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Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine,
Maebashi; and 2Gunma University Faculty of Health Sciences, Gunma 371-8511, Japan
Submitted 21 August 2003; accepted in final form 26 April 2004
2
ALTHOUGH INTRACELLULAR calcium concentration Ca
([Ca2]i)
is the primary regulator of smooth muscle contraction, Ca2
sensitivity of the contractile apparatus can change in response
to agonists. An increase and a decrease in muscle tension at a
constant Ca2 concentration are correspondingly referred to as
Ca2 sensitization and desensitization of smooth muscle con-
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of page charges. The article must therefore be hereby marked advertisement
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L641
L642
RESULTS
Effects of 8-BrcAMP and Y-27632 on CCh- or GTPSinduced Ca2 sensitization in -toxin-permeabilized tracheal
smooth muscle. After obtaining the maximum contraction at
pCa 5.0, we incubated the strip in G1 containing 8-BrcAMP,
Y-27632, or saline for 20 min. In the continuous presence of
the reagents, the tracheal smooth muscle was precontracted in
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Fig. 3. Inhibition of 4-phorbol 12,13-dibutyrate (PDBu)-induced Ca2 sensitization by 8-BrcAMP in the presence of Y-27632. After obtaining the
maximum contraction at pCa 5.0, we treated tracheal smooth muscle with
Y-27632 (30 M) or saline for 20 min, and then PDBu (10 M)-induced Ca2
sensitization was evoked at pCa 6.5. After we verified that the concentration of
Y-27632 was saturated, 8-BrcAMP dose dependently reversed contraction of
the trachea (A), although Y-27632 tended to decrease PDBu-induced contractions. The contractions before 8-BrcAMP application were not statistically
significant (P 0.147, Mann-Whitney U-test), and the IC50 values with or
without Y-27632 were also comparable; the amplitude of the PDBu response
and the IC50 values for 8-BrcAMP in the Y-27632-treated group (F) were
53.1 7.1% and 5.24 0.8 M (n 7); those in the control group (E) were
70.0 8.1% and 8.21 1.0 M (n 11), respectively (B). Relative force was
normalized to the initial pCa 5.0 (105 M) response in the same strip.
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L645
Half-Time of Relaxation, s
DISCUSSION
-Adrenergic agonists can be understood as a cascade involving activation of adenyl cyclase, elevation of cytoplasmic
cAMP levels, and PKA activation leading to phosphorylation
of target proteins. However, the precise mechanisms are still
unknown. In Fig. 2, the contractile rate (T1/2) was comparable
between 8-BrcAMP-treated strips and control strips. The T1/2
of the strips treated with a phosphatase inhibitor (such as
microcystin-LR or calyculin A) is dependent on the MLCK-
Maximum
Contraction
at pCa 5.0,
%
Reagent
PDBu
GTPS
LTD4
Control
8-BrcAMP
Y-27632
8-BrcAMP Y-27632
41137.4
27415.3*
3528.95
24715.7*
37337.5
38222.2
2697.51
2531.52
70.44.7
43.63.7
33.76.0
9.142.3
Fig. 6. Inhibitory effect of 8-BrcAMP and Y-27632 on leukotriene D4 (LTD4)induced Ca2 sensitization in human bronchial smooth muscle. In -toxinpermeabilized human bronchial smooth muscle, after the maximum contraction at pCa 5.0 and the submaximum contraction from pCa 6.8 were obtained,
LTD4 (1 M) was added to strips. When LTD4 caused contraction became
stable, 8-BrcAMP (100 M) or Y-27632 (3 M) or both were added to the
strips. Developed force was normalized to the initial pCa 5.0 response
(n 4 8).
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signaling. Alternatively, this observation could have mechanistic implications; for example, one of many interpretations
would be that 8-BrcAMP modulates activity of MLCP-associated mechanisms whereas Y-27632 modulates activation of
MLCP-associated mechanisms, because 8-BrcAMP accelerates the rate of relaxation without changing the time of onset,
whereas Y-27632 prolongs the onset of relaxing rate without
changing its rate (Fig. 5, B and C).
Muscarinic receptor signaling for airway smooth muscle
contraction contains Ca2 sensitization mechanisms mediated
by Rho/Rho-kinase (14, 28). Although CCh-induced Ca2
sensitization was inhibited by both 8-BrcAMP and Y-27632 to
a similar extent, GTPS-induced Ca2 sensitization was resistant to 8-BrcAMP. In permeabilized canine tracheal smooth
muscle, both PDBu and acetylcholine induce Ca2 sensitization (3, 4). Rho/Rho-kinase-mediated signaling may be distinct
from the PKC system because the effects of saturating concentrations of GTPS and PDBu were additive (9). Eto et al. (5)
reported that PDBu-induced Ca2 sensitization was partially
inhibited by Y-27632 in -toxin-permeabilized rabbit femoral
artery. However, we previously reported only a minor contribution of PKC to GTPS-induced Ca2 sensitization in rabbit
trachea. In our present study, PDBu-induced Ca2 sensitization
was not inhibited by Y-27632 (13), although the reason for the
discrepancy is unknown.
In the present study, we demonstrated leukotriene-induced
Ca2 sensitization in human bronchial smooth muscle. Leukotrienes, as well as CCh, increased Ca2 sensitivity in human
bronchial smooth muscle, and leukotriene-induced Ca2 sensitivity is reversed by 8-BrcAMP and Y-27632 (Fig. 6). Leukotrienes evoke a potent, sustained contraction of intact human
airway smooth muscle (2, 24). Leukotrienes transiently increased Ca2 sensitivity in -toxin-permeabilized porcine tra-
Fig. 7. Signaling pathway for Ca2 sensitization in smooth muscle. Activation of the muscarinic receptor (M3) initiates signaling
through the illustrated cascades that inhibit MLCP, increasing MLC20 phosphorylation and contraction. The Rho/Rho-kinase and
PKC/CPI-17 pathways inhibit the phosphorylation of MLCP and enhance the Ca2 sensitivity of myosin phosphorylation. GTPS
increases the activity of G-binding proteins, including the heterotrimetric G protein and small G proteins such as Rho/Rho-kinase.
PDBu increases the activity of PKC. GTPS-induced Ca2 sensitization is inhibited by Y-27632, but not by 8-BrcAMP. On the
other hand, PDBu-induced Ca2 sensitization was inhibited 8-BrcAMP, but not by Y-27632. Thus 8-BrcAMP has the potential to
affect the PKC/CPI-17 pathway and upstream of the Rho/Rho-kinase pathway.
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ACKNOWLEDGMENTS
The authors thank Y. Ohtani for preparing human lung tissue samples.
REFERENCES
1. Accomazzo MR, Rovati GE, Vigano T, Hernandez A, Bonazzi A, Bolla
M, Fumagalli F, Viappiani S, Galbiati E, Ravasi S, Albertoni C, Luca
MD, Caputi A, Zannini P, Chiesa G, Villa AM, Doglia SM, Folco G,
and Nicosia S. Leukotriene D4-induced activation of smooth-muscle cells
from human bronchi is partly Ca2-independent. Am J Respir Crit Care
Med 163: 266 272, 2001.
2. Arm JP and Lee TH. Sulphidopeptide leukotrienes in asthma. Clin Sci
(Lond) 84: 501510, 1993.
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