]i levels in the presence and absence of receptor activation should differ. In a recent study, we showed that acute muscarinic stimulation did not alter this relationship in permeabilized canine tracheal smooth muscle (CTSM), providing no evidence for such mechanisms in this preparation (26). Given this result, we questioned whether there are any conditions where Ca2+ sensitivity can be regulated by rMLC-independent mechanisms mediated by G proteins in airway smooth muscle. We speculated that if such mechanisms exist, they may require chronic stimulation to become apparent. For example, any reorganization of the cytoskeleton may take some period of time to occur (23, 31). Such chronic changes may be relevant to clinical states such as asthma, in which CTSM may be chronically exposed to inflammatory mediators and other contractile stimuli (35, 37).
The purpose of the present study was to determine if chronic activation
of G proteins in CTSM, produced by either receptor activation or direct
stimulation with aluminum fluoride (AlF
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental techniques. After Institutional Animal Care and Use Committee approval, we anesthetized mongrel dogs (15-20 kg) of either sex with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated the dogs. The trachea was excised and immersed in chilled physiological salt solution (PSS) of the following composition (in mM): 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors under microscopic observation to make muscle strips of 0.1-0.2 mm width, 1 cm length, and 0.2-0.3 mg wet weight.
Isometric force measurements. Muscle strips were mounted in 0.1-ml cuvettes and continuously superfused at 1.2 ml/min with PSS (37°C) aerated with 94% O2-6% CO2. One end of the strips was anchored via stainless steel microforceps to a stationary metal rod, and the other end was connected via stainless steel microforceps to a calibrated force transducer (model KG4; Scientific Instruments, Heidelberg, Germany). The initial gap between microforceps was set at 5 mm. During a 3-h equilibration period, the length of the muscle strips was increased after repeated isometric contractions induced by 1 µM ACh until maximal isometric force (optimal length) was reached. Each muscle strip was maintained at this optimal length for the rest of the experiment. These tissues produced maximal isometric forces of 1-3 mN when stimulated with 1 µM ACh. After optimal length was set, subsequent experimental protocols were performed at room temperature (25°C) to minimize deterioration of the experimental preparation.
Permeabilization procedure.
CTSM strips were permeabilized with 2,500 U/ml Staphylococcus
aureus -toxin for 20 min at 25°C in relaxing solution
(28, 34). The composition of the relaxing solution was as
follows [using the algorithm of Fabiato and Fabiato
(11)]: 7.5 mM MgATP, 4 mM EGTA, 20 mM imidazole, 1 mM
dithiothreitol (DTT), 1 nM free Ca2+, 10 mM
phosphocreatine, and 0.1 mg/ml creatine phosphokinase. Ionic strength
was kept constant at 200 mM by adjusting the concentration of potassium
acetate. The pH was adjusted to 7.0 at 25°C with KOH or HCl. After
the permeabilization procedure, strips were washed with relaxing
solution without
-toxin for 10 min. The Ca2+ ionophore
A-23187 (10 µM) was added to the relaxing solution and all subsequent
experimental solutions to deplete the sarcoplasmic reticulum
Ca2+ stores and to maintain Ca2+ concentration
([Ca2+]). Solutions of varying free [Ca2+]
used in the subsequent experiment were also prepared using the algorithm of Fabiato and Fabiato (11). After
permeabilization, the strips were contracted repeatedly with 10 µM
Ca2+ until reproducible responses were obtained, as
previously described (26). For rMLC thiophosphorylation
studies, low-Ca2+ rigor solution contained (in mM) 85 K+, 0.01 free Mg2+, 4 EGTA, 20 imidazole, 1 DTT, and 1 nM free Ca2+. High-Ca2+ rigor
solutions contained (in mM) 85 K+, 0.1 free
Mg2+, 4 EGTA, 20 imidazole, 1 DTT, and 10 µM free
Ca2+.
rMLC phosphorylation measurements. Samples for rMLC phosphorylation measurements were prepared separately according to the same procedures as for force measurements but were incubated in wells (without determination of optimal length) instead of being superfused. The strips were pinned at both ends to maintain isometric conditions. After permeabilization, all conditions, including repeated contraction with 10 µM Ca2+, were identical to those present in the strips used to measure isometric force responses. At appropriate times in the experimental protocols, muscle strips were flash-frozen with dry ice-cooled acetone containing 10% (wt/vol) TCA and 10 mM DTT (17). Strips were then allowed to warm to room temperature in the same solution. After TCA was washed out with acetone containing 10 mM DTT, strips were allowed to dry. The dry weight of the strips was 0.07-0.13 mg. rMLC was extracted as described by Gunst et al. (14), and phosphorylation was determined by glycerol-urea gel electrophoresis followed by Western blotting as previously described (5). Unphosphorylated and phosphorylated bands of rMLC were visualized by phosphorimage analysis (Cyclone storage phosphor system; Packard Instrument, Downers Grove, IL), and fractional phosphorylation was calculated as the density ratio of the sum of mono- and diphosphorylated rMLC to total rMLC using OptiQuant software (version 3.0; Packard Instrument).
Materials. The polyclonal affinity-purified rabbit anti-20-kDa rMLC antibody was a generous gift from Dr. Susan J. Gunst (Dept. of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN). Synthetic endothelin-1 (ET-1) was obtained from Phoenix Pharmaceuticals (Mountain View, CA). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). A-23187 was dissolved in dimethyl sulfoxide (DMSO); in all experimental solutions, the final DMSO concentration did not exceed 0.1%. At this concentration, DMSO had no effect on isometric force or rMLC phosphorylation induced by free Ca2+ alone (data not shown). All other drugs and chemicals were prepared in nanopure filtered water.
Statistical analysis. Data are expressed as means ± SD; n is the number of dogs. Isometric force is expressed as a percentage of the maximal response to 10 µM Ca2+. Comparisons between means were performed with paired t-tests as appropriate. Parameters for concentration-response curves were calculated using nonlinear regression fit to a three-parameter Hill equation (Sigma Stat; Jandel Scientific, San Rafael, CA), and parameter coefficients were compared using paired t-tests. In all cases, a P value <0.05 was taken as significant.
We compared the relationship between rMLC phosphorylation and force in the presence and absence of chronic AlF ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of acute application of ACh, ET-1, or
AlF-toxin-permeabilized CTSM strips were stimulated with
increasing concentrations of free Ca2+ (1 nM to 100 µM).
In three strips, 100 µM ACh or 100 nM ET-1 or AlF
|
|
Effect of chronic application of ACh, ET-1, or
AlF-toxin-permeabilized strips were stimulated with increasing
concentrations of free Ca2+ (1 nM to 100 µM). Three
strips were exposed to 100 µM ACh or 100 nM ET-1 or
AlF
Effect of chronic AlF
|
|
Dependence of AlFS) inhibits G protein-mediated responses.
|
|
AlF-toxin, and the response to 10 µM
Ca2+ was determined. After exposure to low-Ca2+
rigor solution for 20 min to remove ATP, the strips were incubated with
a high-Ca2+ rigor solution containing 2.1 mM magnesium
adenosine 5'-O-(3-thiotriphosphate) for 20 min. Preliminary
experiments showed that this regimen produced >95% rMLC
thiophosphorylation (data not shown). The strips were then washed with
low-Ca2+ rigor solution for 20 min with and without
AlF
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major finding of this study in -toxin-permeabilized CTSM is
that chronic (1-h) but not acute exposure to AlF
We confirmed prior observations that the acute activation of G
protein-coupled receptors produces Ca2+ sensitization in
permeabilized airway smooth muscle, decreasing the EC50 for
[Ca2+] without significantly changing maximal force
(21). Prior studies suggest that receptor binding triggers
dissociation of a heterotrimeric G protein in airway smooth muscle
phosphorylation. These subunits in turn activate a monomeric G protein
that subsequently inhibits SMPPs and thus increases rMLC (20, 25,
40). Some evidence suggests that kinases activated by monomeric
G proteins, such as Rho-associated kinase, can also regulate
Ca2+ sensitivity produced by receptor activation by
directly phosphorylating rMLC (30), although evidence for
this mechanism is lacking in airway smooth muscle (20).
Recently, integrin-linked kinase has been shown to phosphorylate rMLC
in the absence of Ca2+ (9); its possible role
in agonist-induced Ca2+ sensitization is unknown. We found
that fluoroaluminate complexes (comprised mainly of
AlF-phosphate of GDP and mimic
the terminal
-phosphate of GTP (3). This eliminates the
normal requirement for GDP-GTP exchange to cause conformational change
and consequent activation of G proteins. The bound
AlF
In contrast to acute activation, chronic (1-h) activation of G proteins
with AlFS. As first noted
by Gong et al. (13) in rabbit portal vein, chronic
exposure of permeabilized smooth muscle to GTP
S inhibits G
protein-mediated responses, including responses to AlF
S exposure
in preliminary experiments (data not shown), although 6 h of
exposure were required for maximal inhibition. The ability of GTP
S
to abolish the effects of chronic AlF
We cannot explain why chronic exposure to AlF
According to the observed relationship between rMLC phosphorylation and
force, this increase in maximal force is produced by mechanisms in
addition to rMLC phosphorylation. It should be noted that, although the
strips used to measure rMLC phosphorylation were pinned at both ends to
maintain isometric conditions, optimal length was not determined in
these strips, in contrast to the strips used to measure force
responses. Even though all strips were otherwise treated in the same
manner after permeabilization, this represents a possible limitation to
the interpretation of our phosphorylation data. Indeed, in intact
(nonpermeabilized) airway smooth muscle, muscle length affects rMLC
phosphorylation via effects on intracellular [Ca2+]
(33). However, in preliminary data obtained for prior
studies (5, 17, 22, 26), we showed that muscle length does
not affect rMLC phosphorylation in permeabilized airway smooth muscle in which intracellular [Ca2+] is controlled. Thus this
factor should not influence our conclusions. We confirmed that these
mechanisms are independent of rMLC phosphorylation by showing that
chronic AlF
At least two classes of mechanisms may regulate force in smooth muscle independent of changes in rMLC phosphorylation. Some have proposed an independent regulatory system that is responsible for force maintenance after initial rapid force development (10, 44). Current candidates include the actin-associated proteins caldesmon and calponin, based on evidence obtained in several types of smooth muscle. A role for these proteins in vascular smooth muscle under some circumstances has been demonstrated (10, 44), although evidence for their functional role in airway smooth muscle is more limited. Both proteins are phosphorylated in response to muscarinic stimulation of intact CTSM (12, 38), although whether this can occur with stimulation under constant [Ca2+]i conditions in permeabilized preparations is not known. Others have suggested that the actin cytoskeleton may remodel in response to contractile stimuli to more efficiently transduce force from contractile elements to the cytoskeleton. Several different proteins involved with the actin cytoskeleton, including paxillin and focal adhesion kinase, are tyrosine phosphorylated during airway smooth muscle contraction (36) independent of changes in [Ca2+]i in the case of paxillin (32). We have recently shown that the kinetics of actin polymerization are also a potential regulatory mechanism in CTSM (23), and muscarinic control of actin polymerization has been demonstrated in cultured airway smooth muscle cells (42). Further studies will be necessary to determine which (if any) of these mechanisms might explain the increase in maximal force produced by chronic activation of heterotrimeric G proteins.
The possible physiological significance of these observations remains
to be determined. As discussed above, it is possible that activation of
G proteins not coupled to ACh or ET-1 by mediators such as leukotrienes
and cytokines produces these effects (1, 2). Parris et al.
(35) have shown that chronic tumor necrosis factor-
(TNF-
) exposure activates a Ca2+ sensitization pathway
in guinea pig bronchial smooth muscle, although this effect appears to
depend on increases in rMLC phosphorylation. Hotta et al.
(19) found that chronic exposure of cultured airway smooth
muscle to TNF-
increased G protein expression and enhanced some
responses to muscarinic stimulation. Airway smooth muscle may be
chronically exposed to a variety of inflammatory mediators in diseases
such as asthma, which are characterized by airway inflammation
(2). Any resultant increases in Ca2+
sensitivity might contribute to clinical hyperreactivity
(39). In particular, increases in maximal force produced
by airway smooth muscle could contribute to the lack of plateau in the
histamine dose-response relationship noted in asthmatic subjects,
producing an increase in maximal airway narrowing (43).
Studies of isolated airway smooth muscle have found inconsistent
effects on maximal force when comparing muscle from asthmatic subjects
(or from sensitized models) with normal subjects (4, 8).
Beyond the technical challenges involved in comparing maximal force
among airways (21), our results suggest that another
source of these discrepancies may be that regulation of maximal force
can be a relatively rapid process (
1 h in our studies). Thus any
changes present in vivo may be diminished by several hours of in vitro
studies in tissue baths where inflammatory mediators are not present.
Finally, we cannot exclude the possibility that the pattern of G
protein activation produced by chronic AlF
In conclusion, we demonstrate that, unlike the acute response to receptor agonists, which is mediated solely by rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in CTSM by additional mechanisms that are independent of rMLC phosphorylation. If also present in vivo, these mechanisms may be relevant to the development of airway hyperactivity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank K. Street for technical assistance.
![]() |
FOOTNOTES |
---|
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-45532 and HL-54757 and by grants from the Mayo Foundation.
Address for reprint requests and other correspondence: D. O. Warner, Anesthesia Research, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: warner.david{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 January 2001; accepted in final form 1 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amrani, Y,
Krymskaya V,
Maki C,
and
Panettieri RA, Jr.
Mechanisms underlying TNF- effects on agonist-mediated calcium homeostasis in human airway smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
273:
L1020-L1028,
1997
2.
Amrani, Y,
and
Panettieri RA, Jr.
Cytokines induce airway smooth muscle cell hyperresponsiveness to contractile agonists.
Thorax
53:
713-716,
1998
3.
Bigay, J,
Deterre P,
Pfister C,
and
Chabre M.
Fluoroaluminates activate transducin-GDP by mimicking the gamma-phosphate of GTP in its binding site.
FEBS Lett
191:
181-185,
1985[ISI][Medline].
4.
Bjorck, T,
Gustafsson LE,
and
Dahlen SE.
Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberation of leukotrienes and histamine.
Am Rev Respir Dis
145:
1087-1091,
1992[ISI][Medline].
5.
Bremerich, DH,
Kai T,
Warner DO,
and
Jones KA.
Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle.
Am J Physiol Cell Physiol
274:
C1253-C1260,
1998
6.
Chase, PB,
Martyn DA,
and
Hannon JD.
Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibbers from rabbit psoas muscle.
J Muscle Res Cell Motil
15:
119-129,
1994[ISI][Medline].
7.
Chase, PB,
Martyn DA,
Kushmerick MJ,
and
Gordon AM.
Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit.
J Physiol (Lond)
460:
231-246,
1993[Abstract].
8.
De Jongste, JC,
Mons H,
Bonta IL,
and
Kerrebijn KF.
In vitro responses of airways from an asthmatic patient.
Eur J Respir Dis
71:
23-29,
1987[ISI][Medline].
9.
Deng, JT,
Van Lierop JE,
Sutherland C,
and
Walsh MP.
Ca2+-independent smooth muscle contraction: a novel function for integrin-linked kinase.
J Biol Chem
276:
16365-16373,
2001
10.
Earley, JJ,
Su X,
and
Moreland RS.
Caldesmon inhibits active crossbridges in unstimulated vascular smooth muscle: an antisense oligodeoxynucleotide approach.
Circ Res
83:
661-667,
1998
11.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Lond)
75:
463-505,
1979.
12.
Gerthoffer, WT.
Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle.
J Pharmacol Exp Ther
240:
8-15,
1987[Abstract].
13.
Gong, MC,
Fujihara H,
Walker LA,
Somlyo AV,
and
Somlyo AP.
Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.
Mol Biol Cell
8:
279-286,
1997[Abstract].
14.
Gunst, SJ,
al-Hassani MH,
and
Adam LP.
Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle.
Am J Physiol Cell Physiol
266:
C684-C691,
1994
15.
Gunst, SJ,
Meiss RA,
Wu MF,
and
Rowe M.
Mechanisms for the mechanical plasticity of tracheal smooth muscle.
Am J Physiol Cell Physiol
268:
C1267-C1276,
1995
16.
Gunst, SJ,
Stropp JQ,
and
Flavahan NA.
Muscarinic receptor reserve and -adrenergic sensitivity in tracheal smooth muscle.
J Appl Physiol
67:
1294-1298,
1989
17.
Hanazaki, M,
Jones KA,
Perkins WJ,
and
Warner DO.
Halothane increases smooth muscle protein phosphatase in airway smooth muscle.
Anesthesiology
94:
129-136,
2001[ISI][Medline].
18.
Hori, M,
Sato K,
Sakata K,
Ozaki H,
Takano-Ohmuro H,
Tsuchiya T,
Sugi H,
Kato I,
and
Karaki H.
Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.
J Pharmacol Exp Ther
261:
506-512,
1992[Abstract].
19.
Hotta, K,
Emala CW,
and
Hirshman CA.
TNF- upregulates Gi
and Gq
protein expression and function in human airway smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
276:
L405-L411,
1999
20.
Iizuka, K,
Yoshii A,
Samizo K,
Tsukagoshi H,
Ishizuka T,
Dobashi K,
Nakazawa T,
and
Mori M.
A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea.
Br J Pharmacol
128:
925-933,
1999
21.
Jiang, H,
Halayko AJ,
Rao K,
Cunningham P,
and
Stephens NL.
Normalization of force generated by canine airway smooth muscles.
Am J Physiol Lung Cell Mol Physiol
260:
L522-L529,
1991
22.
Jones, KA,
Hirasaki A,
Bremerich DH,
Jankowski C,
and
Warner DO.
Halothane inhibits agonist-induced potentiation of rMLC phosphorylation in permeabilized airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
273:
L80-L85,
1997
23.
Jones, KA,
Perkins WJ,
Lorenz RR,
Prakash YS,
Sieck GC,
and
Warner DO.
F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs.
J Physiol (Lond)
519:
527-538,
1999
24.
Kahn, RA.
Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins.
J Biol Chem
266:
15595-15597,
1991
25.
Kai, T,
Jones KA,
and
Warner DO.
Halothane attenuates calcium sensitization in airway smooth muscle by inhibiting G-proteins.
Anesthesiology
89:
1543-1552,
1998[ISI][Medline].
26.
Kai, T,
Yoshimura H,
Jones KA,
and
Warner DO.
Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
279:
L52-L58,
2000
27.
Kamm, KE,
and
Stull JT.
The function of myosin and myosin light chain kinase phosphorylation in smooth muscle.
Annu Rev Pharmacol Toxicol
25:
593-620,
1985[ISI][Medline].
28.
Kitazawa, T,
Kobayashi S,
Horiuti K,
Somlyo AV,
and
Somlyo AP.
Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+.
J Biol Chem
264:
5339-5342,
1989
29.
Kitazawa, T,
Masuo M,
and
Somlyo AP.
G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle.
Proc Natl Acad Sci USA
88:
9307-9310,
1991[Abstract].
30.
Kureishi, Y,
Kobayashi S,
Amano M,
Kimura K,
Kanaide H,
Nakano T,
Kaibuchi K,
and
Ito M.
Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.
J Biol Chem
272:
12257-12260,
1997
31.
Mehta, D,
and
Gunst SJ.
Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle.
J Physiol (Lond)
519:
829-840,
1999
32.
Mehta, D,
Wang Z,
Wu MF,
and
Gunst SJ.
Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle.
Am J Physiol Cell Physiol
274:
C741-C747,
1998
33.
Mehta, D,
Wu MF,
and
Gunst SJ.
Role of contractile protein activation in the length-dependent modulation of tracheal smooth muscle force.
Am J Physiol Cell Physiol
270:
C243-C252,
1996
34.
Nishimura, J,
Kolber M,
and
van Breemen C.
Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle.
Biochem Biophys Res Commun
157:
677-683,
1988[ISI][Medline].
35.
Parris, JRM,
Cobban HJ,
Littlejohn AF,
MacEwan DJ,
and
Nixon GF.
Tumour necrosis factor- activates a calcium sensitization pathway in guinea-pig bronchial smooth muscle.
J Physiol (Lond)
518:
561-569,
1999
36.
Pavalko, FM,
Adam LP,
Wu MF,
Walker TL,
and
Gunst SJ.
Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction.
Am J Physiol Cell Physiol
268:
C563-C571,
1995
37.
Pennings, HJ,
Kramer K,
Bast A,
Buurman WA,
and
Wouters EF.
Tumour necrosis factor-alpha induces hyperreactivity in tracheal smooth muscle of the guinea-pig in vitro.
Eur Respir J
12:
45-49,
1998
38.
Pohl, J,
Winder SJ,
Allen BG,
Walsh MP,
Sellers JR,
and
Gerthoffer WT.
Phosphorylation of calponin in airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
272:
L115-L123,
1997
39.
Rodger, IW.
Asthma. Airway smooth muscle.
Br Med Bull
48:
97-107,
1992[Abstract].
40.
Somlyo, AP,
and
Somlyo AV.
Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II.
J Physiol (Lond)
522:
177-185,
2000
41.
Tang, D,
Mehta D,
and
Gunst SJ.
Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle.
Am J Physiol Cell Physiol
276:
C250-C258,
1999
42.
Togashi, H,
Emala CW,
Hall IP,
and
Hirshman CA.
Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
274:
L803-L809,
1998
43.
Woolcock, AJ,
Salome CM,
and
Yan K.
The shape of the dose-response curve to histamine in asthmatic and normal subjects.
Am Rev Respir Dis
130:
71-75,
1984[ISI][Medline].
44.
Zhang, Y,
Moreland S,
and
Moreland RS.
Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways.
Can J Physiol Pharmacol
72:
1386-1391,
1994[ISI][Medline].